Here I'll point out one concrete example of the way in which the SENS Research Foundation puts our charitable donations to good use in rejuvenation research. You'll find many more in the yearly organizational reports. The in-house MitoSENS research team, focused on allotopic expression of mitochondrial genes to eliminate the contribution of mitochondrial damage to degenerative aging, has achieved considerable progress in the past two years. Allotopic expression is the process of placing copies of mitochondrial genes into the cell nucleus, altered in such as way as to allow the proteins produced from that genetic blueprint to find their way back to the mitochondria where they are needed. When mitochondrial genes become damaged, as happens over the course of aging, the backup source of proteins prevents this damage from starting a chain of events that causes lasting harm to tissues and organs.

Last year's MitoSENS crowdfunding initiative provided the funds needed for the SENS Research Foundation team to finish up the demonstration of allotopic expression of ATP6 and ATP8, the second and third mitochondrial genes for which this has been achieved. The SENS Research Foundation also used philanthropic donations to help fund the allotopic expression of the first such gene, ND4, some years ago. That research is now being carried forward to the clinic by Gensight Biologics, with sizable venture backing. To complete this defense against mitochondrial damage and aging, the same work must be completed for thirteen mitochondrial genes in total, and building upon recent success the SENS Research Foundation is expanding the MitoSENS team. If you happen to know a qualified researcher or biotechnologist, point them in this direction:

MitoSENS is Hiring

SENS Research Foundation (SRF) is hiring a Research Assistant for our research center located in Mountain View, CA. SRF is an exciting, cutting edge non-profit dedicated to transforming the way the world researches and treats age-related disease. We are seeking a research assistant in our MitoSENS group for a research project geared toward discovering a gene therapy approach to treating mitochondrial mutations; for more information see the project page. Qualified candidates will be local residents who have a BS or MS in the chemical/biological sciences and at least 2 years of work experience in either academia or industry. Duties will include mostly bench work in a small team-oriented environment. Candidates with experience in molecular cloning, tissue culture, protein analysis / biochemical assays are encouraged to apply. Experience working with mitochondria is a plus.

Engineering New Mitochondrial Genes to Restore Mitochondrial Function

Mitochondria provide energy for the cell by synthesizing energy in the form of high energy bonds. This energy synthesis occurs through a process called oxidative phosphorylation in which respiratory enzymes in mitochondria convert a molecule called adenosine diphosphate (ADP) into the energy currency of the cell, ATP. One interesting feature of mitochondria is that they contain their own DNA (mtDNA). As cells and mitochondria have co-evolved, most of this genetic information has been transferred to the nucleus, leaving only thirteen protein-encoding genes in the mtDNA. Housing these thirteen genes within the mitochondria themselves is precarious because the conditions required to synthesize ATP create reactive oxygen species. Over time, these toxic free-radical byproducts damage the mitochondrial genes in more and more cells, compromising respiratory chain function and hence energy production. The accumulation of mutations in mitochondrial DNA is implicated in the metabolic derangement of aging and in accelerating the course of the degenerative aging process as a whole. One need only examine clinical manifestations of mitochondrial genetic diseases to see the similarities they share with the maladies of aging. For example, mutations in the gene ND1 have been implicated in the development of Parkinson's disease, and Cytochrome B (CYB) mutations can cause muscle fatigue and exercise intolerance in young patients.

SENS Research Foundation's strategic approach to this problem is to engineer a way to let mitochondria keep producing energy normally, even after mitochondrial mutations have occurred. Although damage to mitochondrial DNA is inevitable so long as it is housed in the mitochondria, the harmful effects of mitochondrial mutations can be bypassed by engineering backup copies of the thirteen protein-encoding genes and housing the copies instead in the nucleus of the cell. These allotopic gene copies could continue to provide the necessary proteins even when mutations have compromised the mtDNA's ability to do so. Moreover, the nuclear gene copies would be better shielded from damaging toxins and better maintained by DNA repair machinery. Since the majority of mitochondrial proteins are naturally nuclear-encoded, the natural mechanism to deliver the allotopically-expressed genes to the mitochondria can be co-opted.

The SENS Research Foundation mitochondrial mutations team is moving forward on a method for targeting engineered nuclear-encoded genes (that could function as "backup copies" for cells with deletion mutations) to the mitochondria, and for furthermore optimizing the precision of this targeting. The "working copy" of the relocated mitochondrial gene in this method is equipped with two special sequences. One "untranslated" sequence is not turned into a protein itself, but helps protect the engineered protein during the import process. The other, called the mitochondrial targeting sequence, is a tag appended to the final protein following expression that allows it to be imported once expressed. Combining the two sequences allows the "backup copies" of genes to be turned into working copies in the cell nucleus; to have the "working copies" targeted to the surface of the mitochondria to be decoded and turned into protein. Even as it is still in the process of being decoded, the emerging protein is quickly directed to the surface of the mitochondria for import and incorporation into the electron transport chain (ETC), restoring mitochondrial function.

In 2013, the SENS Research Foundation mitochondrial mutations group created two new cell lines which are 100% null for two mitochondrially-encoded genes: ATP8 and CYB. Using these two new cell lines, this year the team was finally able to unleash their engineered ATP8 gene in cells whose mitochondria completely lack the ability to generate the corresponding proteins on their own, and announced the dramatic rescue of such "ATP8 null" cells using their protein targeting strategy. They anticipate that these results will deliver the proof-of-concept for the overall approach, which should then be applicable as a rescue platform for all thirteen mitochondrially-encoded proteins. Further work by the team aims to enable delivery of working instructions for building proteins that can keep the ETC intact and functioning in the event of age-related mutations of the original mitochondrial genes for these proteins. This method utilizes a "borrowed" structure already employed by mitochondria to take in RNA from the main body of the cell. The team has now achieved the critical first benchmark - i.e. delivering any RNA into the mitochondria - in this pioneering work using a convenient (but not naturally mitochondrially-expressed) RNA.

This review paper makes a good companion piece to another review on primates in aging research published last year. Perhaps the most well known primate studies of aging are the still ongoing and decades-long studies of calorie restriction in rhesus macaques, unlikely to be repeated given the cost and the debate over the quality of the resulting data and the underlying design of the studies. There are many other studies involving the use of various non-human primate species to study aging and age-related disease, however, some of which are just as interesting.

The choices made in the use of animals in aging research are a matter of economics: longer life spans and species closer to ours lead to studies that are slower and more costly, but the data is more likely to be useful and relevant. In practice the costs are too high, and thus most exploratory research into the biochemistry of aging starts out in very short-lived and evolutionarily distant species such as worms and flies. There is a high rate of failure for the results to translate into mammals, but even then the cost of progress is much lower that would be the case if carrying out that initial exploration in mice or other longer-lived mammals. All research involving primate studies has already passed through stages of exploration and validation in worms, flies, mice, and frequently other mammals as well; only the more established lines of research can justify the time and funding needed for further studies in primates.

Nonhuman primates share similar physiology and a close phylogenetic relationship to humans. The use of nonhuman primates in comparative experimental studies thus contributes to our knowledge about aging processes and translation of applications for improving health span in humans and other animals. With the growing development of antiaging strategies, it is expected that nonhuman primates will additionally be highly relevant for preclinical studies testing antiaging strategies. Correlates of average natural life span of an organism are highly complex, but body size in conjunction with metabolism, reproduction, immunity, and environmental stress, among other factors, is associated with average longevity such that larger animal species tend to live longer. Interestingly, human and nonhuman primates exhibit unusually longer average life spans that are nearly 4-fold higher than those of most other mammals relative to their body sizes. In addition, nonhuman primates exhibit similar key life span metrics as humans, such as higher infant mortality rate, followed by lower mortality during the juvenile stage and then an extended period of increasing age-related morbidity and mortality.

By far, the predominant nonhuman primate species utilized in biomedical research facilities as well as for studies on aging are rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fasicularis). Specifically, among the facilities with nonhuman primates in North America that were recently surveyed, 80% housed rhesus macaques of Indian or Chinese origin, followed closely by cynomolgus macaques housed in 73% of the facilities. Aspects of aging research studies that utilize macaques include neurobiology, anatomy, physiology, cognition, and behavior, as well as reproductive senescence, caloric restriction (CR), and immune senescence. The use of macaques in research appears to represent the best compromise between phylogenetic and physiologic relatedness to humans, cost efficiency, life span, resources, expertise in animal husbandry practices, and adaptability for translation of results to humans. To improve efficiency, accessibility, and applicability, however, increasing emphasis is being placed on purpose-bred animals and further advancing animal husbandry practices so that lower primates also may be included for relevant model development of research on aging.

Prosimians, or "premonkeys," are the most phylogenetically distant nonhuman primates from humans. Among the prosimians, grey mouse lemurs (Microcebus murinus) have been the most extensively studied for relating processes of aging in relation to humans. For example, the mouse lemur was the first nonhuman primate species to demonstrate a relationship between cerebral atrophy and cognitive decline with aging that simulated what was seen in aging humans. Neuroscience studies about memory, behavior, and psychomotor function have utilized both captive and wild mouse lemurs. The use of prosimians in research is more cost-efficient, but limitations include their smaller size that restricts specimen sampling; differences in metabolic, biochemical, and endocrine responses compared with humans; and a need for continued development in animal husbandry techniques to reduce stress-related behaviors of captive prosimians.

Link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027759/

Accumulation of senescent cells is one of the root causes of aging. Now that the scientific community has the means to selectively remove these unwanted cells, such as via the use of senolytic drugs, and now that funding has picked up for this field, researchers are rapidly quantifying specific links to the pathology of age-related disease. For example, earlier this year researchers demonstrated that clearance of senescent cells produces significant benefits to vascular health, slowing or reversing many of the aspects of aging in blood vessels, such as calcification and growth of atherosclerotic plaque. This more recently published open access paper on the same topic adds more details to the picture:

Risk factors for ischemic heart disease include hypercholesterolemia, arterial stiffness, chronic inflammation, hypertension, metabolic syndrome, and aging. Importantly, these risk factors contribute to impaired endothelial function, which can contribute to arterial remodeling and accelerate atherosclerotic plaque formation and expansion. Recent work suggests senescent cell burden can be dramatically increased by chronological aging, and short-term treatment with 'senolytic' drugs alleviates several aging-related phenotypes. However, effects of long-term senescent cell clearance on vascular reactivity and structure with aging or chronic hypercholesterolemia remain unknown. To determine whether senolytic treatment with dasatinib and quercetin (D+Q) reduces senescent cell burden and improves vascular function in aged mice, we maintained C57BL/6J mice on standard chow for 24 months, and then initiated D+Q once monthly for 3 months.

Senolytic treatment resulted in significant reductions in senescent cell markers in the medial layer of aorta from aged and hypercholesterolemic mice, but not in intimal atherosclerotic plaques. While senolytic treatment significantly improved vasomotor function in both groups of mice, this was due to increases in nitric oxide bioavailability in aged mice and increases in sensitivity to NO donors in hypercholesterolemic mice. Senolytics tended to reduce aortic calcification and osteogenic signaling in aged mice, but both were significantly reduced by senolytic treatment in hypercholesterolemic mice. Intimal plaque fibrosis was not changed appreciably by chronic senolytic treatment. This is the first study to demonstrate that chronic clearance of senescent cells improves established vascular phenotypes associated with aging and chronic hypercholesterolemia, and may be a viable therapeutic intervention to reduce morbidity and mortality from cardiovascular diseases.

