Fight Aging!

The Life Extension Advocacy Foundation volunteers have a stack of interview materials piled up from the recent Undoing Aging conference, I'll wager. Today they published a lengthy interview with Steve Horvath, originator of one of the epigenetic clocks that assess age based on patterns of DNA methylation. I have to say that it is a pleasure to see so many researchers now willing to talk openly about therapies for aging and their hopes for the future of the field. For so very many years that just didn't happen; no researcher was willing to speak in public on the topic of treating aging as a medical condition and extending healthy life.

When I first become interested in this field, the research and funding institutions that dominated the study of aging were quite hostile towards anyone who wanted to intervene in the aging process and thereby produce longer lives in patients. Thankfully, times have certainly changed since then, and for that we can thank the hard-working community of advocates, scientists, and philanthropists who have since the turn of the century built funding and support for rejuvenation biotechnology, and for the transformation of medical science through the treatment of aging.

The Horvath epigenetic clock has a commercial implementation, myDNAage, produced by Zymo Research. The folk there claim it to be quite stable over time and circumstances in its assessment, with a margin of error of 1.7 years. As noted in the interview, it is one thing to have a metric that maps to age quite well, and shows some signs of reflecting biological age versus chronological age, but it is quite another to know what it is actually measuring. These changes in DNA methylation are some reflection of the growing damage and dysfunction of aging, but are they a very selective reflection? What if it is found that when a human undergoes a senolytic therapy to remove senescent cells, one of the root causes of aging, that before and after measures of epigenetic age are the same? That would be an interesting outcome, and we're about to find out, but what could we learn from it? This result seems unlikely given that cellular senescence appears to contribute to many aspects of age-related decline, but it isn't impossible. Researchers really don't know whether the epigenetic clock reflects one or many of the underlying aspects of aging.

Science and medicine are often a process of starting at the ends and meeting in the middle. A list of the root causes of aging exist, and measures of the state of aging exist, but there is not good map to link those two. Aging progresses in a very complex way, even though it is caused by comparatively simple and easily understood processes, because our biology is very complex. The fastest way to build rejuvenation therapies and metrics to measure the results of rejuvenation therapies is to start on both lines of research and development at the same time, and compare the results against one another. Iteration on this theme will find the path ahead, and allow unhelpful approaches to be discarded earlier rather than later.

Steve Horvath - Aging and the Epigenetic Clocks

Why is the epigenetic clock more accurate than measuring telomere length?

Yes, it is far more accurate, there is no comparison. Why is a good question. In my opinion, it shows that epigenetic changes are far more important for aging than telomere maintenance. People have studied telomeres for many years, including me, but telomere shortening alone does not explain aging. You may know that mice have perfect telomeres, but they only live three years.

It's known that cells in our body are renewed at different speeds; why does your clock measure the age of the tissue or whole organ and not the age of specific cells?

Actually, it does measure the age of specific cells. You can have liver cells, and the epigenetic clock works beautifully. It also works very well for neurons and glial cells. Even in blood, you can have sorted blood, for example T cells or B cells, and the clock works on those cells.

Does your clock represent aging?

This is a good question with two answers. One way to ask this question is to ask if methylation changes cause aging. And we honestly don't know; there is no data. The other question to ask is if the epigenetic clock is the indicator of a biochemical process that plays a role in aging. Which I think it is; it is a biomarker of a process. There is no question that this process that underlies the clock, that if you target this process, you slow aging; this, we know.

What is going to happen if we influence this methylation process?

With the methylation process, we don't know. Imagine that you have a clock; there is the clock face with the dials, and then there is the clockwork. The discussion with the epigenetic clock is whether methylation is part of the dial or is it part of the clockwork. There is no doubt that it is part of the dial, and if you interfere with the clockwork, there is no question you that rejuvenate people. But it could be that the clockwork might not be the same as methylation; we are not sure. With a clock face, you can just take the hands and move them, but it may do nothing to actual time. Behind the clock, there is the clockwork, and we don't completely understand the clockwork. A lot of people are asking about it, but we just don't know yet.

Can we slow down aging now?

I want to tell you that I am very optimistic and that we will have treatments against aging in a few years. I could be wrong, and I want to be cautious, but I want to tell you that I am very optimistic because we already have encouraging results. We already have treatments that have a huge effect, like the Yamanaka factors in mice, but also in human cells. If you use Yamanaka factors on human cells, it completely reverses their age. The problem is how to make them safe.

My hope is that maybe even our generation will benefit from it; certainly, my daughter should benefit from it. I would be absolutely shocked if the next generation does not live twenty years longer. On that level, I am very optimistic. If you ask me right now what you should do, I can only tell you boring things; immediately stop smoking, avoid obesity, avoid diabetes; if you are a diabetic, manage it; avoid high blood pressure, and if you have it, take action. It is boring, but all my studies show that this is the best thing we can do now.

What are the main challenges in your research in aging?

Scientific challenges, honestly I don't have them. Because there is so much work to do and I have a good plan, it is not a problem. Financially, there is a challenge; research is expensive, especially human trials. I have a very exciting collaboration with a company which has an anti-aging treatment, and to test it will cost three million dollars. So, as you can imagine, money is the challenge.

The research here notes an aspect of mitochondrial biochemistry that declines with age in a way that appears unaffected by fitness and exercise. One of the challenges inherent in investigating the mechanisms of aging in muscle tissue is determining the difference between decline due to disuse (secondary aging) versus decline due to intrinsic processes of damage accumulation (primary aging). We live in a world in which being older tends to mean being wealthier, with greater access to transportation and calories. Near all older adults fail to maintain a good program of exercise and diet, and the difference between those who make the effort to remain fit and slim and those who do not is sizable. That much is demonstrated by the significant gains in cardiovascular health and muscle strength that can be achieved in the elderly through structured exercise programs. So it is interesting to see research results in which the data makes it very clear that a specific measure of aging in muscle tissue is independent of exercise.

Aging is a complex process associated with skeletal muscle and strength loss as well as insulin resistance. The cellular mechanisms causing muscular and/or metabolic dysfunction with aging remain poorly understood. However, one proposed mechanism of action driving the aging process is an increase in mitochondrial-derived reactive oxygen species (ROS). Specifically, increased ROS emission has been associated with motor unit loss and abnormal morphology, muscle fiber atrophy, insulin resistance, inflammation, and apoptosis. Conversely, transgenic and pharmacological approaches that attenuate mitochondrial ROS have been shown to preserve insulin sensitivity, mitochondrial content, and muscle mass in diverse models while also prolonging lifespan. Altogether, these data implicate mitochondrial ROS as a fundamental mechanism of action influencing the aging phenotype.

Although these elegant rodent models provide compelling evidence to link mitochondrial ROS with age-associated pathologies, the data in humans remain ambiguous. Contradictory findings suggest that either mitochondria are not responsible for the increased oxidative stress with aging or, alternatively, contemporary in vitro assessment of mitochondrial ROS emission does not accurately reflect in vivo responses. ADP transport is a highly regulated process that is attenuated with rodent models of insulin resistance and improved following high-intensity exercise. Moreover, there is indirect evidence to suggest that the protein required for ADP transport into mitochondria, adenine nucleotide translocase (ANT), is impaired with aging in housefly and rat skeletal muscle. Therefore, previous assessments of mitochondrial ROS emission in the absence of ADP may not adequately reflect the in vivo environment, and as a result current data from human skeletal muscle may underestimate the importance of mitochondrial ROS in the aging process.

In the present study we re-evaluated mitochondrial bioenergetics by establishing a protocol in permeabilized muscle fibers to simultaneously examine mitochondrial respiration and hydrogen peroxide (H2O2) emission in the presence of various substrates and ADP concentrations. Using this in vitro protocol, we assessed age-related mitochondrial defects by comparing healthy young males to healthy older males. We also examined whether potential age-related defects in mitochondrial bioenergetics could be improved over 12 weeks of resistance exercise training. We provide compelling evidence that although the capacity for mitochondrial ROS emission is not increased with aging, mitochondrial ADP sensitivity is impaired, such that mitochondrial ROS, and the fraction of electron leak to ROS, are increased in the presence of virtually all ADP concentrations examined. In addition, although resistance-type exercise training improved several aspects of muscle health in older individuals, the fraction of electron leak to ROS, mitochondrial H2O2 emission rates in the presence of ADP, and muscle oxidative stress were unaltered, suggesting an increase in mitochondrial ROS accompanies the aging process.

Altogether, the assessment of mitochondrial bioenergetics in the presence of sub-saturating ADP concentrations has revealed that there are age-associated impairments in mitochondrial bioenergetics, which are not fully recovered with prolonged resistance-type exercise training. The mechanism for the attenuation in ADP sensitivity remains unknown, but oxidative damage has been proposed as a likely explanation. Regardless of this knowledge gap, the present data imply that an increase in mitochondrial ROS is associated with the primary aging process. Moreover, despite the inability of resistance training to rectify age-related mitochondrial ROS emission, older individuals experienced favorable changes in muscle mass, strength, and fat mass, reinforcing the importance of a physically active lifestyle throughout the lifespan.

Link: https://doi.org/10.1016/j.celrep.2018.02.069

In this interview, a researcher focused on mitochondrial biochemistry discusses the role of these important cellular structures in aging and neurodegeneration, particularly Parkinson's disease. There are really two ways of looking at mitochondria in aging. The first, the view incorporated into the SENS program, looks at damage to mitochondrial DNA and its consequences. A small but significant number of cells fall into a dysfunctional state because some forms of randomly occurring mitochondrial DNA damage can replicate rapidly within the cell, leading to cells that pollute their surroundings with reactive, harmful molecules. This might be addressed by providing backup copies of mitochondrial genes, a methodology known as allotopic expression.

The second view looks at mitochondrial dynamics and morphology, both of which change considerably in response to differences in the environment between old cells and young cells, old tissues and young tissues. This is a much more complex problem to consider, as no-one has yet mapped the chains of cause and effect that stretch from the fundamental forms of damage at the root of aging to this downstream manifestation of aging. Nor is it entirely clear how to best go about reversing these changes - not to mention whether or not some are adaptive to the damaged environment, protective rather than the cause of even more harm.

How many mitochondria are there within each dopamine producing neuron and how frequently are they created?

The dopaminergic neurons in the pars compacta of the substantia nigra, the ones most related to Parkinson's disease, have enormous axons. If you add up all the branches, it is estimated that you would have several meters of axon coming from each cell. If you take the density of mitochondria in a segment of axon, you can then calculate what the total would be. The number is roughly two million mitochondria in each neuron. That's two million mitochondria frantically consuming oxygen and making ATP, all to keep that one cell alive.

On top of that, the proteins in the mitochondria are not going to stay stable for the 80 to 100 years that we live for. The proteins start to fall apart because of heat and the environment they are in. It turns out the mitochondria are a particularly dangerous place for a protein to be, because the mitochondria, in the process of its respiration, generate reactive oxygen species (ROS) which collide with proteins and chemically alter and damage them. Proteins everywhere in the cell have to be constantly degraded and replaced; in a mitochondrion that is even more true because the proteins get damaged even faster.

So we did a back of the napkin calculation, and asked how many mitochondria that cell would have to create every day in order to keep its two million mitochondria healthy and happy? The answer is something like thirty thousand mitochondria created every day. Most of the cells in our body don't have this problem, skin cells and liver cells are tiny and don't need nearly as many mitochondria. That could be part of the reason why, when something is wrong with our mitochondria, it is our neurons that suffer first, particularly the biggest neurons.

Does all that explain why, in Parkinson's disease, these neurons die and not other neurons?

Well, we don't know for sure yet what makes one cell more sensitive than another, but I think that is an excellent guess. The fact that those nerve cells fire at a very high rate, and that every time they fire it opens up a particular type of calcium channel that lets a lot of calcium in, means that you are going to need a lot of ATP to pump that calcium back out of the cell, as well as pumping sodium and other things. That puts a very strong demand on the cell. Then the fact that it has so many branches and so many synapses on the end of it also means that you are going to need a lot of energy to power those synapses. It is indeed a very energy hungry type of nerve cell, and nerve cells are the most energy hungry type of cell in the body. So it has this dual problem of supplying enough mitochondria and then putting strain on the mitochondria to travel through the axons and pump out enough ATP.

Which therapies that target mitochondrial health are you most hopeful for?

I think there are four ways to try to approach it. If you can figure out what is damaging the mitochondria and stop the damage that would be a great thing. In some cases antioxidants might do that. In cases where there are environmental toxins, like paraquat or rotenone, getting those out of the environment is definitely going to help. But in the case where there is a genetic mutation, you can increase the rate at which damaged mitochondria are removed and hope that the cell compensates by increasing the rate of production of healthy ones. There are also genes that control how mitochondria replicate and how they get new proteins added to them, if we can figure out how the cell controls the number of mitochondria and increase that number, that could improve the health of the cell.

Finally, the one that I am most interested in is the transportation problem. It is one thing to try and get proteins into the mitochondria in the cell body, but that cell body is just a tiny fraction of the volume of the neuron, way less that 1% of the cell. The cell has to somehow get mitochondria all the way out to the periphery of the cell and through all of its many axons. Improving the delivery of mitochondria into the remote regions of the cell should also improve the health of the cell.

Link: https://tmrwedition.com/2018/03/29/interview-with-mitochondrial-dysfunction-expert-prof-thomas-schwartz/

What sort of evidence would it take to challenge my assessment of the data to date that methods of raising NAD+ levels with age, such as nicotinamide riboside supplementation, are not worth pursuing as a major area of focus in research and development? Given the history of work in this area of metabolism, mostly that relating directly to sirtuins and their manipulation, one has to be a little skeptical. Initially promising (and overhyped) results in mice went essentially nowhere, or turned out to make the condition of obesity a little less harmful, while showing little evidence of utility for healthy individuals.

To answer the question, human data showing meaningful benefits that could not be achieved via exercise or calorie restriction would be very interesting. Human data showing some reliable level of reproduction of the benefits of exercise or calorie restriction without side-effects would be good news for the present majority who don't put in the effort to stay in shape. Good news for supplement sellers as well - there is no shortage of people who would pay rather than exercise or eat less, even if the results were mixed or marginal.

In either case, the cost-benefit analysis runs along the lines of (a) as an individual, how much it is worth spending on a supplement that can capture a fraction of the benefits of exercise or calorie restriction, but also (b) is it worth making this a major focus of the research community, versus the rejuvenation biotechnology that can achieve far greater gains? I think (b) is always going to be answered in the negative, for me at least. No calorie restriction mimetic or exercise mimetic can possibly be as good as functional SENS repair biotechnologies. They cannot achieve the results produced by senolytics, or any of the other ways to remove the root causes of aging. If one looks at NAD+ research as the final stage of sirtuin-related calorie restriction research as a whole, it has taken as much funding to get here as it would to completely implement the SENS rejuvenation therapy package in mice. Yet we know that exercise and calorie restriction cannot add decades to healthy life, as is possible in principle for repair therapies.

The data here on human nicotinamide riboside supplementation seems promising in comparison to the results of past sirtuin research, but I'd like to see a larger study group. If that larger group shows similar results, then maybe this is worth it for individuals. Either way, it is appreciated that the authors avoided running a study in overweight individuals - in this part of the field, that just muddies the waters, given the very different effects of sirtuin manipulation on thin versus fat animals. Nonetheless, it still appears to be the case that this is essentially a way to gain some of the beneficial long-term effects of fitness without putting in the physical effort. I expect future NAD+ studies and exercise studies in older individuals to converge in some ways, showing overlapping effects on cellular biochemistry. It is arguable as to whether taking up exercise, eating less, or artificially increasing NAD+ levels should be termed rejuvenation. There is certainly a sizable grey area at the intersection of repair, compensation, and overriding regulatory signals that respond to aging.

A pill that staves off aging? It's on the horizon

Scientists have long known that restricting calories can fend off physiological signs of aging. A new study indicates that when people consume a natural dietary supplement called nicotinamide riboside (NR) daily, it mimics caloric restriction, aka CR, kick-starting the same key chemical pathways responsible for its health benefits. "This was the first ever study to give this novel compound to humans over a period of time. We found that it is well tolerated and appears to activate some of the same key biological pathways that calorie restriction does."

Researchers included 24 lean and healthy men and women ages 55 to 79. Half were given a placebo for six weeks, then took a 500 mg twice-daily dose of nicotinamide riboside (NR) chloride (NIAGEN). The other half took NR for the first six weeks, followed by placebo. The researchers took blood samples and other physiological measurements at the end of each treatment period. Participants reported no serious adverse effects. The researchers found that 1,000 mg daily of NR boosted levels of another compound called nicotinamide adenine dinucleotide (NAD+) by 60 percent. NAD+ is required for activation of enzymes called sirtuins, which are largely credited with the beneficial effects of calorie restriction. It's involved in a host of metabolic actions throughout the body, but it tends to decline with age.

Research suggests that as an evolutionary survival mechanism, the body conserves NAD+ when subjected to calorie restriction. But only recently have scientists begun to explore the idea of supplementing with so-called "NAD+-precursors" like NR to promote healthy aging. "The idea is that by supplementing older adults with NR, we are not only restoring something that is lost with aging (NAD+), but we could potentially be ramping up the activity of enzymes responsible for helping protect our bodies from stress."

The new study also found that in 13 participants with elevated blood pressure or stage 1 hypertension (120-139/80-89 mmHg), systolic blood pressure was about 10 points lower after supplementation. A drop of that magnitude could translate to a 25 percent reduction in heart attack risk. "If this magnitude of systolic blood pressure reduction with NR supplementation is confirmed in a larger clinical trial, such an effect could have broad biomedical implications."

Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults

Nicotinamide adenine dinucleotide (NAD+) has emerged as a critical co-substrate for enzymes involved in the beneficial effects of regular calorie restriction on healthspan. As such, the use of NAD+ precursors to augment NAD+ bioavailability has been proposed as a strategy for improving cardiovascular and other physiological functions with aging in humans. Here we provide the evidence in a 2 × 6-week randomized, double-blind, placebo-controlled, crossover clinical trial that chronic supplementation with the NAD+ precursor vitamin, nicotinamide riboside (NR), is well tolerated and effectively stimulates NAD+ metabolism in healthy middle-aged and older adults.

Our results also provide initial insight into the effects of chronic NR supplementation on physiological function in humans, and suggest that, in particular, future clinical trials should further assess the potential benefits of NR for reducing blood pressure and arterial stiffness in this group.

While I suspect that improvements in energy management coupled with magnetic field technologies are the cost-effective way forward when it comes to engineering defenses against radiation for space travel, it is certainly possible to consider classes of biomedical solution that could in principle greatly improve resistance to radiation in mammals. Insofar as that would require an improved capacity for cells to manage and repair oxidative damage and DNA damage, it seems likely that success would lead to treatments and enhancement biotechnologies that also slow the progression of aging.

The degree of slowing, and how it breaks down into cancer resistance versus other aspect of aging, depends on the degree to which DNA damage and oxidative damage are important in normal aging, versus the contributions of other causes and processes. The evidence of recent decades doesn't provide sufficient support for a definitive view on this topic. That will continue to be the case, I suspect, until such time as effective ways to repair or remove individual contributions to aging in isolation from one another are developed and extensively tested. So far theory and inspection have proven poor approaches to the production of good numbers for the relative contribution of specific processes to the progression of aging. Our biology is too complex to make much headway towards these detailed answers via analysis without intervention at the present time.

While many efforts have been made to pave the way toward human space colonization, little consideration has been given to the methods of protecting spacefarers against harsh cosmic and local radioactive environments. The main components of space radiation are solar particle events (SPE), geomagnetically trapped radiation, and galactic cosmic radiation (GCR). The contribution of the first two to the total dose absorbed by astronauts would obviously be negligible on long-duration missions away from Earth and the Sun. Consequently, GCR consisting mainly of highly-energetic particles would be the primary type of radiation encountered by humans under this scenario. It has been estimated that a return trip to Mars could subject astronauts to radiation doses of 660 mSv. Although great uncertainties exist with respect to health (cancer) risk estimates from exposure to cosmic radiation, this dose alone represents more than half of the total NASA astronaut career limit.

In principle, ionizing radiation interacts along charged particle tracks with biological molecules such as DNA. The process is largely stochastic, and can damage DNA via direct interactions or via indirect interactions such as through the production of reactive oxygen species (ROS). Radioresistance denotes the capacity for organisms to protect against, repair and remove molecular, cellular, and tissue damage caused by ionizing radiation. It is a quality that varies greatly in terms of effectiveness between different organisms. For instance, it is well-known that certain organisms are remarkably resistant to the damaging effects of radiation. The bacterium Deinococcus radiodurans, for instance, possess error-free DNA repair mechanisms and can withstand doses as high as 7 kGy. Similarly, tardigrades can withstand doses as high as 5 kGy, though doses exceeding 1 kGy render them sterile.

All eukaryotic organisms have evolved against a backdrop of constant exposure to endogenous and exogenous mutagens, and as such have developed robust cellular mechanisms for DNA repair and protection against DNA damage. Substantial experimental evidence suggests that low-dose radiation may trigger a variety of protective responses within cells, tissues and organisms that serve to protect them from both exogenous (e.g high doses of radiation) and endogenous (e.g. age-related accumulation of DNA damage) genomic instabilities. Importantly, these responses, collectively termed radioadaptive responses or radiation hormesis, may protect against spontaneous or induced cancer.

Genome instability resulting from DNA damage and mutation in both nuclear DNA and mitochondrial DNA caused by replication errors and exposure to endogenous and exogenous mutagens has long been implicated as one of the main causes of aging. All strategies for enhancing radioresistance in humans, from the expression and overexpression of exogenous and endogenous DNA repair genes, antioxidants, and ROS scavengers, to the expression of exogenous radioprotective genes, would also serve as a means of attenuating DNA damage and mutation implicated in eukaryotic aging. As such, strategies for enhancing radioresistance in humans would also constitute a promising geroprotective strategy and a means of attenuating aging and promoting longevity and extension of both lifespan and healthspan in humans as well.

Link: https://doi.org/10.18632/oncotarget.24461

This recent interview with Gary Hudson of Oisin Biotechnologies covers a range of topics; there is a lot more to it than is quoted here. The company is working on the application of a programmable gene therapy to the targeted destruction of senescent and cancerous cells. Since the approach can be adjusted to kill cells that express significant amounts of any arbitrarily selected target protein, it can in principle be adapted to destroy other types of unwanted cell. The immune system in older individuals or patients with autoimmune diseases, for example, contains any number of problem cells that it would be beneficial to remove. As noted in the interview, destruction is only one possibility, however. Cells could be enhanced or have their behavior changed in other ways: destroying cells is the simple first exploration of a class of genetic technology that will over time grow to power a vast and diverse field of targeted cell reprogramming.

Feinerman: Your last interview was in July 2017, more than half a year ago. What has been accomplished?

Hudson: We have conducted many pre-clinical mouse experiments on both cancer and senescent cell removal. All have been successful and produce very remarkable results. We've also conducted a pilot toxicity and safety trial on non-human primates. The results of that trial were also successful and encourage us to proceed to human safety trials as soon as regulatory authorities approve them. We have also spun-out a cancer-focused company, Oisin Oncology, and raised a seed round for that venture.

Feinerman: Great to hear! However, when can we see some papers?

Hudson: Papers are being prepared now for submission to major journals, but that process takes time, especially the peer review. For the moment, most of our data is only available to investors and partners in pharma and the biotech industry.

Feinerman: You planned human clinical trials, have you carried them out?

Hudson: It takes quite some time to organize a human trial and to get it approved. Before one can be conducted, we have to set up so-called "GMP (Good Manufacturing Practice)" manufacture of our therapeutic, and then we have to conduct "GLP (Good Laboratory Practice)" toxicity studies in two different species. Once that is all completed later this year, then we can begin a human safety trial, or a "Phase 1" trial. All this takes time, but we hope that first safety trials in oncology indications might begin this year, or in early 2019.

Feinerman: When we can expect your therapy available in the clinic?

Hudson: It's very difficult to predict. I believe that our cancer treatment will make it to the clinic first, and that could happen in less than five years. Since the FDA doesn't regard ageing as an indication, it may take longer for our treatment to reach the public, since the regulatory environment will need to change.

Feinerman: Now you use only a suicide gene as an effector, do you plan to use other genes? For example to enhance the cells, give them ability to produce new enzymes, or temporarily shutdown telomerase to help anti-cancer therapy to be more effective.

Hudson: We believe we can express any gene under the control of any promoter we wish to use, so the possibilities are almost endless. If people wish to design their own constructs for particular applications they may contact us for collaboration, though we do have several collaborations active at the moment so we may already be working on similar ideas.

Feinerman: How much resources, finances and personnel, you need to move as quickly as possible? Have you open positions? Maybe, some of our readers have enough finances or experience.

Hudson: We could effectively spend tens of millions of dollars or more, very easily, but it isn't realistic to assume we could raise that amount - and if we did, we'd lose control of Oisin's ageing focus, since investors would most likely want us to aim at quick returns. We are always interested in talking with "mission minded" investors, however. As for hiring, we have to do that slowly and judiciously, since labour is one of the biggest costs to a start up company and over-hiring can sink a project quickly. We already have more potential hires than we can bring on-board.

Feinerman: One person has said, we get what we ask for. Can we now aim high and publicly claim that our main goal is not additional five years of life but LEV - Longevity Escape Velocity and finally unlimited healthy life?

Hudson: This is a difficult public relations problem. Most investors, the scientific community, and the public are not yet ready to embrace the notion of longevity escape velocity. Thus at Oisin we do pitch health span as a primary goal. But personally I don't believe that you can obtain health span improvements without making significant progress towards LEV. So in the end, I think we get LEV by targeting health span, and we reduce the controversy by doing so.

Link: https://medium.com/@arielf/the-rise-of-oisin-d4664ed7e9

The paper I'll point out today reports on the effects of calorie restriction in mice and humans on markers of cellular senescence, one of the contributing cause of aging. Calorie restriction is well known to slow aging and extend life span in a near all species and lineages tested, with that effect being largest in short-lived species. Mice live up to 40% longer when calorie restricted, but in humans it would be surprising to find an effect larger than five years or so - once firm data is in hand, which is not presently the case. Nonetheless, the short term benefits to health and the changes to cellular metabolism produced by the practice of calorie restriction are quite similar across mammalian species of different life spans.

These are sweeping changes: near every measure of metabolic activity and progression of aging is altered by calorie restriction. Given that, it is challenging to identify the size of the contribution of any given mechanism, but it is certainly fair to ask. To what degree does calorie restriction act through a reduction of each of the forms of cell and tissue damage that cause aging? One of the forms of damage is an accumulation of senescent cells. Cellular senescence is a fascinating phenomenon with both positive and negative outcomes; it is beneficial when temporary, as cells briefly become senescent in order to aid in regeneration or reduce the risk of damaged cells becoming cancerous. When senescent cells fail to quickly self-destruct, however, they linger to cause harm to surrounding tissue. Their signals generate chronic inflammation, destroy important molecular structures, and change the behavior of other cells for the worse.

Removing all senescent cells increases mouse life span by 25%, calorie restriction increases mouse life span by 40%, and calorie restricted mice still have some number of senescent cells. From a first glance at the numbers and the existing evidence, reduced cellular senescence can only only account for a modest fraction of the benefits of calorie restriction. In line with that, the paper noted here shows that calorie restricted mice and humans appear to have fewer signs of senescent cell activity, consistent with reductions in all of the other measures of age-related damage under calorie restriction.

The interesting question is how exactly calorie restriction produces this outcome. Fewer cells become senescent? More senescent cells successfully self-destruct? Individual senescent cells are less actively harmful, and their signaling is reduced? One item to bear in mind while thinking about this is the evidence for the benefits of calorie restriction to be absolutely reliant upon autophagy - increased autophagy is a feature of calorie restriction, as well as many other methods of slowing aging, and in animals in which autophagy is disabled, calorie restriction does not improve life span or health. So it seems to me that any consideration of calorie restriction and cellular senescence must in some way involve autophagy.

The effects of graded caloric restriction: XII. Comparison of mouse to human impact on cellular senescence in the colon

While genetic manipulations of model organisms have set important milestones for the understanding of the aging process, calorie restriction (CR) is a well-established nongenetic approach able to improve health span and lifespan in different organisms. However, the precise mechanisms by which CR improves health are not fully understood. More than 50 years ago, cellular senescence was discovered. Subsequent studies demonstrated that senescent cells gradually accumulate with increasing age in various organisms. During aging, senescent cells impair cellular turnover and tissue regeneration due to their inability to proliferate, and stimulate a pro-disease environment by the chronic secretion of various pro-inflammatory and tissue-remodeling factors, a phenotype called Senescence-Associated Secretory Phenotype (SASP).

Genetic and pharmacological elimination of senescent cells is sufficient to improve health span. Interestingly, a previous report suggested that CR prevented accumulation of senescent cells in the mouse liver and intestine. To further explore the potential reduction in senescent cells upon short-term CR, and whether this phenomenon might potentially happen in humans, we analyze various classical transcriptomic markers for senescence and SASP in short-term CR interventions in the mouse and human colon mucosa specimens.

Male mice were aged 20 weeks when they entered four levels of CR for 12 weeks: 10%, 20%, 30%, and 40% restriction from baseline food intake. The colon of these mice was divided into three regions: proximal, medial, and distal. In the proximal colon, the expression levels of two classical markers of senescence-associated growth arrest, p16 and p21, did not change significantly among groups. Selected markers for the SASP also did not significantly change. In the medial colon, while there were no differences among the two controls and the lowest CR interventions (10%-20%), all markers of senescence were downregulated at higher CR regimens. A similar trend was present in the distal colon. These data suggest that short-term CR at higher levels can prevent or decrease the accumulation of senescent cells in the mouse colon, even in adult but relatively young animals on short-term restriction.

We then sought to determine whether CR modifies the expression levels of senescence and SASP markers in the human sigmoidal colon mucosa. To this end, we recruited and studied 12 middle-aged (61.7 ± 8.4 years), weight-stable very lean (BMI = 19.1 ± 1.3 kg/m2) members of the Calorie Restriction Society who have been practicing ~30% CR with adequate nutrition (at least 100% of RDI for each nutrient) for an average of 10.1 years. Levels of p16 were significantly lower in the CR group. Levels of p21 followed the trend observed in p16, but did not reach statistical significance. In accordance with a previous study, we observed significantly lower level of SASP factors, but only three reached statistical significance. These data suggest that CR could potentially prevent the accumulation of age-associated senescent cells in the colon mucosa of human beings, and the reduction in senescence might explain the much lower levels of inflammation observed in CR individuals.

The author noted here sees aging as programmed, in the sense that it is an epigenetic program selected for by evolution because shorter life spans prevent population-level ecological issues. His writing is usually a good illustration of how this concept of aging as a selected epigenetic program leads to very different conclusions on the nature of aging as a whole, as well as on any specific research result. In the case of this post, the topic is the role of telomere length and telomerase in aging, and their relationship to the established DNA methylation biomarkers of aging.

The mainstream view of epigenetic change with age is that it is a reaction to accumulated cell and tissue damage, one that evolved in the limited selection pressure thought to characterize post-reproductive life span. Both damage and epigenetic changes are components of a decline that is an accidental outcome of the aggressive selection for success in early life. Evolution produces biological systems that do well initially, then corrode and fail in a haphazard fashion, because there was no selection for long-term function. Thus systems that generate damage as a side-effect of normal operation, and systems that have limited capacity that fills up and causes issues in later life are found everywhere in our biology.

The debate over programmed versus non-programmed aging, and the ordering of cause and effect between cell and tissue damage versus epigenetic change, will be settled over the next decade or two. If one side produces therapies that revert epigenetic changes and the other side produces therapies that repair cell and tissue damage, then simple observation of the results will determine who is right. The greatest extension of life span and health will point the way to the correct interpretation of the process of aging.

Just a few weeks ago, I learned of a new study linking telomerase to the changes in DNA methylation that the epigenetic clock associates with aging. The implication is that telomerase accelerates aging. It began with an investigation asking what genetic variations are associated with people who age faster or slower than average, according to the epigenetic clock? Researchers performed a genome-wide search for statistical correlates and the standout association was telomerase. People who have small genetic variations that support greater telomerase expression tend to have longer telomeres, but they also tend to age faster, as measured by the epigenetic clock.

The association between telomerase and accelerated aging (measured by methylation) was found in the genetic statistics, and then confirmed in a cell culture. When telomerase was artificially activated in the cell culture, the methylation patterns changed in the cells consistent with older age according to the epigenetic clock. In fact (and remarkably in my opinion) they found no epigenetic aging at all in the cell cultures that lacked telomerase. Could it be that telomerase is the one and only driver of epigenetic aging at the cellular level?

So, what's going on? My inclination is always to think in evolutionary terms. Fixed lifespan, (especially when modified conditions of food stress) is helpful in preventing population overshoot that can lead to famines, epidemics, and extinction. But whenever a trait is good for the community and bad for the individual, there is a temptation for the individual to cheat. In this case, cheating would mean evolving a longer lifespan via selfish genes, such as those enabling greater telomerase expression, that spread rapidly through the population. Individual competition would erase aging if left unchecked. The results would be great for individual fitness, but soon would be disastrous for the population. Thus evolution places barriers in the way of individual selection for ever longer lifespan.

My guess is that the connection between telomerase and epigenetic aging is an example of antagonistic pleiotropy crafted by natural selection in its long-term mode. Limiting lifespan has been so important to the viability of the population that evolution has arranged to protect it from leaking away due to cheating, and antagonistic pleiotropy is one of the ways in which this is arranged. I believe that the preponderance of evidence still indicates that activating telomerase has a net benefit for lifespan, but that probably we can add at most a few years by this route. I think that epigenetics is much closer to the core, the origin of aging, and that interventions to modify epigenetic aging will eventually be our holy grail.

Link: https://joshmitteldorf.scienceblog.com/2018/03/19/telomerase-update-and-downgrade/

One of the primary goals of the aging research community is to determine exactly how aging progresses from moment to moment at the detailed level of genes and cellular biochemistry. This is a sizable task, not particularly driven by any application in medicine, and will be only incrementally more advanced by the time that rejuvenation therapies based on the SENS model of damage repair are a going concern. The big advantage of the damage repair approach is that it bypasses the need to understand exactly how aging progresses: since the root cause damage is known, it is possible to make progress immediately and quantify the resulting benefits along the way.

If one was to go about searching for genetic contributions to longevity, however, then the method here is a decent way to go about it. The standard problem in this space is one of complexity and limited resources: there are a lot of genes, and only so many scientists with sufficient funding to look for the needles in the haystack. The researchers here reduce the size of the problem by comparing the genomes of closely related rodent species with varying life spans; the set of genetic differences, much smaller than an entire rodent genome, should include those genes most influential on life span.

As an adaption to different environments rodents have evolved a wide range of lifespans. While most rodents are short-lived, along several phylogenetic branches long-lived species evolved. This provided us a unique opportunity to search for genes that are associated with enhanced longevity in mammals. Towards this, we computationally compared gene sequences of exceptional long-lived rodent species (like the naked mole-rat and chinchilla) and short-lived rodents (like rat and mouse) and identified those which evolved exceptionally fast. As natural selection acts in parallel on a multitude of phenotypes, only a subset of the identified genes is probably associated with enhanced longevity.

A set of 250 identified positively selected genes (PSGs) in liver tissue exhibited a highly significant pattern of down-regulation in the long-lived naked mole-rat and up-regulation in the short-lived rat, fitting the antagonistic pleiotropy theory of aging. Moreover, we found the PSGs to be enriched for genes known to be related to aging. Among these enrichments were "cellular respiration" and "metal ion homeostasis", as well as functional terms associated with processes regulated by the mTOR pathway: translation, autophagy, and inflammation. Remarkably, among PSGs are RHEB, a regulator of mTOR, and IGF1, both central components of aging-relevant pathways, as well as genes yet unknown to be aging-associated but representing convincing functional candidates, e.g. RHEBL1, AMHR2, PSMG1 and AGER.

We conclude that lifespan extension in rodents can be attributed to changes in their defense against free radicals, iron homeostasis as well as cellular respiration and translation as central parts of the growth program. This confirms aging theories assuming a tradeoff between fast growth and long lifespan. Moreover, our study offers a meaningful resource of targets, i.e. genes and specific positions therein, for functional follow-up studies on their potential roles in the determination of lifespan-regardless whether they are currently known to be aging-related or not.

Link: https://doi.org/10.1371/journal.pgen.1007272

Researchers have been talking about therapies based on enhanced levels of autophagy for about as long as I've been paying attention to the field of aging research. Autophagy is a collection of processes responsible for breaking down and recycling damaged structures and unwanted proteins in cells. More aggressively removing harmful or malfunctioning cellular systems and wastes reduces the amount of time they exist to cause problems, and results in better functioning of cells and tissues. Ultimately, more autophagy modestly slows aging and allows individuals to live longer. Many of the varied methods of manipulating metabolism to slow aging demonstrated over the past few decades appear to either depend on autophagy or include increased autophagy among their mechanisms of action.

Despite all of the talking - and the many papers and years of work examining various controlling mechanisms associated with autophagy - there is as yet no real progress towards therapeutics that work via the deliberate, targeted upregulation of autophagy. That is if we don't count things like calorie restriction mimetics, which improve autophagy along the way of changing many other aspects of metabolism. Calorie restriction itself appears to stop producing benefits to health if autophagy is disabled. Calorie restriction mimetics are not really all that solidified yet as a class of therapeutic, however. The most compelling, such as mTOR inhibitors, have significant side-effects that are still being worked around. The rest are largely so marginal or the data for positive effects in animal studies so unreliable as to be unworthy of serious consideration in a world in which one can just eat less and definitely benefit from it.

The editorial here (still in PDF format only at the time of writing) presents a more recent example of research aimed at identifying targets for the therapeutic enhancement of autophagy. It is similar in tone and scope to a dozen others I've seen over the years, and little has come of them as of yet - even the important work from a decade ago, showing restoration of liver function in old mice. The research community, for reasons that remain unclear to me, seems challenged when it comes to moving beyond mapping and investigation in order to build something of practical use on the foundation provided by this part of the field.

Humanin enhances the cellular response to stress by activation of chaperone-mediated autophagy

Increased oxidative stress and loss of proteostasis are characteristics of aging. Failure to remove the oxidative stress-damaged components has been recognized to play critical roles in the pathophysiology of common age-related disorders including neurodegenerative disease and cardiovascular diseases such as myocardial infarction and heart failure. Strategies to diminish oxidative stress or effectively eliminate oxidative-damaged intracellular proteins may therefore provide novel therapeutic option for many age-related diseases.

Chaperone-mediated autophagy (CMA) allows for selective degradation of soluble proteins in lysosomes, contributing to the cellular quality control and maintenance of cellular energetic balance. CMA substrate proteins are targeted by the chaperone hsc70 to the lysosomal surface where, upon binding to the lysosome-associated membrane protein type 2A (LAMP2A), they are translocated into the lysosomal lumen for degradation. CMA is activated by oxidative stress to facilitate degradation of damaged proteins, thereby eliminating the insults of oxidative stress. Given the fact that CMA activity declines with age, and oxidative damage in cells increases during aging, CMA activators hold the potential for development as a new generation of treatment option for age-related diseases.

