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Contents

• Rejuvenation of Immune Function is One of the More Important Outcomes to Engineer through the Treatment of Aging
• Loss of Autophagy in Hematopoietic Cells Contributes to Osteoporosis
• NOXO1 Knockout as an Example of Counterintuitive Results in Animal Longevity
• Galactose Conjugation Makes Navitoclax a Safer Senolytic Drug
• A TAT Peptide Based Approach to Upregulation of Proteasomal Activity
• Olympic Athletes Have a Lower Mortality than the General Population
• There Are Many Ways to Influence Known Longevity Pathways
• CCN2 Inhibition Reverses Fibrosis in Overuse Injury
• Engineered Stem Cells Survive Longer and Improve Outcomes in a Heart Patch
• The Autophagy-Lysosomal Pathway and Protein Aggregation in Neurodegenerative Disease
• Correlating Autonomic Nervous System Aging and Cognitive Impairment
• The Beneficial Metabolic Adaption Provoked by Consistent Exercise
• Intermittent Fasting Increases Neurogenesis in Mice
• Transcriptomic Analysis of Microglia in Mice Shows Greater Inflammatory Activity with Advancing Age
• Soluable α-klotho Reduces Cardiac Fibrosis in Mice

Rejuvenation of Immune Function is One of the More Important Outcomes to Engineer through the Treatment of Aging
https://www.fightaging.org/archives/2020/04/rejuvenation-of-immune-function-is-one-of-the-more-important-outcomes-to-engineer-through-the-treatment-of-aging/

One would hope that it does not require an ongoing pandemic and related hysteria to point out that old people have poorly functioning immune systems, and thus suffer disproportionately the burden of infectious disease. But perhaps it does. The 2017-2018 seasonal influenza, a modestly more severe occurrence of something that happens every year, killed something like 60,000 people in the US alone, with little notice or comment. There is nothing so terrible that it won't be accepted - ignored, even - if it is normal.

Floodgates of funding for infectious disease research and development have been opened in response to COVID-19, and while no doubt all too little of it will be spent wisely or usefully (public funding being the very definition of waste and corruption) it has certainly prompted many groups to try to position themselves to benefit. Those who have, all along, been working on ways to try to make older people more resilient via improvement in their immune function are perhaps more deserving than others, but it really isn't the case that much of this work is closer than five to ten years away from practical realization and completed human trials.

Of the ways to restore immune function in the old, the worst are the small molecule drugs that show signs of adjusting metabolism in the right direction. For example, the mTOR inhibitors that just failed a phase III trial for reducing influenza incidence in the old. Better drug or drug-like approaches are those that target regrowth of the atrophied thymus. The thymus is where T cells of the adaptive immune system mature, and the production rate is reduced to a trickle in the old - a major cause of immune aging. In humans, there is data for the growth hormone approach of Intervene Immune, and better data for sex steroid ablation, to restore the production of T cells.

Further, regeneration of lymph nodes, vital to coordination of an immune response, and regeneration of the hematopoietic stem cell population that creates all immune cells will be beneficial - but existing approaches to these challenges are by no means close to readiness for clinical trials. Selective destruction of malfunctioning, senescent, and exhausted immune cells is also likely to be beneficial - but only removal of senescent cells via senolytic therapies is a very near term prospect at the present.

At the end of the day, therapies capable of making a 70 year old exhibit the immune profile and response of a 40 year old would be transformative. The world has come to accept that sizable numbers of older people die from infectious disease every year, and that this is set in stone and little can be done about it. That is simply not the case - a great deal can be done about it. It just requires the will and funding to move ahead with the most plausible programs of immune rejuvenation.

It is worth noting that the pandemic statistics referenced in today's open access paper require some interpretation and none should be taken either at face value or as usefully applicable across the board. Context is everything. Testing for COVID-19 is presently very selective for symptomatic, more severe cases. No-one yet has a good grasp on how many mild cases there are, and that is everything for determining actual mortality risk. Further, circumstances such as an enclosed cruise liner are not representative of the way matters progress in the broader population. And so on.

Geroprotective and senoremediative strategies to reduce the comorbidity, infection rates, severity, and lethality in gerophilic and gerolavic infections

Aging is a complex, multifactorial process that leads to loss of function and is the primary risk factor for major human pathologies including cancer, diabetes, cardiovascular disorders, and neurodegenerative diseases. Although there is still much debate in the scientific community, proposals have been made to classify aging as a disease in order to develop therapeutic strategies to prevent or delay the onset of age-related illnesses. Increasing frailty with age leads to an increased risk of many diseases. These diseases are commonly referred to as age-related. Many pathogens are more infectious and prevalent in the elderly, and may be referred to as gerophilic (from Greek, géros "old man" and philia, "love"). Some infections, including COVID-19, are not exclusively gerophilic, as younger people may also become infected. However, these individuals have mild symptoms or remain asymptomatic, while the elderly experience substantially more severe symptoms and lethality. The term gerolavic (from Greek, géros "old man", and epilavís, "harmful") may more appropriately describe infections that cause the most harm in the elderly.

Statistics from the COVID-19 pandemic indicate that COVID-19 is a gerolavic infection, one that disproportionately affects the elderly. According to Worldometers, an online resource aggregating data on COVID-19, of the 139,580 people infected worldwide as of March 13, 2020, 70,733 patients had recovered and 5,120 had died. Based on these data, the mortality rates (number of deaths/number of cases), or the probability of dying if infected by the virus, were determined to be 3.6% for individuals aged 60-69, 8% for individuals aged 70-79, and 14.8% for patients aged 80 years or older. The majority of the infected population are 50 and older, while the majority of fatalities are 60 and older.

An open coronavirus analysis project provides further insight into the mortality rates of COVID-19, specifically using data from the Diamond Princess Cruise, where all passengers were exposed to SARS-CoV-2 for an extended period. Of the approximately 1,690 passengers over 65 years of age, 7 passengers died, suggesting a death rate of 0.41%. This death rate is approximately 4.3 times higher than that of influenza. As more countries start reporting statistics, these death rates are likely to be adjusted. These statistics indicate that the infectivity of SARS-CoV-2, and the severity and lethality of COVID-19, are age-related.

