The development of a reliable and accurate biomarker of biological age is an important step for the longevity science field. Testing potential rejuvenation therapies is at present a drawn-out and expensive process, as the only truly effective way to determine outcomes is to wait and see. That requires years and millions of dollars in funding for mouse studies, a cost that greatly restricts the amount of experimentation and exploration it is possible to carry out, even for the better funded research groups. If instead a biomarker test could be applied shortly before and shortly after a treatment in order to assess its potential, that would greatly accelerate progress in the field. Epigenetic clocks based on assessment of patterns of DNA methylation are presently the most promising candidate for such a biomarker of aging, and here researchers discuss the behavior of presently established clocks in mice and humans.

Epigenetic clocks provide powerful tools to evaluate nutritional, hormonal, and genetic effects on aging. What can we learn from differences between species in how these clocks tick? One of the most fascinating findings in human aging is that it is associated with highly reproducible DNA methylation (DNAm) changes. DNAm levels at age-associated CG dinucleotides (CpG sites) can be integrated into epigenetic age predictors, which provide robust biomarkers to estimate chronological age. With the advent of more and more publically available DNAm profiles, such aging signatures were further developed to facilitate higher precision in age predictions, particularly for blood samples. Probably the most commonly used epigenetic aging signature is based on DNAm levels at 353 CpG sites and facilitates relatively precise age predictions for many human tissues: the median "error" (MAE), defined by the median absolute difference between DNAm age and chronological age, is usually less than 4 years.

Now - about 6 years after the first epigenetic clock paper - similar age predictors have been established for mice. Again, they were initially described for defined murine tissues, specifically liver and blood, taking into account the fact that there are notoriously large differences in the epigenetic makeup of cells from different tissues. However, it is also possible to derive a multi-tissue murine DNAm age predictor, in analogy to the most commonly used human clock. The signature is based on 329 CpGs and has been validated for cortex, muscle, lung, liver, and heart tissue. Overall, the multi-tissue age predictor reached a MAE of less than 4 weeks, although how it performs in other tissues has yet to be shown.

Studies indicate that the epigenetic clocks of mice tick faster than those of humans. This can be anticipated because the maximum life-span of mice (about 2 years) is much shorter than it is in humans (about 85 years). If the molecular changes of aging are linked to life expectancy and generation time, then this might support the notion that aging reflects a controlled evolutionary process. However, there is still an open debate on whether aging is due to an accumulation of cellular defects, or is driven by a developmental mechanism. Either way, comparison of epigenetic clocks in mice and men will provide new insights into the regulation of age-associated DNAm.

Direct comparison of age-associated CpGs in mice and men indicated that there is a moderate but significant association between the two species. It is not always trivial to identify orthologous CpG sites, and further interspecies comparison will be required to better understand similarities and differences of age-associated genomic regions. However, the overlap of age-associated CpGs in age predictors for human and mice seems to be rather low, and hence epigenetic clocks need to be trained specifically for different species. In terms of function, age-associated CpGs in humans and mice seem to be enriched in genes that are involved in morphogenesis and development. However, in both species age-associated DNAm changes are not generally reflected at the gene expression level - and thus the biological relevance remains largely unclear.

Mouse DNAm clocks provide powerful tools to study longevity interventions in one of the most relevant model organisms for aging research. These signatures were initially trained to correlate with the "real" chronological age of mice - but aging rates may differ between individuals. In fact, there is evidence that epigenetic clocks rather reflect the biological age, which is related to the perceived aging process of an organism. In analogy, it was previously demonstrated that human DNAm age is related to life expectancy: accelerated epigenetic age is associated with higher all-cause mortality. This finding has been validated in various additional cohorts and with different epigenetic age predictors. Furthermore, human epigenetic aging rates have been shown to be significantly associated with sex, race/ethnicity, and some disease risk factors. In mice, there was no clear difference in predicted DNAm age of male and females. However, ovariectomy, which reduces the average life span in female rats, results also in significant age acceleration. Caloric restriction or dietary rapamycin treatment, both of which result in increased life expectancy of mice, reduced epigenetic age. In humans, specific diet seems to have a less pronounced impact on epigenetic age, but there is significant association of DNAm age and body mass index (BMI). Apparently, different parameters can affect biological aging in mice and men.

Link: https://doi.org/10.1186/s13059-017-1245-8

Following on from a recent post on the subject, here is another article from the Life Extension Advocacy Foundation (LEAF) on strategies and efforts to persuade the world to support work aimed at greatly lengthening healthy human life spans. For those of us to whom it is obvious that a very large amount of time and funding should be devoted to this goal, because aging is by far the greatest source of suffering and death, because the cost of bringing aging under medical control is small in comparison to what is spent on trying and failing to cope with it, and for a score of other equally good reasons, it can be frustrating to see that others do not presently think that way. They seem happy to march to their deaths, while at the same time happy to avail themselves of the small gains resulting from the comparatively crude medicine for age-related disease that is presently available. If rejuvenation therapies existed, these same people would use them and be thankful for them, but very few will so much as raise a finger to help create these technologies. It is a challenge.

One of the most frequent questions we at LEAF hear is this: when will innovative therapies to delay, stop and eventually reverse age-related damage become available? This is not an easy question to answer, because the pace of progress depends on many factors - predominantly, funding. Fundamental studies on aging are not well-funded and the accumulation of knowledge necessary to proceed from lab work to clinical trials and then clinical practice does not happen fast enough. Government funding is more often allocated to mainstream areas, such as research on single diseases. Business, for its part, does not show much interest in fundamental science, because usually there is no final product that can be sold. The only other source of funding is the general public. But most people are not yet sufficiently informed about the plausibility of bringing the aging processes under medical control and do not share the values of our community. Sometimes (to be frank, pretty often) activists trying to engage the public in an enlightening conversation can encounter resistance or even rejection.

Learning requires active, conscious participation. This means that students will seek and absorb information if they are interested, or they will ignore it if they do not see any personal benefit in it. So what do most people want? It's life extension right? Wrong! Studies show that when people are asked "how long would you like to live?" with no other conditions specified, people added around 5 to 10 years to the average life expectancy for their country and that was it. If we do not first explain the aging processes, the connection with age-related diseases and the repair based solutions that lead to healthy longevity, people think longevity means a longer life of prolonged ill health and frailty. Who wants to spend another ten or twenty years in a wheelchair or in a care home? And this is exactly the image most people have when these words are used. This is what the expression "life extension" means to the majority of people - and this is why we should avoid beginning the conversation with it.

At the same time, sociological studies show that if the possibility of perfect health throughout life is introduced into the equation from the very beginning, people show much more interest and support for the idea of ​​prolonging life. People literally switch from one camp to the other, those who just did not want to live for more than 80 years now decide they want to live to more than a hundred, and those who just wanted to get to 120 are suddenly ready to live to 150 or beyond. People are ready to support the development of new medical technologies, even scary ones like gene therapies or gene editing, under the condition they are going to be used for treatment. However their use for the prolongation of life belongs to the category of "human enhancement" and as such the idea is most often rejected. So it is highly recommended for advocates to add that these technologies will help treat or prevent serious chronic diseases, while extended lifespans will just be a possible nice side-effect.

Link: http://www.leafscience.org/effective-advocacy/

The SENS Research Foundation annual reports tend to arrive in the middle of the following year, and today the 2016 report was published. You can find it in PDF format at the foundation website. The story of SENS rejuvenation research, approaches that aim to repair the cell and tissue damage that causes aging, is one of growth and success over the years. It has been a bootstrapping from idea to reality, powered by the philanthropy and determined support of our community. We have come a long way and achieved a great deal these past fifteen years. Yet there remains the upward curve ahead, and the completion of the vision of an end to aging has yet to be accomplished.

It is true that the first SENS therapies are on the way to the clinic, their commercial development funded by venture capital now, and senescent cell clearance is at the front of the pack. But equally important approaches to removing the damage that causes aging, such as the breaking of glucosepane cross-links, are still in the laboratory, still entirely funded by charitable donations, still building the infrastructure and running the tests in search of the first potential basis for a working therapy. When that first breakthrough is made, matters speed up considerable and funding comes running from many sources - but getting there is a slow grind. The more we can do to help the SENS Research Foundation thrive, the faster they can push forward with this stage of development: planting the seeds that will blossom into vast medical industries, and in doing so bring great benefit to humanity.

SENS Research Foundation 2016 Annual Report (PDF)

Since SENS Research Foundation's founding in 2009, we've worked toward bringing our vision of a world free of age-related disease from concept to reality. In challenging ourselves on this front, we have likewise challenged you, our supporters. We've asked a lot of all of you, and not only have you accepted this challenge, you have delivered. The rejuvenation biotechnology community that has emerged over the past several years owes its existence to each and every one of you. You have become our most vocal advocates. Over 2000 of you have become our funders.

We asked you to help us change how the world researches and treats age- related disease. You did. Through the efforts of our donors, collaborators, and our advisory board, world-renowned institutions are pursuing age- related disease research specifically focused on the damage-repair paradigm. We asked you to help us move from basic research to translational research and clinical trials. You did. In 2016 we launched Project|21, our five-year plan to help move rejuvenation biotechnologies from concept to human clinical trials. Project|21 is now backed by a number of generous and forward-thinking individuals.

You asked us to follow through. We did. In lending your support, you place not only resources in our hands, but trust. We know that a world-changing nonprofit cannot operate on the power of vision alone; and we are here not just to inspire, but to deliver results. The purpose of this report is to demonstrate concrete examples of those results to you. With your help, we have taken great steps toward the establishment of a robust rejuvenation biotechnology industry and the realization of our vision. And every step we are able to take is proof of the power of your community.

Death-Resistant Cells: Toward Neutralizing the SASP

Buck Institute researchers led by Dr. Judith Campisi had shown that the presence of senescent cells alongside cancer cells can stimulate those cells to both multiply more rapidly and to spread to other parts of the body - the metastasis process, which ultimately makes most cancers so deadly. Repeating these studies in cell culture while inhibiting the senescence-associated secretory phenotype (SASP) with apigenin almost completely nullified the proliferation-stimulating and pro-metastatic effects of senescent cells on breast cancer cells. Drugs based on parts of apigenin's structure could dampen some of the harmful effects the SASP in senescent cells. Removing these cells is the ultimate solution to these problems, and in the last year several groups have made rapid progress toward this goal. In the meantime, these studies using apigenin may demonstrate important principles from which senescent-cell-focused rejuvenation biotechnologies may be derived.

Target Prioritization of Tissue Crosslinking

Our arteries slowly stiffen with age, in substantial part because of random crosslinking of the structural proteins collagen and elastin. Developing rejuvenation biotechnologies to break these crosslinks is key to restoring youthful arterial function. To tease out the effects and relative importance of all of these different sources of crosslinking in aging tissues, the Babraham Institute team has been studying the crosslinking process in the tissues of aging mice. This has required the development and validation of new experimental methods and assays, which are now ready for use. The team has evaluated multiple tissues for crosslink presence. Importantly, some of the crosslinks that have been reported by others to accumulate in aging tissues were not detected. While further studies are needed to confirm it decisively, these results suggest that several crosslinks now believed to accumulate in aging tissues may actually be experimental artifacts.

