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- How to Organize and Run a Comparatively Simple Self-Experiment to Assess the Impact of MitoQ and Niagen on Cardiovascular Aging
- For Senolytics Companies, an Effective Piperlongumine would be a Greater Competitive Threat than Dasatinib
- Reporting on the 2018 International Cellular Senescence Association Meeting
- Tau Aggregation in the Aging Brain Disrupts Nuclear Pores, Possibly Explaining Loss of Function in Alzheimer's Disease
- A View of Advanced Glycation End-Products that is Primarily Inflammatory
- Debating the Microbial Hypothesis for Alzheimer's Disease
- Sardinian Population Data Provides Evidence for a Reduced Burden of Infectious Disease to Slow the Pace of Aging
- TXNIP, Associated with Aging in Flies, Shown to Influence Cellular Senescence in Mice
- Might Means of Reversing Atherosclerosis also Prevent Macular Degeneration?
- Evidence for a Ketone Body Produced During Calorie Restriction to Reduce the Creation of Senescent Cells
- Scheduled Feeding Shown to Slow Aging in Mice
- Removing Inflammatory Regulatory T Cells Reverses Aspects of Heart Failure in Mice
- Identifying Genes Responsible for Human Longevity Relative to Other Primates
- Exosomes from Young Mice are Shown to Reverse Changes in Expression of Aging-Associated Genes in Old Mice
- An Update from the CellAge Team
How to Organize and Run a Comparatively Simple Self-Experiment to Assess the Impact of MitoQ and Niagen on Cardiovascular Aging
This post walks through the process of setting up and running a simple self-experiment - a trial of one - with two compounds shown to improve measures of cardiovascular aging, specifically (a) pulse wave velocity, a measure associated with rising blood pressure and stiffening of blood vessels, and (b) prevalence of oxidized lipids, associated with the progression of atherosclerosis. These compounds work via their effects on mitochondria, dampening the impact of aging on these vital components of cellular function, but without actually repairing the underlying damage that causes aging.
The two compounds are MitoQ, a mitochondrially targeted antioxidant that was shown to beneficially impact oxidized lipids and pulse wave velocity in a recently published small human trial, and Niagen, a form of nicotinamide riboside which also has recent data from a small human trial suggesting that it can reduce pulse wave velocity.
This post, unlike others in this series, focuses on compounds that are approved for use as supplements rather than drugs, are easily purchased and widely used, and already have at least initial human trial data for impact on aspects of aging. That makes it much simpler from a logistics point of view, and thus more suitable as an introduction for people who have not yet tried to rigorously self-experiment. The downside is that these compounds don't address root causes of aging, but are instead at best a way to modestly compensate for the consequences of molecular damage. In this case that means specifically damage to mitochondria and changes in the signaling environment that otherwise cause declining mitochondrial function.
A caveat: one might think that "widely used" means "safe". Safe is a slippery word, however, in that nothing is ever truely safe. Older individuals can and do suffer injury and death from everyday actions, foods, and medications that have no such impact on younger individuals. Regardless of the legions using a particular compound, it is always wise to gently ease into any personal attempt to join them, rather than leaping in at a full dose on day one.
- Why Self-Experiment with MitoQ?
- Why Self-Experiment with Niagen?
- Establishing Dosages
- Obtaining MitoQ and Niagen
- Establishing Tests and Measures
- Guesstimated Costs
- Schedule for the Self-Experiment
- Where to Publish?
Why Self-Experiment with MitoQ?
Ordinary antioxidant supplements are thought to be, on balance, modestly harmful to long term health. They block signaling that is important to the beneficial response to exercise, for example. Mitochondrially targeted antioxidants, on the other hand, have been shown to slightly slow aging in short-lived species, and improve measures of health along the way. They also appear to be a viable treatment for some localized inflammatory conditions. The theory here is that mitochondria generate oxidative molecules in the normal course of operation that cause damage within the mitochondria themselves, and that in turn leads to dysfunctional cells in which the mitochondria produce a vastly greater amount of oxidative molecules. Delivering a constant supply of mitochondrially targeted antioxidants may either slow down the pace at which mitochondria damage themselves, or dampen the consequences of cells overtaken by damaged mitochondria, or both.
One of those consequences is the bulk export of oxidative molecules into surrounding tissues and the bloodstream, where they react with lipids. Oxidized lipids can cause further harm in all sorts of cellular processes, but of particular interest is the development of atherosclerosis. Oxidized lipids can cause inappropriate inflammatory reactions in blood vessel walls, and some forms can also cause the cells responding to that inflammation to become overwhelmed and die. This is how the fatty plaques of atherosclerosis form, then grow to weaken and narrow major blood vessels. Statin drugs, that reduce blood cholesterol, succeed in slowing atherosclerosis because they reduce the amount of oxidized lipids in the course of reducing the amount of all lipids.
Further, some degree of dysfunction in the vascular smooth muscle responsible for blood vessel contraction and dilation is thought to be caused by rising levels of oxidative stress in aging - too many dysfunctional mitochondria, too many oxidative molecules. This contributes to vascular stiffness and consequent hypertension, cardiovascular disease, and so forth. Suppressing the oxidative consequences of malfunctioning mitochondria may help here as well.
Mitochondrially targeted antioxidants don't solve the roots of these problems. At best, they somewhat compensate or attenuate ongoing mechanisms. They are cheap, however, and if they can produce effects on risk factors for cardiovascular disease that are, say, somewhere in the same order of magnitude as those achieved by statins or drugs that control blood pressure, with minimal side-effects, then they may well be worth using.
Why Self-Experiment with Niagen?
Niagen is a formulation of nicotinamide riboside, a compound shown to beneficially adjust NAD+ metabolism in cells. The outcome is a general improvement in mitochondrial function. To the extent that loss of mitochondrial function is an issue in aging, regardless of the varied causes of that loss, supplementation with nicotinamide riboside can turn back a fraction of that problem. This loss of mitochondrial function is particularly well studied in neurodegenerative disease and muscle aging, as the brain and muscles are two of the most energy-hungry tissues in the body, but there are consequences in all other tissues as well.
The outcome of nicotinamide riboside supplementation that has the most defensible evidence is much the same as the effects of mitochondrially targeted antioxidants noted above, in that it appears to reduce the dysfunction of vascular smooth muscle cells that is responsible for some fraction of vascular stiffening and hypertension. The results of a small human study provide evidence for a modest reduction in pulse wave velocity in older study participants.
As is the case for mitochondrially targeted antioxidants, Niagen supplementation does not reverse the root causes of aging. It compensates for or attenuates one class of downstream consequence, and is thus of limited utility when considered in the grand scheme of things. But if nicotinamide riboside is both cheap and reliable in the production of that limited utility, while producing few to no side-effects along the way, then it can be worth using.
While both MitoQ and Niagen are approved by regulators, are widely used, and are accompanied by good human data on effects and side-effects, one must still think about personal responsibility in any self-experiment. Firstly, read the papers reporting on human trials - the effects, side-effects, and dosages - and make an informed personal decision on risk and comfort level based on that information. This is true of any supplement, whether or not approved for use. Do not trust other opinions you might read online: go to the primary sources, the scientific papers, and read those. Understand that where the primary data is sparse, it may well be wrong or incomplete in ways that will prove harmful. Also understand that older physiologies can be frail and vulnerable in ways that do not occur in younger people and that are sometimes not well covered by the studies.
Secondly, the state of knowledge regarding any particular set of compounds is not static. The science progresses. This post will become outdated in its specifics at some point, as new knowledge and new compounds with similar effects arrive on the scence. Nonetheless, the general outline should still be a useful basis for designing new self-experiments involving later and hopefully better compounds, as well as tests involving more logistical effort.
The only definitive way to establish a dosage for a supplement or pharmaceutical in order to achieve a given effect is to run a lot of tests in humans. Fortunately those tests are underway, and enough has been published for MitoQ and Niagen to simply follow the existing studies. Little further digging, extrapolation of doses from mouse to human, or other similar work is required.
The 2018 MitoQ human study used a once daily dose of 20mg for six weeks. The 2018 Niagen human study used a twice daily dose of 500mg for six weeks.
