Why do epigenetic changes matter for longevity?

We are finding that age-related epigenetic changes are associated with mortality risk and, perhaps more importantly, with disease incidence. For instance, we have different algorithms that represent levels of DNA methylation that we expect to see for someone of a given age. Individuals who have methylation profiles indicative of someone older than they are have increased risk of morbidity and mortality. For instance, if you compare two 40-year olds and one has the methylation profile of someone who is 45, while the other has the methylation profile of someone who is 35, the former will, on average, live for fewer years and develop disease earlier.

What is the theory/mechanism behind the various epigenetic clocks now available?

This is ongoing work that we are actively pursuing. There are about a dozen epigenetic clocks in the literature - perhaps the most famous being the Horvath clock (although it wasn't the first). However, even though these clocks are all intended to capture the same latent concept (biological aging), they differ in their predictions of age and age-adjusted death and disease risk. Using transcriptomic and proteomic data from both blood and brain, we have found that accelerated aging measured using the most widely known epigenetic clocks seem to relate to mitochondrial dysfunction, PI3K/Akt signaling, and immunosenesence.

There is also some evidence coming out that they may reflect cellular senescence to some degree. That being said, our theory is that the clocks - because they are composites of hundreds of CpGs, cytosine separated from a guanine by one phosphate - represent a grab bag of mechanisms. We are currently working on decomposing the various clocks and are finding that they differ in their proportions of various "types" of methylation changes, each of which may have their own distinct mechanisms. Our hypothesis that breaking the clocks down into constituent parts may facilitate our understanding of the underlying biology that is either driving these age-related changes, and/or the functional implications of such changes.

How far are we from understanding epigenetic changes well enough to be able to turn back the clock by changing the epigenetics in individual organisms?

I think we are quite a long way from that. The epigenome is a complex system, and we are not at the point where we can model these changes very well - there is a lot of room for improvement when it comes to the clocks. That being said, we are even further away from understanding what these changes represent or if they are even causal. I hypothesize that many of these changes are actually reactions to something going wrong in some system. Thus, altering DNA methylation directly will not be beneficial and could, in fact, be harmful if this isn't accompanied by changes to the system/extracellular environment. If some of these changes are effects (read-outs) of aging, then they are not the correct points of intervention. Further, many of the changes may be compensatory, and thus making an old cell epigenetically young but leaving it in an aged organismal environment could be detrimental and possibly contribute to neoplastic transformations.

Researchers collected samples during a time span of one week before the Boston Marathon to one week after the Marathon. They also collected samples from sedentary individuals. They then analyzed them to determine the species of bacteria in both cohorts. "One of the things that immediately caught our attention was this single organism, Veillonella, that was clearly enriched in abundance immediately after the marathon in the runners. Veillonella is also at higher abundance in the marathon runners in general than it is in sedentary individuals."

They confirmed the link to improved exercise capacity in mouse models, where they saw a marked increase in running ability after supplementation with Veillonella. Next, they wanted to figure out how it worked. As they dug into the details of Veillonella, they found was that it is relatively unique in the human microbiome in that it uses lactate or lactic acid as its sole carbon source. Lactic acid is produced by the muscles during strenuous exercise. The Veillonella bacteria are able to use this exercise by-product as their main food source. "Our immediate hypothesis was that it worked as a metabolic sink to remove lactate from the system, the idea being that lactate build-up in the muscles creates fatigue. But talking to people in the exercise physiology field, apparently this idea that lactate build-up causes fatigue is not accepted to be true. So, it caused us to rethink the mechanism of how this is happening."

Researchers ran a metagenomic analysis, meaning they tracked the genetics of all the organisms in the microbiome community, to determine what events were triggered by Veillonella's metabolism of lactic acid. They noted that the enzymes associated with conversion of lactic acid into the short chain fatty acid propionate were at much higher abundance after exercise. "Then the question was maybe it's not removal of lactic acid, but the generation of propionate. We did some experiments to introduce propionate into mice via enema and test whether that was sufficient for this increased running ability phenotype. And it was."

