Fight Aging! Newsletter, November 11th 2019

Fight Aging! publishes news and commentary relevant to the goal of ending all age-related disease, to be achieved by bringing the mechanisms of aging under the control of modern medicine. This weekly newsletter is sent to thousands of interested subscribers. To subscribe or unsubscribe from the newsletter, please visit:

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  • Support Rejuvenation Research by Donating to the SENS Research Foundation Winter 2019 Fundraiser
  • Assessing Gene Therapy to Upregulate Three Longevity-Associated Genes in Mice
  • GDF11 as a Calorie Restriction Mimetic
  • Cardiomyocytes Expressing SOX10 are Vital to Zebrafish Heart Regeneration
  • TRIB1 Regulates Uptake of Oxidized Lipids into Macrophages, and thus Drives Atherosclerosis
  • Exercise is a Benefit at Any Age
  • Researchers Call for Rigorous Classifications of Aging to Assist Development of Therapies to Treat Aging
  • An Investigation of Adverse Effects of Nicotinamide Riboside Supplementation
  • Towards a Rigorous Definition of Cellular Senescence
  • HDAC9 in Vascular Calcification
  • Modulating Gut Microbe Populations to Generate More Butyrate, thus Raising BDNF Levels and Improving Cognitive Function
  • Success for the MitoMouse Crowdfunding Project
  • Extracellular Vesicles from Embryonic Stem Cells Make Mesenchymal Stem Cells More Effective in Therapy
  • 3-D Printing of Skin with Embedded Vasculature
  • Reviewing Leucine Supplementation as a Treatment for Sarcopenia

Support Rejuvenation Research by Donating to the SENS Research Foundation Winter 2019 Fundraiser

Winter is upon us, and thus the yearly winter SENS Research Foundation fundraiser just recently started a few days ago. For those who don't know, the SENS Research Foundation remains one of the most important and influential organizations working on advancing rejuvenation research to the point at which it can be moved into clinical development and funded by venture capital. Through conferences and advocacy, the SENS Research Foundation staff also played a sizable role in ensuring that there is in fact an active venture capital community eager to back new companies working on the treatment of aging.

All of this work is powered by charitable donations. The SENS Research Foundation is near entirely funded by philanthropy, by the donations made by our community of visionaries, looking forward to a future in which the causes of aging can be treated effectively, and the course of aging turned back. Over the past six years, our community has provided more than 7 million to the SENS Research Foundation to advance the cause of rejuvenation research.

That funding has paid off. Ever more of the rejuvenation research programs funded, advocates, coordinated, and otherwise supported by the SENS Research Foundation have made the leap to commercial development. In 2016 the results of a decade of work on ways to clear out harmful metabolic waste from the lysosome, all funded by philanthropic donations, materialized in the form of LysoClear. That company is developing an enzyme therapy to break down forms of metabolic waste in the retina that cause age-related blindness, and as of this year is close to entering the FDA IND process. Also as of this year, SENS Research Foundation funded research programs have led to companies such as Revel Pharmaceuticals to tackle glucosepane cross-link breaking, Covalent Bioscience to break down transthyretin amyloid via the use of catabodies, and Underdog Pharmaceuticals to prevent and reverse atherosclerosis by removing 7-ketocholesterol from the body.

Consider also that thanks in part to fifteen years of advocacy and support on the part of the SENS Research Foundation and its predecessor, the Methuselah Foundation, clearance of senescent cells as a rejuvenation therapy has finally moved from being ignored by the research community to being a hot area of development in just the past few years. The SENS Research Foundation funded one of the laboratories working on cellular senescence for a number of years, and in 2015 helped to launch Oisin Biotechnologies, a company working on a best of class therapy to remove these unwanted cells.

This is the sort of result we aim for when we support the SENS Research Foundation: to see previously languishing fields relevant to rejuvenation therapies take off in this manner, gaining large-scale funding and widespread support in a short period of time once the tipping point is reached. Further, all of this progress has spurred many other groups to independently work on tackling causes of aging. Take a look at the Aging Biotech Info resource for a list of companies in the longevity industry, a sizable fraction of which are addressing aging in ways that are informed by the SENS viewpoint of rejuvenation achieved via repair of underlying molecular damage. Growth continues.

Yet there are still numerous forms of molecular damage underlying aging for which research programs have yet to fully emerge from the laboratory, or for which the best approaches to rejuvenation are underdeveloped. There is still a huge role for philanthropy and for organizations like the SENS Research Foundation to ensure that the development required for success in the treatment of aging is in fact carried out to completion. We can all see the young and growing senolytics industry as a success story, but there are half a dozen or more other industries needed in order to comprehensively address the causes of aging. These industries do not yet exist in any meaningful way, and cannot exist until the research is carried out.

The future of a truly effective longevity industry still depends on present philanthropy. Support the SENS Research Foundation in their 2019 winter fundraiser, and in doing so you will help to produce the rejuvenation biotechnology success stories of the years ahead.

Assessing Gene Therapy to Upregulate Three Longevity-Associated Genes in Mice

Today's open access research materials report on results obtained in mice using gene therapy to upregulates protein production of several longevity-associated genes. As expected from prior research into these genes and their influence on the operation of metabolism, health is improved in mouse models of age-related disease. As might also be expected based on past results, some combinations are not effective for reasons that remain to be explored: metabolism is complicated, and pulling on levers and turning dials rarely does exactly what was expected.