Link: http://onlinelibrary.wiley.com/doi/10.1111/acel.12458/full

One of the differences between old tissue and young tissue is an accumulation of misfolded proteins, normally soluable, into solid aggregates. The best known of these are the varieties of amyloid that are clearly associated with specific diseases and are present in significant amounts in patient tissues. These are far from the only proteins that accumulate in such a way, however, and there are many more types of misfolded or damaged proteins that do not form aggregates. Unfortunately the mapping of aggregates by tissue to specific consequences in the course of degenerative aging is far from complete. In the paper I'll point out today, the authors take an interesting path in their attempts to prove relevance of various aggregates to age-related loss of muscle mass and strength, the condition known as sarcopenia. I think that the approach is indirect enough to taken as a first filter that leads next to further study to evaluate how well it did, rather than being, on its own, any sort of confirming evidence for the participation of specific aggregates in the progression of sarcopenia. Even that is well before we get into questions of causation versus correlation. The challenge inherent in all investigations of aging is that it is a global phenomenon in the body; there are many correlations to be found between processes that in fact have little to do with one another, and spring from entirely separate sources. Still, the road to knowledge must start somewhere.

Loss of muscle mass and strength is one of the most visible signs of aging, and a large component of the frailty of old age. Once you start to lose strength you start to lose the ability to exercise and the benefits that brings, and things go downhill from there. There are a range of potential approaches to delay this process, of which calorie restriction and exercise are the most accessible and proven, and some near future therapies that could compensate for the loss, adding muscle without addressing the molecular damage of aging that produces sarcopenia as a downstream consequence. In the near future category are myostatin or follistatin gene therapies, and forms of temporary myostatin blockade such as the antibodies currently in clinical trials. Compensation is compensation and better than nothing, but what we really want to see is reversal via therapies that address the root causes of sarcopenia. At the present time there is little evidence as yet to definitively tie sarcopenia to specific root causes of aging, the categories of cell and tissue damage outlined in the SENS rejuvenation research programs. That is in comparison to the many studies linking sarcopenia to age-related changes that are most likely consequences of that damage, such as altered processing of leucine, changes in mitochondrial dynamics, and infiltration of fat into muscle tissue. Given that it is interesting to see people working towards links with protein aggregates, which are very definitely on the SENS list as a target for rejuvenation therapies, even if there is clearly a lot of work left to do to prove this connection via the methodology chosen here.

Proteins that accumulate with age in human skeletal-muscle aggregates contribute to declines in muscle mass and function in Caenorhabditis elegans

Age-associated muscle loss, or sarcopenia, results in functional decline that increases the risk for falls, disability, and mortality in older adults. This problem is clearly influenced by factors such as diet, physical activity, genetics, and comorbid health conditions. However, much less is known about the underlying etiology. Aging has detrimental effects on myofibers, satellite cells, and muscle protein synthesis. These effects may be due to dampened levels of growth factors needed for muscle growth and regeneration, or heightened levels of inflammation mediators, which can induce catabolism. Several age-associated diseases, particularly those involving neurodegeneration, feature the accumulation of protein aggregates in affected tissues. Interestingly, similar pathology is also seen for inclusion body myositis, an age-associated degenerative skeletal muscle disease, whose protein aggregates contain the amyloid β peptide characteristic of Alzheimer's disease. In diseased neurons and muscle fibers, aggregation is exacerbated by disruption of proteostasis systems responsible for repair or clearance of misfolded and damaged proteins. Muscle health is expected to be highly reliant on these processes since it reflects a lifetime of continuous mechanical and metabolic stress. However, a causal connection between protein aggregation and muscle aging or sarcopenia has yet to be established.

In the current investigation, we examined protein aggregation that accompanies muscle aging and assessed whether it might contribute to age-associated loss of muscle mass and function. This possibility was suggested by our recent studies which identified and quantified proteins in cardiac muscle aggregates that accrue with aging and hypertension in mice. Our work and that of others has also shown that protein aggregation accumulates with normal aging in the nematode Caenorhabditis elegans and in nematode models of protein-aggregation pathologies. The current study extends our investigation of protein aggregation to human muscle, with three objectives: 1) determine if aging is associated with increased protein aggregation in human skeletal muscle; 2) identify muscle-aggregate proteins that are differentially abundant with age; and 3) identify nematode orthologs of selected human aggregate proteins, and test their mechanistic involvement in protein aggregation and age-associated loss of muscle mass and function in C. elegans.

Previous proteomic comparisons of young and aged muscle for rodents and humans found 3-23% of soluble proteins altered in abundance with age. In the present analysis, 43% of the 515 proteins identified in muscle aggregates differed by at least a 1.5-fold in abundance between age groups, and 15% were significantly different, more than the 5% expected by chance. These results suggest that insoluble protein aggregates may be particularly susceptible to the effects of aging and could play a role in sarcopenia analogous to their role in the pathology of neurodegenerative diseases. This possibility was directly supported by disruptions of gene expression for C. elegans orthologs of human aggregate proteins: six of the seven tested knockdowns reduced protein aggregation and improved muscle-mass retention and resistance to amyloid-induced paralysis in aged nematodes.

By comparing aggregate amounts and compositions across human aging, and assessing functional impacts of aggregate-associated proteins through nematode studies, we were able to demonstrate that age-dependent accumulation of aggregates in muscle can underlie the loss of muscle mass and function that are commonly observed to accompany human aging. Skeletal muscle mass is expected to be influenced by relative rates of protein synthesis and degradation but the current study provides the first evidence that specific proteins are involved in the formation of insoluble protein aggregates that are toxic to muscle. Multiple proteins of diverse function were functionally implicated in protein aggregation, suggesting that the key causal parameter is the aggregate burden itself, rather than an upstream regulator of aggregation. Furthermore, since dampening production of aggregate proteins produced marked improvements in muscle mass and function, we propose that protein aggregation may provide attractive targets for therapeutic intervention in age-dependent sarcopenia. We conclude that protein aggregation is not unique to neurodegenerative disease and genetic myopathies, but is also characteristic of normal muscle aging and may contribute to muscle loss and functional decline with age.

Researchers are investigating a drug candidate that inhibits plasminogen activator inhibitor-1 (PAI-1) as a potential treatment to slow the progression of atherosclerosis, in which fatty deposits build up in blood vessel walls. This leads to narrowing, structural failure of blood vessels, or blockage when the deposits grow unstable and rupture. The publicity materials in this case fail to join some of the dots to explain why this is interesting in the broader context; the evidence points to influence on cellular senescence as a possible mechanism for the effect here. Past research has shown that PAI-1 is involved in steering cells to a senescent state, and in the activities of cells while senescent. Further, senescent cells drive a sizable fraction of the growth and instability in the fatty plaques of atherosclerosis, and removing them slows the development of the condition. So we might take this sort of drug development research as further support for the benefits to be realized from bringing clearance of senescent cells to the clinic.

Approximately 2,200 Americans die each day from heart attacks, strokes and other cardiovascular diseases. The most common cause is blocked blood vessels that can no longer supply oxygen and nutrients to the heart and brain. A recent study has shown that a protein inhibitor drug prevents these blockages, and could be a new therapeutic approach to prevent heart attack, stroke and other diseases caused by blocked blood vessels. "Arteries are living hoses that narrow and enlarge in order to regulate blood flow to organs and muscles. Smooth muscle cells in the artery regulate blood flow by constricting and relaxing. However, when chronic inflammation occurs in a blood vessel - typically in response to diabetes, high cholesterol and cigarette smoking - the smooth muscle cells in the walls of arteries change their behavior. They gradually accumulate inside the artery and narrow the blood vessel. In the case of coronary arteries, which supply blood to heart muscle cells, this process produces blockages that can lead to a heart attack."

Plasminogen activator inhibitor-1, or PAI-1, is a naturally occurring protein within blood vessels that controls cell migration. With diseases such as diabetes and obesity, PAI-1 over-accumulates in blood vessels. This promotes blockage formation. This process occurs not only in arteries, but also in vein grafts in patients who have undergone coronary artery bypass graft surgery. The research team studied PAI-039, also known as tiplaxtinin, an investigational drug not yet used to treat humans. The researchers found that PAI-039 inhibited the migration of cultured human coronary artery smooth muscle cells, and prevented the development of blockages in arteries and bypass grafts in mice. "We found that PAI-039 decreased blockage formation by about 50 percent, which is a powerful effect in the models we used. In addition to reducing vascular blockages, inhibiting PAI-1 also produces a blood thinning effect that prevents the blood clots that trigger most heart attacks and strokes." If future studies are successful, PAI-039 or similar drugs could be used to prevent blockages in arteries and bypass grafts.

Link: http://medicine.missouri.edu/news/20161221-new-drug-could-help-prevent.php

Microglia are a form of immune cell found in the central nervous system, responsible for a range of tasks including defense against pathogens and clearance of unwanted extracellular waste. Like all aspects of the immune system, their performance declines with age. Delivering young microglia to the aging brain has been proposed as a potential therapy by a number of research groups, and there has been some exploratory work in mice in recent years. Here researchers work in aged brain tissue sections rather than animal models, but show that introducing young microglia and the signals they produce enhances the removal of the amyloid-β deposits associated with Alzheimer's disease.

Alzheimer′s disease (AD) is the most prevalent neurodegenerative disorder and is pathologically defined by extracellular amyloid β (Aβ) deposition, neurofibrillary tangles, and neuroinflammation. Neuroimmune changes are tightly linked to the pathology of AD, as well as other neurodegenerative disorders. This link has been strengthened by recent discoveries of genes implicated in microglial function that are also risk factors for late onset AD. Interestingly, these newly identified risk factors may be functionally linked to microglial phagocytosis and Aβ clearance. Although microglia are well known for their phagocytic capacity and are found to surround amyloid plaques in mouse models of amyloidosis as well as in AD patients, their role in plaque clearance is still under debate.

One of the major limitations to study microglial contribution to amyloid plaque phagocytosis is the lack of suitable model systems. Major attempts to study microglial phagocytosis of Αβ come from studies using cultured microglial cells to which Aβ has been exogenously added. A key unresolved question is whether microglial dysfunction in AD is reversible and whether their phagocytic ability can be restored to limit amyloid accumulation. To this end, we developed a novel ex vivo model of amyloid plaque clearance by co-culturing young wild type (WT) brain slices together with brain slices from aged AD mice. We show that functional impairment of aged microglial cells in amyloid plaque-bearing tissue can be reversed through factors secreted by young microglia, resulting in increased amyloid plaque clearance and thus reduced amyloid plaque load. Our results suggest a role of microglia in reducing the amyloid burden and support development of therapeutic approaches modulating microglial activity.

Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis.

Link: http://emboj.embopress.org/content/early/2016/12/20/embj.201694591

The environment surrounding our tissues and various complex systems such as organs incorporates a great deal of microbial life. We are surrounded by microbes, we have a whole cooperative ecosystem on our skins and another in our guts, and are constantly under attack by less friendly species. From the point of view of a great many classes of microbial life, we mammals are just another resource to be exploited as a basis for unfettered replication. Before the advent of modern medicine, life expectancy was largely determined by infectious disease and other environmental pathogens rather than the fundamental processes of aging. In the research paper linked below, the author makes a valid point, which is that we haven't really yet defeated the hostile microbes arrayed against us, just postponed their inevitable victory by decades for most individuals. When we consider aging, we should think about aging in the context of our vulnerability to the microbial world in addition to the failure of our component parts for other reasons.