The human gut microbiome is linked to many states of human health and disease. The metabolic repertoire of the gut microbiome is vast, but the health implications of these bacterial pathways are poorly understood. In this study, we identify a link between members of the genus Veillonella and exercise performance. We observed an increase in Veillonella relative abundance in marathon runners postmarathon and isolated a strain of Veillonella atypica from stool samples. Inoculation of this strain into mice significantly increased exhaustive treadmill run time.

Veillonella utilize lactate as their sole carbon source, which prompted us to perform a shotgun metagenomic analysis in a cohort of elite athletes, finding that every gene in a major pathway metabolizing lactate to propionate is at higher relative abundance postexercise. Using labeled lactate in mice, we demonstrate that serum lactate crosses the epithelial barrier into the lumen of the gut. We also show that intrarectal instillation of propionate is sufficient to reproduce the increased treadmill run time performance observed with V. atypica gavage. Taken together, these studies reveal that V. atypica improves run time via its metabolic conversion of exercise-induced lactate into propionate, thereby identifying a natural, microbiome-encoded enzymatic process that enhances athletic performance.

On October 30, 2018, Alcor performed its 164th cryopreservation. It was an otherwise unremarkable moment for the nonprofit organization, save for the way Norman Hardy of Mountain View, California met his demise. Hardy was diagnosed with terminal metastatic prostate cancer, and it had spread to his bones and lungs. As noted in Alcor's case summary, his "pain had been poorly managed," so he opted for assisted death, which was legalized in California in 2016 through the End of Life Option Act (EOLOA).

For Alcor, the case was the first time EULOA was used to "reduce the potential ischemic damage that can result from a prolonged dying process," as the foundation noted in its official case report. Indeed, the quicker a patient can be put into cryonic suspension following death the better, as the sudden shortage of oxygen starts to destroy tissues. For Alcor, time is trauma. In this case, Hardy's choice of when to die allowed his neural tissues to be rapidly preserved following his death, or at least, preserved as well as modern cryonic technologies allow.

The procedure to prepare Hardy's head for long-term suspension in a vat of liquid nitrogen was "relatively successful," noted Alcor in its case report. CT scans following preservation showed some ice formation in the cerebellum and frontal lobes, which isn't ideal. That said, the quick turnaround from the declaration of death to placement in one of Alcor's stainless steel dewars meant that decompositional damage to Hardy's brain was minimized. In a best case scenario, Alcor's response team begins the cryopreservation process within seconds after death is declared. But that doesn't always happen.

Alcor has published a case report on Norman Hardy, A-1990. This case was the first time the newly enacted California End of Life Option Act (EOLOA) was used to reduce the potential ischemic damage that can result from a prolonged dying process. The cryoprotective perfusion was relatively successful. Perfusion flow rates were high throughout the procedure. However, the post-cryopreservation CT scan showed poor cryoprotection and extensive CT-visible ice formation in the cerebellum, and incomplete cryoprotection with a small amount of CT-visible ice formation in the frontal lobes.

In an attempt to integrate some of the different lines of reasoning and research findings, we submit that there exist three widespread classes of attitudes, expectations, and preferences with regard to a possible extension of human lifetime in modern societies that may reflect different schools of thought, such as essentialism, medicalism, and stoicism. The different primary motives that are associated with the three perspectives on longevity and life-time extension are infinite life (striving to overcome biological degeneration and health declines), healthy life (motivation is conditional on physical and mental health), and dignified life (a wish for dignity and meaning even when there is loss and vulnerability). We submit that these three primary motives can be used to characterize different scientific approaches on longevity, as well as individual attitudes and preferences toward longevity in everyday thinking. We refer to such perspectives as mindsets that involve sets of representations, attitudes, and ways of thinking about the meaning and value of a prolonged life.