Obtaining MitoQ and Niagen
MitoQ is cheap and readily available from MitoQ Limited via any number of reputable online storefronts. The same is true of Niagen, with numerous sellers listed at Amazon. In the latter case, there is a wide difference in price for essentially the same product from different vendors, so comparison shopping is a good idea.
Establishing Tests and Measures
The objective here is a set of tests that (a) match up to the expected outcome based on human trials of MitoQ and Niagen, and (b) that anyone can run without the need to involve a physician, as that always adds significant time and expense. These tests are focused on the cardiovascular system, particularly measures influenced by vascular stiffness, and some consideration given to parameters relevant to oxidative stress and the development of atherosclerosis.
- A standard blood test, with inflammatory markers.
- An oxidized LDL cholesterol assessment.
- Resting heart rate and blood pressure.
- Pulse wave velocity.
- Biological age assessment via DNA methylation patterns.
The cardiovascular health measures in that list are those that are impacted by changes in the elasticity or functional capacity of blood vessels, such as would be expected to occur to some degree in a treatment that compensated in some way for the effects of aging on the smooth muscule cells in blood vessel walls - as is thought to be the case for mitochondrially targeted antioxidants. Positive change of the average values in most of these metrics are achievable with significant time and effort spent in physical training, so movement in the numbers in a short period of time as the result of a treatment should be an interesting data point.
There exist online services such as WellnessFX where one can order up a blood test and then head off the next day to have it carried out by one of the widely available clinical service companies. Of the set of test packages offered by WellnessFX, the Baseline is probably all that is needed for present purposes. But shop around; this isn't the only provider.
Oxidized LDL Cholesterol
The more mainstream blood test services such as WellnessFX don't offer as wide a range of testing as some of the specialists. For example, the Life Extension Foundation maintains a blood test service that includes a test for oxidized LDL cholesterol. Again, shop around. There are others.
Resting Heart Rate and Blood Pressure
A simple but reliable tool such as the Omron 10 is all you need to measure heart rate and blood pressure. It is worth noting here a couple of general principles for cardiovascular measures. Firstly, the further away from the center of the body that the measurement is taken, the less reliable it is - the more influenced by any number of circumstances, such as position, mood, stress, time of day, and so forth. Fingertip devices are convenient, but nowhere near as useful as something like the Omron 10 that uses pressure on the upper arm. Secondly, all of the above-mentioned line items also influence every cardiovascular measure, so when you are creating a baseline or measuring changes against that baseline, carry out each measure in the same position, at the same time of day, and make multiple measurements over a week to gain a more accurate view of the state of your physiology. The Omron 10 is solid: it just works, and seems quite reliable.
Pulse Wave Velocity
For pulse wave velocity, choice in consumer tools is considerably more limited than is the case for heart rate. Again, carefully note whether or not a device and matching application will deliver the actual underlying data used in research papers rather than a made-up vendor aggregate rating. I was reduced to trying a fingertip device, the iHeart, picked as being more reliable and easier to use than the line of scales that measure pulse wave velocity. Numerous sources suggest that decently reliable pulse wave velocity data from non-invasive devices is only going to be obtained by measures at the aorta and other core locations, or when using more complicated regulated medical devices that use cuffs and sensors at several places on the body.
Still, less reliable data can be smoothed out to some degree by taking the average of measures over time, and being consistent about position, finger used for a fingertip device, time of day, and so forth when the measurement is taken. It is fairly easy to demonstrate the degree to which these items can vary the output - just use the fingertip device on different fingers in succession and observe the result. All of this is a trade-off. A good approach is to take two measures at one time, using the same finger of left and right hand, as a way to demonstrate consistency. While testing an iHeart device in this way, I did indeed manage to obtain consistent and sensible data, though there is a large variation from day to day even when striving to keep as many of the variables as consistent as possible. That large variation means that only sizable effects could be detected.
DNA methylation tests can be ordered from either Osiris Green or Epimorphy / Zymo Research - note that it takes a fair few weeks for delivery in the latter case. From talking to people at the two companis, the normal level of variability for repeat tests from the same sample is something like 1.7 years for the Zymo Research test and 4.8 years for the Osiris Green tests. The level of day to day or intraday variation between different samples from the same individual remains more of a question mark at this point in time, though I am told they are very consistent over measures separated by months. Nonetheless, as for the cardiovascular measures, it is wise to try to make everything as similar as possible when taking the test before and after a treatment: time of day, recency of eating or exercise, recent diet, and so forth.
An Example Set of Daily Measures
An example of one approach to the daily cardiovascular measures is as follows, adding extra measures as a way to demonstrate the level of consistency in the tools:
- Sit down in a comfortable position and relax for a few minutes.
- Measure blood pressure and pulse on the left arm using the Omron 10.
- Measure blood pressure and pulse on the right arm using the Omron 10.
- Measure pulse wave velocity on the left index fingertip over a 30 second period using the iHeart system.
- Measure pulse wave velocity on the right index fingertip over a 30 second period using the iHeart system.
Consistency is Very Important
Over the course of an experiment, from first measurement to last measurement, it is important to maintain a consistent weight, diet, and level of exercise. Sizable changes in lifestyle can produce results that may well prevent the detection of any outcome using the simple tests outlined here. Further, when taking any measurement, be consistent in time of day, distance in time from last exercise or meal, and position of the body. Experimentation with measurement devices will quickly demonstrate just how great an impact these line items can have.
The costs given here are rounded up for the sake of convenience, and in some cases are blurred median values standing in for the range of observed prices in the wild.
- Baseline tests from WellnessFX: 220 / test
- Oxidized LDL test from LEF: 170 / test
- MyDNAage kits: 310 / kit
- Osiris Green sample kits: 70 / kit
- Omron 10 blood pressure monitor: 80
- iHeart monitor: 210
- MitoQ capsules from MitoQ Limited: 190
- Niagen capsules from Amazon vendors: 280
Schedule for the Self-Experiment
One might expect the process of discovery, reading around the topic, and ordering materials to take a few weeks. Once all of the decisions are made and the materials are in hand, pick a start date. The schedule for the self-experiment is as follows:
- Day 1-14: Once or twice a day, take measures for blood pressure and pulse wave velocity.
- Day 14: Bloodwork and DNA methylation test.
- Day 15: Start the program of daily doses, and keep that going through the following measurements.
- Day 57-70: Repeat the blood pressure and pulse wave velocity measures.
- Day 70: Repeat the bloodwork and DNA methylation test.
Where to Publish?
If you run a self-experiment and keep the results to yourself, then you helped only yourself. The true benefit of rational, considered self-experimentation only begins to emerge when many members of community share their data, to an extent that can help to inform formal trials and direction of research and development. There are numerous communities of people whose members self-experiment with various compounds and interventions, with varying degrees of rigor. One can be found at the LongeCity forums, for example, and that is a fair place to post the details and results of a personal trial. Equally if you run your own website or blog, why not there?
When publishing, include all of the measured data, the compounds and doses taken, duration of treatment, and age, weight, and gender. Fuzzing age to a less distinct five year range (e.g. late 40s, early 50s) is fine. If you wish to publish anonymously, it should be fairly safe to do so, as none of that data can be traced back to you without access to the bloodwork provider. None of the usual suspects will be interested in going that far. Negative results are just as important as positive results. For example, given the measures proposed in this post it is entirely plausible that positive changes in a basically healthy late 40s or early 50s individual will be too small to identify - they will be within the same range as random noise and measurement error. Data that confirms this expectation is still important and useful for the community, as it will help to steer future, better efforts.
For Senolytics Companies, an Effective Piperlongumine would be a Greater Competitive Threat than Dasatinib
Senolytic treatments selectively destroy senescent cells, and several different approaches have been shown to produce some degree of rejuvenation in mice: reversal of measures of aging; reversal of the progression of specific age-related conditions; extension of life span. Most of these initial senolytics are repurposed pharmaceuticals drawn from cancer research databases, with the exceptions being the engineered peptide FOXO4-DRI, the suicide gene therapy developed by Oisin Biotechnologies, and SIWA Therapeutics' immunotherapy. Where animal study data has been published, the results produced by these varied senolytics are remarkably similar: up to 50% clearance of senescent cells from old tissues in mice, varying widely from tissue to tissue.