This is really, I think, a type of argument for putting the age-related decline of the immune system at the top of the list of things to address when it comes to building rejuvenation therapies. I don't necessarily disagree, but it may be that our present state of knowledge makes it easier for us to join the dots between immunosenescence and inflammaging and all of the harm an age-damaged immune system causes, coupled with it being harder to quantify the specific contributions from other causes of aging. Now that senescent cell clearance is getting a whole lot more attention, for example, people are finding all sorts of links to specific age-related diseases and disease processes. When you can actually do something about a cause of aging, such as by clearing senescent cells, it becomes very much easier to find out how much harm they produce. Remove those cells and measure the outcome. Those experiments are ongoing at the moment, and a great deal is being learned. In the case of immune aging, there are several decades of good studies that compare various degrees of impairment of the aging immune system, and the role of inflammation in particular in aging is very well studied. The immune system plays many important roles beyond defense against pathogens, involved in everything from wound healing to destroying senescent and cancerous cells. All of these roles suffer due to the growing disarray in an aged immune system.

But of course, absent increasingly comprehensive medical support, the microbes will get you in the end. The cell and tissue damage of aging produces frailty throughout all of our biological systems, and it isn't just the immune system that becomes less resilient. The immune response becomes less able to defend against attackers, and at the same time it takes less of a disruption due to infection to produce a fatal decline in already precarious vital organ functions. A very great many old people are tipped over the edge by infections that they wouldn't have even noticed a few decades earlier in life. There is still a great deal of work to do in the control of infectious disease, a goal that will probably be more easily achieved by augmenting our natural immune systems with more efficient molecular nanotechnology than by sterilizing the world, but consider how rare fatal infections are nowadays for younger adults when compared to the old. The biggest gains in the near future will come through rejuvenation of the immune system: destroying and then recreating immune cells to remove misconfiguration; regenerating the thymus to increase the supply of new immune cells; supplying new pools of pristine bone marrow stem cells responsible for creating immune cells; and so on.

Classifying Aging As a Disease: The Role of Microbes

Recent publications have proposed that aging should be classified as a disease. The goal of this manuscript is not to dispute these claims, but rather to suggest that when classifying aging as a disease, it is important to include the contribution of microbes. As recently as ~115 years ago, more than half of all deaths were caused by infectious diseases. Since then, the establishment of public health departments that focused on improved sanitation and hygiene, and the introduction of antibiotics and vaccines allowed for a dramatic decrease in infectious disease-related mortality. In 2010, the death rate for infectious diseases was reduced to 3%. Simultaneously, as infectious disease-related mortality rates have decreased, global life expectancy has increased from ~30 to ~70 years.

Because death rates due to infectious diseases have been reduced to very low levels, we've forgotten about the adverse effects of microbes on our existence. The fact is, we live in a microbial world. Even at a young chronological age, microbes find their way into the blood and tissues. Circulating microbial DNA is found in young, healthy adults. Interestingly, levels of circulating bacterial DNA are not homogeneous: some subjects had 3-fold or more circulating bacterial DNA when compared with others. Moreover, various bacterial species are found in skeletal muscle, heart, liver, adipose tissue, and in the brains of young mice. With the passage of time, the barriers responsible for keeping microbes out of us weaken. For example, tight junctions (TJs) connect epithelial cells, thereby minimizing the space in between the cells, and minimizing the ability of microbes to translocate into the blood. Bacteria and viruses have evolved mechanisms to impair TJ assembly. Whether caused by pathogenic microbes or because of defects in host gene expression, levels of many of these tight junction proteins are decreased in old, when compared with young. Furthermore, although the immune system should protect us against an increase in microbial burden, however, many aspects of the immune response are decreased, whereas others are increased, thereby resulting in dysregulation. This phenotype is known as immunosenescence.

The impact of decreased barrier function and immunosenescence would be expected to lead to an increase in circulating microbes in old, when compared with young. Although circulating levels of bacterial DNA have yet to be reported in older adults, plasma levels of lipopolysaccharide (LPS), which is found in the outer membrane of gram-negative bacteria, and levels of the receptors that bind to LPS (TLR4) and to bacterial flagellin (TLR5), are elevated in older adults, when compared with young. In line with this, the incidence of bloodstream infections with LPS-containing Escherichia coli is increased by more than 10-fold in adults older than 74, when compared with subjects younger than 50 years. Similarly, the incidence of bloodstream infections with gram-positive bacteria is elevated by more than 8-17 fold in older adults.

What are the consequences of an age-related increase in microbial burden? Microbes and/or microbial products are causatively involved in multiple theories of aging, including insulin resistance, oxidative stress, inflammation, and telomere shortening. In support of this, LPS injection into young, healthy subjects causes insulin resistance. Oxidative stress is increased in response to the binding of LPS and bacterial flagellin to their respective receptors. Levels of the pro-inflammatory cytokines IL-6 and TNF-α are increased when LPS binds to TLR4. Telomere shortening occurs at a faster rate in the presence of cytomegalovirus (CMV) infection. Interestingly, the prevalence of CMV infection increases from ~20% in adults younger than 50 years, to ~40% in 50-70 year olds, to 100% in adults older than 70. Collectively, these data support a causative role for microbial burden on mechanisms that have been commonly hypothesized to drive the aging process. Microbial burden is also involved in mechanisms related to age-related disease, including cardiovascular disease (CVD), Alzheimer's disease, cancer, stroke, and diabetes. In support of this, approximately 10-fold more circulating bacterial DNA is found in CVD patients, when compared with healthy controls.

If we are fortunate to avoid the common age-related diseases and live to achieve centenarian status, infectious disease as a major cause of death arises again. In Japan, more than 40% of all centenarian deaths are due to infectious diseases, including pneumonia. Similarly, in a larger study of ~36,000 centenarians from the UK, other than "old age," the leading cause of death was pneumonia. In short, over the past 115+ years, we haven't eliminated the adverse effects of microbes on our health, we've merely delayed them! As an argument against the role of microbes on causing many aspects of aging and age-related disease, it is important to note that host aging does indeed occur in their absence. Although lifespan in microbe-free mice is increased by 20-50%, these animals are not immortal. Nonetheless, as presented here, microbes are involved in mechanisms related to aging and age-related disease, and accordingly, I posit that any classification of aging as a disease should include the contribution of microbes.

In this short interview, the main topic of discussion is the use of nanoscale scaffolding materials in tissue engineering. They act as a temporary substitute for the extracellular matrix that normally supports cells, allowing cells to survive and move in order to form new tissue. Ultimately the cells replace the scaffold with new extracellular matrix structures, and the end result is regrowth of tissue where that regeneration would not normally have occurred.

For tissue engineering and repair, we've been focusing lately on skeletal muscle. There's really a medical need for platforms or scaffolds for muscle fiber regeneration, since after injury the body's abilities to repair skeletal muscle are really quite limited. Skeletal muscle makes up a large part of the human body - 40 to 50 percent by weight. And when damage occurs to skeletal muscle on a small scale, we've seen that skeletal muscle possesses innate repair mechanisms. Through these mechanisms, a new fiber can grow, for example, essentially repairing or replacing the damaged one. But above a critical threshold of damage to skeletal muscle, our bodies no longer employ those effective repair mechanisms. Instead, the body forms scar tissue at the wound site - and then you've essentially lost control of that muscle function. You can't get it back. Surgically, you could graft in skeletal muscle. But that depends on the availability of donor tissue. So we know that the body can repair skeletal muscle. It just doesn't do so beyond a certain threshold of damage.

Natural skeletal muscle is surrounded by a complex extracellular matrix that supports muscle fibers as they form and grow in the body. What we would like to do in this field, which many researchers are working on, is to create an artificial extracellular matrix into which we could introduce a progenitor type of cell - like stem cells or muscle progenitor cells - and then provide them with the proper signals to differentiate into muscle fibers. We believe that scaffold and signals are what is needed to grow new muscle fibers, which you could then transplant to the site of damage. In general, with designing scaffolds for cell growth, the material we work with really depends on the type of cell we'd like to introduce into the scaffold to proliferate. For bone tissue regeneration, which we've worked on in the past, we created a scaffold made of chitosan - a complex polysaccharide, essentially long chains of sugar-like molecules - combined with other materials to create a calcified scaffold. For skeletal muscle, we and other researchers work with a variety of anisotropic materials.

Anisotropic materials have physical properties that differ based on direction or orientation. They form the basis of the scaffolds and are usually complex polymer materials. The innate "directionality" of anisotropic materials helps the progenitor cells grow into three-dimensional forms like a myotube, which is a precursor to a muscle fiber. But there are structural challenges to overcome. The scaffold must be micropatterned to promote cell migration, growth and proliferation in the right direction. This involves nanoscale design details, and some polymers are better for this than others. The production of highly aligned nanofibers in a large area remains a great challenge. We have developed several methods to produce nanofibers made of natural polymers with a high degree of alignment and uniformity over large areas. In addition, we often coat the scaffold with biomolecules that help the cells stick to the scaffold and provide them with the right signals to grow and differentiate: adhesion proteins, growth factors and transcription factors that deliver specific messages to cells depending on their structure and location in the scaffold. By changing what we make the scaffolds out of, the protein messages we coat them with or the nanopore structures within the scaffolds, we can reveal many different properties of cells. We can also test the types of external signals, be it a structural feature of the scaffold or a protein message, that can promote or inhibit cell growth.

Link: http://www.washington.edu/news/2016/12/19/uw-researcher-pursues-synthetic-scaffolds-for-muscle-regeneration/

It has for a while been the consensus theory that usual aspects of naked mole-rat biology, such as its extreme cancer resistance and exceptional longevity for its size, are at least in part the outcome of evolving to thrive in the low-oxygen environment found in underground burrows. In most mammals, lack of oxygen followed by its return is quite damaging, but much less so in naked mole-rats. The nature of the mechanisms linking resistance to low-oxygen environments with longevity, and their relative importance when compared to one another, is still up for debate, however. A number of other similar burrowing rodent species are also long-lived and cancer resistant. Here, researchers survey the biochemistry of the blind mole-rat:

The blind mole rat of the genus Nannospalax (hereafter, Spalax) is a subterranean, hypoxia tolerant rodent, evolutionarily related to murines. The last common ancestor of Spalax, mouse, and rat lived ~46 million years ago. Despite the tight evolutionary relatedness of Spalax and murines, they exhibit profound differences in lifespan, propensity to cancer diseases. Although a very common cause of death in rats and mice is cancer, Spalax resists experimentally induced carcinogenesis in vivo and does not develop spontaneous cancer. While both rat and Spalax have comparable body weights, their maximum lifespan is ~4 years and ~20 years, respectively. The naked mole rat (Heterocephalus glaber), another hypoxia-tolerant subterranean species of the Bathyergidae family, separated by ~85 million years of evolution from Spalax, is also long-lived, and was reported to be less sensitive to spontaneous cancers.

Molecular adaptations to subterranean life and longevity where suggested for this species, in a brain transcriptome study. Noteworthy, we have proved that both Spalax and naked mole rat's normal fibroblast secrete substance/s interacting with cancer cells from different species, including a wide variety of human cancer cells, ultimately leading to the death of the cancer cells. In addition, sequence similarities between distantly related hypoxia-tolerant species (diving- and subterranean- mammals) were found in the protein sequence of p53, a master regulator of the DNA damage response (DDR). These studies indicate that adaptations to hypoxia include changes in the DDR that may be linked to cancer resistance, and longevity traits.

Under laboratory conditions, Spalax survives ~3% O2 for up to 14 hours, whereas rat survives such conditions for only ~2-3 hours. Oxygen levels measured in Spalax's natural underground burrows vary between ~21% and 7%, depending on seasonal and ecological conditions. In its natural habitat, Spalax is exposed to acute and transient hypoxia, such as: (i) long-term periods of hypoxia during seasonal rainfalls, which reduce soil permeability to oxygen, and simultaneously reduce the total space available to the animal; and (ii) short-term periods of hypoxia during extensive digging activity, when burrows are clogged by soil pushed to the rear by the animal, forcing it to perform an energy-consuming activity in a small burrow fragment with a limited amount of oxygen. Hence, in its natural habitat, Spalax faces acute cyclical changes in oxygen levels. By the term "acute hypoxia" we refer to short- or long- term hypoxia for a limited period, followed by reoxygenation, which is in contrast to "mild-chronic hypoxia" characterizing habitats, such as high altitudes.

