Fight Aging!

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Contents

• Connecting NANOG Expression with the Response to Methionine Restriction
• Reviewing Cellular Senescence in Idiopathic Pulmonary Fibrosis
• Improving on the FOXN1-TAT Fusion Protein Approach for Thymic Regeneration
• Increased miR-181a-5p Expression Improves Neural Stem Cell Activity, Learning, and Memory in Old Mice
• As Suspected, Local Clearance of Senescent Cells isn't as Effective as Global Clearance for Osteoporosis
• Starting Out on the Long Road to Tissue Engineering for the Brain
• Biochemical Differences Between the Response to High Intensity versus Moderate Exercise
• The Extended Fertility of Naked Mole-Rats
• Exercise is Medicine
• Regular Physical Activity at Any Time in Life Improves Late Life Brain Health
• A Small Clinical Trial of NMN Fails to Produce Significant Results on Arterial Stiffness
• Risk of Death Due to Heart Attack Has Fallen Considerably Over The Past 20 Years
• Improving 3D Printing of Fine Structures in Artificial Tissue
• Considering Proteostasis and Aging
• Assessing the Spread of Mitochondrial Mutations in Tissue

Connecting NANOG Expression with the Response to Methionine Restriction
https://www.fightaging.org/archives/2023/02/connecting-nanog-expression-with-the-response-to-methionine-restriction/

Calorie restriction is known to slow aging, albeit to a much greater degree in short-lived species than in long-lived species. Finding important mechanisms involved in the beneficial response to calorie restriction continues to be a major focus on the research community, even though it is questionable as to whether this is a good approach to the treatment of aging. A sizable fraction of the response to calorie restriction appears to be mediated by methionine sensing, at least judging by the degree to which reducing methioninine intake can reproduce the benefits of full calorie restriction.

In today's open access paper, researchers connect NANOG expression to the methionine restriction response, certainly an interesting link. NANOG is a pluripotency factor expressed in embryonic stem cells, studied in the contexts of regeneration, cancer, and cell reprogramming. One might not expect it to employ methionine sensing mechanisms to achieve changes on cell metabolism, and yet it does. Everything connects to everything else when it comes to regulation of cell behavior, it seems.

More pertinently, senescent cells are metabolically active, with a high methionine metabolism driving their ability to generate harmful signaling. Expressing NANOG squashes that activity to restore better function. This is perhaps a good idea in severe conditions such as progeria in which a sizable fraction of cells become senescent, but less of a good idea in normal aging, even given a role of cellular senescence in the onset of age-related disease, given the balance between NANOG expression and risk of cancer.

Methionine adenosyltransferase2A inhibition restores metabolism to improve regenerative capacity and strength of aged skeletal muscle

A recent study demonstrated that a methionine-restricted (MR) diet improved mitochondrial function and upregulated autophagy-related genes, resulting in a 45% extension of rodent lifespan. Other studies also demonstrated that MR regulates energy expenditure in the aged musculoskeletal system, activated insulin signaling that improved type II diabetes, and decreased lipid peroxidation that reduced hyperlipidemia. Clearly, decreasing dietary methionine has beneficial physiologic effects. However, a pathway connecting methionine to these pathologies is yet to be elucidated. Methionine breakdown begins with MAT2A, which catalyzes the production of S-adenosyl methionine (SAMe) from methionine.

Previously, our laboratory reported similar pathologies affecting cells from progeria patients and cells undergone replicative senescence. Further, we demonstrated that with ectopic expression of the pluripotency factor, NANOG could effectively reverse aging hallmarks and reestablish young attributes in older cells to restore their myogenic differentiation capacity, decrease senescence-associated beta-galactosidase (SA-βgal), restore mitochondrial function, and repair DNA damage. NANOG also restored the ability of senescent myoblasts to differentiate into healthy skeletal myotubes and ameliorated the hallmarks of cellular senescence including genomic instability, loss of proteostasis, and mitochondrial dysfunction in human skeletal myoblasts and restored the number of myogenic progenitors in a mouse model of premature aging.

However, the mechanism through which NANOG imparts its rejuvenating effects is not known. Using NANOG as an investigative tool, we examined whether metabolic impairments due to senescence or premature aging could be reversed, restoring muscle function. We discovered that senescent myoblasts used methionine to meet their metabolic demands and that increased use of methionine contributed to the loss of skeletal muscle function. Conversely, inhibition of MAT2A catabolism by NANOG expression or chemical inhibition restored glucose-based bioenergetics and the force-generating capacity of aged skeletal muscle in a mouse model of premature aging.

Reviewing Cellular Senescence in Idiopathic Pulmonary Fibrosis
https://www.fightaging.org/archives/2023/02/reviewing-cellular-senescence-in-idiopathic-pulmonary-fibrosis/

Fibrosis is the excessive deposition of extracellular matrix, forming scar-like structures that are disruptive to tissue function. It is a feature of aging in many organs, such as heart, liver, kidney, and lungs, and when particularly pronounced it is declared to be fibrotic disease. So far medical science has struggled to make much headway in the reversal of fibrosis once it is established, which makes these conditions particularly threatening.