One of the repurposed pharmaceuticals is dasatinib, a drug already approved by the FDA for cancer treatment, with a sizable amount of human data by which we can judge side-effects and safety. Dasatinib is a generic drug that is mass produced by numerous manufacturers worldwide, whether with or without approval from the US government, and as a consequence it costs very little. This presents an interesting challenge for those companies attempting to produce senolytic therapies, as new treatments must run through clinical trials at enormous expense. In addition to proving new drug candidates or other classes of treatment, these trials will also provide supporting evidence that will allow physicians to prescribe off-label use of dasatinib at a tiny fraction of the cost that must be charged for new therapies in order to recoup development expenditure.
The principals of senolytic development companies will thus find themselves needing to produce treatments that can clear senescent cells far more effectively than the dasatinib and quercetin combination therapy. Even given the choice between a drug costing 100 that can clear 50% of senescent cells versus a drug costing 20,000 that can clear 80% of senescent cells, a company might struggle to obtain the desired level of sales over the long term. Though in fact the situation is more complex than this overly simplistic example, given the variability of results tissue by tissue, and there will be room for senolytics that turn out to be better for the heart, or lungs, or specific other organ than the competitors. But still, you see the challenge. This is particularly problematic for small molecule development, in which it is very expensive, uncertain, and time-consuming to attempt to improve specific aspects of an existing family of drugs. It is by no means certain that small molecule developers such as Unity Biotechnology will be able to produce drugs that are better enough to justify the price premium over dasatinib.
Dasatinib provides a certain degree of sink or swim encouragement to do better, but this pales before the state of affairs that will result should piperlongumine turn out to be senolytic to much the same level in mammals. Which may well be the case, given recent data, but nothing is yet proven in certainty. If piperlongumine is in fact approximately as good at removing senescent cells as the dasatinib and quercetin combination, then this discovery will unleash the dietary supplement industry and in short order allow them to become the major players in the senolytic marketplace, rather than merely a gaggle of hopeful onlookers. Piperlongumine is a plant extract, a natural product that is regulated in a completely different way from small molecule drugs and other medical biotechnologies. It costs far less in time and funding to bring a new natural product to the marketplace, and the resulting supplements are as a result far cheaper than medicine. Given effectiveness for piperlongumine, established dietary product concerns will be selling low-cost senolytics to much of the world well prior to the point at which the first expensive senolytic therapies emerge from the FDA regulatory process.
One could argue that this particular vision is unlikely to come to pass on the basis that the other potentially senolytic categories of natural product are not in fact capable of killing enough senescent cells to be worthy of the name. The flavonoid quercetin, for example, doesn't do much on its own. Certainly not enough to be an alternative to a real senolytic, no matter how cheap it might be. Is this a valid argument to direct at piperlongumine? Maybe so, maybe not. We shall see when the data arrives. Anyone with a few hundred thousand in funding could run the necessary mouse studies to prove or disprove the senolytic capacity of piperlongumine, and that is not a large number in comparison to what it requires to build a new supplement manufacturing and distribution business. Given this, one might wonder whether or not anyone in the industry is already working on this project.
Reporting on the 2018 International Cellular Senescence Association Meeting
Research into cellular senescence is at present one of the most exciting areas of the science of aging, as it is in this part of the scientific community that the first real, actual, legitimate rejuvenation therapies were discovered. These senolytic treatments, capable of selectively destroying senescent cells, are now in the process of verification in human trials. They offer the possibility of significant reversal of all inflammatory age-related disease, to a far greater degree than can be offered by any past therapy: osteoporosis; the fibrosis that drives dysfunction of the lung, heart, and kidney; neurodegenerative conditions such as Alzheimer's; atherosclerosis; and more. All of these conditions are either largely or partly caused by the accumulation of senescent cells that takes place in later life.
In the community of self-experimenters, many have chosen not to wait for the results of formal human trials. The evidence in mice from the past five years is robust and compelling; researchers have found it easy to reproduce benefits resulting from the removal of senescent cells, and have used a variety of small molecule drug families and other classes of therapy to achieve this goal. To the extent that an approach can destroy senescent cells, it works. The first generation of senolytic pharmaceuticals are both cheap and readily available, and tens of millions of older people in the US alone could benefit, given only the understanding and the proof of the first formal human data. A sweeping change is coming in what it means to be old, a great improvement in health across the board, at a very low cost per patient.
Meanwhile, the scientific community is forging ahead, building the foundations for the next generation of improved senolytic therapies, capable of removing a greater fraction of senescent cells with fewer accompanying side-effects. The near future of this field is bright, as is the future of our health in later life. We are now truly entering the era of human rejuvenation, a milestone in our technological progress as a species that will not soon be forgotten.
Cellular senescence, geroscience, cancer and beyond
More than two hundred scientists gathered in Montreal in July 2018 for the International Cellular Senescence Association (ICSA) Meeting to discuss the biological and medical impact of cellular senescence. In his welcoming speech, Dr. Ferbeyre summarized the key aspects that have attracted so much interest in cellular senescence including its ability to act as a tumor suppressor mechanism but also to promote aging and age-linked diseases.
One of the most exiting trends in senescence research is the concept of senolysis or the specific elimination of senescent cells. Jan Van Deursen (Mayo Clinic, USA) presented recent evidence that the elimination of senescent cells can induce regression of advanced atherosclerosis without any detectable side effects. Jennifer Hartt Elisseeff (Johns Hopkins, USA) showed that clearance of senescent cells using senolytics attenuates osteoarthritis development.
The connection between senescent cells and immune responses to injury and repair was presented. Darren Baker (Mayo Clinic, USA) presented experimental evidence that senescent cells promote neurodegeneration in mutant tau mice and their elimination attenuates disease. James Kirkland (Mayo Clinic, USA), showed that transplanting senescent cells to young mice caused frailty, diabetes, and osteoporosis, accelerating death from all causes. A cocktail of quercetin and dasatinib, a SRC-family kinase inhibitor, can kill senescent cells and revert their pathological effects both in senescent-cells transplanted young mice or in naturally aged mice, extending median life span up to 36%.
Salvador Macip (University of Leicester, UK) found another kinase, BTK, which activates the tumor suppressor p53 inducing senescence. Ibrutinib, a clinically approved inhibitor for this kinase increased life span in flies and in a mouse model of progeria. Irina Conboy (UC Berkeley, USA) used parabiosis to demonstrate the presence of factors in the serum of old mice that can induce senescence in young mice suggesting that some senescent cells in vivo may originate from extrinsic factors. She also presented interesting data on enhanced myogenesis and reduced liver adiposity, but no improvement in hippocampal neurogenesis in the old 3MR mice, when p16-high cells were experimentally ablated.
Myriam Gorospe, (NIH, USA) identified proteins expressed at the surface of senescent cells. SCAMP4 was found to favor the senescence-associated secretory phenotype (SASP) and DPP4 was found to allow the selective elimination of senescent cells using anti-DPP4 antibodies. Maria Almeida (University of Arkansas for Medical Sciences, USA) discussed the role of senescent osteocytes in age-related bone loss via production of increased levels of RANKL and the therapeutic potential of senolytic agents in preventing and treating osteoporosis by targeting senescent cells in the bones.
The promise that clearance of senescent cells with a therapeutic agent may prolong the health span and treat age-related diseases stimulates the research in finding new senolytic agents, therapeutic strategies, and delivery methods. Daohong Zhou (University of Florida, USA) presented some new development of Bcl-xl-targeted senolytic agents using proteolysis targeting chimera (PROTAC) technology. These Bcl-xl PROTACs that target Bcl-xl to an E3 ligase for ubiquitination and degradation exhibit an improved potency against senescent cells but reduced toxicity to normal cells and platelets compared to navitoclax and thus have the potential to be developed as a safer senolytic agent.