Engineering New Mitochondrial Genes to Restore Mitochondrial Function

Free radicals derived from our energy-producing mitochondria can mutate the organelle's DNA, leading to deletions of large stretches of the mitochondrial genome. These deletion mutations prevent the mitochondria from building various pieces of the electron transport chain (ETC), with which mitochondria generate most cellular energy. The accumulation of deletion-mutation-containing cells is a significant consequence of aging. A potential rejuvenation biotechnology to recover ETC function is the allotopic expression of functional mitochondrial genes: placing "backup copies" of all of the protein-coding genes of the mitochondria in the "safe harbor" of the nucleus, thereby giving the mitochondrion all of the proteins it needs to continue producing energy normally even when the original mitochondrial copies have been mutated.

This year, the SRF MitoSENS team reported a tremendous success: for what they believe is the first time, they have used allotopic expression to rescue the complete loss of a mitochondrially-encoded protein in a mammalian cell. A publication announced their success in the fall of 2016. The results show that their targeted and recoded ATP8 protein can be expressed from the nucleus, turned into protein in both normal and mutant cells, and efficiently targeted to the mitochondria. Furthermore, they can demonstrate functional rescue of cells. Under conditions where mutant cells die for lack of ability to produce energy, the cells with engineered allotopically-expressed proteins were able to survive and replicate. In addition to ATP8, the SRF MitoSENS team has further demonstrated expression and targeting of a second re-engineered protein, ATP6. It is proof-of-concept that ATP8 is not a special case.

Identification of the Genetic Basis of ALT in Cancer

Telomeres shorten every time a cell divides, and thus all cancers have to find a way to keep their telomeres long enough to prevent senescence or death. Most cancers use an enzyme called telomerase for this purpose, but about 10-15% of cancers use a telomerase-independent mechanism known as Alternative Lengthening of Telomeres (ALT). The ALT mechanism remains largely a mystery, and therefore the OncoSENS team at SENS Research Foundation is working hard to find new ways to attack ALT cancers. First, the team has developed and established two separate high-throughput assays measuring different ALT-specific biomarkers. These assays will finally enable cancer researchers to screen hundreds of thousands of compounds across multiple drug libraries, or even test every single one of the more than 20,000 genes in the human genome, for ways to shut down ALT cancers. In addition to their biomarker work, the team is also pursuing more targeted methods to kill ALT cancer cells.

Glucosepane Crosslinks and Routes to Cleavage

One major cause of crosslink accumulation in aging is Advanced Glycation Endproducts (AGE), and one AGE in particular, called glucosepane, is currently thought to be the single largest contributor to tissue AGE crosslinking. The Yale AGE team is studying the role of AGEs in aging, and developing novel tools and strategies for reversing AGE-mediated protein damage and develop new antibodies and reagents to enable rejuvenation research. Our pilot lab at Cambridge University found that all of the commercially-available antibodies for the major AGE-related molecules are actually highly unreliable. This is a serious impediment. The Yale glucosepane team is now tackling this problem via the novel chemistry and methods they have developed. In the last twelve months, the Yale team has made exciting progress in their work. Most notably, they have developed the first synthetic route to produce glucosepane. Their novel synthetic strategy is the first ever to provide high yields of pure samples of glucosepane, putting them (and soon other scientists) for the first time in a position to explore mechanisms through which crosslinks can be broken.

In collaboration with a colleague at Yale, they have also developed a high-throughput assay for screening proprietary libraries of organic catalysts for agents capable of breaking synthetic glucosepane. One of these libraries has already been taken forward for proof of concept, which led to the identification of several leads for catalysts that could be capable of breaking glucosepane. Beyond that, the Yale group has successfully generated proteins containing their synthetic glucosepane that can be used to identify antibodies that label glucosepane-containing proteins. These antibodies will enable the immunochemical detection of glucosepane crosslinks for a wide range of applications.

Tissue-Engineered Thymus

The thymus gland is responsible for the development of a class of immune cells called T-cells. As part of the degenerative aging process, the thymus shrinks in size. This process of thymic atrophy prevents the body from maturing new T-cells, progressively weakening the immune system's ability to fight off never-before-encountered infections. Engineering new, healthy thymic tissue would help to restore the vigorous immune response of youth. SENS Research Foundation has therefore funded a Wake Forest Institute for Regenerative Medicine (WFIRM) group to apply tissue engineering techniques to the creation of functional thymic tissue to fortify or replace the aging thymus. Engineering new tissues requires a "scaffold" in which to embed cells to give them structure and functional cues, and the WFIRM group has tested different scaffolding systems: decellularized donor scaffolds and hydrogels.

In the decellularized scaffold paradigm, an organ of the type that is needed is taken from a donor, but is then stripped of its original cells and DNA, leaving behind a protein structure with low potential for immunological rejection that can be repopulated with cells taken from the new organ recipient. The WFIRM group initially began work in this paradigm using mouse organs, but they found that once decellularized, mouse thymuses lacked the rigidity to serve in that role. They accordingly moved on to the pig thymus - a species that not only worked well as an experimental system, but has some clinical potential as well. The pig is closer to humans both immunologically and in terms of size.

Catalytic Antibodies Targeting Transthyretin Amyloid

As part of the degenerative aging process, proteins that normally remain dissolved in bodily fluids become damaged, and adopt a misfolded form called amyloid. Amyloid composed of the transporter protein transthyretin (TTR) deposits in the heart and other organs with age, beginning to impair heart function. With SRF funding, the University of Texas-Houston Medical School (UTHMS) extracellular aggregate team is working to develop novel catalytic antibodies ("catabodies") that would recognize and cleave TTR amyloid deposited in the heart and other tissues. Catabodies have the potential to be safer and more effective than conventional antibody-based immunotherapies: their catalytic activity minimizes the amount of antibody required to clear deposits from tissues, and the fact that they don't form stable complexes with their targets or engage immune cells is expected to minimize the inflammatory side-effects seen with other experimental antibody therapies.

Work has resulted in the identification of two powerful TTR-cleaving catabodies. When tested for their ability to degrade misfolded wild-type TTR, these candidates were able to hydrolyze both soluble aggregates and deposit-like particulates, while having no effect on either TTR in its healthy, normal conformation or on a selection of fourteen other physiologically important proteins. Concentrations required to disintegrate 80% of a sample of amyloid were many hundreds of times lower than those routinely achieved in the blood using other infused antibodies. The establishment of stable cell lines will enable larger-scale production, as the team works to develop these candidates into functional rejuvenation biotechnologies.

Rejuvenation of the Systemic Environment

There might be a misunderstanding of what was really going on in parabiosis. When animals are connected, they are not just given reciprocal blood transfusions, but are surgically joined together. So in addition to receiving young blood, the old animals also have their old blood filtered through the young animals' livers and kidneys, and diluted with the young pairmate's own blood. Might the effects of parabiosis mostly come from the removal of toxic or suppressive factors from the old animals' sluggish circulation instead of from the delivery of active rejuvenating factors?

To test this possibility - and to accelerate identification and testing of potential pro- and anti-rejuvenation factors in the exchanged blood - SENS Research Foundation funded Dr. Conboy and the UC Berkeley systemic environment team's development of a novel technological platform. Using a mixture of off-the-shelf and custom 3-D printed parts, this platform enables the group to easily and safely extract blood from small animals and transfuse it quickly and directly into another animal, without the reciprocal exchange of its blood or the passage of its blood through the pairmate's system. It thus separates the effects of the young animals' metabolic and excretory systems from the pure effects of their blood.

The team then used the new system to repeat key parabiosis experiments from Dr. Conboy's and others' labs. As compared with the impact of full-on parabiosis, the effects of isolated young blood on old muscles' ability to repair an injury were still substantial: the stem cells recovered significant regenerative powers, and less residual fibrosis remained after the wound was resolved. But by contrast, previously-reported benefits of parabiosis in the brain and the liver were either not present, or were far more modest. Another critical finding was the confirmation of suppressive factors in the old animals' blood, which inhibited neurogenesis and other regenerative responses of young animals transfused with it. While this clearer picture of the basis of the "parabiosis effect" indicates a lower likelihood of isolating true pro-rejuvenation factors in the blood of young mice, we are nonetheless closer to being able to filter out factors responsible for suppressing the regenerative potential of an older body.

Commercial Development

Two of the companies SENS Research Foundation has supported are moving to raise funding to move their research from the lab to clinical trials. Ichor Therapeutics announced a Series A offering to bring its Lysoclear product for age-related macular degeneration and Stargardt's macular degeneration through Phase I clinical trials. In 2014, Ichor Therapeutics completed a material and technology transfer agreement for rights to concepts and research pioneered by SENS Research Foundation. Lysoclear, which Ichor announced in 2017, is a recombinant enzyme product based on extending SRF's prior work that selectively localizes to the lysosomes of retinal pigment epithelium cells where A2E accumulates, and destroys it. Ongoing studies suggest that Lysoclear is safe and effective at targeting A2E, the main toxin driving these diseases, eliminating up to 10% with each dose. This product would be the first clinical candidate based on concepts and research pioneered by SENS Research Foundation.

Oisin Biotechnologies is focused on the genetic elimination of unwanted cells, but without involving the immune system. Oisin reports significant progress in showing that their vector works, efficiently transducing cells and delivering a DNA construct which can kill targeted cells on command. Oisin closed a $500K oversubscribed convertible debt round in mid-December and is working towards a substantial Series A in the next few months that would take it towards a Phase 1 clinical trial.

Researchers here provide evidence for Parkinson's disease to have a significant autoimmune component, adding to other factors known to contribute to the death of dopamine generating neurons that is characteristic of this disease. It is certainly the case that the growing dysfunction of the immune system in later life includes a variety of autoimmune aspects, and that those aspects are still poorly mapped. It is reasonable to expect that researchers will continue to uncover ways in which immune system failures contribute to well-known age-related conditions in the years ahead. The disovery here is particularly interesting, as it links autoimmunity to the buildup of metabolic waste and consequent failure of mechanisms of maintenance that is observed in aging. This is a target for the SENS rejuvenation research program, and therapies built on this approach should prove broadly beneficial, precisely because they will halt and turn back many chains of consequences akin to that reported by the researchers here.

Researchers have found the first direct evidence that autoimmunity - in which the immune system attacks the body's own tissues - plays a role in Parkinson's disease, the neurodegenerative movement disorder. The findings raise the possibility that the death of neurons in Parkinson's could be prevented by therapies that dampen the immune response. "The idea that a malfunctioning immune system contributes to Parkinson's dates back almost 100 years. But until now, no one has been able to connect the dots. Our findings show that two fragments of alpha-synuclein, a protein that accumulates in the brain cells of people with Parkinson's, can activate the T cells involved in autoimmune attacks. It remains to be seen whether the immune response to alpha-synuclein is an initial cause of Parkinson's or if it contributes to neuronal death and worsening symptoms after the onset of the disease. These findings, however, could provide a much-needed diagnostic test for Parkinson's disease and could help us to identify individuals at risk or in the early stages of the disease."