Fibrosis is connected to the chronic inflammation characteristic of old age, and in recent years evidence has amassed for senescent cells to drive fibrosis. Senescent cells increase in number with age in tissues throughout the body, and produce pro-growth, pro-inflammatory signaling. When tissue is injured, senescent cells emerge for a short time to help coordinate regeneration. In later life, this constant signaling alters cell behavior for the worse, disrupting normal tissue maintenance to encourage pathologies like fibrosis to develop.

Selectively clearing senescent cells via the use of senolytic drugs has been shown in animal studies to reverse fibrosis in a number of different organs. One of the first small human clinical trials of senolytic drugs targeted patients with idiopathic pulmonary fibrosis. It is this condition, and the senescent cells that may drive its onset and development, that are the subject of today's open access review paper.

Molecular mechanisms of alveolar epithelial cell senescence and idiopathic pulmonary fibrosis: a narrative review

Idiopathic pulmonary fibrosis (IPF) is a commonly diagnosed chronic, progressive, and fibrotic interstitial pneumonia that accounts for 20-30% of interstitial lung diseases. It usually occurs in middle-aged and elderly individuals. It is now generally accepted that persistent alveolar epithelial damage and repair dysregulation are the principal mechanisms leading to progressive pulmonary fibrosis.

Repetitive epithelial cell injury and deficiencies in regeneration result in the release of mediators, including cytokines, chemokines, fibrogenic factors, coagulant proteins, oxidants, and regulators of apoptosis. This leads to the recruitment, proliferation, and activation of interstitial fibroblasts to form fibrotic foci. Additionally, excessive deposition of the extracellular matrix leads to destruction of lung parenchymal structures. Interestingly, a variety of cells, including alveolar epithelial type II cells (ATII) and fibroblasts, can drive IPF. Regardless of the driver cell types, senescence leads to a decrease in the repair capacity of damaged alveolar epithelium. As a result, fibrous tissue replaces the damaged alveolar epithelium.

From a histopathological point of view, IPF formation is a dynamic process involving complex interactions among epithelial cells, fibroblasts, immune cells (such as macrophages and T lymphocytes), and endothelial cells. Alveolar epithelial cells undergo cytoskeletal remodeling and acquire a mesenchymal phenotype through epithelial-mesenchymal transition (EMT), in which epithelial cells lose intercellular attachment, polarity, and epithelial-specific markers, leading to fibrosis.

Some investigators have identified ATII as a major player in the synthesis of transforming growth factor-beta (TGF-β) and tumor necrosis factor-alpha (TNF-α) in lung biopsies from patients with IPF. In the process of organ fibrosis formation, including pulmonary fibrosis, TGF-β acts as the master switch for the induction of the EMT process. In particular, TGF-β mediates fibrous proliferative effects by inducing apoptosis in alveolar epithelial type I (ATI) cells. However, there is no direct evidence that TGF-β promotes IPF by inducing senescence in the alveolar epithelial cells.

In the lungs of patients with IPF, the ability of ATII cells to transdifferentiate into ATI cells is diminished. Emerging evidence also suggests that triggering ATII senescence can promote IPF. Therefore, studying the mechanisms of cellular senescence in the lung microenvironment is crucial to understand IPF pathogenesis and progression. Decreasing senescent alveolar epithelial cells may be a promising strategy for the treatment of IPF. Therefore, further investigations into new treatments of IPF by applying inhibitors of relevant signaling pathways, as well as senolytic drugs, are warranted.

Improving on the FOXN1-TAT Fusion Protein Approach for Thymic Regeneration
https://www.fightaging.org/archives/2023/03/improving-on-the-foxn1-tat-fusion-protein-approach-for-thymic-regeneration/

FOXN1 is the master regulator of thymic growth and activity; the thymus is an unusually straightforward organ in this respect. One can make it grow and perform to a greater degree just by dialing up expression of this one gene in thymic tissue. Thymic regrowth is a desirable goal for the elderly, given that thymic atrophy occurs in everyone, and limits the production of T cells. It is a major contribution to immune aging.

One of the approaches that has been taken to achieve this goal of thymic regrowth is the delivery of a FOXN1 recombinant protein attached to the TAT domain derived from the HIV-1 virus, allowing it to enter the cell. Researchers published a study some years back in which intrathymic injection was used, an approach that is probably too risky to serve as a basis for human therapies. Without direct injection, one can't get enough of the protein into the thymus to be worth the effort.

Today's open access paper discusses an advance on that earlier work by the same team. The researchers further attach an additional binding domain to the FOXN1-TAT fusion protein to optimize uptake in thymic tissue, and inject the protein intravenously instead of directly into the thymus. This necessarily requires higher doses (and recombinant proteins remain expensive), but the team reports good results. This is an approach that could in principle be developed for human use, setting aside the more conservative concerns one might encounter regarding the use of TAT versus the adoption of other options for cell entry.