John Lewis (Oisin Biotechnologies, USA) described a clinically viable gene therapy consisting of a suicide gene under a senescent cell promoter delivered in vivo with fusogenic lipid nanoparticles (LNPs) to deplete senescent cells. This approach represents a first-in-class therapeutic that targets cells based on transcriptional activity, rather than surface markers or metabolism. Guangrong Zheng (University of Florida, USA) identified a dietary natural product, piperlongumine, as a novel senolytic agent. It can selectively kill senescent cells by targeting oxidation resistance 1 (OXR1), an important oxidative stress sensor that regulates the expression of a variety of antioxidant enzymes. His finding may lead to the development of better senolytic agents.
Daniel Munoz-Espin (University of Cambridge, UK) described the design of a new targeted-drug delivery system to senescent cells using the technology of the encapsulation of drugs with galacto-oligosaccharides because of the high lysosomal β-galactosidase activity of senescent cells. He showed that gal-encapsulated cytotoxic drugs can selectively target senescent cells in a tumor xenograft mouse model to improve tumor regression and toxicity. At the end of the meeting Ned David (Unity Biotechnology, USA) delivered a talk summarizing how his company is translating basic research on senescence into clinical trials using several senolytics. Senescence is undoubtedly at the forefront of biomedical research.
Tau Aggregation in the Aging Brain Disrupts Nuclear Pores, Possibly Explaining Loss of Function in Alzheimer's Disease
As the amyloid cascade hypothesis of Alzheimer's disease has it, the condition begins with growing levels of amyloid-β in the brain. The amyloid forms solid deposits with a surrounding halo of harmful biochemistry, degrading the function of nearby cells. Perhaps this is caused by failing drainage of cerebrospinal fluid, perhaps by the innate immune response to persistent infections, perhaps by other mechanisms such as the age-related failure of the immune system to clear up molecular waste as aggressively as it should. The amyloid sets the stage for mild cognitive impairment and the later deposition of altered forms of tau protein into neurofibrillary tangles. It is the tau aggregation that is associated with the real damage of Alzheimer's disease: the inflammation, the major dysfunction, the death of neurons in large numbers.
How exactly is tau wreaking such havoc, however? This is an open question, still awaiting a definitive collection of evidence and consensus. There is the hope that, given a good answer to this question, some form of molecular sabotage could prove to be the basis for a therapy to rescue patients who are far along in the progression of Alzheimer's disease. This would be an alternative to the more mainstream strategy of building ways to clear tau from the brain, analogous to the existing lines of work on anti-amyloid immunotherapies. Could this work? I'm not sure, and my feeling is that it is unlikely to be more cost effective than attempts to remove tau aggregates. Finding and blocking any one mode of damage without removing the neurofibrillary tangles will still leave all of the other modes of damage - and there will always be more than one path to harm. Biochemistry is nothing if not exceedingly complex. This is, more generally, the usual objection to adjusting the state of a diseased metabolism rather than removing the cause of disease.
The research here reports on an association between nuclear pore dysfunction and tau aggregration, and this may prove to be a significant contribution to neuronal dysfunction in tauopathies such as Alzheimer's disease. It is interesting to consider that nuclear pores in neurons contain some of the longest-lived proteins in the body. The very same molecule, the same atoms in the same configuration, might accompany you throughout life from birth to death. There is some speculation regarding these and other extremely long lived proteins as the next frontier of longevity science, the challenge that arises after all of the SENS rejuvenation programs are somewhere near completion, and we can largely repair all of the common forms of damage that cause aging. How to deal with potentially damaged molecules deep within countless vital brain cells that our biochemistry will never replace if left to its own devices? Perhaps there will be good answers to that question sooner rather than later, but it is beyond current capabilities, if not beyond present vision.
Tau interferes with nuclear transport in Alzheimer's disease
Researchers have long known that tau accumulates in the brains of individuals with Alzheimer's disease (AD), a major component of AD's hallmark neurofibrillary tangles. Precisely how tau contributes to the disease has remained a mystery. Now scientists have found that the nuclear pore complex, which controls the transport of molecules into the cell nucleus, is defective in animal and human AD cells and that the defect is associated with tau aggregation inside neurons. In a cell, the nucleus is surrounded by a membrane separating contents inside the nucleus from everything else within the cell. The nucleus communicates with the cell through the protein-rich structures known as nuclear pores. Defects in these pores have been suggested in other causes of dementia, particularly frontotemporal dementia, and in amyotrophic lateral sclerosis (ALS).
The nuclear pore complex includes more than 400 different proteins. Researchers focused on one of the major structural proteins of the pore, Nup98. In the presence of tau, the Nup98 nuclear pores are not evenly spaced throughout the structure as expected. Instead, they were physically disrupted, fewer in number, and coalesced with each other. Nup98 seemed to leak or be mislocalized in the cytoplasm of AD brain cells rather than remaining in the nuclear pore. Whenever it was mislocalized, those same cells tended to have aggregates of tau. The team found that the more extreme the AD disease was while patients were alive, at autopsy they had worse pathology related to Nup98 mislocalization with tau. In mice models, when human tau was added to cultures of living rodent neurons, Nup98 was mislocalized in the cytoplasm and functional nuclear import was disrupted.
Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimer's Disease
Here, we show that phospho-tau-positive cells in human AD and tau transgenic mouse brains, as well as in cellular models of tau-related AD neuropathology, have an impaired nuclear transport. Indeed, we found that tau can interfere with nuclear pore complex (NPC) integrity in different ways. Tau directly interacts with the nucleoporin Nup98 in vitro, leads to cytoplasmic mislocalization of Nup98 in neurofibrillary tangles (NFTs) and in neurons with phospho-tau in vivo, and induces a disruption of the NPC distribution in the nuclear membrane.
Consequently, we observe failure of nuclear pore transport and diffusion-barrier properties, with changes in pore permeability to inert test molecules (dextrans) of various sizes, as well as alterations in active protein import and export, including Ran, an endogenous protein whose localization is known to be sensitive to NPC dysfunction. We further show that tau and Nup98 directly interact as assessed by co-immunoprecipitation from human AD brain tissue and surface plasmon resonance (SPR) of recombinant proteins. In addition, in vitro experiments show that Nup98 triggers tau aggregation and accelerates tau fibrilization and thereby possibly contributes to tau aggregation and tangle formation or stabilization in neuronal somata in AD and tauopathy brains.
In summary, we provide in vivo and in vitro evidence for a pathogenic model in which accumulation of tau in the somatodendritic compartment, as occurs in AD and tauopathies, increases the tau concentration in the perinuclear space and enables abnormal interaction of tau with Nups, which in turn leads to impairment in nuclear transport. These tau:Nup interactions may induce a pathological disruption of NPC function and contribute to tau-induced neurotoxicity. Targeting this pathway could provide a new therapeutic strategy for AD and similar neurodegenerative diseases.
A View of Advanced Glycation End-Products that is Primarily Inflammatory
In the materials noted here, a Buck Institute researcher puts forward a view of just one side of the science of advanced glycation end-products (AGEs) and their role in degenerative aging. AGEs are sugary metabolic byproducts of many different varieties, both present in the diet and generated in the body. In the view of AGEs and aging expounded here, near all of the many types of AGE are important, most are transient and levels will vary in response to day to day circumstances, dietary intake of AGEs probably has a significant negative influence on long-term health, and AGEs present in tissues disrupt metabolism by hammering on a set of receptors that trigger chronic inflammatory signaling and a range of other inappropriate cellular behavior.
This leads to proposals for interventions that run along the lines of eating a better diet, finding ways to block the interaction between AGEs and receptors such as RAGE and RANKL, and so forth. If successful, these approaches could be expected to slightly slow the pace of aging, largely via reduced levels of chronic inflammation. It isn't an unreasonable viewpoint: the evidence for AGEs to cause inflammation is fairly robust; the involvement of RAGE is well demonstrated; inflammation does indeed accelerate the progression of all of the common age-related diseases. The question of whether or not dietary intake of AGEs is important in comparison to the creation of AGEs in the body can be debated. It is hard to separate this one potentially negative contribution to health from the many others associated with the sort of sugary, fatty diet that is high in AGEs.