Evolution does not produce optimal organisms, as seen from the perspective of the individual. This is well illustrated in mice, where any number of single gene alterations, even just dialing up or down the amount of protein produced over time, leads to better health, less disease, longer lives. Why haven't any of these small alterations taken place via random mutation and thereafter prospered and spread through the species over the course of evolutionary time? Because evolutionary competition in the wild is a race to the bottom, in which lineages engineered for early life success at the cost of later life collapse tend to outcompete those with a biochemistry more friendly to the individual.

This applies as much to humans as it does to mice. There are variant human genes that offer much reduced risk of cardiovascular disease, found in a small fraction of the population. Why don't we all have those variants? Because evolution doesn't place much emphasis on late life health and survival. Further, many of the alterations known to improve health and longevity in mice should be expected to at least improve health in humans. So at some point in the years ahead, the use of gene therapies to improve human metabolism so as to reduce age-related disease and improve longevity will be a going concern. It will start with therapies for adults that don't integrate with the genome and are not passed on to children, and at some point, once the will is there, the medical community will start to engineer better human lineages.

This class of approach most likely won't be the most important road to increased longevity in the near future, however. The gains that can be achieved through periodic repair of the human biochemistry that we have today should be far greater than those produced by engineering an improved biochemistry that is more resilient to damage. We know that a youthful mammalian biochemistry works pretty well, and the differences between youth and age emerge from forms of cell and tissue damage, such as accumulation of senescent cells and persistent metabolic waste products. To the degree that the research community can repair that damage, rejuvenation will be the outcome. Yes, that repair may take the form of gene therapies, such as to deliver novel enzymes capable of breaking down metabolic waste, but this is a very different approach in comparison to the type of gene therapy tested in the research noted here, which is an attempt to shift the operation of cellular metabolism into a more resilient state, not to repair damage.

Combination gene therapy treats multiple age-related diseases in mice

Researchers honed in on three genes that had previously been shown to confer increased health and lifespan benefits when their expression was modified in genetically engineered mice: FGF21, sTGFβR2, and αKlotho. They hypothesized that providing extra copies of those genes to non-engineered mice via gene therapy would similarly combat age-related diseases and confer health benefits. The team created separate gene therapy constructs for each gene using the AAV8 serotype as a delivery vehicle, and injected them into mouse models of obesity, type II diabetes, heart failure, and renal failure both individually and in combination with the other genes to see if there was a synergistic beneficial effect.

FGF21 alone caused complete reversal of weight gain and type II diabetes in obese, diabetic mice following a single gene therapy administration, and its combination with sTGFβR2 reduced kidney atrophy by 75% in mice with renal fibrosis. Heart function in mice with heart failure improved by 58% when they were given sTGFβR2 alone or in combination with either of the other two genes, showing that a combined therapeutic treatment of FGF21 and sTGFβR2 could successfully treat all four age-related conditions, therefore improving health and survival. Administering all three genes together resulted in slightly worse outcomes, likely from an adverse interaction between FGF21 and αKlotho, which remains to be studied.

A single combination gene therapy treats multiple age-related diseases

Comorbidity is common as age increases, and currently prescribed treatments often ignore the interconnectedness of the involved age-related diseases. The presence of any one such disease usually increases the risk of having others, and new approaches will be more effective at increasing an individual's health span by taking this systems-level view into account. In this study, we developed gene therapies based on 3 longevity associated genes: fibroblast growth factor 21 (FGF21), αKlotho, soluble form of mouse transforming growth factor-β receptor 2 (sTGFβR2). The gene therapies were delivered using adeno-associated viruses, and we explored their ability to mitigate 4 age-related diseases: obesity, type II diabetes, heart failure, and renal failure.

Individually and combinatorially, we applied these therapies to disease-specific mouse models and found that this set of diverse pathologies could be effectively treated and in some cases, even reversed with a single dose. We observed a 58% increase in heart function in ascending aortic constriction ensuing heart failure, a 38% reduction in α-smooth muscle actin (αSMA) expression, and a 75% reduction in renal medullary atrophy in mice subjected to unilateral ureteral obstruction and a complete reversal of obesity and diabetes phenotypes in mice fed a constant high-fat diet. Crucially, we discovered that a single formulation combining 2 separate therapies into 1 was able to treat all 4 diseases. These results emphasize the promise of gene therapy for treating diverse age-related ailments and demonstrate the potential of combination gene therapy that may improve health span and longevity by addressing multiple diseases at once.

GDF11 as a Calorie Restriction Mimetic

GDF11 was one of the first factors in blood identified as a possible explanation for the outcome of heterochronic parabiosis. When a young and old mouse have their circulatory systems joined, some aspects of aging reverse in the old mouse, and some aspects of aging are accelerated in the young mouse. GDF11 levels decline with age, and it was thought that increased levels of GDF11 provided by the young animal could act to improve function of cells and tissues in the older animal - though it was not well understood as to how GDF11 worked to produce these results.

Since then there has been some debate over whether or not the original GDF11 research was technically correct, as well as some debate over whether or not factors in young blood are in fact responsible for parabiosis effects. Researchers have demonstrated benefits in old mice by delivery of GDF11 as a treatment, however. A company, Elevian, was founded to carry forward the development of GDF11 as basis for clinical therapy.

Meanwhile, research into GDF11 and aging continues elsewhere in the scientific community. Today's very interesting open access research provides evidence for GDF11 to produce benefits in large part through triggering many of the same mechanisms as calorie restriction. If this is the primary mechanism of action, it would make GDF11 much less interesting as a basis for human therapy. Firstly because calorie restriction already exists, and is essentially free, and secondly because the practice of calorie restriction produces much larger effects on life span in short-lived species than it does in long-lived species.

