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- Reviewing Recent Research into the Relationship between Autophagy and Aging
- Recent Work on APOE in Alzheimer's Disease
- Alternate Day Fasting and Calorie Restriction Produce Similar Outcomes in Humans
- Reduced TGF-β and Increased Oxytocin Reverses Measures of Aging in Old Mice
- A Large Polypill Clinical Trial Shows a Third Reduction in Cardiovascular Events
- Lipid Accumulation in Microglia Contributes to Neuroinflammation and Neurodegeneration
- Estradiol Accelerates Liver Regeneration in Mice
- Clinical Trial of a Cross-Link Breaker to Treat Presbyopia in the Aging Eye
- The CellAge Database of Genes Associated with Cellular Senescence
- Clearing Dysfunctional Microglia Prevents Formation of Amyloid-β Plaques in a Mouse Model of Alzheimer's Disease
- Evidence for Mitochondrial Dysfunction in Smooth Muscle to be Important in Age-Related Vascular Stiffness
- Visceral Fat Tissue is Anti-Inflammatory in Lean Mice, Inflammatory in Fat Mice
- Methods of Inducing Cellular Damage are Rarely Relevant to Aging, and the Details Matter
- Talking Up the Potential of the Longevity Industry
- The Decline of Autophagy in Skin Aging
Reviewing Recent Research into the Relationship between Autophagy and Aging
The authors of today's open access review paper focus on recent research into autophagy and aging, specifically work using flies as the model organism. Autophagy is the name given to a collection of cellular maintenance processes responsible for recycling damaged cell components, molecular machinery, and metabolic waste. In chaperone-mediated autophagy, selective chaperone proteins pick up other molecules and carry them to lysosomes for disassembly. In macroautophagy, unwanted cellular components are engulfed by an autophagosome, which then travels to a lysosome and fuses with it. In microautophagy, a lysosome engulfs the material to be recycled directly. A lysosome is always the end of the journey, where a mix of enzymes reduces structures and molecules into component parts suitable for reuse.
A sizable majority of the interventions proven to slow aging in short-lived laboratory species (such as flies) involve increased autophagy. Many, such as calorie restriction, do not produce benefits at all in the absence of functional autophagy. When cells are better maintained over time, with less outstanding molecular damage that can cause further downstream consequences, the outcome is a slower pace of decline into age-related dysfunction and disease. This has led to a wide range of research projects, and at least one startup biotech company, focused on trying to produce therapies capable of boosting the operation of autophagy to improve human health.
The plausible size of benefits resulting from an autophagy-based therapy can be seen by looking at the effects of regular exercise and calorie restriction in humans. Improved health is the outcome, but not a significant increase in life span. For evolutionary reasons, stress response mechanisms have a much greater relative impact on the life span of short-lived species. Mice live 40% longer when calorie restricted, while benefiting from boosted autophagy, while humans most certainly do not. This is most likely because seasonal famines last for a much larger proportion of a mouse life span, and the calorie restriction response evolved to increase fitness in this scenario.
On the Fly: Recent Progress on Autophagy and Aging in Drosophila
Besides the description of the characteristics of the aging process, the most significant finding of aging research is that aging now is considered to be a malleable process. The lifespan of organisms can be extended by both environmental and genetic traits and more importantly, their healthspan can simultaneously be improved during aging, which shows that it is possible to reach the ultimate goal of aging research. Recently, a growing body of evidence shows that alterations of autophagy, the main self-degradative process of eukaryotic cells, likely plays a central role in the aging process.
Observations in various organisms indicate that aging and autophagy have a bidirectional connection with each other. On one hand, autophagic degradation shows an age-dependent decline and impairment of autophagy contributes to the development of age-associated diseases. On the other hand, lifespan-extending interventions largely depend on the autophagy machinery for their beneficial effects on longevity. Strikingly, multiple longevity pathways seem to converge on autophagy, and genetic or environmental factors that affect lifespan through these pathways at least partly exert their effects via the modulation of autophagy. These observations point to the key role of autophagy in aging, and suggest that increased autophagy may compensate for at least some of the cellular hallmarks of aging. Thus, the housekeeping functions of autophagy can counteract the accumulation of cellular damage, which is considered to be a primary mechanism driving aging.
Studies in animal models including Drosophila revealed that autophagy defects lead to the rapid decline of neuromuscular function, neurodegeneration, sensitivity to stress (such as starvation or oxidative damage), and stem cell loss. Of note, recently identified human Atg gene mutations cause similar symptoms including ataxia and mental retardation. Physiologically, autophagic activity is known to decrease during aging, and this defect likely contributes to the development of such age-associated diseases. Many manipulations that extend lifespan (including dietary restriction, reduced TOR kinase signaling, exercise, or treatment with various anti-aging substances) require autophagy for their beneficial effect on longevity, pointing to the key role of this housekeeping process. Importantly, genetic (e.g., Atg8a overexpression in either neurons or muscle) or pharmacological (e.g., feeding rapamycin or spermidine to animals) promotion of autophagy has been successfully used to extend lifespan in Drosophila, suggesting that this intracellular degradation pathway can rejuvenate cells and organisms.
Recent Work on APOE in Alzheimer's Disease
Apolipoprotein E (APOE) is a well studied gene, given that variants are associated with a greater risk of developing Alzheimer's disease. That said, high blood pressure and high blood cholesterol levels are just as important as risk factors for Alzheimer's disease when compared against all but the worst APOE variant, APOE4. Looking beyond Alzheimer's, in most cases lifestyle choices and their consequences on the operation of metabolism, particularly becoming overweight, have larger effects on risk of age-related disease than genetic variants. The common wisdom of a 75%/25% split between environment and genetics respectively in the matter of age-related disease and mortality may be overestimating the contribution of genetics, per more recent data.
The point of investigating the activities of specific protein variants in which risk or scope of age-related is shifted is this: that the work may lead towards points of effective intervention. Not the gene or protein itself, usually, but something in the mechanisms with which it interacts. Later stages of all age-related conditions are enormously complex, and having the example of differences that affect the progression of the condition can help to pin down which of the many, many possible metabolic processes are most important. That is somewhat in evidence in the research materials here, but of course says nothing about how to effectively target those important mechanisms.
