The Next Recommendation on Lowering Cholesterol May be to Start Earlier

The clinical work on lowering blood cholesterol that has taken place over recent years has demonstrated that if there is a lower limit beyond which low cholesterol levels become harmful, then that limit is very low indeed. Certainly below 10% of the normal human level. There are a number of uncommon mutations that produce individuals with up to half of the normal amount of blood cholesterol, people who exhibit significantly reduced risk of cardiovascular disease as a result of this difference from the norm. This is all quite interesting: why did we evolve to have the blood cholesterol that we do, if we need only a small fraction of it?

The reason why lowering blood cholesterol lowers the risk of cardiovascular disease is that atherosclerosis is caused by the dysfunction of macrophage cells in an environment rich in oxidized lipids. Atherosclerosis is age-related because oxidative stress, and thus amounts of oxidized lipids, rise with age. A lesser degree of all blood lipids means a lesser degree of oxidized lipids in the blood stream, entering blood vessel walls to aggravate and kill macrophages. This in turn results in a slowed progression of atherosclerosis. Unfortunately it really doesn't help all that much to remove existing fatty lesions produced by the processe of atherosclerosis, and it doesn't do more than slow the progression of the condition. Lowered blood cholesterol only raises the odds of avoiding the consequences of atherosclerosis, meaning stroke and heart attack when a weakened artery or large lesion ruptures, because a few years of delay are enough to allow some other consequence of aging to kill the patient first.

The advent of new and very efficient tools for lowering blood cholesterol, such as PCSK9 inhibitors, present the medical community with something of a quandary. The technology for lowering blood cholesterol has reached its natural limit. One really can't go much lower, and yet it isn't enough. It still only slows atherosclerosis, and cannot meaningfully reverse the corroded state of arteries and their fatty lesions. The research community is investigating other avenues beyond the lowering of blood cholesterol typified by statins and PCKS9 inhibitors, but in the meanwhile what should the medical community focus on? The next logical step appears to be starting treatment earlier, with an even greater focus on prevention and target metrics in healthier individuals, rather than on treating the manifestations of clinical disease.

Cholesterol lowering: to live longer, start younger?

Cardiovascular disease, particularly coronary heart disease and stroke, is a major cause of death globally. Since older age is an independent predictor of increased cardiovascular risk, the global burden of cardiovascular disease increases as populations age. Lowering low density lipoprotein (LDL) cholesterol has become an important strategy for reducing the risk of atherosclerotic cardiovascular disease (ASCVD). Recent evidence has shown that the benefits of this approach extend to patients over 75 years. Though LDL lowering is beneficial across middle to older age, two questions still arise: how early in the disease process should LDL lowering be initiated; and, are the currently recommended LDL targets ambitious enough?

It has been estimated 10-year ASCVD risk for an untreated 50 year old Caucasian male with a systolic/diastolic blood pressure of 140/90 mmHg and LDL cholesterol level of 3.4 mmol/L, is 5.3%. A 60 year old male with the same risk factors has a 10-year ASCVD risk of 11.8%, which increases to 22.6% at age 70. Older age appears to be a marker of the cumulative exposure to LDL cholesterol, along with other traditional cardiovascular risk factors. Delaying treatment until the cardiovascular risk is above a certain threshold will lead to additional years of exposure to this cumulative burden. Initiating statin therapy at say, the age of 50 rather than 60 years old, will prevent an additional 10 mmol/L/year LDL cholesterol exposure, or in other words, provide an additional 10 mmol/L/year of LDL cholesterol reduction.

More complex and older atheromatous plaques only partially regress with LDL lowering therapies, which means delayed treatment will leave older individuals at considerable residual risk of ASCVD. It makes sense to advocate for a greater focus on the lifetime exposure to elevated LDL and the benefit of LDL cholesterol lowering over longer periods of time. A primordial prevention strategy would target the development of atherosclerosis rather than simply preventing its complications. This hypothesis is supported by cohort studies which showed exposure to elevated blood pressure and cholesterol levels during young adulthood is associated with a greater risk of ASCVD later in life, independent of later adult exposures.

An Epigenetic Clock for Skeletal Muscle

Epigenetic clocks are multiplying year by year. Each is a weighted algorithmic combination of the status of various methylation sites on the genome, built by analyzing the epigenome of many different people at different ages in order to arrive at correlations with chronological age, or, more usefully, with metrics such as mortality risk that reflect biological age, the burden of molecular damage and its consequences. This process of building a new clocks is the easier half of the challenge, however. The hard part, that still lies ahead, is to determine what exactly it is that these clocks measure. What do these characteristic epigenetic changes of age actually reflect, in terms of the underlying processes of aging? That is a challenging question to answer well, but answers are needed if epigenetic clocks are to be used to speed up development of rejuvenation therapies by measuring biological age before and after a short treatment.

Ageing is associated with DNA methylation changes in all human tissues, and epigenetic markers can estimate chronological age based on DNA methylation patterns across tissues. However, the construction of the original pan-tissue epigenetic clock did not include skeletal muscle samples and hence exhibited a strong deviation between DNA methylation and chronological age in this tissue. To address this, we developed a more accurate, muscle-specific epigenetic clock based on the genome-wide DNA methylation data of 682 skeletal muscle samples from 12 independent datasets.

In the current study, we aimed to address the poor performance of the pan-tissue clock in muscle by developing a muscle-specific epigenetic clock. We hypothesise that by using a large number of human skeletal muscle DNA methylation profiles, we can develop a muscle-specific epigenetic clock that outperforms the pan-tissue clock and that can estimate chronological age with high accuracy. We utilised DNA methylation data to estimate epigenetic age in a total of 682 male and female skeletal muscle samples aged 18-89. We also conducted an epigenome-wide association study (EWAS) to discover genes whose methylation change with age in skeletal muscle.

The newly developed clock uses 200 cytosine-phosphate-guanine dinucleotides to estimate chronological age in skeletal muscle, 16 of which are in common with the 353 cytosine-phosphate-guanine dinucleotides of the pan-tissue clock. This new clock significantly outperforms the previous pan-tissue clock and can calculate the epigenetic age in skeletal muscle with a mean accuracy of 4.9 ± 4.5 years across 682 samples. This muscle clock will be of interest and potential use to researchers, clinicians, and forensic scientists working in the fields of skeletal muscle, chronic diseases, and ageing. In the future, we intend to evaluate how environmental factors, such as exercise and diet, could influence ageing via this newly developed clock.


USP7 Inhibition Clears Up to Half of Irradiation Induced Senescent Cells From Mouse Tissues

Researchers here report on the discovery of a novel mechanism by which senescent cells can be selectively destroyed. Short-term senolytic treatments to date seem to cluster tightly into two categories: (a) largely ineffective, and (b) able to destroy between 25-50% of senescent cells in tissues. Few have achieved greater clearance so far, and few lie in between these two outcomes. In the present environment, of ample seed stage funding and enthusiasm for targeting senescent cells as a treatment for aging, it seems likely that someone will pick up this new approach for clinical development in the near future.

Although cellular senescence is an important tumor-suppressive mechanism, emerging evidence demonstrates that the accumulation of senescent cells (SnCs) with age and after genotoxic or cytotoxic cancer therapy can lead to various age-related diseases and pathological conditions. The selective removal of SnCs depends on identifying their Achilles' heels, which can be targeted to selectively kill SnCs. Several senolytic targets have been identified, resulting in the discovery of a series of senolytic agents that can selectively kill SnCs in culture and effectively remove SnCs in mice. Unfortunately, some of these agents exhibit toxicities that may prevent their safe use in the clinic, particularly for systemic therapy. For example, navitoclax, a selective BCL-2/BCL-XL dual inhibitor, is a potent senolytic agent but can induce thrombocytopenia, an on-target and dose-limiting toxicity that has prevented its FDA-approval. Therefore, further studies are needed to identify new senolytic targets that can be exploited for the development of safer senolytic agents.

Here we show that ubiquitin-specific peptidase 7 (USP7) is a novel target for senolysis because inhibition of USP7 with an inhibitor or genetic depletion of USP7 by RNA interference induces apoptosis selectively in SnCs. The senolytic activity of USP7 inhibitors is likely attributable in part to the promotion of the human homolog of mouse double minute 2 (MDM2) ubiquitination and degradation by the ubiquitin-proteasome system. This degradation increases the levels of p53, which in turn induces the pro-apoptotic proteins PUMA, NOXA, and FAS and inhibits the interaction of BCL-XL and BAK to selectively induce apoptosis in SnCs. Further, we show that treatment with a USP7 inhibitor can effectively eliminate SnCs and suppress the senescence-associated secretory phenotype (SASP) induced by doxorubicin in mice. These findings suggest that small molecule USP7 inhibitors are novel senolytics that can be exploited to reduce chemotherapy-induced toxicities and treat age-related diseases.


Forthcoming Longevity Industry Conferences, March to May 2020

If you are interested in joining or investing in the growing longevity industry, there are now a fair few conferences taking place each year at which it is possible to meet people, get involved, and make inroads on building a network. Since this is still a young industry, it remains a very friendly, close-knit community in which many of the participants have been involved in research or patient advocacy in the aging field for quite some time. Aging research is in many ways still a small field of research in which everyone tends to know everyone else in the inner circles, and the present longevity industry is just the first steps on a long road towards become the majority of all medical development. Growth lies ahead, and now is a great time to become involved.

Master Investor, London, March 28th 2020

The Master Investor conference series is one of ways in which Jim Mellon's network acts to promote the longevity industry: that it is a wondrous opportunity to change the human condition for the better and make a return on investment while doing it. It isn't enough to start a venture like Juvenescence and invest in a score or more of biotech startups working interventions in the aging process, one must also sell the rest of the investment community and the broader biotech community on the nature of this opportunity, leading to the establishment of bigger pools of funding for later stage companies as they emerge. None of this automatically happens; every step forward requires someone to do the work of persuasion, networking, coordination. Master Investor is a part of this process, and thus a good place for people who are not already on the inside of the longevity industry to meet people who are.

Longevity Leaders World Congress, London, April 21st 2020

Longevity Leaders World Congress is one of a range of biotech conferences run by the LSX organization in the US and UK. This remains distinctive by virtue of drawing in interested factions not present in large numbers at the other conferences yet, such as the life insurance and pensions industries. This conference is a good example of the dynamism that results when putting scientists, entrepreneurs, and investors in the same room for a few days, all interested in seeing progress towards the medical control of aging. The establishment of a growing community capable of advancing promising research from the lab to the clinic requires exactly this sort of gathering.

Undoing Aging, Berlin, May 21st 2020

Undoing Aging is of course the most important of the conferences in which academia meets industry, to discuss how to move forward towards working rejuvenation therapies. Is is organized by the SENS Research Foundation and Forever Healthy Foundation, and the tone is thus very much more ambitious than is the case elsewhere. The goal is the end of aging, not just slowing it down a little. Being held in central Europe, there is a very different mix of investors and advocates than is found in the US - a different end of the international community. It was quite a large gathering in 2019, overflowing the venue, and promises to be much the same this year.

The Skin Microbiome Better Correlates with Chronological Age than the Gut Microbiome

Researchers here report on their assessment of age-related changes in human skin, oral, and gut microbiomes. They find that the skin microbiome is a better marker of chronological age, which might also be taken as indicating that it is a worse tool for measuring biological age. That adherence to chronological age is, after all, observed despite differences in the pace of aging that exist between individuals. A good biomarker of aging is one that reflects the burden of damage and consequent mortality, and therefore will measure a higher age in someone more burdened, rather than only correlating with the chronological progression of time.

Human gut microbiomes are known to change with age, yet the relative value of human microbiomes across the body as predictors of age, and prediction robustness across populations is unknown. In this study, we tested the ability of the oral, gut, and skin (hand and forehead) microbiomes to predict age in adults using data combined from multiple publicly available studies, evaluating the models in each cohort individually.

Intriguingly, the skin microbiome provides the best prediction of age (mean ± standard deviation, 3.8 ± 0.45 years, versus 4.5 ± 0.14 years for the oral microbiome and 11.5 ± 0.12 years for the gut microbiome). This also agrees with forensic studies showing that the skin microbiome predicts postmortem interval better than microbiomes from other body sites. Age prediction models constructed from the hand microbiome generalized to the forehead and vice versa, across cohorts, and results from the gut microbiome generalized across multiple cohorts (United States, United Kingdom, and China).

Interestingly, microbial taxa enriched in young individuals (18 to 30 years) tend to be more abundant and more prevalent than taxa enriched in elderly individuals (older than 60 yrs), suggesting a model in which physiological aging occurs concomitantly with the loss of key taxa over a lifetime, enabling potential microbiome-targeted therapeutic strategies to prevent aging.


Calorie Restriction Suppresses the Senescence-Associated Secretory Phenotype

The accumulation of senescent cells is one of the causes of aging, important in the chronic inflammation of aging, and disruptive of tissue structure and function. The practice of calorie restriction slows the progression of aging, much more so in short-lived animals than in long-lived species, and thus we should expect it to have some effect on cellular senescence. Calorie restriction is nowhere near as effective as senolytic drugs at reducing the populations of senescent cells present in older individuals, that much is evident from studies carried out in recent years. As noted here, however, it does appear to somewhat reduce the inflammatory signaling generated by senescent cells.

Chronic inflammation, a pervasive feature of the aging process, is defined by a continuous, multifarious, low-grade inflammatory response. It is a sustained and systemic phenomenon that aggravates aging and can lead to age-related chronic diseases. In recent years, our understanding of age-related chronic inflammation has advanced through a large number of investigations on aging and calorie restriction (CR). A broader view of age-related inflammation is the concept of senoinflammation, which has an outlook beyond the traditional view.

Senescent cells produce a proinflammatory senescence-associated (SA) secretome, which is referred to as the SASP. Macrophages are recruited by chemotactic factors in the secretome to clear senescent cells. However, senescent macrophages secrete proinflammatory cytokines and exhibit impaired phagocytosis and chemotaxis, and a downregulated rate of cellular proliferation. It has been proposed that deficiency in the ability of aged macrophages to clear senescent cells leads to increased inflammatory response and results in chronic inflammation as SASP plays a role in the initiation of tissue inflammation. Based on previous observations and evidence of the aging process at molecular and cellular levels, we coined the term senoinflammation to provide an expanded, broader view of age-related chronic inflammation and metabolic dysfunction.

Based on studies on senoinflammation and CR, we recognized that the senescence-associated secretory phenotype (SASP), which mainly comprises cytokines and chemokines, was significantly increased during aging, whereas it was suppressed during CR. Further, we recognized that cellular metabolic pathways were also dysregulated in aging; however, CR mimetics reversed these effects. Oxidative stress leads to improper gene regulation and genomic DNA damage during aging. Such improper gene regulation in aged senescent cells allows them to fall into a proinflammatory state, consequently changing systemic chemokine or cytokine activities. The proinflammatory SASP environment further exerts stress on the intracellular organelles, tissues, and systems, which affects the development and occurrence of metabolic disorders.

It appears that a repetitive vicious cycle occurs between SASP and metabolic dysregulation as proposed in the concept of senoinflammation, and this interactive network forms the basis of the aging process and age-related diseases. However, the secretion of proinflammatory mediators, collectively termed as SASP, in response to internal and external stress leads to the chronic inflammatory condition termed as senoinflammation. Based on CR experiments and observations, cytokine, chemokine, and metabolic pathways are significantly regulated by CR and CR mimetics in the aging process.


Preventing Oligomerization of β-arrestin-2 Improves Clearance of Tau via Autophagy

In today's research materials, scientists report on the discovery of a maladaptive response to the presence of tau aggregates in brain cells, one that makes the situation worse than it would otherwise be. Tau is one of a small number of proteins that can become altered in a way that ensures other molecules of the same protein also alter. They join together and precipitate into solid structures, known as neurofibrillary tangles in the case of tau, accompanied by a halo of disrupted biochemistry that is harmful to cell and tissue function. This spreads, seeding dysfunction as it moves from cell to cell, or throughout a tissue between cells.

Cells do attempt to fight back against the spread of broken proteins and their aggregates. Multiple mechanisms allow cells to ingest and break down aggregates present between cells, and aggregates inside cells are also fed into the same recycling machinery. It is perhaps the case that neurodegenerative conditions are age-related in large part because the machinery of autophagy, an important recycling mechanism in cells, degrades with age. The efforts to reduce molecular waste such as protein aggregates falter.

Here, researchers have found that an oligomerized form of β-arrestin-2 acts to interfere with the processes of autophagy as they attempt to remove aggregated tau protein. Normally cells recycle unwanted protein machinery and damaged structures by delivering these materials to a lysosome to be broken down, but an important component of autophagy is inhibited by oligomerized β-arrestin-2. Interestingly, preventing β-arrestin-2 from adopting this unhelpful form reduces tau pathology in mouse models of tauopathy with no apparent side-effects.

Beta-arrestin-2 increases neurotoxic tau driving frontotemporal dementia

Researchers have discovered that a form of the protein comprised of multiple β-arrestin-2 molecules, known as oligomerized β-arrestin-2, disrupts the protective clearance process normally ridding cells of malformed proteins like disease-causing tau. Monomeric β-arrestin-2, the protein's single-molecule form, does not impair this cellular toxic waste disposal process known as autophagy. The study focused on frontotemporal lobar degeneration (FTLD), also called frontotemporal dementia - second only to Alzheimer's disease as the leading cause of dementia. This aggressive, typically earlier onset dementia (ages 45-65) is characterized by atrophy of the front or side regions of the brain, or both. Like Alzheimer's disease, FTLD displays an accumulation of tau, and has no specific treatment or cure.

Both in cells and in mice with elevated tau, β-arrestin-2 levels are increased. Furthermore, when β-arrestin-2 is overexpressed, tau levels increase, suggesting a maladaptive feedback cycle that exacerbates disease-causing tau. Genetically reducing β-arrestin-2 lessens tauopathy, synaptic dysfunction, and the loss of nerve cells and their connections in the brain. Oligomerized β-arrestin-2 - but not the protein's monomeric form - increases tau.

Oligomerized β-arrestin-2 increases tau by impeding the ability of cargo protein p62 to help selectively degrade excess tau in the brain. In essence, this reduces the efficiency of the autophagy process needed to clear toxic tau, so tau "clogs up" the neurons. Blocking of β-arrestin-2 oligomerization suppresses disease-causing tau in a mouse model that develops human tauopathy with signs of dementia. "We also noted that decreasing β-arrestin-2 by gene therapy had no apparent side effects, but such a reduction was enough to open the tau clearance mechanism to full throttle, erasing the tau tangles. This is something the field has been looking for - an intervention that does no harm and reverses the disease."

β-Arrestin2 oligomers impair the clearance of pathological tau and increase tau aggregates

Multiple G protein-coupled receptors (GPCRs) are targets in the treatment of dementia, and the arrestins are common to their signaling. β-Arrestin2 was significantly increased in brains of patients with frontotemporal lobar degeneration (FTLD-tau), a disease second to Alzheimer's as a cause of dementia. Genetic loss and overexpression experiments using genetically encoded reporters and defined mutant constructs in vitro, and in cell lines, primary neurons, and tau P301S mice crossed with β-arrestin2 knockout mice, show that β-arrestin2 stabilizes pathogenic tau and promotes tau aggregation. Cell and mouse models of FTLD showed this to be maladaptive, fueling a positive feedback cycle of enhanced neuronal tau via non-GPCR mechanisms.

Genetic ablation of β-arrestin2 markedly ablates tau pathology and rescues synaptic plasticity defects in tau P301S transgenic mice. Atomic force microscopy and cellular studies revealed that oligomerized, but not monomeric, β-arrestin2 increases tau by inhibiting self-interaction of the autophagy cargo receptor p62/SQSTM1, impeding p62 autophagy flux. Hence, reduction of oligomerized β-arrestin2 with virus encoding β-arrestin2 mutants acting as dominant-negatives markedly reduces tau-laden neurofibrillary tangles in FTLD mice in vivo. Reducing β-arrestin2 oligomeric status represents a new strategy to alleviate tau pathology in FTLD and related tauopathies.

A Survey of the Evidence for Hormesis to Slow Aging in Nematode Worms

Hormesis is the name given to the process by which lesser degrees of cellular stress and damage can result in long-term benefits to health. Cells react to molecular damage with greater repair and maintenance activities, and when that damage occurs transiently and minimally, the additional efforts to maintain function outweigh any detrimental effects. This can slow aging and extend life in a range of short-lived species. Hormesis depends on complex biochemistry, however, and similar approaches to triggering it can easily fall on the wrong side of the line, causing too much damage, or not enough of a maintenance response. Researchers here review the literature in search of a consensus in the matter of hormesis and aging in the widely studied nematode species C. elegans.

The concept of hormesis arouses great interest, because it is a near-universal and reproducible phenomenon. As a beneficial compensatory response triggered by mild stress, hormetic individuals generally exhibit better performance than the untreated controls, and the potential anti-aging effect of hormesis has attracted more attention. It seems promising to apply hormesis in aging intervention, which is evidenced by multiple studies, like the beneficial effects of moderate exercise-induced hormesis on body function and aging-related diseases. However, there are still considerable debates regarding the origin and mechanisms of aging and hormesis, such as the conflicting evidence related to the role of reactive oxygen species (ROS) in aging.

At present, most researchers take a wait-and-see attitude to hormetic treatment for human health, due to the contradictory evidence. Thus it is meaningful to conduct a systematic assessment on the existed evidences in the absence of large-scale empirical research on the correlation between hormesis and aging/anti-aging. Meta-analysis is a powerful tool to synthesize multiple or even conflicting evidence to get a clear and reliable final-evidence, achieving the purpose of quantitative review. in order to thoroughly assess the effect of hormesis on aging, in this work, 26 papers documenting the changes of aging-related indicators induced by hormesis in Caenorhabditis elegans (a significant model organism in aging research due to its unique biological features like short life cycle, strong reproductive ability and clear genetic background) were meta-analyzed.

Meta-analytic results indicated that hormesis could significantly extend the mean lifespan of C. elegans by 16.7% and 25.1% under normal and stress culture conditions, respectively. The healthspan assays showed that hormesis remarkably enhanced the bending frequency and pumping rate of worms by 28.9% and 7.0%, respectively, while effectively reduced the lipofuscin level by 15.9%. The obviously increased expression of dauer formation protein-16 (1.66-fold) and its transcriptional targets, including superoxide dismutase-3 (2.46-fold), catalase-1 (2.32-fold) and small heat shock protein-16.2 (2.88-fold), was one of the molecular mechanisms underlying these positive effects of hormesis. This meta-analysis provided strong evidence for the anti-aging role of hormesis, highlighting its lifespan-prolonging, healthspan-enhancing, and resistance-increasing effects on C. elegans. Given that dauer formation protein-16 is highly conserved, hormesis offers the theoretical possibility of delaying intrinsic aging through exogenous intervention among humans.


Individual Risk of Dementia is Falling, but an Aging Population Means that a Greater Incidence of Dementia Lies Ahead

The risk of suffering many of the most common age-related conditions is trending downwards over time, thanks to improvements in medical technology and public health, but the ever increasing size of the older segments of the population means that the incidence of those age-related conditions will nonetheless grow over time. Growth in the number of older people outweighs the reduced risk for any given individual, or at least this will be the case without much faster progress towards effective therapies than has taken place over the past few decades.

This is of great concern for those who focus more on socioeconomics than on health. The economic stress placed on centralized, government-run medical systems by this trend is perhaps one of the stronger motivations driving large-scale investment into research and development. Evidently still not strong enough, given that intervening in the aging process remains a tiny field in comparison to the rest of medicine, but we can hope that this will change given concrete results from the first rejuvenation therapies.

Across men and women and across most age groups, there has been a reduction in the prevalence of dementia over the past ten years when compared to 2008 estimates. The number of people living with dementia in the European Union (EU27) is estimated to be 7.8 million and in European countries represented by AE members, 9.7 million. Compared to its earlier estimates, this constitutes a significant reduction from 8.7 million for the EU27 and from 10.9 million for the broader European region. Women continue to be disproportionately affected by dementia with 6.6 million women and 3.1 million men living with dementia in Europe. The numbers of people with dementia in Europe will almost double by 2050 increasing to 14.3 million in the European Union and 18.8 million in the wider European region.

"It is promising to see that healthier lifestyles, better education, and improved control of cardiovascular risk factors seem to have contributed to a reduction of the prevalence of dementia. However, our report also demonstrates that the number of people living with the condition is set to increase substantially in the years ahead, which will only place greater pressure on care and support services unless better ways of treating and preventing dementia are identified. If people with dementia, their families and carers are to receive the high-quality and person-centred care they need, governments must ensure their health and care systems are ready to meet this demand and greater investments in research into the treatment and prevention of dementia are needed."


A Process Used by Cells to Ingest Misfolded Proteins Might be Enhanced to Treat Neurodegenerative Conditions

The aggregation of misfolded proteins is a feature of most neurodegenerative conditions: amyloid-β and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, and so forth. These and a few more of the countless proteins present in the body can become altered, such as via misfolding, in ways that encourage other molecules of the same protein to also alter, forming structures of linked, harmful proteins that can spread through tissue or from cell to cell. Cells, particularly immune cells, are equipped with a range of mechanisms to identify and break down these problem proteins, but, for reasons that are not fully understood at the detail level, damage outpaces maintenance in the aging brain. Aggregates grow and spread, leaving a trail of cellular dysfunction and death in their wake.