Scientists once thought that neurons were protected from autoimmune attacks. However, in a 2014 study, researchers demonstrated that dopamine neurons (those affected by Parkinson's disease) are vulnerable because they have proteins on the cell surface that help the immune system recognize foreign substances. As a result, they concluded, T cells had the potential to mistake neurons damaged by Parkinson's disease for foreign invaders. The new study found that T cells can be tricked into thinking dopamine neurons are foreign by the buildup of damaged alpha-synuclein proteins, a key feature of Parkinson's disease. "In most cases of Parkinson's, dopamine neurons become filled with structures called Lewy bodies, which are primarily composed of a misfolded form of alpha-synuclein."

In the study, the researchers exposed blood samples from 67 Parkinson's disease patients and 36 age-matched healthy controls to fragments of alpha-synuclein and other proteins found in neurons. They analyzed the samples to determine which, if any, of the protein fragments triggered an immune response. Little immune cell activity was seen in blood samples from the controls. In contrast, T cells in patients' blood samples, which had been apparently primed to recognize alpha-synuclein from past exposure, showed a strong response to the protein fragments. In particular, the immune response was associated with a common form of a gene found in the immune system, which may explain why many people with Parkinson's disease carry this gene variant.

Researchers hypothesizes that autoimmunity in Parkinson's disease arises when neurons are no longer able to get rid of abnormal alpha-synuclein. "Young, healthy cells break down and recycle old or damaged proteins, but that recycling process declines with age and with certain diseases, including Parkinson's. If abnormal alpha-synuclein begins to accumulate, and the immune system hasn't seen it before, the protein could be mistaken as a pathogen that needs to be attacked."

Link: http://newsroom.cumc.columbia.edu/blog/2017/06/21/parkinsons-is-partly-an-autoimmune-disease-study-finds/

A good deal of evidence has accumulated to show that the immune cells called macrophages play important roles in regeneration. Further, there are several different classes of macrophage with quite different behaviors, and while all are essential in the bigger picture, one of them tends to hinder regeneration as a side-effect of the accomplishment of its other duties. Researchers have shown in a number of studies that adjusting the proportion of macrophages in a tissue, towards less of the hindering type, can significantly improve outcomes, and perhaps even produce regeneration that would normally not occur with any great reliability, such as regrowth of nerve tissue. This paper is a recent example of this area of research:

After nerve trauma, the standard clinical operating procedure is to oppose the two nerve ends and, when possible, suture them together. Ultimately, even with successful autografting, only 40% of patients regain useful function. Therefore, there is a clear, urgent, and unmet clinical need for an alternative approach that can match or exceed autograft performance. After peripheral nervous system (PNS) injuries, neurons respond rapidly by changing their activities and promoting a regenerative phenotype. At the distal nerve stump, Schwann cells (SCs) adopt a reparative phenotype. SCs, as well as infiltrating and resident macrophages, remove inhibitory debris, enabling new axons to sprout into the degenerated nerve. Although monocytes and their descendants (in particular, macrophages) have long been known to play an essential role in the degenerative process, only recently has their importance in positively influencing regeneration been recognized. Monocytes are abundant during nerve degeneration and regeneration and modulate the sequence of cellular events which can determine the outcome of the healing process.

After inflammatory insult, macrophages that accumulate at the site of injury appear to be derived largely from circulating monocytes. Entry of monocytes into the distal site of an injured nerve is enabled through up-regulation and release of a major monocyte chemokine, monocyte chemoattractant protein (CCL2) by SCs, which reaches its maximum 1 day after injury. Besides CCL2, the CX3CR1 ligand (fractalkine) can also recruit monocytes through the CX3CR1 receptor. In rats, two major subsets of monocytes have been identified. These two subtypes of monocytes can be recruited to injured tissues, where they can subsequently differentiate into classically activated (M1) or alternatively activated (M2) macrophages. These two phenotypes of macrophages represent a simplistic discrete depiction of a continuous spectrum between two activation states. Generally, M1 macrophages produce proinflammatory cytokines as well as high levels of oxidative metabolites, and M2 macrophages make the environment supportive for tissue repair by producing antiinflammatory cytokines that facilitate matrix remodeling and angiogenesis.

The plasticity of monocytes/macrophages makes them an attractive target for modulation in the context of nerve repair. A prior short-term study demonstrated that direct modulation of macrophages toward a prohealing phenotype, using interleukin 4 (IL-4), results in an increase in SC recruitment and axonal growth. The premise herein is that preferential recruitment of anti-inflammatory reparative monocytes to the site of injury will more effectively bias the immune microenvironment toward a prohealing response and in turn set off a regenerative biochemical cascade involving SCs and neuronal processes that leads to improved repair. Since CX3CR1 receptor is mainly expressed on antiinflammatory reparative monocytes, exogenous fractalkine, the ligand for CX3CR1, can be used to preferentially recruit these monocytes to the site of nerve injury and thus increase the subsequent ratio of prohealing to proinflammatory macrophages during the regeneration process.

Thus an immunomodulatory approach to stimulating nerve repair in a nerve-guidance scaffold was used to explore the regenerative effect of reparative monocyte recruitment. Early modulation of the immune environment at the injury site via fractalkine delivery resulted in a dramatic increase in regeneration as evident from histological and electrophysiological analyses. This study suggests that biasing the infiltrating inflammatory/immune cellular milieu after injury toward a proregenerative population creates a permissive environment for repair. This approach is a shift from the current modes of clinical and laboratory methods for nerve repair, which potentially opens an alternative paradigm to stimulate endogenous peripheral nerve repair.

Link: https://doi.org/10.1073/pnas.1705757114

There is every reason to believe that selective destruction of senescent cells in older individuals should improve heart health, lowering the risk of cardiovascular disease and dysfunction. Researchers who demonstrated 25% median life extension in mice engineered to lack senescent cells found improvements in a number of measures of cardiac health. There is a good deal of evidence for senescent cells to reduce stem cell activity and tissue regenerative capacity, perhaps through chronic inflammation and other consequences of changes in immune cell behavior. Senescent cells spur fibrosis, which disrupts small scale tissue structure with the formation of scar-like tissue, and the growth of fibrosis is important in heart tissue aging. And so on, through much of the list of problems in cell and tissue function known to be caused by the presence of senescent cells. In the paper I'll point out here, researchers focus specifically on oxidative signaling and rising levels of oxidative stress in the heart, and how senescent cells might be involved in this facet of heart aging.

Oxidative theories of aging were among the first such views of the causes of aging to be established in the modern era of cellular biochemistry. The original, simple theories that postulated aging as directly driven by oxidative damage to important molecules have since been put to one side in favor of more nuanced views. The failure of antioxidants, when applied generally, to slow aging is considered to disprove the simple view of aging as cellular damage that scales as the presence of oxidative molecules increases with age. It is certainly true that the oxidative molecules and signatures of harmful oxidative modification of vital proteins do increase with age, however. Equally it is also true that oxidative molecules and oxidative damage are used as signals in beneficial processes, such as the response to exercise - antioxidants can actually cause harm by interfering there. In short, oxidative metabolism is a complex, balanced process; that it is disrupted and runs awry with aging doesn't necessarily make it a cause rather than a consequence.

Over the years, attention has focused upon mitochondria, the power plants of the cell, as the prime source of oxidative molecules and possibly the prime source of disruption in oxidative metabolism in old tissues. Modestly increasing or reducing the flux of oxidative molecules generated by these organelles can improve health in short-lived species - in one direction by causing less damage and in the other by spurring greater repair and maintenance activities. Severe disruption of correct mitochondrial function, something that is known to occur in aged tissues, can drive cells into a failure state that results in large quantities of oxidative molecules pumped out into the surrounding tissues. To close the circle, mitochondrial dysfunction goes hand in hand with cellular senescence, though the bigger picture of cause and consequence here is complicated and still incomplete.

Cardiac Cell Senescence and Redox Signaling

Among age-related diseases, cardiovascular disease has an impressive prevalence, considering that the remaining lifetime risk for cardiovascular disease is about 50% at the age of 40. Consistently, the pathophysiologic modifications that are observed in aging hearts and arteries interact with alterations that characterize atherosclerosis progression, concurring to the development of age-associated heart failure. This latter is due to a combined diastolic and systolic dysfunction, caused by cardiac hypertrophy, replacement fibrosis, and myocardial ischemia, even in the absence of atherosclerotic coronary disease.

Morphometric data acquired in the early 1990s suggested that the number of left ventricle cardiomyocytes declines progressively with aging. Consistently, investigators have documented that although cardiomyocyte turnover occurs postnatally, the rate of cardiomyocyte renewal declines as age advances. Intriguingly, while the same investigators have recently suggested that the total number of cardiomyocytes residing in the left ventricle does not change with aging, evidence of myocyte death has been shown to occur both in male primates and in humans. In these latter, cardiac troponin T levels increase with aging and can predict cardiovascular events and death in the general population. This finding is thought to be the consequence of the age-related reduction of expression or activity of proteins that are involved in cardioprotection, a condition that eventually leads to an increased susceptibility of cardiac myocytes to injury.

To understand the mechanisms leading to heart failure, we and other authors hypothesized that the reduced cardiomyocyte turnover observed in aging was a consequence of the reduced cardiac growth reserve. Several independent groups have shown that undifferentiated, primitive cells reside in mammalian hearts and are involved in cardioprotection against heart failure, possibly generating new myocytes. Conversely, different lines of evidence obtained in animal models of heart failure and in humans indicate that senescent and dysfunctional cardiac resident stem/progenitor cells (CS/PC) accumulate as a consequence of cardiac pathology. Furthermore, with organism aging, senescent primitive and differentiated cells accumulate in mammalian hearts.

Although the concept of cellular senescence was introduced more than 50 years ago, the debate around this programmed cellular behavior is still ongoing. Specifically, in relatively recent years, it has been shown that cell senescence may exert positive effects, by promoting tissue healing after injury and protecting young organisms from cancer. However, in line with the antagonistic pleiotropy theory of aging, these beneficial effects exerted by cell senescence in young animals may be also responsible for the occurrence of functional impairment and age-related pathologies. Consistently, "rejuvenation" strategies aimed at reducing the frequency of senescent cells in the organism or designed to modulate those pathways whose activation status is altered in cell senescence can restore cardiac function in aged and failing hearts. Finally, we should emphasize that, while it has been postulated that reactive oxygen species (ROS) play a primary role in the development of cell senescence, the molecular mechanisms responsible for the development and evolution of cellular senescence are still a matter of intense research.