Rare Luck: Two Copies of ApoE2 Shield Against Alzheimer's
Mention ApoE and Alzheimer's, and the conversation turns to the E4 allele, the strongest susceptibility gene for the disease. But ApoE has another side, in ApoE2. Though this isoform protects against AD, scientists have barely studied it. Now ApoE2 is attracting scrutiny as scientists are asking exactly how some people maintain their mental acuity into old age. A study of ApoE genotypes in 5,000 autopsy-confirmed cases of AD revealed that people with two copies of E2 see their risk of dementia plummet by a stunning 90 percent compared with those with the common E3/E3 genotype. Other work suggested that this could be because ApoE2 reduces amyloid and tau pathology, and boosts gray-matter volume in critical brain regions. E2's benefits seem specific to Alzheimer's, not generic to neurodegeneration.
ApoE is the major cholesterol-carrying protein in the brain. It has been studied since its discovery as an AD risk gene in the early 1990s, but is newly emerging as a hub for glial responses to amyloid and tau aggregate deposition. The gene exists as three polymorphic alleles - E2, E3, and E4 - with a worldwide frequency of 8 percent, 78 percent, and 14 percent, respectively. Several mutated forms are also known. ApoE4 receives by far the most attention from AD researchers, because it boosts the risk of AD up to 15-fold depending on the study population, and occurs in 40 percent of people with AD. E2, the protective allele, has received scant attention, because it is the least common of the three and largely absent from AD samples.
ApoE4 Glia Bungle Lipid Processing, Mess with the Matrisome
It is suggested that ApoE4 predisposes people to Alzheimer's disease by modulating astrocytes and microglia. Researchers describe transcriptional differences between iPSC-derived human astrocytes and microglia that express ApoE4/4 or ApoE3/3. The ApoE4/4 glia generated more cholesterol than their E3/3 counterparts. They exported and degraded it poorly, causing lipid to build up inside them. The E4/4 glia also pumped out greater amounts of proinflammatory cytokines and extracellular matrix proteins than E3/3s.
Does this have anything to do with Alzheimer's? Lo and behold, in Alzheimer's disease brains, astrocytes and microglia behaved quite similarly to these ApoE4/4 glia. They accumulated lipid and ratcheted up inflammation. Importantly, they did so regardless of their ApoE genotype. The data imply that ApoE4 may nudge microglia and astrocytes toward an Alzheimer's-like state. Perhaps faulty lipid metabolism is one of the earliest changes on the path to Alzheimer's. If so, restoring glial lipid regulation could be a therapeutic approach.
Alternate Day Fasting and Calorie Restriction Produce Similar Outcomes in Humans
Today's research is a comparison of alternate day fasting and calorie restriction in human subjects. Or rather, I think, one might look on it as an examination of alternate day fasting as an alternative approach to achieving calorie restriction. The type of alternate day fasting here is the better form, in which 36 hours are spent fasting, only eating in a 12 hour window every other day. In practice that means eat normally one day, then fast until the morning two days later. This tends to reduce average calorie intake down to something very similar to a straight calorie restricted diet. That calorie restricted diet might be 1500 kcal/day for an averagely sized human being, and I can assure you that it is very, very hard to eat more than 3000 kal in a 12 hour period, at least not without resorting to heavy duty junk food.
So is alternate day fasting just calorie restriction? In animal studies there are significant differences in gene expression profiles between these two approaches, which is enough to suspect that perhaps fasting and feeding versus a consistent low calorie intake are two different beasts. The effects on metabolism are sweeping in either case, which makes analysis challenging, but the important mechanisms, the upregulation of cellular stress response systems such as autophagy, appear the same. More recent research into fasting mimicking diets has attempted to find the point at which low calorie intake triggers benefits, and quantify how long the low calorie diet must be sustained. The results there suggest that additional benefits emerge after three to four days, in terms of a culling of immune cells. That work also suggests that the process of refeeding after a fast is necessary in order to obtain the full benefits.
So it is possible that neither alternate day nor straight calorie restriction are strictly optimal, and something more intermittent would be better. Still, either alternate day or calorie restriction are such a huge improvement over the dietary choices adopted by most people that it seems almost foolish to spend much time on further optimization. This is particularly true when that time and energy could be put towards advancing the development of rejuvenation therapies capable of turning back aging in ways that no amount of fasting can achieve.
Not Eating for 36 Hours Is Shown to Be a Surprisingly Sustainable Diet, Study Shows
New research outlines a novel way to intermittently restrict calorie intake, a method that achieves the same health benefits while possibly being more manageable than constantly restricting calories. An international team of researchers presented the results of a clinical trial in which "alternate day fasting" resulted in reduced calorie intake, reduced body mass index, and improved torso fat composition. Known as "ADF," it is a diet regimen in which adherents avoid all food and caloric beverages for 36 hours, then eating whatever they want for 12 hours - donuts, cookies, dumpster pizza, whatever.
In this randomized controlled trial, 30 non-obese volunteers who had done ADF for at least six months were compared over a 4-week period to 60 healthy control subjects. While the results of this clinical trial show that ADF had similar health benefits to caloric restriction, even though the "feast days" could include a lot of unhealthy calories. The researchers also write that ADF has some distinct advantages over CR. Mainly, they say it may be easier to maintain the habit.
Previous work on intermittent fasting has shown that restricting an animal's calories - without depriving them of adequate nutrition, of course - can increase their lifespan, though much of the work has been limited to monkeys and other non-human animals. This latest study builds on that existing research by following a mid-sized human cohort for enough time to show not just significant benefits but also no negative side effects.
Alternate Day Fasting Improves Physiological and Molecular Markers of Aging in Healthy, Non-obese Humans
Caloric restriction and intermittent fasting are known to prolong life- and healthspan in model organisms, while their effects on humans are less well studied. In a randomized controlled trial study, we show that 4 weeks of strict alternate day fasting (ADF) improved markers of general health in healthy, middle-aged humans while causing a 37% calorie reduction on average. No adverse effects occurred even after more than 6 months.
ADF improved cardiovascular markers, reduced fat mass (particularly the trunk fat), improving the fat-to-lean ratio, and increased β-hydroxybutyrate, even on non-fasting days. On fasting days, the pro-aging amino-acid methionine, among others, was periodically depleted, while polyunsaturated fatty acids were elevated. We found reduced levels sICAM-1 (an age-associated inflammatory marker), low-density lipoprotein, and the metabolic regulator triiodothyronine after long-term ADF. These results shed light on the physiological impact of ADF and supports its safety. ADF could eventually become a clinically relevant intervention.