Recombinant FOXN1 fusion protein increases T cell generation in aged mice

The thymus is a specified immune organ that provides an inductive environment for the generation of T cells that play a critical role in the adaptive immune system. Although the thymus continues to export T cells throughout life, it undergoes a profound atrophy with age, a process termed thymic involution, resulting in decreased numbers and functional capacity of T cells in the older adult, which has direct etiological linkages with many diseases. Furthermore, T cell immune deficiency in the older adult is exacerbated when the immune system is insulted by chemotherapy, radiotherapy, infections (e.g. HIV), and preparative regimens for foreign tissue or organ transplants. Therefore, restoring thymus function in the older adult has important implications.

We have previously reported that intrathymic injection (i.t.) of a recombinant (r) protein containing FOXN1 and a protein transduction domain embedded in the HIV transactivator of transcription (TAT) protein increases the number of thymic epithelial cells (TECs) in mice that have undergone hematopoietic stem cell transplantation. Consequently, these mice had enhanced thymopoiesis, an improved thymic output and an increased number of naïve T cells in the periphery. However, i.t. injection may not be an ideal choice for clinical applications.

It has been reported that chemokine CCL25 is highly expressed in thymic tissue, especially thymic stroma. CCR9 is the receptor for CCL25. Unlike other CC chemokine receptors, CCR9 shows a strict specificity for its ligand CCL25. It has been shown intramural injection of a fusion protein containing the N-terminal of CCR9 and IL-7 increased the content of IL-7 in the thymus as compared to injection of IL-7 alone.

In this study, we develop a rFOXN1 fusion protein that contains the N-terminal of CCR9, FOXN1, and TAT. We show here that, when injected intravenously (i.v.) into aged mice, the rFOXN1 fusion protein can migrate into the thymus and enhance T cell generation in the thymus, resulting in increased number of peripheral T cells. Our results suggest that the rFOXN1 fusion protein has the potential to be used in preventing and treating T cell immunodeficiency in the older adult.

Increased miR-181a-5p Expression Improves Neural Stem Cell Activity, Learning, and Memory in Old Mice
https://www.fightaging.org/archives/2023/03/increased-mir-181a-5p-expression-improves-neural-stem-cell-activity-learning-and-memory-in-old-mice/

Neurogenesis occurs throughout life to support the changes in neural structure inherent in learning and memory. It also provides some resilience to brain injury, when it comes to maintaining and restoring function. In the process of neurogenesis, new daughter neurons are generated by neural stem cells, and then mature and integrate into existing neural networks in brain tissue. This is all largely studied in mice, due to the difficulties inherent in obtaining access to living human brains, but despite some debate it is reasonable to assume that learning and memory in our species are similarly supported by ongoing neurogenesis throughout life.

Unfortunately, stem cell populations decline in activity with advancing age. This is in part the result of intrinsic damage to the cells themselves, but also a response to altered signaling and dysfunction in stem cell niches. The chronic inflammation of aging, for example, appears to have a strongly negative impact on stem cell function. Thus the pace of neurogenesis is much reduced in old individuals, and experiments in mice indicate that this is an important part of the loss of memory function that occurs with aging. Given this, there is some interest in finding ways to boost neurogenesis, to override the reaction of stem cells to the aged tissue environment, and reduce age-related memory dysfunction. Today's open access paper is one example of this sort of research.

MiR-181a-5p promotes neural stem cell proliferation and enhances the learning and memory of aged mice

The hippocampus is a brain region closely related to spatial learning and memory. Adult neural stem cells (NSCs), which are located in the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus, play important roles in the function of the hippocampus. NSCs have the ability to proliferate, either generating two NSCs to maintain NSC pools through symmetric division or generating one NSC and one neural progenitor cell (NPC) through asymmetric division. NPCs then further differentiate into particular cell types, such as neurons or astrocytes. Studies on rodents have shown that proliferation and neurogenesis of NSCs persist throughout the lifespan; however, adult neurogenesis decreases with age, and this decrease is involved in cognitive and memory declines, indicating that abnormalities in hippocampal NSCs are one of the main causes of age-related deterioration in hippocampus-dependent cognition.

Proliferation and neurogenesis of NSCs are exquisitely regulated by extrinsic and intrinsic factors, including secreted molecules, neurotransmitters, transcription factors, and epigenetic regulators. Among these, miRNAs, which are enriched in the brain, have been shown to be widely involved in the regulation of NSC proliferation and differentiation. In the current study, we found that the expression level of miR-181a-5p was decreased in the hippocampal NSCs of aged mice and that exogenous overexpression of miR-181a-5p promoted NSC proliferation without affecting NSC differentiation into neurons and astrocytes.

The mechanistic study revealed that phosphatase and tensin homolog (PTEN), a negative regulator of the AKT signaling pathway, was the target of miR-181a-5p and knockdown of PTEN could rescue the impairment of NSC proliferation caused by low miR-181a-5p levels. Moreover, overexpression of miR-181a-5p in the dentate gyrus enhanced the proliferation of NSCs and ameliorated learning and memory impairments in aged mice. Taken together, our findings indicated that miR-181a-5p played a functional role in NSC proliferation and aging-related, hippocampus-dependent learning and memory impairments.