For many years it was believed that ROS were produced in an unregulated manner as a byproduct of cellular metabolism. Moreover, their ability to cause damage to macromolecules was thought to be responsible for organism aging (also known as the mitochondrial free radical theory of aging, MFRTA). Consistently, several pieces of evidence have shown an age-dependent decrease in mitochondrial integrity, and a parallel increase in the level of oxidized DNA (including mitochondrial DNA). These alterations have led to the formulation of "the vicious cycle hypothesis of mitochondrial ROS generation," according to which the mitochondrial production of ROS would damage mitochondrial DNA (mtDNA) and lead to mitochondrial dysfunction, thus increasing ROS generation. However, discordant results have been obtained in more recent years, which have either supported or refuted the increased production of mitochondrial ROS with aging.

These seemingly contradictory results can be reconciled if we consider that ROS have a dual nature. In fact, on top of their ability to damage in non-specific fashion biological molecules, ROS can exert useful and beneficial effects, by regulating signaling pathways. According to current models, ROS generation is highly regulated, and therefore oxidative stress would arise from the loss of this architecture. Importantly, redox signaling is a crucial regulator of stem cell quiescence, self-renewal, and differentiation. Conversely, loss of controlled redox signaling (oxidative stress) can obliterate stem cell function and promote cell senescence of stem and differentiated cells, two conditions that have been associated with the progressive loss of tissue renewal and reparative reserve that characterize aging.

Cardiac stem cells are not immune from these pathological processes, becoming dysfunctional and unable to effectively repair cardiac damage with organism aging and pathology. mTOR signaling, which associates with cardiac stem cell senescence, may affect redox signaling at multiple levels, overloading the endoplasmic reticulum, and inhibiting both lysosomal function and autophagy. Therefore, innovative interventions aimed at restoring proper redox signaling in primitive cells have the potential to reverse or attenuate age-associated stem cell dysfunction.

The author of this paper is one of the more outspoken advocates in the research community when it comes to mTOR and rapamycin as a path to slowing the progression of aging. He keeps up quite the output of position papers, such as this one, which calls for immediate human trials of polypills made up of rapamycin and a brace of other drugs broadly used in treatment of age-related conditions, such as statins and metformin. I have to think that the evidence to date suggests this will be less effective than hoped, while still very plausibly being better than doing nothing, even considering the side-effects of the drugs involved. Effects in animal studies usually tend to be much more pronounced than effects in humans when it comes to slowing or preventing specific age-related diseases through pharmaceuticals.

If it was the only game in town, I'd be all for it, but there are far more effective ways forward towards the effective treatment of aging as a medical condition - approaches that aim at rejuvenation, not a mere slowing of aging. Still, I think the author here has the right general idea, in that the research community should move faster, the sooner plausible approaches are trialed the better, and that we should all pitch in to help, it is just that he is advocating a poor approach with a limited upside in comparison to other methodologies.

Inhibitors of mTOR, including clinically available rapalogs such as rapamycin (Sirolimus) and Everolimus, are gerosuppressants, which suppress cellular senescence. Rapamycin slows aging and extends life span in a variety of species from worm to mammals. Rapalogs can prevent age-related diseases, including cancer, atherosclerosis, obesity, neurodegeneration and retinopathy and potentially rejuvenate stem cells, immunity and metabolism. Here, I suggest how rapamycin can be combined with metformin, inhibitors of angiotensin II signaling (Losartan, Lisinopril), statins, propranolol, aspirin and a PDE5 inhibitor. Rational combinations of these drugs can maximize their anti-aging effects and decrease side effects.

At first, the discovery of anti-aging properties of rapamycin was met with skepticism because it challenged the dogma that aging is a decline driven by molecular damage caused by free radicals. By now, rapamycin has been proven to be an anti-aging drug. In contrast, anti-oxidants failed in clinical trials and the dogma was shattered. In the last decade, anti-aging effects of rapamycin have been confirmed. Anti-aging doses and schedules can be extrapolated from animal studies. Well-tolerated doses with minimal side effects can be deducted based on clinical use of rapalogs. So optimal anti-aging doses/schedules can be suggested. Given that rapamycin consistently extends maximal lifespan in mice, rapamycin will likely allow mankind to beat the current record of human longevity, which is 122 years. Yet, rapamycin will not extend life span as much as we might wish to. Now is the time for anti-aging drug combinations. For example, metformin is currently undergoing re-purposing as an anti-aging agent. Several other existing drugs can be re-purposed. Now we can design an anti-aging formula, using drugs available for human use.

Rapamycin (or another rapalog) should be a cornerstone of anti-aging combinations, given its universal anti-aging effect and the ability to delay almost all diseases of aging. Rapamycin and metformin: Both drugs extend lifespan in animals and have non- overlapping effects. In addition, they may, in theory, cancel possible metabolic side-effects of each other. Rapamycin and statins: Rapamycin promotes lipolysis increasing blood levels of fatty acids. This, in turn, increases levels of lipoproteins produced by the liver. Rapamycin-induced hyperlipidemia is benevolent and reversible. Still, statins are already used to prevent rapamycin-induced hyperlipidemia.

The 7-drug combination can be tested in mice, especially in mice on high fat diet and in cancer-prone mice. If started late in life, the experiments will take just several months to evaluate the effect on lifespan and cancer incidence as well as weight, blood pressure, glucose, insulin, triglycerides and leptin. In humans, the treatment program can be initiated regardless of any pre-clinical studies, because all 7 drugs are approved for human use and some of them such as aspirin and statin are widely used for disease prevention anyway. The only what is needed is to watch for side effects. Especially, heart rate, blood pressure, and glucose levels should be monitored.

The anti-aging formula is ready for human use. If one will wait until the life-extending effect will be shown in others, this individual will not be alive by the time of the result. Human clinical trials are needed to optimize the doses and schedules. However, unless we participate in clinical trials ourselves, we will not know how long participants will live because they are expected to outlive non-participants. If we want to live longer we should be participants in clinical trials. In the best scenario, this might allow us to live long enough to benefit from future discoveries of anti-aging remedies.

Link: https://doi.org/10.18632/oncotarget.18033

Genetic engineering is now a fast path to cures for inherited diseases, those in which the cause is mutation in a single gene. Remove or suppress the bad gene, and insert the fixed version. This sort of approach, suppressing or editing a few specific genes, is unlikely to be as broadly and directly effective for age-related conditions, however. This is because, as is the case for aging, the disease state is influenced by thousands of genes, but directly caused by none of them. There are arguments, such as the research noted here, that suggest we should expect discoveries such as myostatin knockout for muscle growth, or ASGR1 and ANGPTL4 for reduced cardiovascular disease risk, to be unusual in the magnitude of their effects. When in search of genetic alterations to beneficially change a disease state, we should expect to come up more or less empty handed most of the time.

Here I am focused on the standard approach of editing or manipulating expression of one gene or a few genes, and not on more extensive engineering projects such as the SENS rejuvenation research program that aims to copy altered forms of mitochondrial DNA into the cell nucleus as a backup to remove the consequences of age-related damage to mitochondrial DNA. Or consider the Oisin Biotechnologies delivery of programmable DNA machinery to destroy a cell depending on its state. There will no doubt prove to be a role for other ambitious and essentially genetic reworkings of the cell, ways to improve redundancy of components or expand capabilities to ensure greater resilience, such as by providing a package of new enzymes capable of tackling problematic forms of metabolic waste.

This is, however, a very different class of approach to the single gene editing efforts that make up most of the field at present. This may be an era of medicine dominated by genetics, but we must, I think, recognize where it is limited as well as where there is the greatest potential. To my mind I see far too much enthusiasm for the sort of focus on genetic personalized medicine and single gene manipulations put forward by the likes of Human Longevity, to pick a representative example, while there just isn't enough of a benefit in their approach to be excited by it.

In a provocative new perspective piece, researchers say that disease genes are spread uniformly across the genome, not clustered in specific molecular pathways, as has been thought. The gene activity of cells is so broadly networked that virtually any gene can influence disease, the researchers found. As a result, most of the heritability of diseases is due not to a handful of core genes, but to tiny contributions from vast numbers of peripheral genes that function outside disease pathways. Any given trait, it seems, is not controlled by a small set of genes. Instead, nearly every gene in the genome influences everything about us. The effects may be tiny, but they add up.

The researchers call their provocative new understanding of disease genes an "omnigenic model" to indicate that almost any gene can influence diseases and other complex traits. In any cell, there might be 50 to 100 core genes with direct effects on a given trait, as well as easily another 10,000 peripheral genes that are expressed in the same cell with indirect effects on that trait. Each of the peripheral genes has a small effect on the trait. But because those thousands of genes outnumber the core genes by orders of magnitude, most of the genetic variation related to diseases and other traits comes from the thousands of peripheral genes. So, ironically, the genes whose impact on disease is most indirect and small end up being responsible for most of the inheritance patterns of the disease.

Researchers have thought of genetically complex traits as conforming to a polygenic model, in which each gene has a direct effect on a trait, whether that trait is something like height or a disease. In earlier work on the genetics of height, researchers were surprised to find that essentially the entire genome influenced height. "It was really unintuitive to me. To be honest, I thought that it was probably wrong. I gradually started to realize that the data don't really fit the polygenic model. We started to think, 'If the whole genome is involved in a complex trait like height, then how does that work?'"

The polygenic model leads researchers to focus on the short list of core genes that function in molecular pathways known to impact diseases. So, therapeutic research typically means addressing those core genes. A common approach to gene discovery is to do larger and larger genome-wide association studies, but the team argues against this approach because the sample sizes are expensive and the thousands of peripheral genes uncovered are likely to have tiny, indirect effects. "After you get the first 100 hits, you've probably found most of the core genes you're going to get through genomewide association studies." Instead, the team recommends switching to deep sequencing the core genes to hunt down rare variants that might have bigger effects. "If this model is right, it's telling us something profound about how cells work that we don't really understand very well. And so maybe that puts us a little bit further away from using genome-wide association studies for therapeutics. But in terms of understanding how genetics encodes disease risk, it's really important."

Link: http://med.stanford.edu/news/all-news/2017/06/thousands-of-genes-influence-most-diseases.html

Aubrey de Grey of the SENS Research Foundation is the advocate and scientist at the center of a diverse network of people and organizations who, collectively, are changing the world when it comes to aging, medicine, and research. It wasn't so very long ago that the research community and its associated sources of funding were hostile towards any effort to consider the treatment of aging as a medical condition. Decades were lost to a scientific culture whose leading members wanted to distance themselves from "anti-aging" snake oil at any cost - including the sacrifice of any real possibility of progress. Change has come but slowly, and required outsiders such as de Grey to enter the research field and raise hell until the existing factions and establishments were forced to acknowledge the potential to extend healthy life and reverse the progression of age-related conditions. Younger researchers now benefit from a field in which they can build a better world, applying biotechnology to the causes of aging in order to alleviate this greatest cause of suffering and death. This field is no longer the poorly regarded backwater it once was, thanks to people like de Grey and his allies, but now one of the most exciting areas of modern life science research, the seed that will blossom into a vast and enormously beneficial industry in the years ahead.