In our recent study, we identified that humanin (HN), an antiapoptotic, mitochondria-associated peptide is an endogenous CMA activator. We demonstrated that HN protects multiple cell types including cardiomyoblasts, primary cardiomyocytes and dopaminergic neuronal cells from oxidative stress-induced cell death in a CMA dependent manner. In fact, this protective effect is lost in CMA-incompetent cells (LAMP-2A knockdown). Both exogenously added HN as well as the endogenously generated HN cooperate in CMA activation. Thus, knockdown of endogenous HN decreases CMA activation in response to oxidative stress. Both endogenous and exogenous HN localize at the lysosomal membrane where they cooperate to enhance CMA efficiency. HN acts by stabilizing binding of the chaperone HSP90 to the upcoming substrates at the cytosolic side of lysosomal membrane.

Our study provided the first evidence that regulatory signals from mitochondria can control CMA. We propose that while generating reactive oxygen species (ROS) from metabolism, mitochondria simultaneously initiates signals such as HN to eliminate ROS by increasing antioxidant enzyme activities, and decrease oxidative insults by activating CMA, and that perturbations in this process could cause accumulation of oxidative damage leading to cell death and human diseases. It is interesting to note that HN and CMA both decline with age and that genetic correction of the CMA defect in livers from old mice was effective in improving hepatic homeostasis, conferring higher resistance to stress and improved organ function.

We propose that interventions aimed to enhance mitochondrial peptide HN levels could have a similar effect, and protect against oxidative stress by enhancing removal of oxidative-damage proteins through CMA. Whether this is a unique function of HN, or is shared by other mitochondria-encoded peptides such as small humanin like peptides (SHLPS) requires future investigation. Efforts should be directed to testing a possible protective effect of HN in age-related diseases with a primary defect on CMA such as Parkinson's disease.

Alzheimer's disease, like most neurodegenerative conditions, has a strong inflammatory component. The importance of inflammation is one possible way to explain why Alzheimer's risk appears to have a significant lifestyle component: Alzheimer's disease is associated with excess visceral fat tissue and all of the choices made along the way of gaining and retaining that fat tissue. Fat tissue is a notable source of chronic inflammation, acting to accelerate all of the common process and conditions of aging. There are numerous other paths to inflammation, of course.

If chronic inflammation is important in Alzheimer's disease, how useful is a chronic anti-inflammatory treatment? Various groups have considered this over the years, but the one noted here appears more optimistic than most - and the data is fairly compelling. As an aside, nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen have been shown to modestly slow aging and extend life in a few laboratory species, though the exact mechanisms are up for debate. I normally point this out to dampen enthusiasm for any novel pharmaceutical shown to have similarly sized effects in short-lived species - one shouldn't expect anything more interesting than NSAIDs to result, and the data to date suggests that NSAIDs don't in fact do a great deal for human life span. Can simple, proven methods of suppressing inflammation help people who are declining into Alzheimer's disease, however? That is a separate question, and it will be interesting to see how this line of research progresses.

In 1990, we wrote a short report indicating a substantial sparing of Alzheimer's disease (AD) in patients with rheumatoid arthritis. We suggested that anti-inflammatory therapy might be the explanation. We chose rheumatoid arthritis for the study since it typically commences at an earlier age than AD, and is universally treated with anti-inflammatory agents. Our report of AD sparing in patients consuming anti-inflammatory agents was soon confirmed in 17 epidemiological studies of patients consuming nonsteroidal anti-inflammatory drugs (NSAIDs) compared with controls. There was one consistent caveat in these epidemiological studies. The NSAIDs needed to have been started at least 6 months, and preferably as long as 5 years, before the clinical diagnosis of AD.

A new field of research had been opened up with these epidemiological studies. It required that some important questions be answered. Why was it necessary to commence taking NSAIDs so long before the clinical onset of AD? What was the appropriate NSAID dose? And was it necessary to take NSAIDs on a continuing basis? New techniques were required to provide answers to these questions. We emphasize here the two most important of these: positron emission tomography revealing that deposits of amyloid-β protein (Aβ) build up in the brain of AD cases; and cerebrospinal fluid (CSF) Aβ levels revealing their consequent reduction. These two techniques are complementary. Since the brain Aβ deposits accumulate over time, the effect is integral. Since CSF turns over every few hours, the effect is differential.

Disease development, as revealed by biomarker studies, follows this sequence of events. It commences with Aβ deposits developing in the brain of AD cases. These deposits can be detected by positron emission tomography (PET). The depositions result in a concomitant decrease of Aβ in the CSF. Years later, less definitive biomarkers become positive. These later biomarkers reveal loss of brain tissue. When they become positive, cognitive deficits have already appeared. Together, these studies indicate that AD onset commences more than a decade before clinical signs develop. The ability to identify the onset of AD a decade or more before clinical signs appear creates a window of opportunity to intervene in the process. Moreover, it explains the epidemiological data in which NSAIDs must be commenced years before clinical detection. The missing link is a simple, non-invasive method for identifying those at risk at an age well below the typical age of AD onset.

Analysis of saliva for Aβ42 may provide the missing link. We first developed a simple method for determining Aβ42 levels in tissues as well as saliva. The results showed that Aβ42 is produced in all tissues of the body, and not just in brain as many have believed. Aβ42 secretion in saliva is a reflection of its production by submandibular glands. The non-AD cases resolved into two distinct categories: those with low levels in the 19-25 pg/ml range, and those with high levels in the AD range of 41-60 pg/ml. Significantly, there were no overlapping cases. Analysis of Aβ42 levels in saliva demonstrates three remarkable facts. Firstly, controls, who are not at risk for AD, secrete levels close to 20 pg/ml, regardless of sex or age. Secondly, this production is constant, being invariant with time of day, and from day to day. Thirdly, those at risk for AD secrete levels comparable to AD cases. Widespread application of this test to detect high levels, followed by NSAID consumption, could substantially reduce the prevalence of AD.

Link: https://doi.org/10.3233/JAD-170706

I point out this interesting open access paper not as a suggestion that anyone should consider trying remote ischemic conditioning - one should adopt some form of calorie restriction and greater levels of regular exercise before embarking upon fancier hobbies - but rather because it is illustrative of the degree to which common stress response mechanisms overlap. Heat, exercise, ischemia, and lack of nutrients all share some of the same channels of signal and response that lead to cells undertaking greater maintenance or building more robust tissue structures. That in turn means that we already have a fairly good idea of the plausible bounds on beneficial results when it comes to therapies that use pharmaceutical or other means to induce stress responses. They will be able to move people closer to the life trajectory of a very healthy, well maintained body, but more than that seems unlikely to be attained via this strategy.

Thirty years ago, researchers first discovered the phenomenon of ischemic pre-conditioning in an animal experiment. The seminal discovery that brief episodes of ischemia followed by reperfusion could significantly reduce myocardial infarct size gave rise to the area of myocardial protection firstly and then propagated to multi-organ protection. Ischemic preconditioning has evolved into remote ischemic conditioning (RIC). Although the underlying mechanisms of RIC are still unclear, it was found to be safe and well tolerated in both patients and healthy volunteers.

The use of long-term repeated RIC comes with the expectation that RIC can play its protective roles consistently; this RIC treatment protocol is now called chronic RIC. Clinical studies have demonstrated that chronic RIC could reduce adverse clinical events and improve neurological function, which was rare in previous studies using a once-only RIC treatment protocol. Intriguingly, some sports specialists, inspired by the favorable effects of RIC on skeletal muscles and endothelial function, applied RIC to exercise training, as intense exercise has been demonstrated to lead to a form of cardiac and skeletal muscles ischemic insult. To date, RIC has been shown to improve the maximal performance in highly trained swimmers, enhance 5-km time trial performance and attenuate the submaximal level of blood lactate during the incremental running test. Therefore, RIC could improve exercise performance as does regular interval exercise training.

Given the similar time window of early and late phase protection seen with both exercise and RIC, and their comparable effects on improving exercise performance, it is reasonable to speculate that the underlying mechanisms of RIC likely overlap with those of exercise. Heat shock protein (HSP) 70 family, especially HSP72, has been demonstrated to be associated with cardioprotection. Previous studies found significantly increased HSP72 levels after acute aerobic exercise. Similarly, increased HSP72 has been reported after RIC stimulus. Studies have demonstrated that exercise promoted endothelial NO synthase activity, increased the production of NO and improved endothelial cell function. Similarly, chronic RIC has been demonstrated to significantly improve flow-mediated dilation and enhance endothelial NO synthase expression in patients with coronary heart disease.

Autophagy is a process for eliminating dysfunctional organelles and protein aggregates, which is a kind of endogenous protection and required for cellular survival and homeostasis in response to stress. Studies have found that protective effects of RIC on cell survival were mediated by autophagy pathway, and autophagy participants in RIC-induced protection. Exercise induces the adaptational response from multiple organs (primarily in skeletal muscle), which will benefit human body. Recently, autophagy has been found to be an essential process involved in conserving and recycling the cellular resources, an important process of the adaptation response.

Exercise has direct beneficial effects on the cellular immune system, and it can mobilize NK and T cells to circulation during exercise. The immune cell activity may also be influenced by the exercise-induced release of immune regulatory cytokines. Similarly, RIC has also been demonstrated to influence inflammatory response and immune cells, and these are the essential underlying mechanisms of RIC-induced protection. RIC reduces inflammatory gene expression and dramatically changes the immune response, which induces protection.

From the data available so far, it appears that chronic RIC mimics regular exercise. Further studies are however urgently needed to validate this phenomenon. However, there are still several hurdles for popularizing chronic RIC. Currently, different chronic RIC protocols are used in various studies, the periods of using chronic RIC vary from 1 week to 1 year, and its frequencies vary from twice daily to once every two weeks. Although chronic RIC benefits health and protects organs from injury, its optimal protocol is still unclear.

Link: https://doi.org/10.14336/AD.2017.1015

Many mechanisms of aging are two-way streets: A accelerates B, but B also makes A worse. Or A leads to B that causes C which aggravates A. Chronic inflammation, a persistent and damaging activation of the immune system, is a player in many of these sorts of circular relationships and feedback loops. The open access paper noted here briefly covers some of the known contributions to increased inflammation in aging. Inflammation is a vital part of the way in which the immune system coordinates with tissues in order to repel invaders and respond to injury; it is beneficial when temporary. When inflammation is constant, however, regeneration and tissue maintenance start to run awry, cancer rates rise, and many disease processes accelerate. Among the inflammatory conditions of aging are found osteoarthritis, the many forms of fibrosis, near all neurodegenerative diseases, atherosclerosis, and more.

What causes the raised level of chronic inflammation found in older people? Well, at root the forms of molecular damage outlined in the SENS view of rejuvenation biotechnology, but the line between root cause and age-related inflammation is only clear and direct in a couple of cases. Aging is a spreading, vastly complex network of many layers of cause and effect, branching out from the few fundamental forms of tissue damage, influencing one another along the way. So there are few simple answers when it comes to the proximate causes of chronic inflammation - they are very complicated in their details. It is easy enough to say that a part of it is the signaling of senescent cells, and a part of it is particular to the way in which the immune system runs down and malfunctions in later life. But those short sentences cover a ferociously complex biochemistry that is only partially understood.

There are a few simple ways forward towards effective control of some of the sources of inflammation in aging, however. Selectively destroying senescent cells removes their inflammatory influence without having to understand the details. Similarly, clearing out all immune cells while using cell therapy to speed their replacement is a viable approach to some of the issues in the aged immune system that result in higher levels of inflammation. Restoring the ability of the thymus to produce a larger supply of new immune cells is probably also useful. These and a few other plausible approaches don't require a great deal of new knowledge and largely bypass the state of ignorance regarding the biochemical details of inflammatory aging. Sometimes it doesn't matter why something takes place, given a comprehensive enough approach to removing it or otherwise dealing with it.

Source of Chronic Inflammation in Aging

At present, chronic inflammation is thought to be a risk factor for a broad range of age-related diseases such as hypertension, diabetes, atherosclerosis, and cancer. The burdens of unhealthy aging associated with lifestyle are increasing, both in developed and developing regions. Therefore, the elucidation of the sources and cellular pathways/processes of chronic inflammation is an urgent task.

There are several possible factors that initiate and maintain a low-grade inflammatory response. These include aging, unbalanced diet, low level of sex hormones, and smoking. In contrast to young individuals, aged individuals have consistently elevated levels of inflammatory cytokines, especially interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which may induce muscle atrophy and cancer through DNA damage. Visceral fat tissue from obese individuals can also produce both IL-6 and TNF-α, affecting systemic metabolism. The accumulation of macrophages in visceral fat seems to be proportional to body mass index and appears to be a major source of low-grade persistent, systemic inflammation and insulin resistance in obese individuals. Chronic smoking increases production of several pro-inflammatory cytokines such as IL-6, TNF-α, and interleukin-1β (IL-1β), increases systemic inflammation and is an independent risk factor for several lifestyle-related diseases.

Acute inflammation is indispensable for immune responses to invading pathogens or acute traumatic injuries. This process enables repair and cell turnover in multiple tissues. In contrast, chronic inflammation normally causes low-grade and persistent inflammation, leading to tissue degeneration. Chronic, low-grade inflammation is a crucial contributor to various age-related pathologies and natural processes in aging tissue, including the nervous and the musculoskeletal system. Many tissues in the elderly are chronically inflamed, and inflammatory cytokines such as IL-6, IL-1β, and TNF-α are known to weaken the anabolic signaling cascade, including insulin and erythropoietin signaling, leading to the development of sarcopenia.

Debris and immunoglobulin accumulation due to inappropriate cell elimination systems in aging trigger the innate immune system activation leading to persist inflammation. Among the complex determinants of aging, mitochondrial dysfunction has attracted attention for some time. The consequences of age-related failing mitochondrial quality control include the release of mitochondria-derived damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs, especially cell-free circulating mitochondrial DNA, have recently become the subject of intensive research because of their possible involvement in conditions associated with inflammation, such as aging and degenerative diseases. Through their bacterial ancestry, these molecules contribute to increasing an inflammatory response by interacting with receptors similar to those involved in pathogen-associated responses.

The barrier of the oral and gut mucosa against bacterial invasion deteriorates with age. Periodontal disease has also demonstrated to cause chronic low-grade inflammation. The gut microbiota of elderly people displays decreased diversity. The abundance of anti-inflammatory microbiota are diminished in aged individuals. Conversely, inflammatory and pathogenic microbiota are increased with age.

Cellular senescence is defined as irreversible cell cycle arrest driven by a variety of mechanisms. It is evident that the number of senescent cells in several organs increases with age; these cells secrete multiple inflammatory cytokines, generating low-grade inflammation. This phenotype of senescent cells is termed the senescence-associated secretory phenotype or SASP, which recently has been proposed as the main origin of inflammaging in both aging and age-related diseases such as atherosclerosis, cancer, and diabetes. Increasing evidence has suggested that the clearance of senescent cells in animal models attenuates the progression of age-related disorders, including atherosclerosis and osteoarthritis. These data strongly support the hypothesis that senescent cell clearance, reprogramming of senescent cells, and the modulation of pro-inflammatory pathways related to the acquisition of SASP might be pursued as potential anti-aging strategies for combating age-related diseases and expanding the health span of humans.

"Immunosenescence", which is the age-related dysregulation of an innate immune system, is characterized by persistent inflammatory responses. Immunosenescence increases the susceptibility to malignancy, autoimmunity, and infections; decreases the response to vaccinations; and impairs wound healing. Conversely, chronic inflammatory disease can accelerate the "immunosenescence" process. The mechanisms that underlie this persistent aging-associated inflammation remain incompletely understood but seem to involve changes in the numbers and functions of innate immune cells. Changes in the expression of pattern recognition receptors (PRRs), activation of PRRs by endogenous ligands associated with cellular damage, and unusual downstream signaling events of PRRs activation have been implicated to induce chronic cytokine secretion. Thus, together with cell senescence, dysregulation of immunological imprinting mediated by trained innate immunity might also contribute to persistent low-grade inflammation that occurs even after the initial stimulus has been removed.

The research here is an interesting view on the relevance of vascular aging in cognitive decline and later dementia. The researchers find similar changes in blood vessels in both old mice and mice engineered to undergo the amyloid and tau aggregation characteristic of human Alzheimer's disease. In humans, a sizable proportion of people suffering Alzheimer's disease also have vascular dementia - one of the many challenges facing any group trying to prove success in a therapy intended to narrowly address aspects of Alzheimer's biochemistry. That success, if it takes place at all, could well be masked in many patients by the loss of function that results from vascular aging.

With age, blood vessels stiffen and are weakened by corrosive fatty deposits. Blood pressure rises, causing an increase in the breakage of small blood vessels and consequent damage to surrounding tissue. The heart weakens. Capillary growth into tissues declines for reasons that are still comparatively poorly understood. Further, the amyloid associated with Alzheimer's can also emerge in blood vessels and cause dysfunctional behavior there. The brain is an energy-hungry organ, and all of these problems combine to reduce the supply of needed oxygen and nutrients. Dementia is the end result. That so many of these processes of harm are accelerated by chronic inflammation, such as that produced by excess fat tissue, is why a number of forms of dementia appear to have a strong lifestyle component - Alzheimer's included.

Researchers have demonstrated for the first time that anxiety and problems with blood vessels present a close relationship with Alzheimer's disease, which particularly affects females. Vascular disease resulting from oxidative stress and inflammation is gaining clinical interest, given that subsequent cardiovascular insufficiency can alter the blood flow distribution to different organs and tissues, including the brain, which can worsen a pathology related to this type of dementia.

The research provides the first evidence that mice of advanced ages suffering from Alzheimer's disease present substantial alterations in small blood vessels, which are essential in nourishing different organs and tissues and in the regulation of blood pressure. "The study demonstrates that the sex of the mice is a determining factor. It is specifically the female mice which show more pronounced vascular alterations, which suggests that women of advanced ages suffering from Alzheimer's disease may suffer more from cardiovascular malfunctions."

The characteristics of small arteries were studied under different physiological conditions. Further research revealed that these vascular changes appear in both the vascular structure and function, which suggests an abnormal distribution in peripheral blood flow. Researchers also assessed animal behaviour. This allowed them to discover the existence of a strong relation between the vascular parameters analysed - structure, elasticity, function - and different patterns of anxiety in mice models of Alzheimer's, but also in mice ageing normally.

"Although we must be cautious with these results, the correlation of behaviours propose the existence of direct or indirect relations between conduct and the function of peripheral arteries. These interactions may be able to explain the anomalies of the neuro-immuno-endocrine system, which regulates the performance of different organs and tissues."

Link: http://www.uab.cat/web/newsroom/news-detail/x-1345668003610.html?noticiaid=1345750175358

The LongLongLife team here reports briefly on their time at the recent Undoing Aging conference. This was the first in a series of conferences, hosted jointly by the SENS Research Foundation and Forever Healthy Foundation, that will mix the scientific and academic focus of the SENS rejuvenation research conferences with the biotechnology industry focus of the Rejuvenation Biotechnology conferences. By all accounts the initial Undoing Aging event was well received.

The very first Undoing Aging Congress was held in March 2018 in Berlin, and was attended by 350 people from a total of 36 countries. Initiated by Aubrey de Grey, co-founder of the SENS Research Foundation, and Michael Greve, founder and CEO of the Forever Healthy Foundation, the conference focused on the most promising advances in anti-aging research. The congress, which lasted for three days, was divided into different sessions, each of which dealt with a specific theme of anti-aging research. Among the major issues were regenerative medicine, the elimination of senescent cells, cancer therapies, and biomarkers of aging.

Dr. Doug Ethell, founder and director of Leucadia Therapeutics, linked the site of the development of Alzheimer's disease in the brain to the presence of a porous bone plate at the same site that drains the cerebrospinal fluid (fluid that cleanses the intercellular space of the brain). With age, this bone plate becomes blocked, no longer allowing the toxic metabolites carried by the cerebrospinal fluid to pass. The accumulation of these wastes would lead to the formation of plaques, at the origin of the development of the disease. Leucadia Therapeutics has developed a therapeutic device which restores the flow of cerebrospinal fluid and the elimination of toxic metabolites. Dr. Ethell hopes clinical trials can begin in 2019.

Transthyrethin amyloidosis is a rare disease in two forms (cardiomyopathy and polyneuropathy) and develops with age. It is caused by the accumulation of transthyretin protein (TTR) incorrectly folded into amyloid plaques. There is currently no FDA approved treatment for this disease. Dr. Isabella Graef, co-founder of Eidos Therapeutics, explained how her laboratory discovered a drug candidate that could work on both forms of the disease. It is a small molecule that can bind and stabilize TTR, preventing the formation of amyloid plaque, and which could stop the progression of the disease. This molecule has been in phase 1 clinical trials (trials on healthy subjects) since 2017.

Regenerative medicine aims to create new tissues or organs to replace defective ones. This medicine is booming and the technological processes that allow its development are constantly progressing. Dr. Eric Lagasse of the University of Pittsburgh presented at the congress the work of his LyGenesis laboratory, which has developed a technology to generate the liver from lymph nodes. The goal of the method is to transplant healthy hepatocyte cells into the lymph nodes, resulting in the generation functional liver tissue. This technology has already shown its effectiveness in mice and pigs.

Dr. Steve Horvath, a professor at the University of California at Los Angeles, has a long history of working on biomarkers of aging. He is behind Horvath's epigenetic clock, which predicts biological age as a function of genome methylation. Predicting biological age using methylation is now the most accurate way, says Dr. Horvath. He proposes that DNA methylation should be measured in clinical trials as part of the fight against aging. Finally, his goal by 2021 is to develop an epigenetic clock that applies to all mammals.

Link: http://www.longlonglife.org/en/longevity/lifespan-news/undoing-aging-2018-initiative-remember/

In today's open access paper, the authors review what is known of the role of cellular senescence in the common cardiovascular and metabolic conditions of aging, with a focus on senescence in the vascular system. The accumulation of senescent cells over time is one of the root causes of aging: a process that takes place as a side-effect of the normal operation of cellular metabolism, and that produces slow decline, damage, and systems failure. Research over the past few years has directly connected the growing number of senescent cells in older individuals with age-related disease of the lungs, vascular system, joints, and most of the major organs. Removing senescent cells has been shown to extend life in mice, and partially reverse a number of age-related conditions in other animal studies. Human studies have started, and will be expanding this year and next.

Senescence is a state in which cells cease to replicate, and begin to generate a range of inflammatory and other signal molecules. These cells appear to be important in embryonic development, helping to define shape and structure of tissue, and also play a transient role in regeneration from injury. All somatic cells in the body ultimately reach the Hayflick limit on cell divisions and become senescent, a part of the grand design of multicellular life in which only a few cells are permitted unlimited replication, the first and most important defense against cancer. Cells also become senescent in response to mutational damage or a toxic environment, another defense against cancer. In all of these scenarios, all but a tiny fraction of newly senescent cells quickly destroy themselves.

Unfortunately, a few senescent cells manage to linger, and the signals generated by those few cells ultimately fatally disrupt the function of organs. They cause chronic inflammation, harmful alterations in the processes of tissue maintenance that induce fibrosis, and many other issues linked to an accelerated progression of aging and age-related disease. In the cardiovascular system, evidence points to cellular senescence to be a driver of the calcification that contributes to stiffness and hypertension, and senescent foam cells accelerate the construction of atherosclerotic plaques that narrow and weaken blood vessel walls. The combination of these two processes - high blood pressure and weakened blood vessels - causes a sizable fraction of all human death. Addressing the varied causes of both will go a long way to pushing back the consequences of aging, and destroying senescent cells is the first such approach to enter earnest development.

Vascular Senescence in Cardiovascular and Metabolic Diseases

In aging societies, the discrepancy between the total lifespan and the healthy lifespan is becoming a major problem. Chronological aging is associated with a higher prevalence of age-related diseases, including heart failure, diabetes, and atherosclerotic disorders with or without various comorbidities, resulting in impairment of the quality of life by limitation of normal activities. Thus, aging is associated with several undesirable processes. The mechanisms of aging and age-associated disorders are complex, and thus cannot be comprehended by a simple approach. However, recent studies have indicated a pivotal role of cellular senescence in the progression of age-related disorders.

p53 signaling is thought to have a central role in cellular senescence. Somatic cells have a finite lifespan and eventually enter a state of irreversible growth arrest termed "replicative senescence." Telomeres are repetitive nucleotide sequences located at the terminals of mammalian chromosomes that undergo incomplete replication during cell division, resulting in telomere shortening. Because telomeres are essential for chromosomal stability and DNA replication, DNA damage is recognized when telomere shortening exceeds the physiological range and this triggers cellular senescence, mainly via the p53 or p16 signaling pathways. "Stress-induced premature senescence" is another type of cellular senescence that is triggered by various stress signals. It is also mediated via the p53 or p16 signaling pathways.

It was reported that p53 is increased in the failing heart, in aged vessels, and in the visceral fat of patients with obesity or heart failure. Studies have indicated a pathological role of p53-induced cellular senescence in aging and age-related disorders, including heart failure, atherosclerotic disease, obesity, and diabetes. However, there is controversy about the role of p53 in aging and age-related diseases. In some settings, p53 signaling has been shown to have a beneficial effect by suppression of aging; various reports suggest that the p53/p21 signaling pathways regulate cellular senescence in a context-dependent manner.

Interestingly, it was recently reported that elimination of senescent cells by genetic manipulation inhibited age-related degenerative changes in several organs of mice, such as the heart and kidneys. Other studies have identified several pharmacological agents that selectively damage and remove senescent cells, and these compounds have been described as "senolytic agents". For example, an inhibitor of anti-apoptotic proteins (ABT263) depletes senescent bone marrow hematopoietic stem cells and senescent muscle cells in a chronological aging model, leading to rejuvenation of these tissues. Studies have shown that senescent cells damage their local environment and promote tissue remodeling in age-related disorders, suggesting that inhibition of cellular senescence and/or elimination of senescent cells could be potential next generation therapies for diseases associated with aging.

Reactive oxygen species and chronic low-grade sterile inflammation are two major contributors to the progression of age-related vascular dysfunction. Senescent cells accumulate in the arteries with aging irrespective of whether or not a person has age-related vascular disorders. Along with aging, vascular tissues of rodents and humans show elevation of the levels of p16, p21, phosphorylated p38, and double-stranded DNA breaks, in association with high SA-β Gal activity. It was reported that expression of p53 and p21 is increased in the arteries of elderly persons, together with structural breakdown of telomeres known as telomere uncapping.

Endothelial cells and vascular smooth muscle cells (VSMCs) from patients with abdominal aortic aneurysm (AAA) have the phenotypic features commonly observed in senescent cells. Hypertension is an established risk factor for atherosclerotic diseases, and it was reported that binding of p53 to the p21 promoter is increased in the arteries of hypertensive patients. While telomere length is comparable between patients with hypertension and controls, telomere uncapping is 2-fold higher in hypertensive patients. A murine model of genomic instability demonstrated senescence of endothelial cells and VSMCs in the aorta, along with impaired vasodilation, increased vascular stiffness, and hypertension.

Endothelial cells are critically important for maintaining vascular homeostasis and are involved in various biological functions, including angiogenesis, blood pressure regulation, coagulation, and systemic metabolism. Aged endothelial cells develop a dysfunctional phenotype that is characterized by reduced proliferation and migration, decreased expression of angiogenic molecules, and low production of nitric oxide (NO), which is synthetized by NO synthase (NOS) and mediates vasodilatation. Senescent endothelial cells have been found in atherosclerotic plaque. An autopsy study of patients with ischemic heart disease revealed that SA-β-gal activity is increased in the coronary arteries. In the coronary arteries, SA-β-gal activity is high in cells located on the luminal surface (probably endothelial cells). Both endothelial nitric oxide synthase (eNOS) and NO activity are reduced in these cells compared to young cells.

One of the problems related to an increase of senescent cells is development of the senescence-associated secretory phenotype, which is characterized by production of pro-inflammatory cytokines with a causal role in tissue remodeling. In human arterial endothelial cells with replicative senescence, levels of H2O2 and O2- are high and NO production is reduced. High ROS levels in senescent endothelial cells are thought to accelerate senescence. Aging is reported to be linked with increased circulating levels of pro-inflammatory cytokines, such as interleukin-6, tumor necrosis factor alpha, and monocyte chemoattractant protein-1. It is highly possible that accumulation of senescent endothelial cells in the arteries of elderly persons induces chronic sterile inflammation and vascular remodeling, increasing susceptibility to atherosclerotic diseases.

In conclusion, senescence of vascular cells promotes the development of age-related disorders, including heart failure, diabetes, and atherosclerotic diseases, while suppression of vascular cell senescence ameliorates phenotypic features of aging in various models. Recent findings have indicated that specific depletion of senescent cells reverses age-related changes. Although the biological networks contributing to maintenance of homeostasis are extremely complex, it seems reasonable to explore senolytic agents that can act on specific cellular components or tissues. Several clinical trials of senolytic agents are currently ongoing.

Survivors of hematopoietic stem cell transplantation are prone to premature aging, and one pilot clinical study is designed to test whether dasatinib and quercetin (D + Q) can suppress aging in these patients (NCT02652052). Another clinical trial is testing whether D + Q reduces pro-inflammatory cells obtained by skin biopsy in patients with idiopathic pulmonary fibrosis (NCT02874989). Furthermore, a clinical trial is ongoing to determine whether D + Q can reduce the senescent cell burden and frailty in patients with chronic kidney disease, as well as improving the function of adipose tissue-derived mesenchymal stem cells (NCT02848131). So far, only D + Q has been assessed in the clinical setting, and none of the current clinical trials are testing whether senolytic agents can inhibit cardiovascular disorders. However, depletion of senescent cells was demonstrated to suppress pathological progression of atherosclerotic plaque in rodents, suggesting that senolytic agents could become a next generation therapy for cardiovascular disorders.

The few formal studies of human calorie restriction continue to produce interesting data on the biochemistry of participants, and the degree to which the human response to lowered calorie intake lines up with the outcomes observed in mice. One of the puzzles to be solved is the way in which short-term effects that look very similar between humans and mice nonetheless lead to a radically different degree of enhanced life span. Mice can live up to 40% longer than normal when calorie restricted, which is certainly not the case for humans - it would be very surprising to find an effect much larger than five years for human life expectancy.

The authors of this paper choose to interpret the results as supportive of rate of living and oxidative theories of aging, which I have to think is a mistaken direction. There is so much evidence against those views of aging at this point that it is probably better to try to fit observations into newer and more robust views on how aging progresses at the detailed level of cellular biochemistry. In particular, the animal studies of longevity of the past twenty years include any number of cases in which sources of oxidative molecules are increased or decreased to produce longer life spans as a consequence - the systems of oxidative signaling and damage and repair are complex, and defy the imposition of any straightforward relationship.

For the past 40 years, aging research has focused on the mechanisms underlying the beneficial health impact of a sustained reduction in caloric intake below usual levels, while maintaining adequate intake of essential nutrients. Observations in a variety of laboratory animals indicate that calorie restriction (CR), beginning early or in mid-life and sustained for a substantial portion of the lifespan, increases longevity in a wide variety of, but not all, species. While the field of CR research eagerly awaits final lifespan data from the two remaining colonies of CR primates, despite differences in study designs, current data support the observation that sustained CR extends life without chronic disease and promotes a more youthful physical and mental functionality. In terms of CR in humans, few controlled clinical trials exist.

A variety of mechanisms have been proposed as mediators of the effects of CR on lifespan. An old but arguably a prevailing theory supporting lifespan extension with CR is a hybrid between two long-standing hypotheses of aging: the "rate of living" and the "oxidative damage" theories of aging. There are data from studies in rodents, non-human primates, and humans indicating that CR results in a decrease in metabolic rate that is greater than that expected on the basis of loss of tissue mass. This phenomenon, referred to as metabolic adaptation, was associated with less oxidative damage to DNA in our 6-month pilot study of CR in humans. The CR field has also focused on the ability for CR to attenuate age-related changes in physiological and endocrine factors that are known to change with age, such as core body temperature, plasma insulin, DHEAS, and thyroid hormones, as well as endocrine mediators of metabolic slowing such as plasma leptin.

Phase 1 CALERIE or the Comprehensive Assessment of the Long-Term Effects of Reducing Intake of Energy studies were the first randomized controlled trials to test the metabolic effects of CR in non-obese humans. Then, the phase 2 CALERIE study, a 2-year 25% CR prescription in non-obese volunteers, was shown to be safe and without any untoward effects on quality of life. Importantly, the study confirmed the presence of a CR-induced decrease in total daily energy expenditure (EE). However, in the CR group compared with the control group, resting metabolic rate adjusted for loss of fat-free and fat masses was only lower during the weight loss phase. Furthermore, reductions in core body temperature were noted in the CR group, but were not different from the controls, and changes in oxidative damage were not assessed.

We hypothesized a reduction in oxidative damage after 1 and 2 years of CR. Taken together with lower EE, such results would speak in favor of the long-standing hypotheses of biological aging stating that prolonged CR enhances energy efficiency at rest and therefore results in less reactive oxygen species production and reduced oxidative damage to tissues and organs, thus a combination of the rate of living and the oxidative damage theories of aging. To test this hypothesis, we delivered a highly controlled and intensive behavioral intervention targeting a 25% CR diet over 2 years and obtained reliable measurements of the most robust component of daily sedentary EE, i.e., energy metabolism during sleep, measured in a room calorimeter. Hormonal mediators of metabolism were measured along with urinary F2-isoprostane excretion as an index of oxidative damage.

According to the rate of living theory, those individuals who are the most efficient at utilizing energy should experience the greatest longevity. Observational studies of human aging have shown higher mass-adjusted metabolic rate (24hEE or resting EE) is associated with disease burden and is a predictor of early mortality. Interventions with the capacity to induce a sustained slowing of energy metabolism such as CR should remain a focus of longevity research because randomized clinical trials and cohort studies are lacking. With careful phenotyping of energy metabolism, biomarkers of aging, and oxidative stress, this modest, 2-year study of human CR identified a reduction in the rate of living along with a reduction in systemic oxidative stress. The duration of imposed CR being for only 2 years clearly limits any extrapolation or speculation of the impact of CR on longevity in humans.

Notably, many biomarkers of aging (that could be a consequence of the overall improved metabolic profile commensurate with adipose tissue loss) were also improved in these young, healthy individuals. There is a clear need for continued investigations of CR in humans, since the non-human primate data are not entirely conclusive on the extension in the average and maximal lifespan but provide strong evidence for extensive health benefits including improved quality of life.

Link: https://doi.org/10.1016/j.cmet.2018.02.019

The evidence to date makes it clear that Alzheimer's disease isn't a condition in which amyloid-β alone drives progression of neurodegeneration. There is significant synergy between the aggregation of amyloid-β and tau protein, and between portions of the surrounding biochemistry. It isn't the solid deposits of amyloid-β and hyperphosphorylated tau that are the direct cause of cell dysfunction and death, but rather complex interactions related to these aggregates. Various studies have provided evidence to suggest that amyloid-β spurs tau aggregation, as well as vice versa, and it may be the case that both are true. The work here adds to the evidence for neurodegeneration to start with amyloid-β accumulation, which increases the pace at which tau aggregation later takes place. When it comes to actual damage to the brain, both cause significant harms, however.

Years ago, researchers noted that people with Alzheimer's disease have high levels of tau in the cerebrospinal fluid, which surrounds their brain and spinal cord. Tau - in the tangled form or not - is normally kept inside cells, so the presence of the protein in extracellular fluid was surprising. As Alzheimer's disease causes widespread death of brain cells, researchers presumed the excess tau on the outside of cells was a byproduct of dying neurons releasing their proteins as they broke apart and perished. But it was also possible that neurons make and release more tau during the disease.

In order to find the source of the surplus tau, researchers decided to measure how tau was produced and cleared from human brain cells. The researchers applied a technique known as Stable Isotope Labeling Kinetics (SILK). The technique tracks how fast proteins are synthesized, released and cleared, and can measure production and clearance in models of neurons in the lab and also directly in people in the human central nervous system. Using SILK, the researchers found that tau proteins consistently appeared after a three-day delay in human neurons in a laboratory dish. The timing suggests that tau release is an active process, unrelated to dying neurons.

Further, by studying 24 people, some of whom exhibited amyloid plaques and mild Alzheimer's symptoms, they found a direct correlation between the amount of amyloid in a person's brain and the amount of tau produced in the brain. Whether a person has symptoms of Alzheimer's disease or not, if there are amyloid plaques, there is increased production of this soluble tau. The findings are a step toward understanding how the two key proteins in Alzheimer's disease - amyloid and tau - interact with each other. "We knew that people who had plaques typically had elevated levels of soluble tau. What we didn't know was why. This explains the why: The presence of amyloid increases the production of tau."

Link: https://medicine.wustl.edu/news/study-finds-link-two-key-alzheimers-proteins/

There was something of a blizzard of publicity materials today for work on calorie restriction mimetics and a mechanism of action by which they improve endurance in old mice, acting to increase the generation of capillaries in muscle tissue via stress response systems related to sirtuins and NAD+. Given the present commercial efforts relating to supplements that enhance NAD+ levels, and given that the people involved are the same as those who popularized sirtuin research and development some years ago, we're probably in for at least a few years of hype related to these compounds and research into NAD+ in general.

It is worth remembering that nothing other than scientific knowledge emerged from all of the excitement surrounding sirtuins - well, that, and some people became wealthier by selling a company to GSK, but that research was later written off as not being a viable path to therapies. I'm not yet convinced that any excitement is justified in the present case either: ways to enhance NAD+ look little better than the past decade of ways to adjust sirtuin levels, and neither captures the full effect of calorie restriction. Marginal adjustments to the trajectory of aging are worth having when they are free, but as a major focus of aging research and development, I think this a poor investment. There are other roads to intervention in the aging process, such as SENS, that have a far better expectation value when it comes to the size of future benefits to human health and longevity. If we're going to put billions in funding and scores of scientists to work for decades, why not on the path that leads to comprehensive rejuvenation, rather than the path that leads to only modest effects on aging?

Anyway, that said, at the level of mechanisms and biochemistry this research is most interesting. It should adjust some of the present thinking regarding the relative contributions of various mechanisms to sarcopenia, for example, a condition with many possible causes. Loss of blood supply to muscles is on that list, and it is worth noting that other possible detrimental effects of a loss of capillaries with aging have also been investigated by researchers in recent years. Since calorie restriction is known to slow the progression of sarcopenia, that might increase the expectation for capillary loss to be significant in a variety of tissues - and thus worthy of a greater focus and further investigation. What are the underlying causes, however? This doesn't just randomly happen. Which of the known root causes of aging underlie this loss? It is far from clear as to why exactly this happens, unfortunately, but given greater interest in the topic, answers will arrive in time.