In today's research materials, scientists report on their exploration of a process by which cells ingest and break down misfolded extracellular proteins. Amyloid-β might be the most interesting example of such proteins, given its role in Alzheimer's disease. The researchers note evidence for upregulation of the operation of this maintenance process to reduce the impact of amyloid-β aggregation on brain tissue; we shall see whether this progresses to the point of producing therapies in the years ahead. Certainly researchers are quite interested in upregulating the operation of processes such as autophagy and proteasomal degradation of proteins that are focused on breaking down problem proteins inside cells, so why not also enhance the ability of cells to maintain their surroundings as well?

Researchers discover how cells clear misfolded proteins from tissues

A number of diseases are believed to be caused by the gradual buildup of misfolded proteins that can aggregate together and damage neurons and other cells in the body. To help prevent this damage, cells have developed numerous quality control systems that recognize misfolded proteins within the cell and either fold them back into their correct shape or else degrade them before they start to aggregate. However, approximately 11% of human proteins exist outside of the cell, where they are subjected to even more stresses that may cause them to misfold. Alzheimer's disease is characterized by aggregates of amyloid-β protein in the extracellular space. Despite this, how aberrant extracellular proteins are degraded remains poorly understood.

A protein called Clusterin can bind to misfolded extracellular proteins and prevent them from aggregating. Researchers discovered that Clusterin can escort misfolded proteins into the cell and deliver them to the cell's garbage-disposal units - the lysosomes - where they can be degraded. The researchers also discovered that, after binding to misfolded proteins, Clusterin enters cells by binding to proteins known as heparan sulfate proteoglycans, which are present on the surface of almost all human cells. Together, Clusterin and heparan sulfate proteoglycans allow many different cell types to internalize and degrade a wide variety of misfolded extracellular proteins.

Intriguingly, the researchers also found that Clusterin and heparan sulfate proteoglycans can import amyloid-β into cells for degradation. Mutations in the gene encoding Clusterin have been linked to an increased risk of developing Alzheimer's disease, and experiments in rats have shown that injecting Clusterin into the brain can prevent amyloid β-induced neurodegeneration. "Our results therefore suggest new avenues for the possible treatment or prevention of disorders such as Alzheimer's disease that are associated with aberrant extracellular proteins."

Heparan sulfate is a clearance receptor for aberrant extracellular proteins

The accumulation of aberrant proteins leads to various neurodegenerative disorders. Mammalian cells contain several intracellular protein degradation systems, including autophagy and proteasomal systems, that selectively remove aberrant intracellular proteins. Although mammals contain not only intracellular but also extracellular proteins, the mechanism underlying the quality control of aberrant extracellular proteins is poorly understood. Here, using a novel quantitative fluorescence assay and genome-wide CRISPR screening, we identified the receptor-mediated degradation pathway by which misfolded extracellular proteins are selectively captured by the extracellular chaperone Clusterin and undergo endocytosis via the cell surface heparan sulfate (HS) receptor.

Biochemical analyses revealed that positively charged residues on Clusterin electrostatically interact with negatively charged HS. Furthermore, the Clusterin-HS pathway facilitates the degradation of amyloid β peptide and diverse leaked cytosolic proteins in extracellular space. Our results identify a novel protein quality control system for preserving extracellular proteostasis and highlight its role in preventing diseases associated with aberrant extracellular proteins.

Wnt/β-Catenin Signaling as a Point of Intervention to Spur Greater Neural Regeneration

Wnt signaling is a complicated but well studied portion of the regulatory systems governing regeneration. Numerous groups are engaged in the commercial development of regenerative therapies that are based on manipulation of this part of mammalian biochemistry. Of the more prominent ventures, Samumed has reached phase 3 trials with treatments of this type. This open access paper walks through the evidence for Wnt signaling to be a useful point of intervention for researchers aiming to increase neurogenesis and regenerative capacity in the aging brain. This will be developed as a means of treating neurodegenerative conditions, but success would lead to treatments beneficial for all older individuals.

A common hallmark of age-dependent neurodegenerative diseases is an impairment of adult neurogenesis. Wingless-type mouse mammary tumor virus integration site (Wnt)/β-catenin (WβC) signalling is a vital pathway for dopaminergic (DAergic) neurogenesis and an essential signalling system during embryonic development and aging, the most critical risk factor for Parkinson's disease (PD). To date, there is no known cause or cure for PD. Here we focus on the potential to reawaken the impaired neurogenic niches to rejuvenate and repair the aged PD brain.

Specifically, we highlight WβC-signalling in the plasticity of the subventricular zone (SVZ), the largest germinal region in the mature brain innervated by nigrostriatal DAergic terminals, and the mesencephalic aqueduct-periventricular region (Aq-PVR) Wnt-sensitive niche, which is in proximity to the substantia nigra pars compacta and harbors neural stem progenitor cells (NSCs) with DAergic potential. The hallmark of the WβC pathway is the cytosolic accumulation of β-catenin, which enters the nucleus and associates with T cell factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors, leading to the transcription of Wnt target genes. Here, we underscore the dynamic interplay between DAergic innervation and astroglial-derived factors regulating WβC-dependent transcription of key genes orchestrating NSC proliferation, survival, migration, and differentiation.

Aging, inflammation, and oxidative stress synergize with neurotoxin exposure in "turning off" the WβC neurogenic switch via down-regulation of the nuclear factor erythroid-2-related factor 2/Wnt-regulated signalosome, a key player in the maintenance of antioxidant self-defense mechanisms and NSC homeostasis. Harnessing WβC-signalling in the aged PD brain can thus restore neurogenesis, rejuvenate the microenvironment, and promote neurorescue and regeneration.


Nicotinamide Mononucleotide Supplementation Improves Neurovascular Function in Aged Mice

Supplementation of nicotinamide mononucleotide (NMN) is one of the approaches demonstrated to restore levels of NAD+ in the mitochondria of aged mice. NAD+ is lost with age, with the proximate causes involving declining synthesis and recycling of the molecule, but as yet there is little understanding as to how this connects to the deeper causes of degenerative aging. NAD+ is an important piece of molecular machinery in mitochondrial function, and its loss appears to be one of the factors causing a failure of mitophagy, the quality control mechanism responsible for removing worn and dysfunctional mitochondria.

Mitochondria are responsible for packaging the chemical energy store molecules, adenosine triphosphate (ATP), used to power cellular operations, and when they falter, the cell suffers. Tissue function is disrupted. This is particularly important in energy-hungry tissues such as muscles and the brain, but has an impact throughout the body. Restoring NAD+ levels appears to help; clinical trials to measure effect size in humans have not yet taken place for NMN, but initial results for nicotinamide riboside (NR) supplementation suggest that the beneficial effects on cardiovascular function in older people are in the same ballpark as those of exercise.

Aging-induced structural and functional alterations of the neurovascular unit lead to impairment of neurovascular coupling responses, dysregulation of cerebral blood flow, and increased neuroinflammation, all of which contribute importantly to the pathogenesis of age-related vascular cognitive impairment (VCI). There is increasing evidence showing that a decrease in NAD+ availability with age plays a critical role in age-related neurovascular and cerebromicrovascular dysfunction. Our recent studies demonstrate that restoring cellular NAD+ levels in aged mice rescues neurovascular function, increases cerebral blood flow, and improves performance on cognitive tasks.

To determine the effects of restoring cellular NAD+ levels on neurovascular gene expression profiles, 24-month-old C57BL/6 mice were treated with nicotinamide mononucleotide (NMN), a key NAD+ intermediate, for 2 weeks. Transcriptome analysis of preparations enriched for cells of the neurovascular unit was performed by RNA-seq. Neurovascular gene expression signatures in NMN-treated aged mice were compared with those in untreated young and aged control mice. We identified 590 genes differentially expressed in the aged neurovascular unit, 204 of which are restored toward youthful expression levels by NMN treatment. The transcriptional footprint of NMN treatment indicates that increased NAD+ levels promote SIRT1 activation in the neurovascular unit. Pathway analysis predicts that neurovascular protective effects of NMN are mediated by the induction of genes involved in mitochondrial rejuvenation, anti-inflammatory, and anti-apoptotic pathways.

In conclusion, the recently demonstrated protective effects of NMN treatment on neurovascular function can be attributed to multifaceted sirtuin-mediated anti-aging changes in the neurovascular transcriptome. Our present findings taken together with the results of recent studies using mitochondria-targeted interventions suggest that mitochondrial rejuvenation is a critical mechanism to restore neurovascular health and improve cerebral blood flow in aging.


Linking Gum Disease with the Progression of Atherosclerosis and Risk of Stroke

Atherosclerosis is a condition characterized by the growth of fatty lesions, atheromas, in blood vessel walls. This narrows and weakens blood vessels, leading to heart disease, and then ultimately the fatal rupture or blockage of a major vessel that causes a heart attack or stroke. This degeneration of the arteries is a universal process. It occurs to various degrees in every older person, and kills perhaps a sixth of humanity at the present time. The only reason that it doesn't kill everybody is that other degenerative process of aging manage to get in first, that data suggesting that this is most likely only a matter of a few years of difference. We are as old as our arteries, as they say.

The pace at which atherosclerosis progresses is strongly driven by chronic inflammation. Excessive and constant inflammation is a feature of aging, with some people much worse off than others, and this variation in inflammatory burden goes a long way towards determining who will die, earlier than might otherwise be the case, due to the consequences of atherosclerosis. To understand why inflammation is important to this age-related condition, one needs to know something about the underlying processes that cause atherosclerotic lesions to form.

Atherosclerosis is, in essence, a condition of dysfunctional macrophages. These immune cells are responsible for clearing out unwanted, excessive, or damaged lipids, such as cholesterols, from blood vessel walls. Those lipids are handed off to HDL particles to be returned to the liver for disposal. This works just fine in youth. With age, however, a greater fraction of circulating lipids become oxidized, and macrophages do not handle oxidized lipids well at all. They become dysfunctional foam cells, packed with lipids, and issuing inflammatory signals that draw in more macrophages to suffer the same fate. An atherosclerotic lesion is a macrophage graveyard, seeded by oxidized lipids, and growing via a feedback loop of inflammatory signaling and failing, dying macrophages.

If there is greater systemic inflammation in the body, more macrophages will be drawn to lesions. It is also the case that macrophages attempting to clean up those lesions will be more prone to adopt an unhelpful inflammatory state if greater inflammatory signaling is present in the environment. Further, inflammation and excessive amounts of oxidizing molecules go hand in hand: more inflammation will thus tend to mean more oxidized cholesterols in circulation. This all contributes to a more rapid pace of progression for atherosclerosis.

What causes one older person to have a greater level of chronic inflammation than another? One important variation between individuals is the status of chronic infection. Is there a greater burden of, to pick one important example, herpesviruses such as cytomegalovirus that cannot be cleared unaided by the human immune system and that corrode the immune system over time? Another feature is the one examined in today's research materials, the health of gums. A range of evidence suggests that the localized inflammation of gum disease does not stay localized, and in fact spreads chronic inflammation throughout the body, harming the heart and the brain - and accelerating the progression of atherosclerosis, thus raising the risk of suffering a stroke.

Gum disease, inflammation, hardened arteries may be linked to stroke risk

Two studies raise the possibility that treating gum disease alongside other stroke risk factors might reduce the severity of artery plaque buildup and narrowing of brain blood vessels that can lead to a new or a recurrent stroke. However, these two studies could not conclusively confirm a cause-and-effect relationship between gum disease and artery blockage or stroke risk. "Gum disease is a chronic bacterial infection that affects the soft and hard structures supporting the teeth and is associated with inflammation. Because inflammation appears to play a major role in the development and worsening of atherosclerosis, or 'hardening' of blood vessels, we investigated if gum disease is associated with blockages in brain vessels and strokes caused by atherosclerosis of the brain vessels."

In the first study, researchers examined 265 patients (average age of 64) who experienced a stroke between 2015 and 2017, analyzing whether gum disease was associated with specific types of stroke. They found that large artery strokes due to intracranial atherosclerosis were twice as common in patients with gum disease as in those without gum disease. Further, patients with gum disease were three times as likely to have a stroke involving blood vessels in the back of the brain, which controls vision, coordination, and other vital bodily functions. Gum disease was more common in patients who had a stroke involving large blood vessels within the brain, yet not more common among those who had a stroke due to blockage in blood vessels outside the skull.

The second study focused on 1,145 people who had not experienced a stroke, selected from the Dental Atherosclerosis Risk in Communities (DARIC) Study. Researchers used two magnetic resonance images to measure blockages in arteries inside the brain. Participants were an average age of 76. Periodontal examinations were used to classify the presence and severity of gum disease. Researchers found that arteries in the brain were severely blocked (50% or more) in 10% of participants, and people with gingivitis, inflammation of the gums, were twice as likely to have moderately severe narrowed brain arteries from plaque buildup compared to those with no gum disease. After adjusting for risk factors such as age, high blood pressure, and high cholesterol, people with gingivitis were 2.4 times as likely to have severely blocked brain arteries.

Longevity-Related Genes are Under Greater Evolutionary Constraint in Large and Long-Lived Mammals

Both the evolution of aging and the comparative biology of aging between species with widely divergent lifespans are fascinating topics, though likely of limited relevance to the near future of rejuvenation therapies. Those treatments will be based on repairing cell and tissue damage as it occurs, using the understanding of the human metabolism that exists now, rather than on attempts to rebuild that human metabolism to age more slowly, a goal that will require a great deal more knowledge. The more distant future will certainly include human populations engineered from birth to exhibit enhanced longevity, however, and a greater understanding of the intersection between metabolism, genetics, and species longevity will be an essential part of that endeavor.

Although lifespan in mammals varies over 100-fold, the precise evolutionary mechanisms underlying variation in longevity remain unknown. Species-specific genetic changes have been observed in long-lived species including the naked mole-rat, bats, and the bowhead whale, but these adaptations do not generalize to other mammals. We present a novel method to identify associations between rates of protein evolution and continuous phenotypes across the entire mammalian phylogeny. Unlike previous analyses that focused on individual species, we treat absolute and relative longevity as quantitative traits and demonstrate that these lifespan traits affect the evolutionary constraint on hundreds of genes.

Specifically, we find that genes related to cell cycle, DNA repair, cell death, the IGF1 pathway, and immunity are under increased evolutionary constraint in large and long-lived mammals. For mammals exceptionally long-lived for their body size, we find increased constraint in inflammation, DNA repair, and NFKB-related pathways. Strikingly, these pathways have considerable overlap with those that have been previously reported to have potentially adaptive changes in single-species studies, and thus would be expected to show decreased constraint in our analysis. This unexpected finding of increased constraint in many longevity-associated pathways underscores the power of our quantitative approach to detect patterns that generalize across the mammalian phylogeny.


Evidence for Better Blood Supply to the Hippocampus to Slow Cognitive Decline

As outlined in the research reported here, the variable physiology of the hippocampus allows for an interesting natural experiment to determine the degree to which blood supply is important in the aging of the brain. It is known that capillary density declines with age throughout the body, and this affects the supply of oxygen and nutrients to tissues. The brain is a particularly energy hungry organ, and reduced supply produces consequences. It isn't just capillary density that is important in aging, however, but also the general decline in physical fitness and ability of the heart to pump blood uphill to the brain. This lost performance becomes particularly profound in heart failure patients, but is no doubt producing detrimental consequences even when present to a lesser degree.

The hippocampus exists twice: once in each brain hemisphere. It is considered the control center of memory. Damage to the hippocampus, such as it occurs in Alzheimer's and other brain diseases, is known to impair memory. But what role does blood supply in particular play? To answer this questions, researchers used high-resolution magnetic resonance imaging (MRI) to examine the blood supply to the hippocampus of 47 women and men aged 45 to 89 years. The study participants also underwent a neuropsychological test battery, which assessed, in particular, memory performance, speech comprehension, and the ability to concentrate.

"It has been known for some time that the hippocampus is supplied by either one or two arteries. It also happens that only one of the two hippocampi, which occur in every brain, is supplied by two vessels. This varies between individuals. The reasons are unknown. Maybe there is a genetic predisposition. However, it is also possible that the individual structure of the blood supply develops due to life circumstances. Then the personal lifestyle would influence the blood supply to the hippocampus. In the cognition tests, those study participants in whom at least one hippocampus was doubly supplied generally scored better. The fact that the blood supply is fundamentally important for the brain has been extensively documented. We were therefore particularly focused on the hippocampus and the situation of a disease of the brain vessels. Little is actually known about this."

Of the study subjects, 27 did not manifest signs of brain diseases. The remaining twenty participants showed pathological alterations in brain blood vessels, which were associated with microbleeding. "In these individuals, sporadic cerebral small vessel disease had been diagnosed prior to our investigations. These individuals exhibited a broad spectrum of neurological anomalies, including mild cognitive impairment. The healthy subjects generally scored better on cognitive tests than the study participants with small vessel disease. Among the participants with disease, those with at least one hippocampus supplied by two arteries reached better scores in cognition. They particularly benefited from the double supply. This may be due to a better supply not only of blood but also of oxygen."


Evidence for Bacterial DNA to Promote Tau Aggregation in Neurodegeneration

The field of Alzheimer's disease research is in the midst of a slow-moving and consequential debate over the role of infection in the development of the condition. The fundamental question is this: in the absence of genetic variants that raise risk, why do only some people progress to full blown Alzheimer's disease? The presence - in only some people - of sufficient degrees of persistent infection is one possible answer to that question. Candidates include herpesviruses, oral bacteria such as P. gingivalis, lyme disease spirochetes, and others.

Alzheimer's is a condition characterized by amyloid-β aggregation in its early stages and tau aggregation in its later, more severe stages. The classic amyloid cascade view of the condition is that amyloid-β aggregation sets the stage for immune dysfunction and chronic inflammation leading to tau aggregation. The debate over whether or not persistent infection instead lies at the root of the condition has so far largely focused firstly on amyloid-β as an anti-microbial peptide, a part of the innate immune system that may be upregulated by infection, and secondly on the chronic inflammation that results from infection, as inflammation in the brain clearly strongly drives tau pathology.

Here, however, researchers offer evidence for the presence of bacterial DNA to accelerate the processes of tau aggregation through mechanisms independent of inflammation. Some forms of bacterial DNA may help to seed the aggregates of tau that can then spread independently. The challenge will be, as ever, to determine which of these various processes is the important one, which has the larger contribution. That is hard to accomplish without selectively blocking each disease process in isolation and observing the results.

Bacterial DNA promotes Tau aggregation

In addition to being one of the most devastating diseases of the 21st century, Alzheimer's disease (AD) remains incurable. The cognitive symptoms and neurodegeneration appear to be mostly related to the extensive synaptic dysfunction and neuronal death observed in the brain. In turn, neuronal loss and synaptic damage appears to be mediated by the progressive misfolding, aggregation, and deposition of amyloid-β (Aβ) and tau proteins forming protein aggregates able to spread from cell-to-cell by a prion-like mechanism. Genetics alone cannot account for the complex process of protein misfolding, aggregation and subsequent neurodegeneration observed in AD, particularly because the large majority of the cases are not associated to genetic mutations. Thus, it is likely that diverse environmental factors and age-related abnormalities play an important role on the initiation of the pathological abnormalities. In this sense, various studies have shown that bacterial infection, as well as alterations in the intestinal microbiome may be implicated in the AD pathology.

Here, we report the first evidence for the capacity of extracellular DNA from certain bacterial species to substantially promote tau misfolding and aggregation. The promoting effect of DNA on tau aggregation was observed in a wide range of concentrations from 10 to 1000 ng. The use of these concentrations were informed by the range of cerebrospinal fluid DNA concentrations observed in patients with different diseases: 1-600 ng/mL. The sources of bacterial and fungal DNA were selected based on the literature and personal data that showed associations of certain microorganisms with AD. Among the bacteria previously cultivated directly from the brains of patients with AD, or those whose components (such as nucleic acids, lipopolysaccharides, enzymes) were identified in the cerebrospinal fluid, amyloid plaques, or brains of patients with AD, we used the DNA from B. burgdorferi, P. gingivalis, C. albicans, and E. coli.

Our data indicate that DNA from various, unrelated gram-positive and gram-negative bacteria significantly accelerated Tau aggregation. One of the best promoters was DNA from E. coli species, which is interesting for several reasons. First, it was demonstrated that some strains of E. coli were detectable immunocytochemically in brain parenchyma and vessels in AD patients more frequently compared to control brains. Second, E. coli and P. gingivalis are known to share properties of facultative intracellular parasites and be localized within hippocampal neurons; the latter finding is significant, as the hippocampus is extensively damaged in AD. The intracellular localization of E. coli introduces unique possibilities regarding the interaction of bacterial DNA with tau proteins inside the neuron; e.g. DNA can be secreted via transportation to the outer membrane or released following prophage induction and directly access the host neuron's cytosol, where tau is normally present. Of note, in brains of patients with AD, P. gingivalis is also localized intracellularly; therefore, as in the case of E. coli the same processes for the intracellular interaction of its DNA with tau are applicable for this microorganism.

Future studies should further investigate the possible role of DNA as an initial seeding factor for protein misfolding using cellular and in vivo models as well as the effect of DNA on inducing misfolding of other proteins, including those associated with neurodegeneration, autoimmune diseases, and cancer. Moreover, subsequent studies should explore the targeting of DNA as a therapeutic strategy to prevent tau aggregation.

Correction of Mitochondrial Dysfunction as an Approach to Treat Heart Failure

Mitochondria are the power plants of the cell, and when their activity falters, cell and tissue function suffers as a consequence. Unfortunately mitochondrial dysfunction is a feature of aging, and is connected to the progression and severity of numerous age-related conditions. The research here examines age-related mitochondrial decline in the context of heart failure. As is appropriate for this new era of intervention in the aging process, the focus is on what might be done about this. At the very least, the evidence suggests that even early approaches that can only somewhat restore mitochondrial function in the old, such as NAD+ upregulation or mitochondrially targeted antioxidants, might produce benefits in heart failure patients. Better methodologies capable of greater restoration of mitochondrial function are very much required, however.

The burden of heart failure (HF) in terms of health care expenditures, hospitalizations, and mortality is substantial and growing. The failing heart has been described as "energy-deprived" and mitochondrial dysfunction is a driving force associated with this energy supply-demand imbalance. Existing HF therapies provide symptomatic and longevity benefit by reducing cardiac workload through heart rate reduction and reduction of preload and afterload but do not address the underlying causes of abnormal myocardial energetic nor directly target mitochondrial abnormalities.

Numerous studies in animal models of HF as well as myocardial tissue from explanted failed human hearts have shown that the failing heart manifests abnormalities of mitochondrial structure, dynamics, and function that lead to a marked increase in the formation of damaging reactive oxygen species (ROS) and a marked reduction in on demand adenosine triphosphate (ATP) synthesis. Correcting mitochondrial dysfunction to enhance the energy supply of the failing heart to meet the desired energy needs offers considerable potential to improve cardiac function, reduce symptoms, and improve exercise tolerance in HF, and ultimately offer improved quality of life and survival for patients, and reduce the overall economic burden of this condition.

Elamipretide (SS-31) is a water-soluble, aromatic-cationic mitochondria-targeting tetrapeptide that readily penetrates and transiently localizes to the inner mitochondrial membrane and associates with cardiolipin to restore mitochondrial bioenergetics. Studies in dogs with coronary microembolization-induced chronic HF showed that 3 months of treatment with daily subcutaneous injections of elamipretide improved left ventricle (LV) systolic function and prevented progressive LV dilation without affecting heart rate, blood pressure, or systemic vascular resistance. Elamipretide also elicited a normalization of mitochondrial function evidenced by improved respiration, normalization of membrane potential, reduced ROS formation, and improved maximum rate of ATP synthesis.


Reduced Generation of New Oligodendrocytes May Contribute to Declining Memory with Age

A number of the aspects of cognitive decline are connected to loss of stem cell activity with age, and thus reduced numbers of new somatic cells created to carry out functions in the brain. This is certainly the case for memory, but most such research is focused on neurogenesis, the process by which new neurons are created and integrated into neural circuits. Researchers here point to a different contributing population and mechanism, a reduced creation of oligodendrocytes and thus a reduced supply of myelin, the protein that sheaths nerves and is essential for their function. It is well known that myelin sheathing deteriorates with age, and this lack of oligodendrocyte cells may be an important proximate cause of that deterioration.

While the vast majority of myelin sheaths in the brain are laid down early in life, new studies reveal that a fresh supply of the fatty axonal conductor is required to establish and maintain memories in the adult brain. One reported that newly minted, myelin-producing oligodendrocytes cemented unpleasant memories in mice. The other reported that the birth of new oligodendrocytes plummets with age - a slowdown that could underlie age-related memory loss. In both, a drug that fosters the growth of new oligodendrocytes improved memory. The studies add to growing evidence that active myelination plays a crucial role in memory function, and mesh with recent studies implicating myelination malfunctions in neurodegenerative disease.

Part and parcel of most functional axons, myelin sheaths speed up the conductance of neuronal signals in the brain. While much of the myelin in the brain is as old as the axons themselves, a fraction of myelin continues to be produced by a small pool of new oligodendrocytes that develop throughout life, in response to new experiences and learning. This experience-dependent myelination is thought to bestow structural plasticity on the brain. Such myelination has been implicated in motor learning, and drugs that interfere with oligodendrocyte function reportedly cause memory deficits in mice.