One of the possible causes of the age-associated increases in COVID-19 infection rate, severity, and lethality is immunosenescence. Immunosenescence is a well-known age-related process contributing to the global burden of disease. Among the factors contributing to immunosenescence is the chronic involution of the thymus with increased age. Indeed, the infection rates of COVID-19, separated by age, are correlated with involution of the thymus. The thymus gland is most active early in life, reaching maximum size within the first year. Its activity then declines with age until an individual reaches 40 to 50, after which there are negligible traces of the thymus remaining, replaced by fibrotic tissue. As a result of thymic involution, the number of naïve T cells exiting the thymus decreases significantly, with substantial declines in older age.

Age-associated immunosenescence leads to a reduced ability to resist infection, while infection produces biological damage and loss of homeostasis. This ultimately contributes to accelerated aging and the development of age-related diseases, and further accelerates immunosenescence. In support of this model, infections and other age-related diseases are among the main causes of death in the developed world and in developing countries.

Due to the gerolavic nature of COVID-19, the classical preventative measures and treatment strategies used for targeting infectious diseases may not be as effective, and there is a need for alternative geroprotective and senoremediative strategies. Here we compare the expected benefit of treatments for elderly populations (60 years and older) that are currently in development, including standard preventative strategies such as vaccines and antivirals targeting SARS-CoV-2, and the potential added benefit of speculative geroprotective strategies such as rapalogs, NAD+ boosters, senolytics, and stem cell treatment. These additional measures may be used in isolation or as adjuvant therapies to reduce infection risk, symptom severity, or improve vaccine efficacy.

Therefore, interventions that enable immunocompromised elderly to mount an immune response to newly developed vaccines are necessary to help eradicate the disease and reduce the associated mortality. To avoid substantial loss of life and quality of life, primarily among the elderly and vulnerable populations, governments and healthcare systems should investigate preventative and intervention strategies stemming from recent advances in aging research. As discussed in this paper, small clinical studies have shown that several geroprotective and senoremediative interventions, such as treatment with sirolimus and rapalogs, can induce immunopotentiation, increase resistance to infection, and reduce disease severity in the elderly, without severe side effects.

Many of these predicted geroprotectors are available as supplements; however, no meta-analysis or metaclinical trials have been performed at scale to evaluate their effectiveness. The COVID-19 pandemic highlights the paucity of clinical trials on the effects of dietary supplements and drugs on aging and immunosenescence. The existence of pseudoscience and anecdotal promotion in the supplement industry does not mean that protective compounds do not exist. Dietary supplement vendors and pharmaceutical companies need to actively engage in preclinical and clinical research to evaluate the effectiveness of the currently available products on immunosenescence and aging.

Loss of Autophagy in Hematopoietic Cells Contributes to Osteoporosis
https://www.fightaging.org/archives/2020/04/loss-of-autophagy-in-hematopoietic-cells-contributes-to-osteoporosis/

Today's open access paper is an example of one of the less well known connections between processes of aging. Loss of efficiency of the cellular maintenance processes of autophagy is a characteristic of cells in old tissues. Here, researchers note that this dysfunction in the hematopoietic cells responsible for creating blood and immune cells also results in structure changes in bone marrow that contribute to the development of osteoporosis, the loss of bone mass and strength that occurs with age. Just because such connections are obscure doesn't mean that they are unimportant.

Osteoporosis is the outcome of an imbalance between osteoblasts that create bone and osteoclasts that break down bone tissue. Both cell populations are constantly active throughout life; bone is a dynamically remodeled tissue. With age a number of processes lead to greater osteoclast activity - easy enough to point out, but difficult to pick apart the causes and decide on a point of intervention. Cellular senescence is a contributing factor, of course, as it contributes to near all age-related conditions. Here, reduced autophagy is proposed to interfere in maintenance of the osteoblast population.

There are a few types of autophagy, involving different ways of identifying and moving unwanted proteins and structures to be engulfed by a lysosome. A lysosome is a membrane-wrapped package of enzymes that can break down most of the molecules a cell will encounter. Exactly why autophagy declines with age is an interesting question. A great many papers cover what is known of proximate causes, changes in expression of protein machinery that regulates or is needed by different parts of the process, all of which seem to fail in their own way, as well as the accumulation of persistent metabolic waste in lysosomes, molecules that cannot be broken down by our biochemistry as it stands. The latter is fairly straightforward, but the changes in regulation and expression of proteins are ever a challenge to chase back to their underlying cause.

Deterioration of hematopoietic autophagy is linked to osteoporosis

Previous studies on osteoporosis overwhelmingly focused on the etiology within bone tissue that locally induces the disease. In this study, we showed that osteoporosis is highly associated with reduction in hematopoietic autophagy activity in humans. We showed that an autophagy defect in the hematological system leads to severe bone loss. The disturbed osteocyte homeostasis is apparently caused by impaired type H blood vessels and possibly an aberrant alteration in the extracellular matrix (ECM) pathways that govern osteocyte homeostasis in hematopoietic autophagy-defective mice. Our results thus suggest that autophagy in the adjacent hematopoietic cells is essential to maintain bone homeostasis, and chronic hematopoietic autophagy deficiency can result in the development of osteoporosis in both mice and humans

While the osteal impact on hematopoiesis, in particular on the formation of bone marrow hematopoietic stem cell niches, is well documented, studies of hematopoietic regulation of osteocyte function have been inadequate. Hematopoietic regulation of osteoblast proliferation and differentiation was previously discussed largely with skepticism speculation. However, a recent study showed that loss of the hematopoietic stem cell factor GATA2 in the osteogenic lineage impairs trabecularization and mechanical strength of bone. Our present study of RNA sequencing revealed that impairment of hematopoietic cells by autophagy defect also led to enhanced iron activity, which may eventually lead to iron overload, a major cause of osteoporosis.

Hematopoietic cells and osteocytes are adjacent in the bone marrow niche environment. Normal hematopoiesis and bone homeostasis are interdependent. Men with low bone mineral density (BMD) or greater BMD loss have decreased circulating erythrocytes and lymphocytes and increased myeloid cells, and anemia or low blood cell counts are associated with declining BMD or increased fracture risk in the aged population. On the other hand, chronic disorders affecting hematopoiesis, such as sickle cell anemia and thalassemia, demonstrate clear skeletal phenotypes, including bone loss and increased fracture risk.