Yet this is a transformation still in progress. The first battles have been won, the first rejuvenation therapies after the SENS vision of damage repair - those involving clearance of senescent cells - are well on their way to the clinic. But the majority of research programs and funding sources remain slow to change course. Funding for aging research remains minimal in comparison to funding for other areas of medicine. Where there is funding, it is still largely directed towards initiatives that cannot possibly do more than slightly slow aging, or merely patch over the symptoms of aging, as little attention is given to the cell and tissue damage that is the root cause of all age-related disease, dsyfunction, and death. Longevity science is a field in which the greatest challenge is not the discovery of great swathes of new information about aging, but rather to persuade the research community to make proper use of what is already known, and then fund that work sufficiently. All of the necessary classes of therapy needed for rejuvenation can be constructed based on the knowledge of twenty years ago; the development plans are set out in some detail. Yet all too much of the field remains focused on continued exploration of the details of aging as it operates in the absence of intervention.

This is where we come in. Our philanthropic support of organizations such as the Methuselah Foundation and SENS Research Foundation helps to move the research forward. Our investment in and support of startup companies working on SENS technologies helps to push meaningful therapies for aging closer towards the clinic. The growth and legitimacy of SENS and SENS-like rejuvenation research is something that our broader community has bootstrapped from an idea to its present state. We have succeeded to no small degree! There is much to do yet, however. Our ability to attract support to the most important lines of research and development has increased greatly in recent years, and will continue to soar as SENS approaches such as senescent cell clearance are proven out in trials and animal studies. Now is not the time to rest upon our laurels: so make a point to tell someone you know about the field of rejuvenation research, and that the promising therapies currently in development are the result of donations wisely made in past years. The more people who know today, the more supporters will join us in the years ahead, and this is far more a challenge of persuasion than a challenge of science at this stage.

Science Isn't The Reason That Humans Can't Live Forever

If humanity were to appoint a general in our war against aging, Aubrey de Grey would likely earn the honor. The British author and biomedical gerontologist has been on the front line for years, researching ways to free the world of age-related disease and, ultimately, extend human life indefinitely. From the SENS Research Foundation Research Center (SRF-RC) in Mountain View, CA, foundation scientists conduct proof-of-concept research with the goal of addressing the problems caused by aging. They focus on repairing damage to the body at the molecular level, and their work is helping advance the field of rejuvenation biotechnology.

SRF-RC teams are currently focusing on two equally complex-sounding research projects, one centered on allotopic expression (a way to bypass the harmful effects of age-caused mitochondrial mutations) and the other on telomerase-independent telomere elongation (a little-researched process by which some cancer cells overcome mortality). Either project could lead to major breakthroughs in anti-aging treatments, but as de Grey explains, the path to immortality doesn't just run through the science lab. While the research being conducted at the SRF-RC is far from simple, de Grey claims DNA mutations and cancer cells aren't the biggest hurdles to anti-aging breakthroughs: "The most difficult aspect of fighting age-related diseases is raising the money to actually fund the research." The nature of most science research is exploratory. Researchers don't know that what they're working on is going to yield the results they expect, and even if it does, turning basic research into income is no easy task. To support their work, most have to rely on funding from outside sources, such as government grants, educational institutions, or private companies.

"It's still an incredibly hard sell," de Grey claims. "We have very limited resources. We only have about 4 million dollars a year to spend, and so we spent it very judiciously." That money isn't going to just the two in-house projects, either. The SENS Research Foundation funds anti-aging research at institutions across the globe and provides grants and internships for students, so raising money to support those endeavors is key to continued success in its fight against aging. The benefits of ending the problem of aging would be tremendous. Not only would we be living longer, we'd be living healthier for longer.

Essential to raising money for anti-aging research is ensuring that those with the funds understands why it's worth the investment - a not-so-easy task given current misconceptions about aging. In 2015, eight major aging-focused organizations, released a report detailing what they call the many "notable gaps" that exist between expert perspectives on aging and the public's perception of the process. If the public isn't well informed on aging, it's even less knowledgeable about anti-aging. Fifty-eight percent of respondents in a 2013 Pew Research study said they had never even heard of radical life extension before. When asked if they would undergo treatments that would allow them to live to the age of 120 or older, the majority of those surveyed said they would not, and 51 percent thought such treatments would be "bad for society."

"There is still a huge amount of resistance to the logic that aging is bad for you and that it's a medical problem that needs to be addressed," explains de Grey. "It's really, really extraordinary to me that it's so hard to get this through to people, but that is the way it is. Aging is not mysterious. We understand it pretty well. It's not even a phenomenon of biology. It's more a phenomenon of physics. Any machine with moving parts is going to damage itself ... and the result is inevitably going to be that eventually the machine fails. It's the same for the human body as it is for a car, for example, and if we think about it that way, it becomes pretty easy to actually see what to do about it."

Researchers are these days feeling more confident in the identification of cytomegalovirus as a significant cause of immune system dysfunction in aging, as the conference report here illustrates. We might hope that this growing interest in cytomegalovirus in the context of aging will lead to more funding of means to repair the situation, aiming to restore some of the youthful capability to respond to pathogens and destroy potentially dangerous cells.

The immune system is an adaptable machine, but one with limits. In adults new immune cells are generated at a very slow pace in comparison to the overall count of such cells in circulation. This effectively produces what looks a lot like a limit on the number of immune cells. As the years pass, that limited population is increasingly taken over by endlessly duplicated memory cells specialized to cytomegalovirus. Uselessly specialized, as the immune system cannot effectively clear this virus from the body. Near everyone is infected by cytomegalovirus by the time old age is reached, but aside from its insidious long-term effects on the immune system, it causes no noticeable problems in the vast majority of people. Thus approaches to tackling it have not been given any great priority in the medical science community of past decades. When much of the immune system is overtaken by cytomegalovirus-specific cells, however, that leaves all too little room for cells capable of productively carrying out other functions, and the result is a failing immune system, characteristic of the old.

The best near-team approach to this problem is probably some form of selective destruction of the unwanted specialized immune cells, in order to free up capacity. That can be coupled with the generation of replacement immune cells from a patient cell sample, returned to the body to quickly make up the numbers. This is a very plausible goal, given the various trials and technology demonstrations of immune cell clearance for therapeutic purposes. The greatest challenge involved is to develop targeted cell destruction approaches that are safe and have minimal side effects for the patient, in comparison to the damaging pharmaceuticals used to date in human trials.

Nearly two decades ago, two key findings connected cytomegalovirus (CMV) with immune senescence. In 1999, researchers showed that CMV-positive and -negative humans exhibit dramatically different T cell subset ratios and that the effect seems to be increasing with aging. Around the same time, others described "memory inflation" driven by CMV in mice. Since then, numerous studies have been published investigating the associations between human, non-human primate or murine CMV with their respective hosts in the course of aging. The interest in the topic has been so sustained that it led to the establishment of CMV and Immunosenescence Workshops. This overview summarizes the state of the field before and the discussions at the 6th International CMV and Immunosenescence Workshop.

CMV, a member of beta-herpesvirus family, is the largest human virus. As is the case for other herpesviruses, following a brief acute infection period that elicits a typical CD8 T cell response, as well as CD4 and B cell responses, CMV establishes persistence that includes latency. Persistence/latency is established in reservoir cells that are distributed broadly across the organism. However, it is clear that the primary CMV infection is followed by a period of viral shedding and it remains unclear whether the virus, that is a master in immune evasion, ever really is truly latent in all its reservoirs or whether some cells produce it as a smoldering infection at low levels at most, if not all times. As a consequence, a large population of CMV-specific CD8, and to a lesser extent, CD4 T cells, is generated in response to cycles of viral reactivation (aptly called memory inflation). How exactly this memory inflation impacts the ability of an older immune system to function and provide defense against other infections is one of the key topics of interest.

The 6th International Workshop on CMV and Immunosenescence was organized with a primary goal to fill a gap identified at several recent meetings. Specifically, in addition to the topics reviewed above from the fifth workshop, it was felt that stronger attention must be focused on the biology of the virus itself and on its interactions with the host in the course of latency and reactivation. In fact, one of the greatest weaknesses of research into HCMV and immunosenescence comes from our inability to control and measure the state of viral activity. Another area of major interest is the impact of CMV on diseases on aging - whether and how the virus may be involved in modulating frequent age-related morbidities, in particular cardiovascular diseases (CVD), where there are strong epidemiological associations between CMV infection and morbidity and mortality from CVD.

Link: https://doi.org/10.1007/s11357-017-9984-8

This paper is an example of the further explorations of DNA methylation and aging presently taking place in the research community. DNA methylation is one of the epigenetic decorations to DNA that alter gene expression, and thus the pace at which specific proteins are generated in the cell. A few different epigenetic clocks have been discovered in recent years, patterns of change in DNA methylation levels that correlate well with biological age, in that people of a given chronological age who are more damaged and impacted by aging than their peers tend to have distinctively different DNA methylation patterns. Moving beyond the existing epigenetic clocks, researchers are now searching for more and better correlations, as well as specific mechanistic links with other cellular processes already known to change with age.

Alteration of the ribosome biogenesis and an overall protein synthesis rate decline have been observed to characterize aging process in many organisms, including humans. This decline could be an effect of the progressive deterioration in most cellular functions usually associated with aging, or it could be a concurrent factor in the process. If to date a general reduction of protein synthesis has been attributed to the decreased frequency of mRNA translation, current studies, reporting an involvement of epigenetic mechanisms in silencing a large fraction of the ribosomal RNA (rRNA) genes, with a consequent impairment of ribosomal DNA (rDNA) function, could lead to a new understanding of the phenomenon.

On the basis of this evidence, we investigated whether changes in the DNA methylation patterns of the rRNA gene promoter take place during the lifetime. DNA samples were extracted from whole blood collected from differently aged human individuals displaying different phenotypes according to cognitive, functional, and psychological parameters. We did not find a consistent statistically significant association between the methylation levels of the analyzed CpG sites with the age of the donor. On the other hand, although it is not associated with chronological aging, in middle/advanced-aged subjects the variability of CpG_5 methylation was found to be significantly correlated with both cognitive performances and survival in the 9-year follow-up period. This last result, which held multiple test correction, was further confirmed in the replication sample.

Our results seem to be particularly attractive, because they show a fine remodeling of the methylation profile associated with the biological aging rather than to the chronological age. The effects at molecular levels of the above association have to be clarified, but it is plausible to hypothesize that the decrease in the rRNA levels we observed late in life may be determined by the methylation of the CpG_5 site that in turn might be driven by multiple factors, including genetic variations, diet, environment, and the interindividual variation of the structure of rDNA cluster itself.

How could the methylation changes of peculiar CpG sites be functionally involved in the functional decline characterizing the aging process? If the epigenetic modification of functional sites may hamper ribosomal biogenesis, this may drastically reduce the cellular protein synthesis, being ultimately responsible of those multisystem deficits occurring over the lifetime. Thus, an interdependence seems to exist between rDNA promoter methylation and the aging process, and in particular with the aging associated decay, and these sites may represent an potential evolutionary conserved biomarker of the rate of the aging process.