A blood factor involved in weight loss and aging

In a previous study using mouse models, scientists observed that injecting aged mice with blood from young mice rejuvenated blood vessels in the brain, and consequently improved cerebral blood flow, while increasing neurogenesis and cognition. Scientists put forward the theory that, since calorie restriction and supplementation with young blood were effective in rejuvenating organs, they most likely have certain mechanisms in common.

They therefore examined the molecule GDF11, which belongs to the GDF (Growth Differentiation Factor) protein family and is involved in embryonic development. GDF11 was already known to scientists for its ability to rejuvenate the aged brain. By injecting this molecule into aged mouse models, researchers noticed an increase in neurogenesis and blood vessel remodeling. The scientists also observed that the mice administered with GDF11 had lost weight without changing their appetite. This observation led them to believe that GDF11 could be a link between calorie restriction and the regenerating effects of young blood.

The next step was to confirm this theory by studying adiponectin, a hormone secreted by adipose tissue which induces weight loss without affecting appetite. In animals that have undergone calorie restriction, the blood levels of this hormone are high. In animals that were administered GDF11, researchers also observed high levels of adiponectin, and this shows that GDF11 causes metabolic changes similar to those induced by calorie restriction. Until recently, there has been controversy over the role of GDF11 in aging, and its mechanisms were largely unknown. The findings of this study show that by inducing phenomena similar to those reported for calorie restriction leading to the stimulation of adiponectin and neurogenesis, GDF11 contributes to the birth of new neurons in the brain.

Systemic GDF11 stimulates the secretion of adiponectin and induces a calorie restriction-like phenotype in aged mice

Here, we present evidence that GDF11 induces a healthy calorie restriction-like phenotype together with brain rejuvenation in aged mice, and it acts by stimulating the secretion of adiponectin directly on adipocytes. We demonstrate a potent role for GDF11 as a metabolic actor in the aged organism based on the following findings: (a) systemic administration of GDF11 induced healthy weight loss as early as 1 week after treatment, (b) this weight loss reached a plateau throughout the rest of the treatment and was maintained for 3 weeks beyond the end of the treatment, (c) GDF11 levels were increased in aged mice that were subjected to calorie restriction, (d) metabolic changes were independent of GDF15 activation or anorexia, but correlated with changes in adiponectin levels and the insulin/IGF-1 metabolic pathway, (e) GDF11 activated adiponectin secretion directly from adipocytes, and (f) all the above changes correlated with a brain rejuvenation phenotype in aged mice.

Cardiomyocytes Expressing SOX10 are Vital to Zebrafish Heart Regeneration

A few higher animal species, such as salamanders and zebrafish, are capable of regeneration of limbs and internal organs, regrowing lost and injured tissue without scarring or loss of function. Numerous research groups are engaged in investigating the biochemistry of proficient regeneration, attempting to find the specific differences between species that might explain how it happens and why adult mammals are largely incapable of such feats of regrowth. Today's open access research is an example of the type, in which the authors narrow down on a specific cell population that appear in zebrafish hearts during regeneration, but not in human tissues.

It may be the case that the mechanisms and capacity for adult regeneration do still exist in mammals, but are suppressed in some way, as suggested by the fact that the human ARF gene can shut down zebrafish regeneration. After all, we all managed to undertake the process of growing organ tissue during embryonic development. Alternatively perhaps a single crucial part of the adult regeneration mechanisms was lost over evolutionary time, and thus there is an opportunity to reinsert it into mammalian tissues via gene therapy or some other form of modern biotechnology. It still remains to be seen as to whether there are simple paths towards enabling greater adult mammalian regeneration, or, as seems equally likely, the situation is a complex mess that will take decades to decipher, and offers no easy path to therapy.

Special cells contribute to regenerate the heart in Zebrafish

In mammals, including humans, the heart muscle has a very limited capacity to recover after injury. After an acute myocardial infarction, millions of cardiac muscle cells, named cardiomyocytes, die, and are replaced by a scar. Unlike mammals, other vertebrates can recover much better from a cardiac damage. This is the case of some fish, including the zebrafish, a well-established animal model in biomedical research which shares with humans most of its genes.

Zebrafish are extremely well suited to study organ regeneration. After heart injury, zebrafish cardiomyocytes can divide and the scar is replaced by new cardiac muscle. Now the researchers show that not all cardiomyocytes in the zebrafish heart contribute equally to regenerate the lost muscle, but that there is a specific subset of cardiomyocytes with enhanced regenerative capacity.

A small subset of cardiomyocytes in the zebrafish heart, marked by sox10 gene expression, expanded more than the rest of myocardial cells in response to injury. These cells differed from the rest of the myocardium also in their gene expression profile, suggesting that they represented a particular cell subset. Furthermore, experimental erasure of this small cell population, impaired heart regeneration. The researchers want to find out whether the absence of such a sox10 cell population in mammals could explain why their heart does not regenerate well. If this is the case, the researchers believe that this finding could be of great importance in stimulating the repair process in the human heart.

Adult sox10+ Cardiomyocytes Contribute to Myocardial Regeneration in the Zebrafish

Like mammals, zebrafish cardiomyocytes (CMs) derive from first and second heart field progenitors. However, in the zebrafish, the neural crest represents a third progenitor population that contributes to the developing heart. Cell transplantation and fluorescent dye tracing experiments suggested that cardiac neural crest cells incorporate not only into the areas of the outflow tract, as in mammals and birds, but also into the atrium and ventricle. Moreover, genetic lineage tracing using sox10 as a neural crest cell marker revealed a cellular contribution of sox10+ cells to the zebrafish heart and suggested that sox10-derived CMs are necessary for correct heart development. Noteworthy, it is still unclear if a sox10+ neural crest population differentiates into CMs or if alternatively, a sox10+ CM subset is relevant for heart development.