Many of the genes that showed higher transcript abundance in Spalax are involved in DNA repair and metabolic pathways that, in other species, were shown to be downregulated under hypoxia, yet are required for overcoming replication- and oxidative-stress during the subsequent reoxygenation. We suggest that these differentially expressed genes may prevent the accumulation of DNA damage in mitotic and post-mitotic cells and defective resumption of replication in mitotic cells, thus maintaining genome integrity as an adaptation to acute hypoxia-reoxygenation cycles.

Link: https://dx.doi.org/10.1038/srep38624

Today's open access research paper outlines the discovery of yet another new candidate drug for the selective destruction of senescent cells. This is an increasingly popular research topic nowadays. Senescent cells perform a variety of functions, but on the whole they are bad news. Cells become senescent in response to stresses or reaching the Hayflick limit to replication. They cease further division and start to generate a potent mix of signals, the senescence-associated secretory phenotype or SASP, that can provoke inflammation, disarray the surrounding extracellular matrix structures, and change behavior of nearby cells for the worse. Then they destroy themselves, or are destroyed by the immune system - for the most part at least. This is helpful in wound healing, and in small doses helps to reduce cancer incidence by removing those cells most at risk of becoming cancerous. Unfortunately a growing number of these cells linger without being destroyed, more with every passing year, and their presence eventually causes significant dysfunction. That in turn produces age-related disease, frailty, and eventually death. Senescent cells are not the only root cause of aging, but they provide a significant contribution to the downward spiral of health and wellbeing, and even only their own would eventually produce death by aging.

The beneficial aspects of senescent cells seem to require only a transient presence, so the most direct approach to the problem presented by these cells is to destroy them every so often. Build a targeted therapy capable of sweeping senenscent cells from tissues, and make it efficient enough to keep the count of such cells low. That is the way to prevent senescent cells form contributing to age-related disease. Working in mice, researchers have produced results such as functional rejuvenation in aged lungs and extended life span through the targeted destruction of senescent cells. Since perhaps only a few percent of the cells in old tissue are senescent, this targeted destruction can be accomplished with few side-effects beyond those generated by off-target effects of the medication itself. There are a range of potential ways to destroy senescent cells while leaving other cells intact: the last twenty years of work on the basis for targeted cell destruction in the cancer research community has produced many useful tools. These include the programmable gene therapy approach adopted by Oisin Biotechnologies, immunotherapies of the sort under development by SIWA Therapeutics, and apoptosis inducing senolytic drugs of the sort championed by UNITY Biotechnology. This last category has a particularly close tie to the cancer research community, and in fact the senolytic drugs we know the most about, such navitoclax, also known as ABT-263, are well-categorized precisely because they have been trialed as cancer therapies in past years.

Senescent cells are in a sense primed for apoptosis, a process of programmed cell death. They need less of a nudge to finish that process than normal cells, and so a large number of the varied drugs that can induce apoptosis to some degree might have a future as plausible senolytic therapies. Cancer research groups have libraries of such compounds, many of which might turn out to be far more useful as senolytics than they ever were as cancer treatments. So we should expect to see a growing number of such drug candidates in the years ahead as various research groups and companies shake their archives to see what falls out. So far the first set of drugs, including navitoclax, are largely based on inhibition of bcl-2 family proteins, and have a range of unpleasant side effects. They are in effect chemotherapeutics, but it is likely that their use as senolytics will require lower doses than were used in cancer trials, but that remains to be established, however. The possible side-effects of repurposed chemotherapy drugs are one good reason to favor an approach like that taken by Oisin Biotechnologies, which is a treatment that has next to no side-effects, or at the very least to put more effort into finding drug candidates with alternative mechanisms and far fewer side-effects, as is the case in the research here.

Discovery of piperlongumine as a potential novel lead for the development of senolytic agents

Cellular senescence, an essentially irreversible arrest of cell proliferation, can be triggered when cells experience a potential risk for malignant transformation due to the activation of oncogenes and/or DNA damage. While eliminating aged or damaged cells by inducing senescence is an effective barrier to tumorigenesis, the accumulation of senescent cells (SCs) over time compromises normal tissue function and contributes to aging and the development of age-associated diseases. Often, SCs secrete a broad spectrum of pro-inflammatory cytokines, chemokines, growth factors, and extracellular matrix proteases, a feature collectively termed the senescence-associated secretory phenotype. These factors degrade the local tissue environment and induce inflammation in various tissues and organs if SCs are not effectively cleared by the immune system.

Studies have shown that the genetic clearance of senescent cells extends the lifespan of mice and delays the onset of several age-associated diseases in both progeroid and naturally-aged mice. These findings support the hypothesis that SCs play a causative role in aging and age-associated diseases and, importantly, highlight the tremendous therapeutic potential of pharmacologically targeting SCs. Consistent with these findings, we have shown that ABT-263 (navitoclax), an inhibitor of the antiapoptotic Bcl-2 family proteins, acts as a potent senolytic agent to deplete SCs in vivo and functionally rejuvenates hematopoietic stem cells in both sublethally irradiated and naturally-aged mice. Complementary studies from other labs have confirmed that the Bcl-2 protein family is a promising molecular target for the development of senolytic drugs. These studies further establish the concept that the pharmacological depletion of SCs is a promising, novel approach for treating age-associated diseases. ABT-263 was identified by screening a small library of structurally diverse, rationally-selected small molecules that target pathways predicted to be important for SC survival. By titrating their cytotoxicity against normal human WI-38 fibroblasts and ionizing radiation (IR)-induced senescent WI-38 fibroblasts, this targeted screen also identified the promising senolytic agent piperlongumine (PL); PL is a natural product isolated from a variety of species in the genus Piper. Here, we report the characterization of PL as a potential novel lead for the development of senolytic agents.

Selective depletion of SCs is a potentially novel anti-aging strategy that may prevent cancer and various human diseases associated with aging and rejuvenate the body to live a longer, healthier life. As such, several senolytic agents, including ABT-263, have been identified recently, demonstrating the feasibility of pharmacologically targeting SCs. However, ABT-263 induces thrombocytopenia, and it remains to be determined whether ABT-263 can be used to safely treat age-related diseases, since individuals may require long-term treatment with a senolytic drug. Thus, it is necessary to identify a safer senolytic drug. In the present study, we evaluated PL as a novel senolytic agent. PL induced caspase-mediated apoptosis in SCs and effectively killed SCs induced by IR, replicative exhaustion, or ectopic expression of the oncogene Ras. Unlike ABT-263, the precise mechanism of action by which PL induces SC apoptosis remains unclear. PL modulates the activity of many cell signaling and survival pathways in cancer cells, and a number of studies have investigated the mechanism of action by which PL induces apoptosis in these cells. Data from these studies may be translatable to PL-induced SC apoptosis because SCs and cancer cells share some common pro-survival pathways. In addition, mass spectrometry-based proteomic approaches using probes derived from PL could be used to identify direct molecular targets of PL in SCs. In this regard, novel anti-senescent protein targets and mechanisms of action could be identified, making it possible to develop promising novel classes of senolytic agents. Importantly, PL appears to be safe; the maximum tolerated dose in mice is very high, and it maintains high bioavailability after oral administration. Furthermore, our initial structural modifications to PL demonstrate that we can develop PL analogs with increased potency and selectivity toward SCs, supporting the use of PL as a lead for further drug discovery and development.

Some of the issues causing progressive age-related failure of immune function result from the low rate of replacement of T cells in adults. T cells are created in the bone marrow but mature in the thymus, an organ that atrophies early in life in a process known as thymic involution. It then declines more slowly thereafter across the course of a life span. The level of activity in the thymus limits the rate at which new T cells arrive, and this in turn effectively puts a limit on the number of such cells supported by the body, and determines the rate of turnover in that population. As we age and are exposed to persistent pathogens, especially cytomegalovirus, ever more of the T cell population becomes specialized in ways that remove the ability to deal with new threats. A flood of new immune cells would help to restore the balance, and in recent years researchers have demonstrated that transplanting a young and active thymus into an old mouse does in fact restore measures of immune function, and extends life span as well. This is an indication that the research community should put more effort into regeneration and tissue engineering of the thymus as a way to partially reverse the age-related loss of immune function and the frailty that follows that loss.

The peripheral T cell compartment of aged individuals is characterized by great modifications, including a higher frequency of regulatory T cells (Treg). A tight balance between regulatory and conventional (Tconv) T cell subsets in the peripheral compartment, maintained stable throughout most of lifetime, is essential for preserving self-tolerance along with efficient immune responses. An excess of Treg cells, described for aged individuals, may critically contribute to their reported immunodeficiency. The relative contribution of alterations in thymic exportation versus changes in the homeostasis of the peripheral compartment affecting the Treg/Tconv lymphocytes balance is not yet clearly established, however. In this work, we investigated if quantitative changes in thymus emigration may alter the Treg/Tconv homeostasis regardless of the aging status of the peripheral compartment. We used two different protocols to modify the rate of thymus emigration: thymectomy of adult young (4-6 weeks old) mice and grafting of young thymus onto aged (18 months old) hosts. Alterations in Treg and Tconv peripheral frequencies following these protocols were investigated after 30 days.

Our results show that peripheral T cell homeostasis is promptly disturbed in the absence of the thymus. This disturbance was characterized by a preferential persistence of Treg cells that occurs independently of the age of either the T cells or the peripheral environment. The excess of Treg cells in aged mice is also very rapidly corrected by the grafting of a young functional thymus, supporting the hypothesis that thymus newly emigrated T cell populations, harboring an adequate physiological proportion of Treg/Tconv lymphocytes, are essential to compensate for an excess of peripheral Treg cell expansion or survival. It is also interesting to observe that the aged T cell precursors are fully able to colonize and differentiate in the young grafted thymus. These results suggest that the continuous output of the young grafted thymus, which is numerically much superior to the small number of cells emigrating from the aged host thymus, may contribute to normalize the peripheral proportions of Treg/Tconv cells. The aged peripheral compartment does not interfere with this homeostasis.

Our results, thus, highlight the importance of the thymus as a permanent source of emigrating populations of recently differentiated lymphocytes harboring an adequate, physiological proportion of Treg/Tconv lymphocytes, essential to keep the peripheral Treg cell balance, regardless of the aging status of the peripheral compartment. The immunosenescence associated with aging, in which an excess of Treg cells may impair the immune response to infections and tumors, highlights the relevance of understanding the peripheral Treg cell homeostasis for the development of adequate clinical strategies.

Link: http://onlinelibrary.wiley.com/doi/10.1002/iid3.132/full

Regeneration rather than removal of damaged teeth lies somewhere in the near future, through some combination of tissue engineering of new teeth versus therapies that spur in situ regeneration of tooth structures. Researchers have been making progress towards this goal for some years, and there are any number of promising studies in laboratory animals reported in the literature. Here is one example of the sort of work presently taking place:

When a tooth is damaged, either by severe decay or trauma, the living tissues that comprise the sensitive inner dental pulp become exposed and vulnerable to harmful bacteria. Once infection takes hold, few treatment options - primarily root canals or tooth extraction - are available to alleviate the painful symptoms. Researchers now show that using a collagen-based biomaterial to deliver stem cells inside damaged teeth can regenerate dental pulp-like tissues in animal model experiments. "Endodontic treatment, such as a root canal, essentially kills a once living tooth. It dries out over time, becomes brittle and can crack, and eventually might have to be replaced with a prosthesis. Our findings validate the potential of an alternative approach to endodontic treatment, with the goal of regenerating a damaged tooth so that it remains living and functions like any other normal tooth."