Some of the research here uses methionine restriction as a way to trigger many of the same stress response mechanisms as calorie restriction. While the two approaches don't produce exactly the same outcome in rodents, they clearly work through overlapping mechanisms. It is thought that much of the calorie restriction response is controlled through methionine sensing rather than mechanisms relating to the many other constituents of diet. It is, however, a very complex phenomenon, in which near everything in metabolism changes. That makes it a challenge to reverse engineer exactly what is taking place under the hood, and why progress towards effective calorie restriction mimetic therapies has been so slow and expensive. It is less an exercise of discovery and more an exercise of mapping large areas of cellular biochemistry so that discovery can take place at all.

Sulfur amino acid restriction diet triggers new blood vessel formation in mice

"The benefits of methionine restriction in rodents are fascinating because they resemble those of calorie restriction, but without enforced restriction of food intake." Previous work has shown that a methionine-restricted diet increases production of the gas, hydrogen sulfide, made in our cells where it functions in myriad beneficial ways. One of these is to promote the growth of new blood vessels from endothelial cells - a process known as angiogenesis. So the researchers decided to test whether there was a direct connection between a methionine-restricted diet and angiogenesis.

They fed mice a synthetic diet containing limited methionine and lacking the only other sulfur-containing amino acid, cysteine. These two amino acids are found in high amounts in protein-rich foods. After two months, the diet-restricted mice had increased the number of small blood vessels, or capillaries, in skeletal muscles compared to mice fed a control diet. The authors identified a requirement for the amino acid-sensing kinase GCN2 and the transcription factor ATF4 in angiogenesis triggered by methionine restriction.

Discovery offers hope for improving physical performance as we age

Researchers found that a decline in the blood flow to tissues and organs with age can be reversed by restoring molecules that improved exercise capacity and physical endurance in mice. The researchers found that the two molecules could replicate the benefits of exercise, a finding that could lead to better athletic performance, improved mobility in the elderly and the prevention of aging-associated diseases like cardiac arrest, stroke, liver failure, and dementia.

For the first time, the study showed that as levels of the metabolite NAD+ decline with age, the body's capacity to exercise decreases because of fewer blood vessels and reduced blood flow. By treating mice with the NAD+ booster NMN and increasing levels of hydrogen sulphide, physical endurance was extended in mice by over 60%. This was the case in both young and old mice. "With exercise, the effect is even more dramatic. We saw 32-month-old mice, roughly equivalent to a 90-year-old human - receiving the combination of molecules for four weeks ran, on average, twice as far as untreated mice. Mice treated only with NMN alone ran 1.6 times further than untreated mice." The scientists identified that this mechanism is due to a restoration of capillary formation in muscle by stimulating the activity of the protein SIRT1, a key regulator of blood vessel formation.

Treatment restores blood vessel growth, muscle vitality, boosts exercise endurance in aging animals

As we age, our tiniest blood vessels wither and die, causing reduced blood flow and compromised oxygenation of organs and tissues. Vascular aging is responsible for a constellation of disorders, such as cardiac and neurologic conditions, muscle loss, impaired wound healing and overall frailty, among others. Scientists have known that loss of blood flow to organs and tissues leads to the build-up of toxins and low oxygen levels. The endothelial cells, which line blood vessels, are essential for the health and growth of blood vessels that supply oxygen-rich and nutrient-loaded blood to organs and tissues. But as these endothelial cells age, blood vessels atrophy, new blood vessels fail to form and blood flow to most parts of the body gradually diminishes. This dynamic is particularly striking in muscles, which are heavily vascularized and rely on robust blood supply to function.

Muscles begin to shrivel and grow weaker with age, a condition known as sarcopenia. The process can be slowed down with regular exercise, but gradually even exercise becomes less effective at holding off this weakening. Researchers wondered: What precisely curtails the blood flow and precipitates this unavoidable decline? Why does even exercise lose its protective power to sustain muscle vitality? Is this process reversible? In a series of experiments, the team found that reduced blood flow develops as endothelial cells start to lose a critical protein known as sirtuin1, or SIRT1. Previous studies have shown that SIRT1 delays aging and extends life in yeast and mice. SIRT1 loss is, in turn, precipitated by the loss of NAD+, a key regulator of protein interactions and DNA repair that was identified more than a century ago. Previous research has shown that NAD+, which also declines with age, boosts the activity of SIRT1.

Study suggests method for boosting growth of blood vessels and muscle

Researchers decided to explore the role of sirtuins in endothelial cells, which line the inside of blood vessels. To do that, they deleted the gene for SIRT1, which encodes the major mammalian sirtuin, in endothelial cells of mice. They found that at 6 months of age, these mice had reduced capillary density and could run only half as far as normal 6-month-old mice.

The researchers then decided to see what would happen if they boosted sirtuin levels in normal mice as they aged. They treated the mice with a compound called NMN, which is a precursor to NAD, a coenzyme that activates SIRT1. NAD levels normally drop as animals age, which is believed to be caused by a combination of reduced NAD production and faster NAD degradation. After 18-month-old mice were treated with NMN for two months, their capillary density was restored to levels typically seen in young mice, and they experienced a 56 to 80 percent improvement in endurance. Beneficial effects were also seen in mice up to 32 months of age (comparable to humans in their 80s).

The researchers also found that SIRT1 activity in endothelial cells is critical for the beneficial effects of exercise in young mice. In mice, exercise generally stimulates growth of new blood vessels and boosts muscle mass. However, when the researchers knocked out SIRT1 in endothelial cells of 10-month-old mice, then put them on a four-week treadmill running program, they found that the exercise did not produce the same gains seen in normal 10-month-old mice on the same training plan. If validated in humans, the findings would suggest that boosting sirtuin levels may help older people retain their muscle mass with exercise. Studies in humans have shown that age-related muscle loss can be partially staved off with exercise, especially weight training.

If it is possible to use machine learning to assess human biological age from a photograph, can that same feat also be repeated for mice? It is reasonable to think that this will be a more challenging task, but the potential benefits are sizable. If a reasonably accurate assessment of biological age in mice could be as simple for a researcher as taking a few photographs, then the cost of exploratory research in aging and rejuvenation could be meaningfully reduced. With that eventual aim in mind, initial software development for the MouseAge project was crowdfunded last year at Lifespan.io. Now that an iOS application is available, data collection can begin.

An international team of longevity and deep learning experts working on the crowdfunded non-profit MouseAge project announce the launch of the MouseAge mobile application on the iOS platform to enable a community of researchers to contribute to the data collection and research. The MouseAge team is working on an exciting crowd-funded and crowd-sourced research project intended to develop the proof of concept for the deep learned photographic aging clock in mice.

Development of a reliable biomarker of aging based on photographic images of mice has the potential to accelerate aging research and help identify new interventions that extend lifespan. We would like to address this need while engaging the broader research community, with the goal of offering a simple, freely available tool to anybody working with mice. The group recently ran a successful crowdfunding campaign and developed a specialized mobile app called MouseAge. The app allows the scientists to take pictures of mice of different age and short videos that will be used for training of the deep neural networks.

Even though there is a great degree of risk with the project and it might not be possible to develop the most accurate predictor of age using the many body parts of a large number of mice, in the case the effort is successful, the team plans to make the results public and publish a research paper describing the effort. Scientists working with C57BL/6 mice are invited to contribute images to the project. A collaboration would entail downloading the app and taking pictures of 200 normal aging mice. Qualified researchers actively contributing to the project are expected to be co-authors on the research paper in the case of a successful project completion.

Link: https://www.eurekalert.org/pub_releases/2018-03/imi-acm032118.php

The three most harmful forms of metabolic waste in the aging brain are amyloid-β, hyperphosphorylated tau, and α-synuclein, all of which precipitate into solid deposits with a complex halo of surrounding biochemistry that damages and ultimately kills cells. They contribute to various age-related neurodegenerative conditions that are classified as amyloidosis, tauopathy, and synucleinopathy, respectively. Looking at just one of these forms of waste in isolation misses the real story, however. An aging brain has some of each, and it is apparent from the study of Alzheimer's disease that amyloid-β and tau interact to produce greater harm together than either does on its own. So should we be surprised to find evidence that tau and α-synuclein also have synergies in well known synucleinopathies such as Parkinson's disease? Perhaps not.

This sort of finding favors approaches to clearance of metabolic waste that tackle all of it at once, not just selective types. The most expensive, and so far failed, immunotherapies for Alzheimer's disease, for example, focus specifically on amyloid-β, or more recently specifically on tau. The more that we see interaction between these forms of damaged protein in the brain, the more we should favor methodologies for clearance of all waste present in cerebrospinal fluid, such as the Leucadia Therapeutics line of development, or various means of restoring the activity of microglial cells responsible for clearing out unwanted proteins and other debris.

Parkinson's disease (PD) and Lewy body dementia (LBD), behind Alzheimer's disease (AD), are the most common neurodegenerative disorders with no effective therapies targeting the cause of disease. The pathological hallmarks of PD are cytoplasmic inclusions called Lewy bodies (LB), comprised primarily of α-synuclein, along with hyperphosphorylated tau and other sequestered proteins, in dopaminergic neurons. However, the importance of LB to the neurotoxicity in disease has been questioned. A number of studies have shown that oligomeric α-synuclein is the toxic species, rather than fibrils comprising LBs, and that α-synuclein oligomers may be the most effective therapeutic target.

In spite of the clear prevalence of α-synuclein pathology in disease, one of the greatest genetic risk factors for PD is tau, the role of which is understudied and poorly understood. Phosphorylated tau aggregates have been reported in numerous synucleinopathy mouse models, suggesting a possible synergistic interaction between α-synuclein and tau in mediating neurodegeneration in PD, as α-synuclein may increase tau aggregation and tau may have a similar effect on α-synuclein. While neurofibrillary tangles (NFTs) characterize tauopathies and are not correlative of synucleinopathies, recent studies suggest that intermediate forms of tau - tau oligomers - that form prior to or independently of NFTs, are the true toxic species in disease and the optimum targets for anti-tau therapies.

We have evaluated the efficacy of targeting the toxic, oligomeric form of tau protein by passive immunotherapy in a mouse model of synucleinopathy. We treated seven-month-old mice overexpressing mutated α-synuclein (A53T mice) with tau oligomer-specific monoclonal antibody (TOMA) and a control antibody and assessed both behavioral and pathological phenotypes. We found that A53T mice treated with TOMA were protected from cognitive and motor deficits two weeks after a single injection. Levels of toxic tau oligomers were specifically decreased in the brains of TOMA-treated mice. Tau oligomer depletion also protected against dopamine and synaptic protein loss. These results indicate that targeting tau oligomers is beneficial for a mouse model of synucleinopathy and may be a viable therapeutic strategy for treating diseases in which tau and α-synuclein have a synergistic toxicity.

Link: https://doi.org/10.1186/s13024-018-0245-9

It is already well known that the immune cells called macrophages are involved in the mechanisms of heart failure, and in the research noted here the details of that role are further explored. Macrophages are important in processes of regeneration and tissue growth throughout the body, but also in the propagation of inflammation in response to damaging circumstances. A growing theme in the research of past years is the polarization of macrophages, meaning their division into several subtypes based on behavior. Some are inflammatory and aggressive, attacking pathogens but also hindering regeneration, while others are not inflammatory and undertake a variety of activities to directly aid tissue regeneration. A useful response to injury requires both behaviors in some proportion, and at different times, but later life and many age-related conditions are characterized by the presence of far too many inflammatory macrophages. Removing these macrophages or adjusting their state shows promise as a basis for therapy.

The researchers here find macrophages displaying a CCR2 receptor, which correlates fairly well with the inflammatory polarization, are necessary for much of the harmful growth of the heart that takes place in later life, as the cardiovascular system becomes damaged and dysfunctional. One of the more important components of heart failure is this hypertrophy of heart tissue. The muscle grows larger and weaker, initially in response to the failure of blood pressure feedback mechanisms that takes place alongside the development of hypertension, but later a range of other mechanisms are also involved. Clearly, inflammatory macrophages are doing their part to generate an unhelpful growth response - and so selectively removing them could be a useful form of therapy.

Prevention of blood pressure issues is probably a better first option for those not already old, however. If rejuvenation therapies can (a) prevent the processes that lead to the stiffening of blood vessels, such as cross-linking and calcification, and (b) prevent the atherosclerotic plaque that narrows blood vessels, such as by clearing out the harmful lipid compounds that cells cannot effectively break down, then hypertension and other blood pressure issues could be largely eliminated. Given a life-long normal blood pressure, the impact of inflammatory processes on the heart will be that much less severe. They must still be dealt with, as the secondary consequence of fibrosis remains an issue, but that can happen in a context of better overall health and physical robustness.

Immune cell target identified that may prevent or delay heart failure after pressure overload

Researchers have found that preventing the early infiltration of CCR2+ macrophages into the heart, after experimental pressure overload in a mouse model, significantly lessened the heart's enlargement and reduced pumping ability that leads to later heart failure. Thus, this infiltration is a required step in the path toward heart failure. Macrophages are immune cells that engulf and remove damaged or dead cells in response to tissue injury or infection. They also may present antigens to other immune cell types. The most common forms of pressure overload are aortic stenosis - a narrowing of the aortic valve of the heart that forces the heart muscle to overwork - and high blood pressure.

The researchers used two different methods to prevent early macrophage infiltration - an inhibitor of the macrophage cell-surface CCR2 chemokine receptor, and an antibody that selectively removes CCR2+ macrophages. Migrating macrophages use the CCR2 receptor to home in on damaged tissues in the body that are releasing chemokines. Preventing early macrophage infiltration may offer a therapeutic target in human disease. Researchers had previously known that pressure-overload heart failure is associated with inflammation caused by activated T-cells. The present study showed the link between infiltrating macrophages and the T-cell response during pressure overload of the heart.

One week after inducing pressure load, that the heart showed increased expression of three attractant chemokines that are able to bind to the CCR2 receptor on macrophages. The researchers also found an increased number of monocytes with the cell-surface markers Ly6C and CCR2 circulating in the blood, and they saw an eightfold increase in CCR2+ macrophages infiltrating into the heart. Those macrophages are derived from the circulating monocytes. Thus increased circulating monocytes might serve as an easily measurable biomarker that reflects cardiac tissue CCR2+ macrophage expansion. The circulating monocytes - along with other clinical, imaging and biochemical biomarkers - could guide patient selection for a prospective clinical trial to find out whether modulating CCR2 macrophages in humans with pressure-overload hypertrophy will delay or prevent later transition to heart failure.

CCR2+ Monocyte-Derived Infiltrating Macrophages Are Required for Adverse Cardiac Remodeling During Pressure Overload

Inflammation is a hallmark of chronic heart failure (HF) initially triggered by nonimmune modes of cardiac injury, such as myocardial infarction, genetic mutations, and mechanical stress (e.g., pressure overload). Moreover, the systemic and myocardial immune cell profiles underlying the inflammatory response in the various etiologies of HF are of considerable importance for disease progression. For example, in chronic ischemic HF, expanded populations of both innate immune cells (e.g., macrophages) and T cells in the heart promote tissue injury and pathological remodeling. Chronic nonischemic HF due to pressure overload is characterized by CD4+ T-cell activation, which has been shown to play a critical role in promoting adverse cardiac remodeling. We recently demonstrated that during cardiac pressure overload, proinflammatory macrophage expansion in the heart occurs early, before sustained systolic dysfunction, but resolves during the chronic stage.

Importantly, although pressure-overload HF is characterized by T-cell activation, prior work also indicates that such activation is dependent on antigen presentation, because the progression of HF is ameliorated upon blockade of T-cell costimulatory molecules on antigen presenting cells (APCs). The requirement for specific antigen recognition implies an essential pathogenetic role for macrophages and other APCs, although their specific function in the development of pressure-overload HF remains poorly defined. Recent studies have characterized cardiac macrophage populations in the heart with disparate functions, including tissue-resident, embryonically derived macrophages and infiltrating monocyte-derived macrophages. The normal heart is seeded with resident macrophages that are not replenished by circulating monocytes under steady-state conditions. Resident cardiac macrophages are minimally inflammatory and promote angiogenesis and tissue repair. However, cardiac injury and aging stimulate the infiltration of monocyte-derived macrophages that are proinflammatory, promote tissue injury, and the death and substitution of resident cells.

Monocyte-derived macrophages can be distinguished by the expression of C-C chemokine receptor 2 (CCR2). Although we and others have documented expansion of cardiac macrophages during the early phase of pressure overload, it is unknown whether the macrophages are monocyte-derived, and whether these cells play an important role in subsequent T-cell recruitment and activation, and associated long-term adverse cardiac remodeling. Accordingly, here we tested the hypothesis that CCR2+ monocyte-derived macrophages infiltrate the heart early following pressure-overload-induced hemodynamic stress, and that this macrophage population plays a critical role in the activation of T cells and the ensuing transition to failure.

Vitalik Buterin is the originator of Ethereum, but also a strong supporter of research and development aimed at bringing aging under medical control. He recently stepped up to make a $2.4 million donation to the SENS Research Foundation to support the scientific programs there, and thus help to hasten the advent of the first generation of working rejuvenation therapies. This is very welcome support at a critical juncture in the development of means of human rejuvenation, biotechnologies that will be based on periodic repair of the forms of cell and tissue damage that cause aging. The Life Extension Advocacy Foundation volunteers arranged this interview with Buterin, one of a number of articles resulting from the recent Undoing Aging conference that they hope to publish soon.

Wealthy people usually donate money towards research into and treatment of cancer, Alzheimer's disease, and other diseases. Why did you decide to donate Ethereum to the fight against aging?

The first reason is just because there are many other people who donate to fight against cancer and other specific diseases, which, of course, is very important and necessary. The second reason is that there is strong scientific evidence that aging is the root of the most serious diseases.

It turns out that if you slow down aging or even reverse it, you can save people from serious illnesses such as malignant tumors, stroke, and Alzheimer's disease.

Exactly. After all, if you do not prevent these diseases by eliminating aging, you will have to provide treatment to people who are already sick and suffering and whose quality of life is worsening, and the economy will be under enormous pressure because the treatment is often expensive, caregiving is needed, etc. These problems could be avoided. Studies of aging are very important right now, yet there are still very few people who invest money in this field, unfortunately.

Why do you think that is so?

Most people simply do not know or do not believe that aging can be successfully manipulated. However, I have read Ending Aging by Dr. Aubrey de Grey, I'm interested in scientific discoveries, and I see that this is plausible. Researchers can already extend the life of laboratory animals significantly, and it is necessary to refine these technologies in order to transfer them to humans. And research and full-scale clinical trials of anti-aging therapies in humans requires money.

Do you have plans to continue supporting research projects on aging and life extension, or is your current contribution of 2.4 million dollars likely to be all?

Of course I'm ready to invest more into it. However, right now, I am mostly investigating what the scientists are working on, what the most promising directions are, and what else should be supported.

What, in your opinion, is the main problem currently hampering the fight against aging on Earth?

There is not enough public support. Huge resources, as I said, are invested in research and treatment of single diseases, but the problem is that if we focus only on specific diseases, this will only slightly improve the lives of people who are already chronically sick. Only a few years will be added to their lives.

Link: https://www.leafscience.org/vitalik-buterin-the-best-thing-to-donate-money-to-is-the-fight-against-aging/

Elastin, as the name might suggest, is an important structural molecule in the extracellular matrix of elastic tissues, such as blood vessels. Elastin content in blood vessel walls falls with age, alongside the stiffening of those blood vessels, though it is an open question as to the degree to which that is secondary to various mechanisms such as chronic inflammation, presence of senescent cells, and so forth. A very interesting study in mice from a few years ago demonstrated improved elasticity in the lung tissue of mice resulting from clearance of senescent cells, for example.

It is also an open question as to whether the reduction in elastin is as important as the cross-linking of molecules in the extracellular matrix when it comes to stiffening of blood vessels - absent the ability to selectively fix just one of these problems, firm answers will remain elusive. And that is before we consider other mechanisms such as calcification, probably also due in large part to the presence of senescent cells, or disrupted signaling that hampers the ability of smooth muscle cells in blood vessels to coordinate vasoconstriction and vasodilation.

The line of evidence constructed in the research results noted here is somewhat tenuous, since it was carried out in animal models of a genetic condition in which elastin levels are abnormally low, and with a focus on young patients rather than older individuals. It doesn't necessarily follow that because a boost in elastin production helped to restore blood vessel elasticity in this situation, then the same result will occur in old patients. Old blood vessels may have reduced elastin to some degree, but also have the range of other problems mentioned above. If there is a suitable drug candidate or other means of increased elastin production ready to go, as appears to be the case, then it would seem cost-effective to try it and see - but I'd wager on better results from cross-link breaking if this turns out to be a matter of significant investment in further research first.

Arteries in young, healthy humans and other mammals stretch easily because they contain a protein called elastin. Elastin is produced only during development, however, and is slowly lost with aging. Stiff arteries contribute to development of high blood pressure and significantly increase the risk of sudden death, stroke, myocardial infarction, and cognitive decline. "We know that genetic conditions, such as Williams-Beuren Syndrome (WS) and supravalvar aortic stenosis (SVAS), lead to abnormally low levels of elastin in developing arteries. As a result, children with WS or SVAS have stiff, narrow arteries and high blood pressure. Like older adults, they are also at increased risk of sudden death and stroke. We therefore tested whether a medicine called minoxidil would not only reduce blood pressure but also would help relax arteries and increase their diameter, thus improving organ perfusion."

Minoxidil is perhaps best known for its potential to improve hair growth when applied to the skin. In a different formulation, minoxidil is sometimes prescribed orally for high blood pressure that has not responded to other medications. Earlier studies have suggested that minoxidil may increase elastin deposition even in mature tissues. The research team conducted the work in experimental models of hypertension and chronic vascular stiffness associated with WS and SVAS. They used ultrasound imaging and magnetic resonance imaging-based arterial spin labeling to gauge minoxidil's impact on vessel mechanics, carotid and cerebral blood flow, and gene expression.

"Minoxidil not only lowered blood pressure, but also increased arterial diameter and restored carotid and cerebral blood flow. Minoxidil also reduced functional arterial stiffness and increased arterial elastin content. Equally important, these beneficial changes persisted weeks after the drug was no longer in the bloodstream. The sustained improvements and the increased elastin gene expression suggest that minoxidil treatment may help remodel stiff arteries. Such remodeling may benefit humans whose elastin insufficiency is due to either advanced age or genetic conditions."

Link: https://childrensnational.org/news-and-events/childrens-newsroom/2018/medicine-that-slows-balding-may-turn-stiff-vessels-supple-helping-vital-organs

There will be no bright dividing line between evolved cellular component and artificial molecular machinery in the future of medicine and human enhancement. It is already possible to produce programmable DNA machinery that can react to the environment in simple ways, or to adjust the programming of cells by altering the production or activities of specific proteins. As understanding of the cell improves, it will be possible to produce nanoscale structures that act in similar ways to cellular components. Researchers are starting down this road with the production of various forms of manufactory, artificial membranes that enclose anything from cells or bacteria to a minimal set of DNA or other molecular machinery that can produce specific proteins or other molecules in response to circumstances. The articles below look at the two ends of this scale: an entire cell wrapped in a membrane on the one hand, versus much smaller components designed to be taken up and used by cells, releasing molecules in response to internal signals.

For the future, it is possible to envisage all sorts of further possibilities. Tweaks to existing structures to make them better: enhanced lysosomes equipped with a better range of digestive enzymes, improving the ability of long-lived cells to break down unwanted molecular waste; mitochondria with a stripped down, best of breed mitochondrial genome, based on the most performant of those evolved in our species; protein production and protein clearance structures based upon those found in other species that are much more efficient than the human model; cultured gut bacteria that are designed from the ground up, with minimal genomes, to be entirely beneficial; and more. Or simple artificial cells that replace or augment some of the simpler functions of evolved cells, such as the production of a needed protein or removal of an unwanted protein. Or wholly new structures within a cell that trickle out signal molecules that permanently increase cellular stress responses. Or sophisticated manufactories capable of producing all of the known cancer suppression genes, delivered by the billion, taken up into all cells, where they lie dormant, waiting to triggered into activity in cancerous cells. There are so very many options for improvement.

Further down the line, machinery that looks very different from cells will start to become a viable proposition. Diamondoid nanotechnology, for example, coupled with molecular manufacturing to mass produce devices that look nothing like cells, but can be vastly more efficient than any cell at a specific task. Nanomachines that can store hundreds as times as much oxygen as a red blood cell; that can identify and destroy pathogens without flagging; that can assist in the repair and maintenance of the inner machinery of living cells. The fusion of machine and biology will become highly sophisticated and varied. The importance of the designation of biological or artificial will fade, and ultimately we will become just as designed and enhanced as any of of the countless component parts in our cells.

Artificial and biological cells work together as mini chemical factories

Researchers have fused living and non-living cells for the first time in a way that allows them to work together, paving the way for new applications. The system encapsulates biological cells within an artificial cell. Using this, researchers can harness the natural ability of biological cells to process chemicals while protecting them from the environment. This system could lead to applications such as cellular 'batteries' powered by photosynthesis, synthesis of drugs inside the body, and biological sensors that can withstand harsh conditions.

Previous artificial cell design has involved taking parts of biological cell 'machinery' - such as enzymes that support chemical reactions - and putting them into artificial casings. The new study goes one step further and encapsulates entire cells in artificial casings. The artificial cells also contain enzymes that work in concert with the biological cell to produce new chemicals. In the proof-of-concept experiment, the artificial cell systems produced a fluorescent chemical that allowed the researchers to confirm all was working as expected.

"Biological cells can perform extremely complex functions, but can be difficult to control when trying to harness one aspect. Artificial cells can be programmed more easily but we cannot yet build in much complexity. Our new system bridges the gap between these two approaches by fusing whole biological cells with artificial ones, so that the machinery of both works in concert to produce what we need. This is a paradigm shift in thinking about the way we design artificial cells, which will help accelerate research on applications in healthcare and beyond."

Tiny implants for cells are functional in vivo

In the cells of higher organisms, organelles such as the nucleus or mitochondria perform a range of complex functions necessary for life. Researchers are working to produce organelles of this kind in the laboratory, to introduce them into cells, and to control their activity in response to the presence of external factors (e.g. change in pH values or reductive conditions). These cellular implants could, for example, carry enzymes able to convert a pharmaceutical ingredient into the active substance and release it "on demand" under specific conditions. Administering drugs in this way could considerably reduce both the amounts used and the side effects. It would allow treatment to be delivered only when required by changes associated with pathological conditions (e.g., a tumor).

Now, researchers have succeeded in integrating artificial organelles into the cells of living zebrafish embryos. The artificial organelles are based on tiny capsules that form spontaneously in solution from polymers and can enclose various macromolecules such as enzymes. The artificial organelles presented here contained a peroxidase enzyme that only begins to act when specific molecules penetrate the wall of the capsules and support the enzymatic reaction. To control the passage of substances, the researchers incorporated chemically modified natural membrane proteins into the wall of the capsules. These act as gates that open according to the glutathione concentration in the cell. At a low glutathione value, the pore of the membrane proteins are "closed" - that is, no substances can pass. If the glutathione concentration rises above a certain threshold, the protein gate opens and substances from outside can pass through the pore into the cavity of the capsule. There, they are converted by the enzyme inside and the product of the reaction can leave the capsule through the open gate.

The researchers chose zebrafish embryos because their transparent bodies allow excellent tracking of the cellular implants under a microscope when they are marked with a fluorescent dye. After the artificial organelles were injected, they were "eaten" by macrophages and therefore made their way into the organism. The researchers were then able to show that the peroxidase enzyme trapped inside the artificial organelle was activated when hydrogen peroxide produced by the macrophages entered through the protein gates.

The trial results announced here represent a promising step forward in efforts to regenerate an age-damaged retina, particularly because the patients were in an advanced stage of their degenerative condition and nonetheless achieved a meaningful degree of restored sight. Macular degeneration has a number of different manifestations, and here the wet form was treated, which involves excessive growth of blood vessels in the retina and consequent death of the retinal cells necessary for vision. Researchers have established an approach involving the generation of a patch of engineered retinal cells that can be implanted to restore some of the lost retinal function. Given the details, it is interesting to speculate on the degree to which the transplanted cells are helping by integrating into the retina versus helping by issuing signals that spur local regeneration. In most cell therapies it is the latter, but here the transplanted cells are more organized into a tissue-like structure.

Human embryonic stem cells (ESCs) represent a promising source for cellular replacement therapies owing to their availability, pluripotency, and unlimited self-renewal capacity. However, they also carry risks of neoplastic change, uncontrolled proliferation, and differentiation to inappropriate cell types. The eye is advantageous in investigating hESC-based cell therapy as it is accessible and confined, and the transplanted cells can be monitored directly in vivo, with the possibility of being removed or destroyed if there is evidence of neoplastic change. Furthermore, long-term immunosuppression can be delivered locally.

Late age-related macular degeneration (AMD) is characterized by irreversible cell loss, initially of retinal pigment epithelium (RPE) cells and subsequently of neuroretinal and choroidal cells, and thus may be amenable to hESC-based cell therapy. Suspensions of hESC-derived RPE (hESC-RPE) cells have been transplanted in human subjects with dry AMD and Stargardt's disease, but the extent of cell survival and restoration of vision remains ambiguous.

We developed a therapeutic, biocompatible hESC-RPE monolayer on a coated synthetic membrane, herein termed a 'patch', for transplantation in wet and early-stage dry AMD. The choice of membrane material and its preparation, including the human vitronectin coating, has not been described previously to our knowledge. In contrast to RPE suspensions, cells on the patch are delivered fully differentiated, polarized, and with the tight junction barrier formed, that is, in a form close to their native configuration. The synthetic membrane allows the patch to be handled easily and robustly. The main disadvantage of the patch is that it requires a purpose-built delivery tool and a more complicated surgery compared to cell suspensions, and the use of hESCs may require immunosuppression, unlike an autologous cell source.

The clinical trial was designed as a phase 1, open-label, safety and feasibility study of implantation of an hESC-RPE patch in two subjects with acute wet AMD and recent rapid vision decline. For safety reasons and to obtain an early efficacy signal, the trial involved patients with severe wet AMD only, although we aim to study the RPE patch in early dry AMD in the future. We reported three serious adverse events to the regulator. These were exposure of the suture of the fluocinolone implant used for immunosuppression, a retinal detachment, and worsening of diabetes following oral prednisolone. All three incidents required readmission to the hospital, with the first two incidents requiring further surgery and the third being treated medically. The three incidents were treated successfully. Both patients achieved an improvement in best-corrected visual acuity of more than 15 letters at 12 months after transplantation.

Although 12 months is sufficient to begin to describe cell survival and clinical outcomes, it is early in terms of safety monitoring, especially for late teratoma formation. The patients will be followed for five years after surgery. These two early cases are also instructive as they show an encouraging outcome despite very advanced disease, which increases the complexity of surgery and involves more damaged neuroretina.

Link: https://doi.org/10.1038/nbt.4114

The destruction of near all immune cells followed by cell therapy to speed recreation of the immune system is a fairly harsh procedure, as the only way to clear a sufficiently high fraction of immune cells at the moment is essentially a form of chemotherapy. It is an effective treatment for autoimmune conditions, however, albeit with a significant risk of death, in line with that for many major surgeries. This makes it suitable in its current form only for more severe autoimmune disorders in which the patients tend to be younger and more robust, but with a very poor prognosis. In past years researchers have demonstrated considerable success with multiple sclerosis, and the article here provides an update on ongoing trials. The results continue to be impressive.

In the future, the chemotherapy approach will be replaced with more targeted, less harmful methods of selective cell destruction - consider the Oisin Biotechnologies cell destruction technology turned against immune system markers, for example. More gentle cell destruction methodologies will make immune system recreation viable as a way to rejuvenate aged immune systems, even in very old, frail individuals, clearing out all of the misconfigured, senescent, exhausted, or otherwise harmful immune cells. That is why it is worth keeping an eye on progress in this line of research.

Doctors say a stem cell transplant could be a "game changer" for many patients with multiple sclerosis (MS). Results from an international trial show that it was able to stop the disease and improve symptoms. It involves wiping out a patient's immune system using cancer drugs and then rebooting it with a stem cell transplant. Just over 100 patients took part in the trial, in hospitals in Chicago, Sheffield, Uppsala in Sweden and Sao Paolo in Brazil. They all had relapsing remitting MS - where attacks or relapses are followed by periods of remission. The interim results were released at the annual meeting of the European Society for Bone and Marrow Transplantation in Lisbon.

The patients received either haematopoietic stem cell transplantation (HSCT) or drug treatment. After one year, only one relapse occurred among the stem cell group compared with 39 in the drug group. After an average follow-up of three years, the transplants had failed in three out of 52 patients (6%), compared with 30 of 50 (60%) in the control group. Those in the transplant group experienced a reduction in disability, whereas symptoms worsened in the drug group. "The data is stunningly in favour of transplant against the best available drugs - the neurological community has been sceptical about this treatment, but these results will change that."

The treatment uses chemotherapy to destroy the faulty immune system. Stem cells taken from the patient's blood and bone marrow are then re-infused. These are unaffected by MS and they rebuild the immune system. "We are thrilled with the results - they are a game changer for patients with drug resistant and disabling multiple sclerosis. This is an interim analysis, but with that caveat, this is the best result I have seen in any trial for multiple sclerosis." The transplant costs around $40,000, about the same as the annual price of some MS drugs. Doctors stress it is not suitable for all MS patients and the process can be gruelling, involving chemotherapy and a few weeks in isolation in hospital.

Link: http://www.bbc.com/news/health-43435868

Hopefully the Fight Aging! audience recalls the years-long hype over resveratrol, driven by the self-serving processes that enabled investors in Sitris Pharmaceuticals to make a sizable profit at the expense of GSK, and supplement sellers to open up a new market for the credulous. The only meaningful results from all of that turned out to be an increased knowledge of the biochemistry of sirtuins, one very thin slice of the broad metabolic response to calorie restriction. Resveratrol and its ilk are not meaningful calorie restriction mimetics, and you are far better off cutting a few hundred calories from your daily intake or exercising a little more.

In light of this history I think it is entirely appropriate to be skeptical of the current hype surrounding the role of NAD+ in metabolism, and the various precursor molecules that can increase levels of NAD+ when taken as dietary supplements. When compared with sirtuins and resveratrol, the publicity here involves many of the same people, similar for-profit companies engineering the news cycle, and the same area of cellular biochemistry, which is to say aspects of calorie restriction closely related to sirtuins. My expectation is that, at the end of the day, this will result in nothing more than another increase in the knowledge of this portion of cellular biochemistry, while all the other claims regarding longevity and health are largely smoke and mirrors. Some people will make a lot of money, supplement sellers will prosper, and nothing will meaningfully change in human health as a result of all of this.

The first study in mice noted below is very similar in outcome to past studies of resveratrol, which is to say little in the way of gains in healthy mice, and some compensation for the detrimental effects of being overweight or obese. It is important to remember that mouse longevity is far more plastic than that of humans in response to calorie restriction and interventions that affect the same portions of cellular biochemistry as are involved in the calorie restriction response. Mice live 40% longer when calorie restricted; in humans the gain is unlikely to be larger than a few years, even though the observed health benefits are sizable. So an alleged calorie restriction mimetic that produces no gain in mouse longevity, or only helps to make overweight mice less metabolically abnormal, is not all that interesting. You might compare this with the second paper, which is a commentary from the usual suspects on how great the prospects are for supplementation related to NAD+ levels.

Nicotinamide Improves Aspects of Healthspan, but Not Lifespan, in Mice

The role in longevity and healthspan of nicotinamide (NAM), the physiological precursor of NAD+, is elusive. In the present study, we aimed to characterize the effects of chronic NAM supplementation on the longevity and healthspan characteristics of male C57BL/6J mice fed a synthetic low-fat diet (SD) and the corresponding high-fat diet (HFD). Because of the liver's importance in maintaining metabolic homeostasis, we carried out histological, biochemical, and untargeted metabolomics surveys to provide an unbiased view of the metabolic impact exerted by 62-week NAM supplementation on liver from SD- and HFD-fed mice.

Protein target validation combined with metabolic flux analysis enabled the identification of the underlying mechanisms of enhanced glucose disposal and reduced oxidative stress in response to NAM supplementation. Surprisingly, our data showed that NAM depresses NAD salvage and has complex effects on sirtuin expression and activity. NAM appears to have greater beneficial effects in mice subjected to HFD than SD, which might provide important clues about its therapeutic potential in the fight against obesity and associated comorbidities.

We report that chronic NAM supplementation improves healthspan measures in mice without extending lifespan. Analysis revealed NAM-mediated improvement in glucose homeostasis in mice on a high-fat diet (HFD) that was associated with reduced hepatic steatosis and inflammation concomitant with increased glycogen deposition and flux through the pentose phosphate and glycolytic pathways. Although neither hepatic NAD+ nor NADP+ was boosted by NAM, acetylation of some SIRT1 targets was enhanced by NAM supplementation in a diet- and NAM dose-dependent manner. Collectively, our results show health improvement in NAM-supplemented HFD-fed mice in the absence of survival effects.

Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence

Nicotinamide adenine dinucleotide (NAD) is one of the most important and interesting molecules in the body. It is required for over 500 enzymatic reactions and plays key roles in the regulation of almost all major biological processes. Above all, it may allow us to lead healthier and longer lives. Much of the renewed interest in NAD over the last decade can be attributed to the sirtuins, a family of NAD+-dependent protein deacetylases (SIRT1-7). Sirtuins have been shown to play a major regulatory role in almost all cellular functions. At the physiological level, sirtuins impact inflammation, cell growth, circadian rhythm, energy metabolism, neuronal function, and stress resistance.

By modulating NAD+-sensing enzymes, NAD+ controls hundreds of key processes from energy metabolism to cell survival, rising and falling depending on food intake, exercise, and the time of day. NAD+ levels steadily decline with age, resulting in altered metabolism and increased disease susceptibility. Restoration of NAD+ levels in old or diseased animals can promote health and extend lifespan, prompting a search for safe and efficacious NAD-boosting molecules that hold the promise of increasing the body's resilience, not just to one disease, but to many, thereby extending healthy human lifespan.

Researchers have identified TREM2 as a target to potentially enhance the ability of immune cells in the brain to remove amyloid beta, solid deposits of misfolded proteins associated with the progression of Alzheimer's disease. Removal of amyloid beta remains the primary focus of the Alzheimer's research community, despite the continued lack of progress towards working therapies based on this approach. An increasing number of researchers are investigating alternatives to the existing approaches to amyloid immunotherapy, so far failing to achieve meaningful results in human trials.