An essentialist mindset views aging as a degenerative process that is inevitably associated with physical loss. It reflects the idea that aging is a determined and undesirable process, and that the human mind is held captive in a deficient biological organism. Accordingly, pathological aging cannot be differentiated from normal or healthy aging. One implication is that aging per se is viewed as pathological and ought to be pushed back, for example, with antiaging medicine. Consequently, radical extensions of the life span are expected when antiaging research is successful. Recent studies suggest that the prevalence of essentialist mindsets can be estimated in the range of 3-10% of respondents who wish to live forever or wish to "overcome" the natural aging process.

A medicalist mindset involves that human aging is viewed as burdened only when pathology occurs, and that pathological aging is different from normal aging. In this perspective, aging is associated with age-related health risks, and defined as a medical challenge. One implication is that successful aging may be defined as an absence of disease and disability. On an individual level, medicalism is reflected in an appreciation for an extended lifetime if health functioning can be maintained, and degenerative diseases such as dementia can be avoided. Another implication might be that when endorsing a medicalist mindset, individuals may prefer to avoid the vulnerability of old age and wish to die rather than to become chronically ill or demented.

The stoicist mindset for living long reflects the idea that withstanding the challenges and risks of a long or prolonged life is part of the conditio humana, which involves striving for meaning in life, and for acceptance of one's actual life condition. The challenges, needs, risks, or tasks within the aging process may thus appear manageable or at least bearable as long as there is meaning in life and a sense of grace. Preserving dignity and meaning in a prolonged life is pivotal to a stoicist mindset. Thus, having a worthy and dignified life may be emphasized over the absence of chronical diseases in late life. Regarding the desired extension of lifetime, we submit that holding a stoicist mindset may involve that individuals express a valuation of life per se and "as it comes." This may also involve a discomfort or unwillingness to reflect about lifetime extension rather than about dignity and meaning in life.

Researchers called the pathway LC3-associated endocytosis or LANDO. They found the pathway in microglial cells, the primary immune cells of the brain and central nervous system. However, preliminary evidence suggests LANDO is a fundamental process that functions in cells throughout the body. Investigators showed that LANDO protected against deposits of neurotoxic β-amyloid protein in mice. Activation of the pathway also guarded against toxic neuroinflammation and neurodegeneration, including memory problems.

β-amyloid protein accumulation in neurons is a hallmark of Alzheimer's. Scientists knew microglial cells take up β-amyloid proteins. Discovery of the LANDO pathway answers questions about what comes next. The researchers compared LANDO to the operator of an automatic carwash. In this case, the cars are the receptors on the microglial cells that bind to neurotoxic β-amyloid proteins and bring the protein into the car wash. And, just as cars return to the streets after the dirt is gone, when the β-amyloid is disposed of, the receptor returns to the microglial surface where it can pick up additional β-amyloid.

Several proteins are required for LANDO functioning. The proteins - Rubicon, Beclin 1, ATG5, and ATG7 - are better known for their roles in a related cell pathway used to recycle unneeded and unwanted cell components. These proteins decline with age as their expression decreases.

The expression of some proteins in the autophagy pathway declines with age, which may impact neurodegeneration in diseases, including Alzheimer's disease (AD). We have identified a novel non-canonical function of several autophagy proteins in the conjugation of LC3 to Rab5 +, clathrin + endosomes containing β-amyloid in a process of LC3-associated endocytosis (LANDO).

We found that LANDO in microglia is a critical regulator of immune-mediated aggregate removal and microglial activation in a murine model of AD. Mice lacking LANDO but not canonical autophagy in the myeloid compartment or specifically in microglia have a robust increase in pro-inflammatory cytokine production in the hippocampus and increased levels of neurotoxic β-amyloid. This inflammation and β-amyloid deposition were associated with reactive microgliosis and tau hyperphosphorylation. LANDO-deficient AD mice displayed accelerated neurodegeneration, impaired neuronal signaling, and memory deficits. Our data support a protective role for LANDO in microglia in neurodegenerative pathologies resulting from β-amyloid deposition.