If new oligodendrocytes are important in memory, might a slowdown in their production underlie age-related memory loss? Researchers started by tracking myelin production in mice with age. Using transgenic mice in which newly formed oligodendrocytes and myelin sheaths can be inducibly labeled, the researchers spotted numerous myelin-producing newbies in four- to six-month-old mice in the corpus callosum, but by 13 months of age, new oligodendrocytes were few and far between. As the spigot of fresh myelin started to pinch, age-related memory deficits emerged. Compared with 4-month-old mice, 13-month-olds took longer to learn the location of a submerged platform in the Morris water maze test of spatial memory. The hippocampus is crucial for storage of spatial memory, and the researchers found far fewer newly minted, myelin-producing oligodendrocytes in the CA1 region of the hippocampus in 13-month-old than in younger adult mice. Expression of myelin basic protein (MBP) - the building block of myelin sheaths - was also lower in the hippocampi of older mice.

Wielding a menagerie of mouse models, the researchers went on to reveal that blocking differentiation of oligodendrocytes led to memory loss in 4-month-old mice, while revving up the maturation of new oligodendrocytes prevented memory deficits in older mice, and even upped the density of synaptic puncta in the hippocampus.


A Gentler Approach to Transplanting Young Hematopoietic Stem Cells into Old Mice Modestly Extends Life Span

Stem cell populations become damaged and dysfunctional with age. Some of this is issues with the stem cells themselves, and some of this results from problem with the signaling environment and function of the stem cell niche. Which of these factors is more important likely varies by stem cell population. Among the best studied of stem cell types, the evidence suggests that muscle stem cells remain capable in old age, but become ever more quiescent, while hematopoietic stem cells become damaged and dysfunctional, unable to perform. Hematopoietic stem cells reside in the bone marrow and are responsible for generating blood and immune cells. Altered and reduced hematopoiesis is an important aspect of immune system decline with age, and thus providing functional replacement cells to older individuals may prove to be a useful form of rejuvenation therapy.

Unfortunately, the introduction of new hematopoietic stem cells at present requires removal of the existing population in order to make space in the stem cell niches of the bone marrow. The options for replacement are somewhat blunt and limited, deriving from the bone marrow transplant field. The standard approach is chemotherapy, which is quite unpleasant to experience, and further comes accompanied by a non-trivial risk of death or failure to adequately reconstitute the immune system following transplantation. That risk profile is considerably worse in older patients, and thus this sort of therapy is largely restricted to treatment of serious disease in the old, such as cancer.

A better, more gentle approach is needed if replacement of hematopoietic stem cells is to become a widespread preventative treatment for older individuals, a way to postpone immunosenescence. In the past, I have suggested the application of suicide gene therapies to the selective destruction of cell populations, as presently being pioneered by Oisin Biotechnologies to target senescent and cancerous cells. Here, researchers apply a different approach, using signals that convince stem cells from the bone marrow to leave their niches and migrate into the bloodstream. This is already widely used as a way to collect cells from donors, and the data here provides compelling evidence for it to leave the niches empty enough to allow a meaningful number transplanted stem cells to engraft and set to work. That this approach modestly extends life in mice, when used to transplant young hematopoietic stem cells into older animals, is a good demonstration of the gentle nature of the technique in comparison to chemotherapy.

Mobilization-based transplantation of young-donor hematopoietic stem cells extends lifespan in mice

Stem cells are critical to tissue regeneration and homeostasis during aging and disease. As a hallmark of aging, stem cell dysfunction is critical to improving the quality of life for people with advanced age. Stem cell-based therapy holds considerable promise for treating aging-related diseases, with hematopoietic stem cells (HSCs) being the most widely used for stem cell therapies. It is becoming increasingly clear that age-related changes in the niche space can induce alterations in hematopoiesis, including myeloid lineage skewing. However, extrinsic stimulation of HSCs with cytokines is highly dependent on intrinsic determinants. To date, the "gold standard" measure of HSC functionality remains an in vivo repopulating assay to determine their ability to re-establish lineage cell production in recipients during hematopoietic stem cell transplantation (HSCT). Unfortunately, conventional HSCT procedures require harsh cytotoxic conditioning - irradiation and/or chemotherapy - that alters HSC niches in the bone marrow, permanently damaging bone architecture. These limitations have confounded efforts to assess health-associated benefits of HSC replacement and rejuvenation.

The majority of HSCs reside in specialized niches within the bone marrow, although some HSCs leave these niches and migrate into the blood, ~1-5% of total HSCs each day. Mobilization of HSCs into the peripheral blood can be achieved through administration of G-CSF, an effect that is dramatically increased when G-CSF is administered in combination with other mobilizers, such as AMD3100. This HSC mobilization strategy constitutes the basic mechanism underlying collection of peripheral blood donor stem cells in the clinic. Critically, this increased mobilization also creates temporarily empty niches in the bone marrow, opening a window of opportunity for donor cell engraftment. Here, we use a novel mobilization-based HSCT procedure to investigate the health-associated benefits of replacing HSCs from aged recipients with young-donor HSCs. Additionally, we take advantage of the niche-preserving properties of this mobilization-based HSCT to investigate the influence of aged niche signaling upon a low percentage of young-donor HSCs.

Using this approach, we are the first to report an increase in median lifespan (12%) and a decrease in overall mortality hazard (hazard ratio: 0.42) in aged mice following transplantation of young-donor HSCs. The increase in longevity was accompanied by reductions of frailty measures and increases in food intake and body weight of aged recipients. Young-donor HSCs not only preserved youthful function within the aged bone marrow stroma, but also at least partially ameliorated dysfunctional hematopoietic phenotypes of aged recipients. This compelling evidence that mammalian health and lifespan can be extended through stem cell therapy adds a new category to the very limited list of successful anti-aging/life-extending interventions. Our findings have implications for further development of stem cell therapies for increasing health and lifespan.

EnClear Therapies Raises $10 Million to Develop a Means to Filter Molecular Waste from Cerebrospinal Fluid

Leucadia Therapeutics and EnClear Therapies are both testing the hypothesis that clearance of molecular waste from cerebrospinal fluid is a viable form of prevention and treatment for many neurodegenerative conditions, though they couldn't be more different in their areas of focus and specific implementations. Most of the common neurodegenerative conditions are characterized by rising levels of various forms of harmful molecular waste in the brain, misfolded proteins, and the like. Cerebrospinal fluid circulates in the brain and drains into the body through a variety of pathways, carrying away this waste. Unfortunately, these channels of drainage atrophy or ossify with age, and this loss contributes to the pathological levels of harmful metabolic byproducts that are present in the aging brain. Leucadia seeks to restore drainage through the cribriform plate pathway, while EnClear seeks to filter out the molecular waste present in cerebrospinal fluid though an approach similar to blood filtration, except carried out via a spinal tap.

EnClear Therapies, a life sciences company developing device-based therapies for the treatment of neurodegenerative disease, today announced a $10 million Series A financing. "We are thrilled to have a strong syndicate committed to our team and platform technology, enabling EnClear to move our therapeutic system to our first-in-human clinical trial in Amyotrophic Lateral Sclerosis, as well as expand our platform into new applications and strategic partnerships. Our differentiated technology has the potential to transform not only the treatment of this devastating disease, but also many other conditions related to the central nervous system."

"The founders of EnClear are focused on producing a technology that could revolutionize the field by allowing fast diagnosis, delivery of any drug directly into the cerebrospinal fluid, and the development of new CNS-focused therapeutics. I am excited to help EnClear grow their business to help the millions of patients living with CNS diseases. We believe that EnClear's technology has the potential to fundamentally change the way neurodegenerative diseases are treated and ultimately improve patients' lives."


A Short Review of the Development of Senolytic Therapies to Reverse Aspects of Aging

This short open access review paper covers some of the high points of the past decade of development of senolytic therapies capable of selectively destroying senescent cells in old tissues, as well as some of the earlier, much more sparse work on cellular senescence, prior to the general acceptance of an important role for senescent cells in aging. Senescent cells are constantly created and destroyed in the body. They are beneficial when present in the short term, acting to coordinate wound healing and in suppression of potentially cancerous cells, for example. Near all are destroyed soon after their creation, via programmed cell death or the activity of the immune system. A lingering population of senescent cells grows in number with age, however, as the processes of clearance falter and the tissue environment becomes more damaged. The mix of signals that these cells generate, the senescence-associated secretory phenotype, causes chronic inflammation and disruption of tissue structure and function, contributing to the progression of age-related disease and mortality.

Aging is defined as a progressive decrease in physiological function accompanied by a steady increase in mortality. The antagonistic pleiotropy theory proposes that aging is largely due to the natural selection of genes and pathways that increase fitness and decrease mortality early in life but contribute to deleterious effects and pathologies later in life. Cellular senescence is one such mechanism, which results in a permanent cell cycle arrest that has been described as a mechanism to limit cancer cell growth. However, recent studies have also suggested a dark side of senescence in which a build-up of senescent cells with age leads to increased inflammation due to a senescence-associated secretory phenotype (SASP). This phenotype that includes many cytokines promotes tumorigenesis and can exhaust the pool of immune cells in the body.

In a 2006 primate study, it was observed that senescent cells, as estimated by ATM activation do accumulate and can reach over 15% of the total cell population in aged individuals. In contrast to the vast majority of in vitro studies, this was one of the first studies showing a clear association between aging and the accumulation of senescent cells in vivo. Although this established a strong correlation, efforts were underway to establish causation between the accumulation of senescent cells and aging in vivo. In 2011, the researchers showed that removing p16Ink4a positive senescent cells delayed age-related disorders and increased healthspan in a BubR1 progeroid accelerated model of aging mice. Later studies confirmed the beneficial effects of senescent cell removal in wild type mice that showed increased median lifespan, delayed tumorigenesis, and attenuated age-related multi-organ deterioration. Removal of senescent cells in mice has also been shown to attenuate markers of age-associated neurodegenerative diseases such as tau hyperphosphorylation and neurofibrillary tangle deposition.

Substantial evidence in the last decade connecting senescent cell accumulation, age-related ailments, and roles in lifespan and healthspan fueled the search for therapeutic compounds that could selectively target senescent cells. A transcriptomic analysis between senescent cells and proliferating cells showed increased expression of pro-survival/anti-apoptotic genes such as Bcl-xL, a member of the Bcl-2 family of proteins that regulates programmed cell death by blocking caspase activation. This provided evidence to support the observation that senescent cells accumulate with age by being resistant to a variety of stresses that would normally induce apoptosis. Consistent with this idea, siRNAs to reduce Bcl-xL expression selectively reduced survival and viability in senescent cells while not affecting proliferating cells. Quercetin and dasatinib were obtained as hits from a drug screen based on these observations.

These compounds form one of the first discovered members of the senolytic class of drugs that selectively induce apoptosis in senescent cells. Four years after their initial identification as candidate senolytics, a dasatinib and quercetin combination was reported to decrease the senescent cell burden in humans as part of a Phase-1 clinical trial in diabetic kidney disease patients. This 2019 study was the first peer-reviewed study to demonstrate the efficacy of senolytics to decrease senescent cell burden in humans. This was carried out after an initial pilot study in early 2019 in 14 idiopathic pulmonary fibrosis (IPF) patients was completed to evaluate the feasibility of implementing a senolytic treatment. What now remains to be determined is whether future clinical trials will demonstrate any positive medical outcomes resulting from decreased senescent cell burden in diabetes and other age-associated ailments.


Nicotinamide Mononucleotide Supplementation Restores Lost Fertility in Aged Female Mice

Studies of the various approaches to raising NAD+ levels in aged mitochondria are a good illustration of the importance of the loss of mitochondrial function in degenerative aging. Researchers have studied this effect in numerous tissues and organs, with most such work examining muscle or the brain, both energy-hungry tissues and thus more dependent on their mitochondria for normal function. Today's open access paper is a study of mitochondrial function in a tissue that is less well studied in this context. The authors reporting that supplementation with nicotinamide mononucleotide (NMN) can restore lost fertility in old mice by improving mitochondrial function in oocytes.

Mitochondria are the power plants of the cell, responsible for packaging the chemical energy store molecule ATP that is used to power cellular operations. For reasons that remain poorly understood, meaning that they are not well connected to the underlying molecular damage of aging, mitochondria become dysfunctional throughout the body with advancing age. Mitochondria are the descendants of ancient symbiotic bacteria, and they normally divide and fuse like bacteria, as well as passing component parts of their molecular machinery from one to another. In cells in old tissues, these dynamics change in ways that make mitochondria resistant to the quality control processes responsible for clearing out damaged structures in the cell. Cells become populated by problematic, poorly functioning mitochondria, and suffer accordingly.

A reduced amount of NAD+, a utility molecule important to a number of processes in mitochondria, is one proximate cause of these issues. The pace of synthesis and recycling of NAD+ falls off due to lowered levels of precursors and other necessary ingredients for the chemical reactions involved. This might be traced back to altered levels of gene expression due to epigenetic changes characteristic of aging, but this is still an exploration of proximate causes, and says little about what the underlying root causes might be in any detail.

To the extent that providing more NAD+ to cells restores mitochondrial function and thus cellular function to some degree, and this outcome is well demonstrated in mice, these benefits may be largely the result of enabling sufficient clearance of worn mitochondria to improve overall ATP production. This is better maintenance rather than better function per se; other lines of research also suggest that quality control is the critical item in mitochondrial function. When it comes to the means of raising NAD+ levels, delivery of NAD+ itself is not very efficient, and most current approaches are thus focused on delivering precursor molecules used in the synthesis or recycling of NAD+. Of these only nicotinamide riboside has even early clinical data to show some form of benefit in aged humans, but that will likely change over the next few years as more groups publish their work.

NAD+ Repletion Rescues Female Fertility during Reproductive Aging

The rate-limiting factor for successful pregnancy is oocyte quality, which significantly declines from late in the third decade of life in humans. Despite the enormous demand, there are no clinically viable strategies to either preserve or rejuvenate oocyte quality during aging, which is defined by the capacity of the oocyte to support meiotic maturation, fertilization, and subsequent embryonic development. A non-invasive, pharmacological treatment to maintain or restore oocyte quality during aging would alleviate a rate-limiting barrier to pregnancy with increasing age that has driven demand for assisted reproduction technologies (ARTs) such as in vitro fertilization (IVF), which is invasive, carries health risks, is expensive, and has a limited success rate.

Although somatic tissues undergo continual regeneration through turnover by a self-renewing population of resident precursor stem cells, oocytes in the ovary are laid down during in utero development in humans, where they form a finite pool that does not undergo self-renewal. Oocytes are therefore highly susceptible to age-related dysfunction. The molecular basis for the decline in oocyte quality with advancing age implicates genome instability, reduced mitochondrial bioenergetics, increased reactive oxygen species (ROS), and disturbances during meiotic chromosome segregation due to compromised function of the spindle assembly checkpoint (SAC) surveillance system. The molecular cause of chromosome mis-segregation in oocytes with advancing age is still unknown, and as a result, there are no pharmacological strategies to correct this problem. Understanding the molecular or metabolic basis of this defect could lead to therapies that could maintain or even rescue female fertility with advancing age.

The metabolite nicotinamide adenine dinucleotide (NAD+/NADH) is a prominent redox cofactor and enzyme substrate that is essential to energy metabolism, DNA repair, and epigenetic homeostasis. Levels of this essential cofactor decline with age in somatic tissues, and reversing this decline through treatment with metabolic precursors for NAD+ has gained attention as a treatment for maintaining late-life health. Here, we show that loss of oocyte quality with age accompanies declining levels of NAD+. Treatment with the NAD+ metabolic precursor nicotinamide mononucleotide (NMN) rejuvenates oocyte quality in aged animals, leading to restoration in fertility, and this can be recapitulated by transgenic overexpression of the NAD+-dependent deacylase SIRT2, though deletion of this enzyme does not impair oocyte quality. These benefits of NMN extend to the developing embryo, where supplementation reverses the adverse effect of maternal age on developmental milestones. These findings suggest that late-life restoration of NAD+ levels represents an opportunity to rescue female reproductive function in mammals.

An Approach that Prevents Earlier than Expected Cell Death in Alzheimer's Disease

Researchers here provide evidence for significant levels of cell death to occur in the brain earlier than expected in the development of Alzheimer's disease, during the stage of mild cognitive impairment thought to be driven by the aggregation of amyloid-β. The researchers identify some portions of a mechanism by which amyloid-β might be triggering this cell death, and propose a novel class of therapeutic approaches that will interfere in this link. Given the artificial nature of animal models in Alzheimer's research, and the comparatively sparse nature of human data, it is good to adopt a cautious wait and see approach in response to this sort of news. It is similar in character to numerous other lines of research in the Alzheimer's field that ultimately didn't translate from mice to humans.

The exact cause of Alzheimer's disease is unknown, but pathological changes in the brain, including neuron loss and an accumulation of protein aggregates called beta-amyloid plaques, are a diagnostic hallmark of Alzheimer's disease. Mild cognitive impairment (MCI) describes the slight but measurable changes in cognitive function that are often a precursor to Alzheimer's disease. However, despite the importance of MCI, very little is known about the changes that occur in the brain during the progression from MCI to Alzheimer's.

Researchers have now found that neuronal death occurs much earlier than originally thought, with higher levels of necrosis seen in patients with MCI than in patients with full-blown Alzheimer's disease. The researchers also observed a significant decrease in the levels of a protein known as YAP in Alzheimer's disease model mice and human patients with MCI. YAP positively affects the activity of a second protein called TEAD, a deficiency of which leads to neuronal necrosis. Microscopic examination revealed that the missing YAP was sequestered within beta-amyloid plaques, which have also been linked to neuronal toxicity.

By directly injecting a gene therapy vector expressing YAP analog into the cerebrospinal fluid of mice that were genetically engineered to provide a model of Alzheimer's, the researchers were able to prevent early-stage neuron loss, restore cognitive function, and prevent the development of beta-amyloid plaques. "Confirming that neuronal necrosis was dependent on YAP was really the pivotal moment for us, but observing the almost transformative effects of YAP supplementation was hugely exciting. By showing that neuronal necrosis is YAP-dependent and begins prior to the onset of most symptoms, we predict that novel Alzheimer's disease therapies will be developed to prevent the initiation of Alzheimer's disease."


An Interview with Lewis Gruber of SIWA Therapeutics

SIWA Therapeutics is one of the few senolytics biotech companies founded prior to the past few years, invigorated with new funding now that the clearance of senescent cells as a basis for rejuvenation is an area of intense interest for the research and development community. The company is also, I believe, running the only senolytics program based the use of monoclonal antibodies. This is a way to encourage the immune system to destroy cells bearing specific surface markers, in this case a form of advanced glycation endproduct that is found on cancerous, senescent, and otherwise dysfunctional cells.

Many of our readers are familiar with CAR-T immunotherapy, which has enjoyed some success, but it's not without considerable challenges. How does your approach differ?

We are using a simpler approach of just manufacturing a monoclonal antibody. Of course, we do that in Chinese Hamster Ovary (CHO) cells and purify and produce a monoclonal antibody product so that we don't have to modify patient cells or any other cells in order to apply our treatment. It's just a straight typical monoclonal antibody product, the same sort of immunotherapy that's used in a variety of cancer therapies. In this case, we've found a marker that's on cancer cells and senescent cells because of the way the markers are produced, and therefore the monoclonal antibody can enable removal of those cancer cells.

Can you summarize a bit more how that antibody SIWA 318H works?

It binds to proteins on the surface of oxidatively damaged cells that may be senescent or cancerous, or just very dysfunctional. By doing so, it provokes an immune response, initially an innate immune response with the natural killer cells. The bottom line is, the immune response not only destroys and removes the cells to which the antibody binds, but immune cells also secrete factors that promote regeneration. So, while we're removing cells that are not going to function properly, we're promoting their replacement with new cells from adult stem cell populations. The interesting thing about the markers is that they are a product of glycolysis, and high levels of glycolysis were associated even back before World War Two by Otto Warburg. They were shown on cancer cells. Cancer cells and senescent cells have their peculiarities, and they are high producers of this particular marker; therefore, we can hone in on those two types of cells.

Is it a coincidence that the marker is present on both senescent and cancerous cells? Or is there a reason for that?

Both types of cells conducted an elevated level of glycolysis and there are various explanations in the scientific literature. Both are highly metabolically active. Some people think of senescent cells as almost dead, but, in fact, they are among the most metabolically active of cells, and cancer cells are as well because of high proliferation. Both types of cells have a high need for ATP. One explanation in the literature is that they both resort to glycolysis to get additional ATP. The senescent cells put all of their efforts into the senescence associated secretory phenotype, so they're producing a lot of cytokines and other molecules, so they need to use glycolysis, but they're not using it to divide. A simplified way of looking at it is that senescent cells grow, and they need energy for growth; they basically get to the size that a cell would normally be when it divides, and then they just don't divide. When you look under the microscope, you see large, flattened cells that are senescent cells, because they've grown but they just didn't divide. Cancer cells, of course, will go ahead and do the division and you'll have two daughter cells. The bottom line is that they both have to grow to that large size.

Does this mean a vaccine might be developed for senescent cells or even cancer?

Yes, and we are working on what we have. We've done preliminary studies in mice, and now we're looking at expanding it into other species, even beyond humans, but we do have a candidate vaccine already in the works. As with any drug, you do want to be careful about certain conditions, pregnancy or other conditions where you don't want to disrupt any things happening. For example, senescent cells have been found in fetuses. The one common thing, strangely enough, with senescent cells is every situation in which they're beneficial, they're removed. After they form the different structures in the fetus, they're eliminated. The same thing is true in wound healing, which is often given as an example of a beneficial effect. Initially, senescence promotes proliferation of repairing cells, but if that's allowed to go on too long, the wound tends to produce scar tissue, fibrosis, and the bottom line is that in the natural healing process, senescent cells appear for a time and then are removed. Although you do have to be somewhat careful, you don't want to interfere with the initial stage of wound healing or with fetal development, otherwise, it's a good rule of thumb that removing a senescent cell or a cancer cell is not a bad thing.


Activating Quiescent ILC2 Immune Cells in the Aging Mouse Brain Improves Cognitive Function

Immune cells play a wide variety of important roles in the normal function of tissues throughout the body. The more familiar tasks, such as chasing down pathogens and clearing up metabolic waste and other debris, are just one slice of a much broader spectrum. Many of the other activities undertaken by immune cells are poorly catalogued and understood, particularly in the brain, where resident immune cells appear critical to the fine details of neural function. As is often the case in cellular interactions, many of the distinct contributions of immune cells to tissue function take the form of secreted molecules (or extracellular vesicles) that act upon other cell types to change their behavior.

In today's open access research, scientists identify a population of immune cells, ILC2 cells, largely concentrated in the choroid plexus in the aging brain, that could be helpful were they not largely quiescent. Given suitable signals to override their quiescence, these cells act to improve cognitive function. This is likely achieved via signal molecules secreted by ILC2 cells when active. The researchers identify IL-5 as an important signal, but it will no doubt be the work of years to more completely understand how benefits are produced in this case.

Activating Immune Cells Could Revitalize the Aging Brain

Group 2 innate lymphoid cells (ILC2s) reside in specific tissues of the body and help to repair them when they are damaged. Recently, for example, ILC2s in the spinal cord were shown to promote healing after spinal cord injury. Researchers examined the brains of both young and old mice and found that ILC2s accumulated with age in a structure called the choroid plexus. This structure produces cerebrospinal fluid and is close to the hippocampus, a region of the brain that plays a key role in learning and memory. Older mouse brains had up to five times as many ILC2 cells as younger brains. Crucially, the researchers also saw large numbers of ILC2s in the choroid plexus of elderly humans.

The ILC2s in old mouse brains were largely in an inactive, or quiescent, state, but the researchers were able to activate them by treating the animals with a cell signaling molecule called IL-33, causing the cells to proliferate and produce proteins that stimulate the formation and survival of neurons. Compared with ILC2s from younger animals, ILC2s from older mice were able to live longer and produce more ILC2 upon activation, the researchers found. Additionally, treating old mice with IL-33, or injecting them with ILC2 cells pre-activated in the lab, improved the animals' performance in a series of cognitive tests designed to measure their learning and memory.

One of the proteins produced by activated ILC2s is the signaling molecule IL-5. The research team found that treating old mice with IL-5 increased the formation of new nerve cells in the hippocampus and reduced the amount of potentially damaging inflammation in the brain. Again, IL-5 treatment improved the cognitive performance of aged mice in a number of tests.

Activation of group 2 innate lymphoid cells alleviates aging-associated cognitive decline

In this study, we report the accumulation of tissue-resident ILC2 in the choroid plexus of the aged brain, with ILC2 comprising a major subset of lymphocytes in the choroid plexus of aged mice and humans. ILC2 in the aged brain are long-lived and capable of reversibly switching between cell cycle dormancy and proliferation. They are relatively resistant to cellular senescence and exhaustion under replication stress, leading to enhanced self-renewal capability. They are functionally quiescent at homeostasis but can be activated by exogenous IL-33 to produce large amounts of IL-5 and IL-13 as well as a variety of other effector molecules in vitro and in vivo.