Link: https://doi.org/10.1111/acel.12603

The progress in various approaches to gene therapy over the past decade has succeeded in reducing cost and increasing reliability. This has reached the point at which researchers can afford, from the point of view of both time and funding, to begin to combine gene therapies with other areas of medicine under development. In particular, reliable gene therapies targeting the controlling switches and dials of cell growth and regeneration should be a way to greatly improve the effectiveness of cell therapies and other, similar forms of regenerative medicine. The research reported below is a good example of the type, in which scientists combine a scaffold-based cell therapy with gene therapy to encourage local cells towards increased, controlled bone regrowth to replace severe fracture damage.

There are many methods of delivery for the introduction of therapeutic genes. The familiar use of viruses as a vector for the transfection of genes into a cell is just one class of approach - perhaps the most obvious one, given that viruses are in essence machines whose primary purpose is to place DNA into cells. But there are other approaches. Considered in the larger context, this diversity is a good thing, as greater competition and exploration always leads to a superior end result once all is said and done. The method used here is one of the pore-forming variations, in which one or another form of stimulus induces cells to open pores in the cell membrane and let in the DNA-bearing particles. In this case, the stimulus is physical, provided by cavitation of ultrasound-created microbubbles. It is worth bearing in mind that all of these methods have the potential to damage and kill cells, some more than others, and thus must be carefully calibrated. When it works, however, the results can be fairly impressive.

Injured Bones Reconstructed by Gene and Stem Cell Therapies

Investigators have successfully repaired severe limb fractures in laboratory animals with an innovative technique that cues bone to regrow its own tissue. If found to be safe and effective in humans, the pioneering method of combining ultrasound, stem cell and gene therapies could eventually replace grafting as a way to mend severely broken bones. "We are just at the beginning of a revolution in orthopedics. We're combining an engineering approach with a biological approach to advance regenerative engineering, which we believe is the future of medicine."

The new technique could provide a much-needed alternative to bone grafts. In their experiment, the investigators constructed a matrix of collagen, a protein the body uses to build bones, and implanted it in the gap between the two sides of a fractured leg bone in laboratory animals. This matrix recruited the fractured leg's stem cells into the gap over two weeks. To initiate the bone repair process, the team delivered a bone-inducing gene directly into the stem cells, using an ultrasound pulse and microbubbles that facilitated the entry of the gene into the cells. Eight weeks after the surgery, the bone gap was closed and the leg fracture was healed in all the laboratory animals that received the treatment. Tests showed that the bone grown in the gap was as strong as that produced by surgical bone grafts.

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area.

Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.

While it certainly seems a long time - and that we have come a long way - since the years in which SENS rejuvenation research was only an idea, and the research community was generally hostile towards the idea of treating aging as a medical condition, these are really still the early stages of the upward curve in the bigger picture. That curve leads to a mainstream research community as consumed by the effort to bring aging under medical control as it is presently consumed by work on cancer and stem cell science - and a public at large who support an end to aging just as greatly as they presently support an end to cancer. We'd all like it to go faster.

As our community of scientists, advocates, and supporters grows, the diversity of opinions on what we should be doing in order to speed up progress towards working rejuvenation therapies will also grow. I think this to be a good thing. The more approaches out there being tried in earnest, the more likely that one or another group will find ways to effectively speed up the present phase of the bootstrapping process. There will be disagreements, of course, and I disagree with some of the details in the piece linked here, but so what? Each to their own. Proof of correctness lies in implementations that effectively move the needle, not in opinion. If you have an idea, get out there and try it.

We have made huge strides in the last decade or so and we know a great deal about the processes and damages aging causes, but sadly this does not mean we know enough. There is much more to be learned in order to develop effective interventions and therapies to address these processes, and this is where basic science comes in. The countdown to accessible therapies against aging will not start unless each mechanism of aging is well understood. If all of them were understood right now, you would still need to wait for another 17 years to get a full range of therapies against aging. If you add 17 years to your current age and don't like the resulting number and its relation to the onset of age-related diseases, then ask yourself this, is supporting basic research on aging now in your interest? You may not be very excited about life extension in mice, but remember, no results in mice equals no translation to humans.

It is true the government funds research institutions and awards research grants. But the idea of preventing age-related diseases by addressing its underlying mechanisms is relatively new. There are not many experts among the decision makers in the grant system who can assess the breakthrough projects aimed at the hallmarks of aging and truly understand their potential. This is why these kinds of projects have less support from the government than the mainstream studies of a single disease like Alzheimer's or cancer.

In the case of government funding, the money goes from the taxpayer to the government treasury, where its future allocation is decided, and then to specific research institutions whose plan of research falls within the mainstream priorities. This makes it very hard for our community to influence the direction of research, which is a serious limitation indeed. Crowdfunding does not have this limitation, because it allows the public to connect with the researchers directly and support only the projects they believe are important. The amount of money collected during a crowdfunding campaign can be as much as a government grant (often even bigger), plus there is no need for the excessive paperwork typical for a government grant. This means that the researchers can focus on what they do best of all: their studies.

The number of ardent supporters of aging and longevity studies is relatively small due to the slow dissemination of information from scientists to the public. Most people still believe there is nothing we can do about biological aging, and so they see these studies as researchers simply feeding their scientific curiosity. Education regarding the plausibility and desirability to defeat aging takes time, patience and a lot of effort. It cannot be done by the scientists themselves (as their job is to work in the lab, not to make shows), and here is where advocacy groups and science popularizers should step in. However, people tend to forget that the best results can only be achieved if a group is well-organized, disciplined and uses evidence-based practices in all activities, from planning and management to crowdfunding, educating and lobbying. Steady progress requires a mindful and responsible approach from each person joining an advocacy group - which is sadly rarely seen.

Many members of our community prefer to profess their desire for indefinite lifespans directly, shocking the public. It is important properly explain the connection between aging and age-related diseases, and the causal relationship between aging prevention, health improvement, and longevity - longevity being a side-effect of better health. Being patient and addressing concerns people may have in relation to longer lives (like overpopulation, unequal access, boredom and others) is another important job which is rarely done properly, with enough supporting data to hand. Despite the fact that most of the sociological studies on public attitudes regarding life extension are available to read and have even been summarized by different members of the community, many people still refuse to explain the basics, or insist on using counterproductive radical messages, provoking additional skepticism and closing doors that would otherwise be open. Before starting a conversation with someone who is not familiar with the idea of healthy life extension, it would be useful to take a look at the existing data regarding how to make such conversation productive.

Link: http://www.leafscience.org/bottlenecks-in-research/

Fatty liver disease, or hepatic steatosis, is both age-related and self-inflicted in the sense that in most sufferers the primary cause appears to be the metabolic dysfunction that accompanies obesity, but the risk also rises with age, and even an exemplary life can sometimes eventually result in the appearance of this condition. Chronic inflammation may play an important role in the development of fatty liver disease without obesity, and whenever that it is the case it is sensible to immediately turn to the age-related accumulation of senescent cells as a potential contributing mechanism, as these cells are a potent source of inflammatory signals. The researchers here do just that, and in the course of their work demonstrate that senescent cells are in fact an important cause of the problem, just as they are for many other age-related conditions. This is good news for patients with fatty liver disease, and those destined to be patients absent an effective treatment, given the present pace of progress towards senolytic therapies capable of safely and selectively destroying these unwanted and harmful cells.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excess hepatic fat (steatosis) in individuals who drink little or no alcohol. NAFLD is more prevalent in older populations. The mechanisms underlying this condition are not understood nor is why its prevalence increases with ageing. It has been speculated that ageing processes may promote NAFLD via different mechanisms, including adipose tissue dysfunction, impaired autophagy, and oxidative stress.

Cellular senescence is a state of irreversible cell-cycle arrest, which can be induced by a variety of stressors, including telomere dysfunction and genotoxic and oxidative stress. Senescent cells frequently have increased secretion of a broad repertoire of proinflammatory factors, collectively known as the senescence-associated secretory phenotype, which can induce tissue dysfunction in a paracrine manner. Senescent cells have mitochondrial dysfunction, with decreased oxidative phosphorylation and concomitantly increased generation of reactive oxygen species (ROS), caused at least partly by failing mitophagy.

A significant fraction of hepatocytes develop a senescent phenotype during the life course of mice and with age-related liver disease in humans. However, the relationship between cellular senescence and liver fat accumulation remains unclear. Here we hypothesized that cellular senescence results in impaired fat metabolism and that removal of senescent cells may diminish liver steatosis.

We found a close relationship between senescence markers and fat accumulation in hepatocytes of mice fed ad libitum (AL), dietary restricted (DR) or following dietary crossover and in a small cohort of NAFLD patients. Furthermore, clearance of senescent cells by suicide gene-meditated ablation of p16Ink4a-expressing senescent cells in INK-ATTAC mice and a senolytic cocktail of dasatinib plus quercetin reduced overall hepatic steatosis in ageing, obese, and diabetic mice. In contrast, hepatocyte-specific induction of senescence by a local DNA repair defect resulted in liver steatosis. Finally, we found that induction of senescence in mouse fibroblasts and hepatocytes resulted in decreased ability to metabolize fat. Our findings suggest that interventions targeting senescent cells may be developed into therapies to reduce steatosis during NAFLD.

Link: https://doi.org/10.1038/ncomms15691

The longest lived laboratory mice, more than a decade after the creation of the first lineage, are still those with impaired growth hormone signaling. That record has yet to be overtaken, and I suspect that it may well stand unbroken until the development of rejuvenation therapies based on damage repair is further advanced, and senescent cell clearance is joined by other types of therapy, their effects adding together. Thus growth hormone and growth hormone receptor genes in humans are one of the places to look for variants and mutations that might improve our understanding of how metabolism influences aging, and as a bonus for those interested in pharmacological or genetic adjustment of metabolism, may lead to ways to modestly slow aging.

With this in mind, attention has fallen on a rare human lineage with a growth hormone receptor mutation that produces dwarfism, the Laron syndrome population. Unfortunately it isn't yet possible to say whether or not these individuals have any advantage over the rest of us when it comes to longevity. They do appear to be resistant to cancer and type 2 diabetes, though the evidence in support of that conclusion isn't completely iron-clad at this point. People with Laron syndrome and, separately, people who practice calorie restriction for its health benefits can be compared with the same processes operating in mice. When doing so, we can see that human longevity is not enormously changed, while mouse longevity does increase by up to 70% for growth hormone disruption, and up to 40% for calorie restriction. That degree of gain is certainly not the case in humans. Evolution has delivered a much more plastic life span to short-lived mammals, responsive to environmental circumstances, and with a biochemistry capable of these large changes in the pace of aging.

Putting growth hormone signaling to one side for the moment, considerable effort has been devoted over the years to the broader search for human genetic variants that are associated with longevity. The consensus on genes and longevity is that individual variations in your genome have little influence over aging until later life, and from that point forward, the older and more damaged you get the more that genetic variation matters. Nonetheless, the search has found very few compelling associations. There is solid evidence for variants in APOE and FOXO3A, and less solid evidence for a few other variants such as in TXNRD1, but these are not large effects. Beyond this there are scores of other associations that are never replicated, showing up in only one study or one population, and again with small effects - by which I mean maybe you have a 1.5% chance of living to 100 instead of a 1% chance if you held one of these variants. The big picture is of hundreds or thousands of individually tiny effects; which of these genes and variants are more or less relevant varies widely between populations and individuals, and is very dependent on environmental factors.