Reduced TGF-β and Increased Oxytocin Reverses Measures of Aging in Old Mice
Numerous research and development initiatives have emerged from the heterochronic parabiosis studies of the past decade or more, in which an old and a young mouse have their circulatory systems linked. Researchers have moved on from the initial experiments to the search for circulating factors in blood that change in ways that are harmful in aged individuals, and which might be adjusted to improve cell and tissue function. This area of research is one of many to explore the question of how much of degenerative aging is the result of (a) direct consequences of molecular damage versus (b) the result of inappropriate cellular reactions to the existence of damage, the second of these mediated to some unknown degree by signaling carried in the bloodstream.
Is it possible to ignore the damage and extend healthy life just by suppressing the reactions to damage? It would be very strange if the answer were that this works comprehensively and damage never has to be repaired. Further, the consequences of any given form of underlying damage can be thought of as a network of diverse chains of cause and effect spreading from a single root: it will require far more work to identify and address all of these reactions to damage than to focus down on a means of repairing the damage. Still, and unfortunately, the concept of damage repair, striking at the root of aging, remains a comparatively unpopular strategy in the research community for some reason. Near all work on the treatment of aging is focused on tinkering with the downstream consequences of damage, and therefore probably a highly inefficient use of funds and time, even given the successes that arise.
One of the more noted scientific teams involved in parabiosis research here report on their recent work, opening this open access paper with a bold statement on the degree to which they believe aging to result from signaling changes, reactions to damage. They are focusing down on just a few signaling factors in the bloodstream, TGF-β and oxytocin, and finding ways to alter amounts in circulation in comparative isolation, without adjusting other factors as well. Given that earlier work on GDF-11 as circulating signal involved in cellular responses to aging has resulted in a great deal of ongoing research and at least one biotech startup, the results here seem interesting enough to drawn in funding for further, similar projects.
Rejuvenation of brain, liver and muscle by simultaneous pharmacological modulation of two signaling determinants, that change in opposite directions with age
We hypothesize that altered intensities of a few morphogenic pathways account for most or all the phenotypes of aging. In heterochronic parabiosis, a young and old animal are surgically connected to share a common blood circulation. Experiments in mice showed this shared circulatory milieu restored tissue health and regeneration of the old partner; and at the same time, the young partner experienced a regenerative decline in a number of tissues. However, parabiosis is not clinically translatable and infusion of young blood or plasma into old mammals is controversial and fraught with multiple side-effects. Blood fractionation is typically cumbersome, and it is inherently complicated by the fact that the rejuvenative activities are likely to be contained in multiple molecularly different fractions. Plus, the assays for determining such activity are themselves complex, thus adding to the hurdles of a screen for active blood molecules. With these observations to consider, what would be the key set of molecular parameters that were changed by the blood heterochronicity and what would be the best translational way forward?
The changes that manifest with aging include altered cell metabolism, increased Reactive Oxygen Species (ROS), inflammation, senescence, and decline in immune function. However, from the viewpoint of tissue maintenance and regeneration, we postulated that these arise from changes in tissue growth and homeostasis and specifically in key signaling networks regulating stem cells and their differentiated niches. In support of this idea, pathway modifier-based approaches for the enhancement of aged tissue repair and maintenance have been reported, for example, by systemic delivery of OT which induces MAPK/pERK signaling, by forced activation of Notch-1, by antagonism of TGF-beta/pSmad signaling, or by antagonism of the Jak/Stat pathway.
The highest risk from modulating key cell-fate regulatory signaling pathways come from changing levels too far above or too far below normal healthy levels. Such drastic alterations result in severe multi-tissue side-effects. But high levels of a single modifier might be required to overcome the many age-specific molecular changes. For example, ectopic oxytocin (OT) might be needed at a considerably high dose to overcome age-elevated TGF-beta 1. And, the Alk5 inhibitor (Alk5i) of the TGF-beta receptor might be needed at high dose to overcome the lack of OT and other hormones with age.
Using a two-prong approach of simultaneously diminishing TGF-beta signaling and adding OT (which activates pERK via the oxytocin receptor (OTR)), we were able to reduce the required dose of Alk5i, shorten the duration of treatment and to achieve a more broad rejuvenation of the three germ-layer derivative tissues: brain, liver and muscle. And, we found that Alk5i+OT downregulated the number of cells that show an age-associated increase of the cyclin dependent kinase (CDK) inhibitor and marker of senescence, p16, thereby representing a pharmacological combination of two FDA approved drugs to normalize this checkpoint protein, which when chronically elevated negatively impacts tissue health.
Translationally, this study points toward a pharmacological approach to rapidly enhance the health and maintenance of multiple old tissues. Here we focused on a few key age-related parameters of the three germ layer tissues: neurogenesis and neuroinflammation of the brain, regeneration and fibrosis of the skeletal muscle and adiposity and fibrosis of the liver. In future work if would be interesting to study how these seemingly unrelated aging features become rapidly rejuvenated by A5i+OT, and if additional phenotypes, such as muscle innervation, neural plasticity, metabolism, etc. also become improved in old animals. The observed rejuvenating effects are at least as robust as, and act faster than, heterochronic parabiosis.
A Large Polypill Clinical Trial Shows a Third Reduction in Cardiovascular Events
The research and medical communities are slow to undertake work on combination therapies. Regulation makes it exceedingly expensive to assess multiple combinations, and there are numerous other perverse incentives to challenge any effort to build combination therapies with components developed and manufactured by different groups. Short of working around the existing system of regulation, and methods of doing this at scale are lacking at the present time, this is a challenging problem to solve. People follow incentives. Given this, it it is entirely plausible that there are many largely unexplored instances in which existing classes of medication for age-related disease might synergize to be more effective together.
In this context, clinicians and researchers have been discussing polypills for quite some time. The term polypill usually means a combination of existing treatments for cardiovascular disease such as statins to reduce blood cholesterol, ACE inhibitors to lower blood pressure, diuretics to reduce fluid retention, and so forth. The data to date strongly suggests that many reasonable polypill combinations will improve upon single medication use, and possibly do so at lower overall doses, and thus with lower side-effects.