As Suspected, Local Clearance of Senescent Cells isn't as Effective as Global Clearance for Osteoporosis
https://www.fightaging.org/archives/2023/03/as-suspected-local-clearance-of-senescent-cells-isnt-as-effective-as-global-clearance-for-osteoporosis/

It has long been suspected that removing senescent cells locally is insufficient to treat age-related conditions in which the pro-inflammatory signaling produced by senescent cells contributes to pathology. These signals enter the bloodstream and are carried widely about the body. While the effect of a distant senescent cell on local pathology is more dilute than that of a local senescent cell, there are a lot more distant senescent cells than there are local senescent cells. This issue is likely why Unity Biotechnology's initial clinical trial of localized removal of senescent cells in osteoarthritic knee joints failed to produce sufficiently large beneficial effects in patients to proceed.

In today's open access paper, researchers demonstrate this issue in mice, in the context of the age-related loss of bone density that leads to osteoporosis. Bone tissue is constantly remodeled through the actions of osteoblast cells, depositing extracellular matrix, and osteoclast cells, breaking down extracellular matrix. Chronic inflammatory signaling generated by senescent cells helps to tip the balance away from osteoblast activity, leading to a steady loss in bone density over time. At least in mice, clearing senescent cells locally doesn't help to prevent this issue to anywhere near as great a degree as clearing senescent cells globally.

Local senolysis in aged mice only partially replicates the benefits of systemic senolysis

Clearance of senescent cells (SnCs) can prevent several age-related pathologies, including bone loss. However, the local versus systemic roles of SnCs in mediating tissue dysfunction remain unclear. Thus, we developed a mouse model (p16-LOX-ATTAC) that allows for inducible SnC elimination (senolysis) in a cell-specific manner and compared the effects of local versus systemic senolysis during aging using bone as a prototype tissue. Specific removal of Sn osteocytes prevented age-related bone loss at the spine, but not the femur, by improving bone formation without affecting osteoclasts or marrow adipocytes.

By contrast, systemic senolysis prevented bone loss at the spine and femur and not only improved bone formation, but also reduced osteoclasts and marrow adipocytes. Transplantation of SnCs into the peritoneal cavity of young mice caused bone loss and also induced senescence in distant host osteocytes. Collectively, our findings provide the first proof-of-concept evidence that local senolysis has health benefits in the context of aging, but importantly, local senolysis only partially replicates the benefits of systemic senolysis. Further, we establish that SnCs, through their senescence-associated secretory phenotype (SASP), lead to senescence in distant cells. Therefore, our study indicates that optimizing senolytic drugs may require systemic instead of local SnC targeting to extend healthy aging.

Starting Out on the Long Road to Tissue Engineering for the Brain
https://www.fightaging.org/archives/2023/02/starting-out-on-the-long-road-to-tissue-engineering-for-the-brain/

Can one replace parts of the brain? In principle, yes. It is a tissue, and tissue engineering is a field intent on regrowth and replacement of lost or damaged tissue. There are parts of the brain immediately vital to life, and parts that hold the memory that defines the self; if those are lost, that is irrecoverable. But much of the brain might be tissue engineered in the same way as muscle or liver might be replaced. Researchers are still in the early stages of the long road towards replacement tissues created to order, as illustrated by the scientific work noted here, but much of the brain will be a part of that field of development.

The transplantation of pluripotent stem cell-derived neural precursors into the cortex is an exciting potential approach to repair the brain. To achieve this goal, grafted cells must re-establish damaged neural circuits that participate in the restoration of lost behavioral function. Significant progress has been made in demonstrating the feasibility of transplanting precursor cells to replace neurons in the cortex. Graft-derived neurons can survive for years in mice and differentiate into appropriate neuronal subtypes that exhibit normal electrophysiological activity, project long distances outside of the graft to appropriate targets, synaptically integrate with surrounding host neurons, and respond to sensory input and participate in motor output.

Despite these significant discoveries, it is unclear whether grafted neurons in the neocortex can encode useful behavior as a result of their electrophysiological activity. Reported behavioral benefits are instead a result of activity-independent functions such as the secretion of anti-inflammatory or neurotrophic factors. The inability to demonstrate that electrophysiological activity of grafted neurons encode useful behavior is not surprising considering there are cortical cell types that are thus far missing in grafts, in addition to these grafts lacking normal cortical cytoarchitecture. While cerebral organoids display a subset of similar characteristics to a normal fetal cortex, their differentiation has thus far been abnormal after transplantation. Therefore, there is currently no method of generating facsimiles of neocortical tissue in adults, whether for the purpose of study or therapy.

The goal of this study is to provide an initial proof of concept for a neocortical grafting platform that supports (1) the survival and differentiation of the major neocortical cell types, (2) vascularization, (3) neuronal integration, and (4) layering. Toward this goal, we tested whether grafting cells in a three-dimensional scaffold could sustain the differentiation of all the major cortical cell types, vascularization, and a layered cytoarchitecture. Using dissociated mouse cortical fetal cells mixed with a commercial scaffold, we found that the neuronal, glial, and vascular components within the graft survived and successfully integrated with the host tissue. Our results suggest that this platform is suitable for future optimization and testing of structured, vascularized, multi-cell type neocortical tissue prototypes.