Bone marrow HSCs have been found to be capable of differentiating to osteoblasts through a mesenchymal intermediate. These findings suggest that the developmental capacity of HSCs is not restricted to hematopoietic lineages, but extends to osteogenic differentiation, possibly via the HSC potential to transdifferentiate to osteocytes. HSCs do not rest passively in their niche, but instead directly participate in bone formation and niche activities. Therefore, HSC functions and bone turnover are coupled in osteoporosis.

Finally, screening for expression of selected genes and an immunohistological assay identifies severe impairments in H vessels in the bone tissue, which results in disconnection of osteocytes from hematopoietic cells in the autophagy-defective mice. We therefore propose that hematopoietic autophagy is required for the integrity of H vessels that bridge blood and bone cells and that its deterioration leads to osteoporosis.

NOXO1 Knockout as an Example of Counterintuitive Results in Animal Longevity
https://www.fightaging.org/archives/2020/04/noxo1-knockout-as-an-example-of-counterintuitive-results-in-animal-longevity/

The balance between oxidative damage and adaptive responses to oxidative damage appears important in the way in which the operation of metabolism determines natural variations in longevity. Not as important as the factors that determine order of magnitude differences in life spans between species, but important enough to cause 10-20% changes in life expectancy in mice when manipulated, for example. That is the case in this study, in which researchers assess what appeared to be a damaging genetic alteration, a knockout of the NOXO1 gene associated with production of reactive oxygen species that has detrimental effects on intestinal function, and found that mice lived 20% longer or so as a result.

There have been a lot of surprising results in the manipulation of oxidative metabolism. It is a very complex system. Oxidative molecules are generated by mitochondria or during inflammation, and they both damage cellular components and act as signals to provoke greater cellular maintenance activities, or other tissue functions such as the adaptive growth of muscle in response to exercise. Too much oxidative damage to the point of overloading antioxidant and repair systems is directly harmful. Too little oxidative damage can also be harmful in the long term because too little cellular maintenance takes place. It is hard to predict how a reactive biological system of countless interacting component parts will behave when one part is broken or enhanced.

This particularly study may also be an example of the point that the effects of calorie restriction are large in comparison to most other studied mechanisms. Because NOXO1 knockout degrades intestinal function, these mice weigh less, and may thus be benefiting more from a reduced calorie intake than they lose from a suboptimal oxidative biochemistry. Though the usual signs of the calorie restriction response appear absent - the researchers did do some digging on this topic. All in all, investigating the complexities of metabolism is a challenging business; given the modest size of the effect observed here, this particularly line of work is unlikely to be worth the effort from anything other than the purely knowledge-driven perspective.

NoxO1 Knockout Promotes Longevity in Mice

According to the free radical theory of aging, reactive oxygen species (ROS) have been proposed to be a major cause of aging for a long time. Meanwhile, it became clear that ROS have diverse functions in a healthy organism. They act as second messengers, and as transient inhibitors of phosphatases and others. In fact, their detrimental role is highly dependent on the context of their production. NADPH oxidases (Nox) have been discovered as a controllable source of ROS. NoxO1 enables constitutive ROS formation by Nox1 by acting as a constitutively active cytosolic subunit of the complex. We previously found that both Nox1 and NoxO1 were highly expressed in the colon, and that NoxO1-/- deficiency reduces colon health. We hypothesized that a healthy colon potentially contributes to longevity and NoxO1 deficiency would reduce lifetime, at least in mouse.

In contrast, here we provide evidence that the knockout of NoxO1 results in an elongated life expectancy of mice. No better endothelial function, nor an improved expression of genes related to longevity, such as Sirt1, were found, and therefore may not serve as an explanation for a longer life in NoxO1 deficiency. Rather minor systemic differences, such as lower body weight occur. One effect of low body weight might be the expression and activity of Sirt1 and PGC1α, which are upregulated in response to caloric restriction. However, neither Sirt1 nor PGC1α was upregulated in NoxO1 deficient organs. Accordingly, the Sirt1 downstream target eNOS was not differentially activated in NoxO1-/- mice. We conclude that Sirt1 is not the preliminary effector to prolong the lifespan of NoxO1-/- mice, when compared to wildtypes. Although not significantly different from wildtype mice, low body weight in NoxO1 mice may have other beneficial effects.

As a potential reason for longer life, we suggest better DNA repair capacity in NoxO1 deficient mice. Although final fatal DNA damage appears similar between wildtype and NoxO1 knockout animals, we identified less intermediate DNA damage in colon cells of NoxO1-/- mice, while the number of cells with intact DNA is elevated in NoxO1-/- colons. We conclude that NoxO1 deficiency prolongs lifetime of mice, which correlates with less intermediate and potentially fixable DNA damage at least in colon cells.

Galactose Conjugation Makes Navitoclax a Safer Senolytic Drug
https://www.fightaging.org/archives/2020/04/galactose-conjugation-makes-navitoclax-a-safer-senolytic-drug/

If given a way to more effectively target senolytic drugs towards senescent cells, reducing off-target effects, then navitoclax is a good drug to test with. Navitoclax is arguably the worst of the first set of drugs found to be meaningfully senolytic; it certainly compares unfavorably with dasatinib. While navitoclax can kill a usefully large fraction of senescent cells in aged tissues, the dose required for that outcome will also kill a lot of normal cells along the way. More unpleasant drugs have since been discovered to be usefully senolytic, such as cardiac glycosides, but there is much less data on their senolytic use at this stage. In comparison, a good deal of data exists for the ability of navitoclax to destroy senescent cells. Its unpleasant side effects are quantified as a result of its development as a potential chemotherapeutic.

Finding ways to target highly toxic drugs to specific cell populations is a well established line of work in the cancer research community. Even if it were not the fact that many senolytic drugs were initially characterized for use as cancer therapeutics, it would not be surprising to find analogous cell targeting research taking place in the field of senolytics. This is a well understood approach to making toxic drugs more useful.

You may recall that researchers have been exploring the possibility of conjugating toxic drugs with galactose, producing a far less harmful prodrug molecule. Senescent cells are characterized by the high expression of senescence-associated β-galactosidase. This enzyme acts to remove the galactose from the prodrug, restoring the original toxic molecules that then kill the cell. In today's open access paper, an variant on this approach is demonstrated to improve the characteristics of navitoclax as a senolytic drug.