Cellular senescence is an important protective process with roles in development, tissue homeostasis, and wound healing. However, senescence is also implicated in multiple diseases including cancer, arthritis, atherosclerosis, and a diminished healthspan during aging. The senescence-associated secretory phenotype (SASP) is an important hallmark of senescence that contributes to normal physiology and disease. The SASP is characterised by the release of inflammatory cytokines, chemokines, growth factors, and proteases. This reinforces senescence through autocrine and paracrine signalling, and recruits and instructs immune cells to clear senescent cells. However, senescent cells can also generate an inflammatory environment. Thus, the SASP is often considered a double-edge sword. Whilst promoting immune-mediated clearance of pre-malignant senescent cells is a powerful barrier against transformation, the SASP from uncleared senescent cells, or those arising during natural aging, can create an inflammatory milieu permissive to disease.

The SASP is regulated by interleukin-1 alpha (IL-1α), but the mechanism of IL-1α activation during senescence is unknown. Previous studies have suggested that NLRP3 inflammasomes modulate the SASP, even though caspase-1 cannot activate IL-1α. However, our recent research has demonstrated that caspase-5, which lies upstream of NLRP3 in the non-canonical inflammasome pathway, induces IL-1α activity and regulates the SASP during oncogene-induced senescence (OIS) in vitro and in vivo. Recent research also implicates the non-canonical inflammasome in sterile inflammation, of which the SASP is an important yet rarely cited example.

Our recent investigation demonstrated that caspase-5 or caspase-11, but not caspase-4 or caspase-1, specifically cleaves human or mouse pro-IL-1α at a highly conserved site. We demonstrated that caspase-5/11 is required for IL-1α release from cells. siRNA-mediated caspase-5 knockdown reduced levels of cell-surface and secreted IL-1α, and impaired release of the common SASP factors IL-6, IL-8, and MCP-1 from senescent fibroblasts. Our work identifying caspase-5 as a novel regulator of IL-1α activity and the SASP raises several important questions for future research. Firstly, it will be important to understand how caspase-5 is activated in senescent cells. We demonstrated that knockdown of CGAS results in reduced caspase-5 expression and an impaired SASP, and hypothesised that cGAS/STING activated by cytosolic chromatin in senescent cells may drive caspase-5 expression via type I interferons.

The discovery of caspase-5 as a novel regulator of IL-1α in sterile and non-sterile inflammation has several important clinical implications. Targeting caspase-5 may be a therapeutic strategy that leaves canonical immune responses via caspase-1 and -4 intact. For instance, radiotherapy and chemotherapy induce DNA damage that can trigger tumour cell senescence. However, these non-selective therapies also induce senescence in the underlying stroma, with IL-6 from senescent fibroblasts shown to be a reprogramming factor that drives pluripotency and proliferation of cancer stem cells surviving treatment. Therefore, caspase-5 inhibition during treatment could lessen the chance of tumour recurrence.

Currently, a major support in the diagnostics of Alzheimer's disease is the assessment of abnormal accumulation of the substance beta-amyloid, which can be detected either in a spinal fluid sample or through brain imaging using a PET scanner. "These are expensive methods that are only available in specialist healthcare. In research, we have therefore long been searching for simpler diagnostic tools." In this study, the researchers investigated whether a simple blood test could identify people in whom beta-amyloid has started to accumulate in the brain, i.e. people with underlying Alzheimer's disease. Using a simple and precise method that the researchers think is suitable for clinical diagnostics and screening in primary healthcare, the researchers were able to identify beta-amyloid in the blood with a high degree of accuracy.

"Previous studies on methods using blood tests did not show particularly good results; it was only possible to see small differences between Alzheimer's patients and healthy elderly people. Only a year or so ago, researchers found methods using blood sample analysis that showed greater accuracy in detecting the presence of Alzheimer's disease. The difficulty so far is that they currently require advanced technology and are not available for use in today's clinical procedures."