Biochemical Differences Between the Response to High Intensity versus Moderate Exercise
https://www.fightaging.org/archives/2023/02/biochemical-differences-between-the-response-to-high-intensity-versus-moderate-exercise/

A range of evidence suggests high intensity exercise to produce different, greater benefits than is the case for more moderate, longer periods of exercise. Researchers here look at the biochemistry of cellular senescence in muscle tissue immediately following exercise, and find the characteristics notably different. The results suggest that high intensity exercise induces more short-term inflammatory cell stress, but removes more of the pre-existing markers of cellular senescence as a result of a greater immune reaction to that stress.

In this study, we asked the question whether the cellular senescence-lowering effect of exercise in human skeletal muscle can occur only at the intensity sufficient to induce DNA damage and inflammation. Biopsied vastus lateralis of 9 sedentary men (age 26.1 ± 2.5 y) were assessed before and after a single bout of moderate steady state exercise (SSE, 60% maximal aerobic power) and high intensity interval exercise (HIIE, 120% maximal aerobic power). Increases in cell infiltration (+1.2 folds), DNA strand break (+1.3 folds), and γ-H2AX+ myofibers (+1.1 folds) occurred immediately after HIIE and returned to baseline in 24 hours. Muscle p16Ink4a mRNA decreased 24 hours after HIIE. SSE had no effect on cell infiltration, p16Ink4a mRNA, and DNA strand break in muscle tissues.

The major findings are as follows: (1) Cellular senescence-lowering effect of aerobic exercise can occur only at high intensity. SSE with similar exercise work failed to lower the p16INK4a mRNA in human skeletal muscle within the 24-h recovery period; (2) HIIE triggered immediate increases in cell infiltration and γ-H2AX+ myofibers, followed by a decreased p16INK4a mRNA in human skeletal muscle 24 hours after recovery; (3) By further examining the individual responses, the senolytic effect of HIIE were contributed solely from those participants with high pre-exercise p16INK4a mRNA in skeletal muscles.

High intensity exercise is known to cause greater levels of lactate production and acidosis than low intensity exercise. Decreased pH has been reported to be a danger signal to activate innate immune response and immune cells are functioned to recognize and clear senescent cells by phagocytosis in humans. Therefore, the acute changes in the microenvironment during and after exercise may be a selection pressure to aged stem cell population resided in the skeletal muscle. Exercise intensity determines the magnitude of cell renewal during a brief period of inflammation.

Taken together, decreased cellular senescence 24 hours after HIIE is best explained by senescent cell clearance following a brief increase of bone marrow cell infiltration into challenged skeletal muscle to participate in the early phagocytic and late regenerative phases of inflammation. DNA damage is a potent stimulator of inflammation. In this study, a fast resolution of DNA damage/repair response 24 hours following HIIE suggests an efficient clearance of senescent cells with DNA damage in human skeletal muscle to resolve the inflammation.

The Extended Fertility of Naked Mole-Rats
https://www.fightaging.org/archives/2023/02/the-extended-fertility-of-naked-mole-rats/

Naked mole-rats exhibit few signs of aging across a life span. Only the queens bear young, but they can continue do so into old age. As this study notes, they achieve this feat via a number of mechanisms that ensure a continued supply of egg cells. This isn't just a matter of minimizing damage to these cells and their supporting tissues, but also generating new egg cells in adult life, unlike other mammals. It remains an open question as to whether there is any great realization yet to be found in this comparative biology that will benefit efforts to extend human fertility into later life. It doesn't hurt to look.

Unlike humans and other mammals, which become less fertile with age, naked mole-rats can reproduce throughout their remarkably long lifespans. For most mammals, including humans and mice, females are born with a finite number of egg cells, which are produced in utero via a process called oogenesis. Because this limited supply of egg cells depletes over time - some are released during ovulation, but most simply die - fertility declines with age. In contrast, naked mole-rat queens can breed right through old age, suggesting the rodents have special processes to preserve their ovarian reserve and avoid waning fertility. "There are three possibilities for how they do this: They are born with a lot of egg cells, not as many of these cells die, or they continue to create more egg cells after birth. My favorite hypothesis is that they use a cocktail of all three." Sure enough, researchers found evidence for each of the three processes.

The researchers compared ovaries from naked mole-rats and mice across different stages of development. Despite their similar sizes, mice live four years at most and start to show a drop in fertility by nine months, whereas naked mole-rats have a life expectancy of 30 years or more. They found that naked mole-rat females have exceptionally large numbers of egg cells compared to mice and that death rates of these cells were lower than in mice. For example, at 8 days old, a naked mole-rat female has on average 1.5 million egg cells, about 95 times more than mice of the same age. Most remarkably, the study found that oogenesis happens postnatally in naked mole-rats. Egg precursor cells were actively dividing in 3-month-old animals, and these precursors were found in 10-year-old animals, suggesting that oogenesis could continue throughout their lives.