The slow accumulation of amyloid beta and other metabolic waste in the brain looks a lot like the consequence of a slow failure of clearance mechanisms, as amyloid levels are actually quite dynamic from moment to moment. One candidate for this failure is the age-related deterioration of immune activity, in and of itself a very complex topic - which is one reason to think that therapies based on improved immune function might be helpful. Other candidates include failure of filtration of cerebrospinal fluid in the choroid plexus, or more recent views on the failure of cerebrospinal fluid drainage. Alzheimer's is a complex condition, and the brain is a complex organ.

Two new studies describe how TREM2, a receptor found on immune cells in the brain, interacts with toxic amyloid beta proteins to restore neurological function. The research, performed on mouse models of Alzheimer's disease, suggests boosting TREM2 levels in the brain may prevent or reduce the severity of neurodegenerative disorders including Alzheimer's disease. "Our first paper identifies how amyloid beta binds to TREM2, which activates neural immune cells called microglia to degrade amyloid beta, possibly slowing Alzheimer's disease pathogenesis. The second study shows that increasing TREM2 levels renders microglia more responsive and reduces Alzheimer's disease symptoms."

One of the hallmarks of Alzheimer's disease is the accumulation of amyloid plaques that form between neurons and interfere with brain function. Many drug companies have been working for years to reduce amyloid beta production to thwart Alzheimer's - but with minimal success. "TREM2 offers a potential new strategy. Researchers have known that mutations in TREM2 significantly increase Alzheimer's risk, indicating a fundamental role for this particular receptor in protecting the brain. This new research reveals specific details about how TREM2 works, and supports future therapeutic strategies to strengthen the link between amyloid beta and TREM2, as well as increasing TREM2 levels in the brain to protect against pathological features of the disease."

The first study showed that TREM2 binds quite specifically to amyloid beta. In particular, it connects with amyloid beta oligomers (proteins that bind together to form a polymer), which are the protein's most toxic configuration. Without TREM2, microglia were much less successful at binding to, and clearing out, amyloid beta. Further investigation showed that removing TREM2 downregulated microglial potassium ion channels, impairing the electrical currents associated with the activation of these immune cells. In addition, TREM2 turned on a number of mechanisms associated with the amyloid beta response in microglia.

In the second study researchers added TREM2 to a mouse model with aggressive Alzheimer's disease. They found that the added TREM2 signaling stopped disease progression and even restored cognitive function. As they learn more about how TREM2 modulates the amyloid signals that put microglia to work, the researchers have their work cut out for them. "It could be beneficial in early stages to activate microglia to eat up amyloid beta, but if you over-activate them, they may release an overabundance of cytokines (causing extensive inflammation) damaging healthy synaptic junctions as a side-effect from overactivation." Still, the ability to use the brain's existing immune mechanisms to clear amyloid offers intriguing possibilities.

Link: https://www.sbpdiscovery.org/press/brains-immune-system-may-be-key-to-new-alzheimers-treatments

Biological age, as opposed to chronological age, is driven by the intrinsic processes of primary aging, the accumulation of molecular damage outlined in the SENS rejuvenation research proposals, but also by the influence of the environment, secondary aging. The important contributions to secondary aging are excess visceral fat tissue as a consequence of diet, burden of infectious disease, lack of exercise, and smoking, acting through a range of mechanisms that overlap with the intrinsic processes of primary aging. There are others, but their effects are smaller and it is harder to see them in the data in comparison to the points above.

In the paper here, researchers make an effort to map recent changes in secondary aging, picking combinations of metrics from past data that might offer insight into the biological age of patients. I would say that there is little reason to expect primary aging to have altered significantly in the past few decades, given the landscape of medical technology, but it is certainly up for debate as to whether medications that control blood pressure and cholesterol levels might have some effect. They have certainly become more prevalent and effective over the time covered by the study data.

Overall this is an interesting exercise, but of little relevance to the future of aging. Gains from here on out will increasingly arise from the development of rejuvenation therapies that can repair the damage of primary aging, rather than from lifestyle improvements such as reduction in smoking or obesity. Greater potential gains in health and life span might be achieved through addressing primary aging; the scope of increased longevity through better lifestyle choices is far more limited. Our remaining healthy life span will be determined ever more by progress in rejuvenation biotechnology as time passes.

A new study suggests that at least part of the gains in life expectancy over recent decades may be due to a change in the rate of biological aging, rather than simply keeping ailing people alive. "This is the first evidence we have of delayed aging among a national sample of Americans. A deceleration of the human aging process, whether accomplished through environment or biomedical intervention, would push the timing of aging-related disease and disability incidence closer to the end of life. Life extension without changing the aging rate will have detrimental implications. Medical care costs will rise, as people spend a higher proportion of their lives with disease and disability. However, lifespan extension accomplished through a deceleration of the aging process will lead to lower healthcare expenditures, higher productivity, and greater well-being."

Using data from the National Health and Nutrition Examination Survey (NHANES) III (1988-19994) and NHANES IV (2007-2010), the researchers examined how biological age, relative to chronological age, changed in the U.S. while considering the contributions of health behaviors. Biological age was calculated using several indicators for metabolism, inflammation, and organ function, including levels of hemoglobin, total cholesterol, creatinine, alkaline phosphatase, albumin, and C-reactive protein in blood as well as blood pressure and breath capacity data.

While all age groups experienced some decrease in biological age, the results suggest that not all people may be faring the same. Older adults experienced the greatest decreases in biological age, and men experienced greater declines in biological age than females; these differences were partially explained by changes in smoking, obesity, and medication use. Slowing the pace of aging, along with increasing life expectancy, has important social and economic implications. The study suggests that modifying health behaviors and using prescription medications does indeed have significant impact on the health of the population.

Link: http://news.usc.edu/138495/older-americans-experiencing-delayed-aging-better-health/

The Undoing Aging conference in Berlin is presently underway, a gathering of everyone who is anyone in the rejuvenation research community. It is hosted jointly by the SENS Research Foundation and the Forever Healthy Foundation, and is a unification of the varied themes of the past fifteen years of SENS conferences: the science of aging and its treatment from the earlier SENS conference series mixed with the industry, startup, and commercial development focus of the Rejuvenation Biotechnology series of recent years.

The first rejuvenation therapies to be implemented and shown to work, those based on clearance of senescent cells, are presently entering human trials and being carried forward to the clinic by startups. The next set of rejuvenation therapies, targeted at different mechanisms of aging such as cross-links and mitochondrial damage, are still in the laboratory, working their way towards completion. This is a time of transition, the birth of a new field of applied biotechnology, one that will grow to subsume most of the present medical community and provide services and products to every adult human being.

To commemorate the occasion, the Life Extension Advocacy Foundation volunteers have published a three-part interview with Aubrey de Grey of the SENS Research Foundation, the person who did the most to start this ball rolling back at the turn of the century. Later joined by a range of allies within and outside the scientific community, and then by a community of advocates and supporters, a bootstrapping process of growth towards the industry needed to bring an end to aging has been underway since. These are fairly lengthy interviews, and I'm not quoting more than a fraction of the whole here - you'll certainly find further interesting comments if you read the whole thing.

Undoing Aging With Aubrey de Grey Part One

Why did you choose Berlin and not California or elsewhere in the USA for the event?

Basically, because the suggestion came from our main German donor, Michael Greve, who is also the conference's main sponsor. Hard to argue with that!

Is SRF planning to make Undoing Aging into a recurring event, much like the Rejuvenation Biotechnology conferences in America?

We'll certainly be continuing to do both more science-centered events like Undoing Aging and the SENS Conferences, as well as more rejuvenation biotechnology industry-oriented events like the Rejuvenation Biotechnology series, but we haven't yet decided on the sequence and orientation of future meetings. We certainly want to maintain a strong conference presence in California, but it may be best to do that with smaller, more frequent events, such as the one we did with the California Life Sciences Association.

Recently, SRF has received significant donations amounting to over 7 million dollars. What priorities does SRF plan to address with this money?

First and foremost, we will be gearing up our existing programs in mitochondrial gene replacement, scaling up glucosepane research, rejuvenation biotechnology against cytosolic aggregates, and so on. We will also be initiating new ones; those are still being discussed with potential extramural collaborators, but you can expect some announcements later this year. They will all be within the same seven-strand framework that has defined SENS since the beginning. And after having sometimes in the past allocated nearly all of our available research budget at the beginning of the fiscal year and thereby limiting our ability to take advantage of new opportunities that arose later in the year, we will be maintaining a research reserve fund so that we are always poised to get good work funded year-round.

For anyone reading this who is thinking about doing the same as our recent donors, I will just say that we are a very long way from running out of productive ways to invest more money.

Undoing Aging With Aubrey de Grey Part Two

Regarding the use of senolytics, are you concerned about their potential to remove highly specialized cells like cardiomyocytes, which do not divide or do so very slowly?

Cells that don't divide (like cardiomyocytes and neurons) are far less likely to become senescent in the first place than cell types that divide; many of the main drivers of senescence are related to cell division. In the specific case of cardiomyocytes, there's already significant evidence in rodents that senolytics improve cardiac function overall. However, there is some reason for concern here, which is why we're already working to develop the next generation of senescent cell ablation therapies. The selectivity of senolytic drugs for senescent cells comes from the fact that they target the activity or expression of genes involved in cell survival, on which senescent cells are much more reliant than healthy cells under normal, unstressed conditions. But during times in which the cell is under stress, normal cells also rely on those same pathways to carry them through and give them time to recover. Future therapies can target truly senescent cells more selectively, and SENS Research Foundation is helping to advance those next-generation senescent cell therapies, even as UNITY Biotechnology prepares for human testing, through our investment in Oisín Biotechnologies.

Senolytic drugs gave mice increased healthy lifespan in experiments. Given that every living organism produces senescent cells the same way, could this mean that it may translate to humans?

Interventions that lead to gains in median lifespan only in laboratory mice, with no corresponding effect on a robust maximum lifespan (tenth-decile survivorship), still need to be heavily discounted when speculating on effects in humans. Interventions that only affect median lifespan primarily affect deaths in the first half of the lifespan - and here there is a critical difference between mice in a lab and modern humans, for whom medicine has already eliminated many causes of such early deaths, from vaccines (which also impact late-life mortality by reducing lifelong inflammatory burden), to surgery, to antibiotics, to drugs that more obviously affect middle-aged people.

The force of this reasoning is somewhat attenuated in the case of interventions like senescent cell clearance, which actually repair aging damage, than with interventions affecting environmental or metabolic risk factors driving "premature" disease (obesity, inflammation, cardiovascular risk factors, environmental toxins, etc). Still, you have to assume that the effect on lifespan of any single damage-repair intervention in isolation will be modest, based on the principle of the "weakest link in the chain": all the links are weakening over time, and shoring up only one of them still leaves the rest of the links damaged and ready to shear, whereupon the whole chain is broken. To move the needle on lifespan in modern humans, we have to push back on all of the cellular and molecular damage of aging, not just one form.

Regarding the breakdown of extracellular aggregates, what will you do if the first wave of treatments using antibodies is unable to repair the whole system?

Certainly, it's guaranteed that no first-generation SENS therapy will be able to repair every single contributor to any given category of aging damage - and it doesn't have to. All we have to do to reach "longevity escape velocity" is to remove or repair the specific forms of cellular and molecular aging damage within each category that meaningfully restrict our lives to the extremes of current lifespans. During the extra decades of healthy life that we'll then enjoy, scientists can then work to identify the constraints that limit life- and healthspan to those newly-expanded horizons. Accordingly, all SENS therapies will need to be iteratively improved; we will want safer and more effective ways to repair the damage targeted by earlier iterations of rejuvenation biotechnologies and also to repair additional specific targets within each category. It's only once those first therapies are developed and in use that we'll know what their specific limitations will be

Have you reviewed you position that nuclear mutations matter only in cancer in light of recent research results suggesting that certain ominous mutations in hematopoietic stem cells increase the risk of developing not only blood cancers (50 fold) but dying of all causes by 40%?

The research on this "clonal hematopoiesis" phenomenon is certainly provocative but doesn't ultimately change our view on this question. Remember first that it has never been our position that nuclear mutations matter only in causing cancer; at a minimum, they also matter in causing apoptosis ("cellular suicide," which denudes the body of functional cells with age, most importantly stem cells) and cellular senescence (ditto, plus the baleful effects of the senescence-associated secretory phenotype). And then remember that SENS is fundamentally an engineering approach to aging, focused on practical solutions rather than acquiring a full understanding of mechanistic details. Our position has been, therefore, that all the effects of nuclear mutations that meaningfully constrain current human lifespan/healthspan can be obviated by removing, repairing, or obviating the effects of mutations that are relevant to our health over the course of currently-normal lifespans: clearing senescent cells, replacing cells lost to apoptosis and senescence and other causes, and making the body impervious to cancer.

In clonal hematopoiesis, blood stem cells with one of a small number of mutations gain a selective advantage over blood stem cells with other genotypes, which allows them to "take over" the stem cell compartment. This isn't exactly what an oncologist would call "cancer," but it is a clear case of "too many cells" caused by nuclear mutations proliferating at the expense of their neighbors, which fits the operational criteria for the oncoSENS category. And the periodic purging of all native bone marrow stem cells and their wholesale replacement with fresh, mutation-free, cancer-proof ones - which would immediately eliminate clonal hematopoiesis - is already planned to be the very first clinical phase of the whole body interdiction of lengthening of telomeres (WILT) plan to pre-emptively shut down cancer.

Undoing Aging With Aubrey de Grey Part Three

Has your position on the relevance of telomere attrition changed since you first devised SENS, especially in the light of the recent results with fibrosis and your involvement with AgeX Therapeutics?

No. Let's start with the big picture. Neither I nor anyone sensible has ever suggested that telomere attrition has no functional effects in aging: telomere attrition causes cells to become senescent and runs down the proliferative capacity of stem cells, amongst other things. Nor have I suggested that there wouldn't be some short-term health benefits to activating telomerase or telomerase gene therapy in aging animals or animal models of age-related disease. The issue is rather that those short-term benefits come with the longer-term (and sometimes not so long-term) risk of increased rates of cancer.

So, why don't we see a plague of excess cancers in animal studies that show the benefits of telomerase-based treatments? Depending on the study, it's one or more of several reasons. The most common one is that such studies are usually too short-term. A related issue is that many of these studies involving animal models of age-related disease are actually done in quite young animals that have been damaged in some way that simulates aspects of an age-related disease. Because such animals are still quite young, they haven't yet lived long enough to have accumulated a high burden of the kinds of mutations that predispose cells to become cancerous. A third reason why many animal studies of telomerase treatments don't result in high reported rates of cancer is that the animals may actually be deficient in telomerase to begin with, such that telomerase gene therapies actually just restore the normal activity of telomerase in the animals.

The solution to problems caused by age-related attrition of telomeres is not to juice up telomerase to lengthen them again in often-damaged stem cells, but to take telomerase out of the picture, purge those defective stem cells, and replenish stem cell pools periodically with cancer-proofed, pristine replacement cells that are unable to replicate out of control.

You have been engineering glucosepane-eating bacteria that use enzymes effectively 'gifted' to them. Have the enzymes you identified demonstrated specificity to glucosepane?

We can say that Dr. David Spiegel's SRF-funded lab at Yale has identified some candidates, but we can't go into the details at this time. Still, expect some news on the commercialization front in the glucosepane space in coming months.

Given the state of immunotherapy, and taking into account the rate of progress in the field, how confident are you that OncoSENS may be unnecessary?

The recent progress in cancer immunotherapy has certainly made me much more optimistic than I was five years ago that new cancer therapies might hold off cancer for more than a very small number of years - but not that it might make whole body interdiction of lengthening of telomeres (WILT) redundant. If we had all the other components of a comprehensive panel of rejuvenation biotechnologies assembled and deployed, ongoing progress with these therapies might well give us a slightly longer runway along the path to "longevity escape velocity" than I had expected at the time. But only slightly; within an all-too-short few additional years, I expect that without WILT, the surging rocket of "longevity escape velocity" will still run headlong into a wall of cancer until we have a way to definitively defeat its evolutionary engine of selection and replication. At present, WILT is the sole foreseeable approach to doing that.

Which rejuvenation treatments can we reasonably expect to reach the clinic first?

If you don't count stem cell therapies (some of which are in clinical use, but not as rejuvenation biotechnology), it's a race between ablating senescent cells with senolytics (with UNITY Biotechnology expected to perform their first-in-human trials early next year) and one of the many immunotherapies targeting the intracellular or extracellular aggregates that drive the neurodegenerative diseases of aging.

There are many people who subscribe to the idea that accurately preserving the fine structure of the brain on death, having that brain scanned and discarded, and the data of that scan later used to run a whole brain emulation is essentially no different from cold water drowning followed by successful resuscitation. There is a stop, and then a start. That the same pattern is running in a completely different system, and the original is destroyed, is immaterial: the pattern is the self. The rest of us would say that this individual died permanently with the destruction of the preserved brain, and the emulation is a copy - and possibly not even a continuous, surviving, single entity, depending on the implementation.

Which of these views you or I hold is entirely unimportant right up to the point at which it is possible to preserve the brain on death and have some choice about what happens next. Since we do presently live in the era of brain preservation by vitrification or, recently, vitrifixation, whether one holds a pattern identity view (the self is the pattern) or a continuity view (the self is the pattern as embodied in this particular set of matter) can turn out to be important. The former will kill you, if you let it steer your choices. Clearly I'm not the only one who feels that pattern identity beliefs have the potential to be dangerous to those who subscribe to them, as illustrated by this article on the options for near future development of improved methods of brain preservation.

As someone who is fully supportive of the ultimate goals of the cryonics enterprise, but still views the current state of the practice with some degree of skepticism, I make a point of acquainting myself with the latest evidence regarding the quality of cryonics procedures and their ability to preserve the foundations of a person's identity through time. Over the past two years or so, I have increasingly seen a recent achievement by 21st-Century Medicine (21CM) cited by some cryonics supporters as demonstrating the scientific validity of those procedures: namely 21CM's research on aldehyde-stabilized cryopreservation (ASC). This new technique has allowed them to win the Technology Prize awarded by the Brain Preservation Foundation (BPF) by demonstrating excellent preservation of brain ultrastructure. Were I to follow this line of reasoning, I could happily set aside my concerns about the adequacy of today's cryopreservation procedures, which had now been verified by scientific experts; the proper focus would now need to be on how to responsibly introduce those procedures into a clinical setting, for patients at the end of their lives who might request them.

It turns out, however, that things are not so simple. ASC is no doubt a step forward for the field of brain banking, and as its name indicates, it it is indeed a form of cryopreservation, since it involves vitrification of the brain at -135°C. Nonetheless, ASC does not count as cryonics, insofar as it uses a fixative solution prior to vitrification and cooling, which could potentially preclude revival of the original biological brain (an essential part of cryonics as traditionally understood). And indeed, biological revival with the help of future technology is not a priority for the Brain Preservation Foundation (BPF)'s president, Dr. Kenneth Hayworth. Rather, he envisages brain preservation as conducive to life extension via mind uploading: a process that would involve cutting the preserved brain into thin slices, scanning each slice, and feeding the resulting data to an advanced computer that would thereby be able to map out the entire network of neural connections in the person's original brain, and ultimately to emulate that person's mind. This is quite different from cryonics.

The BPF's commitment to holding brain preservation research to the highest standards of scientific rigour is laudable, and worth emulating. Nonetheless, for those interested in brain preservation with a view to enabling life extension, supporting cryonics-specific research remains the safer bet. We should not simply rely on the BPF's approach if our goal is to try and save those whom medicine in its current state cannot restore to life and health.

To see why this is so, let us begin by noting the two main philosophical theories of personal identity through time that are relevant when discussing the respective merits of cryonics and mind uploading in this context. The first one, which we can call the "Physical Continuity" (PhyCon) theory, asserts that a person is identical with the physical substratum from which her mind emerges: that is to say, her brain, with its intricate web of neurons and synaptic connections. The second relevant theory can be referred to as the "Psychological Continuity" (PsyCon) theory. Roughly speaking, it says that you are to identical with the set of psychological features (memories, beliefs, desires, personality traits, etc.) that constitutes your mind. On this view, preserving you after you have been pronounced dead requires ensuring the persistence of enough of those psychological features, in an embodied mind of some sort (but one that need not be embodied in your current biological brain).

If that is the case, what is the prudent choice to make for those who wish to promote life extension through brain preservation? I submit that traditional cryonics is the more prudent option to pursue. This can be demonstrated using a simple argument that considers what the implications are if we assume that PhyCon and, respectively, PsyCon are true. Suppose first that PhyCon is true. If so, a cryonics procedure carried out properly will save a person's life, whereas using a technique like ASC that compromises the brain's potential for viability, followed by destructive scanning and uploading, will kill that person. If PsyCon is true, on the other hand, both methods can ensure survival. Indeed, adequate cryonic preservation of a person's brain would also preserve the ultrastructure grounding the various psychological features that defined that person.

None of this is meant to imply that the work of the BPF is without merit. On the contrary, the Foundation's approach demonstrates a number of virtues that can provide a model for the cryonics movement to follow. These include a commitment to rigorously and impartially evaluating the quality of brain preservation procedures, in accordance with the standards of scientific peer-review. Another example is the BPF's successful effort at crowdfunding its incentive prizes for brain preservation research, such as the two prizes won by 21CM.

Link: http://www.evidencebasedcryonics.org/2018/03/14/brain-preservation-and-personal-survival/

Reinforcing the SENS rejuvenation biotechnology view of the importance of lysosomal aggregates in aging, researchers here demonstrate a link between lyososomal function and the ability of neural stem cells to support brain tissue. Lysosomes inside cells are recycling machines, packed with enzymes capable of breaking down near everything they will encounter. They are the ultimate destination for damaged proteins and other broken cellular structures. Unfortunately, lysosomes do encounter molecular waste that they cannot handle, and long-lived cells become ever more burdened by damage as their lysosomes falter and become bloated. The processes of recycling and cellular maintenance back up and run down, and the cells become dysfunctional.

The solution envisaged by the SENS Research Foundation is to build therapies capable of safely breaking down the unwanted contents of lysosomes. The most promising way forward appears to be mining the bacterial world for enzymes that might serve as a starting point. The known resilient lysosomal wastes do not accumulate in graveyards, so we know those bacteria and their useful molecular tools are out there, waiting to be discovered. The first SENS program to work along these lines successfully discovered a number of candidate enzymes that proceeded to further development, and are currently at various stages in that process.

Young, resting neural stem cells in the brains of mice store large clumps of proteins in specialized cellular trash compartments known as lysosomes. As the cells age, they become less proficient at disposing of these protein aggregates, and their ability to respond readily to "make new neurons" signals wanes. Restoring the ability of the lysosomes to function normally rejuvenates the cells' ability to activate, the researchers found. "We were surprised by this finding because resting, or quiescent, neural stem cells have been thought to be a really pristine cell type just waiting for activation. But now we've learned they have more protein aggregates than activated stem cells, and that these aggregates continue to accumulate as the cells age. If we remove these aggregates, we can improve the cells' ability to activate and make new neurons. So if one were able to restore this protein-processing function, it could be very important to bringing older, more dormant neural stem cells 'back to life.'"

Researchers isolated several populations of cells for study from the brains of both young and old mice, including resting neural stem cells, activated neural stem cells, and the neural cell progenitors that arise from activated stem cells. They found that resting stem cells expressed many lysosome-associated genes, while activated stem cells expressed genes associated with a protein complex involved in protein destruction called a proteasome. Strict control of production and disposal allows cells to maintain the necessary protein inventory to carry out needed cellular functions.

"The fact that these young, pristine resting stem cells accumulate protein aggregates makes us wonder whether they actually serve an important function, perhaps by serving as a source of nutrients or energy upon degradation." Old resting stem cells express fewer lysosome-associated genes and begin to accumulate even higher levels of protein aggregates. "It's almost as if these older cells lose the ability to store, or park, these aggregates. We found that artificially clearing them by either activating lysosomes in older cells or subjecting them to starvation conditions to limit their protein production actually restored the ability of these older resting stem cells to activate. We'd like to know whether the aggregated proteins are the same in the young and old cells. What do they do? Are they good or bad? Are they storing factors important for activation? If so, can we help elderly resting stem cells activate more quickly by harnessing these factors? Their existence in young cells suggests they may be serving an important function."

Link: http://med.stanford.edu/news/all-news/2018/03/clearing-clumps-of-protein-in-aging-neural-stem-cells-boosts-activity.html

Cellular senescence is one of the root causes of aging. Cells enter a senescent state in response to damage or the end of their replicative life span, and near all quickly self-destruct or are destroyed by the immune system. Others enter senescence to assist in regenerative processes following wounding, again being destroyed soon afterwards. Senescent cells that linger are a real problem, however. They generate harmful signaling that produces chronic inflammation, destructively remodels tissue structures, and changes the behavior of nearby cells for the worse. The accumulation of senescent cells over the years directly contributes to the progression of age-related dysfunction, disease, and risk of death.

Just how many senescent cells is any given individual burdened with, however? What should we expect from this cause of aging at a specific age? Is it negligible at 40 or 50, with a sudden leap to significant levels at 60? Does the answer vary by tissue type? How do the usual health-associated lifestyle choices affect these numbers? Are senescent cells significantly different from tissue to tissue in terms of the signals they generate and the harm done?

The answers to these questions are not yet established in any robust way, but the development of therapies capable of destroying senescent cells is proceeding regardless - there is plenty of evidence to show that removing these cells is beneficial, even without the greater insight into the fine details. This more detailed information is important, however, when it comes to the energy with which any particular individual should pursue access to the first generation of senolytic therapies capable of destroying senescent cells, and where those groups involved in therapeutic development should focus most of their attention.

One of the paths to a better understanding of how the burden of cellular senescence progresses with age, and how that progress varies by tissue type, is the production of a more detailed mapping of biomarkers of senescence. The open access paper here is an example of this sort of work, initially focused on mice. Better and more discerning markers of cellular senescence and the harms it creates will help to validate existing senolytic therapies and steer the development of new and better approaches.

Age- and Tissue-Specific Expression of Senescence Biomarkers in Mice

Cellular senescence plays a complex role, both beneficial and deleterious, in biological processes such as embryonic development, wound healing, tissue regeneration, and tumor suppression, as well as age-related disorders. Senescent cells accumulate within aged tissues and at sites of age-related pathology in vivo, and potentially contribute to the age-related decline of tissue function by affecting the growth, migration and differentiation, of neighboring cells, impacting overall tissue architecture, and promoting chronic inflammation. Indeed, studies on both progeroid and naturally aged mice showed that selective elimination of p16Ink4a-expressing senescent cells increased healthspan and lifespan. Thus, the selective elimination of senescent cells (senolytics) or the disruptions of the senescence-associated secretory phenotype (SASP) program have been developed as potential therapeutic strategies against aging.

However, while p16Ink4a expression has been used as a classical senescence biomarker, no biomarker of senescence identified thus far is entirely specific to the senescent state. Thus, due to the lack of robust biomarkers of cellular senescence in vivo, the precise extent of senescent cell accumulation in aged animals and the functional outcome of such an accumulation, along with the exact target cells of, and removal by, senolytics, remain unclear. Surprisingly, a systematic multi-tissue in vivo study of senescence markers during aging has not been conducted in wild-type animals.

In the era of senolytics, it becomes imperative to develop robust biomarkers of senescence in vivo for preclinical trials, especially with several senolytics now nearing human clinical studies. As a first step, in this study we profiled the expression of a panel of known molecular markers of senescence in multiple tissues in mice at multiple ages, ranging from young (4 months) to very old (30 months). The results demonstrate that the secretory profiles and classical hallmarks of cellular senescence in aged tissues are highly variable and complex, suggesting that a systematic and concerted effort is needed to develop robust biomarkers of senescence for the identification, quantification, and monitoring of senescent cells in vivo.

The wide diversity in tissue-specific profiles we observed was striking. Nevertheless, the matrix metalloproteinase Mmp12 represents a robust SASP factor that showed consistent age-dependent increases in expression across all tissues analyzed in this study. It has been demonstrated that mice lacking Mmp12 are protected from vascular injury, M2 macrophage accumulation, and perivascular heart fibrosis. Together with our data, this finding suggests that Mmp12 upregulation with age has a deleterious impact on heart function.

In this study, we did not observe significant age-dependent upregulation of the prominent SASP cytokine Il6 in any tissue, although an upward trend was observed that was consistent in magnitude with previous observations in the heart and kidney. This modest age-related upward trend could be explained by a previous report which demonstrated that senescent cell-secreted IL-6 acts in an autocrine manner, reinforcing the senescent state, rather than inducing senescence or promoting dysfunction in neighboring cells.

The decreased expression of Il6 with age we observed in the hypothalamus could be indicative of a lack or loss of senescent cells in that tissue with age. In support of this interpretation, p16Ink4a expression was non-detectable in the hypothalamus at any age. Taken together, these results suggest that some other age-related process results in the increased expression of the pro-inflammatory factors Il1b, Mmp12, Cxcl1, and Cxcl2 observed in the aged hypothalamus. Conversely, p16Ink4a expression was upregulated with age in all other tissues analyzed, consistent with previous reports, and thus reinforcing the importance of p16Ink4a as a biomarker of tissue aging.

Questions still remain, however, regarding the ultimate identity of the cells targeted for senolytic elimination in previous studies, as it has been demonstrated repeatedly that p16Ink4a expression is not exclusive to senescent cells, and thus does not represent an unequivocal target for senolytic therapies. Interestingly, however, CDKN2A (the gene that encodes p16Ink4a) was one of the top human genes that exhibited elevated expression with age, in 6 out of 9 tissues, including subcutaneous adipose, tibial artery, lung, skeletal muscle, tibial nerve, and whole blood, as detected by RNA-seq analysis. Thus, utilizing p16Ink4a-expressing cells as a biomarker of tissue aging and a target of senolytic therapies could still prove to be an effective strategy in the future treatment of age-related diseases in humans.

Why do gums heal more rapidly than skin? These research results follow that question down into the cellular biochemistry of regeneration and stem cell activity, in search of the important differences between gums and skin. The authors have uncovered a potentially interesting mechanism in the signaling of stem cells present in gum tissue, one that might be exploited to speed up healing of wounds elsewhere in the body. Investigations of stem cell signaling and its role in regeneration are a growing focus in the research community. Many classes of future regenerative therapies may well do away with cell transplants in favor of delivering only the signals generated by those cells.

Ever notice how a cut inside the mouth heals much faster than a cut to the skin? Gum tissue repairs itself roughly twice as fast as skin and with reduced scar formation. One reason might be because of the characteristics of gingival mesenchymal stem cells, or GMSCs, which can give rise to a variety of cell types. "This study represents the convergence of a few different paths we've been exploring. First, we know as dentists that the healing process is different in the mouth; it's much faster than in the skin. Second, we discovered in 2009 that the gingiva contains mesenchymal stem cells and that they can do a lot of good therapeutically. And, third, we know that mesenchymal stem cells release a lot of proteins. So here we asked, how are the gingival mesenchymal stem cells releasing all of these materials, and are they accelerating wound healing in the mucosal tissues?"

From earlier work it was clear that mesenchymal stem cells perform many of their functions by releasing signaling molecules in extracellular vesicles. So to understand what distinguishes mesenchymal stem cells in the gingiva from those in the skin, researchers began by comparing these extracellular vesicles between the two types. They found that the GMSCs contained more proteins overall, including the inflammation-dampening IL-1RA, which blocks a proinflammatory cytokine.

Next the team zoomed in to look at what might be controlling the release of IL-1RA and other cytokines. They had a suspect in the protein Fas, which they had earlier connected to immune regulation. They found that in gingival MSCs had more Fas than skin MSCs, and that mice deficient in Fas had reduced IL-1RA as well as reduced secretion of IL-1RA. Further molecular probing revealed that Fas formed a protein complex with Fap-1 and Cav-1 to trigger the release of small extracellular vesicles. To identify the connection with wound healing, they examined wound tissue and found that IL-1RA was increased in GMSCs around the margins of wounds. Mice lacking IL-1RA or in which the protein was inhibited took longer to heal gingival wounds. In contrast, when the researchers isolated IL-1RA that had been secreted from GMSCs and injected it into wounds, it significantly accelerated wound healing.

These findings may have special significance for people with diabetes, a major complication of which is delayed wound healing. In the study, the researchers found that GMSCs in mice with diabetes were less able to secrete extracellular vesicles compared to GMSCs in healthy mice, and their GMSCs also had less IL-1RA secretion. Introducing extracellular vesicles secreted from the GMSCs of healthy mice reduced wound healing time in diabetic mice. "Our paper is just part of the mechanism of how these stem cells affect wound healing, but I think we can build on this and use these cells or the extracellular vesicles to target a lot of different diseases, including the delayed wound healing seen in diabetic patients."

Link: https://news.upenn.edu/news/unraveling-how-mesenchymal-stem-cells-gum-tissue-accelerate-wound-healing

This short, interesting interview is with one of the Buck Institute neuroscience researchers with an interest in cellular senescence as a component of degenerative aging. Exhibited here is perhaps the most optimism that I recall seeing in public comments from any of the Buck Institute faculty - but if I were involved in cellular senescence research, I'd be fairly optimistic as well. This part of the field is progressing rapidly, producing solid evidence of the association of cellular senescence with the development of age-related disease, and of the benefits that can be obtained by removing these unwanted cells.

The field seems to have agreed upon nine hallmarks of aging, do you believe it is feasible for us to one day be able to treat all of them?

Ten years ago I would have wondered how feasible this was, but based on the progress that has been made in the last few years I do think it is plausible that we will be able to address each of those pillars of aging and that by addressing these underlying mechanisms that drive aging we are going to be able to treat age-related disease. I think we have a tendency to view age-related diseases in silos but many of these disorders have a lot in common. I think we are on the brink of solutions to these problems, not in the next decades, but within years.

Could you briefly describe senescence and its impact on neurodegenerative diseases?

Senescence is a process in cells that stops cells from dividing, it gets activated when certain types of damage occurs. From an evolutionary perspective, cellular senescence is there to prevent damaged cells from undergoing the kind of rapid division that leads to tumors. This is great in the short term, but if they persist they give off toxic pro-inflammatory factors which can damage neighboring tissues. A lot of research in the field goes into understanding this process and what we can do to prevent this toxic effect.

For a long time the field of neuroscience ignored senescence because everyone just looked at the neurons, which don't divide. However we also started looking at the other cell types in the brain that do divide, namely astrocytes. They are a major support cell in the brain that also secrete growth factors that help neurons grow and communicate, they are also much more abundant than neurons. We then discovered that these cells do undergo senescence by looking at post-mortem tissue from Parkinson's disease brains and found astrocytes that had become senescent. We showed in animal models that if we could remove aggregations of these cells we could slow some of the disease process. This is very exciting because it means we can push this strategy forward into human clinical trials as it is a possible therapeutic strategy that has not been explored before. We were one of the first labs to look into this but now a lot of other labs around the world are jumping into cellular senescence to try and tackle age-related disorders.

You also explore the protein TFEB to boost lysosomal function and autophagy?

We were looking at a young-onset model of Parkinson's disease that has a mutation in the Parkin gene which marks damaged mitochondria for disposal via autophagy. We then learned that one of the major factors in that process is this transcription factor called TFEB, which is a master regulator of autophagy. This has now become a potential target for treating Parkinson's and Alzheimer's because these diseases are the result of protein build ups and dysfunctional mitochondria. It is thought that if we boost TFEB then cells will be able to better dispose of these protein build ups. We screened a number of compounds that boosts TFEB in the brains of these animals are now trying to move this forward to clinical trial.

Link: https://tmrwedition.com/2018/03/14/interview-with-aging-expert-dr-julie-k-andersen/

A few years ago, the Brain Preservation Foundation awarded the small mammal brain preservation prize to a team working with a vitrifixation method: chemical fixation combined with low temperature storage. It produces excellent preservation of the fine molecular structure of the brain, and of particular interest are those areas in which the data of the mind is thought to be encoded. It is not surprising to see the same approach working for a larger brain. While this isn't a completely straightforward step, as working with larger tissue sections is always harder in many ways than working with smaller tissue sections, it was expected.

The Brain Preservation Foundation is representative of a faction of our broader community who are (a) in favor of preserving brains, and thus individuals, from death and oblivion, (b) harshly critical of the technologies and methods of the present cryonics providers, and (c) inclusive of a fair number of pattern identity theorists. The latter viewpoint means that the self is identified with the pattern of information, not the location or matter used to store that pattern. So these are people comfortable with the idea of the data of the mind being read from a stored brain, used to run an emulation of consciousness in software, and the stored brain then discarded. In their view, the resulting artificial intelligence is still the self, rather than a copy. They are alive, not dead.

For those of us who adhere to the alternative viewpoint, the continuity theory of identity, the self is the combination of the pattern and its implementation in a specific set of matter: it is this mind as encoded in this brain. A copy is a copy, a new entity, not the self. Discarding the stored brain is death. The goal in the continuity theory view is to use some combination of future biotechnology and nanotechnology to reverse the storage methodology, repair any damage accumulated in the brain, and house it in a new body, restoring that individual to life.

I point this out because adoption of pattern versus continuity views of identity should determine an individual's view of the utility of vitrifixation for brain preservation. The primary point to consider here is that chemical fixation is a good deal less reversible than present day vitrification, low temperature storage with cryoprotectants. The reversible vitrification of organs is a near-future goal for a number of research groups. But reversing chemical fixation would require advanced molecular nanotechnology at the very least - it is in principle possible, but far, far distant in our science fiction future. The people advocating vitrifixation are generally of the pattern identity persuasion: they want, as soon as possible, a reliable, highest quality means of preserving the data of the mind. It doesn't matter to them that it is effectively irreversible, as they aren't hoping to use the brain again after the fact.

From a technical point of view, better high quality vitrifixation is an achievement. It will be of use in many areas of life science research, and is an important step forward. But in the matter of preservation of the self, for the countless people who will age to death prior to the advent of rejuvenation therapies, this is an excellent example of why philosophy matters. Wherever technological capacity catches up to desire, beliefs start to result in life or death choices. I think that pattern identity views of the world, just like much of religion, will lead to a great deal of unnecessary death and oblivion. This is one small sample of the development choices that lie ahead.

Before quoting some of the publicity materials, I'd like to revisit the point about the Brain Preservation Foundation folk being harshly critical of cryonics methodologies. It is one thing to say that there is considerable room for improvement. That is certainly true. Vitrification is currently irreversible in large tissues. It is challenging to correctly and sufficiently perfuse cryoprotectant into post-mortem brains; more and better automation would be very helpful. The cryopreservation services operate with too little funding for complete comfort, and would benefit from a greater connection to wealthier areas of biotech industry. This is something that will hopefully arise as reversible vitrification for organ storage becomes a reality. It is quite another thing, however, to claim that everyone stored is irreversibly dead, because the fine structure of the mind is no longer there. That is clearly not the case for a well conducted preservation, given the studies showing vitrification and and thaw of nematodes to preserve memory. The question is the degree of damage. Criticism is only useful when it is reasonable rather than a polemic.