Here, researchers report on a recent large clinical trial of a polypill, carried out in a comparatively poor population who are largely without access to the panoply of medications available in wealthier regions. The effect size is about what one would expect: in the world of the immediate past in which no-one was trying to tackle the causes of aging, and thus comparatively little can be achieved, reductions in blood cholesterol and blood pressure have been outstanding successes. Reducing cardiovascular mortality by a third without in any way addressing the underlying causes of cardiovascular mortality is quite the feat. In the immediate future, when senolytic drugs and other therapies that do address the causes of aging start to become widely used, we should expect to see much larger beneficial changes in population health.
Four-in-one pill prevents third of heart problems
A daily pill containing four medicines can cut the number of heart attacks and strokes by a third, a study shows. The polypill contains blood-thinning aspirin, a cholesterol-lowering statin, and two drugs to lower blood pressure. The researchers said the pill had a huge impact but cost just pennies a day. They suggest giving it to everyone over a certain age in poorer countries, where doctors have fewer options and are less able to assess individuals.
The study was based in more than 100 villages in Iran and about 6,800 people took part. Half the people were given the polypill and advice on how to improve their lifestyle, with the other half just getting the advice. After five years there were 202 major cardiovascular events in the 3,421 people getting the polypill and 301 in the 3,417 not getting the pill. The polypill led to large reductions in bad cholesterol but had only a slight effect on blood pressure, the study showed. The drug was given to people over the age of 50 whether they had had a previous heart problem or not.
In the UK and other wealthier countries doctors have the time to assess the needs of individual patients and a wide choice of different drugs, such as statins, to chose from. "In the UK, the advantages would be more marginal and you would probably want a clinical trial to see any benefits over what is offered at the moment."
Effectiveness of polypill for primary and secondary prevention of cardiovascular diseases (PolyIran): a pragmatic, cluster-randomised trial
The PolyIran study was a two-group, pragmatic, cluster-randomised trial nested within the Golestan Cohort Study (GCS), a cohort study with 50,045 participants aged 40-75 years from the Golestan province in Iran. Clusters (villages) were randomly allocated (1:1) to either a package of non-pharmacological preventive interventions alone (minimal care group) or together with a once-daily polypill tablet (polypill group). Randomisation was stratified by three districts (Gonbad, Aq-Qala, and Kalaleh), with the village as the unit of randomisation.
The non-pharmacological preventive interventions (including educational training about healthy lifestyle - eg, healthy diet with low salt, sugar, and fat content, exercise, weight control, and abstinence from smoking and opium) were delivered by the PolyIran field visit team at months 3 and 6, and then every 6 months thereafter. Two formulations of polypill tablet were used in this study. Participants were first prescribed polypill one (hydrochlorothiazide 12.5 mg, aspirin 81 mg, atorvastatin 20 mg, and enalapril 5 mg). Participants who developed cough during follow-up were switched by a trained study physician to polypill two, which included valsartan 40 mg instead of enalapril 5 mg. Participants were followed up for 60 months. The primary outcome - occurrence of major cardiovascular events (including hospitalisation for acute coronary syndrome, fatal myocardial infarction, sudden death, heart failure, coronary artery revascularisation procedures, and non-fatal and fatal stroke) - was centrally assessed by the GCS follow-up team.
We enrolled 6838 individuals into the study - 3417 (in 116 clusters) in the minimal care group and 3421 (in 120 clusters) in the polypill group. During follow-up, 301 (8.8%) of 3417 participants in the minimal care group had major cardiovascular events compared with 202 (5.9%) of 3421 participants in the polypill group (adjusted hazard ratio [HR] 0.66). When restricted to participants in the polypill group with high adherence, the reduction in the risk of major cardiovascular events was even greater compared with the minimal care group (adjusted HR 0.43). The frequency of adverse events was similar between the two study groups.
Lipid Accumulation in Microglia Contributes to Neuroinflammation and Neurodegeneration
Researchers have found that microglia in the aging brain have a tendency to accumulate lipids, and that those that do are harmful. This is a fascinating discovery, given that microglia are essentially the central nervous system version of macrophages elsewhere in the body, and lipid accumulation in macrophages leading to senescence and inflammatory behavior is an important mechanism in atherosclerosis. Further, it is well established that microglia in the brain become inflammatory, senescent, and dysfunctional in later life, and this behavior contributes to the progression of neurodegenerative conditions. It has been demonstrated that removing senescent microglia can turn back Alzheimer's pathology in mouse models of the condition, for example. This lipid accumulation might be an important aspect of dysfunction in microglia, though it is anyone's guess at this point as to where it sits in the web of cause and effect.
Microglia in the brain assume a dizzying array of states. Now researchers describe a new one: lipid droplet-accumulating microglia (LAM). These lipid-stuffed cells resemble the foamy macrophages seen in atherosclerotic lesions. They accumulate in the hippocampus of the aging brain and appear to be bad news, hiking inflammation and reactive oxygen species while having little ability to phagocytose debris. Notably, inflammatory stimuli induce LAM, as do some genetic variants associated with neurodegenerative disease.
Previous studies have identified a smorgasbord of distinct transcriptional profiles that delineate subtypes of microglial states. A handful of these have been correlated with neurodegenerative disease. These include disease-associated microglia (DAM), which cluster around plaques in mouse models of amyloidosis, and the similar microglial neurodegenerative phenotype (MGnD) found in multiple mouse disease models. A recent study characterized human Alzheimer's microglia (HAM), which were isolated from the Alzheimer's brain. It is still unclear how all these types relate to each other and what they do.
While examining hippocampal sections from aged wild-type mice by electron microscopy, researchers were struck by the accumulation of lipid droplets inside microglia. These microglia resembled cells first described by Alois Alzheimer, who reported lipid-stuffed glia clustering around amyloid plaques in the Alzheimer's brain more than 100 years ago. Researchers quantified the phenomenon in mice, finding that more than half the hippocampal microglia in 20-month-old wild-type animals contained from one to three lipid droplets. Droplets were rare in other brain regions, and nearly absent in 3-month-old mice.