Exercise is Medicine
https://www.fightaging.org/archives/2023/02/exercise-is-medicine/

It remains the case that all too little in medical science is demonstrated to be better than exercise for improved long-term health. Much of the early work on ways to slow aging, pioneered by the supplement industry, has proven to be less effective than structured exercise programs when finally evaluated in clinical trials. One conclusion is that the research and development communities must do better, aim higher. Another conclusion, the subject of this open access paper, is that perhaps the practicing medical community should become much more serious about exercise.

Lack of exercise is a health concern worldwide. According to WHO, 31% of the world's population does not attain the minimum required level of physical activity. This unhealthy lifestyle is the fourth leading cause of death worldwide with approximately 3.2 million deaths annually. This situation is continuously worsening and poses a substantial burden on health systems and societies. A considerable portion of health conditions can be attributed to physical inactivity. Sedentary behavior and physical inactivity are the leading risk factors for cardiovascular disease and all-cause mortality. Compared with people who have previously been physically active, those who are inactive have a higher risk of developing neurological diseases.

Recognizing that many chronic diseases are closely related to poor lifestyle habits and that exercise plays a role in a variety of health conditions, the American College of Sports Medicine and the American Medical Association suggested that "exercise is medicine". Subsequently, a multinational collaboration on "exercise is medicine" began and this initiative was centered on global awareness. In this initiative, it was suggested that patients' level of physical activity be added to their medical records, and behavioral physical activity counseling should be provided through a clinical decision support system.

Specifically, the exercise situation of each patient should be considered as a vital sign in each visit, then healthcare workers should provide professional physical activity guidance for patients' exercise situation and health needs. This initiative emphasized that exercise should be used as medical advice in clinical settings. Hence it is not just improving patients' exercise awareness that is significant, but more so the formation of normative medical work for exercise guidance and the healthcare workers' awareness of exercise effects in health.

Regular Physical Activity at Any Time in Life Improves Late Life Brain Health
https://www.fightaging.org/archives/2023/03/regular-physical-activity-at-any-time-in-life-improves-late-life-brain-health/

Researchers here look at epidemiological data on physical activity and brain function in old age. While the presence of any period of life in which physical activity was a regular occurrence correlates with improved late life brain health, the best option is to remain active throughout life. When it comes to established human data, the effects of exercise and calorie restriction remain the bar to beat for any attempt to improve healthspan and longevity across a broad population of varied individuals. We might hope that at least the use of senolytics to clear senescent cells will improve on this, as well as some of the following biotechnologies aimed at other fundamental mechanisms of aging.

Using data from the population-based 1946 British birth cohort, which has followed people born in the same week of 1946, previous studies have demonstrated beneficial effects of midlife physical activity on midlife verbal memory and search speed decline. Here, we extend this work by taking a life course approach to evaluate the effects of physical activity timing, frequency and maintenance, spanning over 30 years, with later-life cognitive function. We assess three measures of later-life cognitive function including a measure of cognitive state, verbal memory, and processing speed. We further aim to investigate to what extent, these effects are explained by pathways including earlier-life influences, cardiovascular health, and mental health.

To investigate the effect of timing of physical activity, we investigated the strength of associations between a range of cognitive tests at age 69 with participation in physical activity at the ages of 36, 43, 53, 60 and 69. We then investigated whether any associations observed are best explained by physical activity in specific 'sensitive' periods across the life course, or being physically active across multiple time periods.

Being physically active, at all assessments in adulthood, was associated with higher cognition at age 69. For cognitive state and verbal memory, the effect sizes were similar across all adult ages, and between those who were moderately and most physically active. The strongest association was between sustained cumulative physical activity and later-life cognitive state, in a dose-response manner. Thus being physically active at any time in adulthood, and to any extent, is linked with higher later-life cognitive state, but lifelong maintenance of physical activity was most optimal.

A Small Clinical Trial of NMN Fails to Produce Significant Results on Arterial Stiffness
https://www.fightaging.org/archives/2023/03/a-small-clinical-trial-of-nmn-fails-to-produce-significant-results-on-arterial-stiffness/

Nicotinamide adenine dinucleotide (NAD) is involved in mitochondrial function, but levels decline with age for reasons that are not fully understood, alongside a loss of mitochondrial function. Thus there is some interest in delivering NAD precursor molecules, largely derived from vitamin B3, that can increase NAD levels. One might compare this trial of nicotinamide mononucleotide (NMN) with a similar trial of nicotinamide riboside (NR) a few years ago, which produced a better outcome, but still nothing to write home about. People who advocate for upregulation of NAD in mitochondria might say that the dosing is too low, but equally the long history of trying to increase NAD levels, accompanied by dozens of clinical trials, has little to show for it in terms of effect sizes that are any better than those produced by exercise. A good exercise program does still outperform NAD precursor supplementation, at least in clinical trial data.

