Fight Aging! Newsletter, March 2nd 2020

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.

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."

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.

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."

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.

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.

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."

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.

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.

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.

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."

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.

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.

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.

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.

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.