To characterize these LAM, the authors isolated microglia from aged mouse hippocampi and sorted out those with high lipid content. Transcriptional profiling revealed 692 genes that were differently expressed between cells with low and high lipid content. In particular, genes involved in the production of reactive oxygen species, lipids, and pro-inflammatory cytokines were up in LAM, while genes responsible for phagocytosis were down. Notably, this transcriptional profile was in many respects the opposite of DAM, which turn up phagocytotic genes.
Functional studies of LAM reinforced these transcriptional findings. When the authors injected myelin debris into aged mouse hippocampus, microglia without lipid droplets engulfed it, but few LAM did. LAM isolated from brain produced more reactive oxygen species (ROS) than did low-lipid microglia, and they secreted higher levels of several pro-inflammatory cytokines such as CCL3, CXCL10, and IL-6. How do these cells arise? Because many of the LAM genes are regulated by inflammation, researchers speculate that they are products of an inflammatory response.
Estradiol Accelerates Liver Regeneration in Mice
The liver is the most regenerative organ in mammals, capable of regrowing lost sections, albeit imperfectly in comparison to the capabilities of highly regenerative species such as salamanders. Researchers here demonstrate that the sex hormone estradiol is involved in the regulation of liver regeneration, and that regeneration can be accelerated via artificially increased levels of estradiol. This is particularly interesting in the context of recent work showing that loss of estradiol with aging is involved in loss of muscle mass, due to effects on stem cell activity. One might wonder if this sort of mechanism will show up in other tissues as well.
The endogenous hormone estradiol is widely recognized as a stress signaling molecule. It is mainly produced by the ovary, and its production can be induced under certain contexts and stimuli such as during the estrus cycle and in late pregnancy. Estradiol exerts its multiple functions by binding to GPR30, estrogen receptor (ER) α, or ERβ, which are members of the nuclear receptor super family.
Estradiol production is also induced after liver resection/injury, suggesting this hormone plays a role in liver regeneration. Several genes that participate in liver regeneration have been identified, including those encoding the inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6). In particular, we have previously focused on the molecules essential for triggering liver regeneration after partial hepatectomy (PH) using mouse models. In addition to these cytokines, the production of the chemical hormone estradiol is also induced in the acute phase of liver injury after PH, via the ovary and testes.
We have further demonstrated that estrogen induces hepatocyte proliferation after PH, which was delayed by ovariectomy. This estradiol induction after PH was in turn found to induce ERα expression in the mainly periportal hepatocytes. Moreover, the WT mice showed transient steatosis during liver regeneration after PH. Therefore, we hypothesized that PH initially triggers estradiol production, leading to elevated ERα expression, to consequently initiate the processes of β-oxidation enzyme expression for anti-steatosis.
In the present study, we tested this hypothesis by analyzing the liver regeneration process in ERα knockout (KO) mice compared with that in their wild-type (WT) littermates. Estradiol administration accelerated liver regeneration through ERα, indicating the feasibility of the estrogen-ERα axis as a target. These findings establish the foundation for the therapeutic application of estradiol to accelerate liver regeneration after resection in clinical settings.
Clinical Trial of a Cross-Link Breaker to Treat Presbyopia in the Aging Eye
Presbyopia in the aging eye manifests as a difficulty in focusing on close objects. It is caused by hardening of the lens, which is in part the result of cross-linking in the extracellular matrix of that tissue, though other mechanisms are involved as well. Cross-links are hardy metabolic byproducts resulting from the normal operation of metabolism, capable of degrading the structural properties of tissue, particularly elasticity, by linking proteins together and restricting their motion. Cross-linking is likely of great importance in skin aging and cardiovascular aging. The primary age-related cross-links of the lens are not the same as those of other soft tissues in the body, however: disulphide bonds rather than glucosepane. So this research is interesting for all of us heading towards older age and dysfunctional vision, but only in the context of dysfunctional vision. As a first attempt, there is clearly some room for improvement in the degree to which the approach taken breaks cross-links, but, given this proof of principle, that further improvement should follow in the years ahead.
A new topical agent is coming closer than ever to improving the accommodative range for presbyopes. The agent, lipoic acid choline ester (UNR844, Novartis, formerly EV06), is a reducing agent that is purported to reduce the disulfide bonds that form between lens proteins, thus increasing the deformability of the crystalline lens. "This chemical was designed to improve the internal rheology of the cytosol within the lens fibers inside the lens capsule. It is safe, well-tolerated, and showed statistically significant near visual acuity improvement in clinical trials compared to placebo. The widespread use of this drug stands to radically alter the visual performance of humans within our lifetimes."
Presbyopia is not just a matter of lens compliance. It is caused by a few different events, each of which constitutes a potential treatment target: the crystalline lens enlarges over time (ectoderm), the ciliary body undergoes atrophic changes, the vitreous becomes less viscous, and the lens loses its flexibility. The hypothesis that drove the development of UNR844 addressed lens flexibility or the lack thereof in presbyopia. When lens proteins become oxidized over time, disulfide bonds form, rendering them less able to move relative to one another during the act of accommodation.
"The theory was that if we had a way to chemically reduce these disulfide bonds, the proteins would regain increased degrees of freedom and allow a greater range of deformation of the lens, translating into a greater dynamic range of accommodation." Lipoic acid is a naturally occurring antioxidant and reducing agent. To allow the reducing agent to achieve sufficient concentration within the eye, researchers developed a prodrug to improve the compound's penetration, allowing it to metabolize and convert to its active form (dihydrolipoic acid [DHLA]) once within the lens. DHLA reduces disulfide bonds between lens proteins and restores lens microfluidics. Proof of concept was confirmed in vitro with human cadaver lenses and in vivo in rabbit eyes, where in both trials the drug produced lens softening and an increase in lens deformability.
The Phase 1/2 clinical study evaluated safety and efficacy of EV06 ophthalmic solution 1.5% in improving distance corrected near visual acuity (DCNVA) in subjects with presbyopia. The prospective, randomized, double-masked, placebo-controlled study included 75 patients (45-55 years) with hyperopia, myopia, or emmetropia, and a diagnosis of presbyopia. At baseline, the study patients had DCNVA below 20/40 in each eye. The study drug was given for 91 days and patients were monitored during a 7-month follow-up period. Visual acuity improvements were most pronounced when subjects employed bilateral vision, with 84% achieving 20/40 bilateral vision or better versus 52% in the placebo group.