Many animal studies have shown that oral administration of the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide mononucleotide (NMN) prevents the reduction of NAD+ levels in organs and tissues, helping alleviate aging-related diseases. However, there are very few clinical reports of NMN supplementation in humans. Thus, this study aimed to investigate the influence of a 12-week NMN oral supplementation on biochemical and metabolic health parameters.

A 12-week randomized, double-blind, placebo-controlled, parallel-group clinical trial was conducted. A total of 36 healthy middle-aged participants received one capsule of either 125 mg NMN or placebo twice a day. Among the NAD+ metabolites, the levels of nicotinamide in the serum were significantly higher in the NMN intake group than in the placebo group. Pulse wave velocity values indicating arterial stiffness tended to decrease in the NMN intake group. However, no significant difference was found between the two groups. Long-term NMN supplementation at 250 mg/day was well tolerated and did not cause adverse events. NMN safely and effectively elevated NAD+ metabolism in healthy middle-aged adults. Additionally, NMN supplementation showed potential in alleviating arterial stiffness.

Risk of Death Due to Heart Attack Has Fallen Considerably Over The Past 20 Years
https://www.fightaging.org/archives/2023/03/risk-of-death-due-to-heart-attack-has-fallen-considerably-over-the-past-20-years/

Atherosclerosis leading to heart attack is one of the more amenable issues in aging to control through lifestyle choice and available medications. A very rigorous commitment to diet, exercise, and lowering LDL cholesterol will greatly reduce the odds of developing atherosclerotic plaque sufficient to produce heart attack or stroke. A combination of better treatment and better lifestyle choices has led to a sizable reduction in mortality due to heart attack in recent decades. Eliminating atherosclerosis entirely is going to require new developments in medical science, however, as lowering LDL cholesterol doesn't do much to help those people who have already developed significant atherosclerotic plaque.

New findings, based on an analysis of data from the Centers for Disease Control and Prevention (CDC) from 1999-2020, indicate that age-adjusted rates of death attributed to acute myocardial infarction (the medical term for heart attack) fell by an average of over 4% per year across all racial groups over the two-decade period. "Researchers often highlight the bad news, but people should know that even if we're not there yet, we're making progress in the right direction. I think the reasons are multifactorial, spanning all the way from health-promoting and prevention activities through treatment during and after a heart attack."

Researchers found the overall rate of death from heart attack, adjusted for age, fell from about 87 deaths per 100,000 people in 1999 to about 38 deaths per 100,000 people in 2020. It is difficult to definitively determine whether the decline is the result of fewer heart attacks occurring or better rates of survival when they do occur because of new diagnostic strategies and treatment options, researchers said. For example, hospitals now frequently test for troponin in the blood when a heart attack is suspected, which can help clinicians diagnose a heart attack at an earlier stage than was possible with previous diagnostic strategies. This change has led to earlier and more sensitive heart attack detection but also makes it challenging to compare data on heart attacks over time.

On the prevention side, the public has become more aware of the need to reduce cardiovascular risk factors through steps such as quitting smoking and managing cholesterol. Clinicians also have a better understanding of the signs of a heart attack and improved tools to quickly diagnose and treat them when they occur. More hospitals are also equipped with mechanical support devices to assist with heart attack treatment and new medications such as potent antiplatelet drugs have become available, which may have improved survival rates and reduced the likelihood of a second heart attack.

Improving 3D Printing of Fine Structures in Artificial Tissue
https://www.fightaging.org/archives/2023/03/improving-3d-printing-of-fine-structures-in-artificial-tissue/

The biggest challenge in tissue printing is the achievement of sufficient control over small scale structure to produce a vasculature that can supply the tissue as it develops. Without that capacity, tissue growth is limited to thin sheets and tiny organoids. Advances have been made in recent years, such as the work of Volumetric, but there is still a way to go before large tissue sections are regularly generated for use in medicine. This is in large part why work on decellularization continues to proceed apace, taking donor tissue and stripping the cells from it to leave the extracellular matrix structure, with all of its fine-scale detail and chemical cues to guide new, patient-matched cells into the correct locations and development activities.

Bioprinting is based on 3D-printing technology, using cells and biopolymer to create biological structures and tissues. One of the most promising types of 3D-bioprinting is called digital light processing (DLP) bioprinting. Within this branch of 3D-bioprinting, progress has been impeded by practical and technical impediments. It has proven difficult to print tissues with high cell densities and finely resolved structures.

DLP-based 3D bioprinting uses a digital micromirror device (DMD) to project a 2D cross-section of the 3D model to the photo-crosslinkable bioink. When exposed to light, the photocrosslinkable bioink, which can be either synthetic or natural, solidifies. Then, a motorized stage lifts up the bioink by a few tens microns to 200 microns, which allows uncured bioink to refill the gap. When the next cross-section is projected to the bioink, a new layer solidifies and the process repeats.