Researchers examined the safety and efficacy of gelatin methacrylate (GelMA) - a low-cost hydrogel derived from naturally occurring collagen - as a scaffold to support the growth of new dental pulp tissue. Using GelMA, the team encapsulated a mix of human dental pulp stem cells - obtained from extracted wisdom teeth - and endothelial cells, which accelerate cell growth. This mix was delivered into isolated, previously damaged human tooth roots, which were extracted from patients as part of unrelated clinical treatment and sterilized of remaining living tissue. The roots were then implanted and allowed to grow in a rodent animal model for up to eight weeks. The researchers observed pulp-like tissue inside the once empty tooth roots after two weeks. Increased cell growth and the formation of blood vessels occurred after four weeks. At the eight-week mark, pulp-like tissue filled the entire dental pulp space, complete with highly organized blood vessels populated with red blood cells. The team also observed the formation of cellular extensions and strong adhesion into dentin - the hard, bony tissue that forms the bulk of a tooth. The team saw no inflammation at the site of implantation, and found no inflammatory cells inside implanted tooth roots, which verified the biocompatibility of GelMA.

Control experiments, which involved empty tooth roots or tooth roots with only GelMA and no encapsulated cells, showed significantly less growth, unorganized blood vessel formation, and poor or nonexistent dentin attachment. The results support GelMA-encapsulated human dental stem cells and endothelial cells as part of a promising strategy to restore normal tooth function. "A significant amount of work remains to be done, but if we can extend and validate our findings in additional experimental models, this approach could become a clinically relevant therapy in the future."

Link: https://www.eurekalert.org/pub_releases/2016-12/tuhs-nsc121916.php

As the clock ticks on this year's SENS rejuvenation research fundraiser - less than two weeks to go now, and plenty left in the matching fund for new donations - it is good to be reminded of the progress that the SENS Research Foundation has accomplished with the charitable funding of recent years. With that in mind, today I'll point you to a recent Google Tech Talk that provides a layperson's introduction to one of the projects that our community has funded, fixing the problem of mitochondrial damage in aging. The point of the SENS (Strategies for Engineered Negligible Senescence) research programs is to accelerate progress towards specific forms of therapy that can bring aging under medical control. To the extent that degenerative aging and age-related disease is caused at root by a few classes of molecular damage, it follows that control of aging - halting and reversing the decline - can be achieved by periodically repairing the damage. The more of it that is repaired, the better the outcome. If all of the fundamental forms of damage could be kept below the levels present in a typical 30 year old, sustained there by a package of treatments undertaken every few years, then individuals would no longer age, no longer suffer disease and frailty, and no longer suffer increased mortality with the passage of time. That, at least, is the goal. It will become clear as the research and development progresses to what degree edge cases and unforeseen issues exist.

One of the forms of damage that causes aging occurs to mitochondrial DNA. Every cell has a swarm of hundreds of mitochondria, the evolved descendants of symbiotic bacteria that over time have become fully integrated as a component part of our cells. They still divide and multiply like bacteria, and have a little of their original DNA left, completely separate from the DNA of the cell nucleus. Mitochondrial DNA encodes a few vital pieces of molecular machinery, such as portions of the electron transport chain that is used in the process of producing chemical energy store molecules to power the cell. Mitochondria are power plants, effectively, among the many other essential tasks they have adopted over the course of evolution. Unfortunately mitochondrial DNA is more vulnerable to damage and its repair mechanisms are less capable when compared to the DNA of the cell nucleus. Damage accumulates. Equally unfortunately, some forms of damage, such as large deletions that hamper the electron transport chain by denying it necessary parts, produce mitochondria that are both dysfunctional and better able to replicate and resist destruction by cellular quality control systems. A minority of cells become overtaken by these broken mitochondria as we age, and themselves become broken, generating and exporting harmful reactive molecules into our tissues. This causes enough further damage to be a significant cause of age-related disease.

The SENS Research Foundation proposes the use of gene therapy to copy these vulnerable genes into the cell nucleus, altered in order to enable the proteins produced to find their way back to the mitochondria. This produces a backup source of the proteins, and thereby eliminates the contribution of mitochondrial DNA damage to aging. This is hard work: there are thirteen genes to copy, and every one of them requires its own complicated solution to the challenge of getting the proteins back to the mitochondria. Equally, most of these genes are associated with inherited disorders, in which a patient has a damaged copy in all mitochondria. So it is possible to produce a proof of principle for a single gene and do some good at the same time. Nearly a decade ago, the SENS Research Foundation started to support work on one of these genes, NH4, that enabled a treatment for Leber's hereditary optic neuropathy (LHON). That area of research was very poorly funded at the time, and as a direct result of that SENS Research Foundation support a well-funded company is now bringing a therapy to the clinic, and looking at doing the same for other related genes. This year, the SENS Research Foundation in-house team demonstrated the same outcome for two more mitochondrial genes, ATP6 and ATP8. That work was funded by donations from people like you and I, and the researcher leading the effort recently gave a presentation in the Google Tech Talk series:

Google TechTalk: Rejuvenating the Mitochondria

"Engineering Approaches to Combating the Diseases and Disabilities of Aging: Rejuvenating the Mitochondria." This is a talk for a general audience on the work of the SENS Research Foundation to fight age related diseases with a focus on repairing the damage that accumulates as we age. The SENS Research Foundation recently published a paper on their research into repairing cells that lack two of the thirteen essential mitochondrial proteins. The SRF scientists were able to reengineer the two mitochondrial genes and move them to the nucleus of the cell, restoring the missing proteins. This work is significant for both its impact on treating age related diseases but also on childhood diseases resulting from a lack of certain mitochondrial proteins.

Researchers here provide evidence to show that lowering blood cholesterol levels to a large degree via new treatments is more beneficial for patients than the more modest targets for lower cholesterol, achieved via lifestyle choices and drugs such as statins, previously set by the research community:

Reducing our cholesterol levels to those of a new-born baby significantly lowers the risk of cardiovascular disease, according to new research. Although previous studies have suggested lowering cholesterol levels may be associated with a lower risk of heart attack, recent evidence has questioned whether very low levels are beneficial. In the latest study, researchers analysed data from over 5,000 people taking part in cholesterol-lowering trials. These studies utilised a new therapy to reduce cholesterol to much lower levels than previously possible. The team wanted to assess whether reducing cholesterol as low as possible is safe, and whether it was more beneficial than the current levels achieved with existing drugs. The scientists found that dropping cholesterol to the lowest level possible - to levels similar to those we were born with - reduced the risk of heart attack, stroke or fatal heart disease by around one third. "Experts have long debated whether very low cholesterol levels are harmful, or beneficial. This study suggests not only are they safe, but they also reduced risk of heart disease, heart attack and stroke."

In the paper, the scientists examined levels of low density lipoprotein (LDL) cholesterol. This is considered to be 'bad' cholesterol, as it is responsible for clogging arteries. LDL carries cholesterol to cells, but when there is too much cholesterol for cells to use, LDL deposits the cholesterol in the artery walls. Official advice suggests most people should aim to keep their LDL cholesterol at 100 mg/dL or below, though this number can vary depending on a person's risk of cardiovascular disease. In the study, the team analysed data from 10 trials, involving 5000 patients. Most had cardiovascular disease, and already had some furring of the arteries or were at very high risk of furred arteries. All of the patients had previously been diagnosed with high cholesterol, and many were slightly overweight. The average age was 60, and the researchers tracked the patients for between three months and two years. The average cholesterol reading was around 125 mg/dL, and they were all deemed at risk of heart problems or stroke.

Mostly patients were taking a cholesterol-lowering statin therapy, but just over half were also taking an additional novel drug, called alirocumab, every two weeks via a small injection, to further lower cholesterol levels. This drug may be needed when patients' cholesterol levels are not sufficiently lowered by statins. Some patients find their cholesterol levels aren't adequately reduced by statins, possibly because they carry a faulty gene. The combined effect of the new drug and the statin in the trials meant that patients reached very low cholesterol - lower than 50mg/dL. This is comparable to the levels we are born with, but is only achievable in adulthood through medication - lifestyle and exercise alone would not drop levels so low. The researchers found lowering levels of cholesterol reduced the risk of heart attack, stroke, angina or death from heart disease, and that for every 39mg/dL reduction in LDL, the risk reduced by 24 per cent.

Link: http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_16-12-2016-12-53-13

The Life Extension Advocacy Foundation is in the process of reworking their online presence and adding a lot more content. One of the new items is this discussion of the likely trajectory of cost for near future therapies that slow aging or produce rejuvenation, such as the panoply of SENS therapies presently under development. There is a tendency for people to assume, without giving it much thought, that rejuvenation therapies will always be enormously expensive and thus restricted to the wealthy, but this is basically nonsense. Once proven and packaged as a product, the projected types of therapy will be mass manufactured infusions and injections, the same for everyone. They will be administered by bored clinicians, needing little in the way of time from expensive medical staff, and only undertaken once every few years or so. If you look at comparable technologies today, even given the way in which a dysfunctional and highly regulated medical industry piles on unnecessary costs, this class of medicine is not expensive once it gets to the point of widespread availability and standardized manufacture in bulk. Further, consider that this is the case is when the number of patients, while large, is only a tiny fraction of the overall population. When the target market is instead everyone over the age of 40, enormous economies of scale will come into play.

The concern that rejuvenation biotechnologies might cause social disparity and further widen the gap between rich and poor is one of the most commonly raised ones, probably second only to concerns of overpopulation. Like many others, this concern may appear valid at first, but it does not survive careful analysis. The underlying assumption of the argument we are discussing is that rejuvenation therapies would be so very expensive that only rich people would be able to afford them, thus fracturing the world into the ever-young, ever-healthy rich ones, and the poor, sick, old ones with no access to these technologies. It is very likely that rejuvenation therapies will be quite expensive initially due to a number of factors. However, even if we can initially assume a high cost for rejuvenation biotechnologies, we need to keep in mind that new technologies generally start off as very expensive and eventually become affordable and widespread.

For instance, it took only 15 years for full genome sequencing cost to drop from $100 million to $300, making personalised medicine a reality globally. In the field of medicine, there are several other examples of this same trend of falling cost and prices. The drug metformin, used for the treatment of type 2 diabetes (and probably the first drug to slow down aging in healthy people, which is currently the subject of the TAME clinical trial), was initially expensive but eventually its price plummeted to a few dollars. Its price fell from $1.24 per tablet in 2002 to 31 cents in 2013. Similarly, improvements in technology have drastically reduced the costs of research diagnostics, and the advent of remote technology has allowed a cost reduction for both patients and hospitals as specialists can be contacted at a distance. As an example, this means hospitals do not need to have radiologists in location all the time, but can instead remotely send them patient data for analysis and thus only pay for each individual service; this, in turn, implies potentially cheaper services for the patients as well.

Technology typically becomes much cheaper as time goes by; there is no reason to believe the same would not be true of rejuvenation technologies, especially when one takes into account an extremely strong economic motivator: The market for rejuvenation biotechnologies would be the largest in history. Every single person in the world has aging and is thus a potential customer. It is of course very likely that those with wealth and therefore greater means will obtain cutting edge technology first (as we have seen repeatedly historically) before everyone else. However, one should consider that those early adopters are playing "guinea pig" and in effect are paving the way for the masses and helping developers offset the costs incurred during the development process due to paying premium prices for early access to these technologies.