The CellAge Database of Genes Associated with Cellular Senescence
The accumulation of lingering senescent cells is a cause of aging, via the inflammatory and other signals secreted by these cells. This is now widely accepted in the research community, and the first senolytic drugs that can selectively clear some of the burden of senescent cells already exist. Unfortunately it is not yet widely appreciated that these first low cost rejuvenation therapies do in fact exist, and are easily obtained and used. Hundreds of millions of people suffer from inflammatory conditions of aging that can likely be effectively treated via even just a single dose of senolytic drugs. Producing more human data for these existing treatments and bringing them to the vast patient population who would benefit should be much more of a priority than it is today.
Given that any new understanding of the biochemistry of senescent cells might lead to a novel basis for therapies that can greatly improve health in old age, there is a great deal of funding these days for efforts to map the biochemistry of the senescent state. These efforts are giving rise to new startup biotech companies, a few every year, and new candidate small molecule senolytics at an accelerated rate. Here, researchers announce a new database of genes associated with cellular senesence, one of a number of scientific initiatives likely to accelerate progress towards a full understanding of the biochemistry of senescent cells.
Cellular senescence, a permanent state of replicative arrest in otherwise proliferating cells, is a hallmark of ageing and has been linked to ageing-related diseases like cancer. Senescent cells have been shown to accumulate in tissues of aged organisms which in turn can lead to chronic inflammation. Many genes have been associated with cell senescence, yet a comprehensive understanding of cell senescence pathways is still lacking. To this end, we created CellAge, a manually curated database of 279 human genes associated with cellular senescence, and performed various integrative and functional analyses.
We observed that genes promoting cell senescence tend to be overexpressed with age in human tissues and are also significantly overrepresented in anti-longevity and tumour-suppressor gene databases. By contrast, genes inhibiting cell senescence overlapped with pro-longevity genes and oncogenes. Furthermore, an evolutionary analysis revealed a strong conservation of senescence-associated genes in mammals, but not in invertebrates.
Using the CellAge genes as seed nodes, we also built protein-protein interaction and co-expression networks. Clusters in the networks were enriched for cell cycle and immunological processes. Network topological parameters also revealed novel potential senescence-associated regulators. We then used siRNAs and observed that of 26 candidates tested, 19 induced markers of senescence. Overall, our work provides a new resource for researchers to study cell senescence and our systems biology analyses provide new insights and novel genes regarding cell senescence.
Clearing Dysfunctional Microglia Prevents Formation of Amyloid-β Plaques in a Mouse Model of Alzheimer's Disease
It is becoming clear that dysfunction in the supporting immune cells of the brain, the microglia, is important in the progression of neurodegenerative conditions such as Alzheimer's disease. This certainly involves microglia becoming senescent, as demonstrated by the ability of senolytic treatments to reverse pathology in animal models of Alzheimer's disease. But it most likely also involves a more subtle shift in the behavior of microglia, from a more regenerative M2 polarization to a more inflammatory and aggressive M1 polarization.
Both classes of microglial behavior are necessary in the grand scheme of things, but aging appears to be accompanied by an excess of M1 and too few M2 microglia (and macrophages as well, which have a similar set of behaviors) in most circumstances and tissues examined to date. The causes of this shift in cell behavior are barely explored at this point; it is unclear how it relates to the underlying molecular damage that drives aging. Nonetheless, it is certainly harmful.
Alzheimer's disease (AD) is a progressive, age-related neurodegenerative disorder thought to be triggered by the appearance and build-up of amyloid-β (Aβ) plaques in the cortex. Genome-wide association studies have identified numerous genes that confer increased risk for developing the disease; however, the mechanisms underlying plaque formation remain unclear. Within the central nervous system (CNS), microglia perform homeostatic maintenance, immune-related, and phagocytic functions. Their reported capacity for Aβ phagocytosis and clearance suggested that age-related changes in microglial function reduce clearance of neuronally derived Aβ from the brain, thus allowing plaque formation.
We and other groups report that following the initial period of plaque formation, microglia surround the plaques and subsequently mount a harmful and non-resolving inflammatory response. Despite this response, however, Aβ clearance and plaque modulation/dynamics is unaffected, yet the removal of the microglia at advanced stages of pathology protects against synaptic and neuronal loss.
Here, we set out to explore the contributions of microglia to plaque formation in the initial stages of the disease, which requires prolonged depletion of microglia throughout the plaque-forming period. To that end, we designed, synthesized, and optimized a potent, specific, orally bioavailable, and brain-penetrant CSF1R inhibitor, PLX5622, to deplete microglia for more than 6 months in 5xFAD mice. With the elimination of microglia, we uncovered critical roles of these cells in plaque formation, compaction, and growth, mitigating neuritic dystrophy, and modulating hippocampal neuronal gene expression in response to Aβ pathology.
Ultimately, these data demonstrate that microglial elimination is associated with the prevention of plaque formation and the downregulation of hippocampal neuronal genes that occur in a preclinical model of AD progression. These results indicate that microglia appear to contribute to multiple facets of AD etiology - microglia appear crucial to the initial appearance and structure of plaques, and following plaque formation, promote a chronic inflammatory state modulating neuronal gene expression changes in response to Aβ/AD pathology.
Evidence for Mitochondrial Dysfunction in Smooth Muscle to be Important in Age-Related Vascular Stiffness
Mitochondria in cells throughout the body become dysfunctional with age, with the proximate cause of this issue being a decline in the quality control mechanisms responsible for clearing out damaged and worn mitochondria. Researchers here show that the increased levels of reactive oxygen species produced by mitochondria in aged smooth muscle cells is important in the stiffening of blood vessels that occurs with advancing age. This loss of the ability of blood vessels to appropriately constrict and relax in response to circumstances leads to hypertension, a chronic state of raised blood pressure that is very damaging over the long term. In this context, it is worth noting that a clinical trial of a mitochondrially targeted antioxidant showed improvement in smooth muscle function and consequent reduction in blood vessel stiffness.
Aging is characterized by increased aortic stiffness, an early, independent predictor and cause of cardiovascular disease. Oxidative stress from excess reactive oxygen species (ROS) production increases with age. Mitochondria and NADPH oxidases (NOXs) are two major sources of ROS in cardiovascular system. We showed previously that increased mitochondrial ROS levels over a lifetime induce aortic stiffening in a mouse oxidative stress model. Also, NADPH oxidase 4 (NOX4) expression and ROS levels increase with age in aortas, aortic vascular smooth muscle cells (VSMCs), and mitochondria, and are correlated with age-associated aortic stiffness in hypercholesterolemic mice.