Here, we assessed the contribution of sox10-derived cells to the adult zebrafish heart. We found that embryonic sox10-derived cells contributed to significant portions of the adult heart. We also identified adult sox10+ CMs that expanded to a higher degree upon injury than other CMs and significantly contributed to cardiac regeneration. Their transcriptome differed from other CMs in the heart, and their genetic ablation impaired recovery from ventricular injury.

TRIB1 Regulates Uptake of Oxidized Lipids into Macrophages, and thus Drives Atherosclerosis

Atherosclerosis is a condition of dysfunctional macrophages. Macrophages are responsible for clearing out lipids that end up in blood vessel walls, ingesting these misplaced lipid molecules and handing them off to HDL particles to be carried to the liver for excretion. This works just fine in youth, in an environment of low oxidative stress and few oxidized lipids. Aging brings chronic inflammation, oxidative stress, and oxidized lipids, however. Macrophages cannot process oxidized lipids all that well, and so become pathological, turning into inflammatory foam cells packed with lipids, and unable to do more than send signals for help. The plaques that form to narrow and weaken blood vessels in atherosclerosis might start out as lipid deposits, but become macrophage graveyards as they grow, as ever more macrophages arrive to try and fail to clear the damage.

A number of new approaches to atherosclerosis based on interfering in this process are under development. My company, Repair Biotechnologies, works on a way to allow macrophages to break down oxidized cholesterol in situ. Underdog Pharmaceuticals works on sequestration of the worst oxidized lipid, 7-ketocholesterol. And so forth. The work here, in which researchers identify the gene TRIB1 as a regulator of macrophage uptake of oxidized lipids, offers a new avenue of attack. The evidence presented is fairly compelling for some form of inhibition of TRIB1 to be the basis for a therapy. If this could be done for an extended period of time, then in principle atherosclerosis could be reversed, as macrophages become able to go about their duties and meaningfully clean up atherosclerotic lesions.

Sheffield scientists identify new potential treatment pathway for cardiovascular disease

Research has shown for the first time that a protein expressed in a subset of immune cells contributes towards the build-up of fatty deposits in arteries, which leads to cardiovascular disease. These fatty deposits are caused by macrophages, a subset of immune cells known to take up surplus cholesterol. When this is present in excess, they mature into larger cholesterol-laden cells known as foam cells which accumulate and cause blockages inside arteries. The study shows for the first time that levels of a protein called Tribbles-1 (TRIB1) inside macrophages controls the amount of oxidized cholesterol taken up by foam cells. The research shows that higher levels of TRIB1 increased specific cholesterol uptake receptors, promoting arterial disease, whereas decreasing TRIB1 reduced disease. The findings of this study suggest that inhibiting TRIB1 in macrophages could be a viable therapeutic target in treating cardiovascular disease.

Myeloid Tribbles 1 induces early atherosclerosis via enhanced foam cell expansion

Atherosclerosis, a progressive disease of arterial blood vessels and the main underlying cause of stroke, myocardial infarction, and cardiac death, is initiated by the conversion of plaque macrophages to cholesterol-laden foam cells in the arterial intima. In the early-stage atherosclerotic plaque, this transformation is induced by the uptake of both low density lipoprotein-cholesterol (LDL-C) and oxidized LDL (oxLDL), which may serve a beneficial purpose; but unrestrained, the crucial function of plaque macrophages in resolving local inflammation is compromised, and the development of unstable, advanced lesions ensues.

Tribbles 1 (Trib1) has been detected in murine plaque-resident macrophages, and variants at the TRIB1 locus have been associated with increased risk of hyperlipidemia and atherosclerotic disease in multiple populations. However, no study had examined the macrophage-specific cellular processes dependent on myeloid-specific Trib1 expression and how these tally with the assumed atheroprotective properties of this pseudokinase. At the whole-body level, one study has shown that Trib1-deficient mice have markedly reduced numbers of M2-like macrophages in multiple organs, including adipose tissue. Hence, these studies strongly implicated that loss of macrophage-Trib1 expression within the arterial wall would lead to excessive atherosclerotic plaque inflammation and/or impair inflammation resolution and promote atheroma formation.

In the current study, we found that contrary to expectations, myeloid-specific knockout of Trib1 is atheroprotective, while myeloid-specific Trib1 expression is detrimental. In brief, Trib1 increased OLR1 RNA and protein expression, oxLDL uptake, foamy macrophage formation, and atherosclerotic burden in two distinct mouse models of human disease. The expression of these two genes, as well as those of LPL and SCARB1 (which mediates selective HDL-cholesterol uptake), is also tightly linked in human macrophages. Collectively, our studies reveal an unexpected beneficial effect for selectively silencing Trib1 in arterial plaque macrophages.

Exercise is a Benefit at Any Age

In our modern societies of convenience and luxury, and the overwhelming majority of human societies today are exceedingly convenient and luxurious in comparison to the hunter-gatherer existence of our ancestors, it is the case that most people do not exercise to the degree needed to maintain function with advancing age. If exercise is seen to produce benefits in near all patients of any later age, and it is, that is the case because those patients are not maintaining themselves sufficiently.

Elderly patients are at a higher risk for complications and accelerated physical deconditioning after a cardiovascular event, yet older patients are largely underrepresented in rehabilitation programs. Studies have shown that this might be due to a lack of referral and encouragement to attend cardiac rehabilitation in older patients. Several studies have looked at the effects of cardiac rehabilitation in older adults. However, these data often focus on patients above the age of 65 with no distinction between old and very old patients and examine either physical or psychological outcomes but not both.