Galacto-conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity

Recent research has identified targetable vulnerabilities of senescent cells that can be exploited by a novel group of drugs called senolytics. These compounds preferentially kill senescent cells by different mechanisms. Senolytics include the BCL-2 family inhibitors Navitoclax (ABT-263) and ABT-737; the flavonoid fisetin; combinations of tyrosine kinase inhibitors and flavonoids (e.g. dasatinib and quercetin); FOXO4-p53 interfering peptides; HSP90 chaperone inhibitors; and other compounds such as piperlongumine and cardiac glycosides. Senolytics have emerged as promising agents for treatment of pulmonary fibrosis, atherosclerosis, osteoarthritis, type 1 diabetes mellitus, type 2 diabetes mellitus, and neurocognitive decline. They can also rejuvenate aged hematopoietic stem cells and muscle stem cells and extend the lifespan of naturally aged mice.

Despite successful preclinical proofs-of-concept for senolytics, their potential translatability is hampered by their associated toxicities, necessitating the development of more specific, and less toxic, second-generation senolytics. Navitoclax has been validated in a variety of preclinical models showing high potency in killing senescent cells - however, it also has significant on-target haematological toxicity, including thrombocytopenia.

One consistent feature of senescent cells is their enrichment in lysosomes and lysosomal proteins, including senescence-associated β-galactosidase (SA-β-gal) which is widely used as a marker of senescence. We previously showed that the encapsulation of nanoparticles with galacto-oligosaccharides (GalNPs) is an efficient method to preferentially deliver cytotoxic drugs and tracers to the lysosomes of senescent cells where SA-β-gal activity digests the galacto-oligosaccharides, thereby releasing the cargo. We have also shown that a fluorescent probe covalently linked to multi-acetylated galactose is preferentially digested by senescent cells, releasing the free fluorophore.

Here, we have modified Navitoclax with an acetylated galactose to exploit the enriched SA-β-gal activity of senescent cells. Using a variety of model systems, we show that galacto-conjugation of Navitoclax, which we name Nav-Gal, results in a prodrug with selective, pro-apoptotic senolytic activity released in senescent cells that is dependent on GLB1 activity. Galacto-conjugation of Navitoclax reduces thrombocytopenia in treated mice at therapeutically effective doses, as well as apoptosis of platelets in human blood samples treated ex vivo. Overall, we propose galacto-conjugation of cytotoxic drugs as a versatile methodology for developing second-generation prodrugs with high senolytic activity and reduced toxicity. We provide evidence of the efficacy of combining senescence-inducing chemotherapies with senotherapies in cancer, with potential for clinical application.

A TAT Peptide Based Approach to Upregulation of Proteasomal Activity
https://www.fightaging.org/archives/2020/04/a-tat-peptide-based-approach-to-upregulation-of-proteasomal-activity/

The proteasome is a construct in cells that shreds damaged, misfolded, or unwanted proteins, reducing them to component parts that can be reused. It is a part of the ubuiquitin-proteasome system: molecules to be destroyed are tagged with ubiquitin, and drawn into a proteasome for recycling. Greater proteasome activity is thought to be a good thing, improving cell function. This is of particular relevance to aging, as proteasomal function declines with age, contributing to faltering cell and tissue function, particularly in the long-lived cells of the nervous system.

While established drugs exist to inhibit activity of the proteasome, useful in cancer therapies in which cell death is a goal, improving proteasomal activity is less well explored. The one approach shown to work well to date is to increase expression of some of the individual rate-limiting proteins that make up proteasomal structure. This has been shown to extend life in short-lived laboratory species, an enhancement therapy that partially compensates for the progressive loss of proteasomal function with age. Researchers here outline their discovery of a different methodology, one that may be more amenable to the production of a drug capable of upregulating proteasome function.

New Peptide-Based Pharmacophore Activates 20S Proteasome

As the central protease of the ubiquitin-proteasome pathway, the proteasome has long been considered an attractive target for drugs potentially affecting multiple aspects of cell physiology. Indeed, small molecules targeting the proteasome have entered the clinic with great success. However, their scope at present is very limited: all proteasome-modifying compounds currently approved or clinically tested as drugs are competitive inhibitors and all are used to treat advanced blood cancers. Here we turn to the opposite side of pharmacological intervention into the proteasome: augmentation of catalytic activity. Since dysfunction of proteasome-mediated controlled protein degradation is a hallmark of both cellular aging and neurodegenerative diseases, enhancement of the enzyme's activity should be considered an attractive therapeutic option.

The complex structure of the catalytic core 20S proteasome (the "core particle") presents fascinating options for allostery-based augmentation. The peptidase responsible for post-hydrophobic (chymotrypsin-like, ChT-L) cleavages is considered a rate-limiting "workhorse" and is the major target for inhibitors and activators alike. Indeed, overexpression of a catalytic subunit harboring the active site of the ChT-L peptidase has been shown not only to extend lifespan but also to reduce age-related cognitive decline in animal models. However, reports on pharmacological augmentation are limited to in vitro and cell culture studies.

Here, we describe a series of short, modified peptides based on the basic domain of the viral Human Immunodeficiency Virus-1 (HIV-1) Tat protein. Among many intracellular effects, the HIV-1 Tat protein inhibits the core proteasome. In our previous studies, we noted that short peptide fragments of HIV-1 Tat displayed peculiar in vitro properties: they inhibited detergent-treated core particle but mildly activated the latent core. However, treatment with detergent, although convenient for in vitro assays, yields mildly denatured proteasome and, under these far-from-physiological conditions, likely with destroyed natural allosteric routes.

Therefore, we turned our attention to the activating properties of HIV-1 Tat protein-derived "TAT peptides". After observing a strong in vitro proteasome augmentation by modified HIV-1 Tat-derived peptides, we tested selected compounds in cell culture. In a separate study, we found that proteasome stimulation by TAT peptides partially prevented the cognitive deficits and mortality in animal models of Alzheimer's disease. The very encouraging results included increased proteasome-mediated turnover of amyloid precursor protein (APP) and β-secretase (which cleaves APP to generate β-amyloid peptide), concomitant with lowered levels of β-amyloid, lowered mortality and protection against cognitive decline. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.