The present study investigated whether young mice (4 months-old) with increased mitochondrial NOX4 levels recapitulate vascular aging and age-associated aortic stiffness. We generated transgenic mice with low (Nox4TG605; 2.1-fold higher) and high (Nox4TG618; 4.9-fold higher) mitochondrial NOX4 expression. Young Nox4TG618 mice showed significant increase in aortic stiffness and decrease in phenylephrine-induced aortic contraction, but not Nox4TG605 mice. Increased mitochondrial oxidative stress increased intrinsic VSMC stiffness, induced aortic extracellular matrix remodeling and fibrosis, a leftward shift in stress-strain curves, decreased volume compliance and focal adhesion turnover in Nox4TG618 mice.
Nox4TG618 VSMCs phenocopied other features of vascular aging such as increased DNA damage, increased premature senescence and replicative senescence and apoptosis, increased proinflammatory protein expression and decreased respiration. Aortic stiffening in young Nox4TG618 mice was significantly blunted with mitochondrial-targeted catalase overexpression. This demonstration of the role of mitochondrial oxidative stress in aortic stiffness will galvanize search for new mitochondrial-targeted therapeutics for treatment of age-associated vascular dysfunction.
Visceral Fat Tissue is Anti-Inflammatory in Lean Mice, Inflammatory in Fat Mice
Excess visceral fat tissue leads to chronic inflammation via a range of mechanisms that include the creation of more senescent cells than would otherwise exist. Senescent cells secrete a potent mix of inflammatory and other signals that degrade tissue function in many ways. Since the accumulation of lingering senescent cells is a cause of aging, being overweight doesn't just increase risk and severity of age-related disease, and shorten life expectancy, but also literally accelerates aging. The more fat tissue, the worse the outcome over the long term. As this paper points out, however, this is only the case for excess visceral fat tissue. When lean, the normal, smaller amounts of this tissue are actually anti-inflammatory and beneficial.
Adipose tissue is host to various immune cells and it is well established that during obesity, the amount of inflammatory macrophages increase in adipose tissue. Visceral adipose tissue (VAT), surrounding the inner organs, has been shown to be more inflammatory active than subcutaneous adipose tissue (SAT), as increased amounts of visceral/abdominal fat associates with high levels of circulating inflammatory markers and a high number of pro-inflammatory cells in their adipose tissue.
Interestingly, in human and rodent studies, ageing is associated with an increase in the amount of visceral adipose tissue and/or level of inflammation. It is, however, unclear to what extent these age-related changes are a result of ageing per se or rather the result of changes in life-style with e.g. reduced levels of physical activity without a corresponding reduction in caloric intake. A human cross sectional study reported that whereas ageing is associated with increased inflammation, life-long endurance training resulted in lower circulating levels of inflammatory markers in both young and elderly individuals.
In the current study, we wanted to investigate the inflammatory status and tissue integrity of VAT in an exercise-training model of lean adult and old mice. We randomized adult (11 months; n = 21) and old (23 months; n = 27) mice to resistance training or endurance training, or to a sedentary control group. Strikingly, we observed an anti-inflammatory phenotype in the old mice, consisting of higher accumulation of anti-inflammatory M2 macrophages and IL-10 expression, compared to the adult mice. In concordance, old mice also had less VAT mass and smaller adipocytes compared to adult mice. In both age groups, exercise training enhanced the anti-inflammatory phenotype. In conclusion, in the absence of obesity, visceral adipose tissue possesses a pronounced anti-inflammatory phenotype during aging which is further enhanced by exercise.
Methods of Inducing Cellular Damage are Rarely Relevant to Aging, and the Details Matter
One of the major challenges in aging research is determining whether or not models of cellular or organismal damage and its consequences are in any way relevant to the natural processes of aging. One can hit a brick with a hammer, but that says very little about how bricks weather over the years. One can hit the brick very carefully with the hammer in ways that produce results that look weathering-like, but can that be used to tell us anything about weathering? In cells the line between artificial and natural damage can be hard to pin down, but the fine details of the processes involved always matter. It is easy to break cells and see them become dysfunctional as a result, but hard to determine the relevance of that breakage to natural aging. Even in the example here, in which researchers are trying to achieve something very similar to the consequences of excessive oxidative damage in mitochondria that is observed in aging, it is possible to argue that the methodology used has little relevance to the actual damage of aging in its details, and therefore may not be a useful model.
Researchers have carried out a causal experiment to kick off a mitochondrial chain reaction that wreaks havoc on the cell, all the way down to the genetic level. "I like to call it 'the Chernobyl effect' - you've turned the reactor on and now you can't turn it off. You have this clean-burning machine that's now polluting like mad, and that pollution feeds back and hurts electron transport function. It's a vicious cycle." The researchers used a new technology that produces damaging reactive oxygen species - in this case, singlet oxygen - inside the mitochondria when exposed to light. "That's the Chernobyl incident. Once you turn the light off, there's no more singlet oxygen anymore, but you've disrupted the electron transport chain, so after 48 hours, the mitochondria are still leaking out reactive oxygen - but the cells aren't dying, they're just sitting there erupting."
At this point, the nucleus of the cell is being pummeled by free radicals. It shrinks and contorts. The cell stops dividing. Yet, the DNA seems oddly intact. That is, until the researchers start looking specifically at the telomeres - the protective caps on the end of each chromosome that allow them to continue replicating and replenishing. Telomeres are extremely small, so DNA damage restricted to telomeres alone may not show up in a whole-genome test, like the one the researchers had been using up to this point. So, to see the genetic effects of the mitochondrial meltdown, the researchers had to light up those tiny endcaps with fluorescent tags, and lo and behold, they found clear signs of telomere fragility and breakage. Then, in a critical step, the researchers repeated the whole experiment on cells with inactivated mitochondria. Without the mitochondria to perpetuate the reaction, there was no buildup of free radicals inside the cell and no telomere damage.