If, for the sake of the argument, we assumed that rejuvenation biotechnologies could somehow be an exception to the trend of falling prices in technology, we would need to decide whether people ending up paying for their own rejuvenation therapies is more a realistic scenario than governments subsidizing the treatments, partly or wholly. The majority of countries in the world have universal healthcare systems that take care of their citizens or residents health needs either for free or for a nominal fee. These costs are offset by taxes which ensure the health service is able to provide this level of care to all. Presently, health expenditures for the elderly constitute a considerable burden on a country's economy. Although the elderly have already contributed wealth to society when they were younger, they often stop doing so when they retire. The desired result of rejuvenation therapies leads to a much better scenario. If rejuvenation therapies are reapplied with proper timing, no individual would ever reach a state of age-related decay and poor health that could make him or her unfit for work. Consequently, the costs of treating age-related diseases using current medicine could be reduced with the arrival of more robust therapies offered by rejuvenation biotechnology. Such rejuvenation therapies aim to prevent a plethora of diseases before they manifest, potentially saving money. However, even if the costs are the same and we are simply trading one set of medicines for another, the benefit to health, quality of life and productivity makes it more than worth it regardless.

Link: http://www.lifeextensionadvocacyfoundation.org/education/only-the-rich/

You, your self, consists of the slowly shifting structural pattern of matter that holds the data of the mind. That structure is thought to reside in the synapses that connect neurons in the brain, though there is some debate on this topic and final confirmation still lies somewhere in the future. Survival after cold water drowning, in which the brain ceases all activity for a time but nonetheless carries on after rescue, adequately demonstrates that the basis of the mind is physical, not ephemeral, however, no matter where exactly it is to be found in the fine structure of brain tissue. This is important, because it means that an individual is only finally, absolutely dead and gone when that structure is destroyed. A person can be minutes past present definitions of clinical death, but still exist, still be dying in the sense that the structures of the mind are being destroyed by ischemia. Past that span of minutes it gets far more sketchy and unknown as how much of the self remains. That is a hard question to answer absent a definitive location for that data. What if the destruction can be halted, the brain preserved, however?

Preservation of the brain, the self, is the point of the cryonics industry. As soon as possible following death, the brain is cooled by stages and perfused with cryoprotectant. The result is vitrification with minimal ice crystal formation, a method demonstrated to preserve the fine structure of brain tissue, assuming a sufficiently comprehensive perfusion was achieved. There are examples of vitrified and thawed nematode worms retaining memory, and the cryobiology field is working towards reversible vitrification of organs to improve the logistics of organ transplantation and tissue engineering. When a patient is cryopreserved by a cryonics provider, the vitrified body and brain is stored in liquid nitrogen, awaiting a future with sufficiently advanced technology to undertake restoration. The individual is clinically dead, but not gone. The mind still exists, paused, and while that remains true we can envisage future combinations of molecular nanotechnology and regenerative medicine that could achieve a restoration to active life. Will that come to pass? Hopefully so, but nothing is certain. You roll the dice and look for the best odds, just as in any other decision. The odds following cryopreservation are infinitely better than those following burial or cremation. No-one comes back from oblivion.

A potential alternative to cryonics is plastination. This uses chemical fixation at room temperature instead of low-temperature vitrification, halting all biological processes by binding them up in fixative molecules while preserving the original molecular structure of the tissues. The technology needed to restore plastinated tissue is likely to be much more advanced than that needed to restore a vitrified brain: there are many more chemical reactions that need to be undone, molecule by molecule, and at the same time as kick-starting the normal cellular processes. At the present time there is no plastination industry akin to the cryonics industry that preserves people following death, but this may be nothing more than a historical accident. If the founders of the cryonics movement in the 1960s and 1970s had the chemistry background to settle on plastination, then we'd be looking back at decades of increasing experience in that technology instead. As the Brain Preservation Technology Prize contest of recent years illustrated, there isn't any great difference between the two approaches in terms of preservation of fine structure in brain tissue. Both can achieve the goal given a good methodology and absence of complications - and in both cases the burden and the challenge of restoration is placed upon future researchers. Which is fine; preserved patients can wait it out for as long as the preservation organizations continue.

How do we assess the quality of a preservation method, however? The primary methodology at the moment is the use of electron microscopy to assess the small-scale structure of neurons and synapses. It is possible to raise objections to this as a measure of success, but it is a good starting point. If significant disruption is seen here, then there is little point in looking any closer until we have a much better idea as to exactly which structures encode data. Another possible approach is to work with studies in lower species that can be preserved and restored, and assess their cognitive function after the process. That isn't possible for plastination, but has been done for vitrified nematode worms, as I mentioned above. Beyond this, what else can be attempted? In the research linked below, a novel approach is assessed in one of the common forms of plastinated brain tissue. The researchers manage to exercise some of the functionality of the preserved brain cells despite the chemical fixation process. If it can be replicated, this strikes me as a very compelling demonstration, and one that should certainly be expanded upon. I would be most interested to learn whether or not this sort of approach could be attempted in vitrified brain tissue at liquid nitrogen temperature - unfortunately I know far too little about this area of science to even guess at how one would go about such a task, or the degree to which it is possible at that temperature.

When Is the Brain Dead? Living-Like Electrophysiological Responses and Photon Emissions from Applications of Neurotransmitters in Fixed Post-Mortem Human Brains

The fundamental principle that integrates anatomy and physiology can be effectively summarized as "structure dictates function". This means the functional capacities of biological substrata are determined by the chemical composition, geometry, and spatial orientation of structural subcomponents. As the heterogeneity of structure increases within a given organ, so does the functional heterogeneity. Nowhere is this more evident than in the human brain. It can be described as a collection of partially-isolated networks which function in concert to produce consciousness, cognition, and behaviour. It also responds to its multivariate, diversely energetic environment by producing non-isotropic reflections within its micrometer and nanometer spaces. The specific spatial aggregates of these dendritic alterations result in processes that have been collectively described as memory: the representation of experience.

When structures of the brain undergo changes sufficient to terminally disrupt these functional processes and the individual is ultimately observed to lose the capacity to respond to stimuli, the brain is said to be clinically dead. This state has been assumed to be largely irreversible. It should be noted that the specific criteria which must be achieved in order to ascribe death to an individual are not universal and exhibit a significant degree of non-consensus. The precise point beyond which the brain is no longer "living", a threshold which remains unidentified, is perhaps less definite than has been historically assumed. Without life support systems, either endogenously in the form a cardiovascular network or exogenously in the form of mechanical aids, the brain degenerates progressively until full decomposition and dissolution. Complete loss of structure is strongly correlated with the complete loss of function. When the brain is dead and the tissue has lost its structural integrity, the individual is assumed to no longer be represented within what remains of the organ.

If, however, the brain is immersed within certain chemical solutions before degeneration and decomposition, the intricate and multiform structures of the human brain can be preserved for decades or perhaps centuries. The gyri and sulci which define the convex and concave landscapes of the brain's outer surface as well as the cytoarchitectural features of the cerebral cortex remain structurally distinct. The deep nuclei and surrounding tract systems remain fixed in space, unchanging in time. Though structurally intact, the functions of the brain are, however, still considered to be absent. It has been assumed that the chemical microenvironment (e.g., pH, nutrient content, ionic gradients, charge disparities, etc.) of both cells and tissues within the preserved brain must be altered to such a degree to prevent degradation that these spaces no longer represent those which underlie the cellular processes which give rise to normal human cognition and behaviour.

The principle of anatomy and physiology which describes the relationship between structure and function would hold that in the presence of structural integrity so too must there be a functional integrity. If the structure-function relationship is a physical determinant, functional capacities should scale with structural loss and vice versa. Therefore the maintenance of structure subsequent to clinical death by chemical fixation could potentially regain some basic function of the tissue to the extent to which structure and function are intimately related. Here we present lines of evidence that indicate brains preserved and maintained over 20 years in ethanol-formalin-acetic acid (EFA), a chemical fixative, retain basic functions as inferred by microvolt fluctuations and paired photon emissions within the tissue. They are both reliably induced and systematically controlled by the display of electrical and chemical probes which include the basic inhibitory and excitatory neurotransmitters or their precursors. Each of these profiles exhibit dosage-dependence and magnitude dependences that are very similar to those displayed by the living human brain. As neuroscientists we have been taught or have assumed that the fixed human brain is an unresponsive mass of organic residual that has replaced what was once a vital, complex structure that served as the physical substrate for thought, consciousness, and awareness. The results of the present experiments strongly suggest we should at least re-appraise the total validity of that assumption.

A cell might be considered a state machine whose state and state transitions are determined by the amounts of various proteins present. The process of gene expression by which genetic blueprints are converted into proteins is enormously complex, and a large fraction of the various types of molecule assembled inside a cell have much more to do with manipulating the steps involved in gene expression than with other cellular activities. Every facet of gene expression, from the pace at which proteins are produced to which protein is produced when there are multiple options for a given stretch of DNA, is subject to a constant, ever-changing set of interactions, feedback loops between production and influence over production. Researchers are these days putting a great deal of effort into mapping the classes of protein machinery involved in regulation of gene expression, such as microRNAs (miRNA), and some of that work is focused on aging:

Biomarkers of aging are biological parameters that change in a predictable direction with aging in most individuals and, when assessed early in life, may predict subsequent longevity better than chronological age alone. Beyond their prognostic utility, the discovery of biomarkers of aging is attractive because they may shed light into the intrinsic mechanism of aging as a biological process. Identifying biomarkers of aging may also provide insight into the biological mechanisms that accelerate or decelerate aging. miRNAs have emerged as important regulators of biological mechanisms that are relevant for aging. miRNAs are short non-coding RNAs that regulate gene expression. With over 1800 human miRNAs reported, miRNAs influence a wide range of biological functions, such as stem cell self-renewal, cell proliferation, apoptosis, and metabolism.

Profiles of miRNAs found in plasma and serum have been linked to numerous cancers, cognitive impairment, Alzheimer's disease and other neurodegenerative disorders, and other pathologies, indicating that miRNAs are a new class of biomarkers of human diseases present in blood. Because of the close relationship between these diseases and longevity, miRNAs may also serve as biomarkers of human aging. Our prior work has shown that miRNAs can serve as genetic biomarkers of aging in the nematode C. elegans. Because miRNAs and aging genetic pathways are conserved from nematodes to humans, an increasing number of human miRNA studies have been carried out over the past several years. These studies have shown differential abundance of multiple miRNAs in peripheral blood mononuclear cells (PBMCs) or serum/plasma when comparing younger and older adults. We used miRNA PCR arrays to measure miRNA levels in serum samples obtained longitudinally at ages 50, 55, and 60 from 16 participants of the Baltimore Longitudinal Study of Aging (BLSA) who had documented lifespans. We compared miRNA expression changes not only across (i.e., between older and younger participants) but also within participants (using the three samples taken at different ages from each individual). In accordance with recent research that found a strong association between circulating miRNAs and human aging, our study suggests that circulating miRNAs are biomarkers of longevity.