The goal of this study was to compare the effects of an exercise-based cardiac rehabilitation program on physical and psychological parameters in young, old, and very old patients. It also aimed to identify the features that best predicted cardiac rehabilitation outcome. Investigators examined 733 patients who completed a 25-session cardiac rehabilitation program. They were divided into three subgroups: less than 65 years old; between 65 and 80 years old; and 80 years or older. Physical and psychological variables such as scores of anxiety and depression were evaluated for all patients before and after cardiac rehabilitation.

Following the intervention, all patients experienced improvements. "We found a few weeks of exercise training not only significantly improved exercise capacity, but also decreased anxiety and depression. Patients with the greatest physical impairments at baseline benefited the most from exercise. Another interesting result was that patients younger than 65 who were very anxious before rehabilitation benefited the most from exercise training. A similar result was found for depressed patients older than 65. These improvements will surely have a great positive impact on patients' independence and quality of life and might help both clinicians and patients to realize how beneficial exercise rehabilitation can be."

Researchers Call for Rigorous Classifications of Aging to Assist Development of Therapies to Treat Aging

The first rejuvenation therapies exist, in the form of senolytic drugs that selectively destroy senescent cells, but no regulatory bodies yet recognize aging as a legitimate target for therapy. A variety of efforts are underway to change this state of affairs, many of which focus on the contents of the International Classification of Diseases (ICD) maintained by the World Health Organization (WHO), which is presently in the process of revision to ICD-11. Incorporating aging into the ICD in a rigorous way would lead, in time, to medical service providers and regulatory bodies worldwide adopting the concept of aging as a condition that can be treated.

Beyond updating the ICD to include aging, other initiatives include the TAME metformin trial, which is an attempt to get the FDA to agree upon trial endpoints that are close enough to aging for practical purposes. Other factions, and I fall into this camp, are of the opinion that real progress will emerge from battles with the regulators over widespread off-label use of cheap rejuvenation therapies that are approved for other conditions, with senolytic treatments such as the dasatinib and quercetin combination leading the way there.

An international team of researchers has put forward a call to action to governments, the WHO, and the scientific and medical communities to come together and develop classifications and staging systems utilizing the framework as the basis for diagnosing and treating age-related diseases, including directly treating all aging tissues and organs. Age-related diseases without adequate diagnostic criteria and severity staging limit the ability for prevention or treatment and the ability to develop new drugs and interventions. Ultimately, this impacts the quality of life for older members of society.

Currently the classification and severity staging of age-related diseases is limited because it is inconsistent, incomplete, and non-systematic. Some types of disease that can be found in many organs, such as intrinsic organ aging, or organ atrophy or wasting, are classified in one organ but not others. Experts, scientists, and physicians have created a position statement which lays out a framework for properly and comprehensively classifying and staging the severity of age-related diseases. Importantly the statement includes aging at the tissue and organ level as well as organ atrophy, pathologic remodelling and calcification, and age-related systemic and metabolic diseases.

While aging is classified as a condition within the WHO International Classification of Diseases (ICD-11) in relation to intrinsic skin aging and photoaging, the framework proposes the classification of aging as conditions in all organs, along with the comprehensive classification of all aging-related diseases and syndromes. As part of this work, initial classification submissions related to age-related diseases in line with the framework have already been submitted to the latest version of WHO ICD-11.

An Investigation of Adverse Effects of Nicotinamide Riboside Supplementation

Nicotinamide riboside is so far the only approach to NAD+ upregulation for which there is published human trial data, though other trials for other approaches are underway at the present time. NAD+ declines with age, for reasons that remain comparatively poorly understood, and this has a negative impact on mitochondrial function. Thus there is considerable enthusiasm at the moment for intervening in this known manifestation of aging by tackling the proximate causes, raising NAD+ levels, but without addressing underlying causes.

Researchers here find the potential for adverse effects on glucose metabolism and white adipose tissue function to result from nicotinamide riboside supplementation, but there are a great many details involved: dietary differences and genetic differences in mice appear important as to whether problems arise, and the final sections of the discussion in the paper are worth reading closely. It is hard to say whether or not the discoveries made in mice that are reported in this open access paper will apply to humans, but the specific details suggest that investigation is warranted.

Nicotinamide riboside (NR) is a nicotinamide adenine dinucleotide (NAD+) precursor vitamin. The scarce reports on the adverse effects on metabolic health of supplementation with high-dose NR warrant substantiation. Here, we aimed to examine the physiological responses to high-dose NR supplementation in the context of a mildly obesogenic diet and to substantiate this with molecular data. An 18-week dietary intervention was conducted in male C57BL/6JRccHsd mice, in which a diet with 9000 mg NR per kg diet (high NR) was compared to a diet with NR at the recommended vitamin B3 level (control NR). Both diets were mildly obesogenic (40 en% fat). Metabolic flexibility and glucose tolerance were analyzed and immunoblotting, qRT-PCR, and histology of epididymal white adipose tissue (eWAT) were performed.

Mice fed with high NR showed a reduced metabolic flexibility, a lower glucose clearance rate and aggravated systemic insulin resistance. This was consistent with molecular and morphological changes in eWAT, including sirtuin 1 (SIRT1)-mediated PPARγ (proliferator-activated receptor γ) repression, downregulated AKT/glucose transporter type 4 (GLUT4) signaling, an increased number of crown-like structures and macrophages, and an upregulation of pro-inflammatory gene markers. In conclusion, high-dose NR induces the onset of WAT dysfunction, which may in part explain the deterioration of metabolic health.