The new results are based on studies of blood analyses collected from 842 people in Sweden (the Swedish BioFINDER study) and 237 people in Germany. The participants in the study are Alzheimer's patients with dementia, healthy elderly people and people with mild cognitive impairment. The method studied by the researchers is a fully automated technique which measures beta-amyloid in the blood, with high accuracy in identifying the protein accumulation. "The next step to confirm this simple method to reveal beta-amyloid through blood sample analysis is to test it in a larger population where the presence of underlying Alzheimer's is lower. We also need to test the technique in clinical settings, which we will do fairly soon in a major primary care study in Sweden. We hope that this will validate our results."

Our body is in continuous contact with viruses and various defense mechanisms are used to prevent the entry or to eliminate the invader within the body. There is ample evidence demonstrating that the aging-associated decline of the immune system, i.e. immunosenescence, significantly weakens these mechanisms. This is often observed in the case of common viral pathogens, e.g. influenzavirus. On the other hand, it is known that at least some viruses may induce or modify immunosenescence and in this respect cytomegalovirus (CMV) is the classical and extensively investigated example.

However, there is now emerging evidence showing that the number of viruses or virus-like entities is much larger than expected. i) Next generation (NGS) RNA/DNA sequencing based approaches have shown that within our body there are large amounts of various viruses even without known clinical or biological significance, forming the virome, i.e. the classical concept about the "sterility" of the inner body should be rejected; ii) Our genome contains mobile genetic elements, retrotransposons, endogeneous retroviruses (HERV), some of which may still be active and might modify the immune system.

Analysis of the virome (including bacteriophages) is technically more challenging than that of the bacteriome, but the first virome analyses have now been published. It seems that the different compartments of the body harbor distinct viral communities. However, the total number of viruses is highly variable, 10^9 particles per gram in the intestinal content, 10^7/ml in the urine and 10^5/ml in the blood. Studies on gut virome have shown, that the most common viruses are not those infecting eukaryotic cells, but those infecting prokaryotic cells, bacteriophages, form a clear majority.

Thus far the relationship between virome composition and immunosenescence is not known. However, there are several reports demonstrating changes in the gut virome compostion in diseases of immunological nature, e.g. type I diabetes. Based on these, it could be expected that immunosenescence would have an influence on virome composition. However, its possible role in the aging-associated pathologies can presently only be speculated. Does the weaker immunity allow the presence of potentially pathogenic viruses in the blood of elderly individuals? It is also possible that this viral "normal flora" would have a protective effect, in analogy with the bacterial normal flora in several compartments of the human body.

The data shown here indicate that the relationship between viruses, virosphere, and immunosenescence is more complex than previously thought.

The classical hallmarks of Alzheimer's disease (AD) include the formation of amyloid-beta (Aβ) plaque deposits and neurofibrillary tangles (NFT) containing abnormal hyperphosphorylation of tau. The mechanisms triggering the deposition of the Aβ or the formation of NFTs are currently under investigation. However, several mechanisms and factors have been suggested to be involved in the initiation and the progression of the disease, including activation of the innate immune system, environmental factors and lifestyle. The innate immune system has been widely studied and has been implicated in several neurodegenerative diseases. Over the last few years, several studies have suggested that inflammation plays a major role in the initiation and progression of AD.

The inflammatory process in the central nervous system (CNS) is generally referred to as neuroinflammation. Glial cells have a leading role in propagating neuroinflammation. Among glial cells, microglia are considered the main source of proinflammatory molecules within the brain. It is believed that sustained release of proinflammatory molecules such as cytokines, chemokines, nitrogen reactive species (NRS) or reactive oxygen species (ROS) can create a neurotoxic environment that drives the progression of AD.

One of the key molecules involved in microglial activation is galectin-3 (gal3), and we demonstrate here for the first time a key role of gal3 in AD pathology. Gal3 was highly upregulated in the brains of AD patients and 5xFAD (familial Alzheimer's disease) mice and found specifically expressed in microglia associated with Aβ plaques. Gal3 deletion in 5xFAD mice attenuated microglia-associated immune responses, particularly those associated with TLR and TREM2/DAP12 signaling. In vitro data revealed that gal3 was required to fully activate microglia in response to fibrillar Aβ. Gal3 deletion decreased the Aβ burden in 5xFAD mice and improved cognitive behavior. Overall, our data support the view that gal3 inhibition may be a potential pharmacological approach to counteract AD.