Olympic Athletes Have a Lower Mortality than the General Population
https://www.fightaging.org/archives/2020/04/olympic-athletes-have-a-lower-mortality-than-the-general-population/

A number of studies have shown that elite athletes have a significantly lower mortality and longer life expectancy than the general population. It is unclear as to whether this is due to physical activity and training for fitness versus other mechanisms, as elite chess players appear to have similar advantages. This study is par for the course, looking at Japanese Olympic participants. Interestingly, it hints at the upper end of the dose-response curve for physical activity, in that a longer career as a professional athlete may be detrimental in comparison to lesser degrees of exercise and training.

From this large, retrospective cohort study targeting 3546 Japanese Olympic athletes, we observed significant lower mortality among Olympians compared with the Japanese general population. The overall standardised mortality ratio (SMR) was 0.29. The results were consistent with previous studies conducted in other non-Asian countries, but the SMR was lower than in previous studies. A retrospective cohort study targeting 203 French Olympic rowers reported that the SMR for all causes of death was 0.58. Another retrospective cohort study targeting 2403 French Olympic athletes reported a SMR of 0.49 among women and 0.51 among men. A third retrospective cohort study of 233 Croatian male Olympic medalists reported a SMR of 0.73.

In the analysis by total number of participation in the Olympic Games, significantly higher mortality was observed among those who participated in the Olympics more than twice compared with those who participated only once. The underlying reason for this may be that those who participated in Olympic Games many times may have had long careers as elite athletes. To be continuously successful, they could have been exposed to exercises with high intensity for long periods, which may have led to higher mortality compared with those who participated only once.

We conducted cohort classification by sport discipline according to the nine combinations of static and dynamic intensity of sports disciplines, and demonstrated the association between intensity of sports disciplines and mortality. We did not observe significantly higher mortality among athletes who participated in disciplines of highest static and highest dynamic intensity. This could be explained in part by their exercise habits after retiring from international competitions. Although a study reported that those capable of prolonged vigorous physical exercises lived longer compared with the general population, Olympic athletes involved in sports disciplines with high intensity may not necessarily continue sports activities after retirement. The habit of sports activities after retiring from athletics may have more influence on mortality than the static/dynamic intensity of each sports discipline.

There Are Many Ways to Influence Known Longevity Pathways
https://www.fightaging.org/archives/2020/04/there-are-many-ways-to-influence-known-longevity-pathways/

The paper here is an example of the point that there are many ways to influence any given longevity-related mechanism. The vast majority of the diverse approaches shown to slow aging in short-lived laboratory species, such as nematode worms, in fact act through a small number of core mechanisms. These are related to cellular stress responses, such as an increased operation of maintenance processes that include, prominently, autophagy. Unfortunately they also have diminishing returns as species life span increases: it is possible to produce large gains in short-lived species via manipulation of these mechanisms, but not in humans. Other approaches are needed if the goal is rejuvenation of the old.

Physiological aging is a complex process, influenced by a plethora of genetic and environmental factors. While being far from fully understood, a number of common aging hallmarks have been elucidated in recent years. Among these, transcriptomic alterations are hypothesized to represent a crucial early manifestation of aging. Accordingly, several transcription factors (TFs) have previously been identified as important modulators of lifespan in evolutionarily distant model organisms.

Based on a set of TFs conserved between nematodes, zebrafish, mice, and humans, we here perform a RNA interference screen in C. elegans to discover evolutionarily conserved TFs impacting aging. We identify a basic helix-loop-helix TF, named HLH-2 in nematodes (Tcf3/E2A in mammals), to exert a pronounced lifespan-extending effect in C. elegans upon impairment. We further show that its impairment impacts cellular energy metabolism, increases parameters of healthy aging, and extends nematode lifespan in a reactive oxygen species dependent manner.

We then identify arginine kinases, orthologues of mammalian creatine kinases, as a target of HLH-2 transcriptional regulation, serving to mediate the healthspan-promoting effects observed upon impairment of hlh-2 expression. Consistently, HLH-2 is shown to epistatically interact with core components of known lifespan-regulating pathways, i.e. AAK-2/AMPK and LET-363/mTOR, as well as the aging-related TFs SKN-1/Nrf2 and HSF-1. Lastly, single-nucelotide polymorphisms (SNPs) in Tcf3/E2A are associated with exceptional longevity in humans. Together, these findings demonstrate that HLH-2 regulates energy metabolism via arginine kinases and thereby affects the aging phenotype dependent on ROS-signaling and established canonical effectors.

CCN2 Inhibition Reverses Fibrosis in Overuse Injury
https://www.fightaging.org/archives/2020/04/ccn2-inhibition-reverses-fibrosis-in-overuse-injury/

Fibrosis is a characteristic feature of aging, degrading tissue and organ function in the lungs, kidneys, heart, and elsewhere. It is a failure of regeneration and tissue maintenance, involving the inappropriate formation of scar-like collagen structures. Fibrosis is also found in overuse injury in muscle. In the case of aging, recent research has shown that cellular senescence has a prominent role in fibrosis, most likely mediate by inflammatory signaling. Removal of senescent cells reverses fibrosis in animal models, making senolytic therapies interesting in this context. Here researchers do not discuss senescent cells, but do show that inhibition of CCN2 reverses muscle fibrosis due to overuse injury. CCN2 inhibition underwent a successful human trial for lung fibrosis, which in turn suggests that perhaps senolytic therapies would usefully treat overuse injuries.

Overuse-induced musculoskeletal disorders are widely understood to be injuries and disorders affecting the musculoskeletal system. Tissue fibrosis is a pathological hallmark of overuse-induced muscle injuries and is considered to play key roles in associated motor dysfunction. Such fibrosis is thought to distort dynamic properties of tissue and contribute to functional declines due to adherence of adjacent structures. We have shown that inflammation is a key driver of further fibrosis, and that early use of anti-inflammatory drugs, ergonomic task reduction and manual therapy treatments are able to prevent their development. However, treatments aimed at reducing established muscle and other tissue fibrosis have proved to be more difficult, because once deposited and cross-linked, the extracellular matrix becomes resistant to degradation.