Towards a Rigorous Definition of Cellular Senescence

The accumulation of lingering senescent cells is a significant cause of aging, disrupting tissue function and generating chronic inflammation throughout the body. Even while the first senolytic drugs capable of selectively destroying these cells already exist, and while a number of biotech companies are working on the production of rejuvenation therapies based on clearance of senescent cells, it is still the case that much remains to be settled when it comes to the biochemistry of these errant cells. More rigorous consensus definitions relating to the mechanisms, manifestations, and tissue specificity of senescence have yet to be pinned down. This process will proceed in parallel with the development of more effective therapies, as is often the case.

Cellular senescence is a cell state implicated in various physiological processes and a wide spectrum of age-related diseases. Recently, interest in therapeutically targeting senescence to improve healthy aging and age-related disease, otherwise known as senotherapy, has been growing rapidly. Thus, the accurate detection of senescent cells, especially in vivo, is essential. Here, we present a consensus from the International Cell Senescence Association (ICSA), defining and discussing key cellular and molecular features of senescence and offering recommendations on how to use them as biomarkers.

Over the last decade, improved experimental tools have significantly advanced our knowledge about causes and phenotypic consequences of senescent cells. However, specific markers and a consensus on the definition of what constitutes senescent cells are lacking. Further, although a link to organismal aging is clear, aging and senescence are not synonymous. Indeed, cells can undergo senescence, regardless of organismal age, due to myriad signals including those independent of telomere shortening. Consequently, senescent cells are detected at any life stage from embryogenesis, where they contribute to tissue development, to adulthood, where they prevent the propagation of damaged cells and contribute to tissue repair and tumor suppression. Thus, cellular senescence might be an example of evolutionary antagonistic pleiotropy or a cellular program with beneficial and detrimental effects.

Cellular senescence is a cell state triggered by stressful insults and certain physiological processes, characterized by a prolonged and generally irreversible cell-cycle arrest with secretory features, macromolecular damage, and altered metabolism. Senescent cells secrete a plethora of factors, including pro-inflammatory cytokines and chemokines, growth modulators, angiogenic factors, and matrix metalloproteinases (MMPs), collectively termed the senescent associated secretory phenotype (SASP) or senescence messaging secretome (SMS). The SASP constitutes a hallmark of senescent cells and mediates many of their patho-physiological effects. The SASP composition and strength varies substantially, depending on the duration of senescence, origin of the pro-senescence stimulus, and cell type.

The role of senescence in human disease is clear from cellular studies, while in vivo evidence is only now catching up. Evidence linking senescence to other common age-associated human diseases has recently emerged. These diseases include neurodegenerative disorders, glaucoma, cataract, atherosclerosis and cardiovascular disease, diabetes, osteoarthritis, pulmonary, and renal, and liver fibrosis. In most studies, senescence is assessed in ex vivo cultures or fresh samples by SA-β-gal staining or indirect markers in formalin-fixed tissues. Since SA-β-gal is not suitable for fixed tissues, analyzing senescence in human samples is challenging. Despite promising results from other individual markers, no marker is completely senescence specific. We recommend combining cytoplasmic (e.g., SA-β-gal, lipofuscin), nuclear (e.g., p16INK4A, p21WAF1/Cip1, Ki67) and SASP, context, and/or cell-type-specific markers.

HDAC9 in Vascular Calcification

Researchers here show that HDAC9 plays a role in the calcification of blood vessel walls, a process that contributes to the stiffening of blood vessels that leads to hypertension and all of the damage that chronic raised blood pressure causes to delicate tissues throughout the body. That mice lacking HDAC9 are more resistant to calcification suggests that there may be a mechanism here that can serve as the basis for a therapy to slow down the progression of calcification in human tissues. That said, it is worth comparing effort such as this with the potential for senolytic drugs to achieve similar results, based on the evidence for senescent cells to contribute to vascular calcification.

Arterial wall calcification is the buildup of calcium in the blood vessel walls, which can often be a predictor of serious cardiovascular events like heart attacks and strokes. A new study looked at more than 11,000 people and found patients with significant blood vessel calcification were more likely to have a specific variant of HDAC9. This high-risk variant of HDAC9 is present in about 25 percent of the population. In follow-up mouse studies, the researchers also found HDAC9 caused abnormal changes in the cells of the vessel walls, resembling that of human bone cells.

"Our research proved HDAC9 is not just associated with cardiovascular disease but can actually cause it by changing the makeup of those vascular cells. We then investigated it at the molecular level and looked at what would happen if we knocked out HDAC9." The researchers found that inhibiting HDAC9 in mice preserved normal function in vascular cells and prevented vascular calcification, therefore identifying HDAC9 as a target for the potential treatment of cardiovascular disease. "Currently, there are no heart drugs available to patients that would prevent this type of hardening of the arteries. These findings are exciting in that they harness genetics to open the door for future pathways to heart disease prevention."

Modulating Gut Microbe Populations to Generate More Butyrate, thus Raising BDNF Levels and Improving Cognitive Function

The microbial populations of the gut have an influence on health and the progression of aging via the molecules that they generate, and which our cells react to. It isn't entirely clear as to the ordering of cause and effect in the detrimental changes that take place with aging in intestinal tissue, immune system, diet, and microbial populations. Studies have shown, however, that restoring more youthful populations can influence the function of tissues throughout the body, including the brain. The authors of this open access paper discuss modulating gut microbial populations in rats so as to upregulate butyrate production and BDNF levels, thereby improving some aspects of cognitive function. Similar examples exist in the literature for a range of other organs and tissues; it is an interesting area of research, though ultimately the size of the effects are probably not all that different from those relating to exercise or diet.