All of this, however, is just the one side of considerations of AGEs and aging. In the materials here there is no mention of the other side, which is that in humans, the overwhelming majority of persistent cross-links formed by AGEs involve glucosepane rather than any of the other varieties of AGE. So when it comes to damage to the material properties of the extracellular matrix, leading to structural change in skin and blood vessels due to loss of elasticity, or structural change in bone and cartilage due to loss of tensile strength, only one type of AGE really counts. In this view of AGEs and aging, the vast majority of short-lived AGEs ebb and flow, while age-related degeneration is driven by the glucosepane AGEs that persist to shackle molecules of the extracellular matrix to one another, weakening and stiffening tissues.
A key challenge in this area of research is that the important classes of persistent AGEs and cross-links are completely different between mammalian species, and hence (a) past attempts to remove cross-links failed to translate from mice to humans, while (b) the ability to work with glucosepane at all was only developed comparatively recently, as this compound isn't a focus for groups working primarily in mice, and (c) ongoing work on AGEs in short-lived species is of little relevance to cross-links and aging in humans. That said, give it another five to ten years or so and I'd imagine we'll have solid evidence to back a declaration regarding which of these views of AGEs is the more important in aging. Glucosepane cross-link breaker development at the Spiegel Lab and elsewhere has been nearing the leap from laboratory to startup company for a few years now. If the Buck Institute is signaling interest in the other side of the AGE field, then approaches on that side of the house may also start to emerge in the near future.
Advanced Glycation End Products As Drivers of Age-Related Disease
An inevitable by-product of metabolism, advanced glycation end products (AGEs) are toxic molecules formed when proteins, DNA, and fats become bound after exposure to sugar. They are also in some of the foods we eat. Some Buck Institute researchers think the research community has neglected the importance of AGEs because they are challenging to study. Now they are on a mission to get scientists to focus on them as a driver of many age-related diseases. AGEs affect nearly every cell type and our bodies have inherent defense mechanisms that can clear them. But the production of AGEs really ramps up when blood sugar is high, and eating a typical high-carbohydrate, highly processed Western diet can overwhelm those natural defenses. Further, some of us are likely to be genetically prone to develop more of them, no matter what we eat.
AGEs make our cells old before their time, and over time the molecules accumulate in our tissues. The AGEs cause chronic inflammation, make proteins lose their shape, and send our metabolism into a sugar burning state, making it hard to lose weight. To make matters worse, the molecular damage from AGEs is irreversible. AGEs contribute to obesity and metabolic syndrome. They've long been implicated in insulin-resistant type 2 diabetes and are linked to its complications. In addition, AGEs are now seen as potential players in neurodegeneration. Recent findings associate AGEs with familial, early-onset and sporadic forms of Parkinson's disease, and with proteins linked to Alzheimer's disease. In one study, plaques extracted (post-mortem) from brains of patients with Alzheimer's show a 3-fold increase in AGEs content compared to age-matched individuals who died from other causes. AGEs are even found in cataracts.
The chemistry behind the formation of AGES was discovered in 1912 and an AGEs-based theory of aging was proposed more than three decades ago. Interest in the then red-hot field flagged when a drug designed to clear AGEs in diabetic kidney disease failed in clinical trials in 1998. But it's nearly impossible to study the biological development of AGEs and their implications in humans because they take decades to accumulate and there are obvious ethical concerns in encouraging the development of the toxic molecules in test subjects. So how to get researchers excited about understanding and exploiting the biology of AGEs?
The Buck Impact Circle, a donor group that pools its resources to support collaborative early-stage research at the Institute, has chosen to fund many projects involving AGEs. In addition to supporting research on the complications of diabetes and the link between AGEs and Parkinson's disease, the group has also funded projects aimed at determining if a ketogenic diet can protect against the complications of diabetes. This year they put their money toward research that tests compounds that show promise in lowering AGES associated with Alzheimer's disease pathology.
The Role of Advanced Glycation End Products in Aging and Metabolic Diseases: Bridging Association and Causality
Accumulation of advanced glycation end products (AGEs) on nucleotides, lipids, and peptides/proteins are an inevitable component of the aging process in all eukaryotic organisms, including humans. To date, a substantial body of evidence shows that AGEs and their functionally compromised adducts are linked to and perhaps responsible for changes seen during aging and for the development of many age-related morbidities. However, much remains to be learned about the biology of AGE formation, causal nature of these associations, and whether new interventions might be developed that will prevent or reduce the negative impact of AGEs-related damage. To facilitate achieving these latter ends, we show how invertebrate models, notably Drosophila melanogaster and Caenorhabditis elegans, can be used to explore AGE-related pathways in depth and to identify and assess drugs that will mitigate against the detrimental effects of AGE-adduct development.
Debating the Microbial Hypothesis for Alzheimer's Disease
Why do only some older people develop the elevated levels of amyloid-β that start the amyloid cascade of Alzheimer's disease, leading to tau aggregation and consequent death and dysfunction of brain cells? If amyloid-β is the result of persistent infection by pathogens such as herpesviruses and lyme spirochetes that are, collectively, only present in 20% or so of the population, then perhaps that is the answer. This is the core of the microbial hypothesis of Alzheimer's disease, that amyloid-β is a feature of the innate immune system, and thus persistent infection of brain tissue will result in higher levels of amyloid over time.
The microbial hypothesis can be balanced against other views on the rise of amyloid-β aggregation with age, such as the contribution of immune aging, in which the immune cells responsible for clearing out these aggregates falter in that work. Or consider the evidence for drainage of cerebrospinal fluid to decline due to age-related changes in fluid passages, and thus aggregates can no longer be effectively removed from the brain via these routes. It is plausible that all of these theories, each backed by a good amount of evidence, are to some degree correct. Alzheimer's will turn out to be a condition with multiple significant causes, and addressing all or most of those causes will be required to produce reliable benefits across the patient population.
The "germ theory" of Alzheimer's has been fermenting in the literature for decades. Even early 20th century Czech physician Oskar Fischer - who, along with his German contemporary Alois Alzheimer, was integral in first describing the condition - noted a possible connection between the newly identified dementia and tuberculosis. If the germ theory gets traction, even in some Alzheimer's patients, it could trigger a seismic shift in how doctors and understand and treat the disease. For instance, would we see a day when dementia is prevented with a vaccine, or treated with antibiotics and antiviral medications? Some researchers think it's worth looking into.
The hallmark pathology of Alzheimer's is accumulation of a protein called amyloid in the brain. Many researchers have assumed these aggregates, or plaques, are simply a byproduct of some other process at the core of the disease. Other scientists posit that the protein itself contributes to the condition in some way. Researchers have shown that amyloid is lethal to viruses and bacteria in the test tube, and also in mice. Evidence suggests that the protein is part of our ancient immune system that like antibodies, ramps up its activity to help fend off unwanted pathogens.
So does that mean that the microbe is the cause of Alzheimer's, and amyloid a harmless reaction to it? It's not that simple. In many cases of Alzheimer's, microbes may be the initial seed that sets off a toxic tumble of molecular dominos. Early in the disease amyloid protein builds up to fight infection, yet too much of the protein begins to impair function of neurons in the brain. The excess amyloid then causes another protein, called tau, to form tangles, which further harm brain cells. The ultimate neurological insult in Alzheimer's is the body's reaction to this neurotoxic mess. All the excess protein revs up the immune system, causing inflammation - and it's this inflammation that does the most damage to the Alzheimer's-afflicted brain.
So what does this say about the future of treatment? Possibly a lot. Researchers envision a day when people are screened at, say, 50 years old. "If their brains are riddled with too much amyloid, we knock it down a bit with antiviral medications. It's just like how you are prescribed preventative drugs if your cholesterol is too high." Any treatment that disrupts the cascade leading to amyloid, tau, and inflammation could theoretically benefit an at-risk brain. The vast majority of Alzheimer's treatment trials have failed, including many targeting amyloid. But it could be that the patients included were too far along in their disease to reap any therapeutic benefit.
Sardinian Population Data Provides Evidence for a Reduced Burden of Infectious Disease to Slow the Pace of Aging
Aging is defined as an increase over time in the risk of death due to intrinsic causes. By this measure, aging has slowed over the past 150 years in most populations, particularly during the transition from an era of expensive calories and high rate of infection to an era of cheap calories and widespread use of effective antibiotics. A 60-year old today is far less impacted by aging and exhibits a far lower mortality risk than was the case for a 60-year old of two centuries past. To what degree is this late life outcome the result of improved nutrition versus a reduced burden of infection?
