"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.

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.

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.

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.

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.

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.

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. Its price fell from 1.24 per tablet in 2002 to 0.31 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.

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.

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."

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.

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.

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.

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.

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.

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.

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.