Blocking CCN2 signaling has shown promise for many fibrotic disorders. Downregulation of CCN2 reduces liver fibrosis and limits hypertrophic scarring without affecting wound healing. We recently found that CCN2 is critical to the early progression of chronic overuse-induced muscle fibrosis and grip strength declines in rats that performed an operant reaching, grasping, and lever-pulling task at high repetition high force (HRHF) levels for three weeks. CCN2 inhibition reduced this early progression of fibrosis and improved motor declines. However, continued performance of the HRHF task for 18 weeks, untreated, induces even greater muscle fibrosis and motor declines than at earlier weeks. Therefore, we examined for the first time whether inhibition of CCN2 using this antibody is able to reduce established skeletal muscle fibrosis in our operant rat model of overuse injury.

We show here that 6 weeks of rest combined with systemic inhibition of CCN2 significantly reduced established skeletal muscle fibrosis and improved motor function, compared to control rat levels. We again show that increased muscle fibrosis was mirrored by increased serum levels of CCN2, adding further support to its use as a serum biomarker of underlying tissue fibrosis occurring with overuse injuries as well as other diseases associated with enhanced fibrogenic activity.

Engineered Stem Cells Survive Longer and Improve Outcomes in a Heart Patch
https://www.fightaging.org/archives/2020/04/engineered-stem-cells-survive-longer-and-improve-outcomes-in-a-heart-patch/

In most cell therapies, the transplanted cells do not survive for long, or in large numbers. They produce beneficial effects, such as reduced inflammation or enhanced regeneration, via signaling that changes the behavior of native cell populations. Considerable effort is going into finding ways to make cells used in therapy survive for a longer period of time following transplantation. The approach taken here is to engineer a fraction of the transplanted cells to produce a growth factor that improves the survival of the others. The results are demonstrated in an animal model, showing a greater regeneration of heart muscle.

Human mesenchymal stem cells (hMSCs) have been considered as one of the most promising cell sources for cell-based cardiac regeneration therapy because of their proven safety and notable paracrine effects to secrete numerous antiapoptotic and angiogenic growth factors, which enabled them to be a more competitive agent for clinical applications. However, unlike promising results obtained from preclinical models of myocardial infarction (MI), recent multiple meta-analyses have debated whether the therapeutic potential of hMSC treatment is sufficient. While these clinical trials successfully demonstrated the feasibility and safety of hMSC treatment, the researchers were unable to show significant functional benefit.

In response, diverse approaches have been attempted to enhance the therapeutic efficacy of hMSCs in treating MI. For instance, genetically engineered hMSCs overexpressing a number of antiapoptotic proteins, growth factors, or prosurvival genes - such as vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), and hepatocyte growth factor (HGF) - showed increased survival and retention in vivo resulting in improved cardiac function and myocardial angiogenesis in MI-induced hearts. However, these approaches require genetic modification and, therefore, are incompatible with clinical applications.

Another strategy to bolster the therapeutic potential of hMSCs is priming/preconditioning the hMSCs - which exposes them to physical treatments (e.g., hypoxia and heat shock), pharmacological agents, growth factors, distinct types of biomaterials, modified culture conditions, or other various molecules, including microRNAs - in vitro before transplantation into the hearts. However, it appears that the priming application only provides a short-term benefit.

Consequently, for hMSCs to be used more effectively for comprehensive cardiac repair, an innovative method that can maintain the priming effect of hMSCs more consistently and effectively must be developed. In the present study, we sought to develop a strategy, namely, in vivo priming, which could prime hMSCs in intact hearts in vivo. To induce and maintain the beneficial effects of priming persistently in situ, we loaded MSCs isolated from human bone marrow (BM-MSCs) together with genetically engineered HGF-MSCs (HGF-eMSCs) that continuously secrete HGF within a three-dimensional (3D) cardiac patch, which was implanted in the epicardium of MI-induced hearts. Subsequently, we demonstrated that the primed BM-MSCs had a higher survival rate compared with unprimed BM-MSCs in the patches while they were attached to the MI hearts, which led to a significant improvement in cardiac function and an enhancement of vessel formation after MI.

The Autophagy-Lysosomal Pathway and Protein Aggregation in Neurodegenerative Disease
https://www.fightaging.org/archives/2020/04/the-autophagy-lysosomal-pathway-and-protein-aggregation-in-neurodegenerative-disease/

Researchers here review some of the evidence for a two-way relationship between the age-related failure of autophagy and the accumulation of harmful protein aggregates that characterizes many neurodegenerative conditions. Autophagy is a collection of cellular maintenance processes responsible for recycling unwanted or damaged proteins and structures, but isn't just the case that failure of autophagy can lead to the spread of protein aggregates. It is also possible that the presence of these aggregates can cause a loss of autophagy, implying a vicious cycle of progression of these age-related conditions.

A hallmark of many neurodegenerative diseases is the progressive formation of insoluble protein aggregates. There are two main factors that cause protein aggregation in neurodegenerative diseases: mutations in genes encoding aggregate-prone proteins and the decline of cellular degradation functions, in particular of the autophagy-lysosomal pathway (ALP).

ALP is a major process for degrading intracellular macromolecules and generating energy or building blocks to make other macromolecules. ALP relies on the engulfment of cargos to be degraded (macromolecules or damaged organelles) in double-membrane vesicles (autophagosomes), which, therefore, fuse with endosomes/lysosomes to form autolysosomes, where autophagosome contents are degraded by lysosomal enzymes. ALP plays a key role in protein homeostasis and in the clearance of protein aggregates (processes that are particularly important in non-dividing neurons).

Mounting evidence also shows that protein aggregation itself may affect ALP, thus generating a vicious cycle, which boost protein aggregation and toxicity. Different works have shown that the aggregated forms of α-synuclein can bind the lysosome, thus impairing the chaperone-mediated autophagy or inducing lysosomal rupture. The interplay between protein aggregation and ALP dysfunction is crucial in driving neurodegenerative processes in a number of neurological conditions.