When activated in vitro and transferred intracerebroventricularly, they revitalized the aged brains and enhanced cognitive function of aged mice. Administration of IL-5, a major ILC2 product, repressed aging-associated neuroinflammation and alleviated aging-associated cognitive decline. Together, these results suggest that aging may expand a unique population of brain-resident ILC2 with enhanced cellular fitness and potent neuroprotective capability. Targeting ILC2 in the aged brain may unlock therapies to combat aging-related neurodegenerative disorders.

A Comparison of Biological Age Measurement Approaches

Researchers here assess the performance of a range of approaches to measuring biological age, including a number of epigenetic clocks based on DNA methylation changes characteristic of aging. The ideal measurement of biological age is one that is quick and cheap to undertake, and that accurately reflects the underlying burden of damage and consequence that drives aging. Such a measure could be used to determine the effectiveness of potential rejuvenation therapies far more rapidly than is presently possible, and would thus accelerate development efforts. Unfortunately none of the existing approaches are quite ready for this, as it is far from clear as to whether they do actually measure the full range of damage and consequence in aging. Their effectiveness will have to be proven in conjunction with the development of each new class of rejuvenation therapies, starting with senolytics.

Everyone ages, but how aging affects health varies from person to person. This means that how old someone seems or feels does not always match the number of years they have been alive; in other words, someone's "biological age" can often differ from their "chronological age". Scientists are now looking at the physiological changes related to aging to better predict who is at the greatest risk of age-related health problems. Several measurements of biological age have been put forward to capture information about various age-related changes. For example, some measurements look at changes to DNA molecules, while others measure signs of frailty, or deterioration in cognitive or physical abilities. However, to date, most studies into measures of biological age have looked at them individually and less is known about how these physiological changes interact, which is likely to be important.

In a new study, researchers have looked at data on nine different measures of biological age in a group of 845 Swedish adults, aged between 50 and 90, that was collected several times over a follow-up period of about 20 years. The dataset also gave details of the individuals' birth year, sex, height, weight, smoking status, and education. The year of death was also collected from national registers for all individual in the group who had since died. The nine measures were telomere length, four different epigenetic clocks, physiological age derived from a list of age-correlated biomarkers, chronological age, functional aging index, and frailty index. Researchers found that all nine biological age measures could be used to explain the risk of individuals in the group dying during the follow-up period. In other words, when comparing individuals with the same chronological age in the group under study, the person with a higher biological age measure was more likely to die earlier. The analysis also revealed that biological aging appears to accelerate as individuals approach 70 years old, and that there are noticeable differences in the aging process between men and women.

Lastly, when combining all nine biological age measures, some of them worked better than others. Measurements of methylation groups added to DNA (known as DNA methylation age) and frailty (the frailty index) led to improved predictions for an individual's risk of death. Ultimately, if future studies confirm these results for measures from single individuals, DNA methylation and the frailty index may be used to help identify people who may benefit the most from interventions to prevent age-related health conditions.


Greater Height Correlates with a Lesser Risk of Dementia

Being taller is associated with a shorter life span, for reasons that are far from fully explored. The role of growth hormone in longevity is no doubt close to the roots of this correlation, but there are plenty of questions remaining, such as why lung disease plays a sizable role in greater mortality for taller people in later life. As illustrated by the research here, there is a bright side to being taller, which is that epidemiological studies show taller people to have a lesser incidence of dementia. Again, why exactly this is the case is far from fully explored. These and other natural variations between people are interesting, but we should expect them to vanish with the introduction of the first rejuvenation therapies in the near future, swamped by the benefits that might be achieved by directly addressing the causes of aging.

This study examined the relationship between body height and dementia and explored the impact of intelligence level, educational attainment, early life environment, and familial factors. A total of 666,333 men, 70,608 brothers, and 7388 twin brothers born 1939-1959 and examined at the conscript board were followed in Danish nationwide registers (1969-2016). Cox regression models were applied to analyze the association between body height and dementia. The findings of this current study provide substantial support to previous evidence of a link between body height and dementia. All previous studies had accounted for educational level and other socioeconomic indicators, yet none of these studies had adjusted for intelligence level earlier in life. Intelligence level has been suggested to be a stronger marker of brain and cognitive reserve than educational level. Intelligence level is furthermore correlated with body height and by itself associated with dementia.

In contrast to previous studies, we also investigated the impact of other potential early-life familial factors including genetics and socioeconomic resources in the family that may influence both body height and later risk of dementia. Body height has been shown to have a strong genetic component with around 80% of the variation in populations being explained by genetic differences between individuals. The genetic component of height has furthermore been found to be consistent across countries independent of living standards. The genetic and environmental variation influencing body height, but not risk of dementia, is smaller within brothers than between men in general, which may weaken the association between body height and dementia in the latter compared to the former group. Through this mechanism, the finding of a stronger association within brothers may be explained by less dilution of the effects of different harmful exposures early in life influencing both body growth and risk of dementia. These findings furthermore suggests that genetics has a minor role in the association of body height and dementia.

In conclusion, taller body height at the entry to adulthood, supposed to be a marker of early-life environment, is associated with lower risk of dementia diagnosis later in life. The association persisted when adjusted for educational level and intelligence test scores in young adulthood, suggesting that height is not just acting as an indicator of cognitive reserve.


Request for Startups in the Rejuvenation Biotechnology Space, 2020 Edition

This is the latest in a series of yearly posts in which I suggest areas of development for biotech startups I'd like to see actively developed as a part of the longevity industry in the near future. Today, this year, is a good time to be starting a company focused on the production of a novel therapeutic approach to intervening in the aging process. There is a great deal of funding for seed stage investment, and many compelling projects lacking champions, yet to be carried forward from academia into preclinical development. Numerous scientific and industry crossover conferences are now held every year, at which it is possible to meet a mix of entrepreneurs, scientists, and investors, all interested in advancing the state of the art. The industry, and its pool of potential funds for later stage investment, are both growing rapidly, driven by the energetic activities of patient advocates such as Aubrey de Grey and activist investors such as Jim Mellon and his allies. The public at large is becoming ever more aware of the potential to change the progression of aging. This will become a very large industry in the years ahead, and rejuvenation will eventually become the largest portion of medicine as a whole. There is tremendous opportunity here, both for returns on investment, and to change the human condition for the better.

A Viable Approach to Medical Tourism for the Era of Rejuvenation Therapies

Rejuvenation therapies, by their nature, have a far greater market size than any existing medical technology intended to treat the clinically ill. Many more people will undergo rejuvenation treatments than presently undergo medical procedures or take medications. This opens the door for radical change and improvement in the poorly organized, scattered, and unhelpful medical tourism industry. A population of potential customers an order of magnitude larger than is the case today is a great opportunity for any company to successfully smooth the road of regulatory arbitrage, allowing people in restrictive regulatory regions to effectively make use of reputable services available in other countries. The first rejuvenation therapies already exist, but discovery, validation, and access are all challenging. There is considerable room for improvement.

Restoration of Hematopoietic Stem Cell Function

Hematopoietic stem cell populations are responsible for producing blood and immune cells, as well as other important cell types, such as endothelial progenitor cells that help to maintain blood vessel integrity. All of this degrades with aging, but the standard approach to bone marrow transplantation, involving chemotherapy to destroy existing stem cells, is too harsh to be used as a basis for any therapy based on replacement of cells. There are potential alternatives, however, a fair number of them. For example, mobilizing stem cells to leave the bone marrow produces enough spare room in stem cell niches to allow a meaningful fraction of transplanted stem cells to engraft, resulting in extension of life and improved function in mice. The function of the immune system is so vital in aging that treatments to restore hematopoiesis are of great importance.

A Low Impact Method of Destroying the Peripheral Immune System

The peripheral immune system becomes cluttered with senescent and dysfunctional cells with advancing age, one of the numerous issues that must be addressed in order to repair an aged immune system. There is plenty of evidence for the selective destruction of specific dysfunctional cells to be beneficial. The entire B cell complement can be cleared to remove dysfunctional and harmful B cells, for example. The lost B cells are quickly replaced with functional B cells even in later life. What is needed is a way to clear out the peripheral immune system in entirety without undue side-effects, a form of therapy that should be useful not just to clear out the problem cells from an aged immune system, but also to effectively treat autoimmune disease. This goal can at present be accomplished via the application of high dose immunosuppressive drugs, but with significant side-effects that make it unsuitable for widespread use in a frail population. Thus something akin to suicide gene therapies or other targeted means of low-impact cell destruction is needed instead.

Build a Physician Network to Bring Low-Cost Senolytics to the Masses

The evidence for dasatinib and quercetin to meaningfully clear senescent cells, one of the causes of degenerative aging, is presently compelling. Soon we will know whether or not fisetin performs well in humans. All of these substances cost little. As yet, only a few groups are trying to build physician networks or services that will deliver these and other actual or potential senolytic treatments to patients. To my eyes this is an important exercise in logistics and exercising the right to off-label use of approved drugs. Functional senolytics have the capacity to greatly improve the state of health for every older person, and it is reasonable to believe, based on the evidence, that at least a few of the portfolio of potentially senolytic low-cost drugs and supplements can achieve this goal right now. Yet they are not being widely used. Tens of millions of patients in the US alone are suffering when their situation could be improved. At the very least, many more people should be made aware that senolytics exist, so as to be able to make a decision based on present evidence as to whether or not to try this form of therapy.

A Competitor for Revel Pharmaceuticals in Glucosepane Cross-link Breaking

Persistent cross-links between extracellular matrix proteins are likely very influential on both late life mortality, due to stiffening of blood vessels and consequent hypertension, and on the loss of elasticity in skin, a sizable component of skin aging. One of those topics is much more important than the other, but, as any survey of the community will tell you, opinions differ on which one it is. The market size for methods of reversing either outcome of aging is very large, and breaking cross-links is a plausible way forward given what is known of their biochemistry. Revel Pharmaceuticals is presently the only startup biotech company working on development of ways to break down the primary form of persistent cross-link in human tissues, those based on glucosepane. Competing with Revel in the discovery of compounds that can break glucosepane cross-links is a very feasible prospect: this part of the field is presently at the same point that senolytics were five to ten years ago, and most likely has a similar trajectory ahead of it.

Interfere with Telomere Lengthing to Defeat All Cancers

The requirement for telomere lengthening is the Achilles' heel of cancer. All cancer cells must abuse at least one of the two available mechanisms of telomere lengthening, telomerase and alternative lengthening of telomeres (ALT), in order to continue unfettered replication. Successfully sabotage this process and any cancer will wither as a result, no matter how advanced it is in its progression. This is truly the best basis for the development of a single, universal cancer therapy, and numerous potential approaches exist at various stages of development. Some have made it to the point of preclinical work, such as the program at Maia Biotechnology, but most have yet to make the leap from academia to industry. There is considerable opportunity here to revolutionize the treatment of cancer.

Break the Link Between DNA Repair and Epigenetic Change

One of the more interesting of recent discoveries in the field of aging research is that DNA double strand break repair causes epigenetic changes characteristic of aging. This opens the door to investigations of the intricate and complex DNA repair mechanism of the cell nucleus, in search of points of intervention that might stop this process of epigenetic change from occurring, or slow it down, or perhaps even reverse it. A sizable literature on DNA repair exists, and this part of our biochemistry is comparatively well mapped. Somewhere in there are the starting points for therapies that might be very influential on the state of degenerative aging - perhaps the basis for reversing epigenetic changes and cellular dysfunction in ways other than the approach of in vivo reprogramming that is growing in popularity.

Restore Lost Mitochondrial Function to a Much Greater Degree than can be Achieved via NAD+ Upregulation

At present NAD+ upregulation is a popular topic, as is the application of mitochondrially targeted antioxidants. Both approaches appear to sufficiently restore the quality control mechanism of mitophagy in old cells to allow some degree of restored mitochondrial function throughout an aged body. How sizable is the effect? In the same ballpark as exercise, judging from the few small human trials conducted to date, focused on the cardiovascular system: blood pressure, blood vessel compliance, vascular smooth muscle function, pulse wave velocity. We might take this as an encouraging sign that if mitochondrial function was actually fully restored, the benefits could be sizable. There are any number of possible approaches that might prove to be much more effective than NAD+ upregulation: tinker directly with gene expression changes that appear to impair mitophagy; epigenetic reprogramming in vivo; delivery of whole mitochondria to tissues; targeted destruction of damaged mitochondrial DNA; and so forth. Any group able to demonstrate significantly better outcomes in animal models than have been obtained from NAD+ upregulation should have no issues in raising funds for commercial development.

Restore a Youthful Human Gut Microbiome

Work on the human gut microbiome and the changes that take place with age has picked up considerably in past years. A number of groups have identified specific metabolites that are produced at lower levels by the aged microbiome, as well as changes in the balance of beneficial and harmful gut microbes that lead to greater chronic inflammation. In animal studies, transplantation of a young microbiome into old animals results in a lasting restoration of the microbiome in those older animals. In human medicine, fecal microbiota transplantation is well developed for use in a number of pathological conditions. It has not yet been applied to aging, but it should, or variants that dispense with the donor and just provide the appropriate mix of microbes directly. Further, it is not unreasonable to build probiotic-like treatments that deliver the right mix of microbes in sufficient volume to achieve the same effect. Given the diverse influences of the gut microbiome and the metabolites it produces, this may be a way to meaningfully reduce chronic inflammation, restore stem cell activity, and generally improve health in older people.

Reverse the Loss of Capillary Density with Age

Capillary density declines throughout the body with advancing age, reducing delivery of oxygen and nutrients, and thereby leading to dysfunction in cells and tissues, particularly in the energy hungry brain and muscles. The underlying causes of this manifestation of aging are not well understood, but the processes of angiogenesis in general, the regulatory mechanisms governing generation of blood vessels, are fairly well explored. There is an opportunity here to take what is known and apply it to this challenge. Approaches that restore lost capillary density may prove to be a useful means of reversing the loss in tissue function that occurs with age, but it requires a successful methodology to be demonstrated in at least animal models in order to understand just how useful. Loss of capillary density is directly implicated in neurodegeneration and heart failure, providing well-understood indications to target for any company working towards this form of repair biotechnology.

An Interview with Hanadie Yousef of Juvena Therapeutics

The Life Extension Advocacy Foundation volunteers here interview Hanadie Yousef of Juvena Therapeutics. Her team is mining the secretions of pluripotent stem cells to find factors that can improve regeneration and stem cell activity in older individuals. Juvena represents one small slice of a broad trend in the regenerative medicine community, many teams building on the past decades of work on stem cell transplantation by seeking to understand and manipulate the cell signaling thought to produce benefits in patients undergoing these first generation therapies. In near all such stem cell therapies, the transplanted cells die rapidly rather than integrate into patient tissues, but benefits such as reductions in chronic inflammation and improved regeneration are nonetheless observed, albeit quite unreliably. Using the signals rather than cells as a basis for treatment should, in principle, turn out to be a more controllable, reliable approach.

Can you describe in more detail Juvena's approach to developing protein therapeutics that promote tissue regeneration in the elderly?

We are utilizing the secretome of human embryonic stem cells. We know that human embryonic stem cells have the capability to develop every tissue in the body, an entire human being. I and my colleagues discovered, nearly a decade ago, that by isolating a sub-fraction of the proteins that they themselves secrete and produce in order to signal to stem cells to develop every tissue in the body, concentrating these proteins, and then adding them directly onto old muscle precursor cells isolated from humans over the age of 65, we could enhance their regenerative potential. When we injected this cocktail of proteins into injured old mice, we saw muscle regeneration returned to levels of younger animals, two-month old mice that are like people in their 20s, and this is a cocktail of human proteins.

The way that Juvena Therapeutics is taking this discovery into the clinic is by establishing a very efficient identification, high-throughput screening, and preclinical development pipeline, which has become ever more predictive and accelerated with the use of AI tools in order to identify what proteins in this original cocktail are actually driving the rejuvenation process, which ones are master regulators of signal transduction and key regulatory pathways involved in tissue differentiation and regeneration. By identifying those proteins and their sequences and exactly what they are compositionally, we can then test them individually and in combinations for their ability to promote human muscle precursor cell function and promote tissue regeneration in mouse models of human aging and human diseases.

Why did you choose to focus on muscle cell regeneration?

Interesting fact about muscle: It's the largest internal tissue organ in the body. One of the first hallmarks of aging is the fact that once we hit our 30s, everybody, for the rest of our lives, heads downhill. We're losing muscle strength and mass every year, but it accelerates with every decade so that by the time we're in your 60s, everyone has some form of muscle wasting, some people more severe than others, so severe enough, in fact, that it prevents their daily functions and daily living and can be so severe that they can be clinically diagnosed with the disease of sarcopenia. Because there is now an ICD-10 code for sarcopenia, which was only issued at the end of 2016, meaning it's an age-related disease that has a clinical indication, we can actually make therapies to target it. There's zero FDA approved therapies, so it's a huge unmet need and a huge market.

Excitingly, one of the best experimental models that we have today to really understand how stem cells decline and function with age is the muscle system. Key discoveries made by my former co-thesis advisor, Irina Conboy, and other pioneers in the field, really paved the way for us to understand mechanistically how stem cells decline and function with age in muscle and develop methods to repair and rejuvenate them, so it's a great first tissue to focus on. Juvena will use this as a way to then launch into other tissue types. Laser-like focus on muscle first; once we find the proteins that are secreted by human embryonic stem cells that can drive muscle regeneration, we'll then apply our platform and our technology and our approach to identifying therapeutics, approaching candidates that can act as therapeutics to promote the brain and prevent things like dementia, really targeting regenerative diseases, as well as go after other tissue types, such as the heart, the skin, and other ones that are really affected with age and decline in function in part by loss of stem cell function.


The Epigenetic Profile of Werner Syndrome is Very Different from that of Aging

The research community has long used progeroid syndromes such as Hutchinson-Gilford progeria syndrome and Werner syndrome as tools in the investigation of aging. This category of conditions are colloquially thought of as accelerated aging, but are in fact only a little similar to aging. The various underling genetic causes of progeria result in accelerated accumulation of cellular damage of various sorts, different in each case, leading to tissue dysfunction and outcomes that resemble a range of normal age-related conditions. Aging is itself a process of damage accumulation, so it isn't surprising to find some degree of similarity. The forms of cellular damage and their proportions are quite different, however, which makes it challenging to draw any specific lesson from progeroid syndromes and apply it to normal aging.

Werner syndrome (WRN) is a canonical member of a family of genetically determined disorders that include multiple phenotypes consistent with their characterizations as segmental progeroid syndromes. It is important to note, however, that these syndromes may include discordances with the usual phenotypic features of aging. For example, the ratio of epithelial to non-epithelial cancers in WRN is 1:1, whereas the ratio seen in usual aging is 10:1. Moreover, while WRN research has contributed to the widespread acceptance of genomic instability as one of the hallmarks of aging, features such as variegated translocation mosaicism and a preponderance of large deletions are particularly characteristic of WRN.

Epigenetic signatures of Werner syndrome occur early in life and are distinct from normal epigenetic aging processes. The vast majority (more than 90%) of differentially methylated CpGs and regions (DMRs) in WRN were not affected by aging, consistent with the view that WRN is not merely accelerated normal aging. A particularly striking finding was that DMRs were enriched in genes associated with transcription factor activity, leading us to hypothesize that WRN might best be conceptualized as a disease based upon aberrant controls of the expressions of a wide array of genetic loci, some of which are plausibly related to clinical phenotypes of WRN. Moreover, given that the methylation changes in the highest ranking DMR in the promoter region of the HOXA4 gene as well as in other DMRs preceded disease manifestation, it seems likely that these transcriptional aberrations began early in development. That finding reinforces the concept that how well one builds an organism makes a great deal of difference on how long it lasts and how well it functions!

The epigenome of an individual is most plastic during early development and is shaped by a plethora of stochastic, internal (i.e. genetic variation), and external factors (i.e. environmental exposures). The epigenetic changes associated with WRN and other segmental progerias are widespread but are all of small effect size. Both premature and normal aging phenotypes may manifest when the adverse factors exceed a critical threshold. Individuals with WRN may be endowed with an epigenome early in life which lowers their threshold for developing a number of specific aging phenotype.


PTTG1 as Prompt for Discussion of the Evolutionary Genetics of Aging

Aging is under genetic control in the sense that species have different genomes, different life spans, and different manifestations of aging within those life spans. Within any given species, it is far from clear that genetic variation has a large enough influence to care about. Individuals vary, but the evidence strongly suggests that this is near all due to environmental rather than genetic differences. Where there are genetic differences, the old who survive to benefit from them are still old people, greatly impacted by aging and trapped in a downward spiral of dysfunction.

The author of this commentary uses the gene PTTG1 as a starting point for a discussion of the genetics and evolution of aging across species, but again, this really doesn't have to mean that PTTG1 and its effects on metabolism are necessarily a place to start the development of therapies to treat aging in humans. Relevance in the former does not automatically lead to relevance in the latter. The research community should look less to differences between individuals and more to addressing the known causes of aging, mechanisms that are the same in all individuals of a given species.

Of mice, genes and aging

Why do we get old? How much of aging is genetic? And in what genes? There is clearly a genetic basis of aging, as demonstrated from yeast to worms to humans. As one example, different mouse strains have different potential lifespans. Much effort has been invested in understanding the genetic underpinnings of lifespan differences between the long-lived C57Bl/6 strain and the short-lived DBA/2 strain, with 50% mortality in captivity by 914 and 687 days, respectively. Quantitative trait loci mapping in C57Bl/6 X DBA/2 (BXD) recombinant inbred strains identified a locus on chromosome 11 that is linked to lifespan. Subsequent analyses revealed that this locus confers differential expression of the pituitary tumor-transforming gene-1 (Pttg1)/Securin gene. PTTG1 level-dependent impacts on chromosomal segregation during mitosis could influence longevity.

Natural selection only acts to promote longevity to the extent that it benefits the passage of genetic material to subsequent generations. Different animals have evolved different strategies for somatic maintenance that maximize reproductive success, and the extension of youth through additional investment in tissue maintenance would be disfavored if the costs (often manifested through reduced investment in reproduction) outweigh benefits. For a small vulnerable animal like a field mouse that faces high extrinsic hazards (such as predation), natural selection has favored a "fast" life history - a breed early, breed often strategy with little investment in longevity. For larger animals like humans, elephants, and whales, or for animals like tortoises, moles, bats and birds that have evolved other strategies to greatly reduce extrinsic hazards, natural selection has favored a "slow" life history, with greater and/or prolonged tissue maintenance leading to longer potential lifespans.

While we understand how natural selection has shaped the pathways that control longevity, we know less about what these pathways actually are. Studies from model organisms have clearly demonstrated that modulation of the insulin-like growth factor-1 (IGF-1) pathway, which positively regulates the mTOR pathway and negatively regulates autophagy, can significantly impact longevity. Decreases in IGF-1 and mTOR, or increases in autophagy, have been shown to prolong lifespans in organisms ranging from yeast to mammals. Additional studies have shown how inflammation can contribute to aging-associated phenotypes, and polymorphisms in genes controlling the IGF-1 pathway and inflammation are enriched in human centenarians, but the extent to which these polymorphisms and their impact on inflammation are contributing to differences in longevity has not been established.

While genetic screens in model organisms have revealed key pathways that regulate lifespan, the mechanisms employed by natural selection in the evolution of lifespans largely remain a mystery. Although one could argue that the selective breeding to generate different mouse strains over the last couple of centuries may not qualify as "natural" selection, the studies focused on PTTG1 reveal at least one potential (and novel) mediator of lifespan control. Key questions remain: Do variations in PTTG1 expression or activity contribute to lifespan differences across species, and perhaps within a species (including variability in the human population)? Would modulation of PTTG1 expression or activity promote the extension of healthspan or lifespan? How do activities known to modulate lifespan, such as dietary restriction and exercise, influence PTTG1 activity? Are there links between known aging pathways such as via IGF-1 and PTTG1? Good science generates good questions, leading to new insights (and sometimes even solutions). As a senior colleague once told me after I had told him that I worked on aging - "Hurry up".

Transplantation of Young Bone Marrow into Old Mice Produces Systemic Benefits

Researchers here report that transplanting bone marrow from young donor mice into old recipient mice produces a range of benefits, such as improvement in the behavior of macrophage cells. Bone marrow stem cells are responsible for producing blood and immune cells, among other important populations, and this capability is degraded in a number of ways with age. Introducing younger stem cells and their supporting structures is a plausible means to at least partially reverse this process. That said, this sort of approach is unlikely to arrive in human medicine in exactly the same form, given the challenges involved in bone marrow transplantation. It is not a procedure one would want to undergo unless there were no other options, and deploying it widely as a preventative therapy doesn't seem feasible in the present environment. The more likely outcome is for researchers to continue to work in mice so as to better identify specific mechanisms involved in bone marrow aging, those that might be manipulated with small molecule drugs, gene therapies, and the like.

The bone marrow is an important reservoir of stem cells and progenitor cells which cross-talk with peripheral organs to help maintain tissue function. Hematopoietic stem/progenitor cells (HSCs) are responsible for producing blood cells throughout life and these downstream cells play an active role in maintaining tissue homeostasis. With aging reduced function of bone marrow cells correlates with dysfunction of peripheral organs. For example, the decline in immune function with age, referred to as immunosenescence, contributes to the accumulation of senescent cells, persistent low grade inflammation, and reduced responses to injury. The bone marrow is also an important source of endothelial progenitor cells (EPCs) which participate in the generation and repair of vasculature endothelium; aging leads to a decline in circulating EPC number and function.