Large Mammal BPF Prize Winning Announcement

Using a combination of ultrafast glutaraldehyde fixation and very low temperature storage, researchers have demonstrated for the first-time ever a way to preserve a brain's connectome (the 150 trillion synaptic connections presumed to encode all of a person's knowledge) for centuries-long storage in a large mammal. This laboratory demonstration clears the way to develop Aldehyde-Stabilized Cryopreservation into a 'last resort' medical option, one that would prevent the destruction of the patient's unique connectome, offering at least some hope for future revival via mind uploading. You can view images and videos demonstrating the quality of the preservation method for yourself at the evaluation page.

The Brain Preservation Foundation's (BPF) Large Mammal Brain Preservation Prize has been won by the cryobiology research company 21st Century Medicine (21CM) and lead researcher Robert McIntyre (an MIT-trained scientist who is now co-founder of the startup Nectome) and senior author Greg Fahy (Fellow of the Society for Cryobiology). The Prize required the successful preservation of synaptic connectivity across an entire pig brain in a manner compatible with centuries-long storage. To accomplish this, McIntyre's team scaled up the same procedure they used to previously preserve a rabbit brain, for which they won the BPF's Small Mammal Prize.

The first step in the ASC procedure is to perfuse the brain's vascular system with the toxic fixative glutaraldehyde, thereby instantly halting metabolic processes by covalently crosslinking the brain's proteins in place, and leading to death by contemporary standards (but not necessarily information-theoretic standards). Glutaraldehyde is sometimes used as an embalming fluid, but is more commonly used by neuroscientists to prepare brain tissue for the highest resolution electron microscopic and immunofluorescent examination. It should be obvious that such irreversible crosslinking results in a very, very dead brain making future revival of biological function impossible. So, it is reasonable to ask: "What is the point of a procedure that can preserve the nanoscale structure of a person's brain when biological revival is impossible?" The answer lies in the possibility of future non-biological revival.

A growing number of scientists and technologists believe that future technology may be capable of scanning a preserved brain's connectome and using it as the basis for constructing a whole brain emulation, thereby uploading that person's mind into a computer controlling a robotic, virtual, or synthetic body. The Brain Preservation Prize challenged the scientific community to develop a 'bridge' to that future mind uploading technology.

Implications of the BPF Large Mammal Brain Preservation Prize

Traditional cryogenic preservation faces two conflicting challenges: rapid decay and ice crystal formation. The brain begins decaying immediately upon death, and therefore must be chilled quickly to halt the decay. However, the water contained in the brain is at risk of freezing into ice crystals, which would slice through the organic matter. Consequently, a process known as vitrification is preferred, in which water descends below freezing without crystallizing, instead forming what is called an amorphous solid. Vitrification is achieved by perfusing the brain with cryoprotectants before lowering the temperature.

However, the perfusion and chilling process, better performed slowly and carefully, cannot be allowed an optimal timeframe in which to occur due to the rapid decay. The necessary frenzied approach can, therefore, still result in tissue damage. Another problem arises as well. To expedite the process, the method currently used by cryonics organizations forces the cryoprotectants into the brain so aggressively, and at such high concentrations, as to actually osmotically pull water out of the cells. The brain is literally dehydrated like a raisin, and with similar results: significant shrinkage and deformation. It is frankly difficult to imagine the large-scale, region-to-region connective relationships of the brain surviving such trauma.

This problem of getting to low temperatures quickly underlies the most serious challenge currently facing the cryonics industry, and gives many neuroscientists pause about the best interpretation of the standard practice, namely that it is quite likely destroying the patients' brains, rendering future revival impossible. As such, cryonics "patients" or "subjects" might be better called by a different word: cadavers. To their credit, advocates for traditional cryonics acknowledge this problem, expressing their hope that futuristic technologies will repair both the micro- and macroscale damage. However, if the damage is truly information-destroying in nature, then no future technology, regardless of advancement, can ever recover the information. That fact is a fundamental trait of information theory.

So we can summarize the problem with current cryonics in the following way: Since the brain decays rapidly upon death, it must be chilled quickly to initiate preservation, but this hasty approach prevents adequate cryoprotectant perfusion, thereby risking partial ice crystal damage, while furthermore, the aggressive perfusion process used to accelerate the timeline additionally causes shrinkage and deformation.

Alcor Position Statement on Brain Preservation Foundation Prize

Many people are wondering whether Alcor plans to adopt the "Aldehyde-Stabilized Cryopreservation" (ASC) protocol used to win the prize and what the win means for cryonics in practice. Alcor's position is as follows: We are pleased that vitrification, the same basic approach that Alcor Life Extension Foundation has utilized since 2001, is finally being recognized by the scientific mainstream as able to eliminate ice damage in the brain during cryopreservation. Alcor first published results showing this in 2004. The technology and solutions that Alcor currently uses for vitrification (a technology from mainstream organ banking research) were actually developed by the same company that developed ASC and has now won both the Small Mammal and Large Mammal Brain Preservation Prize.

Current brain vitrification methods without fixation lead to dehydration. Dehydration has effects on tissue contrast that make it difficult to see whether the connectome is preserved or not with electron microscopy. That does not mean that dehydration is especially damaging, nor that fixation with toxic aldehyde does less damage. In fact, the M22 vitrification solution used in current brain vitrification technology is believed to be relatively gentle to molecules because it preserves cell viability in other contexts, while still giving structural preservation that is impressive when it is possible to see it. For example, note the synapses visible in the images at the bottom of this page.

While ASC produces clearer images than current methods of vitrification without fixation, it does so at the expense of being toxic to the biological machinery of life by wreaking havoc on a molecular scale. Chemical fixation results in chemical changes (the same as embalming) that are extreme and difficult to evaluate in the absence of at least residual viability. Certainly, fixation is likely to be much harder to reverse so as to restore biological viability as compared to vitrification without fixation. Fixation is also known to increase freezing damage if cryoprotectant penetration is inadequate, further adding to the risk of using fixation under non-ideal conditions that are common in cryonics. Another reason for lack of interest in pursuing this approach is that it is a research dead end on the road to developing reversible tissue preservation in the nearer future.

Alcor looks forward to continued research in ASC and continued improvement in conventional vitrification technology to reduce cryoprotectant toxicity and tissue dehydration. We are especially interested in utilizing blood-brain barrier opening technology such as was used to win the prize.

It is possible to think that (a) FDA regulators are not all that interested in much other than protecting their own positions, and their actions impose a terrible cost on health and longevity by suppressing progress in medicine, (b) that some degree of reviews and trials and data and proof are a great idea, necessary to the development of new therapies, and can be handled in a distributed way in a free market, and (c) people who run so far from the FDA that they drop the reviews and trials and data and proof, replacing them with marketing and wishful thinking, are not doing anyone any favors.

This collection of sensible ideas is, sadly, somewhat distant from the mainstream position these days, which appears to be that anything that isn't the full and complete FDA process (twice as lengthy and expensive today as it was ten years ago) is so dangerous as to be unworthy of consideration. This absolutism is unhelpful, to say the least. It is particularly pernicious when biased against patient paid trials, as is the case in the article here, for no particularly solid reason. Patient paid studies are a powerful tool when arranged well, carried out in search of large and reliable effects. The rest of the population is the control group, and it is sometimes possible to find funding in this way for very useful studies that would otherwise be overlooked.

When it comes to balancing scientific rigor against other influences, the anti-aging movement has long been a mixed bag. In many cases, the heart is in the right place, yearning after therapies that will help restore health to the aged, to make the world better, to help end suffering and death. But where this manifests in concrete actions, in all too many places the result is nothing but varieties of snake oil: a melange of outright nonsense, cherry-picked studies, the selling of nostrums and supplements that cannot possible have any significant effect. The sellers who found that they were too early became corrupted by the opportunity to make profit, or deluded themselves regarding the value of what was available. The financial success of this industry, while failing to achieve any of its original aims, is a noteworthy cautionary tale.

As time goes on and efforts progress in the production of calorie restriction mimetics, senolytics, and various other legitimate first generation attempts that either slightly slow or modestly reverse aging, the line between snake oil and marginal but real treatment will become indistinct and fuzzy. That has been going on for some years now. The Life Extension Foundation and its principals have given far more funding than I will ever manage to SENS rejuvenation research and various other legitimate scientific studies, yet this is also an organization that pumps out the traditional forms of misinformation about supplements and the value of dubious health gurus. Just as Isaac Newton was as much an alchemist as a scientist, so does the nonsense of the anti-aging marketplace at its worst and the promise of modern rejuvenation research at its best merge in many members of the community. Their projects cheerfully move back and forth across any line I'd care to draw between useful work and snake oil, between viable levels of testing and mere wishful thinking.

For the crowd of mostly baby boomers the warning could not have been more dire: You're running out of time. "We can't sit still. We don't have the time to do that," bellowed Bill Faloon, the 63-year-old former mortician addressing them from the stage. To his left and right, giant screens projecting government actuarial tables reminded the group of the "projected year of our termination." Men of Faloon's age could expect to die in 2037. Any 83-year-old women in the room? They've got until only 2026. "Take that initiative," Faloon urged his audience of about 120 people who had flown in from as far as California, Scotland, and Spain. How? Paying to participate in a soon-to-launch clinical trial testing transfusions of young blood "offers the greatest potential for everyone in this room to add a lot of healthy years to their life," Faloon said. "Not only do you get to potentially live longer ... but you're going to be healthier. And some of the chronic problems you have now may disappear."

The symposium attendees complained about ailments that hadn't bothered them when they were younger: Back problems. Bad hips. The aftermath of a stroke. Parkinson's disease. Arthritis. Many of them voiced frustration with the medical establishment and pharmaceutical companies, which they said pay too little attention to fixing the root cause of disease. Others voiced fears of spending their final days hooked up to machines in a hospital bed.

This $195-a-head symposium was held last month in this wealthy beachside community. It offered a striking view of how promoters aggressively market scientifically dubious elixirs to aging people desperate to defy their own mortality. Eight independent experts reviewed informational handouts about the clinical trial, and all sharply criticized the study's marketing, design, and scientific rationale. "It just reeks of snake oil," said Michael Conboy, a cell and molecular biologist at the University of California, Berkeley, who's collaborated on studies sewing old and young mice together and transfusing blood between them. "There's no evidence in my mind that it's going to work."

Beyond the questionable science, participants have to pay big money to join the trial. Faloon, an evangelist of anti-aging research who cut a slim figure in his black suit and had the thick dark hair of a younger man, acknowledged during his talk that it would be "expensive" to sign up for the trial. People considering enrolling said they had been told they would have to pay $285,000. But the Florida physician running the trial said the final price tag is still being discussed in consultation with the Food and Drug Administration and is likely to change. To evaluate whether the experimental treatment is safe and whether it might be able to reduce frailty, it is planned to run a battery of baseline testing on each clinical trial participant before they get their first infusion of young plasma and then monitor their changes for two years: That means cognitive exams, questionnaires about their quality of life and their indicators of frailty, and tests to measure biomarkers believed linked with aging, such as telomere length and DNA methylation.

Experiments like this operate on the fringes of science, yet they have captured the public imagination. The trial wouldn't be the first to transfuse plasma from young donors into older people. A biotech startup called Alkahest, spun out from a Stanford lab, reported results in November from a placebo-controlled safety trial testing the effect of plasma from young donors on 18 patients with mild to moderate Alzheimer's. The patients who got the plasma didn't suffer serious side effects, but the group didn't see a statistically significant improvement in their scores on a widely used cognitive exam. Meanwhile, a company called Ambrosia recently completed a clinical trial that charged about 80 people over the age of 35 a sum of $8,000 to get an infusion of plasma from a donor between the ages of 16 and 25. Ambrosia plans later this year to try to publish those results in a peer-reviewed journal.

Clinical trials that charge enrollees to participate are ostensibly aimed at giving patients early access to promising therapies - often in the fields of stem cells or aging reversal - that are too unusual or have too little profit potential to get funding from traditional sources such as companies, foundations, or the National Institutes of Health. But critics worry that such trials too often exploit desperate patients, offering them false hope of restored health while doing little or nothing to advance scientific research.

Link: https://www.statnews.com/2018/03/02/young-blood-anti-aging-study/

It is sometimes helpful to look back at recent history in order to see just how far we have come in terms of progress in medicine, wealth, and health. Ours is an era of rapid, profound change in technology and its capabilities, and that is very apparent in mortality statistics, such as the charts provided in the article noted here. The numbers change dramatically every few decades, the result of the scientific and medical communities turning their attention to the most pressing issues of their time, generation after generation.

The past century is a story of success due to advancing medical technology on the one hand and the will, wealth, and understanding to address environmental causes of mortality on the other. Yet at the same time improvements in wealth, comfort, and longevity created new forms of bad lifestyle choice and new challenges in health. Over the course of the 20th century, infectious diseases gave way to lifestyle diseases and age-related diseases. As ever more people had the opportunity to live longer, the medical conditions of old age increased as a cause of mortality - and then the medical community turned to address those newly prominent causes of death. Some lines on the chart of mortality fell and others rose. A line may rise for decades until it reaches the point of perceived crisis, then it falls as greater efforts are made to prevent and treat the conditions responsible.

One of the goals of treating aging as a medical condition is to break this cycle - to have no more rising lines for age-related disease on the chart of causes of mortality. If the medical community tries to control diseases of old age one by one, firstly they will fail to reduce the incidence to zero because the only way to prevent age-related disease is to address the root causes of aging, and secondly partial and limited success in controlling age-related conditions, already achieved for heart disease, just means another condition will cause aged, damaged patients to die. One line on the chart falls, another rises to take its place. The way out is to repair the root causes of aging, the biochemical damage that produces age-related dysfunction and disease. To the degree that this damage is repaired successfully, the incidence of all age-related disease will fall.

From the beginning of the 20th century to 2010, the life expectancy at birth for females in the United States increased by more than 32 years. However, new causes of death have emerged with changes in technology and the built environment (eg, the automobile and highways), emerging infections (eg, HIV), and behavior (eg, cigarette smoking). We analyzed trends in mortality rates among females at each decade from 1900 through 2010, focusing on major causes of death, and examined differences by age and by race. Historical trends may indicate future trends, contributing factors, opportunities for intervention when interventions are known, and research needs when they are not.

We analyzed all-cause unadjusted death rates (UDRs) for males and females and for white and nonwhite males and females from 1900 through 2010 in decadal years to indicate mortality burden. We analyzed UDRs for black persons beginning in 1970 when the data were first made available. We also computed age-adjusted all-cause death rates (AADRs) by the direct method using age-specific death rates and the 2000 US standard population.

From 1900 to 2010, the UDR among females in the United States decreased from 1,646.9 per 100,000 to 787.4 per 100,000, an overall decrease of 52.2%. Among males, the UDR decreased from 1,791.1 per 100,000 in 1900 to 812.0 per 100,000 in 2010, an overall decrease of 54.7%. The male UDR exceeded the female UDR in all decadal years except 2000; by 2010, the male excess had decreased to 24.6. From 1970 to 2010, death rates increased by 5.5% among white females and decreased by 22.5% among black females. Rates decreased by 20.3% among white males and by 38.9% among black males.

From 1900 to 2010, the AADR among females decreased from 2,410.4 per 100,000 to 634.9 per 100,000, a decrease of 73.7%. Among males, the AADR decreased from 2,630.8 per 100,000 in 1900 to 887.1 per 100,000 in 2010, a decrease of 66.3%. The male AADR exceeded the female AADR in all decades, with the greatest excess in 1970 at 570.7 per 100,000; the male excess was higher in 2010 than in 1900.

The 5 major causes of death for females in 1900 (46.3% of all deaths) were pneumonia and influenza (198.5 per 100,000), tuberculosis (187.8 per 100,000), enteritis and diarrhea (134.9 per 100,000), heart disease (133.7 per 100,000), and stroke (107.7 per 100,000). Of these causes, only heart disease and stroke were among the 5 major causes in 2010. In 2010, the 5 major causes (59.7% of all deaths) were heart disease (184.9 per 100,000), all cancers (168.2 per 100,000), stroke (49.1 per 100,000), chronic lower respiratory diseases (46.3 per 100,000), and motor vehicle accident (21.8 per 100,000).

Twenty years of the 30-year increase in female life expectancy from 1900 to 2010 occurred between 1900 and 1950, affected principally by social and environmental factors. During the first half of the 20th century, sanitation improved substantially, with greater benefits for blacks than for whites. Sanitation, the provision of clean drinking water and safe disposal of sewage and solid waste, affected rates of infectious and chronic diseases and was associated with almost half the total decrease in mortality rates in major US cities between 1900 and 1940, three-quarters of the decrease in infant mortality rates, and almost two-thirds of the decrease in child mortality rates.

Three major nonexclusive explanations for increased heart disease mortality rates from 1900 to 1950 are possible. First, as understanding of diseases improved, the apparent rise may have partly resulted from changes in classifying and assigning causes of death during the first half of the century. Second, the rise has also been attributed to a reduction in "competing causes" of death, most notably the reduction of deaths due to infectious and diarrheal diseases. Third, cigarette smoking was a major influence on trends in female chronic disease mortality rates. The prevalence of cigarette smoking among females rose rapidly in the 1930s, peaked from about 1965 to 1975, and decreased thereafter.

Much of the decrease in mortality rates among females in the past 110 years is attributable to improvements in major social and environmental determinants of health - education, income, housing, and sanitation. The rapid decrease in mortality rates from infectious by mid-century largely preceded the widespread use of antibiotics or immunization. The extent and specific causes of increased heart disease mortality rates among females in the first half of the century remain uncertain. The decrease of heart disease mortality rates during the second half of the century may be the result of multiple factors.

Trends in mortality rates during the past century reflect major patterns of health determinants. Sanitary and safety improvements along with understanding of and therapies for infectious diseases led to great reductions in infectious causes of death. With increasing longevity and more sedentary lifestyles, chronic diseases increased as major causes of death. Although some of these causes, particularly heart disease and stroke, decreased as a result of behavior change and effective health care, decreases in mortality rates are slowing.

Link: https://www.cdc.gov/pcd/issues/2018/17_0284.htm

Here I'll point out a commentary from the SENS Research Foundation on one of the many changes that is needed in medical regulation in order to smooth the path ahead towards clinical availability of the first rejuvenation therapies. It is not a cynical viewpoint, and is focused on working within the system to make incremental beneficial alterations to one of the many regulatory positions that currently hold back progress. Accordingly, I'll follow it with my much more cynical view of the state of regulation, the harm it does, and the prospects for change - that I think must come from outside the system, not within.

Pathway To New Therapies

Substantial regulatory reform is needed to create a pathway for investors and pharma to put the necessary time and money into researching and developing rejuvenation biotechnologies such that licensable therapies can come out the other end. The most important regulatory reform would entail acceptance of novel biomarkers of the removal, repair, replacement, or rendering harmless of specific forms of cellular and molecular aging damage as sufficient basis to grant rejuvenation biotechnologies preliminary licensure. This would then be followed up by further monitoring of patients to ensure that the therapy actually does bend the curve on diseases of aging over the longer term. This standard would mark a break with regulators' usual insistence (which has been getting more entrenched, rather than less, in recent years) that therapies prove an effect on "hard outcomes" to get approval: things like heart attacks, amputations, or blindness. But people should ideally begin to receive rejuvenation biotechnologies well before patients are in near-term danger of such acute threats to life and health, making it extremely expensive and time-consuming to run a trial.

In 2012, it looked as if significant progress had been made on this front and several others during major stakeholder meetings amongst Alzheimer's disease (AD) patient and caregiver advocates, researchers, and FDA regulators. In particular, there was consensus on the need to work toward the development of new clinical trial designs and regulatory reforms to advance previously-untested combination therapies for AD into clinical testing. Draft guidance acknowledged that therapies for AD are unlikely to have substantial effects in "full-blown" dementia, because by that point, the brain has suffered either irreversible damage, or too many kinds of damage for any one therapy to be useful anymore. Then, despite having climbed up to the top of the diving tower, with advocates and scientists cheering at every step, FDA and pharma seemed to hesitate. The same FDA leaders who had previously expressed their openness to bold initiatives instead adopted a more conservative stance on using biomarkers as outcomes for early-stage disease.

This February, however, FDA finally took the plunge with a revised Draft Guidance, which would represent a tremendous step in the right direction if finalized as official FDA policy. The results of imaging tests or relevant biomarkers could then be considered sufficient to identify so-called "Stage 1" Alzheimer's patients as eligible candidates for clinical trials or new therapies, or to test existing therapies that had failed in people with frank AD. These "Stage 1" candidates are described in terms that identify people at an even earlier stage along the insidious path to dementia than the 2013 guidance: outwardly healthy but aging people without very-high-risk mutations or apparent cognitive or functional impairments, but who are nonetheless identified as being at higher risk than most. In short, these individuals would now be eligible to participate in trials of new and old therapies that might prevent them from ever tipping over into major cognitive impairments.

The use of biomarkers and imaging directly reflecting the key cellular and molecular damage driving AD and other neurodegenerative diseases of aging should be extended to testing of therapies in people who are in even greater danger, showing early signs of cognitive problems but still not suffering from full-fledged dementia. Analogously, biomarkers of the cellular and molecular damage that accumulates in our tissues as we age and that drives other age-related illness and debility should also be acknowledged as the best targets for new therapies that would prevent, arrest, and reverse those conditions. And because AD and other diseases of aging involve multiple kinds of cellular and molecular damage, it's critical that regulators allow the testing of combination therapies potentially capable of attacking multiple kinds of cellular and molecular aging damage, without first needing the constituent therapies to be tested individually.

Medical regulation is a huge problem of misaligned incentives. Regulators act as though the most important thing is to avoid bad press, and they thus trample over the freedom of individuals to assess risk and make their own choices. Regulators impose ever greater requirements on groups seeking regulatory approval of new therapies, and approve ever fewer applications, as a way to (a) make bad press go away, and (b) make them look better when the inevitable small number of problems occur. All medicines bear risk. Over the past few decades, the already enormous regulatory cost imposed on medical development has doubled, and the number of approved therapies fallen dramatically. Countless lines of development have not been carried forward to the clinic because the cost has become prohibitive. Rigorous experimentation and exploration has declined. Regulatory agencies, but in particular the FDA, appear to be following the road of eliminating the appearance of risk by minimizing all progress. This causes far greater actual harms, but those costs are invisible, rarely reaching the press: the new medicines that never appeared, the lives that would have been saved, the suffering that would have been eliminated.

This must change. We stand on the verge of the development of real, working rejuvenation therapies, technologies that will bring radical improvement to the health of older individuals, and offer the potential to indefinitely extend healthy life spans. This is too great a gain to let regulators follow the usual playbook for padding their own nests at the expense of everyone else. The system must be changed, or responsible clinical development must take place outside the system. There is nothing magical about clinical trials: any group can run and publish and and replicate and review tests. Existing law on fraud and harm is more than capable of handling fraudulent or harmful behavior. The onerous requirements forced upon development by the FDA are far greater than the activities needed to obtain a useful assessment of risk and results, and medical development worked just fine before those requirements came into being over the past few decades.

Many groups object to the FDA or its present heavy-handed suppression of progress, and are working within the system to change it. They have been doing this with considerable vigor for quite some time, and very little has changed as a result. I'm not optimistic about the near future of such efforts either: if something as self-evidently humanitarian as allowing terminal patients to choose their treatments has struggled to reach its present position, what hope is there for a general reduction in the FDA presence in medicine? The only approach that has proven to work is to move clinical development and availability overseas, into areas with a lesser regulatory burden, as happened for stem cell medicine. The only reason any stem cell therapies are presently approved for use in the US is that they were widely available elsewhere for years, and the FDA was gaining worse publicity for being obstructionist than from approving some of these approaches.

Within the scientific and established medical development community, the public bias of opinion tends to be towards reforming the system from within. One reason for this is that the regulators at the FDA have proven themselves vindictive when it comes to those who rock the boat - which follows from the observed first principle of minimizing bad press. Make a lot of noise about the FDA and expect the result to be increased attention, cost, and risk of rejection when the time comes to put a therapy through regulatory approval. Another reason is that the publicly funded research community is quite hierarchical and conformist; it doesn't select for career scientists who find that irksome, or who see going outside the system as a viable choice. Revolutionaries tend to be thin on the ground. Nonetheless, I'm much more in favor of development outside the present system as a way to force change - it has a better track record of success.

All age-related neurodegenerative conditions appear in conjunction with rising levels of chronic inflammation. The immune system runs awry with age, and while the immune cells of the central nervous system are significantly different in type and character from those of the rest of the body, inflammation is still a major consequence of age-related immune failure. In turn, that inflammation accelerates other ongoing degenerative processes. Calorie restriction is the most reliable and well-studied way of modestly slowing aging, and here researchers demonstrate that it is more effective than exercise when it comes to postponing the rise of inflammation in the brain.

Practicing both calorie restriction and regular exercise is a great idea, but only because these options are free. Calorie restriction results in sizable health benefits in humans, and even though it doesn't extend human life spans by anywhere near the same proportion as is observed in mice, it is still something for nothing. But should be we supportive of research efforts that expend billions and decades on attempts to recreate slices of the calorie restriction response? Probably not, when that is a poor alternative to building rejuvenation therapies after the SENS model of damage repair. Why spend large amounts of time and funding on trying to slightly slow aging rather than trying to halt and reverse aging? Both are equally plausible goals at the present time. Why pick the worse option?

Microglia are brain cells that help maintain the integrity and normal functioning of brain tissue. Dysfunction of these cells, as may occur in disease, is linked to neurodevelopmental disorders and neurodegenerative conditions. Aging is also associated with inflammation driven by microglia in specific regions of the brain, but it is unclear whether diet or lifestyle can influence this process.

Researchers investigated the impact of high- and low-fat diets on inflammation and microglial markers in a specific brain region - the hypothalamus - of 6-month-old mice. They further looked at the effect of low- or high-fat diets on the microglia of 2-year-old mice, which were also given a lifelong exercise regime (voluntary running wheel) or lifelong restricted diets (a 40% reduction in calories). "Aging-induced inflammatory activation of microglia could only be prevented when mice were fed a low-fat diet in combination with limited caloric intake. A low-fat diet per se was not sufficient to prevent these changes."

The researchers also found that exercise was significantly less effective than caloric restriction at preventing these changes, although work by others has shown that exercise is associated with reducing the risk of other diseases. There is still much more work needed to understand the meaning of these findings. In the study, mice were only given one type of diet throughout their lives. It remains unclear how changing between diets would alter these results - for example whether switching to a low-fat diet could undo the negative consequences of a high-fat, unrestricted diet. Further studies are also needed to determine how these changes correspond to the cognitive performance of the mice.

Link: https://www.eurekalert.org/pub_releases/2018-03/f-cri030718.php

You might recall that aldehyde-stabilized cryopreservation was the approach that won the Brain Preservation Prize a few years back. The prize sought to encourage progress in the field, and particularly development of the means to proof high quality preservation of the fine molecular structure of brain tissue. Somewhere in there, the data of the mind is stored. Thus cost-effective preservation of that fine structure offers the chance at a renewed life in the future for the countless multitudes who will age to death prior to the advent of comprehensive rejuvenation biotechnology. It is welcome to see signs of greater research, development, and growth in the cryonics community.

That said, this advance comes from the side of the community that is more interested in storing the pattern than the flesh. Their end goal for the more distant future is to scan the brain as though it were a recording, and then run an emulation of the stored mind in suitable software. This is creating a copy and discarding the original - not a wonderful outcome from my point of view. It is the same as death for the preserved individual. Only restoration and repair of the stored brain itself is sufficient for personal continuity. Alcor, for example, doesn't intend to adopt aldehyde-stabilized cryopreservation because it will be much harder to carry out restoration in comparison to present day vitrification, and because it isn't a stepping stone technology on the way to near future reversible vitrification.

What if we told you we could back up your mind? Imagine a world where you can successfully map and pinpoint a specific memory within your brain. Today's leading neuroscience research suggests that it is possible by preserving your connectome. The connectome is all the connections called synapses between neurons in your brain. Researchers are now learning to manipulate individual memories, building advanced brain prosthetics, and reverse-engineering the brain.

Our mission is to preserve your brain well enough to keep all its memories intact: from that great chapter of your favorite book to the feeling of cold winter air, baking an apple pie, or having dinner with your friends and family. We believe that within the current century it will be feasible to digitize this information and use it to recreate your consciousness. Our process of vitrifixation (also known as aldehyde-stabilized cryopreservation) has won the Brain Preservation Prize for preserving a whole rabbit connectome, and we are currently hard at work to scale our preservation process to larger brains.

We currently need help with developing tissue staining protocols for richer connectome imaging. We also are starting to develop a computational neuroscience program. If you are an interested research institution, or are interested in joining our engineering department, please get in touch.

Link: https://nectome.com/

Sarcopenia is the name given to the characteristic loss of muscle mass and strength with age. It is one of the better conditions to use in order to illustrate the point that the research community often approaches complex aspects of aging in the manner of the blind men and the elephant. Every group is specialized, and focused on one specific aspect of the overall situation. So one can look at a recent paper on stem cell decline as the dominant cause of sarcopenia and come away quite convinced, and then read the paper I'll point out today, that paints issues with the interface between nerves and muscles - the motor unit - as an important cause of sarcopenia, and start to think that perhaps it isn't all stem cells.

The same is true of many other possible causes of sarcopenia. Some researchers have run studies of strength training and suggest that substantial fractions of the loss of muscle with age are due to lack of exercise. Others have investigated age-related defects in the processing of amino acids such as leucine necessary for the construction of proteins in muscle tissue, or the falling dietary intake of protein that seems common in older individuals, or the contribution of cellular senescence. At some point, these and other views of the problem must be synthesized into a complete understanding, and the contradictory evidence reconciled.

Given the pace of progress in applied biotechnology, it seems that the best approach to determining the important causes of sarcopenia is to work towards a fix for each potential cause, one by one, and in isolation from one another. The degree to which any particular fix improves or reverses sarcopenia is the metric by which the related potential cause can be judged primary or secondary, an actual cause or a secondary consequence of some other, more important mechanism. As implementing therapies becomes easier in comparison to reverse engineering all of the details of any specific slice of cellular biochemistry, this approach will only become more attractive. Efforts to restore muscle stem cell activity are far enough advanced to make this an area worth keeping an eye on.

Can we turn back time? Muscles' own protective systems could help reduce frailty

As people grow older, their leg muscles become progressively smaller and weaker, leading to frailty and disability. While this process inevitably affects everyone living long enough, until now the process has not been understood. New research suggests that muscle wasting follows on from changes in the nervous system. By the age of 75, individuals typically have around 30-50% fewer nerves controlling their legs. This leaves parts of their muscles disconnected from the nervous system, making them functionally useless and so they waste away.

However, healthy muscles have a form of protection, in that surviving nerves can send out new branches to rescue some, but not all, of the detached muscle fibres. This protective mechanism is most successful in older adults with large, healthy muscles. When the internal protective mechanism is not successful and nerves are unable to send out new branches, it can result in extensive muscle loss. This can result in a condition called sarcopenia, which affects an estimated 10-20% of people aged over 65 years.

The researchers are currently looking at whether regular exercise in middle- and older-age slows the process of muscles becoming disconnected from the nervous system, or improves the success of nerve branching to rescue detached muscle fibres. The goal is to identify the best type of exercise - strength training or endurance - and to understand the physiology of why the nerve-muscle changes occur as we get older.

Failure to expand the motor unit size to compensate for declining motor unit numbers distinguishes sarcopenic from non-sarcopenic older men

Sarcopenia results from the progressive loss of skeletal muscle mass and reduced function in older age. It is likely to be associated with the well-documented reduction of motor unit numbers innervating limb muscles and the increase in size of surviving motor units via reinnervation of denervated fibres. However no evidence currently exists to confirm the extent of motor unit remodelling in sarcopenic individuals. The aim of the present study was to compare motor unit size and number between young (n = 48), non-sarcopenic old (n = 13), pre-sarcopenic (n = 53) and sarcopenic (n = 29) men.

Motor unit potentials (MUPs) were isolated from intramuscular and surface electromyographic recordings. The motor unit numbers were reduced in all groups of old compared with young. Motor unit potentials were enlarged in non-sarcopenic and pre-sarcopenic men compared with young, but not in the vastus lateralis of sarcopenic old. The results suggest that extensive motor unit remodelling occurs relatively early during ageing, exceeds the loss of muscle mass, and precedes sarcopenia. Reinnervation of denervated muscle fibres likely expands the motor unit size in non-sarcopenic and pre-sarcopenic old, but not in the sarcopenic old. These findings suggest that a failure to expand the motor unit size distinguishes sarcopenic from pre-sarcopenic muscles.

The present dominant approach to the development of therapies to treat aging is not, sadly, the SENS rejuvenation research agenda, but instead efforts to persistently activate evolved responses to cellular stress. These mechanisms normally start up in response to exercise, calorie restriction, raised temperature, and the topic here, hypoxia, among other sources of stress. The responses generally lead to some period of more aggressive cellular maintenance, particularly autophagy, responsible for identifying and recycling damaged molecules and structures within the cell.

There is comprehensive evidence to support the idea that running these mechanisms at a higher level all the time, in the absence of stress, is beneficial. It is an aspect of numerous approaches shown to modestly slow aging in various short-lived species over the past few decades. As the authors of this short commentary note, in the case of hypoxia the situation is more complex, however. A number of age-related conditions involve disarray or excessive activation of mechanisms of the hypoxia response. That must be in some way reconciled with the evidence for overactivation of the hypoxia responses to modestly slow aging life in various animal studies.

Regardless, it is the case that enhancement of stress responses doesn't come with the expectation of sizable benefits to life span in humans. We know what the results of exercise and calorie restriction look like in our species, and they don't produce anywhere near as large an effect as additional decades added to our life spans. They are means to slow aging just a little. Slightly slowing aging is only worth it if the cost of developing the necessary therapies is low. Unfortunately, it is not low. The past twenty years have seen enormous sums and the careers of many scientists poured into the effort to understand cellular metabolism sufficiently well to recreate only thin slices of the response to calorie restriction, or exercise, or other stresses. If this level of effort is to be expended, then why is it being expended on a strategy that cannot produce meaningful gains, versus something more along the lines of SENS, that can in principle result in rejuvenation and lives extended by decades or more?

Cells in metazoan species produce energy via oxidative phosphorylation, a process that requires a carbon source and oxygen (O2). O2 homeostasis is therefore of utmost importance and is maintained by intricate circulatory and respiratory systems. When the function of these is compromised, cells in the afflicted areas experience lower than optimal physiological O2 levels, a condition termed hypoxia. To cope with hypoxia, cells employ an evolutionarily conserved pathway controlled by hypoxia-inducible factors (HIFs).

Proteins encoded by hypoxia-inducible genes are functionally diverse, their primary role is to reprogram the cell towards survival under a hypoxic microenvironment and trigger specific physiological responses to help organisms adapt to conditions such as high altitude by inducing synthesis of erythropoietin, a hormone that stimulates production of red blood cells, or wound healing by activating secretion of angiogenesis-stimulating factors such as VEGF. This fine-tuned physiological response to hypoxia can, however, also be co-opted and contribute to age-related diseases.

For example: hypoxia and HIF-1 may participate in the pathogenesis of atherosclerosis; overactivation of the HIF pathway in cancer has raised significant interest in its targeting with small-molecule inhibitors; HIF-1 activates mPGES-1 gene expression in chondrocytes and contributes to the excessive catabolism underlying cartilage destruction and osteoarthritis. In the context of aging-associated diseases, more work is required to establish whether activation of HIF plays a causative role or is the consequence of some other underlying changes. In either case, there is evidence that targeting HIF-1, rather than its targets, e.g. VEGF, in at least some of these conditions may provide broader effect and eventually translate into greater therapeutic efficacy. As the number of age-associated maladies with activated HIF increases, it is rational to consider whether combining early detection with a HIF inhibitory pill could be of benefit for preventive treatment.

However, the relationship between HIF and aging is more complex. Genetic studies mainly in invertebrates have shown that HIF might control normal physiological processes that both promote and limit longevity. Life span extension imparted by stabilized HIF-1 occurs by a mechanism genetically distinct from both insulin-like signaling and dietary restriction. On the other hand, increased life span of C. elegans hif-1 deletion mutants was explained in terms of either activation of stress-regulated transcription factor DAF-16 or reactivating endoplasmic stress resistance downstream of mTOR. Further studies are warranted to understand the role of HIF-1 in longevity in mammals before merit of therapeutic modulation of its activity for age-related disease can be assessed.

Link: https://doi.org/10.18632/aging.101395

Given the past few years of research results, it is becoming quite clear that wherever researchers observe inflammation and scarring in the body, senescent cells should be high on the list of suspected causes. Senescent cells are created constantly in large numbers, the normal fate for cells that become damaged or reach the end of their replicative life span. Near all quickly self-destruct, or are destroyed by the immune system, but some few manage to linger - or more, in medical conditions that create a harmful tissue environment that encourages senescence. Over time a population of lasting senescent cells contributes greatly to the progression of aging and age-related disease.

Senescent cells cause issues through the potent mix of signal molecules that they generate; even a comparatively small number can degrade the function of surrounding tissue. To pick one example, there is a good deal of evidence linking the accumulation of senescent cells to the development of fibrosis in various tissues, this being a harmful process by which tissue maintenance runs awry, disrupted by inappropriate levels of inflammation, and scar-like deposits form in place of normal, healthy structures. Targeted removal of senescent cells can help to reverse these conditions, and this is one of many reasons to speed the clinical development of senolytic therapies that can destroy these unwanted cells.

Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are the most prevalent type of cholangiopathies, a diverse group of genetic and acquired disorders that affect the biliary population of the liver. PSC/PBC have variable prognoses but frequently evolve into end-stage liver disease, with limited treatment options. The aetiologies remain unclear, although a role of cellular senescence in the development of PSC/PBC has been suggested.

Senescence is an irreversible cell cycle arrest, driven by dominant cell-cycle inhibitors and characterized by the activation of the senescence-associated secretory phenotype (SASP). The SASP is a pro-inflammatory response that activates and reinforces the senescent phenotype in the surrounding cells, modulates fibrosis, and promotes regeneration. The SASP is composed by a variable set of secreted cytokines and chemokines, responsible for the beneficial and deleterious effects of senescence within the tissue. However, despite a number of studies suggesting a potential link between senescence and biliary disease, it has not been shown whether senescence is actually a driver of the damage rather than solely a consequence. We have therefore investigated the relationship between senescence and biliary disease, focusing on SASP-related mechanisms to explain part of the pathophysiology of PSC/PBC.

Here, we present a model of biliary disease, based on the conditional deletion of Mdm2 in bile ducts under the control of the Krt19 cholangiocyte promoter. In this model, senescent cholangiocytes induce profound alterations in the cellular and signalling microenvironment. The presence of senescent cholangiocytes in this model promotes ductular reaction, increases deposition of collagen, and impairs liver regeneration after injury. The presence of αSMA-positive cells in the proximities of the senescent cholangiocytes suggests that senescence might have a role in the development of fibrosis, characteristic of human PSC/PBC.