That said, there are genetic variants with sizable effects on resistance to specific age-related disease, such as those in ASGR1 or ANGPTL4, both of which reduce blood cholesterol and cardiovascular disease risk. Nothing is published on their effects on longevity at this time, but give it time. Should we believe that there are human genetic variants that meaningfully increase life expectancy in the carriers based on what we've seen to date? The association studies with their poor catch of results suggest no. The existence of the variants mentioned above suggest maybe, but equally it is the case that aging has many facets. Being resistant - or even immune - to one thin facet, such as cardiovascular disease, is thought unlikely to do a great deal to overall longevity. It just means that something else gets you in the end, perhaps a couple of years later.

Returning to growth hormone metabolism, I see that researchers are claiming that a common human gene variant of growth hormone receptor, per their statistics, may result in a ten year difference in life expectancy. This is replicated in multiple study populations, but the effect appears only in men. It is interesting, but there is every reason to be cautious with this sort of very statistical genetic association study. I'd say read the paper and put it aside until someone replicates the result. Would a ten year gain from a growth hormone signaling genetic variant be surprising if this turns out in fact be the case? Maybe not, given what we know about the relative sizes of effects in mice and humans. Ten years is about on the outside end of variations that can plausibly exist in some numbers and yet blend in with broader population data, and the effects in mice are considerably larger on a relative basis.

The GH receptor exon 3 deletion is a marker of male-specific exceptional longevity associated with increased GH sensitivity and taller stature

Growth hormone (GH) and insulin-like growth factor (IGF) play a central role in development, differentiation, growth, and metabolism among divergent taxa. Dwarf individuals appear to live longer among many species, suggesting a role for the GH/IGF-1 axis in modulating aging and life span. A considerable body of in vitro experimental evidence also suggests an important role for the IGF axis in human longevity and aging-related processes in a tissue-specific manner. Furthermore, several studies in selected human populations lend support on the role of this axis in health and life span.

For instance, we have previously identified a cluster of functional mutations in the IGF-1 receptor in centenarians. We showed that Laron dwarfs, who are naturally short, have decreased prevalence of diabetes, cancer, and stroke, suggesting increased health span although life span in this small sample size cannot be determined accurately. Also, we previously established that centenarians with lower levels of IGF-1 had significantly longer survival. Clearly, individuals with severe GH deficiency have reduced life expectancy, suggesting that some GH is necessary for survival. On the other hand, interventional GH therapy in humans is commonly used to reverse age-related morbidities; hence, the kind of deficiency that will be most beneficial for health span and longevity needs to be further established.

GH production is decreased with age; however, it is never completely diminished. That said, there is accumulating evidence that GH may play a crucial role in modulating aging. Surprisingly, GH deficiency or diminished secretion has been linked to longevity phenotypes both in mice models and in humans with familial longevity. The GH receptor (GHR) gene has nine coding exons and consists of two common isoforms: (i) full-length GHR-flGHR and (ii) a shorter form with a deletion of exon 3, d3-GHR. The allele frequencies of these isoforms among human populations range from 68-90% for flGHR and 10-32% for d3-GHR.

Investigations of the effects of GHR isoforms on human health have provided mixed results. In two Genome Wide Association Studies (GWAS) based on single-nucleotide polymorphisms (SNPs), the GHR locus showed association with final height. However, to our knowledge, the association of d3-GHR with final height has not been examined, possibly because individuals with d3-GHR are expected to maintain normal GH action despite lower GH production. It is reasonable to hypothesize that increased GH sensitivity can also alter IGF-1 secretion and therefore regulate longevity. Given the potential role of the GH/IGF axis in longevity, we hypothesize that low IGF-1 levels will assure longevity of the d3-GHR carriers. To address this hypothesis, we genotyped the d3-GHR locus in four human cohorts with long-lived participants, and we tested its association with longevity-related phenotypes and stature with a relatively common GHR variation.

In Ashkenazi males, but not in females, a marked difference in allele frequency for the exon 3 deletion polymorphism (d3-GHR) was found between centenarian and control, as well as offspring and control groups. Whereas the male control group carried only 4% homozygote deletions, male offspring of centenarians and male centenarians carried 11 and 12%, respectively. We further validated these results in three independent cohorts - the Old Order Amish (OOA), Cardiovascular Health Study (CHS), and the French Long-Lived Study (FLLS). These results demonstrate a consistent relationship between homozygosity for the d3-GHR deletion allele and longevity among the cohorts studied. However, this observation was limited only to males; the frequency of d3-GHR deletion homozygosity among females did not differ with age in any of the cohorts studied. On average, d3/d3 homozygotes were 1 inch taller than the wild-type (WT) allele carriers and also showed lower serum IGF-1 levels. Multivariate regression analysis indicated that the presence of d3/d3 genotype adds approximately 10 years to life span.

It appears that deletion of the GHR gene exon 3 might have originated from complex genomic events taking place after the emergence of Old World monkeys, followed by homologous recombination between two retro-elements in Homo sapiens. Thereafter, it spread throughout the human clades to be present now in approximately 25% of Caucasian chromosomes. In centenarians, most IGF-1 regulation seems to respond to caloric and protein nutritional signals, not from GH. IGF-1 is not lower in carriers of d3-GHR during childhood, adolescence, and adulthood despite several reports showing that the GHR genotype may influence circulating IGF-1 under basal conditions. People with d3-GHR or fl-GHR alleles produce comparable amounts of circulating IGF-1. That said, we suspect people with d3-GHR alleles to have a decreased GH secretion.

A fair amount of aging research starts in yeast (or worms, or flies) for reasons of cost, only later moving to mammals such as laboratory mice. A surprisingly large fraction of the cellular mechanisms relevant to aging are much the same in all of these species. Most of the ways in which metabolism determines natural variations in longevity, both between individuals and between species, were established very early in the evolution of cellular life. With this in mind, you might recall researchers demonstrating earlier this year that life span in yeast can be extended via provision of lithocholic acid. The open access paper here follows that with a consideration of the mechanisms involved: it appears to work via alteration of the composition of mitochondria, making them more resistant to damage.

The membrane pacemaker theory of aging puts mitochondrial composition front and center, based on comparisons of mitochondria between species with very different life spans. Longer-lived species tend to have mitochondria built out of more resilient lipids - though in this paper the researchers suggest that such differences in lipid composition are far more influential in the cell than simply a matter of damage resistance. Mitochondrial function and mitochondrial damage are any case considered to be very important in aging. Loss of mitochondrial activity is seen in many age-related diseases, and some forms of damage to mitochondria appear capable of creating dysfunctional cells that export harmful reactive molecules into the surrounding tissues. Restoring mitochondria to youthful function in the aged is an important component of rejuvenation research.

Mitochondria are indispensable for organismal physiology and health in all eukaryotes. The efficiencies with which these organelles generate the bulk of cellular ATP and make biosynthetic intermediates for amino acids, nucleotides, and lipids are known to deteriorate with age. Such age-related deterioration of mitochondrial functionality is the universal feature of aging in evolutionarily distant eukaryotic organisms.

A number of mechanisms underlie the essential roles of some traits of mitochondrial functionality in both modes of yeast aging. These traits in replicatively and chronologically aging yeast include mitochondrial electron transport chain and oxidative phosphorylation, membrane potential, reactive oxygen species (ROS) homeostasis, protein synthesis and proteostasis, iron-sulfur cluster formation, and synthesis of amino acids and NADPH. Until recently, it was unknown if such trait of mitochondrial functionality as the composition of mitochondrial membrane lipids can influence aging in yeast. Our recent studies have revealed that lithocholic bile acid (LCA) can delay the onset and decrease the rate of yeast chronological aging. We demonstrated that the robust geroprotective effect of exogenously added LCA is due to its ability to cause certain changes in lipid compositions of both mitochondrial membranes. These changes in mitochondrial membrane lipids enable mitochondria to establish and maintain an aging-delaying pattern of the entire cell.

This LCA-driven remodeling of mitochondrial lipidome triggers major changes in mitochondrial abundance and morphology and also alters mitochondrial proteome. These changes in the abundance, morphology, and protein composition of mitochondria lead to specific alterations in mitochondrial functionality. Our recent unpublished data indicate that the LCA-dependent alterations in mitochondrial lipidome, proteome, and morphology can also elicit changes in lipidomes of other organelles and in concentrations of a specific set of water-soluble metabolites. By sensing different aspects of mitochondrial functional state, a discrete set of ten transcription factors orchestrates a distinct transcriptional program for many nuclear genes. The denouement of this cascade of consecutive events is the establishment of a cellular pattern that delays the onset and slows the progression of yeast chronological aging.

Of note, the proposed mechanism here for how the LCA-dependent remodeling of mitochondrial lipidome in the yeast S. cerevisiae allows to establish an aging-delaying cellular pattern is reminiscent of the mechanism in which the mitochondrial unfolded protein response causes remodeling of the mitochondrial lipidome in the nematode C. elegans, and then triggers a cascade of events that institute an aging-delaying cellular pattern. Moreover, the essential role of mitochondrial lipid metabolism in defining the pace of yeast chronological aging further supports the notion that the vital role of lipid homeostasis in healthy aging has been conserved in eukaryotes.

Link: https://doi.org/10.1155/2017/2916985

Researchers here outline a novel method of searching for longevity-related mechanisms in nematode worms: mutate the bacteria that the worms eat. The researchers worked their way through a selection of bacterial mutations, and along the way uncovered a few items of interest for further exploration. I think the leap made by press and publicly materials to supplements for human consumption containing mutated bacteria is getting far ahead of the science, however. This is, as of the moment, really only a demonstration of a new method of discovery in a commonly used laboratory species.

Scientists have identified bacterial genes and compounds that extend the life of and also slow down the progression of tumors and the accumulation of amyloid-beta, a compound associated with Alzheimer's disease, in the laboratory worm C. elegans. "The scientific community is increasingly aware that our body's interactions with the millions of microbes in our bodies, the microbiome, can influence many of our functions, such as cognitive and metabolic activities and aging. In this work we investigated whether the genetic composition of the microbiome might also be important for longevity."

This question is difficult to explore in mammals due to technical challenges, so the researchers turned to the laboratory worm C. elegans, a transparent, simple organism that is as long as a pinhead and shares essential characteristics with human biology. During its 2 to 3 week long lifespan, the worm feeds on bacteria, develops into an adult, reproduces, and progressively ages, loses strength and health and dies. Many research laboratories around the world work with C. elegans to learn about basic biological processes.