Different strategies have been proposed to rejuvenate the aged bone marrow such as pharmacological treatments, gene therapy, and dietary interventions. However, most approaches have focused on the effect of rejuvenation on HSC differentiation and EPC colony formation rather than effects on peripheral tissues. Therefore, we hypothesized that reconstituting aged mice with young bone marrow leads to stable engraftment of young cells in aged mice and rejuvenates tissue repair responses.

We recently utilized this bone marrow rejuvenation approach to study the effect aging has on the repair processes initiated post-myocardial infarction. Aged mice were reconstituted with young Sca-1+ bone marrow stem cells and examined 4 months later to allow cross talk between the bone marrow and heart. Young bone marrow reconstitution rejuvenated cardiac endothelial cells which contributed to improved repair and better outcome following myocardial infarction. In addition to improved angiogenesis, our lab has shown that rejuvenation using reconstitution of young cells improves multiple repair processes. Young bone marrow cell transplantation increases the proliferation of resident cardiac cells, increases epicardial derived cell migration/activation, and enhances the acute inflammatory response following myocardial infarction in aged mice.

Beyond cardiac repair, we have shown that bone marrow cells interact with other tissues and that bone marrow rejuvenation can benefit multiple organ systems. Reconstituting aged mice with young cells leads to the repopulation of the retina with young bone marrow derived microglia. Within the retina these cells secrete cytoprotective factors such as fibroblast growth factor-2 and insulin-like growth factor-1 which limit cell death following ischemia/reperfusion injury. More recently, we also demonstrated that bone marrow rejuvenation leads to the introduction of young bone marrow-derived microglia in the brain and that these cells act to improve learning and memory responses compared with mice receiving old bone marrow. Mechanistically, young bone marrow-derived microglia adopt a neutral or anti-inflammatory phenotype while old bone marrow-derived microglia adopt a pro-inflammatory phenotype. These results are consistent with studies which have linked increased neuroinflammation to a decline in cognitive function.


Transplantation of Senescent Cells is an Issue in First Generation Stem Cell Therapies

Researchers here demonstrate that comparatively simple regenerative cell therapies, of the sort presently widely used, in which stem cells are derived from adipose tissue, will tend to introduce senescent cells into the recipient in the case of older donors. Senescent cells are constantly created and destroyed in the body, but the processes of clearance decline with age, and these cells are harmful when they linger for the long term: their secreted signals cause chronic inflammation, while also contributing to tissue dysfunction in a number of other ways. The presence of senescent cells in older individuals is one of the contributing causes of degenerative aging, and adding more such cells is something to be avoided.

Adipose-derived mesenchymal stem cells (ADSCs) or "preadipocytes" have been increasingly suggested for use in regenerative medicine as a treatment for a wide range of diseases due to their multipotency and accessibility. Older adults represent most likely recipients of ADSC therapies given the high burden of diseases in this population. Since autologous ADSCs are preferred in the clinic, it is essential to understand age-related changes influencing these cells. Emerging evidence suggests that ADSCs from aged donors have reduced regenerative potential, leading to diminished therapeutic efficacy. However, it is unknown whether transplanting ADSCs from aged donors might cause unexpected or even harmful effects in recipients. This is especially important for older adults, since they tend to be more vulnerable and less resilient to such stresses.

To examine this, we isolated ADSCs from 12 young (6-7 months, referred to as young ADSCs) and 12 old (28-31 months, referred to as old ADSCs) C57BL/6 male mice. We transplanted ADSCs from young or old donors into syngeneic 20-month-old C57BL/6 male mice. Four to six weeks after transplantation, we tested maximal walking speed, grip strength, physical endurance, daily activity, food intake, and body weight change to assess overall physical function in recipients, based on criteria used in clinical practice. ADSCs from old donors significantly impaired walking speed, grip strength, endurance, and daily activity of older recipient mice after transplantation, compared with mice transplanted with the same number of ADSCs from young donors.

Using single-cell transcriptomic analysis, we identified a naturally occurring senescent cell-like population in ADSCs primarily from old donors that resembles in vitro-generated senescent cells with regard to a number of key pathways. Overall, these findings suggest that ADSCs from old donors can induce physical frailty, which is highly associated with morbidity and loss of independence. Our study potentially begins new avenues of research to discover whether pharmacological interventions, such as senolytic drugs or anti-inflammatory drugs, can prevent or reverse dysfunction caused by transplanting ADSCs or even organs from old donors and improve clinical outcomes of transplantation for older patients.


Video and Transcript of Aubrey de Grey Presenting to the Effective Altruism Community

Aubrey de Grey administers the scientific programs at the SENS Research Foundation, and is a leading figure in the rejuvenation research community and newly formed longevity industry. Here find a transcript of his present commentary on the state of rejuvenation research, lightly tailored for delivery to an audience of effective altruists. Effective altruism is a useful movement, I feel, if nothing else for the pressure that advocates might bring to bear on the corruption and ineffectiveness of much of present day institutional philanthropy. Further, while it might seem self-evident to much of the Fight Aging! audience that the most efficient use of charitable donations, if one aims to reduce suffering in the world, is to fund rejuvenation research programs, the public at large is still far removed drawing this same conclusion. More persuasion is needed, and effective altruists are already engaged in exactly that sort of effort.

The effective altruism community has a culture of running the numbers and arguing from data, and this is attached to a culture of advocacy for their view on how to conduct philanthropy in a more efficient way. Once one starts to run the numbers and argue from data, it is quite hard to avoid the conclusion that aging is by far the worst problem affecting humanity, and thus working to treat aging, in an era in which this is a plausible goal, is by far the most effective form of philanthropy. Aging is the greatest cause of suffering and death in the world, far more so than even infectious disease, war, and poverty. Given this, effective altruists have the potential to become vocal advocates for the cause of human rejuvenation.

Aubrey de Grey: Rejuvenation Technology - Will "Age" Soon Cease to Mean "Aging"?

What I'm going to do today is try to explain why I believe it makes sense for effective altruists (EAs) to prioritize the issue of aging. To make that case, there are a number of questions that need to be answered in the affirmative. First, is aging a really big problem? I believe that it is, by a good distance, the world's biggest problem. But I understand that this group thinks very carefully about such statements, so I need to justify my opinion. The second argument I need to make is that we have a sufficient understanding today of what aging is, and generally how we might go about addressing it. Therefore, if we throw money at this problem, there's a good chance of having a significant impact. This is not trivial. Other times that I've spoken at EA events, I've received a lot of pushback. Many EAs believe we understand so little about aging that what we do at the SENS Research Foundation is essentially random, and therefore spending money on it is unjustified. The third argument I need to make is very new. It's really only arisen over the past few years, and it is this: philanthropy is still critical, even though private investment in the study of aging has exploded.

To address the first point - why aging is important - I'm just going to tell you why I think that is clearly true. To me, it's just a fact that aging causes far more suffering than anything else in the world today or in the foreseeable future. It's not just the death part. We're talking about effective altruism here, and altruism means worrying about other people. People dying makes other people unhappy. That's not arguable. But what might be much more important is that when people die of aging, they do it slowly. They do it as a consequence of a chronic, progressive accumulation of damage in the body, a decline in mental and physical function. So, to me, it's self-evident that aging is, by far, the source of the largest amount of suffering in the world today. You could even argue that it was true a long time ago.

Now I'm going to address the second question. In order to do that, I'm going to fill in a lot of background information. Aging is the combination of two processes, metabolism and damage, which together result in pathology. A network of processes keeps us alive - that's what metabolism means - and, over time, generates damage. Currently, the overwhelming majority of money and effort spent to prevent the pathologies of late life is spent wrongly. It is spent on trying to break the link between damage and pathology. Damage, by definition, is accumulating, which means that efforts to stop it from causing pathology are bound to become progressively less effective as someone gets older. It's obvious. What we might be able to do is separate the component processes of metabolism and damage from each other. That's the maintenance approach - it's damage repair. We might be able to periodically repair some of the damage that metabolism generates, so that even though it continues to generate it, the damage does not reach a level of abundance that causes pathology. I think it is reasonable to call this the common sense alternative.

Seventeen years ago, I described the damage of aging in only seven words, as seven types of damage: cell loss or atrophy, division-obsessed cells, death-resistant cells, mitochondrial mutations, intracellular waste products, extracellular waste products, extracellular matrix stiffening. What's most important is the fact that for each of these types of damage, we can describe a generic therapy that could potentially represent the maintenance approach - the way to repair this type of damage. However, one thing I want to emphasize is that I'm not the only one saying this anymore. Five or 10 years ago, this was an argument that still needed to be made. But now it has been made. As an illustration, the Hallmarks of Aging paper came out only six years ago, and will be by far the most highly-cited paper this decade in the whole of the biology of aging. It's simply a restatement of what I said more than a decade earlier. The important point is that a divide-and-conquer, damage-repair approach is now a completely mainstream, orthodox idea.

On the the third point, that philanthropy still matters despite the growth of a longevity industry, SENS Research Foundation views itself these days as the engine room of that industry. We work on early-stage projects for as long as it takes to establish sufficient proof of concept to spin them out into startup companies. We're not the only ones. The Longevity Research Institute (LRI) is a new organization that's working in a more narrowly defined space. But they may grow, and, of course, there are going to be other organizations like this. Philanthropy matters enormously in this. We believe that we can get all - or at least almost all - of the key technologies in rejuvenation biotechnology into clinical trials within a few years. But the key point here is that the things being funded effectively by the private sector are the low-hanging fruit. And damage repair is an inherently divide-and-conquer concept. You can't just focus on the low-hanging fruit. You've got to address all of the components. It's more important than ever to make progress on the most difficult areas, and that is still a goal for philanthropy.

Deacetylation of the NLRP3 Inflammasome as a Way to Control Chronic Inflammation

Chronic inflammation is an important component of degenerative aging. Excessive inflammatory signaling and activation of the immune system arises due to a combination of many factors, of which some are more important than others, such as the presence of lingering senescent cells. Most of the research focused on controlling inflammation is more interested in sabotaging the mechanisms of control than in removing root causes, however. The work here is an example of the type, in which scientists identify an important feature of the regulatory system controlling inflammation. Forcing a sizable reduction of inflammation via this regulatory system is a fairly blunt tool, as some degree of transient inflammation is vital to health, such as in the response to infection or injury. Nonetheless, the benefits may be large enough to outweigh the side-effects, as is the case for a number of past approaches to limiting inflammation in, for example, the treatment of autoimmune conditions.

Chronic inflammation, which results when old age, stress, or environmental toxins keep the body's immune system in overdrive, can contribute to a variety of devastating diseases, from Alzheimer's and Parkinson's to diabetes and cancer. Researchers now show that a bulky collection of immune proteins called the NLRP3 inflammasome - responsible for sensing potential threats to the body and launching an inflammation response - can be essentially switched off by removing a small bit of molecular matter in a process called deacetylation. Overactivation of the NLRP3 inflammasome has been linked to a variety of chronic conditions, including multiple sclerosis, cancer, diabetes, and dementia. The results suggest that drugs targeted toward deacetylating, or switching off, this NLRP3 inflammasome might help prevent or treat these conditions and possibly age-related degeneration in general.

By studying mice and immune cells called macrophages, the team found that a protein called SIRT2 is responsible for deacetylating the NLRP3 inflammasome. Mice that were bred with a genetic mutation that prevented them from producing SIRT2 showed more signs of inflammation at the ripe old age of two than their normal counterparts. These mice also exhibited higher insulin resistance, a condition associated with type 2 diabetes and metabolic syndrome. The team also studied older mice whose immune systems had been destroyed with radiation and then reconstituted with blood stem cells that produced either the deacetylated or the acetylated version of the NLRP3 inflammasome. Those who were given the deacetylated, or "off," version of the inflammasome had improved insulin resistance after six weeks, indicating that switching off this immune machinery might actually reverse the course of metabolic disease.

"We are asking to what extent can aging be reversed. And we are doing that by looking at physiological conditions, like inflammation and insulin resistance, that have been associated with aging-related degeneration and diseases. I think this finding has very important implications in treating major human chronic diseases. It's also a timely question to ask, because in the past year, many promising Alzheimer's disease trials ended in failure. One possible explanation is that treatment starts too late, and it has gone to the point of no return. So, I think it's more urgent than ever to understand the reversibility of aging-related conditions and use that knowledge to aid a drug development for aging-related diseases."


Aspects of Immune System Aging Proceed More Rapidly in Men

Men do not live as long as women. This is a consistent effect across populations and eras, and there are any number of theories as to why this is the case. This might lead us to expect measurable aspects of aging to be more pronounced in older males than in older females, and researchers here show that this is the case for the age-related dysfunction of the immune system. As we age, the immune system becomes both overactive and less capable, leading to chronic inflammation alongside decreased resistance to infection and cancer. This is an important contribution to age-related frailty, disease, and mortality.

Human peripheral blood mononuclear cells (PBMCs) undergo both cell-intrinsic and cell-compositional changes (i.e., cell frequencies) with age, where certain immune functions are impaired and others are remodeled1. Analyses of human blood samples uncovered significant aging-related changes in gene expression and DNA methylation levels. Recent studies revealed that chromatin accessibility of purified immune cells, especially CD8+ T cells, change significantly with aging, impacting the activity of important receptor molecules, signaling pathways, and transcription factors. Together, these changes likely contribute to aging-related immunodeficiency and ultimately to increased frequency of morbidity and mortality among older adults. However, it is unclear to what extent these aging-associated changes are shared between men and women.

Immune systems of men and women function and respond to infections and vaccination differently. For example, 80% of autoimmune diseases occur in women, who typically show stronger immune responses than males. Stronger responses in women produce faster pathogen clearance and better vaccine responsiveness, but also contribute to increased susceptibility to inflammatory and autoimmune diseases. Although not systematically described, these differences likely stem from differences in both cell frequencies and cell-intrinsic programs. For example, a study in young individuals showed that women have more B cells (percentage and absolute cell counts) in their blood than men. Moreover, hundreds of genes are differentially expressed between young men and young women in sorted B cells.

Despite the importance of sex and age in shaping immune cell functions and responses, it is not known whether men's and women's immune systems go through similar changes throughout their lifespan, and whether these changes occur at the same time and at the same rate. To study this, we profiled PBMCs of healthy adults by carefully matching the ages of male and female donors. These data reveal a shared epigenomic signature of aging including declining naïve T cell and increasing monocyte and cytotoxic cell functions. These changes are greater in magnitude in men and accompanied by a male-specific decline in B-cell specific loci. Age-related epigenomic changes first spike around late-thirties with similar timing and magnitude between sexes, whereas the second spike is earlier and stronger in men. Unexpectedly, genomic differences between sexes increase after age 65, with men having higher innate and pro-inflammatory activity and lower adaptive activity.


The Prospects for Telomerase Gene Therapy as a Treatment for Heart Disease

Telomerase gene therapy is considered in some quarters to be a viable treatment for aging. Telomeres are the caps of repeated DNA sequences at the ends of chromosomes. They are an important part of the mechanism limiting the number of times that somatic cells in the body can divide, the Hayflick limit. A little telomere length is lost with each cell division, and short telomeres trigger cellular senescence or programmed cell death, halting replication. Stem cell populations use telomerase to lengthen their telomeres and thus self-renew to provide a continual supply of new somatic daughter cells with long telomeres to replace those lost to the Hayflick limit. Average telomere length is reduced over the course of aging because stem cell function declines.

This division between a few privileged stem cells and the vast majority of limited somatic cells is the way in which higher forms of life have evolved to reduce the risk of cancer. Somatic cells largely do not last long enough to develop mutational damage sufficient to become cancerous. When cancer does occur, most cancer lineages use expression of telomerase in order to lengthen their telomeres, allowing for unfettered replication. This biochemistry of cancer is the primary reason for caution in the matter of the clinical application of telomerase gene therapies in human medicine. While in mice cancer incidence is actually reduced by telomerase upregulation, possibly because immune system activity is improved to the point at which the destruction of potentially cancerous cells is efficient enough to outweigh risk due to greater replication of damaged cells with lengthened telomeres, it is still a question as to whether human tissues will see the same outcome over time.

Some groups look on telomerase gene therapy as being primarily a form of regenerative medicine, able to improve stem cell and progenitor cell function and thus lead to greater tissue maintenance and regeneration. This is the case in today's open access review paper. It may also act to prevent cells from becoming senescent, and thus lower the burden of cellular senescence in old tissues by allowing slowed and declining clearance mechanisms to catch up. Allowing damaged cells to continue to replicate by lengthening their telomeres may be less harmful than the presence of more lingering senescent cells. It no doubt has other effects on cell function: the mechanisms of action for telomerase gene therapy are far from fully catalogued, but the evidence for benefits to result in mice is quite solid. While a number of humans have undergone forms of telomerase gene therapy via medical tourism and similar arrangements, there is little that can be said of the therapy or the outcomes there, as these applications are few in number, comparatively recent, and undertaken outside the bounds of formal clinical trials.

Telomeres as Therapeutic Targets in Heart Disease

Although there could be asynchrony of telomere length among different tissues, peripheral leukocyte DNA has been most commonly used in clinical studies to measure leukocyte telomere length (LTL). Traditional risk factors for cardiovascular diseases (CVD), such as smoking, diabetes mellitus, dyslipidemia, hypertension, obesity, and shift work, have been associated with short LTL. In the prospective WOSCOPS (West of Scotland Primary Prevention Study) trial, subjects in the lowest tertile of LTL had a 44% increased risk of 5-year major cardiovascular events compared with subjects in the highest tertile of LTL. In a prospective WHI (Women's Health Initiative) study, hen patients developed chronic heart failure, they were also observed to have shorter LTL. Moreover, short LTL was also associated with congestive heart failure severity and clinical outcome.

Robust epidemiological and genetic evidence linking telomere length and CVD risk support the therapeutic hypothesis that genetic manipulations of the telomere system can be a potential treatment target for CVDs. Telomerase gene therapy was first achieved by delivering mouse TERT with an adeno-associated virus (AAV) into young and old mice. This nonintegrative gene therapy resulted in elongated telomeres, extended lifespans, and delayed age-associated pathologies. Importantly, telomerase-treated mice did not develop cancer at a higher rate than the corresponding control group. With the nonintegrative and replication incompetent properties of AAVs, this strategy restricted TERT expression to a few cell divisions and provided a relatively genome-safe TERT activation.

A report for age-associated diseases, such as CVDs, demonstrated improved ventricular function and limited infarct scars after acute myocardial infarction with TERT gene therapy in a preclinical mouse model. TERT gene therapy is a promising candidate that deserves further research efforts for clinical implementation for the treatment of age-associated diseases. Apart from direct TERT delivery by nonintegrative AAV vectors, new gene therapy methods using modified mRNA for in vitro encoding of TERT in human fibroblasts can transiently increase telomerase activity, rapidly extend telomeres, and increase proliferative capacity without the risks of insertional mutagenesis and off-target effects. In addition to proof-of-concept experimental data in mice, the development of safe strategies for transient and controllable telomerase activation in humans can be a subject of future studies.

Evidence for Loss of Capillary Density to be Important in Heart Disease

Loss of capillary density, and thus flow of blood through tissues, is a known feature of aging, though the causes of this change in tissue maintenance are far from completely explored. It is proposed to be quite important in loss of tissue function, particularly in organs with high metabolic demands, such as muscle and the brain. Researchers here provide evidence to suggest that this loss of capillary density is a noteworthy mediating mechanism linking the age-related impairment of heart function with the presence of chronic kidney disease. The latter is already known to correlate with impaired capillary structure in the heart, and here data from patients shows that this is a factor in the progression to heart disease.

People with chronic kidney disease have a higher risk for heart disease and heart-disease death. Coronary microvascular dysfunction, or CMD, is decreased blood flow in the small blood vessels inside the heart muscle that provide oxygen and fuel to feed the pumping heart. A new study links kidney disease to progressive heart disease via this mechanism. In healthy hearts, visualized postmortem, these blood vessels look like a tight filigree network that fills the heart muscle tissue. A diseased postmortem heart has lost much of this network. In living patients, however, those small blood vessels inside the heart muscle cannot be visualized; blood flow scans of living patients visualize only the larger, exterior coronary arteries.

Thus researchers needed an indirect way to gauge CMD. That measure is coronary flow reserve, or CFR, assessed via positron emission tomography. CFR is the maximum increase in blood flow through the coronary arteries above the normal resting volume. In a longitudinal study of 352 patients with chronic kidney disease, all with healthy heart function as measured by ejection fraction and none with signs of overt coronary artery disease, the researchers measured CFR and also measured signs of subclinical heart dysfunction. The patients were then followed a median of 4.4 years for major adverse cardiac events. A total of 108 patients had such major events, including death and hospitalization for non-fatal heart attack or heart failure.

The researchers found that CMD was a significant predictor of abnormal mechanics of the left ventricle - the heart's major pumping chamber - and a significant predictor of clinical risk of adverse cardiovascular outcomes. A statistical model called mediation analysis examined the relationship between impaired kidney function and heart disease. It showed that CMD accounted for 19 to 24 percent of left ventricle diastolic dysfunction, 19 to 42 percent of left ventricle systolic dysfunction and 32 percent of major adverse cardiovascular events. This evidence suggests that the development of severe microvascular dysfunction likely signals the transition from physiological to pathological left ventricle remodeling that increases the risk of heart failure and death in patients with chronic kidney disease.


The Aging Retina, a Mirror of the Aging Brain

Retinal degeneration is a feature of old age, and here researchers show that it correlates quite well with a loss of volume in portions of the visual cortex of the aging brain. These two portions of the nervous system are are connected and related, but it is unclear as to whether there is a direction of causation, or whether this is a case of similar structures being similarly affected by the same underlying mechanism of aging. Chronic inflammation, for example, operates throughout the body, and many aspects of aging are correlated because inflammation accelerates tissue dysfunction in a systemic, whole-body manner.

Age-related retinal diseases, such as late-stage macular degeneration have an impact on cortical morphology. For example, researchers found that the regions of the striate cortex that usually sample the visual input from the injured area of the retina were thinner in participants with macular disease when compared to controls. On the contrary, regions corresponding to non-damaged areas demonstrated a significant increase in cortical thickness. Overall, these findings suggest a strong retinocortical coupling of structural and functional changes in retinal disorders.

Healthy aging is characterized by diverse structural changes in both brain and retina, which association remains to be studied. Concerning the former, there is substantial evidence suggesting that widespread cortical shrinkage takes place with increasing aging. The nature of such effects in early visual areas remains controversial. Whereas some studies have shown volume loss or cortical thinning of visual cortices, others have reported a certain sparing of these areas during aging. Specifically to the primary visual cortex such discrepancy in the literature could be explained by methodological issues, since they demonstrated that different sub-regions of primary visual cortex were unequally affected by aging, depending on their retinotopic eccentricity. Nonetheless, studies directly examining the structure and function of primary visual areas are scarce in healthy aging, in spite of the fact that the organization of early visual areas is well documented in young adults.

The retina also undergoes substantial modifications throughout the lifespan. Histological studies have reported a reduction in the density of photoreceptors, ganglion cells, and pigment epithelial cells with age. Overall, retinal thickness studies using optical coherence tomography (OCT) imaging have revealed regional age-dependent differences in global macular integrity. The improvement of OCT image processing techniques has now allowed for the automatic segmentation of retinal individual layers, which provides a more detailed and specific perspective on such alterations.

we aimed to investigate the association of retinal layer and cortical integrity, in a healthy cohort aged between 20-80 years old. To that end, we performed magnetic resonance structural data imaging measurements of cortical thickness in the primary visual cortex - BA17, the cortical area that receives direct retinal input - and OCT to measure the thickness of the macula and their individual layers, in the same set of participants. We found an age-related decay of primary visual cortical thickness that was significantly correlated with a decrease in global and multiple layer retinal thicknesses. The atrophy of both structures might jointly account for the decline of various visual capacities that accompany the aging process. Furthermore, associations with other cortical regions suggest that retinal status may index cortical integrity in general.


Nicotinamide Riboside Improves Hematopoiesis and Immune Cell Populations in Mice

Mitochondria are the power plants of the cell. They produce the chemical energy store molecule ATP that is used to power cellular operations. Unfortunately, mitochondrial function falters throughout the body with advancing age, and while this is harmful in all tissues, the effects are particularly problematic in energy-hungry tissues such as the muscle and brain. Research of recent years has implicated the loss of nicotinamide adenine dinucleotide (NAD+) in mitochondria in this process. Evidence suggests that loss of effectiveness in mitophagy, the process that recycles worn and damaged mitochondria, is the important issue connected to NAD+ deficiency. NAD+ is largely produced by recycling its products, rather than by synthesis, but both the recycling and synthesis pathways suffer a loss of effectiveness with advancing age.