Given the importance of inflammation in aging, and the known impact of infectious disease on immune health over the long term, the consensus is that infection over a lifetime is more important than nutrition, even if the major contributing factor is persistent infection by just a few pathogens. Researchers here support that view by analyzing historical data from the Sardinian population that transitioned the era in which antibiotics were first introduced without also greatly changing their nutritional status. The results indicate that the use of antibiotics to control infectious disease produced a slowing of aging, and the data allows some insight into details of the period of a few decades over which that slowing took hold.
In biology, the term senescence is used to indicate the progressive accumulation of molecular damage that takes place in an organism as time goes by. This results in a gradual growth in the risk of death (demographic aging). By studying the pace at which mortality accelerates, it is possible to infer the general characteristics of the senescence process and to investigate which factors accelerate or decelerate its progression. So far, three major explanations for the determinants of senescence have been proposed: the constant senescence hypothesis; the inflammaging theory; and the calorie or energy restriction theory.
According to the constant senescence hypothesis, the pace of senescence is a biological constant among humans. As a result, senescence cannot be accelerated or decelerated by exogenous factors. Instead, the inflammation theory claims that the number and intensity of immune system responses to antigenic load in a lifetime is a fundamental factor in regulating the pace of senescence. Thus, individuals who have experienced a higher exposure to antigenic load will also experience a more rapid aging process. Finally, the calorie restriction theory, which is based on a plethora of experiments on a vast range of mammals and non-mammals, explores a reduced daily calorie intake and its positive effect on aging. In particular, a reduction in daily calorie intake is thought to slow senescence.
From a theoretical point of view, the three theories can be empirically tested. This would require a comparison of the aging process in cohorts who have experienced different nutritional regimes and different disease loads. However, the coincidence of the epidemiological transition and advent of antibiotics, from infection as majority cause of death to age-related disease as majority cause of death, with the onset of the nutrition transition, from a low to a high calorie regime, makes it difficult to isolate the effects of these two contrasting forces on mortality acceleration.
The epidemiological transition in Sardinia is unusual in that it started without any substantial modification in nutritional levels. This makes Sardinia a quasi-natural experiment where we can test the constant senescence hypothesis against the inflammation theory, without the confounding effect of changes in nutritional levels. To implement the analysis, the longitudinal life tables from 80 years onwards for Sardinian cohorts born in the period 1866-1908 were reconstituted and used to estimate the Gamma-Gompertz model: this model assumes that the individual hazard function follows the Gompertz model and that frailty is Gamma-distributed. The β parameter of the Gamma-Gompertz model, the so-called rate of aging, measures the relative derivative of the force of mortality, and in this sense, it may be used to measure how fast mortality progresses with age.
The results show that the Sardinian population experienced a dramatic reduction in the rate of aging that coincides with the onset of the epidemiological transition. The reduction in the rate of aging in an epoch characterized by a rapid reduction in infectious disease burden (probably due to quinine) appears to be consistent, at least at first sight, with the inflammation theory. The very low levels of nutrition observed in Sardinia, coupled with the dramatic fall off in the disease burden in the last years of the 19th century, might help to explain why the decline in the Sardinian rate of aging has been so dramatic compared with other European regions. The explanations advanced in the literature to justify the high prevalence of male centenarians in Sardinia have emphasized the role played by genetic factors. The possibility that genetic factors played a role in the evolution of the rate of aging in Sardinia cannot be entirely ruled out then. However, the analysis presented in this paper suggests that the very low Sardinian rate of aging at the beginning of the 20th century may depend on other factors such as nutrition and disease load.
TXNIP, Associated with Aging in Flies, Shown to Influence Cellular Senescence in Mice
Now that the accumulation of senescent cells is broadly accepted to be one of the fundamental causes of aging, ever greater funding is flowing into this part of the scientific ecosystem. Many research groups are investigating aspects of the biochemistry of cellular senescence: how cells become senescent; the harmful signaling they produce; ways to prompt them to self-destruct, thereby removing their contribution to the aging process. One of the results of this expansion of effort is that some proteins previously known to be associated with aging are now being found to either influence or act through cellular senescence. The research here is an interesting example of the type, in which TXNIP, a protein associated with oxidative stress and aging in flies, is now implicated in cellular senescence in mice.
Cells are constantly exposed to metabolic stress, a major cause of cellular senescence. Recent reports have shown that metabolic changes influence aging in model systems, from the budding yeast to mouse models. One of the prominent cellular senescence markers is the accumulation of reactive molecules, such as reactive oxygen species (ROS), a product of an essential energy production. Glucose serves as an energy source in virtually all eukaryotic cells. A high concentration of glucose increases the metabolic input into cells and consequently induces oxidative stress via ROS production, thereby inducing DNA, protein, and lipid damage, causing premature senescence.
Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that is induced by a rise in glucose and oxidative stress and is known to be a tumor suppressor and inhibit thioredoxin (TRX), an antioxidant protein, via a direct interaction. Many studies have examined the role of TXNIP in glucose uptake and metabolism. TXNIP expression is related to mitochondrial fuel switching under conditions of starvation, diabetes, and exercise in skeletal muscle. Previously, we suggested that TXNIP is highly expressed and acts as an antioxidant protein to regulate cellular ROS by activating p53 activity or by inhibiting p38 mitogen-activated protein kinase (MAPK) activity.
AKT is a serine-threonine kinase that is involved in a variety of cellular processes including cell survival, proliferation, and metabolism. AKT plays an essential role in the insulin-regulated transport of glucose and in whole-body glucose homeostasis. Activation of the AKT pathway is directly correlated with increased rates of glucose metabolism. The activation of AKT induces intracellular ROS by inducing oxygen consumption or inhibiting the forkhead box O (FOXO) family of transcription factors, in turn, promoting cellular senescence and apoptosis. AKT also activates the mechanistic target of rapamycin (mTOR) and induces cellular senescence.
In this study, we found that TXNIP deficiency induces accelerated senescent phenotypes of mouse embryonic fibroblast (MEF) cells under high glucose condition and that the induction of cellular ROS or AKT activation is critical for cellular senescence. Our results also revealed that TXNIP inhibits AKT activity by a direct interaction, which is upregulated by high glucose and H2O2 treatment. In addition, TXNIP knockout mice exhibited an increase in glucose uptake and aging-associated phenotypes including a decrease in energy metabolism and induction of cellular senescence and aging-associated gene expression. We propose that TXNIP is a critical regulator of AKT-mediated cellular senescence under glucose-mediated stress in vitro and in vivo.
Might Means of Reversing Atherosclerosis also Prevent Macular Degeneration?
Atherosclerosis is characterized by growing lipid deposits that weaken and narrow blood vessels. Age-related macular degeneration is also characterized by a deposition of lipids in and around the retina in its early stage. One might therefore speculate as to whether age-related problems with the mechanisms responsible for clearing lipids might be at the root of both conditions. Macrophages are the cell responsible for gathering up unwanted lipids, which they then hand off to HDL particles for the journey back to the liver and consequent excretion, a process known as reverse cholesterol transport. This system works well in enough in youth, but falters with age. Macrophages become dysfunctional, with one theory being that this is due to increasing levels of oxidized lipids that cannot easily be broken down, and thus clog up the vital functions of macrophage cells.
A sizable amount of research into reverse cholesterol transport has taken place in the context of atherosclerosis, and this has given rise to a varied set of attempts to increase the flow of cholesterol through macrophages. So far this has resulted in failed clinical trials and limited benefits to patients, but efforts continue on the next generation of potential therapies. Less work has taken place in the context of macular degeneration. The authors of the open access paper here disable reverse cholesterol efflux in mice and observe the resulting deposition of lipids in the retina, making the argument that the results indicate that the situation is much the same as in atherosclerosis. Thus methods of treating atherosclerosis that are based on improved rates of reverse cholesterol transport may also turn out to prevent macular degeneration.