When all goes well, a newly formed layer precisely matches the shape of the projected cross-section. However, with existing methods, the incorporation of cells in the bioink can cause severe light scattering, which blurs the projected light in the bioink. As a result, the newly formed layers cannot replicate the fine details of the projected cross-sections. The researchers reduced this light-scattering effect by tenfold, allowing them to print with high cell densities and high resolution thanks to the contrast agent iodixanol, a new ingredient in the bioink.

Tuning the refractive index of the bioink minimizes the scattering effect and significantly improves the fabrication. The new research shows that a ~50 µm feature size can be achieved in a refractive-index-matched gelatin methacrylate (GelMA) bioink with a cell density as high as 0.1 billion/mL. This approach introduces a few novel technical innovations, including a hollow organic vascular network embedded in a cell-laden thick tissue, enabling it for perfused and long-term culture, and a snow-flake and spoke shape to showcase the high resolution for both positive and negative features.

Considering Proteostasis and Aging
https://www.fightaging.org/archives/2023/03/considering-proteostasis-and-aging/

Proteostasis is the normal maintenance of protein levels and protein structure in a cell. This is disrupted with age, the result of failing quality control, epigenetic change, and other issues. Loss of proteostasis is a hallmark of aging, but has the look of a consequence of aging, not a cause to be addressed. It is also highly complex, and thus progress towards practical therapies is probably better served by a focus on causes of aging rather than the fine details of age-related changes in the cell. Fix the causes, see how well those repair efforts improve long-term health, and then worry about the fine details of the biochemistry of aging.

Proteostasis is the sum of reactions and signalling pathways related to the synthesis, folding, trafficking, disaggregation, and degradation of proteins. One of the hallmarks of aging is a decline in proteostasis. Unsurprisingly, defects in all major steps of proteostasis are related to the accumulation of toxic aggregates and misfolded proteins, a key feature of neurodegenerative diseases. Throughout evolution, a range of protein quality-control mechanisms have emerged, some of which are specialised in monitoring the proteome within specific subcellular compartments. Examples are the cytosolic heat-shock response (HSR), the mitochondrial unfolded protein response (UPRmt), and the unfolded protein response of the endoplasmic reticulum (UPRER). The mechanisms underlying age-related proteostasis collapse are still not completely understood, but studies using Caenorhabditis elegans and mice suggest that it initiates during early adulthood preceding the emergence of age-related diseases.

A fundamental question in biogerontology is why animals lose the ability to maintain proteostasis with aging. A new study observed that an age-related increase in ribosome pausing occurs driven by a reduced activity of the ribosome quality control (RQC) pathway. Another provocative study, in mice, argued that error-prone translation caused by ribosomal ambiguity mutations induces phenotypes that more closely match the progression of Alzheimer's disease than amyloid-β overexpression models do. Their findings suggest that the accumulation of random mutations in DNA over time may induce increased protein misfolding, sequestering away key components of the proteostasis maintenance machinery, such as chaperones, ultimately causing a collapse in proteostasis.

Assessing the Spread of Mitochondrial Mutations in Tissue
https://www.fightaging.org/archives/2023/03/assessing-the-spread-of-mitochondrial-mutations-in-tissue/

There is evidence for mitochondrial DNA mutations to spread throughout a tissue, though the degree to which each of the possible mechanisms contribute to this outcome is unknown. Mitochondrial DNA mutations in stem cells will spread in the same way as nuclear DNA mutation, producing mosaicism. Cells can also transfer mitochondria, however. Further, mitochondria are subject to selection effects based on their continued replication and removal by quality control mechanisms. Thus it is far from clear as to exactly how any observed snapshot of mitochondrial mutations came about. Researchers have taken a swing at this challenge, as noted here, but pay much more attention to substitutions than to deletions that completely disable mitochondrial genes. We might expect that deletions are the more important form of mutation. Nonetheless, the researchers find some evidence for quality control to bias the spread of substitution mutations in favor of those that are less harmful to mitochondrial function.

Accumulation of somatic mutations in the mitochondrial genome (mtDNA) has long been proposed as a possible mechanism of mitochondrial and tissue dysfunction that occurs during aging. A thorough characterization of age-associated mtDNA somatic mutations has been hampered by the limited ability to detect low frequency mutations. Here, we used Duplex Sequencing on eight tissues of an aged mouse cohort to detect more than 89,000 independent somatic mtDNA mutations and show significant tissue-specific increases during aging across all tissues examined which did not correlate with mitochondrial content and tissue function.

G→A/C→T substitutions, indicative of replication errors and/or cytidine deamination, were the predominant mutation type across all tissues and increased with age, whereas G→T/C→A substitutions, indicative of oxidative damage, were the second most common mutation type, but did not increase with age regardless of tissue. We also show that clonal expansions of mtDNA mutations with age is tissue and mutation type dependent. Unexpectedly, mutations associated with oxidative damage rarely formed clones in any tissue and were significantly reduced in the hearts and kidneys of aged mice treated at late age with Elamipretide or nicotinamide mononucleotide. Thus, the lack of accumulation of oxidative damage-linked mutations with age suggests a life-long dynamic clearance of either the oxidative lesions or mtDNA genomes harboring oxidative damage.