Many interesting expression profiles were observed between study participants with different lifespans. For example, when comparing samples analyzed at age 50 between the long-lived and short-lived subgroups, we identified the 10 most differentially higher and lower expressed miRNAs. The most upregulated miRNA in long-lived participants, miR-373-5p, is part of the miR-373 family, which functions as a tumor suppressor in breast cancer. The most downregulated miRNA in long-lived participants, miR-15b-5p, has been found to be upregulated in oral cancer cells. Because lifespan is a complex trait characterized by escaping, delaying, or surviving fatal age-related diseases, including cancers, further scrutiny of the potential roles of the identified miRNAs in human aging is of great importance and interest. Six of the nine miRNAs (miR-211-5p, 374a-5p, 340-3p, 376c-3p, 5095, 1225-3p) may serve as useful biomarkers, as each of the six miRNAs were correlated with lifespan and were significantly up- or downregulated. Future studies can identify how examining expression of multiple miRNAs simultaneously versus one or a few miRNAs individually would affect these correlations. While some miRNA biomarker or disease-association studies have found significant correlations only by analyzing a profile of expression of multiple miRNAs, our study did identify miRNAs that individually correlate with lifespan. Further, it is striking that miRNA expression at ages 50, 55, and 60 correlates with the eventual, quite varied lifespans of the 16 participants in our pilot study.

Link: http://dx.doi.org/10.18632/aging.101106

Growth in the clonal expansion of immune cells, the creation of many similar cells of the same lineage, and a reduction in the diversity of such lineages, is characteristic of the aged, dysfunctional immune system. The context in which this is usually discussed is the way in which the proportion of memory T cells, particularly those devoted to persistent pathogens such as cytomegalovirus that cannot be effectively cleared from the body, expands at the expense of other types of immune cell. An immune system burdened with too many memory cells focused on just a few pathogens is one that cannot effectively carry out all of its other tasks. In the research here, however, the authors argue that some forms of this age-related clonal expansion represent an attempt by the immune system to compensate for the damage and disarray of aging. Interestingly the class of cells examined here are senescent, and most other evidence suggests that various forms of senescent immune cells are not beneficial - they produce harmful effects, just like other cells do when they fall into a senescent state.

Inasmuch as immunity is a determinant of individual health and fitness, unraveling novel mechanisms of immune homeostasis in late life is of paramount interest. Comparative studies of young and old persons have documented age-related atrophy of the thymus, the contraction of diversity of the T cell receptor (TCR) repertoire, and the intrinsic inefficiency of classical TCR signaling in aged T cells. However, the elderly have highly heterogeneous health phenotypes. Studies of defined populations of persons aged 75 and older have led to the recognition of successful aging, a distinct physiologic construct characterized by high physical and cognitive functioning without measurable disability. Significantly, successful agers have a unique T cell repertoire; namely, the dominance of highly oligoclonal αβT cells expressing a diverse array of receptors normally expressed by NK cells. Despite their properties of cell senescence, these unusual NK-like T cells are functionally active effectors that do not require engagement of their clonotypic TCR.

The accumulation of NK-like CD28null T cells with advancing age represents a remodeling of the immune repertoire as a compensatory mechanism for the general age-related losses in conventional T cell-dependent immunity. There is thymic atrophy with age leading to impaired production of new naïve T cells, making older adults unable to respond to new and emerging pathogens in an antigen-specific manner. With antigenic exposure through life, there is progressive contraction of the naïve T cell compartment, with corresponding expansion of memory and senescent T cell compartment. These events over the lifespan result in the contraction of diversity of the clonotypic TCR repertoire. With cycles of expansion and death of T cells during antigenic challenges, the phenomenal accumulation of apoptosis-resistant CD28null NK-like T cells is likely a protection against clinical lymphopenia, which is very rare among older adults.

The acquisition of a diverse array of NK-related receptors on CD28null T cells maintains immunologic diversity in old age. There is co-dominant expression of diverse NK-related receptors along clonal lineages of CD28null T cells in late life. This is in stark contrast to the conventional clonotypic TCR diversity that is characteristic of the young. Signaling of these NK-related receptors effectively imparts an innate function to aged T cells; hence, we had originally introduced the term "NK-like T cells" to emphasize their NK-related receptor-driven, TCR-independent effector function. NK-like T cells compensate for the corresponding age-related functional loses in the NK cell compartment. Induction of NK-related receptors on T cells may not be surprising since T cells and NK cells originate from a common lymphoid progenitor. Thus, inducibility of NK-related receptors in senescent CD28null NK-like T cells is consistent with functional plasticity of T cells. Although the intricacies of T cell plasticity are still being investigated, such plasticity re-directs the elaboration of effector activities to ensure a vigorous immunity. In old age, signaling of effector activities of NK-like T cells through NK-related receptors is an adaptation of the aging immune system. Such adaptation is a way to maintain immune homeostasis despite the inefficiency of classical TCR signaling and the contraction of diversity of the repertoire of clonotypic TCRs. NK-like T cells are highly resistant to cell death and may represent Darwin's "fittest" lymphocytes that contribute to immune function into old age.

The expression of NK-related receptors along clonal lineages of CD28null T cells with aging clearly represents a reshaping or remodeling of the immune repertoire. T cell signaling through these receptors independent of the TCR also illustrates the emerging theme that cell senescence may not necessarily be synonymous with dysfunction. One scientific challenge is to determine what drives the induction of diversity of expression of NK-related receptors on T cells with advancing age. Another is to determine whether the TCR-independent effector function of NK-like T cells translates into vigorous immune defense and/or immune surveillance in late life.

Link: http://dx.doi.org/10.3389/fimmu.2016.00530

Today's interesting news, doing the rounds in the popular press and being gleefully misinterpreted along the way, is that, working in mice, researchers have induced temporarily increased levels of the proteins used to reprogram normal cells into pluripotent stem cells. This produced a number of short term benefits to regeneration and metabolism, though the long-term results on life span remain to be assessed. Cancer and regeneration are two sides of the same coin, and it is thought that the characteristic decline in stem cell activity with age is part of an evolved balance between risk of cancer and risk of tissue failure. Many of the methods of globally spurring greater regeneration either definitely or theoretically carry the risk of cancer. Stem cell therapies and telomerase gene therapies fall into this categories, though on the whole the cancer risk in practice has so far turned out to be lower than the cancer risk in theory. The reasons for this remain to be fully explored. Nonetheless, the whole complex system of a few stem cells with unlimited replication supporting a tissue of many somatic cells with tightly limited replication that exists in near all species came into being in the evolutionary context of cancer. We depend upon biological structures that are self-repairing and resilient in many ways, but that are very vulnerable to cellular malfunctions of uncontrolled growth that distort the structure and disrupt correct function. So where we are less self-repairing and resilient than we might be, cancer is the first and most obvious culprit when considering the evolutionary history that created us.

It has been a decade since researchers first figured out how to reprogram normal adult cells into induced pluripotent stem cells, capable of forming any cell, but likely to do who knows what if put into the context of living tissue. Reprogramming occurs in a cell culture, using a cell sample, not in a living organism. This reprogramming actually involves surprisingly few changes, dialing up the gene expression of a few specific proteins, with the first attempts using Oct4, Sox2, cMyc, and Klf4. The use of induced pluripotent stem cells in medicine is a matter of developing a methodology that will differentiate the pluripotent cells into the desired type of stem or progenitor cell appropriate to the tissue in question, using the patient's own cells as a starting point so that the resulting therapeutic cells are matched perfectly. That is fairly safe, given suitable testing, and will eventually provide a cost-effective source of all the cells needed for the next generation of regenerative medicine and tissue engineering. The cells put in place match those already present in the tissue, and should pick up on the same environment of signals and undertake the appropriate work of regeneration. Delivering pluripotent stem cells as-is, on the other hand, is just asking for cancer: it is more or less the same thing as putting precancerous cells into the patient. There is no control or guidance, and what happens next is up to the hand of fate.

Given this, one would think that taking the next step and using gene therapy to upregulate the reprogramming proteins in a living individual would be even worse. In addition to a whole bunch of newly pluripotent cells, you have newly pluripotent cells appearing in random locations and changed from random cells with random levels of preexisting damage. None of this sounds particularly safe. In fact, that experiment has been carried out in mice, and as you might expect the result is the development of cancers. The newly created pluripotent cells start building whatever springs to mind, wherever they happen to be. However, there are several examples we can point to in which dialing up protein production permanently is disastrous, but turning it up intermittently is quite beneficial. One good example is the tumor suppressor gene p53, which if producing proteins all the time will, in addition to even more effectively reducing cancer risk, accelerate aging by suppressing processes that are also necessary to regeneration and tissue maintenance. Cancer and regeneration use the same mechanisms - one is simply more regulated than the other. Most of the tumor suppression genes that have been cataloged target these shared mechanisms. Yet producing additional p53 only when regulatory processes determine it is needed, suppresses cancer more effectively without accelerating aging.

In this context the researchers here use a methodology of temporarily increasing expression of the pluripotency genes Oct4, Sox2, cMyc, and Klf4 in mice. They do this in cell cultures, then in mice with an accelerated aging disorder - actually a dysfunction of the cellular structure akin to that in human progeria - and then in normal aged adult mice. I think it a good idea to ignore the first two of these. Cell cultures are not living animals, and we should usually pay little attention to studies on accelerated aging models for the same reason we should pay little attention to studies in poisoned mice. Progeria and poisoning are both conditions that have little relevance to normal aging, being an accumulation of cell damage that doesn't occur to any large degree in normal aging, so it is often very hard to determine whether or not the results are in any way useful. If you produce a lot of damage and then help work around that damage, but none of the above actually happens in the course of ordinary aging, what does that say? The answer depends on details specific to each case that most of us are not knowledgeable enough to assess.

Fortunately here the researchers did undertake a study in normal mice. Unfortunately it was only a short-term study, so considerations of life span and longer term outcomes, such as cancer rate, will have to wait. Still, as an additional data point in the larger picture of what can be done to enhance regeneration in mammals, it is interesting. We can consider all sorts of plausible candidate mechanisms that have been explored in past years and likely overlap with those involved in the outcomes produced by stem cell transplants and telomerase gene therapies. That said, this upregulation of pluripotency factors is certainly something that I'd put in the "very unwise" bucket, should you find yourself in the position to undergo such a gene therapy in the years ahead. It is much more risky than telomerase gene therapy, and that in and of itself looks like something to skip until more data on the outcomes in larger mammals arrives.

Turning back time: Salk scientists reverse signs of aging

As people in modern societies live longer, their risk of developing age-related diseases goes up. In fact, data shows that the biggest risk factor for heart disease, cancer and neurodegenerative disorders is simply age. One clue to halting or reversing aging lies in the study of cellular reprogramming, a process in which the expression of four genes known as the Yamanaka factors allows scientists to convert any cell into induced pluripotent stem cells (iPSCs). Like embryonic stem calls, iPSCs are capable of dividing indefinitely and becoming any cell type present in our body. "What we and other stem-cell labs have observed is that when you induce cellular reprogramming, cells look younger. The next question was whether we could induce this rejuvenation process in a live animal."

While cellular rejuvenation certainly sounds desirable, a process that works for laboratory cells is not necessarily a good idea for an entire organism. For one thing, although rapid cell division is critical in growing embryos, in adults such growth is one of the hallmarks of cancer. For another, having large numbers of cells revert back to embryonic status in an adult could result in organ failure, ultimately leading to death. For these reasons, the team wondered whether they could avoid cancer and improve aging characteristics by inducing the Yamanaka factors for a short period of time. To find out, the team turned to a rare genetic disease called progeria. Both mice and humans with progeria show many signs of aging including DNA damage, organ dysfunction and dramatically shortened lifespan. Moreover, the chemical marks on DNA responsible for the regulation of genes and protection of our genome, known as epigenetic marks, are prematurely dysregulated in progeria mice and humans. Importantly, epigenetic marks are modified during cellular reprogramming.