We suggest that senescence may contribute to the development of biliary disease through complementary mechanisms. First, through an impaired regenerative response of cholangiocytes, unable to compensate biliary damage, and second, through SASP expression, that can induce paracrine senescence in hepatocytes (thus diminishing the regenerative capacity of the liver during injury), promote collagen deposition, and enhance fibrogenesis. Overall, we have shown that cellular senescence is likely to be a driver of biliary injury by affecting the microenvironment, impairing liver parenchyma regeneration, and impairing biliary function.

Link: https://doi.org/10.1038/s41467-018-03299-5

It seems plausible that one of the first major mainstream areas of development for human gene therapy will involve disabling genes that sustain levels of lipids in the bloodstream. There are a number of credible targets, including those with sizable numbers of human loss of function mutants who seem to do quite well as a result of their mutation: PCSK9, ANGPTL3, ANGPTL4, and ASGR1 for example. Reducing lipid levels in the bloodstream has the effect of slowing the development of cardiovascular disease, reducing the risk of heart attack, stroke, and other related issues. The success of statin drugs is based on exactly this effect, and gene therapies would be much more effective than statins - a one time treatment producing a larger and permenant benefit.

How does this work under the hood? Why does lowering blood lipids - cholesterol, triglycerides, and so forth - have this beneficial result? The primary mechanism of interest relates to the development of atherosclerosis through damaged lipid molecules. The normal operation of metabolism produces reactive molecules that can oxidize lipids, so some small fraction of the lipids in the bloodstream are damaged in this way. With the progression of aging, various forms of cell and tissue damage and their consequences lead to the generation of many more reactive molecules, and thus greater numbers of damaged lipid molecules entering the bloodstream - the problem becomes worse over time. You might look at the progression of cause and effect that starts with mitochondrial DNA damage, for example, but there are also more systemic issues such as chronic inflammation, which goes hand in hand with greater levels of oxidative damage.

Oxidized lipids can irritate blood vessel walls, giving rise to a feedback loop of inappropriate cellular reactions that produce inflammation and draw in ever more immune cells to try to clean up the mess. Many of these cells die, overwhelmed by forms of oxidized lipid that mammalian biochemistry is not well equipped to handle. The result is a growing, fatty plaque of dead cells and harmful lipids, the signature of atherosclerosis. These plaques weaken and narrow blood vessels, and eventually something ruptures or a blood vessel is blocked - an occurrence that is often fatal, and at best disabling. Interfering in this feedback loop at any point can slow it down: cut down the amount of all lipids entering the bloodstream, make immune cells more resilient or capable, or remove just the problem lipids through some other mechanism.

I think that the latter two are better than the former, as they can in principle be made close to 100% efficient without altering the way in which cellular metabolism functions in ways that are yet to be fully understood over the long term, as is the case for dramatic reductions in blood lipid levels. It isn't the case that all lipids can be removed from the bloodstream, and it isn't the case that all oxidative damage can be prevented. In general, periodic repair is a good deal more useful than partial prevention, as repair can help those already damaged and in late stages of disease. Still, we'll be getting a more effective implementation of the worse option in the near future, it seems, as that is where the bulk of the attention is focused.

A CRISPR edit for heart disease

Consider this scenario: it's 2037, and a middle-aged person can walk into a health centre to get a vaccination against cardiovascular disease. The injection targets cells in the liver, tweaking a gene that is involved in regulating cholesterol in the blood. The simple procedure trims cholesterol levels and dramatically reduces the person's risk of a heart attack. Although antibody-based therapies have been launched to help those most at risk, the cost and complexity of the treatments means that a simpler, one-off fix such as a vaccine would be of benefit to many more people around the world.

The good news is that a combination of gene discovery and the blossoming of genome-editing technologies such as CRISPR-Cas9 has given this vision of a vaccine-led future for tackling heart disease a strong chance of becoming reality. The breakthrough came in 2003, when researchers investigated three French families with members who had potentially lethal levels of low-density lipoprotein (LDL) cholesterol and who harboured a mutation in the gene PCSK9. PCSK9 encodes an enzyme that regulates levels of LDL - or 'bad' - cholesterol. Sensing the possibilities, investigators sought to determine whether naturally occurring mutations in PCSK9 could also have the effect of lowering LDL cholesterol. After combing the data from about 3,600 individuals who provided a blood sample, the researchers sequenced DNA from the 128 participants with the lowest levels of LDL cholesterol. They discovered that about 2% of African-American participants had one broken copy of PCSK9. A follow-up study of a different, larger population similarly found mutations in almost 3% of African Americans, which was associated with an 88% reduction in the risk of ischaemic heart disease.

The liver is a preferred target organ of gene therapy for companies such as Editas Medicine, Sangamo Therapeutics, and CRISPR Therapeutics; it is straightforward to deliver genes to the liver, and the CRISPR-Cas9 tool is especially efficient in the organ, editing a greater proportion of cells than it does in most other tissues. The liver is also an excellent place from which to tackle cholesterol - it clears LDL cholesterol from the blood and is also a main engine of lipid synthesis. Researchers have shown that more than half of Pcsk9 genes in the mouse liver could be silenced with a single injection of an adenovirus containing a CRISPR-Cas9 system directed against Pcsk9. This led to a roughly 90% decrease in the level of Pcsk9 in the blood and a 35-40% fall in blood LDL cholesterol.

The approach is "absolutely plausible, even feasible", from a technological point of view. But there is also a philosophical barrier to negotiate. "You don't necessarily want to treat people who haven't got a disease yet." Others go further. "Changing lifestyle may be much more effective for a population than focusing on high-cost interventions." They worry that a gene therapy for individuals at high risk would hinder efforts to help people to help themselves. "It is the way the human mind works. Take a pill and we think we are protected."

There is certainly a reluctance to follow through in permanent gene therapies for prevention and enhancement at the moment - the work could be proceeding much more rapidly than it is, given the rapidly falling costs of genetic biotechnology. It will probably require more adventurous groups such as BioViva Sciences or Ascendance Biomedical to break down that door by simply going ahead and offering the gene therapies that are technologically plausible outside the mainstream regulatory system. These treatments will initially have a low effectiveness, in terms of the proportion of cells transfected by the therapy, but that is a challenge that will be solved with increasing efficiency over the next decade. Someone has to get started, go first, show the way. If regulatory systems as they presently exist make that hard, then the start will occur outside the regulatory framework, just as it did for stem cell therapies - and a good thing too, as that is pretty much the only circumstance that might help to make current medical regulation less oppressively terrible.

One of the better ways to dampen down the unhelpful hype generated by one or another new supplement or drug alleged to modestly slow aging on the basis of animal data is to point out that aspirin does just as good a job in animal studies. We all know what aspirin does for human life span, which is to say pretty much nothing, while still managing to be a useful tool in the pharmaceutical toolbox. Chasing marginal outcomes in human longevity will at best achieve marginal outcomes - and that is the major problem with the mainstream focus on trying to recapture the beneficial effects of calorie restriction through any number of candidate calorie restriction mimetic drugs. We need to do better, to aim higher. This means more work focused on the development of therapies after the SENS model, those that repair the molecular damage that causes aging and thus are capable in principle of achieving rejuvenation and significant extension of healthy human life.

The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs). Here, we show that aspirin or its active metabolite salicylate induce autophagy by virtue of their capacity to inhibit the acetyltransferase activity of EP300.

We demonstrate that aspirin fails to modulate autophagic flux in cells lacking EP300 or cells in which EP300 has been engineered to avoid aspirin binding to the enzyme. As a confirmation of the evolutionarily conserved nature of this process, we demonstrate that aspirin failed to further induce autophagy in Caenorhabditis elegans strains deficient for the EP300 homolog CBP-1 or the essential autophagy gene products ATG-7 and BEC-1.

Based on the results described in this paper, aspirin may be classified as a CRM. Indeed, aspirin fulfills all the criteria of a CRM as it (1) reduces protein acetylation by virtue of its ability to inhibit the acetyltransferase activity of EP300, (2) stimulates autophagic flux, and (3) has no cytotoxic activity. At this point, it remains to be determined to which extent EP300 inhibition and autophagy activation may effectively contribute to these aspirin effects that apparently transcend its well-established anti-inflammatory effects.

Pre-clinical evidence suggests that a brain-permeable aspirin derivative can reduce tau-mediated neurodegeneration in an EP300-dependent fashion. However, the role of autophagy has not been explored in this setting. Epidemiological and experimental data indicate that a high nutritional uptake of the EP300 inhibitor spermidine counteracts cardiac aging, both in humans and rodents. In addition, spermidine reduces arteriosclerosis and colon carcinogenesis in mouse models. These spermidine effects hence show a notable overlap with those of aspirin, in accord with the observation that both compounds inhibit EP300.

Link: https://doi.org/10.1016/j.celrep.2018.02.024

Researchers have found a reason to distrust the results of past nematode life span studies with modest effect sizes, even those that controlled for the effects of dietary intake on longevity, a now well-known issue in animal studies that has caused plenty of problems in the past. The researchers have found that light exposure affects the life span of the commonly used Caenorhabditis elegans species of nematode. Their data shows a sizable difference between conditions of permanent light and permanent darkness, but the problem would arise more subtly in comparison between studies where duration, intensity, and type of lighting varied - say, by season, by employee hours, by building fixtures, by nematode housing, and so forth. By the sound of it, this is bad news for near all past life span studies carried out with nematodes, casting doubt on a large amount of exploratory data in the field of aging research.

Historically, the nematode Caenorhabditis elegans was believed to lack the ability to sense light due to the absence of a bona fide photoreceptor system and its original isolation in soil samples. However, recent work in C. elegans has identified the LITE-1 taste receptor homolog as a UV-specific photoreceptor. Interestingly, high-energy UV and blue wavelength light trigger escape behavior and feeding inhibition in C. elegans. In contrast to animals with external pigmentation, the transparent body of nematodes allows light to penetrate their body, making them particularly vulnerable to the mutagenic effects of UV.

We used C. elegans to test whether the photoperiod (the interval in a 24-h period during which an animal is exposed to light) could impact its physiology and lifespan. This is also of special interest since standard laboratory manuals and practices for C. elegans handling completely ignore random exposure to light versus dark. Here, we demonstrate that daily exposure to white light decreases C. elegans lifespan and alters development. Importantly, these effects are not mediated through known photoreceptor pathways or through a proper disruption of circadian rhythms. Our results indicate that the effect of light on C. elegans lifespan is not specific to a particular wavelength of the visible spectrum, but is photon energy dependent.

We find that light exposure causes oxidative stress and induces canonical stress responses. Several long-lived mutants that ectopically activate these stress-responsive pathways are resistant to light stress. Furthermore, we find that treatment of wild-type worms with antioxidants is sufficient to rescue their short lifespan due to light exposure. Such findings strongly invite a reconsideration of the standard methods of C. elegans handling, especially in the context of aging research and stress biology.

Link: https://doi.org/10.1038/s41467-018-02934-5

This age of biotechnology is also an age of comparative indolence and comfort. As the research community measures specific biochemical aspects of aging, such as the decline of the cardiovascular system, or metrics relating to immunosenescence in the immune system, we might question the degree to which the results are peculiar to our era. How much of aging is the result of our choices - to eat more and exercise less than our ancestors - rather than the result of inexorable processes of biochemical damage that we, as yet, have little influence over? (Conversely, how much of past aging was due to infectious disease, malnutrition, and other adverse external circumstances that are controlled to a much greater degree today?) This topic crops up fairly often in research into the effects of exercise on health, and the research noted here is a particularly striking example of the type.

The study authors find that the age-related decline of new T cells maturing in the thymus is negligible in some people, those who exercise much more than the rest of us. This diminished supply of new T cells is thought to be an important component of immune system aging, and the failure of the immune system is very influential over many other aspects of aging: senescent cell accumulation, frailty, loss of regenerative capacity, chronic inflammation, cancer risk, and so on. Yet when we look at the demographic evidence for spread of life span based on exercise, it appears to be, at most, 6 or 7 years (with a much larger divergence when it comes to state of health over time). What does this tell us about the likely gains resulting from rejuvenation therapies seeking to regenerate the thymus? Less than we would like, I suspect, and not just because it is hard to evaluate any one contribution to aging in isolation of all of the others.

The thymus atrophies over adult life, with active tissue necessary for the production of T cells being replaced by fat. The first major loss of active thymic tissue occurs at the end of childhood, however, in a process known as involution. Immune cells are generated at a tremendous rate in children in comparison to young adults; evolution selected for a system that would be highly effective at the outset, at the cost of later issues. When it is observed that old people in their 60s and 70s who maintained a high level of fitness throughout life exhibit much the same thymic output as young people in their 20s, that tells us little regarding the outcome were the thymus restored to the same level of active tissue as is present in children. Only a mild restoration, to move thymic activity from typical aged to typical young adult, would be comparable - and why would we stop there?

Exercise can slow the ageing process

Prehistoric hunter-gatherer tribes were highly active, spending a lot of time and energy sourcing their food. If they weren't successful, they would also spend days with or little or no food. By contrast, today we are a highly sedentary society. As we get older, our physical activity levels decline even further. In our research, we have tried to determine how much this low level of physical activity contributes to the ageing of many body systems, including muscle, bone and the immune system.

We examined 125 male and female cyclists, aged 55 to 79, who had maintained a high level of cycling throughout most of their adult lives. These were not Olympians, but very keen cyclists who were able to cycle 100km in under 6.5 hours for the men, and 60km in under 5.5 hours for the women. At mid-life, people start losing muscle mass and strength at a rate of 1% to 2% per year, making it harder to carry out their normal activities such as climbing stairs. Our bones also become thinner with age and this can eventually lead to diseases such as osteoporosis. We showed that the cyclists did not lose muscle mass or strength as they aged, and their bones only became slightly thinner. We then went on to examine a body system that was not so obviously influenced by physical activity - the immune system.

When we compared the immune system of the cyclists to older adults who had not done regular exercise, and to young people in their twenties, we found that their immune systems looked most like the young persons'. In particular, we found that the cyclists still made lots of new immune T cells, produced by an organ called the thymus, which normally starts to shrink after we reach puberty. The older cyclists seemed to have a thymus that was making as many new T cells as the young people's. We investigated why this happened and found that the cyclists had high levels of a hormone called interleukin 7 in their blood, which helps to stop the thymus shrinking. Interleukin 7 is made by many cells in the body, including muscle cells, so we think that active muscles will make more of this hormone and keep the immune system, and especially the thymus, young.

Major features of immunesenescence, including reduced thymic output, are ameliorated by high levels of physical activity in adulthood

What confounds human studies of immunosenescence is that physical activity is not taken into account in either cross-sectional or longitudinal studies of immune aging. The majority of older adults are largely sedentary and fail to meet the recommended guidelines for physical activity of 150 min of aerobic exercise per week. Regular physical activity in older adults has been associated with lower levels of pro-inflammatory cytokines such as IL-6, TNFα, improved neutrophil chemotaxis and NK cell cytotoxicity, increased T-cell proliferation and improved vaccination responses. Thus, the current literature on immunesenescence is not able to determine which aspects of age-related immune change are driven by extrinsic factors and which may be the consequence of a constitutive aging programme.

Here, we studied several aspects of the adaptive immune system in highly physically active older individuals (master cyclists) in which we have shown the maintenance of a range of physiological functions previously reported to decline with age. We show that compared with more sedentary older adults, the cyclists show reduced evidence of a decline in thymic output, inflammaging and increased Th17 cell responses, although accumulation of senescent T cells still occurred. We reveal high serum levels of IL-7 and IL-15 and low IL-6, which would together provide a environment protective of the thymus and also help to maintain naïve T cells in the periphery. We conclude that maintained physical activity into middle and old age protects against many aspects of immune aging which are in large part lifestyle driven.

The results here will cause some upheaval in the research community if verified, and will do doubt lead to considerable debate regardless of the outcome. For decades it is has been considered that neurogenesis, the production and integration of new neurons in the brain, continues past childhood, albeit at a lower rate. This is based largely on studies in mice, but also on a range of human evidence. The researchers here suggest that this is wrong, and in fact humans are not like mice in this regard: we do not generate new neurons at any detectable level as adults. This question of adult neurogenesis has great influence on the strategies adopted in the development of therapies that might enhance maintenance of the brain. This is a topic of considerable importance to the future of human rejuvenation: we are our brains, and damage and loss must be repaired in situ. If there are no naturally occurring mechanisms to achieve that goal in some or all of the brain, this suggests that the task will be that much harder to safely achieve.

One of the liveliest debates in neuroscience over the past half century surrounds whether the human brain renews itself by producing new neurons throughout life, and whether it may be possible to rejuvenate the brain by boosting its innate regenerative capacity. Now scientists have shown that in the human hippocampus - a region essential for learning and memory and one of the key places where researchers have been seeking evidence that new neurons continue to be born throughout the lifespan - neurogenesis declines throughout childhood and is undetectable in adults.

The findings present a challenge to a large body of research which has proposed that boosting the birth of new neurons could help to treat brain diseases such as Alzheimer's disease and depression. But the authors said it also opens the door to exciting new questions about how the human brain learns and adapts without a supply of new neurons, as in seen in mice and other animals. It was once neuroscientific dogma that the brain stops producing new neurons before birth. In the 1960s, experiments in rodents first suggested that new neurons could be born in the adult mammalian brain, but these results remained highly controversial until the 1980s, it was shown that new neurons are born and put to use throughout life in several parts of the songbird brain.

These findings launched a whole field of research. Much work has focused on a region of the hippocampus called the dentate gyrus (DG), where rodents produce newborn neurons throughout life that are thought to help them form distinct new memories, among other cognitive functions. Rodent studies have shown that DG neurogenesis declines with age, but is otherwise quite malleable - increasing with exercise, but decreasing with stress, for example - leading to popular claims that we can boost brain regeneration by living a healthy lifestyle. Beginning in the late '90s, a handful of studies reported evidence of adult neurogenesis in the human brain, either by estimating the birth dates of cells present in postmortem brain specimens or by labeling telltale molecular markers of newborn neurons or dividing neural stem cells. However, these findings, some of which were based on small numbers of brain samples, have remained controversial.

In the new study, researchers collected and analyzed samples of the human hippocampus. They analyzed changes in the number of newborn neurons and neural stem cells present in these samples, from before birth to adulthood, using a variety of antibodies to identify cells of different types and states of maturity, including neural stem cells and progenitors, newborn and mature neurons, and non-neuronal glial cells. The researchers also examined the cells they labeled based on their shape and structure - including imaging with high-resolution electron microscopy for a subset of tissue samples - in order to confirm their identity as neurons, neuronal stem cells, or glial cells.

The researchers found plentiful evidence of neurogenesis in the dentate gyrus during prenatal brain development and in newborns, observing an average of 1,618 young neurons per square millimeter of brain tissue at the time of birth. But the number of newborn cells sharply declined in samples obtained during early infancy: dentate gyrus samples from year-old infants contained fivefold fewer new neurons than was seen in samples from newborn infants. The decline continued into childhood, with the number of new neurons declining by 23-fold between one and seven years of age, followed by a further fivefold decrease by 13 years, at which point neurons also appeared more mature than those seen in samples from younger brains. The authors observed only about 2.4 new cells per square millimeter of DG tissue in early adolescence, and found no evidence of newborn neurons in any of the 17 adult post-mortem DG samples or in surgically extracted tissue samples from 12 adult patients with epilepsy.

Link: https://www.ucsf.edu/news/2018/03/409986/birth-new-neurons-human-hippocampus-ends-childhood

There is a constantly replicating herd of mitochondria in every cell, the evolved descendants of ancient symbiotic bacteria now well integrated into cellular mechanisms. They still bear a small remnant of the original bacterial DNA, however, and this is prone to mutational damage. Some forms of this damage cause mitochondria to both malfunction and become more resilient or more able to replicate than their peers. As a result, the cell is quickly overtaken by broken mitochondria and becomes broken itself, exporting damaging reactive molecules into surrounding tissues, the bloodstream, and the body at large.

This process is one of the root causes of aging, so it is a matter of considerable interest to the research community to understand exactly how it is that these damaged mitochondria can so quickly replicate to fill a cell with their descendants. That said, the beauty of the SENS rejuvenation research approach to the problem is that it really doesn't depend on how the damage occurs or spreads. It aims to place backup copies of mitochondrial genes into the cell nucleus, thus ensuring that there is always a supply of the proteins encoded in mitochondrial DNA. So if mitochondrial DNA does become damaged, then there are no further consequences, and mitochondria will nonetheless continue to function correctly.

An intriguing hallmark of aging in mammals is the appearance of cells carrying significant burdens of mitochondrial DNA (mtDNA) mutants. Unlike the mtDNA mutations which cause inherited diseases, those associated with aging appear to be somatically acquired. Within a given tissue, there is often considerable heterogeneity in the burden of mtDNA mutations, such that affected cells co-exist side by side with healthy cells that carry few, if any, mutations. Furthermore, the frequency of affected cells tends to increase with age and there is evidence that within individual cells, the mitochondrial population is commonly overtaken by a single mutant type, very often a deletion in which a part of the normal mtDNA genome has been lost. The precise mutations tend to differ from one affected cell to another, suggesting that individual mtDNA mutations arise at random. How these mtDNA mutations undergo clonal expansion is a question of longstanding interest.

The possibilities that they multiply either because of a so-called vicious cycle such that defective mitochondria simply generate more reactive oxygen species (ROS), which in turn cause more mutations, or because of random drift, have both been considered but found to be unsatisfactory. Instead, it seems most likely that new mtDNA mutations are acted upon by some form of intracellular selection, causing the expansion of a clone of mutant mitochondria that may come to dominate or entirely exclude the wild type population.

Among the various possibilities to account for a selective advantage favoring mtDNA deletions are that: (i) in a cell where wild type and deleted mtDNA molecules co-exist, there may be a selection advantage for deletion mutants since they have a smaller genome size, which might result in a shorter replication time; (ii) if mitochondria that are compromised by a high burden of mutations have a slower rate of metabolism, they may be less damaged by ROS and so relatively spared from deletion by mitophagy, thereby resulting in survival-based selection through a process that has been termed survival of the slowest; (iii) the selection advantage of mtDNA deletions might be based on features relating to some aspect of the machinery for mtDNA replication, of which several possibilities exist, at least hypothetically.

Possibility (i) has been closely examined but found to be implausible, chiefly because the time required for replication of an mtDNA molecule is only a tiny fraction (less than 1%) of the half-life of mtDNA, which drastically diminishes any scope for a size-based replication advantage to be important. Possibility (ii) has also been found to be unlikely, since not only is it incompatible with mitochondrial dynamics, but it also appears that dysfunctional mitochondria are degraded preferentially rather than more slowly than intact ones By a process of elimination, it appears probable, therefore, that the enigma of clonal expansion of mtDNA deletions requires explanation in terms of the machinery for DNA replication.

Recently, we noticed that when the locations of mtDNA deletions, which had been reported from rats, rhesus monkeys, and humans, were compared, there was a stretch of mtDNA that was overlapped in nearly every instance. Based on this observation and noting that the primer required for DNA replication is provided by processing an mRNA transcript, we suggested a novel mechanism based on this intimate connection of transcription and replication in mitochondria. If a product inhibition mechanism exists that downregulates the transcription rate if sufficient components for the respiration chain exist, then deletion events removing a region of the genome involved in this feedback-loop would confer to such deletion mutants a higher rate of replication priming, leading to a substantial selection advantage. In this article, we report additional data from mice that are strongly consistent with our previous analysis of rats, monkeys, and humans, and we further examine the implications of the hypothesis that a shared sequence, falling within the common overlap of these many individual deletions, might throw light on the underlying mechanism for clonal expansion.

Link: http://dx.doi.org/10.3390/genes9030126

Today I'll point out a couple of recently published research results that add to the understanding of Parkinson's disease and its progression. Parkinson's disease is comparatively straightforward as neurodegenerative diseases go - which is to say that its biochemistry is still enormously complex in detail, but it hasn't proven as hard to identify the important aspects as is the case for Alzheimer's disease. At root, this is a synucleinopathy, a condition caused by the accumulation of α-synuclein deposits. This results in mitochondrial dysfunction and cell death in a small but important population of dopamine-generating neurons connected to motor function, but also a more widespread disruption of normal function in the brain. The challenge in Parkinson's is less a matter of knowing where to intervene, meaning the targeted removal of α-synuclein, but rather the construction of an effective methodology. You might look at one of the SENS Research Foundation reviews on the topic to get a sense of just how difficult it is to safely clear a specific form of metabolic waste from the brain.

Why do only some people develop Parkinson's disease? In a small number of cases, it is due to mutated genes, particularly those like parkin that are important in the processes of cellular maintenance. Impairment of autophagy directed at quality control of mitochondria appears to be an important facet of Parkinson's disease, but in those patients without evidence of mutation, the path to Parkinson's may be a random one of small differences in age-related damage and declining cellular maintenance that snowball and accelerate over time. A little less removal of metabolic waste leads to a little more α-synuclein and a little more mitochondrial dysfunction, which in turn further impacts maintenance systems, and so the feedback loop progresses, ever faster over time. Given enough time, everyone would suffer Parkinson's disease eventually - but as things stand, other processes of aging kill most people before that can happen.

Beyond clearance of α-synuclein, cell therapy is the other major area of effort in the production of therapies for Parkinson's disease. The goal there is to replace lost dopamine-generating neurons with new cells capable of taking over the same function in the brain. Since the process of loss is gradual, this should provide lasting benefit, even though it doesn't address the causes of cell loss - the new cells will be destroyed in time, just like the old ones. This situation is similar in near any proposed use of cell replacement therapy in older individuals: the tissue environment is typically hostile and damaged, and the details of how and why existing cell populations are no longer working matter greatly when it comes to the potential effectiveness of introducing new cells. Will they function correctly at all, or will they quickly succumb?

Researchers uncover culprit in Parkinson's brain cell die-off

If we could peer into the brains of Parkinson's patients, we'd see two hallmarks of the disease. First, we'd see a die-off of the brain cells that produce a chemical called dopamine. We'd also see protein clumps called Lewy bodies inside the neurons. Researchers believe a key to treating Parkinson's is to study possible links between these two phenomena. "This study identifies the missing link between Lewy bodies and the type of damage that's been observed in neurons affected by Parkinson's. Parkinson's is a disorder of the mitochondria, and we discovered how Lewy bodies are releasing a partial break-down product that has a high tropism for the mitochondria and destroys their ability to produce energy."

Lewy bodies were described a century ago, but it was not until 1997 that scientists discovered they were made of clumps of a misfolded protein called α-synuclein. When it's not misfolded, α-synuclein is believed to carry out functions related to the transmission of signals between neurons. Researchers looked at cell cultures of neurons that were induced to accumulate fibrils made of misfolded α-synuclein, mimicking Lewy bodies in patients with Parkinson's. They discovered that when α-synuclein fibrils are broken down, it often creates a smaller protein clump, which they named pα-syn* (pronounced "P-alpha-syn-star").

It turns out that the result of that partial degradation, pα-syn*, is toxic. Researchers made this discovery by labeling the pα-syn* with an antibody so they could follow it throughout the cell after it was created. They observed that pα-syn* traveled and attached itself to the mitochondria. Further investigation revealed that once the pα-syn* attached, the mitochondria started to break down. These fragmented mitochondria lose their ability to carry an electrochemical signal and produce energy. "The Lewy bodies are big aggregates and they're sitting in the cell, but they don't come into direct contact with the mitochondria in the way pα-syn* does. With this discovery, we've made a direct connection between the protein α-synuclein and the downstream effects that are observed when brain cells become damaged in Parkinson's."

Study Uncovers New Insights Into Cause of Cell Death in Parkinson's

Researchers have found that cardiolipin, a molecule inside nerve cells, helps ensure that a protein called alpha-synuclein folds properly. Misfolding of this protein leads to protein deposits that are the hallmark of Parkinson's disease. These deposits are toxic to nerve cells that control voluntary movement. When too many of these deposits accumulate, nerve cells die. "Identifying the crucial role cardiolipin plays in keeping these proteins functional means cardiolipin may represent a new target for development of therapies against Parkinson's disease."

The study revealed that inside cells, alpha-synuclein binds to mitochondria, where cardiolipin resides. Cells use mitochondria to generate energy and drive metabolism. Normally, cardiolipin in mitochondria pulls synuclein out of toxic protein deposits and refolds it into a non-toxic shape. The researchers found that in people with Parkinson's disease, this process is overwhelmed over time and mitochondria are ultimately destroyed. Understanding cardiolipin's role in protein refolding may help in creating a drug or therapy to slow progression of the disease.

Some cancerous cells express signatures normally associated with senescent cells, so why not try senolytic compounds against them? This is something of a full circle, given that most of the current senolytic drug candidates were originally characterized and tested as potential chemotherapeutics. The open access paper here is interesting for two points: firstly, that senolytic drugs didn't kill cancerous cells with a senescent signature, and secondly that a suicide gene therapy targeting that signature does work against both normal senescent cells and cancerous cells with a senescent signature. The gene therapy approach reported here is conceptually similar (at a very high level) to the Oisin Biotechnologies gene therapy used to destroy senescent cells, but less flexible. The Oisin Biotechnologies founders have shown that targeting p53, a cancer suppressor, rather than p16 / p16Ink4a, a signature of senescence, is highly effective against cancer, but it appears that p16 is also a viable trigger for cell killing gene therapy mechanisms in many cancers.

p16Ink4a arrests cell cycle progression by inhibiting the S phase. Cellular senescence, a tumor suppressive mechanism defined as irreversible growth arrest and induced by accumulation of DNA damage, is often associated to induction of p16Ink4a. Consequently, p16Ink4a is considered a strong tumor suppressor. Loss-of-function mutations affecting p16Ink4a are a common mark of various human tumors, and considered an essential step towards tumor progression. However, in the presence of mutations affecting RB or CDK4/CDK6, p16Ink4a activity is not sufficient to arrest cell cycle progression. Moreover, p16Ink4a overexpression has been observed at the invasive front of endometrial, colorectal and basal cell carcinoma and correlated with high aggressiveness. Thus, under these conditions targeting p16Ink4a-overexpressing cells could be a potent anti-cancer intervention.

Despite the mutation-enabled bypass of the senescence program, sarcoma cells overexpressing RAS and with inactive p53 induced high level of p16Ink4a. We then hypothesized that treatment with compounds shown to selectively eliminate senescent p16Ink4a-overexpressing cells could be an efficient strategy. Two of the most effective compounds with senolytic properties (i.e. selectively toxic against senescent cells) are ABT-263 and ABT-737, well-known anti-cancer agents inhibiting the BCL2 family of anti-apoptotic proteins. However, neither treatment was toxic for these cancerous cells. This suggests that p16Ink4a overexpressing tumor cells are resistant to currently available compounds with specificity against p16Ink4a+ cells.

We then reasoned that an alternative strategy for elimination of p16Ink4a-overexpressing tumor cells could make use of gene targeting therapy. Suicide gene therapy has been investigated in various types of cancers because of its superior specificity compared to standard genotoxic therapies. A previous effort in testing a suicide gene therapy under the regulation of the p16Ink4a promoter - the so-called INK-ATTAC system - failed to kill p16Ink4a+ cancer cells, despite being effective in eliminating p16Ink4a+ senescent cells. We have recently developed a similar suicide system, called p16-3MR. The major difference is that the p16-3MR gene is under the regulation of the full p16Ink4a promoter, while the INK-ATTAC is regulated by a small portion proximal to the transcription starting site of the INK4a locus.

Our strategy, which we have shown being highly effective in non-proliferating cells, showed high toxicity for cancerous cells both in cell culture and in vivo. Additionally, since it has been shown that in some instances p16Ink4a+ cells are precursor of malignant cells, the 3MR system could allow reduction of tumor incidence via removal of p16Ink4a+ pre-malignant cells. At this stage, extensive research should to be done to test the toxicity of a p16Ink4a-driven suicide gene therapy strategy against additional tumor types.

Link: https://doi.org/10.18632/oncotarget.23752

The research noted here is a representative example of efforts to reverse engineer the mechanisms by which exercise produces benefits, with an eye to achieving the same result with pharmaceutical compounds rather than exertion. Exercise works to grow muscle, improve endurance, and maintain long-term cardiovascular health through some set of mechanisms, as yet far from fully explored. The future of efforts to develop exercise mimetic drugs will no doubt be as laborious and difficult as the past fifteen years of work on calorie restriction mimetics, and for all the same reasons. Both are enormously broad and complex swathe of cellular biochemistry, poorly mapped, and expensive to explore.

In this area of research, even incremental advances in understanding have required years and a great deal of funding to achieve - just look at ongoing work on sirtuins, for example. As yet none of these programs have delivered meaningful approaches to therapy, treatments that might capture a sizable fraction of the effects of either exercise or calorie restriction. This will all change at some point, as biotechnology becomes ever more capable, but it seems foolish to imagine that it will happen in the next few years, given the past record. Even when it does, "so what?" we might ask. Exercise and calorie restriction cannot add decades to healthy life spans. We need a different approach to produce far longer healthy life.

If you've ever wondered how strenuous exercise translates into better endurance, researchers may have your answer. "ERRγ helps make endurance exercise possible. It gears up the energy-creating cellular power plants known as mitochondria, creating more blood vessels to bring in oxygen, take away toxins, and help repair damage associated with muscle use. This makes ERRγ a really interesting potential therapeutic target for conditions with weakened muscles."

The story starts with the PGC1α and PGC1β proteins, which stimulate 20 other proteins associated with skeletal muscle energy and endurance exercise, including ERRγ. In turn, ERRγ, a hormone receptor, acts to turn on genes. Researchers wanted to precisely understand ERRγ's role in skeletal muscle energy production and how that impacts physical endurance. To unravel this relationship, the team studied mice without PGC1α/β. In some, they increased ERRγ selectively in skeletal muscle cells. This approach allowed them to measure how ERRγ and PGC1 act independently, as well as how they function in combination.

Losing PGC1 had a negative impact on muscle energy and endurance. However, boosting ERRγ restored function. The team found ERRγ is essential to energy production, activating genes that create more mitochondria. In other words, they found the power switch for skeletal muscles. The researchers also showed that increased ERRγ in PGC1-deficient mice boosted their exercise performance. By measuring voluntary wheel running, they found that increasing ERRγ produced a five-fold increase in time spent exercising compared to mice with no PGC1 and normal ERRγ levels. "Now that we have detected this direct target (ERRγ) for exercise-induced changes, we could potentially activate ERRγ and create the same changes that are being induced by exercise training."

Link: https://www.salk.edu/news-release/salk-scientists-find-power-switch-muscles/

The Undoing Aging conference is coming up later this month in Berlin, the latest in a long series of conferences focused initially on the science and later on commercial development of SENS rejuvenation biotechnology. In addition to prominent researchers from many parts of the field, this year we'll see a much greater presence of startup companies and investors. The first legitimate rejuvenation therapies, those based on the SENS model of periodic repair of the cell and tissue damage that causes aging, have reached the stage of clinical development. Some of the companies are a few years old now. More will arrive in the years ahead, as support for this cause grows.

The Undoing Aging conference is co-hosted by the SENS Research Foundation and the Forever Healthy Foundation. The former we're all familiar with by now, I'd hope, as an umbrella organization that coordinates and funds a wide range of scientific programs to unblock important lines of rejuvenation research. The latter is investor and philanthropist Michael Greve's non-profit organization. You might recall his support for the SENS Research Foundation in past years of fundraising; in 2016 he pledged $10 million to SENS rejuvenation research programs and the startups that will emerge from those programs. Hosting Undoing Aging is a part of his continued material support for SENS research and the development of practical near-future rejuvenation therapies. Here the Life Extension Advocacy Foundation volunteers interview Michael Greve on his work and his views on the field.

Undoing Aging With Michael Greve

You have spoken about your own advocacy efforts. How you think the public's perception of the subject has changed over the years?

In general, public opinion has already changed significantly over the past two or three years. Nowadays, you read much more often and positively on extending the healthy human lifespan. I firmly believe that once the first working rejuvenation therapy is out there, the whole discussion will immediately change. It will turn from abstract arguments about over-population and such to a very personal one. Do I want to live twenty more years in good health or not? At that point, I guess nobody will say, "Well no, I won't use that treatment and rather get cancer because of, you know, overpopulation." So, the best thing we can all work on is to make this very first therapy happen and then really promote it.

Many people in our community are hoping to see more wealthy people engaged because they have more resources at their disposal and could have a greater impact by donating even a small share of their wealth. However, we don't see that happening much. What messages might be more convincing to these wealthy people? Are they any different from what we usually say when we are trying to convince someone?

Large-scale philanthropy in a very early market such as rejuvenation biotech is hard and only for a few very forward-thinking individuals. I think the most straightforward and effective message to rich people, in general, is to show them a way to become even richer. I see the acceleration of the development of actual rejuvenation therapies as a three-stage process. First, motivate scientists to enter the field and work on the underlying science, then spin out promising research results as early as possible into fundable startups and finally bring in private capital to fuel development of the actual therapies. This last step will allow those high net-worth individuals to both put their money to good use and benefit from it at the same time.

That is why we are organizing the Undoing Aging conference, funding basic research and working hard to move promising research into fundable startups, allowing private capital to fuel the journey from there on. In terms of startups, we have done this already a few times and are seeing a lot of positive effects there.

What do you expect out of UA2018? What do you expect it to impact the most? Public awareness, investors' interest, or networking among scientists?

First and foremost, we are focussed on the science itself. We want to provide a platform for the existing scientific community that already works on damage repair and strengthens the community itself. At the same time, Undoing Aging offers interested scientists and students a first-hand understanding of the current state of affairs to attract new scientists to our exciting field. Apart from that, we have invited the broader longevity community to enable extended networking and support all advocates that do public work. Since we have a lot of interest from journalists, bloggers and several TV stations, there is going to be a public aspect as well. So, yes, you could say it's networking on all levels to advance our cause.

Are there any plans to make Undoing Aging into an annual event?

Yes, we are in this for the long run. This year, we have already received so much positive feedback and even more registrations than we expected. That is very encouraging.

Many people are concerned about affordability. Do you imagine that governments will necessarily have to step in and subsidise rejuvenation therapies that are otherwise difficult to afford?

There is no need to worry about that. We are talking about a market with billions of customers, numerous possible approaches to each aspect of aging, such as clearance of senescent cells. And you can't patent an approach in general, e.g. clearing senescent cells, just the particular implementation. In such a market, the fundamental economic forces as in any other industry will apply, and healthy competition and a multitude of products in combination with a massive customer base will force quality up and prices down as products quickly mature and become a general commodity.

You are currently supporting several biotech startups that are taking rejuvenation treatments into clinical trials. Is there an estimate of the baseline cost of these therapies, and do you know what the companies are planning to do to make them more affordable?

At the end of the day, these therapies are going to be an extremely affordable commodity.