Correlating Autonomic Nervous System Aging and Cognitive Impairment
https://www.fightaging.org/archives/2020/04/correlating-autonomic-nervous-system-aging-and-cognitive-impairment/

Heart rate variability is known to be a good way to assess the function of the autonomic nervous system. (That said, most of the commercially available tools for those who want to measure heart rate variability at home are quite unreliable; it is challenging for a self-experimenter to obtain results that are as useful as those provided by medical equipment used by medical staff). The autonomic nervous system, like all aspects of our biology, is negatively impacted by the progression of aging. The same mechanisms of molecular damage that drive autonomic nervous system aging will be involved in cognitive decline and neurodegenerative conditions, so it should be no great surprise to see that these forms of age-related degeneration correlate with one another.

Changes in cognitive performances and cardiovascular disorders represent a normal phenomenon of the aging process. Cardiovascular risk factors, such as smoking, obesity, diabetes mellitus, increased cholesterol, and systemic blood pressure (BP) levels, and an inadequate lifestyle may compromise also cerebral blood flow, which in turn can negatively affect cognitive performance. Moreover, the same age-related anatomical and functional cardiac changes, including also the autonomic nervous system (ANS), determine cardiac output alteration, causing cerebral blood flow modulation. This variation could interfere with microcirculation and cause cerebral ischemia, particularly in those brain sites that control the different cognitive domains.

Evidence supports the relevance of ANS study by heart rate variability (HRV) assessment as a tool for the noninvasive analysis of cardiovascular autonomic function. HRV, defined as a marker identifying the balance between sympathetic and parasympathetic tone, predicts total mortality, sudden death, cardiovascular disease risk, as well as other morbidities. It represents the measure of physiological variation in the interval between consecutive heart sinus beat or in the fluctuations between instantaneous heart rates and provides the importance that the ANS has regarding cardiovascular health and prognosis.

We assessed the relationship between long-term heart rate variability (HRV), as a measure of autonomic nervous system (ANS) functioning, and cognitive performance in elderly patients representative of outpatients in a real-life setting. 117 patients underwent anthropometric evaluation, cardiac assessment by 12-lead electrocardiogram, 24-hour electrocardiogram recording, and blood pressure (BP) measurement, as well as global cognitive evaluation. Our results show that an increased sympathetic activity, but not decreased vagal activity, is associated with better cognitive performances. These results support the sympathetic autonomic function, in that the relationship between better cognitive performances and a moderate prevalence of autonomic function appears dependent on long-term changes in heart rate, mediated by sympathetic activation.

The Beneficial Metabolic Adaption Provoked by Consistent Exercise
https://www.fightaging.org/archives/2020/04/the-beneficial-metabolic-adaption-provoked-by-consistent-exercise/

Consistent exercise produces sweeping changes in metabolism. It is clearly beneficial at any age, and there is a mountain of data to support that assertion. With more modern tools of analysis, greater efforts are being made to catalog the beneficial changes in cellular metabolism that result from exercise, rather than just the improvements to health at a high level. The open access paper noted here is an example of this sort of work.

Exercise provides many health benefits, including weight loss, improved lipid profiles, and improved insulin sensitivity. It is particularly relevant in the era of high-prevalence childhood and adult obesity and cardiometabolic disease. Exercise is a core tenet of all cardiovascular prevention guidelines, and degree of physical fitness is a strong predictor of cardiovascular mortality.

Metabolites are a diverse array of biochemicals that together capture an individual's metabolic state. They are particularly useful in the investigation of cardiometabolic diseases. Furthermore, they can characterize response to both acute and chronic exercise. Several studies have revealed key changes in lipolysis, glycolysis, glycogenolysis, citric acid cycle, and amino acid metabolism after a single/acute aerobic exercise session and identified differences in metabolite substrate use between fit and unfit individuals. However, much less work has been done with respect to metabolic changes following chronic exercise training. These studies reported increases in microbiome-derived tryptophan metabolites and acylcarnitines, and decreases in adenine nucleotides.

We analysed changes in metabolomic profiles at the end of an 80-day exercise intervention compared to baseline, and the association of metabolite changes with changes in clinical parameters. Global metabolism was dramatically shifted after the exercise training programme. Fatty acids and ketone body substrates, key fuels used by exercising muscle, were dramatically decreased in plasma in response to increased aerobic fitness. There were highly significant changes across many classes of metabolic substrates including lipids, ketone bodies, arginine metabolites, endocannabinoids, nucleotides, markers of proteolysis, products of fatty acid oxidation, microbiome-derived metabolites, markers of redox stress, and substrates of coagulation.

For the first time, therefore, we were able to provide an accurate report of the degree of increased consumption of fatty acid and ketone body substrates by trained, energy-efficient muscle. We also captured heretofore unseen, in terms of scale and scope, shifts in metabolism across many different substrates. These findings have important implications in cardiovascular disease prevention and risk reduction regimes.

Intermittent Fasting Increases Neurogenesis in Mice
https://www.fightaging.org/archives/2020/04/intermittent-fasting-increases-neurogenesis-in-mice/

Neurogenesis is the process by which neurons are created and then integrated into neural circuits. It is essential to the processes of memory and learning, but also vital to the maintenance of brain tissue over a lifetime. It may or may not take place throughout the brain, versus only in areas connected to memory function, and mice may or may not be a good model for human neurogenesis. Most of the work to date has taken place in mice, as working with human brain tissue is beyond the reach of most research groups.

Neurogenesis declines with age, likely largely because the stem cell populations responsible for generating new neurons become less active, as is the case for stem cells throughout the body. Greater neurogenesis is generally considered to be a good thing, but the research community has yet to produce therapies reliably proven to achieve this outcome in humans. A number of approaches work in mice to varying degrees. Fasting, calorie restriction, and exercise all appear to improve neurogenesis, but a larger effect size than is produced via better lifestyle choices would be desirable.

Dietary restriction (DR) is defined as a decrease in energy consumption without reducing nutritional value. This simple dietary intervention has been shown in a wide range of experimental animals to extend lifespan and decrease the incidence of several age-related diseases. The definition of DR has been expanded from an alternative description of caloric restriction (CR) to also encompass a broader scope of interventions, including short-term starvation, periodic fasting, fasting-mimetic diets, and intermittent fasting (IF).