Subcutaneous adipose tissue (SAT) is the largest and best storage site of excess fat in the body provided that new cells can be recruited as needed (hyperplastic obesity). Inappropriate expansion of the adipose cells (hypertrophic obesity) promotes insulin resistance and other obesity-associated metabolic complications and is a consequence of inability to recruit new adipose cells. This has been shown both in vitro and in direct studies of human adipose cell turnover in vivo.

Our previous extensive studies have shown large inter-individual differences in ability of human SAT stromal vascular fraction (SVF) cells to undergo adipogenesis. Furthermore, markers of reduced SAT adipogenesis are associated with genetic predisposition for type 2 diabetes (T2D) and first-degree relatives (FDR), like individuals with manifest T2D, are characterized by hypertrophic obesity. Human SAT contains a pool of adipose progenitor cells but the detailed signals for recruiting new adipose cells and the reasons for the large individual differences are unclear. Bone morphogenetic protein 4 (BMP4) is important for the commitment of mesenchymal progenitor cells into the adipogenic lineage and BMP-signalling is regulated by different secreted inhibitors. We found Gremlin-1 (GREM1) to be an important BMP4 antagonist and increased in hypertrophic obesity. Thus, impaired commitment of progenitor cells could be one reason for the reduced adipogenesis.

Cell senescence is, in part, a consequence of repeated progenitor cell mitogenic expansion and there is a 10% annual cell turnover in human SAT. Cell senescence leads to permanent cell cycle arrest, secretion of different senescence-associated proteins and inhibited cell differentiation. We here characterize mechanisms for the impaired SAT adipogenesis in adult human subjects with hypertrophic obesity including FDR/T2D. A key mechanism for the impaired adipogenesis in hypertrophic obesity / T2D is increased progenitor cell senescence, dysregulated p53 and P16ink4 and secretion of senescence-associated secretory phenotype (SASP) factors antagonizing normal cell adipogenic differentiation.

Under normal circumstances, proteins destined for the secretion are translated directly into the endoplasmic reticulum (ER) via ribosomes embedded in the ER membrane, bound by chaperone proteins, folded, and then packaged into vesicles for secretion. In some cases, however, this pathway can go awry; proteins may become misfolded or unfolded in the ER, and unable to be recovered by the protein quality control machinery. In this instance, the improperly folded protein is targeted for degradation, exported into the cytosol, and degraded by a proteasome. Again, however, this process is imperfect. Some environmental, cellular, or molecular factors can cause disruptions in this pathway, preventing the proper turnover of misfolded or unfolded proteins, potentially leading to their accumulation and aggregation. This generates a cellular condition known as ER stress.

Endoplasmic reticulum stress and the failure to correctly fold proteins are associated with loss of protein function and cell death. To avoid this, the cell resolves misfolded protein stress via two major stress response pathways: the heat shock response (HSR), which handles misfolded proteins in the cytoplasm, and the unfolded protein response (UPR), which takes place in the ER. These protein quality control mechanisms are essential for maintaining the function and integrity of cellular processes. When perturbed, they can lead to whole-cell dysfunction and toxicity. Under normal conditions, both lead to resolution of the cellular stress caused by the presence of misfolded proteins.

The UPR is a complicated signaling pathway which works to resolve ER stress and allow protein synthesis and folding to continue and has been shown to interact with multiple cellular pathways and processes to do so, including (but not limited to) those occurring in the ER. It has also been shown to be impacted by several seemingly unrelated external influences, including aging and lipid metabolism, and dysfunction in this pathway has been linked with shortened cellular lifespan and cell death. Because of this, the study of the molecular mechanisms behind ER stress and the UPR is essential to the understanding of how protein homeostasis impacts the entire cell and its processes, including response to stressors, aging, and cell death.