Various approaches to boost levels of NAD+ have been assessed in animals and are readily available for application to humans. Delivering NAD+ directly is inefficient in comparison to providing precursors and metabolites used in the synthesis and recycling pathways. Nicotinamide riboside supplementation is at present the only approach to upregulation of NAD+ in mitochondria with human trial data. The results from a small trial show a modest reduction in blood pressure in older hypertensive individuals, comparable with what can be achieved through lifestyle choices, due to improved smooth muscle function in blood vessels. One would expect there to be many more forms of benefit resulting from systemic improvement in mitochondrial function, but it is always hard to predict the size of effect in advance, and thus whether or not a particular approach to aging is actually worth it.

The research here is interesting for suggesting that NAD+ upregulation via nicotinamide riboside will lead to gains in immune function via improving the generation of immune cells in bone marrow. This is something that can be tested and quantified in humans without too much trouble, via examination of immune populations in a blood sample. That sort of effort is well within the reach of the self-experimentation community - though, as ever, it is more likely that the research community will get around to running a formal trial before self-experimenters organize sufficiently to produce robust data. The question at the end of the day is the size of effect: is it actually larger than that produced by exercise, and how does that vary by age?

Targeting mitochondria to stimulate hematopoiesis

Hematopoietic stem cells (HSCs) consist of a small cell-population in the bone-marrow (BM) that are responsible for lifelong production of all mature blood cells in an organism. A delicate balance of different HSC fates, namely, quiescence, self-renewal, and differentiation is decisive in maintaining the HSC pool and blood cell homeostasis. Cellular metabolism has emerged as one of the fundamental regulators of HSC fate decision process. HSCs rely primarily on anaerobic glycolysis while downstream progenitors use mitochondrial metabolism to fulfil their energy requirements.

In a recent study we have tested the mitochondrial modulator and NAD+ boosting agent, Nicotinamide Riboside (NR), in the context of regenerative hematopoiesis. One week of NR dietary supplementation to wild type mice resulted in increased BM cellularity and expansion of hematopoietic progenitor cells, this reflected in a significant increase in terminally differentiated circulating blood and immune cells. Importantly, mitochondrial profiling of HSCs derived from mice supplemented with NR revealed significant reduction of mitochondrial membrane potential (an indirect readout on mitochondrial activity), indicating that NR has a direct effect on HSC metabolism when administered systemically.

To understand the molecular mechanisms driving the effect of NR, we performed transcriptome analysis (by RNA sequencing) on ex vivo cultured HSCs. We found upregulation of autophagy (and mitophagy) and of NAD salvage pathway genes upon NR treatment, and a concomitant downregulation of mitochondrial metabolism pathway genes (TCA cycle and Oxidative Phosphorylation). NR-induced mitophagy was confirmed by image analysis of in vitro cultured HSCs and by bone marrow analysis of mitophagy reporter mice (mito-QC). We discovered that mitophagy induction was coupled with activation of mitochondrial unfolded protein response (UPRmt), that has been recently proposed as a conservation mechanism of the HSCs pool during aging. We hypothesize that NR-induced mitochondrial stress leads to the clearance of damaged mitochondria unable to coop with the metabolic stress. This complex mechanism initiates an instruction process where cells are primed toward asymmetric self-renewing cell division via differential distribution of active mitochondria in daughter cells.

Given that previous studies have implicated the importance of mitophagy and autophagy in HSC function and aging, we believe that NAD boosting strategies could be used to improve functionality of the hematopoietic stem cell pool in the elderly, where HSCs lose their capacity to produce a balanced immune system, being strongly primed toward a myeloid fate.

Evidence for PASK Deficiency to Reduce the Impact of Aging in Mice

There are many ways to slow aging to a measurable degree in short-lived species such as mice, and the work noted here is a recently discovered example. Mice have evolved to have a sizable variability of life span in response to environmental circumstances, and thus the cellular machinery relating to various stress responses has an equally sizable influence on health and longevity. Since there are many ways to adjust the operation of that machinery, by increasing or decreasing levels of specific proteins, there are also many ways to slow aging. Few of them are going to be all that useful, unfortunately, as longer-lived species such as our own have a far less plastic life span. An increased operation of stress response mechanisms does not increase human life span by anywhere near as great a proportion as is the case in mice.

Several reports indicate that caloric restriction and intermittent periods of fasting may reduce the risk of complications associated with aging. Cells use nutrient sensing to identify and respond to differences in nutrient levels; the sensing mechanisms are dysregulated during the aging process. AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) pathways are nutrient sensors that have been involved in lifespan. Additionally, PASK (a serine/threonine kinase that contains PAS domains) can sense intracellular oxygen, redox state, and various metabolites.

We have previously described how PASK is a critical regulator of AMPK and mTOR pathways in the hypothalamus and liver, as well as a key regulator of oxidative stress and glucose and lipid liver metabolism. PASK-deficient mice are protected against the development of obesity and insulin resistance induced by a high-fat diet (HFD). PASK has recently been described as a target of mTORC1 during regenerative myogenesis in muscle stem cells.

To investigate PASK's role in hepatic oxidative stress during aging, we analyzed the mitochondrial function, glucose tolerance, insulin resistance, and lipid-related parameters in aged PASK-deficient mice. Hepatic Pask mRNA decreased in step with aging, being undetectable in aged wild-type (WT) mice. Aged PASK-deficient mice recorded lower levels of reactive oxygen species and reactive nitrogen species compared to aged WT. The regulators of mitochondrial biogenesis, PGC1a, SIRT1, and NRF2, decreased in aged WT, while aged PASK-deficient mice recorded a higher expression of NRF2, GCLm, and HO1 proteins and CS activity under fasted conditions. Additionally, aged PASK-deficient mice recorded an overexpression of the longevity gene FoxO3a, and maintained elevated PCNA protein, suggesting that hepatic cell repair mechanisms might be functional. PASK-deficient mice have better insulin sensitivity and no glucose intolerance. PASK may be a good target for reducing damage during aging.


Correlations of Mitochondrial DNA Copy Number and Epigenetic Age Measures

Mitochondria are the power plants of the cell, a herd of bacteria-like organelles that produce the chemical energy store molecule ATP. They have their own DNA, a circular genome distinct from that of the cell nucleus, sometimes several copies per mitochondrion. The number of those mitochondrial DNA copies in a cell is a measure of mitochondrial health that declines with age, as mitochondria become dysfunctional throughout the body. The proximate causes of this dysfunction involve changes in mitochondrial structure and dynamics that inhibit the quality control process of mitophagy, responsible for recycling worn and damaged mitochondria. Connections to deeper causes are not well understood, but these issues must in some way result from the underlying damage of aging.

The DNA methylation (DNAm) based estimator of biological age, DNAm-Age, has become a well-known molecular measure of human aging. DNAm-Age has been associated with cancers, cardiovascular diseases, neurological diseases, and chronic inflammation diseases. Subsequently, another DNAm based marker, DNAm-PhenoAge, was developed to be an improved predictor of mortality and health span using phenotypic age estimated from a range of aging-related clinical measures. Most recently, another metric, DNAm-GrimAge, has been developed to predict all cause mortality and health span.

Unfortunately, the underlying biological and molecular processes that drive these epigenetic age biomarkers are still unknown. Despite the observation that the DNAm-Age is associated with metabolic processes, the relationship between mitochondrial health and DNAm-Age remains understudied. Mitochondria are vital for metabolic processes as they are responsible for ATP production and are known to be involved in the aging process, become larger and less numerous with age. In addition, mitochondrial function may be related to DNAm aging. Activity of DNA methyltransferases (DNMT), as with any cellular enzyme, depend on ATP levels and impaired energy production as a result of mitochondrial dysfunction may influence normal function of DNMTs.

Mitochondrial DNA copy number (mtDNAcn), a measure of mitochondrial genome abundance, is commonly used as a reflection of the mitochondria's response to oxidative stress as well as general dysfunction. Mitochondria DNA (mtDNA) is sensitive to oxidative stress because it lacks a robust DNA repair system to restore oxidative stress induced damage and mtDNA damage persists longer compared to genomic DNA. Typically, mtDNA will increase when the endogenous antioxidant response is no longer able to recover its redox balance, possibly as a compensatory response. Previous studies have shown that mtDNAcn decreases with age and is positively associated with telomere length.

Recently, our group has shown that mtDNAcn is negatively correlated with DNAm-Age and hypothesized mtDNAcn may be a proxy of mitochondrial buffer capacity. Reduced mtDNAcn may be a consequence of exhausted mitochondrial buffering capacity, leading to adverse outcomes such as aging. In a population of 812 aging male veterans, we found contrasting results between cross-sectional and prospective analyses of mtDNAcn with aging biomarkers DNAm-Age, DNAm-PhenoAge, DNAm-GrimAge and leukocyte telomere length. We observed that mtDNAcn is negatively associated with cross-sectional measures of DNAm-Age and DNAm-PhenoAge. We found suggestive evidence that mtDNAcn is positively associated with prospective measures of DNAm-PhenoAge and negatively associated with prospective measures of leukocyte telomere length. These results suggest that while the negative cross-sectional associations reflect the opposing time-trends of mtDNAcn and aging biomarkers, it may be driven by unmeasured confounders such as underlying biological processes that drives both the decrease of mtDNAcn over time and the increase of DNAm-Age and DNAm-PhenoAge over time.


Notes on the 2020 Longevity Therapeutics Conference in San Francisco

I recently attended the 2020 Longevity Therapeutics conference in San Francisco. I presented on the work ongoing at Repair Biotechnologies, but as is usually the case the more important parts of the visit took place outside the bounds of the conference proper. Longevity Therapeutics is one of the four or five core conferences for the longevity industry, at which you'll meet many of the early participants - a mix of scientists, entrepreneurs, and investors, and patient advocates. As such, most of the conference goers have already seen my updates, or are otherwise aware of the Repair Biotechnologies programs aimed at thymic regeneration and reversal of atherosclerosis. This year was heavily biased towards the entrepreneurial component of the community. It was even the case that most of the scientists attending were presenting in the context of a company that is advancing their work towards the clinic. As the longevity industry expands, ever more researchers in the aging field are finding the opportunity to start a company, or otherwise hand off their work for clinical development.

The first day was a lightly populated set of workshops prior to the conference proper. In the morning, Aubrey de Grey of the SENS Research Foundation and AgeX Therapeutics gave his usual overview of the state of rejuvenation research and development, with a little more emphasis than usual on clinical development and investment in the field. Irina Conboy discussed the plasticity of aging; she is one of the more noted researchers involved in the modern investigations of parabiosis, in which old and young mice have their circulatory systems linked. She gave a tour of differences observed in old mice during parabiosis, such as improved liver regeneration. The argument of beneficial factors in young blood versus detrimental factors in old blood has resolved, by the sound of it, to the conclusion that both mechanisms are relevant - there are a lot of different factors, of different importance. She noted that she is starting a company to push forward some of her work on upregulation or downregulation of factors identified in parabiosis, particularly the combination of oxytocin and TGF-β. Michael Fossel talked about the hallmarks of aging and what to do with them. His point was that metabolism and aging are enormously complicated, forming a system that exhibits risk factors rather than deterministic behaviors. The focus should be on finding the best point of intervention, which is not the same thing as understanding the system. Greater understanding only makes finding the best point of intervention easier, it isn't absolutely required.

The afternoon was more focused on clinical translation, with presentations from companies further along in the process of conducting trials with the FDA. Mark Allen of Elevian talked about indication choice as a challenging process for companies targeting aging. Elevian is a GDF11 company, and they presently think that prior issues with contradictory results for GDF11 delivery in animal models were due to poor manufacture of the protein. Indication choice is challenging for therapies intervening in aging because so many different indications can be considered, but most are dead ends. It is very important to consider how the choice of indication affects time to market: one is looking for short treatments that can produce large effects. Further, if you want the FDA on your side, you really have to go after large unmet needs for serious diseases. The Elevian team used a matrix/scoring approach to assess different indications. Outside expertise is vital; you can't do this yourself.

Elizabeth Jeffards and Erin Newman from Alkahest further elaborated on this process of indication selection, and then moved on to talk about how to run trials. Their high level point was that the operation of trials becomes your whole company, determining everything about how you are seen and how you proceed. The two talked about the central matter of payer willingness to pay for your therapies - whether insurance giants, Medicare, and other entities will toe the line. This is a very important matter, at all stages of the process of figuring out which indication to pursue. They also emphasized the need to build a very specific target product profile, the exact cost and performance of your therapy, well in advance of any sort of data. Another vital issue is manufacturing: getting the timing right, given the lengthy duration of GMP manufacture, and the huge cost of that process. This is challenging and needs very careful management. Peter Milner of Retrotope talked about their orphan disease trials, and reinforced the points already made. Retrope uses deteurium stabilized lipids to treat neurodegenerative conditions in which lipid peroxidation is a serious concern. In talking about about the Retrotope clinical trials, he again pointed out that cost and time are very important in their choices of indications - one has to to look for large effects achieved in quick trials.

The first day of the conference opened with a keynote by Nathaniel David of UNITY Biotechnologies. He surveyed the common approaches to research aimed at intervention in aging, that small changes between species biochemistry leads to large changes in species life span, and so forth. Regarding UNITY, he discussed their human data on the performance of senolytics for osteoarthritis and for degeneration of the retina, such as dry macular degeneration. They are in phase II for osteoarthritis, with data coming out late in 2020. For the eye, they are still working on phase 1 safety data, also coming out late 2020. They are also in the earlier stages of developing senolytic treatments for lung and kidney diseases.

Following that, Joan Mannick of ResTORbio opened her presentation by lauding mTORC1 as a target, pointing to the large body of research in short-lived species. Following failure on their phase III trial for reducing influenza incidence, they are now focusing on neurodegenerative disease, particularly Parkinson's disease. They believe that raised autophagy via mTORC1 inhibition may help with aggregates in these conditions, and discussed some of the supporting evidence in animal models. Peter Fedichev of Gero presented on their AI program for small molecule drug repurposing and discovery. Based on their models of biochemical data from mice and humans, they divide aging into two overlapping processes that they call "aging" and "frailty" - these are names for portions of a data model, and don't necessarily map well to the common meanings of the words. Mice and humans have quite different proportions of "aging" versus "frailty". Gero has new data from lifespan and rejuvenation studies using compounds that they intend to repurpose: they have achieved some degree of slowing or reversal of aspects of aging via their drugs in mice. This essentially shows they can pick drugs that perform comparably to some of the historical efforts to achieve this sort of outcome, and can do so faster than was possible in the past.

Gino Cortopossi of UC Davis is working on new approaches to upregulate mitochondrial function. He discussed how his group carried out the discovery of drug candidates to try to target mitochondrial function, SHC, and MTORC1. This presentation was an exercise in thinking about how to test interventions of this nature, what sort of a path leads forward from there to the clinic, and how to organize a handoff from academia to Big Pharma. Hanadie Yousef of Juvena Therapeutics talked about their AI-driven program of mining the secretome of pluripotent cells. The Juvena staff are searching for secreted molecules that can delivered as therapies to upregulate regenerative and stem cell capacity in old people. Their initial focus is on muscle regeneration in the context of age-related sarcopenia. In one of the more interesting presentations of the day, Matthias Hackl of TAmiRNA talked about biomarker development in the microRNA space. The TAmiRNA folk think that they should be able to use a blood sample to produce simultaneous measures of senescent cell burden in many different tissues via assessment of circulating miRNAs from the senescence-associated secretory phenotype (SASP): each tissue has a signature. They are not quite there yet, but this will be very useful if it works out. Dana Larocca of AgeX Therapeutics talked on the topic of exosomes. That AgeX is focused on production of useful cell lines via induced pluripotency gives them a good head start on the production of useful exosomes via harvesting cell cultures of those cell lines. They are presently engaged in the search for interesting exosomes that might form the basis for therapies that make adult stem cells more active.

Jay Sarkar of discussed their approach to transient epigenetic reprogramming in order to force cell function to become more youthful. They use mRNA for reprogramming, as they feel it gives them greater control, and precise control is very important in their work - they must not push cells all the way into pluripotency, just shock them into better operations, and there is a fine line between those two outcomes. The staff are using in vitro cell data to suggest that they can affect various hallmarks of aging: changing certain cell properties and the overall transcription landscape. The approach doesn't lengthen telomeres, which is interesting; the most important thing it does, I would say, is to restore mitochondrial function. He showed data for chondrocytes, relevant to osteoarthritis, and the team are also trying a cell therapy approach on this front, to reprogram cells and then transplant them to see if they help. Additionally, they have worked in skin models to show reversal of aspects of aging there. is one of a growing number of companies working with Entos Pharmaceuticals to produce a non-toxic lipid nanoparticle vector to deliver their therapy in vivo. Given that, it isn't surprising that that they are also working with Oisin Biotechnologies, who also use the Entos Pharmaceuticals platform, to see how senolytics plus reprogramming work in synergy.

Rich Allsop of University of Hawaii talked on the role of FOXO3, one of the few robustly longevity associated genes in humans, in influencing telomere shortening and inflammaging. It touches on the IGF-1 pathway, and a variant is associated with greater longevity in humans. These researchers think that the behavior of the variant is more to do with enhancer or promoter effects on gene expression, not functional differences in the protein, as the difference is in a non-coding region of the gene. There is some question as whether inflammation causes a difference in the pace of telomere shortening, such as via faster replication of immune cells in response to inflammatory signaling, or whether the relationship functions in a different way. Michael Fossel of Telocyte discussed his view on telomeres, cellular senescence, and telomerase gene therapy. He argues that the data shows that you need to increase telomere length to a large degree in order to see reductions in cancer risk, meaning lots of telomerase, not just a little - too little and there will be more cancer. His company is presently looking for funding to run an Alzheimer's disease trial of telomerase gene therapy; they have everything planned, and just need the backing.

Steve Turner of InVivo Biosystems presented on a system that can be used to determine quickly, say in 3 months, whether or not a therapy will extend lifespan and healthspan. To achieve this result they use C. elegans and zebrafish, and assess omics results, with some degree of automation in their platform. In a related presentation, Gordon Lithgow of the Buck Institute outlined their work on small molecule discovery with a C. elegans platform. A fair number of varied approaches to cost-effectively use these short-lived species in conjunction with automation, omics, and AI are out there under development these days. Kristen Fortney of Bioage Labs talked on their AI-driven discovery in human aging omics data, in search of pathways that can be drugged. They take a holistic view of aging: don't study age-related diseases, study aging as a whole, look for important processes. Given pathways, they perform screening in vitro, then take drug candidates to a sizeable vivarium of 3,000 mice (expanding to 12,000 all too soon), and test the outcome there. They outlined a few example targets and the data supporting their ongoing work, including approaches to reduce neuroinflammation. Andrea Maier of the University of Melbourne talked at a high level on the development of potential aging-targeting repurposed drugs in Australia. This was a very nuts and bolts outline regarding how one plans and conducts human trials for specific age-related diseases. They were largely focused on lifestyle intervention, and are only now starting to think about drugs. Rounding out the first day, Wim von Schooten of Teneobio presented on the use of a CD38 inhibitor as a way to upregulate NAD+ levels and mitochondrial function. CD38 is somewhat connected to the proximate causes of NAD+ reduction in aging mitochondria, but it has other roles as well. It is also anti-inflammatory. CD38 is upregulated with age, in concert with NAD+ drop and inflammation rise, and the position in this presentation is that CD38 is causal of NAD+ decline.

The second day of the conference kicked off with a presentation by Sergio Ruiz on the topic of the Methuselah Fund and their progress to date in supporting new and important companies in the longevity industry. He gave a general overview on the state of investment in early stage companies in the field: what investors are looking for; how to transition from lab to clinic; the recent evolution of the longevity industry and the field of aging research. He noted that this is a huge opportunity for changing the human condition, not just a chance for a sizable return on investment. The team is presently working on raising their second fund. The first fund writes $50k-$500k checks, second fund will be much larger and write $1m-$5m checks.

Ronald Kohanski of the National Institute on Aging gave the NIA/NIH perspective on rejuvenation and accelerated aging as therapeutic targets. They see the Interventions Testing Program and other programs as ways in which the NIA supports industry. He noted a range of ongoing work at the NIA that connects to the hallmarks of aging. They are starting to think about using omics data from the Interventions Testing Program and other studies to better understand what is taking place in aging-related pathways, as well as to develop ways to measure rejuvenation and aging. The presentation mostly dwelled on parabiosis and small molecules that slow aging as interventions to consider in this context. Nir Barzilai of the Albert Einstein College of Medicine followed to talk about the challenges inherent in making therapies to target aging or age-related diseases. The first problem is that animal models are not great, there is too much failure in translation to human medicine. Then there is the issue of payers (insurance companies, medicare, and so on) that don't want to pay for interventions that slow aging, which is related to the challenge of there being no FDA-approved indication for aging. The lack of an indication is largely why payers will not pay, even if therapies could be approved in some useful way. He mixed this in with his usual talk about centenarians and data on their health habits, genetics, and so forth.

Kevin Perrott of OpenCures presented on collecting data from people who are trying interventions themselves, self-experimenters, to try to reduce the time taken to develop new therapies. He is conducting proteomic analysis of blood samples from people in the self-experimentation community to measure outcomes, and the OpenCures team are also carrying out volunteers studies of supplement-regulated compounds, somewhat similar to phase 1 trials in organization, with proteomic measurements to assess effects. Julie Andersen of the Buck Institute talked about cellular senescence as a driver of Alzheimer's disease - something I would like to see a lot more work on, given the potential for meaningful benefits to patients. She noted the evidence for senescent glial cells, such as astrocytes, to contribute to neurodegenerative pathology. It is now thought possible for post-mitotic neurons to undergo senescence as well, contrary to earlier dogma. That might present a challenge, but equally obvious issues with cognitive function haven't manifested yet in animal studies of senolytics. She presented in vitro evidence for amyloid-β to cause senescence in brain cells, and suggested that the spread of senescence via the SASP occurs without amyloid-β in the later stages of the condition. The initial presence of amyloid-β is required, but not thereafter, and might be why removing amyloid-β doesn't help once this process is underway.

Richard Marshak of Torcept Therapeutics undertook a discussion on how to go about rational drug development with aging as the target. The company conducts drug discovery of mTORC1 inhibitors, and he talked about their pipeline and evidence. There is still skepticism from Big Pharma regarding the whole of the longevity industry: there are no clear endpoints; the technical and regulatory risk is far greater than Big Pharma entities are usually prepared to engage with; and the expense of testing against aging as a target is believed to be high. Once again this included a discussion of payers versus regulators, and what these two groups are looking for. Payers are interested in extending healthy longevity, it is worth bearing this in mind - there are strong economic incentives here that may help to overcome other issues. The development of endpoints for interventions in aging is important, since we can't use aging itself right now. Yet surrogate outcomes (measurements of biomarkers rather than patient outcomes) are not popular with anybody in the regulatory system at this time.

Marco Quarta at Rubedo Life Sciences presented on their small molecule discovery of senolytic and anticancer compounds. They are at the preclinical stage and would like to start looking at other cell changes that occur with age as well, such as loss of stem cell function. They are claiming a 60-70% clearance of senescent cells in multiple tissues via their lead senolytic, which is larger than most of the published literature to date - but it is hard to say how this compares with the state of the art in the various companies working on new senolytics. A range of other mouse model data on toxicity, safety, and effectiveness was presented. Andy Schile of Jackson Laboratory gave a plug for their aged mice, a source for studies. He surrounded that with examples of some of their studies of mice at different ages, presenting data on their usefulness in various models of age-related disease and dysfunction. Pan Zheng from the University of Maryland Baltimore talked about the role of CD24 in the inflammatory response to tissue damage, such as in the context of graft versus host disease, for example. This research group is attempting to influence the CD24 pathway to reduce inflammation in bone marrow grafts, HIV patients, and during immunotherapy. They have a CD24 fusion protein that works via affecting immune checkpoints to dampen the response.

Jean-Marc Brondello of ISERM discussed cellular senescence as a cause of osteoarthritis, with a focus on the details of the manifestations of the condition and how senescence contributes to these issues. This team is processing omics data to identify possible new senotherapeutics that might address the issue. John Lewis of Oisin Biotechnologies gave the usual presentation on the Entos Pharmaceuticals lipid nanoparticle platform and its application as a senolytic therapy when delivering a suicide gene therapy triggered by expression of p16 or p53. An important point emphasized here is the exceptional safety profile of these nanoparticles - massive doses can be supplied to mice and other mammals with no signs of toxicity. Andrei Gudkov at Genome Protection discussed retrotransposons and their role in aging. Of particular interest is that retrotransposon activity drives chronic inflammation via cellular senescence. This team is developing therapies to try to ameliorate these issues. He presented an interesting view of aging as a species-specific cliff of mortality, and argues that DNA damage (i.e. retrotransposon activity) is the cause of the cliff, via production of chronic inflammation at a time dictated by loss of suppression of retrotransposon activity. Genome Protection studies retrotransposons in dogs, as breed variations in lifespan may be largely caused by retrotransposon based changes - the differences in genetics between dog breeds tend to cluster appear in locations connected to retrotranspon activity. Lastly, Lewis Gruber from SIWA Therapeutics presented on their program focused on a senolytic monoclonal antibody. They originally started out by targeting oxidative stress and glycolysis: these aspects of cell dysfunction have a common advanced glycation endproduct surface marker for a monoclonal antibody to bind to. Given that binding, immune cells then destroy the errant cell. He pointed out that these marked cells are largely senescent, but others might only be dysfunctional. That can include cancerous cells.