Neuroinflammation is correlated with a decline in cognitive function and memory, primarily because inflammation of the hippocampus tends to cause deleterious changes in synaptic transmission and plasticity. Because BDNF helps to sustain and enhance long-term potentiation (LTP) induction, it serves an essential role in cognitive function. Aging is associated with decreased levels of BDNF, suggesting that the maintenance of adequate BDNF concentrations could potentially help to preclude or delay the onset of cognitive impairment.

One convenient way to raise BDNF levels is supplementation of butyrate, a short-chain fatty acid (SCFA) that functions as a histone deacetylase inhibitor. Butyrate maintains the relaxation of chromatin and thereby enhances BDNF expression in the hippocampus. Secretion of pro-inflammatory cytokines may also be inhibited by BDNF, as the latter molecule interferes with activation of nuclear factor-kappa beta (NF-κβ). In addition, the expression of enzymes involved in the production of glutathione (GSH) may also be triggered by butyrate secretion. GSH is an antioxidant enzyme that relieves oxidative stress - another neurodegenerative risk factor.

The intestinal microbiota is responsible for a significant proportion of SCFA production. However, levels of SCFA decline with age due to dysbiosis, a microbial imbalance that often results in a considerable increase in pathological bacteria (Proteobacterium) at the expense of mutualistic ones (Bifidobacterium). Progression of gut dysbiosis has been linked to chronic systemic inflammation, including inflammation of the brain. Supplementation with probiotics and prebiotics may counteract the damaging effects that aging has on the brain by not only lessening inflammation and oxidative stress but also by increasing neurotrophic factors and neuronal plasticity.

A study was conducted to test how probiotic and prebiotic supplementation impacted spatial and associative memory in middle-aged rats. The results showed that rats supplemented with the symbiotic (both probiotic and prebiotic) treatment performed significantly better than other groups in the spatial memory test, though not in that of associative memory. The data also showed that this improvement correlated with increased levels of BDNF, decreased levels of pro-inflammatory cytokines, and better electrophysiological outcomes in the hippocampi of the symbiotic group. Thus, the results indicated that the progression of cognitive impairment is indeed affected by changes in microbiota induced by probiotics and prebiotics.

Success for the MitoMouse Crowdfunding Project

The latest mitochondrial rejuvenation research project to be crowdfunded by the and SENS Research Foundation teams focused on proving out allotopic expression of mitochondrial gene ATP8 in mice with a loss of function mutation in that gene. I'm pleased to note that the community rallied around and the project was fully funded, including its stretch goals.

Mitochondria have their own small genome; allotopic expression is the process of placing a copy of a mitochondrial gene into the nuclear genome, suitably altered to enable the proteins produced to find their way back to the mitochondria where they are needed. This backup source of proteins allows mitochondria to function normally even when their own DNA is damaged. The technique, when applied to single genes, allows for the treatment of inherited mitochondrial conditions, as demonstrated by Gensight Biologics. More importantly, however, when applied to all thirteen mitochondrial genes it will prevent mitochondrial DNA damage from contributing to the aging process.

The MitoMouse campaign has ended, and what a final few days it has been! Thanks to the efforts of the community, an amazing total of 77,525 has been raised in support of this mitochondrial repair project of the SENS Research Foundation. There were 319 people who backed the project and helped to make this the most successful fundraiser on to date, even higher than the previous record breaker, the NAD+ mouse project. This is very impressive and shows that support for the field is growing and that the tide has really turned.

We would like to give special thanks to, which generously stepped in once we reached the first stretch goal and agreed to fully fund the project all the way to the second stretch goal! Thanks to LongeCity, the project not only hit the final stretch goal, which greatly expanded the scope of the project, the total funds raised went well over that goal. We are confident that the extra money will be put to good use by the MitoMouse team, and a few more boxes of mouse food and laboratory supplies are sure to come in handy.

Big thanks to everyone who supported the project, including the team at SENS Research Foundation, John Saunders, the Foster Foundation, Patrick Deane, and the volunteers and staff at LEAF for pulling together to make this happen. Hopefully, MitoMouse will enjoy the same success as the previous MitoSENS project, and we will be one step closer to having a solution to mitochondrial damage and potential cures for inherited mitochondrial conditions as well as age-related diseases.

Extracellular Vesicles from Embryonic Stem Cells Make Mesenchymal Stem Cells More Effective in Therapy

Mesenchymal stem cell (MSC) is a category so broad as to be near meaningless, but many varieties are widely used for therapeutic purposes. MSCs are taken from any one of a variety of sources, expanded in culture, and introduced to the patient. Researchers here show that applying extracellular vesicles from embryonic stem cells to the cultured MSCs reduces the usual issues that arise when expanding cells in culture, such as senescence, and improves the effectiveness of MSCs as a therapy when tested in mice. On a practical basis, one would imagine that induced pluripotent stem cells would serve just as well as a source of extracellular vesicles with this capability.

Mesenchymal stem cells (MSCs), derived from several kinds of tissues such as placenta, umbilical cord, bone marrow, and adipose tissue, are multipotent stem cells that can differentiate into many cell types. MSCs have been recognized as important candidates for the treatment of many degenerative diseases or injuries. Furthermore, MSCs can be expanded by continuously passage in vitro, to obtain a sufficient number of cells that can be used for clinical applications. Along with the continuous passage in vitro, MSCs exhibit the senescence-associated features, including enlarged morphology, irreversible growth arrest, enhanced SA-β-gal activity, decreased stemness of stem cells, increased cell apoptosis and DNA damage foci, and telomere attrition.