Aging cells have been shown to have decreased total levels of a number of ER proteins, including protein chaperones which normally supervise and ensure proper protein folding, and assist in targeting misfolded proteins for degradation. This usually prevents the accumulation and aggregation of misfolded proteins and prevents them from having toxic effects on the cell. In addition, the limited chaperones that are still present in the aging ER appear to be impaired. This is possibly due to an increased rate of oxidation of these chaperones in aged cells, leading to structural changes and consequently decreased function.

Aging is accompanied by a range of DNA modifications. Telomere length, which shortens as a consequence of DNA replication, has been widely accepted as a biomarker of aging. While being inversely correlated with chronological age, telomere length is also associated with a range of age-associated phenotypes and clinical diseases. Recently, a novel candidate epigenetic biomarker of aging has been shown to predict an individual's chronological age with high accuracy: the epigenetic clock is based on the weighted DNA methylation fraction of a number of cytosine-phosphate-guanine sites (CpGs).

Interestingly, DNA methylation (DNAm) age correlates with cell passage number in vitro and can be predicted across different tissues, including non-proliferating ones in vivo, suggesting that DNA methylation is not exclusively reflecting mitotic age.

This is in line with the finding that DNAm age and relative leukocyte telomere length (rLTL) were independently associated with chronological age and mortality. The few existing studies found no supporting evidence of a significant association between rLTL and DNAm age or reported a weak association. Moreover, rLTL was reported to have a lower predictive power in estimating chronological age in comparison to the epigenetic clock.

While the number of studies reporting a positive correlation between DNAm and chronological age in a range of different study populations rises, there is accumulating evidence suggesting that DNAm age somewhat reflects biological age. Under the assumption that DNA methylation age reflects biological age, calculating the deviation of the epigenetic age estimate and the chronological age gives rise to a second potentially clinically relevant measure: DNAm age acceleration. Here we aim to explore the association of rLTL and the epigenetic clock variables, DNAm age and DNAm age acceleration, in the context of cardiovascular disease in the LipidCardio cohort.

Both rLTL (0.79 ± 0.14) and DNAm age (69.67 ± 7.27 years) were available for 773 subjects (31.6% female; mean chronological age 69.68 ± 11.01 years). While we detected a significant correlation between chronological age and DNAm age, we found neither evidence of an association between rLTL and the DNAm age nor rLTL and the DNAm age acceleration in the studied cohort, suggesting that DNAm age and rLTL measure different aspects of biological age.

In studies of aging, changes in the length of life are usually analyzed by comparing average (mean) or median longevity. Frequently, some estimate of maximal longevity is also considered. While values of the standard deviations or standard errors of the mean are routinely reported, the distribution of individual age at death is rarely analyzed or discussed. In a recent publication based on analysis of demographic data, it was reported that socio-economic status influences not only the mean longevity but also the variability of human life-span. Using an example of Finnish women, these investigators showed that reduced mean longevity of less educated and less affluent people is associated with greater variability of life-span.

Because of the potential significance of this relationship for the analysis of mortality data and physiological biomarkers in studies of aging as well as for various public health considerations, we thought that it would be of interest to determine whether interventions known to extend the average (or the average and the maximal) longevity of experimental animals have any effect on the variability of life-span. We hypothesized that extension of longevity by genetic, dietary or pharmacological means leads to reduction of life-span variability.

However, inspection of data from the National Institute of Aging Interventions Testing Program (ITP) and from our studies of the interactions of murine longevity genes with calorie restriction (CR) indicated that reciprocal changes of longevity and its variability are not consistently observed. This suggested that our hypothesis would most likely need to be rejected and brought up a new question, namely, what factors influence variability of the lifespan. Here we report results of a study aimed at analyzing the effects of sex, strain, life-extending interventions and their interactions on life-span variation.