All in all it was a interesting event, a good chance to catch up with existing members of the community and meet some new faces. If one has an interest in joining the longevity industry in some way, Longevity Therapeutics should be on the list of conferences to attend, along with Undoing Aging in Berlin, Ending Age-Related Diseases in New York, and Longevity Leaders and the Longevity Week events in London.

The Potential for Exosome Therapies to Treat Sarcopenia

The authors of this open access review walk through some of the evidence for delivery of exosomes, such as those derived from stem cells, to be a basis for treating sarcopenia. Sarcopenia is the progressive loss of muscle mass and strength that takes place with aging. While a fair degree of sarcopenia is preventable, being the consequence of an age of comfort, leisure, and too little exercise, the rest of it is still inevitable absent some way to interfere in the mechanisms of aging that disrupt muscle tissue maintenance. The delivery of cell signals encapsulated in exosomes might be capable of forcing muscle stem cells into greater activity, overriding their natural reaction to an aged tissue environment. While this class of therapy doesn't address the underlying causes of the problem, the benefits may still be large enough to be worth chasing.

Sarcopenia is one of the hallmarks of the aging process. Human muscle undergoes constant changes with the most alterations taking place with age. As shown by different studies, on average, the prevalence of sarcopenia in older adults aged 60-70 years lies at 5-13%, increasing to 11-50% in people aged 80 or older. Sarcopenia is closely related to negative outcomes in older adults, such as an increased risk of falls and fractures and impaired cognitive function and physical performance. Researchers estimated that the economic costs for sarcopenia in the USA were about $18.5bn in 2000. Strikingly, if the prevalence of sarcopenia would be reduced by only 10%, it would save $1.1bn in medical costs per year in the US health care expenditures. Therefore, it is urgent to develop more effective research strategies and therapeutic approaches for preventing sarcopenia based on a better understanding of the potential mechanisms of this disease.

Over the years, many researches have investigated physiological and pathological conditions related to poor muscle regeneration in sarcopenia. However, the underlying molecular mechanisms associated with sarcopenia remain not completely understood. Some evidence suggests that such factors as anabolic resistance and endothelial dysfunction may contribute to the development of sarcopenia. Another one of the recent studies reported that exosomes released by muscles into the bloodstream may also play an essential role in muscle regeneration. This opens a novel field of research in preventing muscle loss. It is hypothesized that exosomes as carrier of a cargo of proteins, mRNA, miRNA, and other non-coding RNAs play a crucial role in myogenesis and muscle development. It has been shown in a mouse model of muscle injury that human skeletal myoblasts-derived exosomes containing all sorts of signal molecules can promote muscle regeneration.

Although the treatment of sarcopenia remains challenging, it is widely accepted that such strategies as nutritional supplementation and physical training (both aerobic exercise and resistance exercise) are the key interventions that can help maintain skeletal muscle mass. However, the molecular mechanisms behind the prevention from the age-related muscle loss by nutrition and exercise are still poorly understood. More recent data indicate that exercise attenuates sarcopenia mainly through increasing peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) signalling, which can be activated by heat shock protein 60-bearing exosomes released after physical training. This connection between sports and release of exosomes supports the assumption about the key role of exosomes in muscle regeneration.

Other recent data also indicates that exosomes, secreted by skeletal muscle cells and carrying miRNAs and other factors, may act as vital modulators of skeletal muscle function, and may have the potential in the research strategies of sarcopenia. Owing to their diverse pathological and therapeutic effects, exosomes has attracted more attention in the scientific community in recent years. It has been shown that exosomes are related to organ crosstalk and can be beneficial in research of many diseases, including kidney injury, myocardial infarction, Parkinson's disease, and cancer. Importantly, the present data suggest that exosomes may mediate and enhance the beneficial effects of exercise. Emerging evidence has presented that exosomes as carriers can increase muscle regeneration after skeletal muscle injury and improve muscle protein synthesis and hypertrophy, which could support exosomes as vectors for future research strategies of sarcopenia. All these mechanisms are interconnected, but the underlying pathways are still not understood completely and should be examined more thoroughly in future studies.


Thymic Involution Contributes to Immunosenescence and Inflammaging

The thymus is an underappreciated organ, responsible for the complex process of generating mature T cells of the adaptive immune system. Unfortunately it atrophies with age in a process called thymic involution. By age 50 most people have little active thymic tissue left. They must coast for the rest of their lives on the adaptive immune cells that they have at that point, replicating in the periphery of the body without a meaningful supply of new reinforcements. This inevitably leads to an immune system made up of damaged, overspecialized, and malfunctioning cells, incapable and inflammatory.

That part of the overall decline in immune function that is driven by thymic atrophy is a noteworthy component of aging, and this is why restoration of thymic tissue and activity is an important goal for the rejuvenation biotechnology research and development community. Since there are a number of us working on this, including Repair Biotechnologies, Lygenesis, and others, we might hope that a viable rejuvenation therapy for thymic function will arrive sooner rather than later in the years ahead.

The aged immune system has various characteristics. One of which is immunosenescence, which describes the vast and varied changes in the structure and function of the immune system as a result of age. Many of the early observations, such as reduced ability to fight new infections, diminished vaccine immunity, and reduced tumor clearance are generally categorized as immune insufficiencies. Immunosenescence is not due to the lack of immune cells, but due to reduced immune repertoire diversity, attributed to insufficient production of naïve immune cells and amplified oligo-clonal expansion of memory immune cells. Immunosenescence is therefore linked to the thymus. Natural aging causes the thymus to progressively atrophy, a process called thymic involution. This phenomenon is readily observed in most vertebrates and results in structural alterations, as well as functional decline, ultimately resulting in significantly decreased thymic output of naïve T cells that reduces the diversity of the T cell antigen receptor (TCR) repertoire, culminating in disrupted T cell homeostasis.

The second characteristic of aged immunity is termed inflammaging. Inflammaging describes the elevated self-reactivity in the elderly, resulting in the typical chronic, low-grade, but above baseline, systemic inflammatory phenotype observed in the absence of acute infection. Although immunosenescence and inflammaging appear to be opposing phenotypes, they comprise two sides of the same coin when attempting to holistically understand age-related immune dysfunction. It has been proposed that the basal inflammatory state in the elderly, defined by inflammaging, greatly contributes to many age-related degenerative diseases.

T lymphocyte (T cell) development and selection occurs in the thymus. Included in this process is central tolerance establishment. First is thymocyte negative selection, during which the majority of self (auto)-reactive T cells are depleted from the repertoire via apoptosis. Second is the generation of CD4 single positive FoxP3+ regulatory T (Treg) cells, whose primary function is to suppress T cell-mediated self-reactivity and preserve immune homeostasis in the periphery. These arms of central T cell tolerance work in tandem, and Treg cells most likely compensate for imperfections of negative selection, as some self-reactive T cells escape negative selection. With age, however, the atrophied thymus declines in its capacity to establish central tolerance, thereby, causing increased self-reactive T cells to escape to the periphery and participate in the process of inflammaging.


Two Examples of UK Biotech Startups Focused on the Treatment of Aging

The biotech startups of the core longevity industry, founded by the entrepreneurs who are regulations in the English-language conference circuit, are largely US companies. This is the way of the world in the biotech industry in general. The non-US participants include startups of British, Russian, and other origins, though it is often the case that when a company achieves some success it moves to where its backers and allies are found. For reasons relating to the historical interests of local research communities, and current interests of local venture funds, companies tend to be clustered in a few countries rather than being more evenly spread. The United Kingdom includes a number of research communities working on portions of aging relevant to therapeutic development. It is also the case that Juvenescence is based in the UK, and London is the center of the sizable advocacy and funding network associated with its principals.

In these articles, takes a brief look at a couple of early stage UK-based biotech startups that are focused on the treatment of aging. They are not the only ones; I am aware of a few other groups at earlier stages in their progression from academic laboratory to launching a startup. It isn't unusual that both of the startups noted here have an interest in senescent cells: the role of cellular senescence in driving degenerative aging is a very active area of development. It is also the basis for a market that is potentially so large that dozens of companies could find success here in the years ahead. The animal data for elimination of senescent cells as a basis for rejuvenation therapies is very compelling, and the human data obtained to date is promising.

Five Alarm Bio targets Alzheimer's and skin aging

Five Alarm Bio is founded by Dr William Bains, a scientist and entrepreneur with a 30-year track record in research into the fundamentals of biology and commercialisation of those discoveries. Dr Bains attended an anti-aging conference in November 2014, which is where he had his eureka moment that led to the creation of Five Alarm Bio. "While thinking why the speaker in a talk had got it all wrong, I had a flash of insight how the chemistry and the aging fitted, and the basic idea was born. It took another year to flesh out why this was a business, and we incorporated in 2016."

"We are in the research stage, about to embark on a major program of chemical synthesis to optimise our initial probe molecule. All the data so far has been seed-funded research in vitro. We have got good data to show that the probe molecule we are using a) is non-toxic on prolonged use, b) slows the rate at which cells age in vitro, and c) may reduce the damaging effects that senescent cells can have on the cells around them."

Dr Bains explains that all the work is done on primary human cells, which he feels is the best model for human cellular aging (other than people), but it is still in the petri dish, not in an animal. Five Alarm Bio's next steps are to secure broad patent coverage of its mechanism of action and on the optimum molecules to take forward. In terms of specific targets for the company's technology, its initial proof of concept experiments were on cell senescence, and so is looking at other targets where cell senescence is important, but Dr Bains is also keen to explore its potential benefit in Alzheimer's.

Biosens raising £5m to support commercialisation

The market for the development of anti-senescence therapeutics is on fire and we've spoken to a number of companies working in this field in recent months. The latest of these to come to our attention is Biosens, a British-German biotech start-up focused on the discovery and production of performance and longevity enhancing products. Founded in 2013, Biosens originally focused on agricultural applications, but refocused on human therapeutics early 2017 with the goal of making longer and healthier lives an attainable and affordable reality.

While still relatively early stage, the company already has three therapeutic products in its pipeline, including therapies for cell rejuvenation, muscle regeneration and cognitive improvement. "Aging is characterized by accumulated damage in stem cells and somatic cells causing their senescence as well as pathogenic factors in blood causing chronic inflammation. Reactive oxygen species accumulation, DNA damage, epigenetic alterations, protein aggregation, and telomere shortening are major causes of cell senescence. Our lead candidate works by rejuvenating the stem cell pool while simultaneously removing damaging senescent cells."

Exceptionally Long Lived Humans Exhibit Slower Epigenetic Aging, Measured by DNA Methlyation Clocks

Epigenetic clocks are produced by examining age-related changes in DNA methylation, finding combinations of such changes that are consistent across populations, and predict chronological age. These clocks also predict mortality, in the sense that people with higher epigenetic than chronological age tend to have a higher mortality risk, or be more burdened by chronic age-related disease. The challenge here is that it remains very unclear as to what these epigenetic clocks are actually measuring, which of the underlying processes of aging they reflect, and to what degree. That makes it hard to use epigenetic clocks in any meaningful way - the results are not actionable.

There are other issues to be debugged as well. For example, that the first generation epigenetic clocks are unaffected by fitness differences, or that they appear to systemically underestimate age in older individuals. Given that second point, when looking at the results in the paper here, in which slower epigenetic aging is claimed for a cohort of exceptionally long lived individuals, we are left somewhat in the dark regarding the relevance of the data. These and other issues are not insurmountable problems, but they are standing in the way of broader application of epigenetic measures of biological aging.

Many studies are aimed at biomarker discovery and improvement for aging. The need for such characterization is of upmost importance in light of efforts to achieve longer health and lifespans across the world. Such biomarker detection would enable tracking and even reversal of aging processes and allow for drug targeting and development to benefit the already graying population. Molecular and genomic biomarkers for aging are still sparse and inaccurate with the exception of the very recent development of DNAmGrimAge. This DNA methylation biomarker outperforms all previously reported methylation age estimators and serves as a very accurate estimate of chronological age. Although this is expected due to the use of chronological as a surrogate for the age prediction, DNAmGrimAge also serves as an evaluation of health status, indicative of the rate of epigenetic aging. Use of such biomarkers as indication of rate of age acceleration could promote better understanding of the processes underlying progression of aging and replace use of chronological age in clinical assessments relating to those conditions.

We show here that, although accurate in offspring of exceptionally long lived individuals (ELLI) and unrelated controls, DNAmGrimAge underestimates the chronological age of our ELLI participants, predicting a younger epigenetic age. We believe that this represents a slower rate of aging processes occurring in ELLI, enabling them to reach such exceptional chronological age. This is in agreement with the methylation profile of semi-supercentenarians and their offspring, and replicates earlier results in our independent cohort.

Further, the DNA methylation based estimator of telomere length, DNAmTL, showed no correlation with qPCR measurement of telomere length, until adjusted by DNAmGrimAge. This masking effect of the physiological age (measured by DNAmGrimAge) adds support to the slower rate of aging. Telomere length has long been argued for and against use as an age indicator, but it is well-established to be decreased with age. Our qPCR measurements are consistent with previous observations of longer telomeres in ELLI. While telomere length of ELLI was expected to shorten in respect to offspring and controls because of their relatively advanced age, it remained unchanged, indicating a similar telomere length despite almost 30 years average age difference between group participants, demonstrating once again, a decreased aging rate. Taken together with the juvenile methylation rates in ELLI, we suggest that ELLI age slower than the general population through a beneficial methylation profile that may affect telomere length and other aspects of the hallmarks of aging.


Reviewing Efforts to Develop NAD+ Therapies to Reverse Age-Related Loss of Mitochondrial Function

Increasing levels of NAD+ in mitochondria, is a class of therapy that probably produces most of its benefits in animal models and human trials by restoring mitophagy. This may well be true of mitochondrially targeted antioxidants as well. Mitophagy removes damaged mitochondria, but is hampered by age-related changes in mitochondrial dynamics, among other reasons. Mitochondria are responsible for packaging chemical energy store molecules to power cellular operations. Mitochondrial function is critical to tissue function throughout the body, but is of particular note in the energy-hungry tissues of muscle and brain.

NAD+ declines with aging for causes that are not well understood, not well linked to the underlying molecular damage that causes aging. Methods of increasing NAD+ are operating on proximate causes at best. They can reverse some degree of the decline, as demonstrated in human trials focused on the function of smooth muscle in major blood vessels. Not all of these trials produced benefits, however, and in those that did, NAD+ upregulation so far doesn't achieve more than "some degree" of improvement. Thus assessment of the field of prospective NAD+ interventions is still very much an ongoing project.

Over the last decade, the importance of NAD+ in healthy ageing and longevity has been recognised, detailed molecular mechanisms unveiled, and many clinical trials explored. Studies from laboratory animals, such as in nematodes and mice, and in human primary cells and post-mortem tissues, as well as human brain imaging, indicate that there is an age-dependent reduction of NAD+ in cells and tissues. Mechanistically, it is suggested that ageing-induced NAD+ reduction may result from reduced production - as there is an age-dependent reduction of key enzymes involved in NAD+ metabolism - or increased consumption by NAD+-consuming enzymes, such as PARPs, CD38, and Sirtuins. All three classes of enzymes compete for NAD+ during ageing, ultimately leading to a bioavailability level insufficient to sustain all NAD+-requiring cellular activities.

Intriguingly, NAD+ repletion, by the supplementation of NAD+ precursors, such as nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), nicotinamide (NAM), or even NAD+ itself, delay ageing phenotypes and promote healthy longevity in both normal and accelerated ageing models in Caenorhabditis elegans (roundworms), Drosophila melanogaster (fruit flies), and mice. Encouraged by animal studies, more than 20 clinical studies exploring whether NR may alleviate pathological ageing and age-predisposed diseases have been initiated.

At least 5 clinical trials have been completed showing that 1-2 g/day of NR for up to 1-3 months is safe. While there were encouraging results in some NR-based phase I clinical trials aiming to reduce blood pressure in healthy middle-aged and older adults and to slow disease progression in amyotrophic lateral sclerosis (ALS) (NR + pterostilbene), no effect was reported in trials of short-term (up to 2-3 months) NR supplementation in obese, insulin-resistant men and nondiabetic males with obesity, nor muscle-mitochondrial bioenergetics in aged men. Of note, all three reports were from the same study and reported different outcomes from the same set of obese men. Possible considerations include a much higher dose of NR (2 g/day) than other trials (mostly 1 g/day) and the sensitivity of the enzymatic, assay-based NAD+ detection method. Thus, these studies emphasise some of the challenges with clinical trials of NAD+-boosting compounds with regards to dose and assessment of NAD+ bioavailability.


More Data on the Tissue Specificity of Senescent Cell Accumulation with Age in Mice

As work progresses on the clinical development of senolytic therapies to selectively destroy harmful senescent cells in old tissues, it is becoming ever more necessary to have a better understanding of just how many senescent cells are present in any given tissue with age. Not all tissues acquire lingering populations of these cells at the same pace. Further, most current senolytic therapies are quite tissue specific, either because of the biodistribution characteristics of the drug, or because effectiveness varies in destroying senescent cells of different cell types.

Prioritization of development efforts requires some idea as to which tissues are more burdened by senescent cells, and thus more subject to the senescence-associated secretory phenotype in producing dysfunction and age-related disease, at least in the small molecule portion of the field. It is possible that Oisin Biotechnologies at least could just power through this challenge by saturating all tissues in the body with their non-toxic, highly selective suicide gene therapy vector. Brute force is sometimes an option.

Non-invasive ways of quantitatively assessing the presence of senescent cells in different tissues are also much needed, because we'd all like some idea as to how effective a given therapy might be. The dasatinib and quercetin senolytic combination is readily available, and you'll bump into people who have used it at longevity industry conferences, but few of those have undergone the biopsies that are presently the only viable way to make before and after comparisons of senescent cell burden. Better methods are on the horizon, such as the circulating microRNA approach under development at TAmiRNA, but they are not on the market yet. These tools will be needed to enable a more rational design of the next generation of senolytics, and they would certainly help in the clinical development of the present generation of senolytics.

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice

In this study, we provide a comprehensive measure of senescence in aged wild type (WT) mice. Senescence was quantified in multiple tissues, using numerous methods and numerous molecular endpoints, and we compared measures with that of young adult WT mice. We used this as a benchmark to determine whether Ercc1-/∆ mice, that exhibit accelerated aging, accumulate senescent cells in physiologically relevant tissues.

As measured by qRT-PCR and p16LUC signal, levels of p16Ink4a were significantly increased in aged WT mice compared with younger adult mice, as expected, p16Ink4a and p21Cip1 expression are found in peripheral T cells and numerous tissues (10 of 14 total tested) with the exception of heart and skeletal muscles. The differences in senescent cell burden in tissues could be reflective of different levels of genotoxic stress and/or different responses to that stress (e.g., selection of cell fate decisions: senescence or apoptosis). Near complete concordance was found between the expression of senescence markers in aged WT (2.5 years) and progeroid Ercc1-/∆ (4-5 month) mice, in terms of tissue specificity and expression levels.

The systemic burden of senescent cells was equivalent at the halfway point of lifespan in each of Ercc1-/∆ and WT mice, although the strains have vastly different lifespans. This supports the notion that senescent cell burden correlates with organismal health and may prove to be useful in predicting health span, or the remaining fraction of life that is disease-free. The data also support the conclusion that Ercc1-/∆ mice spontaneously develop senescent cells in the same tissues and at similar levels as WT mice, albeit more rapidly, supporting the notion that these animals represent a model of accelerated aging.

Endothelial Cell Senescence can Impair Insulin Sensitivity

The growing presence of senescent cells contributes to near all of the declines and tissue dysfunctions of aging, judging by the results produced in extensive research carried out in animal models of age-related disease. Senescent cells secrete a mix of inflammatory and other signals that, when present for the long term, cause considerable harm to tissue structure, function, and maintenance. The research here is focused on just one form of dysfunction, but is illustrative of many other studies in the field of senescence carried out in recent years.

Endothelial cells (ECs) line the inner surface of blood vessels, and plays an essential role in vascular biology, such as vasodilation, hormone trafficking, and neovessel formation. Moreover, EC produces many secreted angiocrine factors that are crucially involved in maintaining tissue homeostasis. Aging causes cellular senescence in various types of cells including EC, and cellular senescence plays an important role in age-related organ dysfunction.

Senescent cells produce senescence-messaging secretomes that have deleterious effects on the tissue microenvironment, referred as the senescence-associated secretory phenotype (SASP); therefore, cellular senescence is considered to be a primary cause for age-related diseases, such as diabetes, stroke, and heart attack. Because of the crucial roles of EC in tissue homeostasis, EC senescence is presumed to play significant roles in age-related organ dysfunction; however, whether and the mechanism by which EC senescence causes age-related diseases remained unknown.

Here we show that EC senescence induces metabolic disorders through the SASP. Senescence-messaging secretomes from senescent ECs induced a senescence-like state and reduced insulin receptor substrate-1 in adipocytes, which thereby impaired insulin signaling. We generated EC-specific progeroid mice. This EC-specific progeria impaired systemic metabolic health in mice in association with adipose tissue dysfunction. Notably, shared circulation with EC-specific progeroid mice by parabiosis sufficiently transmitted the metabolic disorders into wild-type recipient mice. Our data provides direct evidence that EC senescence impairs systemic metabolic health, and thus establishes EC senescence as a bona fide risk for age-related metabolic disease.


Long Non-Coding RNAs and Macrophage Senescence in Age-Related Disease

Here, researchers review cellular senescence in macrophage cells and the biochemistry of long non-coding RNAs in macrophage senescence, a topic of great relevance to a number of age-related conditions, such as atherosclerosis. Cellular senescence takes place in most cell populations, in response to reaching the Hayflick limit on replication or in response to stress and damage. Senescent cells have important short-term roles to play, and near all destroy themselves or are destroyed by immune cells soon after entering the senescent state. These cells become harmful when they linger over long term, however, even in comparatively small numbers. They secrete a mix of signals, the senescence-associated secretory phenotype, that encourages other cells to become senescent, rouses the immune system to chronic inflammation, destructively remodels surrounding tissues, and more. The accumulation of senescent cells is one of the driving causes of degenerative aging.

Cellular senescence is a particularly stable state of permanent cell cycle arrest. Macrophages, although terminally differentiated cells, do not undergo this type of replicative senescence and may hence undergo stress-induced senescence. In healthy conditions, macrophages maintain homeostasis; however, in pathological states, different stresses including DNA damage, telomere shortening, oncogene activation, impairment of some key proteins, and infections activate the p53, AIM2, and NF-κB signal pathways, initiating macrophage senescence.

When these damage-associated molecule patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) are highly intensive or temporally irreversible, the balance between the production and clearance of proinflammatory factors is disrupted. At later stages of macrophage senescence, the net effect is senescence-associated secretory phenotype (SASP) expression. These factors not only aggravate macrophage senescence but are also extracellularly released, thus impairing the functions of surrounding cells. This process is called "paracrine senescence" and causes a wider range of inflammaging. With steady accumulation of senescent cells, senescence eventually occurs at the cellular level and then at the organ level, causing organ malfunction, consequently resulting in corresponding aging phenotypes.

Emerging data suggest that long non-coding RNA (lncRNA) plays a key role in regulating inflammatory responses. Alterations in various lncRNA expression levels are associated with a proinflammatory phenotype in various age-related diseases (ARDs). This leads to modification of cellular senescence through several diverse approaches, whether by mediating gene expression or protein function or functioning as competing endogenous RNA (ceRNA). Changes in lncRNAs in ARDs and the corresponding consequences have been widely studied, especially in cancer. However, the association between lncRNA and cellular senescence in ARDs remains an interesting and complex issue.

Atherosclerosis is a chronic inflammatory disease. Macrophages have been recently reported to display marked inflammatory plasticity, particularly polarization. They perpetuate chronic inflammation and growth of atherosclerotic plaques, thus being central to the initiation, growth, and ultimately the rupture of arterial plaques. Studies on atherosclerosis and macrophages have reported that lncRNAs majorly function as ceRNA in causing atherosclerosis. By sequestering microRNAs, MITA, GAS5, HOTAIR, and UCA1 promote M1 polarization, inducing proinflammatory cytokine, matrix metalloproteinase, and reactive oxygen species (ROS) levels. Atherosclerosis contributes to various lesions, especially cardiovascular disease. Current evidence suggests that the effect of lncRNAs on macrophages in coronary artery disease is the same as that on atherosclerosis, highlighting the consistency of its function and prompting its potential as a therapeutic target.


Macrophages Implicated in the Scarring of Heart Tissue Following Injury

A potentially important faction within the regenerative medicine community is engaged in trying to understand exactly how highly regenerative species such as salamanders and zebrafish can regenerate organs following injury, and do so repeatedly without scarring. There are also a few examples of adult mammals capable of regenerating a limited number of body parts without scarring, such as African spiny mice and the MRL mouse lineage. It seems plausible that mammalian species still carry much of the machinery of proficient regeneration, but that this machinery is suppressed in some way, possibly because that suppression acts to reduce cancer risk. Evidence in support of that thesis includes the ability of human tumor suppressor ARF to block zebrafish regeneration.