For senescent MSCs, the characteristics of stem cell are lost, and their therapeutic effects are limited. Therefore, researchers attempt to find a better way to block the cellular senescence. Mouse embryonic stem (ES) cells, derived from the blastocyst stage embryos, are distinguished by their ability to self-renew and differentiate into all cell types. The major barriers to the possible transplantation of ES cells into patients are immune rejection and the risk of forming tumors. It has been reported that conditioned medium from ES cells (ES-CM) has beneficial effects on cell proliferation and tissue regeneration via the factors secreted from ES cells.

Recently studies suggested that extracellular vesicles (EVs), which are biological particles released by many cell types, could be considered for therapeutic utility. The EVs transfer proteins and nucleic acids between cells and play an important role in the target cells. Moreover, EVs isolated from various types of stem cells have different properties such as anti-apoptosis, pro-angiogenesis, and anti-fibrosis. In this study, we explored the effects of EVs derived from ES cells (ES-EVs) on the senescent MSCs. Our results indicated that ES-EVs rejuvenated the senescent MSCs and enhanced their therapeutic effects in a mouse cutaneous wound model.

3-D Printing of Skin with Embedded Vasculature

Researchers continue to take incremental steps towards the creation of engineered living tissues containing the vascular networks needed to support it. Absent blood vessels, numerous varieties of functional tissue can be generated from cell samples: lung, liver, kidney, and so forth. These organoids are limited in size to a few millimeters, however, the distance the nutrients and oxygen can perfuse. Generating blood vessel networks is a serious technical challenge, and the major obstacle to the production of entire organs for transplantation. Consider that natural capillary networks exhibit a density of hundreds of vessels passing through every square millimeter of tissue cross-section. Even the best of present efforts are distant from that scale, though in laboratory demonstrations they suffice to produce essentially functional larger tissue sections.

Researchers have developed a way to 3D print living skin, complete with blood vessels. The advancement is a significant step toward creating grafts that are more like the skin our bodies produce naturally. "Right now, whatever is available as a clinical product is more like a fancy Band-Aid. It provides some accelerated wound healing, but eventually it just falls off; it never really integrates with the host cells."

A significant barrier to that integration has been the absence of a functioning vascular system in the skin grafts. Researchers have been working on this challenge for several years, previously showing that they could take two types of living human cells, make them into "bio-inks," and print them into a skin-like structure. Researchers now show that if they add key elements - including human endothelial cells, which line the inside of blood vessels, and human pericyte cells, which wrap around the endothelial cells - with animal collagen and other structural cells typically found in a skin graft, the cells start communicating and forming a biologically relevant vascular structure within the span of a few weeks.

"As engineers working to recreate biology, we've always appreciated and been aware of the fact that biology is far more complex than the simple systems we make in the lab. We were pleasantly surprised to find that, once we start approaching that complexity, biology takes over and starts getting closer and closer to what exists in nature." Once the team grafted the engineered tissue onto a mouse, the vessels from the printed skin began to communicate and connect with the mouse's own vessels. In order to make this usable at a clinical level, researchers need to be able to edit the donor cells using something like the CRISPR technology, so that the vessels can integrate and be accepted by the patient's body. "We are still not at that step, but we are one step closer,."

Reviewing Leucine Supplementation as a Treatment for Sarcopenia

Sarcopenia is the name given to the characteristic loss of muscle mass and strength that takes place with advancing age. A surprisingly large fraction of this loss is self inflicted: few people undertake the necessary exercise and strength training to maintain muscle in later life. But the rest of the losses are to some degree inevitable, a consequence of damage and disarray in muscle stem cells, neuromuscular junctions, and various processes necessary to muscle tissue maintenance. There is evidence for one those issues to be a growing inability to process leucine, an essential amino acid. Leucine supplementation may thus slow the onset of sarcopenia, even while being a compensatory approach that in no way addresses the underlying causes of this form of age-related degeneration.

One of the main ways in which sarcopenia contributes to disease is that it alters muscular turnover and metabolism. Moreover, older adults exhibit a decreased anabolic response to protein feeding, which is a mechanism underpinning the loss of muscle mass in sarcopenic individuals. Compared to younger adults, those aged over 65 years require ∼70% more protein per meal to maximally stimulate muscle protein synthesis. Furthermore, at a global level, only 40% of older adults meet the recommended daily allowance for protein (0.8 g/kg/day) and 10% of older women do not even meet the estimated average requirement of 0.66 g/kg/day.

One strategy to increase the muscle protein synthesis that has been investigated is the supplementation of diets with leucine, an essential branched-chain amino acid with important regulatory actions in muscles, which are at least partially mediated by the mammalian target of the rapamycin pathway. Leucine modifies protein turnover in skeletal muscles, by decreasing proteolysis and by increasing protein synthesis. Physiological research reports have shown that leucine can enhance muscle protein-synthesis. Furthermore, leucine can stimulate insulin release by pancreatic cells, showing that besides its beneficial effect in enhancing skeletal muscle glucose uptake, it is also an important anabolic signal in skeletal muscle.

Based on the above, administration of leucine-containing supplements is therefore a promising approach for treating sarcopenia. We took a systematic approach to analysing the current scientific evidence in this area, and to ascertaining whether the administration of leucine-containing supplements is effective in the treatment of sarcopenia. We also included interventions that used whey protein as a supplement, because these contain large amounts of leucine (approximately 13 g leucine/100 g protein) and the consumption of whey protein appears to be the most effective at increasing muscle protein synthesis.

In overall terms, published study results show that administration of leucine or leucine-enriched proteins (in a range of 1.2 g to 6 g leucine/day) is well-tolerated and significantly improves sarcopenia in elderly individuals, mainly by improving lean muscle-mass content and in this case most protocols also include vitamin D co-administration. The effect of muscular strength showed mixed results, however, and the effect on physical performance has seldom been studied.

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