The relationship of changes in longevity and in longevity variance was found to depend on sex and treatment and apparently also on strain. Increased longevity of male mice treated with effective anti-aging drugs was accompanied by reduced variance of age at death and apparent reduction of early life mortality. Life extension induced by growth-hormone related mutations and calorie restriction tended to increase longevity variance in females only. We conclude that impact of anti-aging interventions on the variance of age at death and distribution of individual lifespans in laboratory mice is treatment-dependent and sexually dimorphic.

The E(z) histone methyltransferase heterozygous mutation in Drosophila is known to increase lifespan and stress resistance. However, the longevity mechanisms of E(z) mutants have not been revealed. In the present research we studied the effects of E(z) histone methyltransferase heterozygous mutation on lifespan, stress-resistance, fecundity, and genome-wide transcriptional profile dynamics in Drosophila imagoes. We observed 22-23% lifespan extension in both sexes, and E(z) mutants were significantly more resistant to hyperthermia, oxidative stress, and endoplasmic reticulum stress, and demonstrated enhanced fecundity.

The genome-wide transcriptome analysis identified 239 genes, which expression level was altered more than 2 times by E(z) mutation. Several of the most differentially expressed genes had never been described before as pro-longevity genes. Most likely, these genes may be associated with E(z) mutation, but not related to aging and longevity. The exception is a differential expression of antimicrobial peptides and the Turandot family of genes. These humoral innate immunity factors have been previously discussed in the context of aging and stress-resistance.

A mutation in the E(z) gene surprisingly neither activated nor repressed canonical pro-longevity or anti-longevity genes like mTor or insulin/IGF-signaling elements and either determinants of DNA repair, Sod, Sirtuins, etc. We also did not find strong changes in the expression of the Hox family of genes, for which gene repression by polycomb group proteins such as E(z) was previously shown.

We observed that the E(z) mutation leads to modulation of many genes related to the immune response, ribosome biogenesis, and cell cycle. Although age-dependent changes in the expression of these genes are similar to changes in control flies, it is most likely that mutations in E(z) lead to positive perturbations in the pathways for which age-associated gene expression changes are shown. In addition, the gene expression is sex-specific, despite the fact that the increase in median lifespan for both sexes was broadly similar, which emphasizes the importance of separate preparation and analysis of females and males.

Heart muscle cells called cardiomyocytes hold on to the capacity to reprogram themselves and alter their fate in response to heart damage. Although several signalling cues are known to be involved in this regeneration activity, it is not well understood how heart injury switches on these pathways to initiate heart cell reprogramming. "Recent studies suggest that biomechanical forces generated by blood flow can contribute to heart development through modulating cell signalling. We wanted to explore this further by seeing whether mechanical forces caused by altered blood flow during heart injury also activate these signalling pathways to control heart cell reprogramming and regeneration."

The team first looked at how heart injury affects signalling of an important heart development molecule called Notch in zebrafish. They found that injury-induced Notch activity peaks at 24 hours after injury but diminishes as the heart regenerates, so that by 96 hours it has returned to normal. If Notch is blocked, however, this prevents heart cell growth and stops heart precursor cells from reprogramming and maturing into cells that can replace the damaged cells.

They next explored whether heart injury could alter blood flow forces and, in turn, control injury-induced Notch signaling. Klf2a is a molecule that responds to changes in blood flow and switches on certain genes in response. In regions of the injured heart where blood flow was most disrupted, they found that levels of Klf2a were increased. In addition, they found that levels of Klf2a overlapped with the levels of Notch.

Further experiments revealed that, when mutated, Trpv4 - a molecule that is known to 'sense' changes in blood flow and can switch on the gene for Klf2a - led to a reduced amount of genes that drive heart cell growth and fewer cells maturing to replace the damaged tissue. Additionally, the team found that changes in blood flow controls heart cell reprogramming and growth via another two molecules, BMP and Erbb2. As these molecules are important for heart regeneration in mammals, the changes observed in the zebrafish may also hold true for other organisms, including humans.