Advanced age-related macular degeneration (AMD), the leading cause of blindness among people over 50 years of age, is characterized by atrophic neurodegeneration or pathologic angiogenesis. Early AMD is characterized by extracellular cholesterol-rich deposits underneath the retinal pigment epithelium (RPE) called drusen or in the subretinal space called subretinal drusenoid deposits (SDD) that drive disease progression. However, mechanisms of drusen and SDD biogenesis remain poorly understood. Although human AMD is characterized by abnormalities in cholesterol homeostasis and shares phenotypic features with atherosclerosis, it is unclear whether systemic immunity or local tissue metabolism regulates this homeostasis.
Here, we demonstrate that targeted deletion of macrophage cholesterol transporters ABCA1 and ABCG1 leads to age-associated extracellular cholesterol-rich deposits underneath the neurosensory retina similar to SDD seen in early human AMD. These mice also develop impaired dark adaptation, a cardinal feature of RPE cell dysfunction seen in human AMD patients even before central vision is affected. Subretinal deposits in these mice progressively worsen with age, with concomitant accumulation of cholesterol metabolites including several oxysterols and cholesterol esters causing lipotoxicity that manifests as photoreceptor dysfunction and neurodegeneration.
These findings suggest that impaired macrophage cholesterol transport initiates several key elements of early human AMD, demonstrating the importance of systemic immunity and aging in promoting disease manifestation. Polymorphisms in genes involved with cholesterol transport and homeostasis are associated with a significantly higher risk of developing AMD, thus making these studies translationally relevant by identifying potential targets for therapy.
Evidence for a Ketone Body Produced During Calorie Restriction to Reduce the Creation of Senescent Cells
Researchers here report on the identification of a fairly direct link between the biochemistry of calorie restriction and a reduced accumulation of senescent cells, one of the root causes of aging. All aspects of aging are slowed somewhat by the practice of calorie restriction, though far less so in humans than is the case in short-lived mammals such as mice. Since calorie restriction changes near everything in the operation of cellular metabolism, finding the few important links between those changes and the mechanisms of aging has proven to be a slow and expensive task. Still, as this example demonstrates, evidence emerges eventually.
"As people become older, they are more susceptible to disease, like cancer, cardiovascular disease and Alzheimer's disease. Age is the most important so-called risk factor for human disease. How to actually delay aging is a major pathway to reducing the incident and severity of human disease. The most important part of aging is vascular aging. When people become older, the vessels that supply different organs are the most sensitive and more subject to aging damage, so studying vascular aging is very important. This study is focused on vascular aging, and in old age, what kind of changes happen and how to prevent vascular aging."
Researchers identified an important small molecule that is produced during fasting or calorie restriction conditions. The molecule, β-Hydroxybutyrate, is one type of a ketone body, or a water-soluble molecule that contains a ketone group and is produced by the liver from fatty acids during periods of low food intake, carbohydrate restrictive diets, starvation, and prolonged intense exercise. "We found this compound, β-Hydroxybutyrate, can delay vascular aging. That's actually providing a chemical link between calorie restriction and fasting and the anti-aging effect. This compound can delay vascular aging through endothelial cells, which line the interior surface of blood vessels and lymphatic vessels. It can prevent one type of cell aging called senescence, or cellular aging."
Senescent cells can no longer multiple and divide. The researchers found β-Hydroxybutyrate can promote cell division and prevent cells from becoming senescent. Because this molecule is produced during calorie restriction or fasting, when people overeat or become obese this molecule is possibly suppressed, which would accelerate aging. In addition, the researchers found when β-Hydroxybutyrate binds to a certain RNA-binding protein, this increases activity of a stem cell factor called Octamer-binding transcriptional factor (Oct4) in vascular smooth muscle and endothelial cells in mice. Oct4 increases a key factor against DNA damage-induced senescence. "We think this is a very important discovery, and we are working on finding a new chemical that can mimic the effect of this ketone body's function."
Scheduled Feeding Shown to Slow Aging in Mice
The metabolism of mice and rats is very sensitive to the stress of hunger; cells dial up their recycling and maintenance activities in response, and over time this adds up to a significant benefit to health and longevity. Calorie restriction has a sizable effect on longevity in short-lived rodents, but then so does intermittent fasting, even if the overall calorie intake is kept to the same level. Researchers here explore the lower end of this effect, using scheduled feeding to create comparatively short daily fasts between meals. This still produces health benefits.
Increasing time between meals made male mice healthier overall and live longer compared to mice who ate more frequently, according to a new study. Health and longevity improved with increased fasting time, regardless of what the mice ate or how many calories they consumed. "This study showed that mice who ate one meal per day, and thus had the longest fasting period, seemed to have a longer lifespan and better outcomes for common age-related liver disease and metabolic disorders. These intriguing results in an animal model show that the interplay of total caloric intake and the length of feeding and fasting periods deserves a closer look."
The scientists randomly divided 292 male mice into two diet groups. One group received a naturally sourced diet that was lower in purified sugars and fat, and higher in protein and fiber than the other diet. The mice in each diet group were then divided into three sub-groups based on how often they had access to food. The first group of mice had access to food around the clock. A second group of mice was fed 30 percent less calories per day than the first group. The third group was meal fed, getting a single meal that added up to the exact number of calories as the round-the-clock group. Both the meal-fed and calorie-restricted mice learned to eat quickly when food was available, resulting in longer daily fasting periods for both groups.
The scientists tracked the mice's metabolic health through their lifespans until their natural deaths and examined them post-mortem. Meal-fed and calorie-restricted mice showed improvements in overall health, as evidenced by delays in common age-related damage to the liver and other organs, and extended longevity. The calorie-restricted mice also showed significant improvement in fasting glucose and insulin levels compared to the other groups. Interestingly, the researchers found that diet composition had no significant impact on lifespan in the meal fed and calorie restricted groups.
Removing Inflammatory Regulatory T Cells Reverses Aspects of Heart Failure in Mice
The progression of heart failure following a heart attack is driven by sustained levels of chronic inflammation. Researchers have now demonstrated the importance of this inflammation through the targeted removal of a critical population of T cells in mice, cells that become inappropriately inflammatory after injury to heart tissue. This selective destruction of immune cells produces a reversal of detrimental remodeling of heart muscle, as well as improvement in other inflammation-linked aspects of heart failure, such as fibrosis in heart tissue. Interestingly, this approach seems to result in lasting effects, as the replacement T cells, newly generated by the body, do not provoke further inflammation. All in all, this is a very promising set of data.
A heart attack triggers an acute inflammatory response, followed by resolution of inflammation and wound healing. A severe heart attack, however, can cause chronic and sustained inflammation that leads to heart failure and death. Researchers have found that a group of immune cells called regulatory T-lymphocyte cells, or T-regs, appear to go rogue in heart failure. Instead of their normal job to resolve inflammation, the dysfunctional T-reg cells become pro-inflammatory and prevent the growth of new capillaries. Experimental removal of those dysfunctional T-reg cells from heart-failure mice acted as a reset button to reverse heart failure, and the replacement T-regs that the mice produced resolved inflammation.
In a previous study, researchers had seen that CD4+ T-cells - which include T-regs - were globally expanded and activated in mouse heart failure, and there was persistent inflammation and activation of effector T cells, despite the increased numbers of T-reg cells that normally should help resolve inflammation. This led to the hypothesis for the present work - that the T-reg cells in heart failure themselves become dysfunctional, pro-inflammatory and tissue-injurious, and that that altered phenotype contributes to sustained inflammation and the pathologic enlargement of the heart's main pumping chamber. Such enlargement is known as left-ventricular remodeling.
The current study shows that dysfunctional T-reg cells are essential for adverse left-ventricular remodeling. Researchers selectively ablated the dysfunctional T-reg cells four weeks after heart failure. Ablation was accomplished by giving diphtheria toxin to genetically engineered mice that have the diphtheria toxin receptor inserted into T cells at the Foxp3 gene site, or by giving the mice anti-CD25 antibodies. T-reg ablation reversed left-ventricular remodeling over the next four weeks. Also, ablation with antibody halted further increase in left-ventricular remodeling, while remodeling in the heart failure mice given a non-specific antibody continued to worsen. Ablation alleviated fibrosis and systemic inflammation in the heart, and it enhanced growth of new capillaries.