Stem cells are responsible for tissue maintenance, delivering replacement somatic cells and a variety of signals that help to keep organs and other biological systems running. There are many varieties of stem cell, at least one for every tissue type, and all have significant differences in their biochemistry. Unfortunately, one of the shared behaviors in all stem cell populations is a slowing of activity with advancing age, an evolved response to rising levels of damage in cells and tissues that probably serves to reduce the risk of cancer, but at the cost of a decline into organ failure, as essential maintenance shuts down.

The research here is characteristic of a wide range of initiatives that seek to find signal and regulator proteins that can override the evolved reduction in stem cell activity. The aim is to increase activity to youthful levels, and thus avoid the slowdown. Evidence from stem cell therapies and a variety of other approaches to regenerative medicine suggest that this will not cause as great a risk of cancer as feared, even though it doesn't address the underlying damage in cells. Forcing damaged cells in a damaged environment into greater activity must have adverse consequences at some point, but it seems there is nonetheless some leeway to do just that within the present natural state of stem cell aging.

Cells in the brain are constantly dying and being replaced with new ones produced by brain stem cells. As we age, it becomes harder for these stem cells to produce new brain cells and so the brain slowly deteriorates. By comparing the genetic activity in brain cells from old and young mice, the scientists identified over 250 genes that changed their level of activity with age. Older cells turn on some genes, including Dbx2, and they turn off other genes.

By increasing the activity of Dbx2 in young brain stem cells, the team were able to make them behave more like older cells. Changes to the activity of this one gene slowed the growth of brain stem cells. These prematurely aged stem cells are not the same as old stem cells but have many key similarities. This means that many of the genes identified in this study are likely to have important roles in brain ageing.

The research also identified changes in several epigenetic marks - a type of genetic switch - in the older stem cells that might contribute to their deterioration with age. Epigenetic marks are chemical tags attached to the genome that affect the activity of certain genes. The placement of these marks in the genome change as we age and this alters how the cells behave. The researchers think that some of these changes that happen in the brain may alter causing brain stem cells to grow more slowly. "We hope this research will lead to benefits for human health. We have succeeded in accelerating parts of the ageing process in neural stem cells. By studying these genes more closely, we now plan to try turning back the clock for older cells. If we can do this in mice, then the same thing could also be possible for humans."

Link: https://www.babraham.ac.uk/news/2018/03/genes-for-agelinked-brain-deterioration-identified

Recently Y Combinator announced their intent to fund companies working on treatments for aging. It is one of the many signs of a growing interest in this area of development in the venture community. One of the early results appears to be more funding for computational methods of improving drug discovery, with therapies for aging as the rallying cry, after the established Insilico Medicine model. It makes sense that a primarily software-focused part of the venture community would move into a new area, biotechnology, by funding ventures that apply computational technology to the space. That says nothing about the effectiveness of the approach, of course, just that it is a natural evolution of established knowledge and interests.

There is certainly a lot of room for improvement when it comes to the cost and effort required to find and prove out small molecule and other drugs to treat specific conditions or target specific biological mechanisms with minimal side-effects. It is reasonable to think that established deep learning approaches can be fruitfully applied here, to focus attention on molecules in the standard libraries that might otherwise be overlooked, and to design new therapeutic molecules based on existing data and desired characteristics. There is, however, a sizable difference between, say, applying this technology to the search for senolytics and cross-link breakers, approaches that can in principle produce rejuvenation, or applying it to the search for more geroprotectors like metformin, rapamycin, and so forth. The latter can only marginally slow the progression of aging, and the research community is struggling to produce anything in that part of the field that can do any better than exercise and calorie restriction. It remains to be seen as to the direction taken by this venture.

Over the past few decades, an unignorable amount of evidence has piled up that we are able to slow the biological processes of aging in animals. This evidence has been accumulating along multiple lines of research covering many different therapies. We're left with the same conclusion: by understanding and directly treating the biological damage accumulated while aging, we can find powerful new therapies for fighting disease and living healthier, longer lives.

At Spring Discovery, we're accelerating the discovery of these therapies with our machine learning-based drug discovery platform. And we're proud to announce that we've raised a $4.25M seed round from a team of biotech funders who support our long-term vision. Why do therapies focused on aging present such a profound opportunity? Because aging is the single greatest risk factor for the most detrimental diseases on Earth - cardiovascular disease, neurodegenerative disease, pulmonary disease, cancer, muscle wasting, and more - and drugs that slow the biological damage accumulated while aging have the potential to reduce the incidences of these diseases, possibly simultaneously.

Combined, aging represent A) one of the most important problems facing humanity and B) a problem that looks increasingly possible to tackle. The diseases of old age don't discriminate, but they can be fought. We believe that in the not-too-distant future the discovery of therapies for aging will provide some of the most effective tools in history for reducing our burden of disease and extending our healthy lifespan. Spring Discovery's mission is to dramatically accelerate the realization of that future. And we're bringing a new set of machine learning tools to bear on this challenge.

Link: https://medium.com/spring-discovery/accelerating-the-discovery-of-therapies-for-aging-and-its-related-diseases-7c6a2109189f

This lengthy post covers the topic of setting up and running a self-experiment, a human trial of a single individual, to assess the senolytic effects of the peptide FOXO4 D-Retro-Inverso (FOXO4-DRI). This is the protein produced from the FOXO4 gene, with D-amino acids substituted for L-amino acids, reversing the chirality of the molecule. This means it cannot be processed in the usual way by cellular metabolism, and the consequence of interest is that this sabotages the survival efforts of lingering senescent cells in old tissues, causing them to self-destruct. This peptide was evaluated in aged mice in 2017, showing destruction of senescent cells without side-effects, and producing the usual array of benefits to measures of health and age-related decline as a result. This is quite interesting when compared to the various chemotherapeutic senolytic drug candidates that exibit similar degrees of senescent cell destruction in mice, but an array of unpleasant side-effects.

Of course, the chemotherapeutics have extensive human data that catalogs the side-effects at various doses in our species, while at the time of writing there is no human data for FOXO4-DRI whatsoever. This is a very important point! It is perhaps the most important consideration here.

The purpose in publishing this outline is not to encourage people to immediately set forth to follow it. If you come away thinking that you should do exactly that, and as soon as possible, then you have failed at reading comprehension. This post is intended to illustrate how to think about self-experimentation in this field: set your constraints; identify likely approaches; do the research to fill in the necessary details; establish a plan of action; perhaps try out some parts of it in advance, such as the measurement portions, as they never quite work as expected; and most importantly identify whether or not the whole plan is worth actually trying, given all that is known of the risks involved. Ultimately that must be a personal choice.

Contents

• Why Self-Experiment with Senolytics?
• Caveats in More Detail
• Summarizing FOXO4-DRI
• Establishing Dosage
• An Introduction to Injections
• Considering Autoinjectors
• Obtaining a Needle-Free Injection System
• Obtaining Vials of the Correct Size
• Preparing FOXO4-DRI for Injection
• Obtaining FOXO4-DRI
• Storing FOXO4-DRI
• Validating the Purchased FOXO4-DRI
• Establishing Tests and Measures
• Guesstimated Costs
• Practice Before Working with FOXO4-DRI
• Schedule for the Self-Experiment
• Where to Publish?
• Final Thoughts: Why Not Wait?

Why Self-Experiment with Senolytics?

Senolytic therapies are those that selectively destroy senescent cells. The build up of senescent cells is one of the causes of aging. So obviously, one hope is to benefit personally from such a therapy sooner than would otherwise be the case, balancing that against incurring some unknown degree of risk of failure or harm. The first human trials, those that establish numbers for that risk, will take another few years to wind through to robust conclusions, and further years beyond that will be required for the medical community to become willing to prescribe senolytics generally. Further, those trials will almost all test only a single candidate therapy, and the evidence to date in mice suggests that different senolytics with different mechanisms are tissue-specific in their effects on senescent cells. Multiple different compounds may be more effective than one - but that won't be discovered in the formal trial process. Lastly, well run self-experimentation carried out by a number of people, where the results are published, can help to guide the direction of later, formal studies.

All of these reasons must be balanced against a sober assessment of the risks involved in obtaining and using an injected peptide that has no published human data whatsoever, and an acceptance of personal responsibility for consequences should one choose to run those risks.

Caveats in More Detail

There are two areas of personal responsibility to consider here. Firstly, this involves injecting a peptide that has no published data on human use at all. The anecdotal data from people claiming to have used FOXO4-DRI should be ignored, as it only covers the most serious short-term consequences, and few if any of these individuals are verifying that they are indeed obtaining the right compound, and nor are they carefully checking their own biochemistry for outcomes. Anything is possible in the long-term, from cardiovascular failure to cancer to subtle increases in disease or organ failure risk, no matter how compelling the animal data might appear to be. The absence of any human data should be far more concerning to any rational individual than the case of a compound with extensive human data showing serious side-effects. The latter can be planned and accounted for. The former cannot: it is a leap into the unknown.

Secondly, obtaining and using arbitrary novel peptides such as FOXO4-DRI in the manner described here is potentially illegal: not yet being a formally registered medical treatment, it falls into a nebulous area of regulatory and prosecutorial discretion as to which of the overly broad rules and laws might apply. In effect it is illegal if one of the representatives of the powers that be chooses to say it is illegal in any specific case, and there are few good guidelines as to how those decisions will be made. The clearest of the murky dividing lines is that it is legal to sell peptides that are not defined as a therapy for research use, but illegal to market and sell them for personal use in most circumstances. This is very selectively enforced, however, and reputable sellers simply declare that their products are not for personal use, while knowing full well that this is exactly what their customers are doing in many cases.

Choosing to purchase and use FOXO4-DRI would therefore likely be a matter of civil disobedience, as is the case for anyone obtaining medicines or potential medicines outside the established national system of prescription and regulation. People are rarely prosected for doing so for personal use in the US - consider the legions of those who obtain medicines overseas for reasons of cost, despite the fact that doing so is illegal - but "rarely" is not "never." If you believe that the law is unjust, then by all means stand up against it, but accept that doing so carries the obvious risks of arrest, conviction, loss of livelihood, and all the other ways in which the cogs of modern society crush those who disagree with the powers that be.

Lastly, senolytics is a fast-moving field. This post will become outdated quite rapidly in its specifics, as new FOXO4-DRI research arrives on the scene, and FOXO4-DRI may well be obsoleted by better options. Nonetheless, the general outline should still be a useful basis for designing new self-experiments involving later and hopefully better compounds, as well as tests involving more logistical effort.

Summarizing FOXO4-DRI

You might look at an earlier post for a high level overview of how FOXO4-DRI works to selectively destroy senescent cells. The short version of the story is that functional FOXO4 interacts with p53 in order to suppress the cell self-destruction mechanisms that are primed for activation in all senescent cells. This appears to be the primary method by which a small fraction of senescent cells manage to linger in order to cause age-related dysfunction in tissues. Since FOXO4-DRI does not function correctly in cellular metabolism, the FOXO4-p53 interation fails when FOXO4-DRI is present in sufficient amounts to replace enough of the native FOXO4, and the cell destroys itself.

From studies in mice, FOXO4-DRI appears to cause no issues in other cell types; it is simply ignored - at least in the short-term, and in mice. In the longer term, high levels of non-functioning FOXO4 in calls might indeed cause problems, but the treatment are intended to last only a short time, with the FOXO4-DRI being broken down in a matter of a day or so. There is no human data to show that any of this also applies to our species, but given a few years for the research community to make progress, that will change.

Establishing Dosage

The only definitive way to establish a dosage for a pharmaceutical in order to achieve a given effect is to run a lot of tests in humans. Testing in mice can only pin down a likely starting point for experiments to determine a human dose, but the way in which you calculate that starting point is fairly well established for most cases. That established algorithm is essentially the same for most ingested and intravenously (or intraperitoneally in small animals) injected medicines, but doesn't necessarily apply to other injection routes. More on that in the next section of this post. Some compounds - as always - are exceptions to the rule, and the only way that scientists discover that any specific compound is an exception is through testing at various doses in various species.

When considering dosage of any substance, it is important to emphasise that more is not better; this cannot be approached in the way people tend to naively approach the (over)use of dietary supplements. The primary goal, if self-experimenting, is to take as little as necessary of any senolytic compound. The first look at FOXO4-DRI by the research community suggests that higher doses should do nothing more than is achieved by the therapeutic doses, and with the same absence of immediate side-effects - but that is only the result of an initial examination. No long-term assessment has taken place, even in mice. If FOXO4-DRI does turn out to produce lasting or subtle side-effects, then following the maxim of using as little of it as possible should help to lower the impact. That the dose makes the poison is an ancient adage, but no less true today.

The steps to figure out a suitable starting point for a human test of an injected senolytic pharmaceutical are as follows: firstly read the mouse studies for the senolytic compound in question, in order to find out how much was given to the mice and for how long. Doses for most pharmaceuticals are expressed in mg/kg. Secondly apply a standard multiplier to scale this up to human doses, which you can find in the open access paper "A simple practice guide for dose conversion between animals and human". Do not just multiply by the weight of the human in kilograms - that is not how this works. The relative surface area of the two species is the more relevant scaling parameter. Read the paper and its references in order to understand why this is the case. Again, note that the result is only a ballpark guess at a starting point in size of dose. The duration of treatment translates fairly directly, however. For the period of treatment, start with the same number of doses, spacing of doses, and duration as takes place in senolytic studies in mice.

In the case of FOXO4-DRI, the mice in the single study were injected intraperitoneally with 5 mg/kg of FOXO4-DRI dissolved in phosphate buffered saline, three times, taking place every other day. For a 60kg human, that translates to a 25mg dose via intravenous injection, carried out three times on alternate days.

An Introduction to Injections

The relationship between different forms of injection, dosage, and effects is actually a complicated and surprisingly poorly mapped topic. There are four type of injection to consider, here listed in descending order of difficulty to carry out safely: (a) intraperitoneal, through the stomach muscle into the abdominal body cavity, which is rare in human medicine but common in studies using small animals; (b) intravenous, into a vein, which requires some practice to get right; (c) intramuscular, into the muscle beneath the skin; and (d) subcutanous, into the lower levels of the skin.

The amount of fluid that can be easily injected varies by type. In humans, effectively unlimited amounts of fluid can be introduced via intraperitoneal or intravenous injection. The subcutaneous route is limited to something less than 1ml, and intramuscular is limited to 2-3ml depending on location. These are all very fuzzy numbers - some sources, for example, give an upper limit of 5ml for intramuscular injections, but I can't say as I would be lining up to be on the receiving end of that. Measure out 5ml and take a look at it. Ouch.

The different injection routes can alter the character of the injected medicine; how much is required to gain a given effect, how long it takes to get into the system and how fast it does it. A rare few types of medication cannot be injected subcutaneously, because the metabolism of the skin will degrade them, while some are better given subcutaneously. If you root through the literature looking for comparisons between performance and dosage for different injection types, you'll find a very ragged collection of examples showing that there are few coherent rules. Some compounds have no discernible differences between injection route, some see altered peaks of concentration, some require higher doses when subcutaneous, some require lower doses when subcutenous. Oil-based solutions can produce a very slow uptake of medication when injected into muscle or skin in comparison to an intraveous injection, while water-based solutions result in just as rapid an uptake into the bloodstream.

Do we know how FOXO4-DRI will be affected by different injection routes? No. That data has yet to be established. So it seems acceptable to say that a self-experimenter should try to use the much easier paths of subcutaneous and intramuscular injection, rather than attempting intravenous administration, and just keep the same dose as was established for intravenous injection. For most people, intraveous injections require a helper or a lot of painful practice. For subcutaneous and intramuscular injections, there is a market of autoinjection tools that can remove many of the challenges inherent in managing injections.

Considering Autoinjectors

Sticking a needle into one's own flesh is not an easy thing to do, and this is the rationale for the range of autoinjection systems that have been developed by the medical community. They are most easily available for subcutaneous injections; spring-based devices that accept a standard needle and syringe, and that are trigged by a button push. Intramuscular autoinjectors do exist, but unfortunately not in a general or easily available way. All of the needle-based intramuscular autoinjectors are regulated devices that come preloaded with a particular medicine, and are not otherwise sold in a more generally useful way. Unfortunately, there is no automation that can help with intravenous injections. You are on your own there.

Option 1: Subcutaneous Autoinjection with Needle and Syringe

If intending to carry out subcutaneous injections it is easy enough to order up a supply of disposible needles and syringes, an autoinjector device that accepts the standard needle and syringe arrangement, and other necessary items such as sterilization equipment from the sizable diabetes-focused marketplace. Such injections are relatively easy to carry out, a wide range of vendors sell the materials, and there is a lot of documentation, including videos, available on how to carry out subcutaneous injections. All of the equipment is cheap. Buying these materials will probably put you on a list in this era of the drug war, but there are many people out there doing it.

Option 2: Subcutaneous or Intramuscular Needle-Free Autoinjection

Are there viable alternatives to needles? As it turns out, yes, and some can solve the problem of missing general intramuscular autoinjectors as well. Needle-free autoinjectors that use a thin, high-pressure fluid jet to punch medication through the skin are a growing area of development. These systems have numerous advantages over needles, but they are more expensive, most can only manage subcutanous injections, and all are limited in the amount of fluid they can inject in comparison to the traditional needle and syringe. Nonetheless, for the purposes of this outline, I'll focus on needle-free systems. The biggest, primary, and most attractive advantage of a needle-free system is in the name: it means not having to deal with needles in any way, shape, or form.

Obtaining a Needle-Free Injection System

There are a fair number of needle-free injectors on the market, but most are hard to obtain unless you happen to be a regulated medical facility running through the standard regulated purchase model, and are looking for large numbers of units in a bulk purchase. Some systems use compressed gas, others use springs. The spring-based systems tend to be less complicated and more reliable. From my survey of the marketplace, the two systems worth looking at are (a) PharmaJet, which can be purchased in the US via intermediary suppliers such as Moore Medical, and (b) Comfort-in, which is sold directly to consumers in most countries by an Australian group. So far as I can tell, PharmaJet is the only available needle-free system that is capable of intramuscular rather than subcutaneous injection.

PharmaJet is the better engineered and more expensive of these two systems, and its specialized syringes are very definitely built to be one-use only. Further, loading fluid into the syringes requires the use of vials and a vial adaptor. First the vial is loaded with the fluid to be injected, then the vial is connected to the syringe via the adaptor to transfer the fluid. Comfort-in has a similar setup, but is more flexible, and on the whole more consumer-friendly when considering the entire package of injector and accessories. It is has a wider range of vial and other adaptors. Further, the Comfort-in syringes can in principle be reused given sterilization, though of course that is not recommended.

The instructions for both of these systems are extensive, and include videos. They are fairly easy to use. One caveat is that needle-free systems produce a puncture that more readily leaks some of the injected material back out again than is the case for needles. It is a good idea to have a less absorbent plaster ready to apply immediately after injection, such as one of the hydrocolloid dressings now widely available in stores.

Obtaining Vials of the Correct Size

If using the insulin needle and subcutaneous injection approach, then any variety of capped glass vial will do when it comes to mixing and temporarily holding liquids for injection. It does, however help greatly to either use preassembled sterile vials or assemble your own vials with rubber stoppers and crimped caps, as described below, as that sort of setup makes it easier to take up small amounts of a liquid into a syringe. If using the needle-free systems, then vials of a specific type and size are necessary in order to fit the adaptors. The rest of this discussion focuses on that scenario.

There are many, many different types of vial manufactured for various specialized uses in the laboratory. The type needed here is (a) crimp-top vial, also called serum vials by some manufacturers, with (b) a 13mm (for PharmaJet and Comfort-in) or 20mm (for Comfort-in only) diameter open top aluminium cap, one that has a central hole to allow needles and adaptor spikes through, and (c) a rubber or rubber-like stopper that is thin enough in the center to let a needle or adaptor spike past. The cap is crimped on over the rubber seal to keep everything in place - this requires a crimping tool, and removing it requires the use of another tool.

There are two options here. The first option is to purchase preassembled empty sterile vials of the right size and a set of disposable needles and syringes to transfer liquid into the vials. In order to continue to bypass the whole business of needles, however, the other alternative is to purchase vials, stoppers, and aluminium caps separately, or in a kit, and assemble your own vials. A crimping tool is also needed in order to seal the cap. That tool, like the vials and the caps, must be of the right size. Be careful when purchasing online. Vials are categorized by many different dimensions, and descriptions tend to mix and match which dimensions of the vial they are discussing, or omit the important ones. For sterile vials, it is usually only the cap diameter that is mentioned. For crimp-top vials, there are any number of dimensions that might be discussed; the one that needs to match the cap diameter is the outer diameter of the mouth or crimp.

It is usually a good idea to buy a kit where possible, rather than assembling the pieces from different orders, but if taking the assembly path, it is best to buy all the pieces from the same company. Wheaton is a decent manufacturer, and it is usally possible to find much of their equipment for sale via numeous vendors. One can match, say, the crimp-top 3ml vials #223684 with 7mm inner mouth and 13mm outer mouth with snap-on rubber stoppers #224100-080 of the appropriate dimensions and 13mm open top caps #224177-01. Then add a 13mm crimping device #W225302 and pliers #224372 to remove 13mm crimped caps.

Preparing FOXO4-DRI for Injection

The objective is to wind up with the right amount of FOXO4-DRI dissolved in phosphate buffered saline in a sealed vial, ready to be used with the injection system, and with as little contamination as possible from the environment. Depending on the size of the vial, it might contain doses for multiple injections - in fact it is much easier to set things up this way. FOXO4-DRI dissolves very readily in saline, so placing a single human dose into 0.5ml or 1ml is quite feasible. A 3ml vial can hold three doses for the treatment without issue.

One approach is to measure out FOXO4-DRI by weight using a suitable microscale, then transfer that dose to a mixing container, such as larger glass vial. Pipette in the desired amount of saline and stir with a rod to ensure it is fully dissolved. Then either inject the mix into a sterile vial using a standard needle and syringe, or pipette the mix into an clean open vial that is then sealed, capped, and crimped. You will probably find that it is much easier to make up multiple doses at once and measure them out in liquid form rather than using a microscale, however, as greater precision is possible that way. Small amounts tend to be hard to weigh accurately, but pipetting them when in solution is feasible.

Keeping Things Sterile is Very Important

Keeping hands, tools, vials, and surfaces clean and sterile is important: wash everything carefully and wipe down surfaces with an alcohol solution before and after use. Laboratories use autoclaves, which sterilize with steam. These are largely expensive devices, but a range of smaller, cheaper options exists. There are many best practices guides and summaries available online. This extends to the injection itself. Even with needle-free systems, an injection site should still be wiped down with alcohol first. It is all too easy to infect an injection site if skipping the precautions, and this can have severe consequences.

Further, it is important to ensure that the FOXO4-DRI mix is sterile, as you have no control over how it was handled prior to shipping. One approach to doing this is to filter the peptide solution after making it up. This is fairly easily accomplished using a sterile syringe filter rated to block bacteria, and for use with peptides. Some filters may leach peptides from the solution, so caution is needed in choosing the right product - read the descriptions carefully. You might look for polyethersulfone 0.2 micron filters, for example, which are intended to filter out bacteria without binding appreciably to proteins. A good practice is to make up a double concentration solution, pass it through the filter, then flush the filter with another, equal amount to make up the final solution for injection.

Obtaining FOXO4-DRI

There are a small number of peptide synthesis companies worldwide that advertise their willingness to sell FOXO4-DRI, as an established recipe for synthesis rather than a special order. Any other reputable peptide synthesis company will be able to manufacture FOXO4-DRI to order from the description provided in the 2017 paper.

H-ltlrkepaseiaqsileaysqngwanrrsggkrppprrrqrrkkrg-OH. Molecular weight: 5358.2 It was manufactured by Pepscan (Lelystad, the Netherlands) at more than 95% purity and stored at -20°C in 1mg powder aliquots until used to avoid freeze-thawing artifacts. For in vitro experiments FOXO4-DRI was dissolved in Phosphate Buffered Saline to generate a 2mM stock. For in vivo use, FOXO4-DRI was dissolved in Phosphate Buffered Saline to generate a 5mg/ml stock solution, which was kept on ice until injection. Before injection the solution was brought to room temperature.

An advantage of the companies that advertise FOXO4-DRI in their catalog is that they will have established mass spectra and other data sheets for the compound that can be compared with one another, or used as a basis for evaluating the quality of the product. The disadvantage, in at least the case of NovoPro Bioscience, is that the price charged is outrageous - possibly a rational choice made to discourage amateur purchasers. Chinese companies, on the other hand have a comparatively low price for their offerings, significant lower than that of even the most aggressively competitive companies in the US and Europe. Among European and US synthesis companies out there, Pepscan and Genscript are options, with Genscript coming recommended by a number of sources.

Without focusing on any of the specific vendors mentioned above, the easiest way to obtain cost-effective synthesis of peptides is to follow the process outlined in the last post on self-experimentation, which is to use Alibaba to find and connect with smaller-sized suppliers in China. There are a good many reputable peptide synthesis concerns in that part of the world, even if only one or two explicitly advertise FOXO4-DRI as a product at this time.

As noted at the outset of this post, all of these efforts to obtain, ship, and use a peptide are to some degree illegal - it would be an act of civil disobedience carried out because the laws regarding these matters are unjust, albeit very unevenly enforced. Many people regularly order pharmaceuticals from overseas, with and without prescriptions, for a variety of economic and medical reasons, and all of this is illegal. The usual worst outcome for individual users is intermittent confiscation of goods by customs, though in the US, the FDA is actually responsible for this enforcement rather than the customs authorities. Worse things can and have happened to individuals, however, even though enforcement is usually targeted at bigger fish, those who want to resell sizable amounts of medication on the gray market, or who are trafficking in controlled substances. While the situation with an arbitrary peptide isn't the same from a regulatory perspective, there is a fair amount written on the broader topic online, and I encourage reading around the subject.

Avoid Trifluoroacetate (TFA) Salt

The most common method of producing peptides to order involves the use of trifluoroacetate (TFA) salt, and the manufactured peptide will have residual TFA salt in it. This stuff harms cells and can cause painful, lasting, inflammatory reactions if injected. If you don't specify in an order that you wish TFA salt to be exchanged or removed, then you will get TFA salt. Ordering without TFA salt residuals will raise the price a little, but it is both the recommended course of action and well worth it.

Open a Business Mailbox

A mailbox capable of receiving signature-required packages from internal shipping concerns such as DHL and Fedex will be needed. Having a business name and address is a good idea. Do not use a residential address.

Use Alibaba to Find Manufacturers

Alibaba is the primary means for non-Chinese-language purchasers to connect to Chinese manufacturers. The company has done a lot of work to incorporate automatic translation, to reduce risk, to garden a competitive bazaar, and to make the reputation of companies visible, but it is by now quite a complicated site to use. It is a culture in and of itself, with its own terms and shorthand. There are a lot of guides to Alibaba out there that certainly help, even if primarily aimed at retailers in search of a manufacturer, but many of the specific details become obsolete quickly. The Alibaba international payment systems in particular are a moving target at all times: this year's names, user interfaces, and restrictions will not be the same as next year's names, user interfaces, and restrictions.

Start by searching Alibaba for peptide synthesis companies. There are scores of biotech companies in China for any given specialty. Filter the list for small companies, as larger companies will tend to (a) ignore individual purchasers in search of small amounts of a peptide, for all the obvious economic reasons, and (b) in any case require proof of all of the necessary importation licenses and paperwork. Shop around for prices - they may vary widely, and it isn't necessarily the case that very low prices indicate a scam of some sort. Some items and services are genuinely very cheap to obtain via some Chinese sources. Remember to ask the manufacturer for mass spectra and liquid chromatography data if they have it.

Many manufacturers will state that they require a large (often ridiculously large) minimum order; that can be ignored. Only communicate with gold badge, trade assurance suppliers with several years or more of reputation and a decent response rate. Make sure the companies exist outside Alibaba, though for many entirely reliable Chinese businesses there are often sizable differences between storefronts on Alibaba, real world presence, and the names of owners and bank accounts. Use your best judgement; it will become easier with practice.

Arrange Purchase and Shipping via Alibaba

Given the names of a few suppliers, reach out via the Alibaba messaging system and ask for a quote for a given amount of FOXO4-DRI; you will have to provide the sequence and a reference to the paper in which it is described. Buy more than you'll think you need, as a small amount of it will be used to validate the identity and quality of the compound batch. Payment will most likely have to be carried out via a wire transfer, which in Alibaba is called telegraphic transfer (TT). Alibaba offers a series of quite slick internal payment options that can be hooked up to a credit card or bank account, but it is hit and miss whether or not those methods will be permitted for any given transaction. Asking the seller for a pro-forma invoice (PI), then heading to the bank to send a wire, and trusting to their honesty should work just fine when dealing with companies that have a long-standing gold badge.

To enable shipping with tracking via carriers such as DHL, the preferred method of delivery for Chinese suppliers shipping to the US or Europe, you will need to provide a shipping address, email address, and phone number. Those details will find their way into spam databases if you are dealing with more than a few companies, and will be, of course, sold on by Alibaba itself as well. Expect to see an uptick of spam after dealing with suppliers via Alibaba, so consider using throwaway credentials where possible.

Chinese manufacturers active on Alibaba are familiar with international shipping practices. On their own initiative may or may not decide to declare the true cost and contents of the shipped package. This is another form of widely practiced civil disobedience, but is much more common in the shipping of pharmaceuticals than in the shipping of synthesized peptides such as FOXO4-DRI. The former are likely to be confiscated by customs officials, while the latter are not. If the true cost is declared, then expect to pay customs duty on that cost; payment is typically handled via the carrier. Note that different carriers tend to have different processes and rates at which shipments are checked for validity.

Storing FOXO4-DRI

Peptides are usually shipped in powder form, and while in this form are easily stored in a refrigerator for the short-term, or in a freezer for the long term. It has a much shorter life span once it has been mixed with liquid for injection, however, and should be kept on ice, and used within days or or at most weeks.

Validating the Purchased FOXO4-DRI

A peptide of a given sequence may have been ordered, but that doesn't mean that what turns up at the door is either the right nondescript powder or free from impurities or otherwise of good quality. Even when not ordering from distant, infrequent suppliers, regular testing of batches is good practice in any industry. How to determine whether a peptide is what it says it is on the label? Run it through a process of liquid chromatography and mass spectrometry, and compare the results against the standard data for a high purity sample of that compound. Or rather pay a small lab company to do that.

Note that there isn't a cheap way to validate that the peptide in question does in fact have D-amino acids substituted for L-amino acids. They look the same in mass spectrometry, and largely the same in liquid chromatography, or at least without some additional work. Ask the vendor for the approach they would take or are familiar with, and shop around for prices. One approach is stability profiling, which relies on the different half-life of L- and D-amino acids in some circumstances.

Obtain the Necessary Equipment

Since this process will involve weighing, dividing up, and shipping powders in milligram amounts, a few items will be necessary: spatulas or scoops for small amounts of a substance; a reliable jeweler's scale such as the Gemini-20; sealable vials; small ziplock bags; labels; and shipping and packing materials. All of these are easily purchased online. The recommended shipping protocol is to triple wrap: a labelled vial, secured within a ziplock bag and tape, and then enclosed within a padded envelope.

Use Science Exchange to Find Lab Companies

Science Exchange is a fairly robust way to identify providers of specific lab services, request quotes, and make payments. Here again, pick a small lab company to work with after searching for LC-MS (liquid chromatography and mass spectrometry) services. Large companies will want all of the boilerplate registrations and legalities dotted and crossed, and are generally a pain to deal with in most other ways as well. Companies registered with Science Exchange largely don't provide their rates without some discussion, but a little over $100 per sample is a fair price for LC-MS to check the identity and purity of the compound.

Work with the Company to Arrange the Service

The process of request, bid, acceptance, and payment is managed through the Science Exchange website, with questions and answers posted to a discussion board for the task. Certainly ask if you have questions; most providers are happy to answer questions for someone less familiar with the technologies used. Service providers will typically want a description of the compounds to be tested and their standard data sheets, as a matter of best practice and safety. Here provide the mass spectra and other data sheets from the vendor, or use those published by NovoPro or other sources.

Ship the Samples

Measure out 1-5mg or so of FOXO4-DRI as a distinct sample, label it carefully, and package it up. More in the sample is better than less, as several attempts might be needed to get a good result out of the machines used, but each attempt really only needs a very tiny amount of the compound. Ship the sample via a carrier service such as DHL, UPS, or FedEx. Some LC-MS service companies may provide shipping instructions or recommendations. These are usually some variety of common sense: add a description and invoice to the package; reference the order ID, sender, and receiver; clearly label sample containers; and package defensively with three layers of packing; and so forth.

Examine the Results

Once the LC-MS process runs, the lab company should provide a short summary regarding whether or not the compound is in fact the correct one and numbers for the estimated purity. Also provided are the mass spectra, which can be compared with the existing spectra from the vendor, and from other sources such as NovoPro.

Establishing Tests and Measures

Unfortunately there is no available, established, proven, useful test that can directly assess senescent cell level in humans or human biopsies. It is possible to use immunohistochemistry to assess cellular senesence in tissue samples, which is a standard approach in animal studies, but no-one appears to have yet validated that in humans, given biopsies taken from a living individual. Since senescent cells are generated temporarily by wounding, it is quite possible that anything that starts with a biopsy will prove to be unhelpful as a before and after comparison measure for senolytic trials - the levels measured may not bear any resemblance to the normal levels absent a wound.

Without a direct measure, we must fall back on indirect assessments of the detrimental effects of senescent cells. The objective here is a set of tests that anyone can run without the need to involve a physician, as that always adds significant time and expense. Since we are really only interested in the identification of large and reliable effects as the result of an intervention, we can plausibly expect a collection of cheaper and easier measures known to correlate with age to be useful. Once that hill has been climbed, then decide whether or not to go further - don't bite off more than is easy to chew for a first outing.

From an earlier exploration of likely tests, I picked the following items on the basis of a likely connection to the actions of senescent cells, reasonable cost and effort, and ability to carry out the test without a physician's office being involved. Note that this does rule out, to pick one example, the interesting and relevant examination of kidney and liver function, as it would have to be carried out via the radioactive tracer methods of nuclear medicine to obtain decent results. That leaves the tests below quite focused on (a) the cardiovascular system, particularly measures influenced by vascular stiffness, and (b) inflammatory and other markers in the bloodstream:

• A standard blood test, with inflammatory markers.
• Resting heart rate and blood pressure.
• Heart rate variability.
• Pulse wave velocity.
• Biological age assessment via DNA methylation patterns.

The cardiovascular health measures in that list are those that are impacted by changes in the elasticity or functional capacity of blood vessels, such as would be expected to occur to some degree following any rejuvenation therapy that addresses senescent cells, chronic inflammation, or other factors that stiffen blood vessels, such as calcification or cross-linking. Positive change of the average values in most of these metrics are achievable with significant time and effort spent in physical training, so movement in the numbers in a short period of time as the result of a treatment should be an interesting data point.

Bloodwork

There exist online services such as WellnessFX where one can order up a blood test and then head off the next day to have it carried out by one of the widely available clinical service companies. Of the set of test packages offered by WellnessFX, the Baseline is probably all that is needed for present purposes. But shop around; this isn't the only provider.

Resting Heart Rate and Blood Pressure

A simple but reliable tool such as the Omron 10 is all you need to measure heart rate and blood pressure. It is worth noting here a couple of general principles for cardiovascular measures. Firstly, the further away from the center of the body that the measurement is taken, the less reliable it is - the more influenced by any number of circumstances, such as position, mood, stress, time of day, and so forth. Fingertip devices are convenient, but nowhere near as useful as something like the Omron 10 that uses pressure on the upper arm. Secondly, all of the above-mentioned line items also influence every cardiovascular measure, so when you are creating a baseline or measuring changes against that baseline, carry out each measure in the same position, at the same time of day, and make multiple measurements over a week to gain a more accurate view of the state of your physiology. The Omron 10 is solid: it just works, and seems quite reliable.

Heart Rate Variability

Surprisingly few of the numerous consumer tools for measuring heart rate variability actually deliver the underlying values used in research papers rather than some form of aggregate rating derived by the vendor; the former is required for any serious testing, and the latter is useless. Caveat emptor, and read the reviews carefully. As an alternative to consumer products, some of the regulated medical devices are quite easy to manage, but good luck in navigating the system to obtain one. The easiest way is to buy second hand medical devices via one of the major marketplaces open to resellers, but that requires a fair-sized investment in time and effort - which comes back to the rule about keeping things simple at the outset.

After some reading around the subject, I settled on the combination of the Polar H10 device coupled with the SelfLoops HRV Android application. I also gave the EliteHRV application a try. Despite the many recommendations for Polar equipment, I could not convince either setup to produce sensible numbers for heart rate variability data: all I obtained during increasingly careful and controlled testing was a very noisy set of clearly unrealistic results, nowhere near the values reported in papers on the subject. However, plenty of people in the quantified self community claim that these systems work reasonably well, so perhaps others will have better luck than I. Take my experience as a caution, and compare data against that reported in the literature before investing a lot of time in measurement.

Pulse Wave Velocity

For pulse wave velocity, choice in consumer tools is considerably more limited. Again, carefully note whether or not a device and matching application will deliver the actual underlying data used in research papers rather than a made-up vendor aggregate rating. I was reduced to trying a fingertip device, the iHeart, picked as being more reliable and easier to use than the line of scales that measure pulse wave velocity. Numerous sources suggest that decently reliable pulse wave velocity data from non-invasive devices is only going to be obtained by measures at the aorta and other core locations, or when using more complicated regulated medical devices that use cuffs and sensors at several places on the body.

Still, less reliable data can be smoothed out to some degree by taking the average of measures over time, and being consistent about position, finger used for a fingertip device, time of day, and so forth when the measurement is taken. It is fairly easy to demonstrate the degree to which these items can vary the output - just use the fingertip device on different fingers in succession and observe the result. All of this is a trade-off. A good approach is to take two measures at one time, using the same finger of left and right hand, as a way to demonstrate consistency. While testing an iHeart device in this way, I did indeed manage to obtain consistent and sensible data, though there is a large variation from day to day even when striving to keep as many of the variables as consistent as possible. That large variation means that only sizable effects could be detected.

DNA Methylation

DNA methylation tests can be ordered from either Osiris Green or Epimorphy / Zymo Research - note that it takes a fair few weeks for delivery in the latter case. From talking to people at the two companis, the normal level of variability for repeat tests from the same sample is something like 1.7 years for the Zymo Research test and 4.8 years for the Osiris Green tests. The level of day to day or intraday variation between different samples from the same individual remains more of a question mark at this point in time, though I am told they are very consistent over measures separated by months. Nonetheless, as for the cardiovascular measures, it is wise to try to make everything as similar as possible when taking the test before and after a treatment: time of day, recency of eating or exercise, recent diet, and so forth.