Talking Up the Potential of the Longevity Industry
One of the Juvenescence founders is here enthusiastic about the potential for treating aging as a medical condition. While one should always filter the remarks of people who run companies via a cynical view of their incentives, as talking up the company, the industry, and the prospects is very much expected, it is in fact the case that the longevity industry as a whole has tremendous potential. It will up-end the whole of healthcare, all expectations of what it means to be older, and will most likely become the largest industry on the planet. It will alleviate more suffering, pain, and death than any other human endeavor to date, by a very large margin.
Exactly which of the specific projects and companies will turn out to produce the lion's share of the benefits is hard to predict in advance. That said, I am of course willing to argue that following the SENS methodology of repairing underlying damage is going to be far more effective, on balance, than interventions that target downstream metabolic states or processes. Thus of the present set of approaches, senolytic therapies to clear senescent cells seem far more likely to change the world significantly than is the case for, say, mTOR inhibitors that mimic some of the effects of calorie restriction.
Earlier this year, an executive from Juvenescence-backed AgeX predicted the field of longevity will eventually "dwarf the dotcom boom." Greg Bailey, the UK-based anti-aging biotech's CEO, certainly hopes so. The business of anti-aging is gaining steam - Bank of America has forecast the market will balloon to 610 billion by 2025, from an estimated 110 billion currently - but investors are cautious.
"I think there's a huge amount of skepticism. There's an enormous number of charlatans ... I understand why they would be thinking you know, is this real? Walk into your local drugstore, you're going to see about 50 products that claim to be anti-aging, and I can assure you that none of them are." Bailey suggested that investors are not quite as enthusiastic about placing bets on anti-aging, as they are in the tech world. "Institutions tend to move in lockstep when they're investing. VCs are astonishing, you know, if one of them buys the yellow halter top, all of them have to buy a yellow halter top. We're dramatically underserved. It's not getting the exposure that tech gets, considering the size of the market. There is a disconnect on what investors - sophisticated investors - institutions, how they're viewing this, I don't think they quite grasp how fast this is going to happen, and how big it's going to be."
Juvenescence has now raised 165 million in the last 18 months - in January it unveiled the first 46 million tranche of the Series B - and the money is being used to fund longevity projects with the lofty goal of extending human lifespans to 150 years. It is a popular vision. Inspired by Juvenescence, venture capitalist Sergey Young - who is in charge of all things longevity at the non-profit XPRIZE and VC fund BOLD Capital Partners - unveiled a 100 million fund with the same goal in February. Google-owned stealthy biotech Calico is after the same prize - and has partnered with AbbVie.
Juvenescence has been busy, collaborating with different groups and setting up joint ventures, such as Alex Zhavoronkov's AI shop at Insilico Medicine - and has invested in firms including AgeX and LyGenesis. In February, Juvenescence debuted an anti-aging joint venture with the Buck Institute dedicated to inducing ketosis. In recent months, it spawned a new biotech called Souvien Therapeutics, which is developing medicines to address the epigenetic underpinnings of neurodegenerative diseases, and injected 6.5 million in equity financing into a preclinical metabolic disease biotech dubbed BYOMass. Juvenescence will maintain a focus on regeneration. "I'm mindful that if you live to 150, you know, people don't want to be all wrinkled, and in a wheelchair. So what we want to be able to do is regenerate tissues."
The Decline of Autophagy in Skin Aging
The maintenance processes of autophagy recycle damaged structures and protein machinery in the cell. Autophagy is influential on the course of aging, as illustrated by the fact that many of the interventions capable of slowing aging in animal models involve increased autophagic activity. Some, like calorie restriction, have been demonstrated to require autophagy in order to extend healthy life span. Further, autophagy declines with age, and this is associated with the progression of a range of age-related diseases. Better maintenance of cells means better function of tissue and a slower onset of age-related dysfunction. The research community spends a great deal of time and effort in the investigation of autophagy and how to adjust its operation, but for all that, comparatively little concrete progress has been made towards clinical therapies that upregulate autophagy in humans.
Changes of the skin belong to the most recognizable signs of aging. Accordingly, skin aging is a major area of interest for cosmetic and skin care industries. From the medical viewpoint, aging of the skin is associated with health problems including increased skin fragility, delayed wound healing, and the increased occurrence of skin cancers, the most abundant types of malignancies in humans. For a long time it has been recognized that the rate of skin aging is determined by intrinsic and extrinsic drivers, but only recent advances in skin gerontology have helped to dissect the molecular and cellular processes that underlie the aging of the skin.
Several of the aging processes are triggered or enhanced by the presence of damaged molecules and organelles within cells, and their turnover is controlled partly by autophagy. Besides proteostasis and organelle maintenance, other factors that are accepted hallmarks of aging, such as nutrient sensing and genomic instability are under the control of or elicit the activation of autophagy, making autophagy a major counter-regulatory process that supports skin homeostasis and healthy aging.
The skin provides several examples to illustrate the two main interactions between autophagy and aging: (1) Autophagy decreases the rate of aging and (2) the activity of autophagy declines during aging. Autophagy suppresses aging in a cell-autonomous manner by maintaining intracellular homeostasis and in a non-autonomous manner by contributing to various cell features that protect other cells. For instance, autophagy supports the differentiation of epithelial cells which allows them to protect other cells against the external environment. Since autophagy achieves the removal and recycling of intracellular material only to a certain extent, potential toxic cell components and dysfunctional lysosomes tend to accumulate during the life-time of cells. Some of the compromised cells succumb to cell death whereas others remain alive but lose their capacity to execute intracellular processes, including autophagy, with full efficiency. Loss and dysfunction of cells manifest in aging.
Long-lived and mostly quiescent stem cells require autophagy for intracellular homeostasis and for continuous ability to supply functional progeny cells. Inherent decline or exogenous suppression of autophagy leads to stem cell loss by competition, differentiation, or cell death. In short-lived differentiating cells, autophagy also contributes to intracellular homeostasis, however, autophagic activity needs to be maintained only over a short time for these cells to be functional. Nevertheless, autophagy defects can be inherited from the long-lived precursor cells (stem cells) and potentially compromise processes such as the defense against microbes, the release of cytokines, and most importantly, the protection against stress factors from the environment. In long-lived differentiated cells, autophagy contributes to the maintenance of cell survival and function. A decrease of autophagy leads to the accumulation of damaged or even toxic components and/or energy crisis. These disturbances of intracellular homeostasis impair the processes essential for cell functions and eventually lead to a loss of these cells.