Importantly, the new T-reg cells produced by the mice after an ablation pulse were no longer pro-inflammatory - instead, they showed restoration of normal T-reg immunosuppressive capacity. Thus, ablation of the pathogenic and dysfunctional T-reg cells acted, in effect, as a reset that restored the mouse T-reg cells back to their normal immunomodulatory function.
Identifying Genes Responsible for Human Longevity Relative to Other Primates
We humans are unusually long-lived for our size, as compared to other mammals. This is particularly noticeable in comparison to our nearest primate relatives. Since our exceptional longevity among primates arose only comparatively recently in evolutionary time, coincident with intelligence, culture, and modernity, it is thought feasible to identify genetic changes likely involved in this process. That effort proceeds in tandem with more theoretical considerations regarding how it is that natural selection produced this gain in species life span, such as the Grandmother hypothesis, and the two lines of work can inform one another as they progress.
Aging is a complex process affecting different species and individuals in different ways. Comparing genetic variation across species with their aging phenotypes will help understanding the molecular basis of aging and longevity. Although most studies on aging have so far focused on short-lived model organisms, recent comparisons of genomic, transcriptomic, and metabolomic data across lineages with different lifespans are unveiling molecular signatures associated with longevity. Here, we examine the relationship between genomic variation and maximum lifespan across primate species.
We used two different approaches. First, we searched for parallel amino-acid mutations that co-occur with increases in longevity across the primate linage. Twenty-five such amino-acid variants were identified, several of which have been previously reported by studies with different experimental setups and in different model organisms. The genes harboring these mutations are mainly enriched in functional categories such as wound healing, blood coagulation, and cardiovascular disorders. We demonstrate that these pathways are highly enriched for pleiotropic effects, as predicted by the antagonistic pleiotropy theory of aging.
A second approach was focused on changes in rates of protein evolution across the primate phylogeny. We show that some genes exhibit strong correlations between their evolutionary rates and longevity-associated traits. These include genes in the Sphingosine 1-phosphate pathway, PI3K signaling, and the Thrombin/protease-activated receptor pathway, among other cardiovascular processes.
To our knowledge, this is the first systematic report providing direct evidence of gene-phenotype evolution of aging-related traits in primates. Genes and biological processes reported in this study could be added to the list of genes that increase lifespan when overexpressed or mutated (gerontogenes) and represent a valuable resource for examination of new candidate interventions that mimic gene evolution associated with natural changes in lifespan. Although our results may reflect local adaptive responses of species to their environment, we observed nonrandom association of gene evolution with pathways mainly related to wound healing, coagulation, and many cardiovascular processes. This would make sense from a biological perspective, since flexible and adjustable control of coagulation mechanisms is required for species that live longer.
Exosomes from Young Mice are Shown to Reverse Changes in Expression of Aging-Associated Genes in Old Mice
Work on heterochronic parabiosis, in which an old and young mouse have their circulatory systems joined, has led to a wide variety of investigations into which signal molecules present in the bloodstream might be important in aging. The signaling environment changes in response to rising levels of molecular damage with age, leading to alterations in cellular behavior, some of which help to compensate, and some of which cause further harm.
At the same time, there is a rising level of interest in the roles played by various forms of extracellular vesicle in intracellular communication. These membrane-wrapped packages contain a diverse set of signal molecules, and are passed promiscuously back and forth between cells. That vesicles are conveniently packaged and distinguishable by size makes it comparatively easy to harvest them from cell cultures or blood samples, and from there they can be analyzed, or perhaps used as the basis for a therapy to change the behavior of cells in old tissues.
Changing the signaling environment may produce benefits large enough to be worth chasing, as the stem cell research community has demonstrated over the past twenty years. Most first generation cell therapies work because of the signals generated by transplanted cells, not because the cells manage to survive and integrate into tissue. Unfortunately, this approach doesn't target the underlying damage that causes aging, and thus will always be limited as to how great the benefits can be at the end of the day. If the molecular damage of aging remains unrepaired, it will continue to cause pathology.
Understanding the regulatory mechanisms and the involved molecules underlying aging has aroused interest to prevent or delay aging or aging-associated diseases. It has been reported that the upregulated or downregulated miRNAs induce cellular senescence. In cell-to-cell signaling in systemic aging, miRNAs are reported to be released in circulation and transferred to remote tissues. The released miRNAs can affect their levels in circulation in aged individuals, and in a recent study, they served as regulatory molecules to control aging speed. Therefore, they are strongly considered as aging-associated biomarkers, possibly determined by minimally invasive or noninvasive methods. So far, several studies comparing miRNA expression profiles from the blood of young and old animals have revealed differences in the expression levels of several miRNAs with aging.
One of the ways by which miRNAs are released in circulation is via vesicles blebbed out from cellular membranes. A representative type of these vesicles is exosomes, which are tens to hundreds of nanometers in diameter. The exosomes released from parent cells enter systemic circulation, which thus explains the signaling process among remote tissues. Cells under stress would release more exosomes in vitro to dispose unnecessary molecules or communicate their signals to the surrounding cells. Actually, aging is a type of cellular stress; thus, exosomes are secreted at higher levels from senescent cells than from normal cells. However, limited information is available on changes in the miRNA contents of exosomes in naturally aged individuals and their effects in the aging process. Therefore, the identification of miRNA molecules deregulated in exosomes in the aging process would be required to understand the mechanisms underlying aging and may have potential applications in evaluating or reversing the aging status of an individual.
In this study, we primarily identified differentially expressed miRNAs in exosomes from aged mice and compared them with those from young mice. If the miRNAs in exosomes have regulatory capability in systemic aging, their increased levels in young exosomes were expected to exert a reversing effect on tissues of old mice. Therefore, after intravenously injecting exosomes from young mice to aged mice, changes in aging-associated molecule levels were analyzed in aged mice. In the aged tissues injected with young exosomes, mmu-miR-126b-5p levels were reversed in the lungs and liver. Expression changes in aging-associated molecules in young exosome-injected mice were obvious: p16Ink4A, mTOR, and IGF1R were significantly downregulated in the lungs and/or liver of old mice. In addition, telomerase-related genes such as Men1, Mre11a, Tep1, Terf2, Tert, and Tnks were significantly upregulated in the liver of old mice after injection of young exosomes. These results indicate that exosomes from young mice could reverse the expression pattern of aging-associated molecules in aged mice. Eventually, exosomes may be used as a novel approach for the treatment and diagnosis of aging animals.
An Update from the CellAge Team
The Life Extension Advocacy Foundation staff note a recent update from the CellAge team. That company was partially funded by a crowdfunding event, held at Lifespan.io, that completed in early 2017. The founders are now moving forward with their work on synthetic promoters as a way to identify senescent cells and quantify the burden of senescence in specific tissues. The senolytics development community has spent the past few years forging ahead with ways to destroy senescent cells, but improvements in the state of assays for senescence has lagged behind.
Staining a tissue sample for simple markers of senescence, such as expression of p16, is the present standard procedure. It is good enough for development, but really not acceptable for either commercial use or more sophisticated research in the years ahead. If someone wants to assess on a month to month or year to year basis just how many senescent cells are in specific tissues, a much better approach will be needed. That demand will arise rapidly enough once human data starts to arrive from trials of early senolytic therapies. The microfluidics approach to counting senescent cells by size that was published last year is a step in the right direction, and hopefully the CellAge work will in the fullness of time lead to still better options.
We have been quiet for a while so we thought it was time for a small update about the Cellage project. We are working with Circularis to screen for new senescent cell promoters using a unique technological platform never used before with human or senescent cells. A promoter is a region of DNA that initiates the expression of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA. In this case, we are searching for gene expression relating to cellular senescence and using p16 and CMV promoters as our positive controls.
If this is successful we will then move onto screening for synthetic promoters from a library of over 100,000 novel synthetic promoters. The objective being to identify suitable promoters so we can develop a highly accurate way to detect the presence of senescent cells that surpasses the current state of the art methods such as p16.