IF has been proven to be advantageous to various organ systems in the body and acts as a mild metabolic stressor. It has been postulated that IF is able to cause powerful changes in the metabolic pathways in the brain via an increase in stress resistance, and breakdown of ketogenic amino acids and fatty acids. Experimental studies have also shown that IF is neuroprotective against acute brain injuries such as stroke, and neurodegenerative diseases. In addition, recent studies have also shown that IF can lead to an increase in neurogenesis levels in the hippocampus.

We evaluated the impact of 3 months of IF (12, 16, and 24 hr of food deprivation on a daily basis) on hippocampal neurogenesis in mice using immunoblot analysis. We investigated the expression levels of molecular and cellular components of the hippocampal region, focusing specifically on Notch activation and associated proteins that are known to promote hippocampal neurogenesis such as brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB).

Three-month IF significantly increased activation of the Notch signaling pathway, neurotrophic factor BDNF, and downstream cellular transcription factor, cAMP response element-binding protein (p-CREB). The expression of postsynaptic marker, PSD95, and neuronal stem cell marker, Nestin, was also increased in the hippocampus in response to 3-month IF. These findings suggest that IF may increase hippocampal neurogenesis involving the Notch 1 pathway.

Transcriptomic Analysis of Microglia in Mice Shows Greater Inflammatory Activity with Advancing Age
https://www.fightaging.org/archives/2020/04/transcriptomic-analysis-of-microglia-in-mice-shows-greater-inflammatory-activity-with-advancing-age/

Microglia are innate immune cells of the brain, akin to macrophages elsewhere in the body, but equipped to undertake an additional set of tasks relating to neural function. A range of evidence strongly suggests that the progression of neurodegenerative conditions is strongly driven by greater inflammatory activity in the microglia of older individuals. This is perhaps largely due to cellular senescence, perhaps largely due to greater adoption of the aggressive M1 phenotype. Underlying causes include greater leakage of the blood-brain barrier due to the molecular damage of aging, allowing unwanted compounds and cells into the brain that will rouse an inflammatory response.

Accordingly, there is greater interest nowadays in strategies that might reduce inflammation in the brain, whether senolytic drugs targeting senescent cells, small molecules that might force microglia into the more helpful M2 phenotype, or other approaches to selectively sabotaging mechanisms of the immune response. Repair of underlying damage beyond cellular senescence that causes the chronic inflammation of aging is still a fairly low priority in the research community, alas.

Aging and Alzheimer's disease (AD) are both associated with diminished blood-brain barrier (BBB) integrity and an opening for T cell migration into the central nervous system (CNS). In the parenchyma, bidirectional crosstalk occurs between the infiltrating cells and the resident glial cells; activated microglia impair BBB function by releasing several inflammatory modulators and thus lead to hyperpermeability; and the resulting T cell infiltration, in turn, favors increased microglial activation by secreting proinflammatory cytokines or acting in a protective manner toward senescent microglia.

We performed RNA-seq analyses on microglia and astrocytes freshly isolated from wild-type and APP-PS1 (AD) mouse brains at five time points to elucidate their age-related gene-expression profiles. Our results showed that from 4 months onward, a set of age-related genes in microglia and astrocytes exhibited consistent upregulation or downregulation (termed "age-up"/"age-down" genes) relative to their expression at the young-adult stage (2 months). Most age-up genes were more highly expressed in AD mice at the same time points. Bioinformatic analyses revealed that the age-up genes in microglia were associated with the inflammatory response, whereas these genes in astrocytes included widely recognized AD risk genes, genes associated with synaptic transmission or elimination, and peptidase-inhibitor genes.

The results of this study indicate that microglia exhibit an increase in responsiveness to inflammation stimuli with age, which is reflected by the consistently elevated expression of inflammatory-response genes, whereas astrocytes appear to function as "preservers" of inflammation, which is reflected by the upregulation of peptidase-inhibitor genes upon aging.

Soluable α-klotho Reduces Cardiac Fibrosis in Mice
https://www.fightaging.org/archives/2020/04/soluable-%ce%b1-klotho-reduces-cardiac-fibrosis-in-mice/

Klotho is a longevity-related gene. In mice, greater expression extends life while reduced expression shortens life. Most study has focused on beneficial effects on cognition and kidney function, with some debate over where in the body it acts - it may be that benefits to brain function are entirely the consequence of improved kidney function. That isn't all that klotho does, however. The delivery of soluble α-klotho has emerged as a basis for building therapies based on klotho biochemistry, and Unity Biotechnologies is one of the groups working on clinical development of this line of work. Here, researchers show that the scarring of fibrosis in heart tissue, a feature of aging, can be reduced via delivery of α-klotho. The same questions apply here as for the brain; is this a local mechanism of action, or something mediated by effects in another organ and systemic signaling throughout the body?

Heart disease is the leading cause of death worldwide. The major cause of heart failure is the death of the myocardium caused by myocardial infarction, detrimental cardiac remodeling, and cardiac fibrosis occurring after the injury. This study aimed at discovering the role of the anti-aging protein α-klotho (KL), which is the co-receptor of fibroblast growth factor-23 (FGF23), in cardiac regeneration, fibrosis, and repair.

FGF23 is the most recently discovered fibroblast growth factor and functions as an endocrine hormone that regulates phosphate homeostasis through binding to FGFR and KL, its coreceptor in the kidney and parathyroid glands. Elevated levels of circulating FGF23 have been associated with left ventricular hypertrophy, and it has been suggested that FGF23 exerts a direct effect on the myocardium. Interestingly, the co-receptor of FGF23, KL, has been shown to exhibit renal protective functions independent of FGF23/FGFR signaling.

We found that the anti-apoptotic function of soluble KL in isoproterenol-treated cardiomyocytes was independent of FGF23 in vitro. In vivo, isoproterenol-induced cardiac fibrosis and cardiomyocyte and endothelial cell apoptosis were reduced by KL treatment. Moreover, the number of Ki67-positive endothelial cells and microvessel density within the isoproterenol-injured myocardium were increased upon KL treatment. However, by using genetic fate-mapping models, no evident cardiomyocyte proliferation within the injured myocardium was detected with or without KL treatment. Collectively, the cardioprotective functions of KL could be predominantly attributed to its anti-apoptotic and pro-survival activities on endothelial cells and cardiomyocytes. KL could be a potential cardioprotective therapeutic agent with anti-apoptotic and pro-survival activities on cardiomyocytes and endothelial cells.