An Example Set of Daily Measures

An example of one approach to the daily cardiovascular measures is as follows, adding extra measures as a way to demonstrate the level of consistency in the tools:

• Put on the Polar H10; this is involved enough to increase heart rate a little for a short period of time, so get it out of the way first.
• Sit down in a comfortable position and relax for a few minutes.
• Measure blood pressure and pulse on the left arm using the Omron 10.
• Measure blood pressure and pulse on the right arm using the Omron 10.
• Measure pulse wave velocity on the left index fingertip over a 30 second period using the iHeart system.
• Measure pulse wave velocity on the right index fingertip over a 30 second period using the iHeart system.
• Measure heart rate variability for a ten minute period using the Polar 10 and Selfloops.

Consistency is Very Important

Over the course of an experiment, from first measurement to last measurement, it is important to maintain a consistent weight, diet, and level of exercise. Sizable changes in lifestyle can produce results that may well prevent the detection of any outcome resulting from a first generation senolytic therapy using the simple tests outlined here. Further, when taking any measurement, be consistent in time of day, distance in time from last exercise or meal, and position of the body. Experimentation with measurement devices will quickly demonstrate just how great an impact these line items can have.

Guesstimated Costs

The costs given here are rounded up for the sake of convenience, and in some cases are blurred median values standing in for the range of observed prices in the wild. The choice to use needles for subcutaneous injection is obviously much cheaper than exploring the world of needle-free injections and vial assembly.

• Business mailbox, such as from UPS: $250 / year
• Baseline tests from WellnessFX: $220 / test
• MyDNAage kits: $310 / kit
• Osiris Green sample kits: $70 / kit
• Omron 10 blood pressure monitor: $80
• Polar H10 heart monitor: $100
• iHeart monitor: $210
• American Weigh Gemini-20 microscale: $90
• Miscellenous equipment: spatulas, labels, vials, a vial rack, etc: $60
• Subcutaneous autoinjector for use with needle and syringe: $45
• Needles and syringes: $40
• Small pack of 13mm sterile serum vials: $35
• PharmaJet Needle-free Injection Kit: $1020
• Comfort-in Needle-free Injection Kit: $470
• Bulk 13mm serum vial parts and capping tools: $750
• 200mg of FOXO4-DRI via Alibaba: $2000
• Customs import duty on FOXO4-DRI: $150
• Shipping and LC-MS analysis of a sample: $200
• Shipping and stability profiling of a sample: $900

Practice Before Working with FOXO4-DRI

Do you think you can measure and move milligrams of powder and crystals around between containers without dropping it or otherwise losing a sizable fraction of it? Or reliably pipette fluid in 0.5ml amounts between small vials? Or cap vials or connect adaptors or fill syringes or carry out an injection without messing it up somewhere along the way? Perhaps you can. But it is a very good idea to practice first with salt and saline solution rather than finding out that your manual dexterity and methods are lacking while handling the expensive peptide. You will doubtless come to the conclusion that more tools or different tools are needed than was expected to be the case. It is possible to get by with a spatula small enough to fit into vial mouths, vials, labels, a vial rack to keep vials in place while hands are doing something else, and pipettes sized for moving small amounts of fluid. Other items may be helpful, such as suitably sized powder funnels, though there is considerable utility in a small, singly folded piece of paper for moving non-sticky powders from a measuring device to a small-mouthed container.

Schedule for the Self-Experiment

One might expect the process of discovery, reading around the topic, ordering materials, and validating an order of FOXO4-DRI to take a couple of months. Once all of the decisions are made and the materials are in hand, pick a start date. The schedule for the self-experiment is as follows:

• Day 1-10: Once or twice a day, take measures for blood pressure, pulse wave velocity, and heart rate variability.
• Day 10: Bloodwork and DNA methylation test.
• Day 11: Test a 1/10 dose of FOXO4-DRI and abandon the effort if issues are experienced.
• Day 12: Inject FOXO4-DRI.
• Day 14: Inject FOXO4-DRI.
• Day 16: Inject FOXO4-DRI.
• Day 46-55: Repeat the blood pressure, pulse wave velocity, and heart rate variability measures.
• Day 55: Repeat the bloodwork and DNA methylation test.

Note the initial low dose test. This is important, not least as a test of the ability to carry out an injection safely and competently. Consider that peptides tend to be delivered at 95% to 98% purity. It is quite possible for the remaining 2-5% of impurities in a manufactured peptide - whether there due to manufacturer error or as a natural side-effect of producing the peptide in question - to produce unwanted effects. The effects might include localized immune reactions, or the immune system becoming permanently sensitized to the peptide because of an impurity present in one dose. You will find discussions of this in the literature. If any untoward effects occur, it is wise to stop. You might refer back to the initial notes on risk and responsibility.

That said, the exact timing in the schedule is not really important, but it is a good idea to allow enough time following the end of the dosage for things to settle down. In animal studies, senolytic effects occurred fairly rapidly, as did the benefits, but allowing a few weeks of time in a human self-experiment still sounds like a good idea. Certainly it costs nothing to take that step.

Where to Publish?

If you run a self-experiment and keep the results to yourself, then you helped only yourself. The true benefit of rational, considered self-experimentation only begins to emerge when many members of community share their data, to an extent that can help to inform formal trials and direction of research and development. There are numerous communities of people whose members self-experiment with various compounds and interventions, with varying degrees of rigor. One can be found at the LongeCity forums, for example, and that is a fair place to post the details and results of a personal trial with senolytics. Equally if you run your own website or blog, why not there?

When publishing, include all of the measured data, the doses taken, duration of treatment, and age, weight, and gender. Fuzzing age to a less distinct five year range (e.g. late 40s, early 50s) is fine. If you wish to publish anonymously, it should be fairly safe to do so, as none of that data can be traced back to you without access to the bloodwork provider. None of the usual suspects will be interested in going that far. Negative results are just as important as positive results. For example, given the measures proposed in this post it is entirely plausible that positive changes as a result of present senolytic treatments in a basically healthy late 40s or early 50s individual will be too small to identify - they will be within the same range as random noise and measurement error. Data that confirms this expectation is still important and useful for the community, as it will help to steer future, better efforts.

Final Thoughts: Why Not Wait?

Given all of the cautions above, why not wait? Waiting can be a very sensible strategy. The state of senolytic therapies is progressing rapidly. New senolytics are emerging, and more data is being published for existing senolytics. At some point in the next few years, reliable direct tests for senescence will arrive on the scene, allowing a much better view of whether or not these treatments are actually achieving the claimed results. That said, it doesn't hurt to plan, and it doesn't hurt to tinker with some of the component parts of a plan. That is how we can determine whether or not it is worthwhile to experiment now versus waiting to experiment later with better tools.

A sizable fraction of the regenerative medicine community is interested in finding ways to improve existing repair systems in the body, and particularly in the central nervous system, which exhibits little ability to recover from injury in mammals. Initiatives in progress include efforts to increase the rate at which new neurons are created and integrated into the brain, work on ways to encourage more glial cells to adopt a pro-regenerative state, and the usual range of approaches based on delivering signal molecules found to be significant in stem cell therapies or heterochronic parabiosis studies. This open access review paper looks over some of the areas of present research. One of the more interesting points made by the authors is that the window of time for a successful regenerative intervention to restore function is very long, years or more. Any significant advance in the field will bring benefits to a large number of existing patients.

A stroke is caused by a sudden interruption of cerebral blood supply to a specific region of the brain, resulting in regional brain tissue death. Once a stroke occurs, brain tissue that is located inside and outside the infarct/lesion area undergoes significant changes over time. The major pathological cascades include primary neuron loss, secondary neuron loss, brain edema, neuroinflammation, dead cell removal, neuron functional reorganization, blood vessel regeneration, and neural network rewiring. Stroke represents a very serious medical condition and causes huge medical and financial burdens throughout the world. It remains the leading cause of long-term disability and the second leading cause of death worldwide.

Over the past few decades, major advances have been made in understanding of the pathophysiology of stroke, while there has not been much progress in the development of stroke treatment, especially for stroke recovery. Extensive efforts have been devoted to developing neuroprotective therapies to rescue dying neurons within the limited hours post-stroke, and this approach has been shown effective in animal models; unfortunately, the neuroprotective agents have all failed in clinical trials.

Despite the permanent brain tissue damage, spontaneous recovery occurs days, weeks, and months after stroke onset. This type of recovery occurs during the first 3-6 months after stroke with the most dramatic recovery from neurological impairments in the first 30 days. The mechanism underlying the spontaneous recovery after stroke has not been fully understood. Early recovery post-stroke is associated with resolution of edema and reperfusion of the ischemic tissue. Later recovery is related to brain plasticity. Brain plasticity is an intrinsic ability of the brain to reorganize its function and structure in response to stimuli and injuries from both internal and external sources. Brain plasticity is centered on neuronal plasticity, which is coupled with the changes of other types of cells in the brain such as astrocytes, microglia, and blood vascular cells. Convincing evidence shows that brain plasticity exists throughout a person's lifespan.

The involvement of astrocytes and microglial cells in neuroinflammatory responses during the early stage of stroke has been intensively studied. Microglia, the brain tissue resident macrophages, are classified into pro-inflammatory phenotype (M1 type) and anti-inflammatory phenotype (M2 type) based on their responses to local environment. The pro-inflammatory phenotype microglia release destructive pro-inflammatory cytokines, whereas the anti-inflammatory phenotype microglia produce molecules and trophic factors that participate in anti-inflammatory and tissue repair. M2 type microglia have shown beneficial effects in neurogenesis, axonal regeneration, angiogenesis, and vascular repair.

Neural stem cells (NSCs) or neural progenitor/precursor cells (NPCs) are multipotent cells that have the capacity for self-renewal and differentiation into neurons, astrocytes, and oligodendrocytes. Although extensive research has been done over the past decade, understanding the role of endogenous NSCs/NPCs in brain self-repair and spontaneous functional recovery after stroke still remains incomplete. The original hypothesis has been proposed that the NSC/NPC-generated new neurons may replace the stroke-damaged neurons, leading to brain self-repair and functional recovery after stroke. The vast majority of studies have been directed by this hypothesis and are searching for evidence that stroke-induced NSC/NPC proliferation, migration, differentiation, and survival/integration are linked to spontaneously functional recovery.

In total, convincing evidence supports that the brain has the intrinsic ability to repair itself, which is the foundation of spontaneous functional recovery after stroke. However, the capability of brain self-repair post-stroke is limited, especially in severe stroke, as spontaneous recovery is often incomplete in most stroke patients. Clearly, the brain needs more help for reinforcing the repair process. Can we provide extrinsic interventions or treatments to enhance the intrinsic ability of brain self-repair for further strengthening stroke recovery? Emerging evidence has renewed our knowledge on the time window for stroke recovery, which is much longer than previously thought. By contrast to the limited several-hour-effective window of thrombolytic/thrombectomy treatment, the therapeutic window of restorative/rehabilitative interventions is much broader, from years after stroke to lifelong applicability. Recognizing this unique feature of restorative approach will direct stroke research into a fruitful direction and provide great opportunities to develop more treatments for maximizing stroke recovery.

Link: https://doi.org/10.1016/j.pneurobio.2018.01.004

Researchers here make an interesting discovery in the genetics of fly aging. Old flies lose repeated DNA sequences in the genome that encode for RNA related to the ribosome, a cellular structure important in the intricate, multi-stage process by which proteins are created from their genetic blueprints. Protein creation changes in numerous ways in later life, better ribosomal function is associated with greater species longevity, and it is known that ribosomal RNA genes acquire epigenetic markers in a characteristic way with age. How exactly this all links together is yet to be determined in detail.

The more interesting part of the report here is that young flies regain lost ribosomal DNA, if they were the offspring of old parents and thus inherited a genome with few repeats of ribosomal DNA. This suggests that, whatever is going under the hood, the loss of ribosomal RNA genes is a secondary aspect of aging, driven by some other process. We might ask whether this observation in flies is relevant to mammals. It may not be, judging from the results of an older study in aged mice that examined this part of the genome and found no great losses - but that was sufficiently long ago that revisiting the topic is certainly on the agenda.

Studies in fruit flies have shown how cells in the offspring of older fathers can replace copies of genes that have been lost due to aging. The findings provide clues as to how some cells could overcome genomic shrinkage that appears to occur as an organism ages. If the same results can be confirmed in humans, they could offer a new level of understanding about how cells deteriorate with time.

The team looked specifically at ribosomal DNA (rDNA) loci that contain the genes for ribosomal RNA (rRNA). These loci are repeated at multiple sites on different chromosomes. For example, five human chromosomes contain regions in which rDNA genes are repeated hundreds of times. However rDNA is very unstable. "rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. The end result is that some copies are lost every cycle. They are popping out of the chromosome."

Studies have confirmed that in yeast cells, at least, this rDNA instability and gene copy loss underlies aging, via a process known as replicative senescence. What isn't yet known, however, is whether rDNA instability contributes to aging in multicellular organisms. To investigate this further, the researchers turned to the fruit fly, Drosophila melanogaster. Their studies looks more closely at the dynamics of rDNA loci and rDNA loss during aging in male Drosophila germline stem cells (GSCs), which continue to divide throughout adulthood. The results of their cell analyses showed that in comparison with younger male fruit flies, older males had fewer copies of the rDNA genes on the Y chromosome in their GSCs-in effect their Y chromosomes had shrunk. These older fathers then passed the reduced amount of rDNA on to their male offspring.

However, rather make do with fewer rDNA genes, the offspring were able to rebuild the number of rDNA copies in the Y chromosomes of their GSCs. By the time they had reached about 10 days of age, the sons of aged fathers had comparable amounts of rDNA to those male offspring of younger fathers that had passed on less depleted Y chromosomal rDNA. Interestingly, recovery of of rDNA copy number was limited to young adults, suggesting that the mechanisms at work might only occur under certain conditions. The results indicate that rejuvenation of rDNA in sons plays a key role in the persistence of stem cells from father to son. What isn't known yet is whether the same rebuilding of lost rDNA can also occur in female stem cells in the ovary.

Further analysis indicated that the process of rDNA copy number recovery uses the same factors that are needed for a phenomenon known as rDNA magnification, in which DNA copy number is rapidly expanded in the male germline of animals that are deficient in rDNA due to large rDNA deletions. "Our study also indicates that the phenomenon classically regarded as 'rDNA magnification' might be a manifestation of a general 'maintenance' mechanism that operates in the population that experiences normal fluctuations in rDNA copy number." The researchers suspect that mechanisms allowing cells to reset gene copy number may also be present in some types of human cells, but this has yet to be demonstrated.

Link: https://www.genengnews.com/gen-news-highlights/aging-clues-found-in-stem-cell-ribosomal-dna/81255490

Today's open access paper reports on a new senolytic drug candidate, with good-looking data on its effects on the bone marrow environment in aged mice - reducing inflammation, and improving the hematopoietic stem cell pool, among other benefits. Senolytic drugs are those that selectively destroy senescent cells. Cells become senescent constantly, but near all either self-destruct or are destroyed by the immune system. Unfortunately, a tiny fraction linger, and their behavior produces chronic inflammation and degrades tissue function in a variety of ways. Their growing presence is one of the root causes of aging, directly implicated in the progression of many age-related diseases. If, however, senescent cells could be periodically culled, safely and efficiently, this contribution to degenerative aging could be removed entirely.

In recent years, researchers have found a dozen or so senolytic compounds. These are largely well-known to the research community, and most have been tested in dozens of studies, usually for anti-cancer effects. Yet next to no-one was looking for effects on cellular senescence much before six or seven years ago, more is the pity. The best of these senolytics have since been demonstrated to clear out between 25% and 50% of senescent cells in some tissues in old mice. A few are proceeding into human studies, the first of which is a pilot conducted by Betterhumans, alongside a fair degree of quiet self-experimentation. Here is a question to consider: given this, just how many more senolytics should we expect to exist in the body of compounds that are already well explored, with good data on side-effects and pharmacokinetics in mice and humans? I think it quite likely that the number is large.

This is a good thing, because we should expect these senolytic pharmaceuticals to be quite varied in their effectiveness by tissue type. The accumulation of evidence is beginning to suggest that senescent cells have their differences, and thus any given mechanism that can tip them over into self-destruction will work well for some tissues, poorly or not at all for others. The best pharmaceutical approach to senescent cells will likely involve a mix of several different classes of compound. This is distinct from the non-pharmaceutical approaches, such as the Oisin Biotechnologies gene therapy or SIWA Therapeutics immunotherapy, that will likely be more broadly effective and reliable, capable of clearly a much higher faction of senescent cells, but at greater expense.

The compound examined here, tetramethylpyrazine, is well studied and widely used in various forms. Take a look at PubMed and you will find a flood of papers from just the past few years, as well as a lengthy period of study over the few decades prior, assessing the benefits of tetramethylpyrazine for a range of age-related conditions. Researchers think it promising for stroke, neurodegeneration, reduction of chronic inflammation, and more. Like a number of the other established compounds that have turned out to be meaningfully senolytic, it is inexpensive and widely available for purchase in the open marketplace. If a compound this well studied can turn out to be senolytic to a significant degree, what else is right underneath the noses of the scientific community, lurking in the sizable batch of promising compounds that are under evaluation at any given time? Equally, if a widely used compound can be senolytic to this degree, that should perhaps temper our expectations on the size of the gains that this approach alone can achieve in humans.

Local delivery of tetramethylpyrazine eliminates the senescent phenotype of bone marrow mesenchymal stromal cells and creates an anti-inflammatory and angiogenic environment in aging mice

During aging, bone homeostasis is interrupted with the chaos of the marrow microenvironment, including a disrupted hematopoietic stem cell (HSC) niche, decreased vessel formation and abnormal inflammation factor release. As a result, increased cellular senescence in bone marrow can be induced by cellular damage or environment changes. It is reported that senescent cells (SnCs) accumulate in bone marrow with aging and contribute to age-related pathologies through their secretion of factors contributing to the senescence-associated secretory phenotype (SASP). Although cell senescence has been well studied in recent decades, the mechanisms and local treatment targets for SnCs-induced bone degenerative disease are not well understood.

Mesenchymal stromal cells (MSCs), including mesenchymal stem/progenitor cells (MSPCs), play an essential role in bone metabolism and HSC maintenance. MSC senescence during aging markedly impairs the HSC niche, decreases osteoblast numbers and disrupts epithelial-mesenchymal transition. LepR+ cells in bone marrow were a major source of MSPCs in adult and formed bone, cartilage, and adipocytes in culture and upon transplantation. Additionally, LepR+ cells are essential for maintaining the HSC niche. However, little is known about whether LepR+ cells are senescent and dysfunctional during aging.

Tetramethylpyrazine (TMP), the bioactive component extracted from Ligusticum wallichii Franchat (Chuanxiong) which is widely used for the treatment of ischaemic stroke, cerebral infarction, and degenerative diseases of the central nervous system, has been reported to have anti-inflammatory and anticancer effects in certain cell types. In this study, we aimed to investigate the local effect of TMP on the bone marrow of aging mice and to determine whether the senescent phenotype of MSCs could be eliminated. Our findings revealed that local delivery of TMP eliminates the senescent phenotype of LepR+ MSCs via epigenetically modulating angiogenic environment in aging mice.

Senescent cell (SnC) accumulation in bone marrow with aging leads to aging-related pathologies, and local ablation of SnCs attenuates several pathologic processes and extends a healthy lifespan. In this study, we found that senescent LepR+ MSPCs accumulated in the bone marrow of aging mice with bone degeneration and that local delivery of TMP in bone marrow inhibited LepR+ MSPC senescence. In this study, we just began to understand that local elimination of senescent MSPCs in bone marrow is critical to aging-related bone degenerative change and microenvironment disruption. Identification of the local treatment for cellular senescence and the underlying mechanism of the crosstalk between SnCs and niche cells in maintaining whole bone homeostasis remain interesting for further investigation, which will provide insight into extensive clinical studies in use of local treatment for bone degenerative and regenerative applications.

The endosomal network is a complex system of many parts responsible for moving endosomes within cells. Endosomes are membrane-bound packages used to transfer material in the cell to destinations such as lysosomes, where it is broken down, or a variety of other locations. Dysfunction in the overall system of autophagy, in which wastes and broken structures are sent to the lysosome for recycling, is a feature of aging and neurodegenerative diseases in particular. The researchers here focus on failure in the endosomal network, and find a way to patch it up a little - though it is unclear as to how far removed their point of intervention is from fundamental forms of damage that cause aging. This approach appears to improve the situation in Alzheimer's disease, probably by allowing cells to somewhat better dismantle the amyloid and tau protein aggregates that are associated with the condition. It is, in any case, an interesting take on the problem of declining autophagy with aging, and may turn out to be relevant in many other tissues and conditions.

Brain tissue from people with Alzheimer's disease shows clumping of two types of proteins. One, amyloid beta, accumulates outside of brain cells; the other, called tau protein, collects within the cells. Both of these toxic proteins are thought to cause the brain cell death seen in Alzheimer's. Recent research suggests that these proteins accumulate because of a defect in the system that ferries proteins within the cell. The proteins are shipped in membrane-bound packages, called endosomes. The system that shuttles them around the cell is the endosomal network. For proteins to be properly processed, eliminated or recycled, this system must function correctly.

Researchers used human brain cells created from stem cells to investigate whether enhancing the function of the endosomal network, in a laboratory setting, would affect amyloid beta and tau protein in these human cells. The scientists tested a compound that had been shown in animal studies to stabilize and boost the function of a protein assembly called the retromer. The retromer is a key player in directing how the endosomal "packages" are shuttled about in the endosomal network to be delivered to the right destination.

The researchers found that the compound, called R33, did enhance the function of the retromer. This led to considerable reduction in the production of both the amyloid beta and the form of tau protein that readily aggregates, phosphorylated tau. The findings suggest that targeting defects in the endosomal network, through the discovery of drugs or other therapeutics, such as gene therapy, may be a promising strategy against Alzheimer's disease. "This also suggests that something upstream is affecting the production of amyloid beta and phosphorylated tau independently. So one thing we're going to work on going forward will be to identify what this upstream defect might be and whether it, too, could be a target for new therapeutics to treat Alzheimer's."

Link: https://newsroom.uw.edu/news/stem-cell-study-points-new-approach-alzheimers-disease

The heart is not a very regenerative organ in mammals, its cells comparatively reluctant to multiply to make up losses or repair injuries - and mammals are a good deal less regenerative than many other species. Zebrafish can regenerate entire missing sections of the heart to completely restore normal function without scarring, for example. Is it possible for the biochemistry of mammals to be adjusted so as to approach this feat? If so, this could make a sizable difference to the trajectory of heart disease and heart failure in later life, even though it doesn't address the root causes of age-related cardiovascular disease. Researchers here report on an important step in this direction, inducing replication in heart muscle cells, and showing that their approach results in significant regeneration in rodents.

In the embryo, human heart cells can divide and multiply, allowing the heart to grow and develop. The problem is that, right after birth, cardiomyocytes (heart muscle cells) lose their ability to divide. The same is true for many other human cells, including those of the brain, spinal cord, and pancreas. "If we could find a way to get these cells to divide again, we could regenerate a number of tissues." For decades, the scientific community has been trying to do just that, with limited success. Until now, attempts have been ineffective and poorly reproducible.

Researchers have now developed the first efficient and stable method to make adult cardiomyocytes divide and repair hearts damaged by heart attacks, at least in animal models. The team identified four genes involved in controlling the cycle of cell division, these being cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1. They found that when combined - and only when combined - these genes cause mature cardiomyocytes to re-enter the cell cycle. This results in the cells dividing and rapidly reproducing.

The scientists tested their technique in animal models and cardiomyocytes derived from human stem cells. They used a rigorous approach to track whether the adult cells were truly dividing in the heart by genetically marking newly divided cells with a specific color that could be easily monitored. They demonstrated that 15-20 percent of the cardiomyocytes were able to divide and stay alive due to the four-gene cocktail. "This represents a considerable increase in efficiency and reliability when compared to previous studies that could only cause up to 1 percent of cells to divide."

To further simplify their technique, the team looked for ways to reduce the number of genes needed for cell division while maintaining efficiency. They found they could achieve the same results by replacing two of the four genes with two drug-like molecules. The researchers believe that their technique could also be used to coax other types of adult cells to divide again, given that the four genes they used are not unique to the heart.

Link: https://gladstone.org/about-us/press-releases/unlocking-cell-s-potential-regenerate-heart

This three part interview covers the induction of greater numbers of free radicals in tissues as an approach to slow aging. I can't say as I think this is a way to obtain large gains in health and longevity, much greater than those possible through exercise and calorie restriction. Both of those approaches essentially work in a similar way, being beneficial stress responses that include free radical signaling among their mechanisms. Little of the work on recreating these responses via pharmaceutical or genetic means does all that much better in terms of extended healthy life. The background is quite interesting, however.

Over the decades, the scientific understanding of the role that reactive molecules, free radicals, play in aging and metabolism has become much more nuanced. The original formulation of the free radical theory of aging, in which more free radicals are always a bad thing, is clearly not correct and the field has moved on since then. The situation is much more complicated than the presence of free radicals being a straightforward form of damage, reacting with important molecules to break their function. Yes, that breakage happens, constantly, but it is near entirely repaired. Yes, any circumstance that produces very large amounts of free radicals, far more than are produced normally, such as ionizing radiation exposure, is directly harmful (but not all that relevant to what happens during the aging process). Yes, greater amounts of oxidative free radicals and chronic inflammation, which is known to be harmful, go arm in arm.

Nonetheless, it is the case that cells use free radicals as signals in beneficial processes. Mitochondria, the power plants of the cell, are the primary source of free radicals. These free radicals are produced in the progress of producing chemical energy stores to power cellular operations, a necessarily energetic process with a range of byproducts. The benefits of exercise rely upon an increase in free radicals produced by mitochondria. Other forms of mild cellular stress work in a similar way, requiring mitochondrial free radicals in order to instruct the repair systems of the cell to get to work. Numerous methods of modestly slowing aging in short-lived laboratory species involve tinkering with mitochondria to somewhat increase their output of free radicals, and therefore produce a net benefit in cellular maintenance due to increased repair activities.

An adaptation of the original free radical theory of aging, the mitochondrial free radical theory of aging, suggests that mitochondria are both the primary source and the primary important point of damage for reactive molecules. Mitochondrial DNA, distinct from the DNA of the cell nucleus, becomes broken in ways that cause cells to become overtaken by malfunctioning mitochondria. That growing group of faulty cells exports damaged proteins and other molecules to tissues and bloodstream to contribute to degenerative aging. Clearly, given the way in which free radicals can produce benefits, this process of mitochondrial DNA damage has to be somewhat disconnected from the mechanisms of gain through cellular stress - these days some researchers question whether it is free radicals causing the DNA damage, and point instead to replication errors.

Free radicals are not your enemy | An interview with Dr. Michael Ristow (part i)

The "free radical theory of aging" - where is that theory at now?

It's dead. Well, let's be a bit more precise. The free radical theory of aging dates back to the 1950s, and then there were decades of research on it, which was all very good research scientifically speaking, but it was always in artificial settings with high doses of free radicals that never occur in real life. In real life, in healthy model organisms or humans, free radicals occur in very low doses, and they have very different functions from high doses of free radicals, where they serve as signaling molecules that increase our body's defense mechanisms against external stressors. So in the 1990s, evidence emerged that small doses of free radicals serve as signaling molecules in cells. Around 2006 or 2007 we showed that in C. elegans that we could increase free radical production and that would make the worm live longer.

If normal amounts of free radicals aren't harmful, what's the story with antioxidants?

Well, this was an important issue we looked at a bit later in 2009. It's also well known that exercise produces free radicals, which was already surprising because exercise is probably the most healthy intervention a person can use, and that conundrum lead us to the hypothesis that the increase in free radicals would explain the health-promoting effects of exercise. And we tested this by seeing whether the antioxidant supplementation would kill the effects of exercise in humans, and that was exactly the case - the guys who got the placebo showed the expected effects of exercise on metabolism, and the guys who got the antioxidants had almost none of the effects.

About six or seven years later, another group had even more data available from the public domain, and they found that antioxidants increase all mortality. Meanwhile, experimental groups have shown that antioxidant supplementation in mice increases cancer and metastasis rates. The evidence out there is very straightforward and very bad for the antioxidant industry, but it's widely ignored. Consumption has not decreased for years, but at least it's not increasing.

Glucosamine: The new metformin? | Interview with Dr. Michael Ristow (part ii)

So we've discussed this effect called hormesis, where substances that are toxic at high doses can actually be helpful in low doses. There's a related word that pops up a lot in your research, "mitohormesis" - what is that?

It's an abbreviation of mitochondrial hormesis, and it essentially translates this hormesis principle, which normally applies to compounds and drugs, to whatever comes out the mitochondria. So mitochondria send out signals that promote health and lifespan at low doses, and at higher doses these signals do the opposite. The most well-established signal from the mitochondria is reactive oxygen species (ROS), but there are also other signals.

Does free radical production become a problem as mitochondria get older and start producing more free radicals?

While older mitochondria do produce more free radicals, it's unclear whether that really accelerates aging. I think it does at very high, artificial doses. For example, one very artificial mouse model showed that mice with a mutation in their mitochondria aged horribly. For human examples, you can look at the hundreds of mitochondrial diseases that cause premature aging and increased cancer and so on, but again, these are very rare. There wasn't much evidence about the effect of mitochondrial ROS production on normal aging until recently. A paper looked at naturally occurring mutations in mice and compared their lifespans, and, contrary to their expectations, they found that the mice producing more ROS lived longer than the mice producing less ROS. And these mice were otherwise genetically identical. So I think that fits in well with the results we've seen with exercise.

You're a big proponent of a particular intervention that plays off of mitohormesis to increase lifespan - glucosamine.

Back in 2007 when we showed that increased ROS extends lifespan in C. elegans, we used a compound that completely blocks glucose metabolism, deoxyglucose. Since the cell can't metabolize glucose anymore, it enters an energy deficit similar to starvation, and responds by switching on its mitochondria. It turned out to be toxic in mice. Then a student in my lab said, "Why don't we use glucosamine?" Glucosamine only slightly inhibits glucose metabolism (glycolysis), and it's known to be completely harmless to humans. It's like the cell being on a diet: it still activates its mitochondria, still produces a bit more ROS, but not to the excessive level that it would with deoxyglucose. We took two year old mice, which is equivalent to something like 55 or 60 in humans, and gave them glucosamine, which caused both males and females to live longer. The effect was stronger in females, but it was independently detectable in both sexes.

Do you think there will be a trial for glucosamine akin to the TAME trial for metformin?

There should be. I think it's long overdue. It's an ideal supplement because it's cheap, there's no intellectual property attached to it, and it could improve healthspan significantly at almost no cost. The return on investment for both insurers and individuals would be significant. The evidence for glucosamine in C. elegans and mice is about the same as for metformin.

An ounce of prevention | Interview with Dr. Michael Ristow (part iii)

Are there compounds besides glucosamine that you find promising as geroprotectors?

Both my lab and others have been working on lithium, which is found in drinking water, and we've both seen it extend lifespan in C. elegans. Then we did a study with some Japanese colleagues who had data on lithium concentrations in drinking water all over Japan, and they worked out that areas with more lithium in the drinking water lived longer on average, so there was a positive correlation between lithium concentration and lifespan. And then another study from Texas came out a month or two ago saying the same thing, so there's several lines of very independent evidence that all points in the same direction. So that could have a pretty direct impact on supplementation. Low-dose lithium is something people could start to think about incorporating.

And can you tell us what motivates your research?

Well, first of all, I always like to question established concepts. That's probably the most driving source, especially when it's evident that something is wrong and no one is discussing it - like the common wisdom around reactive oxygen species, for instance. So that's one side of things. The other is that I'd ideally like to work on something that impacts quality of life for humans. That's quite distant from questioning fundamental concepts, but on certain occasions they come together - for instance, questioning antioxidants, or finding safe, easily available compounds like glucosamine that could make a huge difference. Of course, there's also increasingly good evidence that preventing age-related diseases while people are still healthy is so much smarter than treating existing diseases later on, and that's a very unique win/win situation. It's one of the rare cases where tremendous increases in individual quality of life and decreases in socioeconomic costs coincide, because normally it's either one or the other. And so I think it's almost mandatory to work on something like that.

What are the biggest challenges you see ahead?

In Western medicine nowadays, you only start taking care of health once it's gone, and I think that approach is totally wrong. That perception will have to change. Instead we need to prevent diseases from occurring in the first place, and people can do that by eating healthily, and exercising, and so on. And individuals are willing to take care of that - but only to a limited extent. We could make the success rate much higher if we came up with a drug or combination that mimicked certain aspects of healthy lifestyle, rather than requesting that everyone follow all of these rules their whole lives. Because the majority of us are simply not willing or capable of following them at all times, and I think that's just human nature. I'm not endorsing it, but I think it's a matter of fact.

Telomerase gene therapy as a treatment for aging is a popular topic these days, given the results in mice from past years, though I still think that more work needs to be done in mammals other than mice to address concerns related to cancer risk and effectiveness. Mice have telomere and telomerase dynamics that are quite different from those in humans, and the details of those differences might turn out to be important in the balance between greater stem cell activity and greater risk of cancer resulting from the activity of age-damaged cells. It is not unreasonable to think that adding a given amount of telomerase to cells might be good, bad, or neutral to varying degrees on a species by species basis.

Telomerase therapies are thought to work because telomerase lengthens telomeres, among other possible activities, and thus causes cells to undertake more replication and other activity than they would otherwise have done. This is particularly the case for the stem cell populations responsible for tissue maintenance. That tissue maintenance normally declines with age, an evolved reaction to rising levels of molecular damage that serves to reduce cancer risk at the cost of a slow failure of tissue function. The research here is early stage, but it suggests there might be ways to produce a telomerase therapy that works by making existing telomerase more efficient at lengthening telomeres, rather than by adding more telomerase. That means it might also be a way to find out whether the other, less well studied activities of telomerase are at all important in the observed results in animal studies of telomerase gene therapy.

Typical human cells are mortal and cannot forever renew themselves. As demonstrated a half-century ago, human cells have a limited replicative lifespan, with older cells reaching this limit sooner than younger cells. This "Hayflick limit" of cellular lifespan is directly related to the number of unique DNA repeats found at the ends of the genetic material-bearing chromosomes. These DNA repeats are part of the protective capping structures, termed "telomeres," which safeguard the ends of chromosomes from unwanted and unwarranted DNA rearrangements that destabilize the genome. Each time the cell divides, the telomeric DNA shrinks and will eventually fail to secure the chromosome ends. This continuous reduction of telomere length functions as a "molecular clock" that counts down to the end of cell growth. The diminished ability for cells to grow is strongly associated with the aging process, with the reduced cell population directly contributing to weakness, illness, and organ failure.

Telomerase lengthens telomeres by repeatedly synthesizing very short DNA repeats of six nucleotides - the building blocks of DNA - with the sequence "GGTTAG" onto the chromosome ends from an RNA template located within the enzyme itself. However, the activity of the telomerase enzyme is insufficient to completely restore the lost telomeric DNA repeats. Understanding the regulation and limitation of the telomerase enzyme holds the promise of reversing telomere shortening and cellular aging with the potential to extend human lifespan and improve the health and wellness of elderly individuals. Researchers recently uncovered a crucial step in the telomerase catalytic cycle that limits the ability of telomerase to synthesize telomeric DNA repeats onto chromosome ends. "Telomerase has a built-in braking system to ensure precise synthesis of correct telomeric DNA repeats. This safe-guarding brake, however, also limits the overall activity of the telomerase enzyme. Finding a way to properly release the brakes on the telomerase enzyme has the potential to restore the lost telomere length of adult stem cells."

This intrinsic brake of telomerase refers to a pause signal, encoded within the RNA template of telomerase itself, for the enzyme to stop DNA synthesis at the end of the sequence 'GGTTAG'. When telomerase restarts DNA synthesis for the next DNA repeat, this pause signal is still active and limits DNA synthesis. Moreover, the revelation of the braking system finally solves the decades-old mystery of why a single, specific nucleotide stimulates telomerase activity. By specifically targeting the pause signal that prevents restarting DNA repeat synthesis, telomerase enzymatic function can be supercharged to better stave off telomere length reduction, with the potential to restore the activity of aging human adult stem cells.

Link: https://asunow.asu.edu/20180223-discoveries-asu-scientists-unveil-immortality-enzyme-telomerase

In cryopreservation of tissues, ice is the primary enemy. Ice crystals destroy cell structures, both during freezing, and again during thawing. Ice is the reason why a great deal of effort has gone into the development of cryoprotectant chemicals that enable vitrification, a low-temperature state with minimal ice formation. Vitrification is what makes it possible for the cryonics industry to store patients while doing their best to preserve the fine structure of the brain in which the data of the mind is encoded. There is an enormous difference in damage between a frozen brain and a vitrified brain.

There is still much that can be done to improve cryoprotectants and vitrification of tissues. For example, thawing remains a challenge - reversible vitrification is a near term goal for organ storage, a capability that will revolutionize the logistics of organ transplantation, but has yet to be achieved in more than proof of principle demonstrations. Researchers have made some inroads in the past few years towards the production of better methods of thawing that can minimize ice crystal formation, such as through the use of nanoparticles. The researchers noted here have a different, small molecule approach to the problem, and are claiming to be able to effectively disrupt ice crystal formation. If validated, this may be a big deal for reversible vitrification, for the transplant industry, and ultimately for the cryonics industry and their efforts to save lives.

Everyone knows that freezing things is an imperfect process. Take frozen food, for example - most have experienced those frozen ice crystals that change the texture and taste of their favourite meal. The medical field experiences a similar problem when freezing cells (stem cells) and tissues, except the result is cellular death or lower quality. Two investigators founded a startup company, PanTHERA CryoSolutions, to commercialize a revolutionary product for the cryopreservation, or freezing, of cells and tissues, resulting in better quality cells for cellular therapies and superior products.

"Cryopreservation is a common strategy, but the technology that was developed to do it is 70 to 80 years old. With current technology, when you freeze something, you get a large amount of cell death that occurs, so you don't recover all those cells. In addition, we're actually adversely affecting the functional capacity of those cells." The process that causes the majority of this cellular damage and death is called ice recrystallization. PanTHERA CryoSolutions has discovered a small molecule inhibitor that prevents ice recrystallization - something that none of the current cryoprotectants available on the market can do - making it a unique technology. "Our technology uses small molecular structures that have the ability to inhibit the ice recrystallization process. They actually prevent that damage from occurring, so when we thaw that product, it's a superior product and it's also functional."

The proof of concept for this technology was established with a project that looked at hematopoietic stem cells. "The results have clearly indicated that this ice recrystallization inhibitor technology really works and makes a superior product where we get faster engraftment and increased incidence of engraftment, which is exactly what you want in a clinical setting." PanTHERA CryoSolutions aims to have a product commercially available in 2018 for a specific therapy, but the founders see the potential to apply this technology to many areas, including cellular therapies, regenerative medicine, reproductive biology, and 3-D bioprinting applications.

Link: http://canadianglycomics.ca/radical-new-technology-prevents-freezer-burn-in-cells-tissues/