Using skin cells from mice with progeria, the team induced the Yamanaka factors for a short duration. When they examined the cells using standard laboratory methods, the cells showed reversal of multiple aging hallmarks without losing their skin-cell identity. Encouraged by this result, the team used the same short reprogramming method during cyclic periods in live mice with progeria. The results were striking: Compared to untreated mice, the reprogrammed mice looked younger; their cardiovascular and other organ function improved and - most surprising of all - they lived 30 percent longer, yet did not develop cancer. Lastly, the scientists turned their efforts to normal, aged mice. In these animals, the cyclic induction of the Yamanaka factors led to improvement in the regeneration capacity of pancreas and muscle. In this case, injured pancreas and muscle healed faster in aged mice that were reprogrammed, indicating a clear improvement in the quality of life by cellular reprogramming.

In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming

The last decade of scientific research has dramatically improved our understanding of the aging process. The notion that cells undergo a unidirectional differentiation process during development was proved wrong by the experimental demonstration that a terminally differentiated cell can be reprogrammed into a pluripotent embryonic-like state. Cellular reprogramming to pluripotency by forced expression of the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc [OSKM]) occurs through the global remodeling of epigenetic marks.

Although in vitro studies have been informative, the physiological complexity of the aging process demands an in vivo approach to better understand how reprogramming may affect cellular and organismal aging. Breakthrough studies have shown that cellular reprogramming to pluripotency, although associated with tumor development (e.g., teratoma formation), can be achieved in vivo in mice by the forced expression of the Yamanaka factors. In addition, we and other groups have demonstrated that partial reprogramming in vitro by transient expression of OSKM can induce a dedifferentiated progenitor-like state. Together, these observations suggest that cellular reprogramming may be used to promote tissue regeneration and led us to hypothesize that in vivo partial reprogramming could slow or reverse the aging process and extend organismal lifespan. Here, we report that cyclic in vivo induction of OSKM in a mouse model of premature aging improves age-associated phenotypes and extends lifespan. In addition, we demonstrate the amelioration of cellular phenotypes associated with aging by short-term induction of the Yamanaka factors in mouse and human cells. Finally, we show that short-term expression of OSKM alleviates pancreatic and muscle injury in older wild-type (WT) mice.

Our observations may reinforce the potential role of epigenetic changes as drivers of aging and highlight the plasticity of the aging process, which might be altered by cellular reprogramming in vivo. In addition, our results suggest that aged cells undergo a process of molecular rejuvenation during the initial stages of cellular reprogramming to pluripotency. Failure to erase critical hallmarks of aging may lead to refractory populations of cells and cellular senescence. Due to the complexity of the reprogramming and aging processes, future studies will be necessary to investigate whether partial reprogramming can ameliorate aging hallmarks during physiological aging and to better understand the molecular mechanisms behind this phenomenon. This information will be necessary if we are to develop accurate and efficient epigenetic remodeling strategies toward maximizing the beneficial effects of in vivo reprogramming while avoiding potential risks associated with the in vivo expression of the Yamanaka factors.

Reading the whole of the analysis in the paper, I have to say that I think these researchers have a lot of the picture back to front. Putting epigenetic changes front and center as a primary mechanism in aging, as opposed to a reaction to rising levels of cell and tissue damage, is the cart in front of the horse. Sure, those epigenetic changes cause further problems, but focusing on targeting them won't remove the primary damage that causes aging. It only forces the damaged engine to work harder. Maybe that produces benefits, as it seems to in stem cell therapies that work via signaling to put existing cells back to work, but it isn't solving the real problem.

Google founded the California Life Company, or Calico Labs, to work on aging, and has put a large amount of money into this project. It is all comparatively secretive, but so far the evidence suggests that this will, sadly, turn out much the same way as the Ellison Medical Foundation, which is to say (a) work on extending the map of all cellular biochemistry relevant to the progression of aging at the most detailed level coupled to (b) attempts to slightly slow aging via pharmaceuticals. The project is headed by someone who has little interest in translational research, the business of bringing therapies to market, and those involved - for the most part - are not people with a track record of paying attention to the SENS program of repairing damage to produce rejuvenation. The SENS research agenda is to my eyes the only viable way forward to produce meaningful extension of healthy life any time soon, and certainly the only way to help older people by turning back aging at later stages. It is also far closer to realization and far less expensive to develop than efforts to safely alter human metabolism to slow the rate at which damage is done. The field of aging research has all too little funding in comparison to its potential, but it doesn't suffer from a lack of fundamental research anywhere near as much as it suffers from a lack of taking what is already well known about the forms of cell and tissue damage that cause aging in order to build therapies here and now.

David Botstein is Calico's chief scientific officer. He is 74, with a grizzled shadow of beard reaching up from his collar. In November, I found him at a lecture hall at MIT, where he offered a rare window onto experiments under way at Calico. Botstein, a well-known Princeton geneticist whom Calico recruited out of near retirement, was in town to celebrate the birthday of a successful former student, now a sexagenarian. "The pleasure is coming to see old friends. The not-so-­pleasure is if these guys are 60, what am I?" In his lecture, Botstein described several technologies - four, in fact - that Calico has for isolating old yeast cells from the daughter cells that bud off them. These old cells are tracked and subjected to a comprehensive analysis of which genes are turned up or turned down, a technique that is Botstein's specialty. Botstein told me Calico is exactly what Google intended: a Bell Labs working on fundamental questions, with the best people, the best technology, and the most money. "Instead of ideas chasing the money, they have given us a very handsome sum of money and want us to do something about the fact that we know so little about aging. It's a hard problem; it's an unmet need; it is exactly what Larry Page thinks it is. It's something to which no one is really in a position to pay enough attention, until maybe us."

Botstein says no one is going to live forever - that would be perpetual motion which defies the laws of thermodynamics. But he says ­Cynthia Kenyon's experiments on worms are a "perfectly good" example of the life span's malleability. So is the fact that rats fed near-starvation diets can live as much as 45 percent longer. The studies Botstein described in yeast cells concerned a fundamental trade-off that cells make. In good times, with lots of food, they grow fast. Under stresses like heat, starvation, or aging, they hunker down to survive, grow slowly, and often live longer than normal. "Shields down or shields up," as ­Botstein puts it. Such trade-offs are handled through biochemical pathways that respond to nutrients; one is called TOR, and another involves insulin. These pathways have already been well explored by other scientists, but Calico is revisiting them using the newest technology. "A lot of our effort is in trying to verify or falsify some of the theories," Botstein says, adding that he thinks much of the science on aging so far is best consumed "with a dose of sodium chloride." Some molecules touted as youth elixirs that can act through such pathways - like resveratrol, a compound in red wine - never lived up to their early hype.

According to Botstein, aging research is still seeking a truly big insight. Imagine, he says, doctors fighting infections without knowing what a virus is. Or think back to cancer research in the 1960s. There were plenty of theories then. But it was the discovery of oncogenes - specific genes able to turn cells cancerous-that provided scientists with their first real understanding of what causes tumors. "What we are looking for, I think above everything else, is to be able to contribute to a transformation like that. We'd like to find ways for people to have a longer and healthier life. But by how much, and how - well, I don't know." Botstein says a "best case" scenario is that Calico will have something profound to offer the world in 10 years. That time line explains why the company declines media interviews. "There will be nothing to say for a very long time, except for some incremental scientific things. That is the problem."

To some, Calico's heavy bet on basic biology is a wrong turn. The company is "my biggest disappointment right now," says Aubrey de Grey, an influential proponent of attempts to intervene in the aging process and chief science officer of the SENS Research Foundation, a charity an hour's drive from Calico that promotes rejuvenation technology. It is being driven, he complains, "by the assumption that we still do not understand aging well enough to have a chance to develop therapies." Indeed, some competitors are far more aggressive in pursuing interventions than Calico is. "They are very committed to these fundamental mechanisms, and bless them for doing that. But we are committed to putting drugs into the clinic and we might do it first," says Nathaniel David, president and cofounder of Unity Biotechnology. This year, investors put $127 million behind Unity, a startup in San Francisco that's developing drugs to zap older, "senescent" cells that have stopped dividing. These cells are suspected of releasing cocktails of unhelpful old-age signals, and by killing them, Unity's drugs could act to rejuvenate tissues. The company plans to start with a modestly ambitious test in arthritic knees. De Grey's SENS Foundation, for its part, has funded Oisin Biotechnologies, a startup aiming to rid bodies of senescent cells using gene therapy.

Link: https://www.technologyreview.com/s/603087/googles-long-strange-life-span-trip/

Researchers have demonstrated a number of genetic and pharmacological approaches that seem to modulate cellular senescence, either by somewhat lowering the number of cells that become senescent or by somewhat reducing the impact of the senescence-associated secretory phenotype (SASP). Senescent cells are one of the root causes of aging. They produce damage and age-related disease through the signals they secrete, which cause inflammation, remodel surrounding tissue structures, and alter the behavior of normal cells for the worse. Many of the methods that over the years have been demonstrated to modestly slow aging in laboratory animals have some sort of effect on the properties of cellular senescence, but a potential therapy based on these methods would have to be far, far more effective in order to compete with selective destruction of senescent cells as an approach to the problem. The cell culture research here is an example of present explorations into altering the processes of senescence rather than simply destroying the unwanted cells, but I can't say that I see it as being all that promising for anything other than the production of greater knowledge of the senescent state.

Cellular senescence is a hallmark of aging and senescent cells accumulate with age in vivo in mammals; this is thought to drive aging by limiting tissue replicative capacity and causing tissue dysfunction. Developing strategies to delay the onset of senescence or remove senescent cells may provide a route to preventing age-related disease. Targeting senescence as a means to combat aging and age-related diseases is, however, challenging due to its antagonistically pleiotropic nature - any treatment needs to limit the deleterious impacts of senescent cells without impacting the potent barrier against tumorigenesis. While caloric restriction has been reported to extend healthspan in macaques, the most promising candidate for a longevity therapeutic in mammals is rapamycin.

Rapamycin mechanistically acts by binding the protein FKBP12, producing a complex which can bind and inhibit mTOR. mTOR constitutes the point at which diverse environmental signals are coordinated into a cellular response, regulating pathways including cell growth, proliferation, survival, motility and protein synthesis. mTOR is present in two complexes in metazoa, mTORC1 and mTORC2, which have different components and functions. Rapamycin inhibits mTORC1, but chronic treatment may also disrupt mTORC2. While rapamycin extends lifespan in mice even when administered in middle age, it has significant side-effects that may limit its use in humans. We have therefore explored the potential of second generation rapalogs i.e. pharmacological agents that inhibit mTORC but act not through binding to FKBP12 but instead as mTORC-specific ATP mimetics. AZD8055 is an ATP-competitive inhibitor of mTOR kinase in both mTORC1 and mTORC2. AZD8055 has anti-proliferative effects similar to those of rapamycin and has been taken forward into clinical trials against various forms of cancer.

Here, we test whether acute mTORC inhibition can alter features of senescence in cells that have already undergone a large number of population doublings (PD) - as they are about to undergo senescence but are currently still proliferating, we term these populations 'near-senescent'. Such high cumulative PD (CPD) near-senescent cells show many signs characteristic of senescence including increased size and granularity, SA-β-gal staining, high lysosomal content and accumulation of actin stress fibers. They are still capable of cell proliferation, albeit with a reduced rate of proliferation compared with cells at lower CPD. Here, we test the effect of inhibiting both mTORC1 and mTORC2 using the TOR-specific ATP mimetic AZD8055. Remarkably, we demonstrate significant reversal of major phenotypes of senescence on short term low dose pan-TOR inhibition. We therefore suggest that AZD8055 may prove useful in modulating health outcomes in late life.

Link: https://dx.doi.org/10.18632/aging.100872