Thus understanding of the fine details of the ways in which highly regenerative species differ from near all mammals might lead to ways to induce limb and organ regrowth in humans. It is still a little early to say whether or not these differences will in fact include anything that could be the basis of a therapy. The differences might be too complex, or too fundamental to easily alter with today's tools. That said, there is compelling evidence for macrophage behavior to be fundamental to exceptional regeneration. All regeneration is an intricate dance between somatic cells, stem cells and progenitor cells of various types, transient senescent cells, and immune cells, particularly macrophages.

In this context, the research here is an interesting exploration of the activities of macrophages in heart injury in mice and zebrafish. Mice scar rather than regenerate from this type of injury, and the heart is one of the least regenerative organs in mammals. Zebrafish normally regenerate heart injuries without scarring, but this isn't the case if the injury is inflicted by freezing. These various circumstances give points of comparison to look into the behavior of macrophages in scarring and regeneration, and perhaps suggest lines of investigation that could lead to therapies to prevent scarring in human patients.

New target identified for repairing the heart after heart attack

Billions of cardiac muscle cells are lost during a heart attack. The human heart cannot replenish these lost cells, so the default mechanism of repair is to form a cardiac scar. While this scar works well initially to avoid ventricular rupture, the scar is permanent, so it will eventually lead to heart failure and the heart will not be able to pump as efficiently as before the damage caused by heart attack.

Zebrafish, a freshwater fish native to South Asia, is known to be able to fully regenerate its heart after damage due to the formation of a temporary scar as new cardiac muscle cells are formed. Researchers have been striving to understand and compare the composition of the cardiac scar in different animals as part of ongoing efforts to investigate whether it can be modulated to become a more transient scar like that of the zebrafish, and therefore potentially avoid heart failure in heart attack patients.

The team focused their efforts on studying the behaviour of macrophages, cells normally associated with inflammation and fighting infection in the body, when exposed to the three post-injury environments. They extracted macrophages from each model to examine their gene expression. In both mouse and fish macrophages, they found that they were showing signs of being directly involved in the creation of the molecules that form part of the cardiac scar, and particularly collagen, which is the main protein involved. "This information is important and quite striking because up to today, only cardiac myofibroblasts have been implicated in directly forming a scar in the heart. By showing that macrophages produce collagen, a key part of scar tissue, this research could lead to new ways to enhance repair after a heart attack."

Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse collagen donors, enhances scar formation via cell autonomous production of collagen.

In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair.

A Mechanism by which Chronic Inflammation Spurs Cancer Metastasis

Chronic inflammation is a risk factor for cancer and cancer mortality. There are numerous reasons as to why this might be the case, some much more proven and settled than others, but the research here is focused on metastasis, the spread of cancerous cells throughout the body. Since cancer mortality is largely determined by whether or not a tumor progresses to the point of metastasis, we should not be surprised that researchers can identify mechanisms linking inflammation with metastasis.

Dysregulated inflammation is recognized as one of the hallmarks of cancer and is involved in tumor initiation, progression, and metastasis. Chronic inflammatory conditions, such as chronic obstructive pulmonary disease or ulcerative colitis, are strongly associated with elevated cancer incidence. Chronic use of aspirin or other non-steroidal anti-inflammatory drugs reduces mortality of esophageal, colorectal, and lung cancers.

Thus chronic inflammation facilitates tumor progression. We discovered that a subset of non-small cell lung cancer cells underwent a gradually progressing epithelial-to-mesenchymal (EMT) phenotype following a 21-day exposure to IL-1β, an abundant proinflammatory cytokine in individuals at-risk for lung cancer, and in the lung tumor microenvironments. Pathway analysis of the gene expression profile and in vitro functional studies revealed that the EMT and EMT-associated phenotypes, including enhanced cell invasion, PD-L1 upregulation, and chemoresistance, were sustained in the absence of continuous IL-1β exposure. We referred to this phenomenon as EMT memory.

Utilizing a doxycycline-controlled SLUG expression system, we found that high expression of the transcription factor SLUG was indispensable for the establishment of EMT memory. High SLUG expression in tumors of lung cancer patients was associated with poor survival. Chemical or genetic inhibition of SLUG upregulation prevented EMT following the acute IL-1β exposure but did not reverse EMT memory.

Although it is well known that EMT endows cells with metastatic capacity, analysis of tissue specimens from metastatic tumors often reveals cells with epithelial features. EMT plasticity therefore is proposed to temporally modify these properties by facilitating cellular responses to the microenvironmental stimuli that lead to mesenchymal phenotypes and metastatic behaviors. In the current study, fading of EMT memory, accompanied by a gradual elevation of E-cadherin expression, is consistent with a profound EMT plasticity. In a case of acquired EMT, increased migration and invasion of tumor cells enable them to travel away from primary tumor sites, which also distance them from EMT-promoting stimuli, such as inflammatory factors in the primary TME. We propose that because of the memorized EMT phenotypes, these migratory cells are able to seed the metastatic spread to distant organ sites.


An Example of Epigenetic Effects on Offspring Longevity

It was discovered only comparatively recently that epigenetic alterations, decorations attached to the genome rather than changes to the genome itself, can produce changes in offspring longevity. Not all epigenetic changes are erased during early embryonic development; some are retained and go on to influence development and metabolism throughout life. This is a mechanism by which species can improve their reproductive fitness via producing offspring better suited to the environment experienced by the parents. One of the best examples is that calorie restriction affects the metabolism and longevity of the offspring of animals, not just the calorie restricted parents. The research here is an example of ongoing investigations into this aspect of epigenetic regulation, focused on a single epigenetic mark that is shown to produce greater longevity in parents and offspring.

It is commonly accepted that genetic sequences coded within DNA are passed down through generations and can influence characteristics such as appearance, behavior, and health. However, emerging evidence suggests that some traits can also be inherited 'epigenetically' from information that is independent of the DNA sequence. One of the ways characteristics may be epigenetically passed down is through the temporary modification of histone proteins which help to package DNA into the cell. Histones are adorned with chemical marks that can regulate how and when a gene is expressed by changing how tightly the DNA is wrapped. These marks are typically removed before genetic information is passed on to the next generation, but some sites escape erasure.

It has previously been reported that genetic mutations in an enzyme complex called COMPASS increase the lifespan of tiny worms called Caenorhabditis elegans. This complex acts on histones and creates a chemical mark called H3K4me, which is typically associated with less compact DNA and higher gene expression. When these mutants mate with wild-type worms they generate descendants that no longer have COMPASS mutations. Although these wild-type offspring recover normal levels of H3K4me, they still inherit the long-lived phenotype which they sustain for several generations.

Previous work showed that one of the COMPASS complex mutants, known as wdr-5, has increased levels of another histone mark called H3K9me2. This epigenetic mark generally promotes DNA compaction and appears to antagonize the action of H3K4me. Researchers found that homozygous wdr-5 mutants, which had descended from ancestors carrying one copy of the mutated wdr-5 gene and one wild-type copy for multiple generations, did not live for longer than their non-mutant counterparts. This indicates that the mutation carried by wdr-5 worms did not immediately cause a lifespan change. However, future generations of worms that maintained the homozygous wdr-5 mutation had an increasingly longer lifespan, suggesting that the accumulation of an epigenetic signal across generations promotes longer living. These late generation wdr-5 mutants had higher levels of H3K9me2, and they were able to pass on this extended longevity to their progeny following mating with wild-type worms as previously reported.


Chronic Inflammation is of Great Importance in the Progression of Aging

Aging is a process of damage and consequence. Damage to the molecular machinery of cells and the molecular structure of tissues accumulates as a normal consequence of the operation of healthy metabolism. This damage degrades function, producing a lengthy chain of downstream consequences that interact with one another, make one another steadily worse, and culminate in age-related disease. Some of these downstream consequences are more important than others. Among the most important are raised blood pressure, which converts low level molecular damage into actual structural pressure damage to tissues, and chronic inflammation, which converts low level molecular damage into sweeping failure and detrimental change of cell and tissue function.

These two downstream consequences of damage are so influential over the development and progression of age-related disease that some progress has been made in lowering age-related mortality by crudely forcing reductions in blood pressure and inflammation, with no effort to eliminate the causes of these issues. Most efforts to tackle inflammation involve sabotaging the cell signaling that drives it. This is a blunt tool, as transient inflammation is quite important to health. Only the inappropriate chronic inflammatory signaling should be suppressed, but the tools of the past are far from being discriminating enough in this matter.

A sizable degree of the chronic inflammation of aging is driven by the presence of senescent cells and the inflammatory signaling that they generate. Numerous mechanisms create senescent cells, and they are useful in the short term, a necessary part of wound healing, cancer suppression, and other mechanisms. The issue is that clearance of these cells fails with age, and thus their numbers grow inexorably. Fortunately, the advent of senolytic therapies to selectively destroy senescent cells offers considerable potential as a way to reduce only the undesirable chronic inflammation of aging, while preserving desirable transient inflammation. Given the importance of inflammation in aging, we might expect considerable benefits to emerge from the use of senolytics.

Scientists have identified the role of chronic inflammation as the cause of accelerated aging

"Today, chronic inflammatory diseases are at the top of the list of death causes. There is enough evidence that the effects of chronic inflammation can be observed throughout life and increase the risk of death. It's no surprise that scientists' efforts are focused on finding strategies for early diagnosis, prevention and treatment of chronic inflammation."

One of the serious results obtained to date has been the concept of immune aging, which enables researchers to characterize the immune function of an individual and to predict the causes of mortality much more accurately than by relying only on chronological age. In addition to well-known inflammation biomarkers, such as C-reactive protein, interleukin 1 and interleukin 6, tumor necrosis factor, scientists note the need to study additional biomarkers of the immune system, which differ very much from person to person, in particular, the subgroups of T-lymphocytes and B-lymphocytes, monocytes, etc.

Scientists have identified certain factors (social, environmental and lifestyle factors) that contribute to systemic chronic inflammation. Taken together, such factors are the main cause of disability and mortality worldwide. An integrative approach to the study of mechanisms of systemic chronic inflammation is being adopted by a growing number of scientists. Research is continuing, and scientists have a long way to go to fully understand the role of chronic inflammation in aging and mortality, and to be able to predict changes in a person's health throughout life.

Chronic inflammation in the etiology of disease across the life span

Although intermittent increases in inflammation are critical for survival during physical injury and infection, recent research has revealed that certain social, environmental and lifestyle factors can promote systemic chronic inflammation (SCI) that can, in turn, lead to several diseases that collectively represent the leading causes of disability and mortality worldwide, such as cardiovascular disease, cancer, diabetes mellitus, chronic kidney disease, non-alcoholic fatty liver disease, and autoimmune and neurodegenerative disorders. We describe the multi-level mechanisms underlying SCI and several risk factors that promote this health-damaging phenotype, including infections, physical inactivity, poor diet, environmental and industrial toxicants, and psychological stress. Furthermore, we suggest potential strategies for advancing the early diagnosis, prevention and treatment of SCI.

Modulating Macrophage Polarization as a Therapy for Atherosclerosis

Macrophages are the cells responsible for removing cholesterols from blood vessel walls, to prevent the formation of fatty lesions. Unfortunately they become dysfunctional and inflammatory with age, as a result of rising levels of oxidized cholesterol. This leads to atherosclerosis, an ultimately fatal condition in which lesions grow to the point of weakening and narrowing blood vessels. This condition is strongly affected by inflammation, as macrophages can adopt different behavioral types, known as polarizations, in response to circumstances. Greater inflammatory signaling will drive more macrophages to adopt the aggressive M1 phenotype, focused on destroying pathogens, rather than the regenerative M2 phenotype that is more useful in removing cholesterol from blood vessel walls.

A number of groups are working on ways to force macrophages to adopt a specific phenotype, overriding their usual reaction to surrounding circumstances. In the research here, an approach is demonstrated to be beneficial in a mouse model of atherosclerosis, presumably by putting more macrophages back to work in lesions, clearing out lipids rather than flailing and adding to the inflammatory environment.

Atherosclerosis-related cardiovascular disease is still the predominant cause of death worldwide. Araloside C (AsC), a natural saponin, exerts extensive anti-inflammatory properties. In this study, we explored the protective effects and mechanism of AsC on macrophage polarization in atherosclerosis in vivo and in vitro. Using a high-fat diet (HFD)-fed ApoE-/- mouse model and RAW 264.7 macrophages exposed to oxidized LDL, AsC was evaluated for its effects on polarization and autophagy.

AsC significantly reduced the plaque area in atherosclerotic mice and lipid accumulation in oxidized-LDL-treated macrophages, promoted M2 phenotype macrophage polarization, increased the number of autophagosomes and modulated the expression of autophagy-related proteins. Moreover, the autophagy inhibitor 3-methyladenine and BECN1 siRNA obviously abolished the antiatherosclerotic and M2 macrophage polarization effects of AsC. Mechanistically, AsC targeted Sirt1 and increased its expression, and this increase in expression was associated with increased autophagy and M2 phenotype polarization. Altogether, AsC attenuates foam cell formation and lessens atherosclerosis by modulating macrophage polarization via Sirt1-mediated autophagy.


Mechanisms of Slowed Muscle Aging via Calorie Restriction in Rhesus Macaques

Many of the readers here will be familiar with the very long-running studies of calorie restriction in rhesus macaques. There was some discussion of the data a few years ago. The research has continued since then, and here researchers report on their investigation of the biochemistry of calorie restriction in connection to the slowed aging of muscle tissue observed in these animals. Calorie restriction produces sweeping changes in the operation of cellular metabolism, and aging is itself a very complex process, even though it stems from simpler root causes. Research into the tissue-specific details of how and why calorie restriction slows specific aspects of aging is thus a slow and complex undertaking.

Our studies of aging in rhesus monkey and calorie restriction (CR) include a comprehensive investigation of age-related change including physical parameters. Similar to humans, muscle mass loss begins in middle age in monkeys at ∼15 years of age, where age-related loss of quadricep bulk from ∼15 to +25 years of age is ∼23% for females and 27% for males. Vastus lateralis (VL) is one of the four constituent muscle groups within the quadriceps, and it is the one that is the most vulnerable to aging, with 40% lower tissue weight for old monkeys (∼30 years of age) at necropsy compared with young adults of full stature (∼8 years of age). VL comprises both oxidative and glycolytic fiber types and succumbs to fiber atrophy and increased fibrosis with age in humans and monkeys. Responses to aging are of a fiber-type-specific nature: slow twitch type I fibers are resistant to age-related atrophy, but fast twitch type II fibers exhibit a gradual decline in cross-sectional area beginning at middle age.

Defects in skeletal muscle energy metabolism with age have been documented in humans, rats, and mice. In humans, skeletal muscle mitochondrial activity declines with age. In rhesus monkeys, age-related changes in mitochondrial and redox metabolism occur in advance of the onset of muscle mass loss and before age-related declines in physical activity are detected, suggesting that metabolism could play a causal role in skeletal muscle aging. A separate study has demonstrated that energy metabolism pathways are uniformly but modestly induced with CR in mice, and pathway level analysis confirmed the same in rhesus monkey skeletal muscle.

To test this, we investigated the molecular and cellular phenotypes of delayed sarcopenia due to CR in rhesus monkeys and related these data to tissue, biometric, and functional outcomes. We show that CR induced profound changes in muscle composition and the cellular metabolic environment. Bioinformatic analysis linked these adaptations to proteostasis, RNA processing, and lipid synthetic pathways. At the tissue level, CR maintained contractile content and attenuated age-related metabolic shifts among individual fiber types with higher mitochondrial activity, altered redox metabolism, and smaller lipid droplet size. Biometric and metabolic rate data confirm preserved metabolic efficiency in CR animals that correlated with the attenuation of age-related muscle mass and physical activity. These data suggest that CR-induced reprogramming of metabolism plays a role in delayed aging of skeletal muscle in rhesus monkeys.


Forcing Macrophages into Greater Clearance of Debris in Atherosclerotic Lesions

Atherosclerosis is the generation of fatty deposits in blood vessel walls, called plaques, atheromas, or lesions, that narrow and weaken important vessels. Sooner or later a vessel ruptures, or a plaque disintegrates and its fragments block the flow of blood, and this results in stroke or heart attack. In the public eye atherosclerosis is considered a disease of cholesterol, of blood lipids, and reducing cholesterol and other lipids in the blood remains the primary focus of treatment. This is despite the fact that this approach can only slow progression - it doesn't reverse existing lesions to a sizable degree.

Atherosclerosis is in fact a condition of macrophage dysfunction, not of cholesterol. Macrophages are the cells responsible for clearing out lipids from blood vessel walls. They ingest cholesterol, and then hand it off to HDL particles that can carry it back to the liver for excretion. This process works just fine in youth, but macrophages are unfortunately vulnerable to oxidized cholesterol. It makes them dysfunctional, inflammatory, and even kills them. As a result of other forms of age-related damage, such as mitochondrial dysfunction, levels of oxidized cholesterol increase significantly. A feedback loop forms in which macrophages are constantly drawn to a lesion, succumb to the oxidized cholesterol present there, and add their corpses to the growing deposit. Atherosclerotic lesions are macrophage graveyards.

Thus macrophages are, to my eyes, the right point of intervention for therapies to effectively treat atherosclerosis - to actually prevent and meaningfully reverse lesions. This might be achieved by making macrophages invulnerable to the oxidized cholesterol that challenge them, as Repair Biotechnologies is working towards, or by clearing out oxidized cholesterols, as Underdog Pharmaceuticals is working towards. The research noted here takes a different view of the opportunities presented by a tissue that is rich in macrophages, and reports on a way to force those macrophages to more aggressively clear debris and destroy harmful cells in a lesion, despite their impediments. The initial data seems promising.

Nanoparticle chomps away plaques that cause heart attacks

Researchers have demonstrated a nanoparticle that homes in on atherosclerotic plaque due to its high selectivity to a particular immune cell type - monocytes and macrophages. Once inside the macrophages in those plaques, it delivers a drug agent that stimulates the cell to engulf and eat cellular debris. Basically, it removes the diseased/dead cells in the plaque core. By reinvigorating the macrophages, plaque size is reduced and stabilized.

The research is focused on intercepting the signaling of the receptors in the macrophages and sending a message via small molecules using nano-immunotherapeutic platforms. Previous studies have acted on the surface of the cells, but this new approach works intracellularly and has been effective in stimulating macrophages. "We found we could stimulate the macrophages to selectively eat dead and dying cells - these inflammatory cells are precursor cells to atherosclerosis - that are part of the cause of heart attacks. We could deliver a small molecule inside the macrophages to tell them to begin eating again."

Pro-efferocytic nanoparticles are specifically taken up by lesional macrophages and prevent atherosclerosis

Atherosclerosis is the process that underlies heart attack and stroke. A characteristic feature of the atherosclerotic plaque is the accumulation of apoptotic cells in the necrotic core. Prophagocytic antibody-based therapies are currently being explored to stimulate the phagocytic clearance of apoptotic cells; however, these therapies can cause off-target clearance of healthy tissues, which leads to toxicities such as anaemia.

Here we developed a macrophage-specific nanotherapy based on single-walled carbon nanotubes loaded with a chemical inhibitor of the antiphagocytic CD47-SIRPα signalling axis. We demonstrate that these single-walled carbon nanotubes accumulate within the atherosclerotic plaque, reactivate lesional phagocytosis and reduce the plaque burden in atheroprone apolipoprotein-E-deficient mice without compromising safety, and thereby overcome a key translational barrier for this class of drugs.

Single-cell RNA sequencing analysis reveals that prophagocytic single-walled carbon nanotubes decrease the expression of inflammatory genes linked to cytokine and chemokine pathways in lesional macrophages, which demonstrates the potential of 'Trojan horse' nanoparticles to prevent atherosclerotic cardiovascular disease.

A Discussion of Recent Work on Allotopic Expression of Mitochondrial Genes at the SENS Research Foundation

A paper published last month outlines recent progress on allotopic expression of mitochondrial genes carried out by the SENS Research Foundation team. Allotopic expression is the name given to the process of putting copies of mitochondrial genes into the nuclear genome, suitably altered to allow proteins to be generated and shipped back to the mitochondria where they are needed. Mitochondria replicate like bacteria, and some forms of stochastic mitochondrial DNA damage can make mitochondria both dysfunctional and able to outcompete their undamaged peers. This is thought to be an important contribution to aging, resulting a small but damaging population of cells that are overtaken by broken mitochondria and which export harmful reactive molecules into the surrounding tissues.

Having a backup supply of mitochondrial proteins can in principle block these consequences of mitochondrial DNA damage, and thus remove this contribution to the aging process. Proof of concept has been demonstrated for a few of the thirteen proteins needed, and work proceeds on the rest. As noted here, one of the challenges in this project is that mitochondrial genetic machinery is of a different evolutionary origin to that of the cell nucleus, and thus the efficient production of equivalent proteins from nuclear genes is a much more challenging process than would otherwise be the case.

While the vast majority of mitochondrial proteins are encoded by the nuclear genome, translated in the cytosol, and imported into the mitochondrion, 13 core subunits of respiratory complexes are encoded by the reduced mitochondrial genome and synthesized within the mitochondrial matrix. Mutations in these 13 genes (or their associated non-protein-coding genes) tend to be especially severe, as all 13 proteins are core subunits of the oxidative phosphorylation chain, and any disruption to subunit structure, stability, or function may have grave biochemical and physiological consequences. Gene therapy to target affected mitochondrial subunits is a promising alternative strategy which circumvents some of the technical challenges faced by the above approaches. One issue that remains, however, relates to the prokaryotic origin of the organelle. Translation within the mitochondrion deviates from the universal genetic code, utilizing machinery and codon frequencies more similar to its α-proteobacterial ancestry than to the mammalian nuclear genome.

Subsequently, allotopic expression has been suggested as a therapeutic tool to genetically remedy deleterious mitochondrial DNA mutations through nuclear complementation of the affected genes. A critical, but often-overlooked consideration in these nuclear relocation studies is the influence of the primary coding sequence on protein production. The vast majority of these previous studies have utilized what may be considered "minimally-recoded" mitochondrial genes. While making these codon changes is essential to maintain amino acid sequence integrity during cytosolic translation, this minimal approach fails to account for other elements of primary sequence which can critically influence both gene and protein expression.

Many commercial algorithms have therefore been developed to determine the optimal sequence and conditions for expression of a gene from a particular host. Though there are concerns regarding the use of codon optimization to increase homologous expression of a nuclear gene, such as the generation of novel or immunogenic peptides or structural perturbations in the encoded protein, codon optimization continues to be widely utilized for the production of biotherapeutics. Applying this principle to allotopic expression, we hypothesize that, given the bacterial origin of the mitochondrial genome, the coding sequences of minimally-recoded mitochondrial genes are dissimilar from nuclear genes and are inefficiently translated by nuclear machinery, therefore resulting in poor allotopic expression.

Here we employed codon optimization as a tool to re-engineer the protein-coding genes of the human mitochondrial genome for robust, efficient expression from the nucleus. All 13 codon-optimized constructs exhibited substantially higher protein expression than minimally-recoded genes when expressed transiently, and steady-state mRNA levels for optimized gene constructs were 5-180 fold enriched over recoded versions in stably-selected wildtype cells. Eight of thirteen mitochondria-encoded oxidative phosphorylation proteins maintained protein expression following stable selection, with mitochondrial localization of expression products. We also assessed the utility of this strategy in rescuing mitochondrial disease cell models and found the rescue capacity of allotopic expression constructs to be gene specific. Allotopic expression of codon optimized ATP8 in disease models could restore protein levels and respiratory function, however, rescue of the pathogenic phenotype for another gene, ND1, was only partially successful. These results imply that though codon-optimization alone is not sufficient for functional allotopic expression of most mitochondrial genes, it is an essential consideration in their design.


Evidence for the Epigenetic Clock to Underestimate Age in Later Life

In recent years, the research community has put in ever more effort into the development of epigenetic clocks capable of assessing biological age. This focus has led to the discovery of various issues, as the nature of epigenetics of aging is further explored, challenges that will need to be understood and addressed in order to enable the practical use of epigenetic clocks. For example that degree of fitness doesn't appear to much affect epigenetic age, which is problematic to say the least, as we know that it affects the progression of aging. Here, researchers identify a systemic issue with assessment of epigenetic age in older individuals.

Subject age is a piece of data available in almost every study in which DNA methylation profiles are obtained. There is thus a huge amount of cross-sectional data in which it can be seen that the methylation level of many CpG sites varies with subject age, which, amongst other processes, could reflect developmental changes, cellular aging, cumulative environmental effects, and changes in cell-type composition.

Horvath used a large collection of publicly available DNA methylation data on multiple tissue types to train and test a model for age prediction from 353 CpG loci. This "epigenetic clock" continues to be widely used and is extremely valuable as a way of estimating ages of samples from unknown donors and possibly as an indicator of whether there are alterations in the aging rate of certain tissues or individuals. Although the epigenetic clock provides an estimate of age, the testing data used in generating this clock did not have a large representation of tissue from elderly individuals and as such it is unclear if the clock is accurate in older age groups, or those with age-related diseases.

The model systematically underestimates age in tissues from older people. This is seen in all examined tissues but most strongly in the cerebellum and is consistently observed in multiple datasets. Epigenetic age acceleration is thus age-dependent, and this can lead to spurious associations. The current literature includes examples of association tests with age acceleration calculated in a wide variety of ways. In conclusion, the concept of an epigenetic clock is compelling, but caution should be taken in interpreting associations with age acceleration. Association tests of age acceleration should include age as a covariate.