Researchers employed a complete gene-deletion library of bacterium E. coli; a collection of E. coli, each lacking one of close to 4,000 genes. "We fed C. elegans each individual mutant bacteria and then looked at the worms' life span. Of the nearly 4,000 bacterial genes we tested, 29, when deleted, increased the worms' lifespan. Twelve of these bacterial mutants also protected the worms from tumor growth and accumulation of amyloid-beta." Further experiments showed that some of the bacterial mutants increased longevity by acting on some of the worm's known processes linked to aging. Other mutants encouraged longevity by over-producing the polysaccharide colanic acid. When the scientists provided purified colanic acid to C. elegans, the worms also lived longer. Colanic acid also showed similar effects in the laboratory fruit fly and in mammalian cells cultured in the lab.

Interestingly, the scientists found that colanic acid regulates the fusion-fission dynamics of mitochondria, the structures that provide the energy for the cell's functions. "These findings are also interesting and have implications from the biological point of view in the way we understand host-microbe communication. Mitochondria seem to have evolved from bacteria that millions of years ago entered primitive cells. Our finding suggests that products from bacteria today can still chime in the communication between mitochondria in our cells. We think that this type of communication is very important and here we have provided the first evidence of this. Fully understanding microbe-mitochondria communication can help us understand at a deeper level the interactions between microbes and their hosts."

Link: https://www.bcm.edu/news/molecular-and-human-genetics/gut-bacteria-slow-aging

Last year some of the researchers associated with the Biogerontology Research Foundation proposed a class of therapy they call whole body induced cell turnover. I noticed a new paper and publicity materials on this topic today. In essence the goal is to augment the normal processes of cell turnover with therapies that remove and replace more cells than would normally be the case, thus clearing out the damage in those cells along the way. Since aging is caused by cell and tissue damage, in the ideal case this approach should act as a form of rejuvenation therapy. Obviously there are some limits here, such as areas of the brain where cells are storing the state of the mind, but in principle all other tissues are amenable to cell replacement. Beyond that, however, there is also the question of damage outside cells, such as waste compounds in tissue fluids and within the extracellular matrix structures that support cells. Further, consider signals propagated by damaged cells in one part of a tissue that affect normal cells elsewhere, such as the inflammation spurred by senescent cell signaling.

That said, it is nonetheless clear that the logical long-term direction for tissue engineering and regenerative medicine strategies must be to move towards more incremental, in-situ, small-scale replacement of damaged parts. Practical tissue engineering in the years immediately ahead will certainly start with tissue sections and organs grown in bioreactors and then transplanted into patients. Surgery is expensive and traumatic, however, and especially so in the old. In order to avoid those costs, all treatments will ultimately involve manipulation and repair of cell populations in the body, with the complexity baked into the therapeutics, and little human supervision of their progression required. This might be accomplished by delivery of signals, delivery of cells, or various other more sophisticated therapeutics, but there will be no surgery and no construction of tissue outside the body.

It is easy enough to point out the underpinnings of this trend, and theorize at the high level as to how to make it a reality, but the implementation details are of great importance - they are the whole of the story, in fact. Replacing cells in a way that is safe, and that also removes cellular damage rather than propagating it, isn't a straightforward task with an obvious solution, for all that there are plenty of starting points in today's biotechnology industry and research community. Strategies will likely vary considerably from tissue to tissue. It is likely that much more of the biochemistry of natural regenerative processes must be mapped and understood, so as to avoid interfering in counterproductive ways. Targeted cell destruction technologies must evolve into more sophisticated and discriminating forms. And so on. It is a big task and an expansive vision for the future of medicine.

Induced Cell Turnover: A proposed modality for in situ tissue regeneration and repair

Researchers originally proposed Induced Cell Turnover (ICT) in 2016. The proposed therapeutic modality would aim to coordinate the targeted ablation of endogenous cells with the administration of minimally-differentiated, hPSC-derived cells in a gradual and multi-phasic manner so as to extrinsically mediate the turnover and replacement of whole tissues and organs with stem-cell derived cells. In a new paper the authors refine the methodological underpinnings of the approach, take a closer look at potential complications and strategies for their deterrence, and analyze ICT in the context of regenerative medicine as an intervention for a broader range of conditions.

"One of the major hurdles limiting traditional cell therapies is low levels of engraftment and retention, which is caused in part by cells only being able to engraft at locations of existing cell loss, and by the fact that many of those vacancies have already become occupied by extracellular matrix (ECM) and fibroblasts (i.e. scar tissue) by the time the cells are administered, long after the actual occurrence of cell loss. The crux underlying ICT is to coordinate endogenous cell ablation (i.e. induced apoptosis) with replacement cell administration so as to manually vacate niches for new cells to engraft, coordinating these two events in space and time so as to minimize the ability for sites of cell loss to become occupied by ECM and fibroblasts. This would be done in a gradual manner so as to avoid acute tissue failure resulting from the transient absence of too many cells at any one time. While the notion of endogenous cell clearance prior to replacement cell administration has become routine for bone marrow transplants, it isn't really on the horizon of researchers and clinicians working with solid tissues, and this is something we'd like to change."

Cell-type and tissue-specific rates of induced turnover could be achieved using cell-type specific pro-apoptotic small molecule cocktails, peptide mimetics, and/or AAV-delivered suicide genes driven by cell-type specific promoters. Because these sites of ablation would still be "fresh" when replacement cells are administered, the presumption is that the patterns of ablation will make administered cells more likely to engraft where they should, in freshly vacated niches where the signals promoting cell migration and engraftment are still active. By varying the dose of cell-type targeted ablative agents, cell type and tissue-specific rates of induced turnover could be achieved, allowing for the rate and spatial distribution of turnover to be tuned to the size of the tissue in order to avoid ablating too many cells at once and inadvertently inducing acute tissue failure.

"ICT could theoretically enable the controlled turnover and rejuvenation of aged tissues. The technique is particularly applicable to tissues that are not amenable to growth ex vivo and implantation (as with solid organs) -- such as the vascular, lymphatic, and nervous systems. The method relies upon targeted ablation of old, damaged and/or senescent cells, coupled with a titrated replacement with patient-derived semi-differentiated stem and progenitor cells. By gradually replacing the old cells with new cells, entire tissues can be replaced in situ. The body naturally turns over tissues, but not all tissues and perhaps not optimally."

Induced Cell Turnover: A novel therapeutic modality for in situ tissue regeneration

Induced Cell Turnover (ICT) is a theoretical intervention in which the targeted ablation of damaged, diseased and/or nonfunctional cells is coupled with replacement by partially differentiated induced pluripotent stem cells in a gradual and multi-phasic manner. Tissue-specific ablation can be achieved using pro-apoptotic small molecule cocktails, peptide mimetics, and/or tissue-tropic AAV-delivered suicide genes driven by cell-type specific promoters. Replenishment with new cells can be mediated by systemic administration of cells engineered for homing, robustness, and even enhanced function and disease resistance. Otherwise, the controlled release of cells can be achieved using implanted biodegradable scaffolds, hydrogels, and polymer matrices. In theory, ICT would enable in situ tissue regeneration without the need for surgical transplantation of organs produced ex vivo, and addresses non-transplantable tissues (such as the vasculature, lymph nodes, skin and nervous system). We have outlined several complimentary strategies for overcoming barriers to ICT in an effort to stimulate further research at this promising interface of cell therapy, tissue engineering, and regenerative medicine.

Ischemic injuries, in which insufficient oxygen is delivered to tissues, can occur in numerous ways, but heart attacks are among the most common, evident, and dangerous. A sizable branch of the research community works on ways to efficiently and quickly provide oxygen to the impacted tissues so as to reduce the long-term damage and speed recovery. At the end of this road lie permanent enhancements such as respirocyte nanomachinery that will provide hours of supplemental oxygen for all tissues, but for now researchers are still working on the first potential advances in emergency oxygen supplementation, such as the example noted here.

The use of photosynthetic microorganisms to provide much needed oxygen to damaged heart tissue could be a feasible approach to treating heart attacks. Recent research describes the injection of the cyanobacterium Synechococcus elongatus into ischemic heart muscles of live rats, where, in response to light exposure, the microbes produced oxygen and improved organ function. Photosynthetic organisms capture energy from sunlight and use it to convert carbon dioxide and water into carbohydrates for growth. The process creates a surplus of oxygen, which the organisms simply expel into the atmosphere, much to the delight of aerobic organisms such as humans. "One day I was thinking: what is the fundamental problem with a heart attack? It's the absence of oxygen being delivered to the heart muscle. And, what in nature makes oxygen for us every single minute? Plants."

"Cardiologists are always thinking about how to deliver more blood to ischemic heart tissue. But, if oxygen is the critical component, what if you could take a plant, or the photosynthetic mechanism of a plant, and put it right next to a heart cell?" To investigate this unusual idea, researchers took an equally unusual approach. "We started grinding up kale and spinach to isolate the chloroplasts and put them with heart cells." But the results of these experiments were disappointing. "What we found is that chloroplasts do not like being outside of a plant cell. They're not very stable."

So, the team instead tried the photosynthetic unicellular microorganism S. elongatus. "We put them with heart cells in a dish and we found that they could live together and, when we shone light on them, they could produce oxygen." The team then tested the idea in live rats. They gave the animals heart attacks and then injected their hearts with S. elongatus and exposed the hearts to light. After just 10 minutes, oxygen in the bacteria-containing and light-exposed hearts had risen approximately 25-fold compared with just a 3-fold rise in oxygen in bacteria-containing hearts kept in darkness. And by 45 minutes, left ventricle pressure and cardiac output had improved, suggesting increased heart contractility. The microbes also provided long-term improvements to heart function. Four weeks after rats were subjected to temporary cardiac ischemia (60 minutes) - during which the animals were, or were not, injected with S. elongatus and exposed to light - analyses of heart function revealed that recipients of the microbe treatment had significantly improved contractility compared with controls.

Link: http://www.the-scientist.com/?articles.view/articleNo/49662/title/Bright-Lights-and-Bacteria-Treat-Rats--Heart-Attacks/

One of the theories relating to causes of sarcopenia, the characteristic loss of muscle mass and strength with age, is that it relates to dysfunction in the processing of amino acids such as leucine. There is evidence for leucine supplementation to help slow the progression of sarcopenia, for example. The research here adds more along these lines, though it seems the authors will have to run a redesigned study to see whether or not the cellular differences observed actually produce meaningful results over a longer period of time:

The loss of skeletal muscle mass and quality is common with aging. This loss highlights the development of sarcopenia, where diminished muscle mass and strength are major contributors engendering loss of independence and quality of life for older adults. Current research suggests that reductions in the ability to stimulate muscle protein synthesis and promote proliferation and differentiation of muscle satellite cells may be important contributors to the development of sarcopenia.

It is well known that exercise and the ingestion of essential amino acids (EAA), in particular the amino acid leucine, are important stimulators of muscle protein synthesis through activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. While an anabolic resistance to the independent effects of exercise and EAA or protein is prevalent with aging, combining the two stimuli shows promise in combating sarcopenia via the ability for this combination to maximally stimulate mTORC1 and upregulate the translation initiation machinery. Indeed, we have recently demonstrated that provision of leucine-enriched EAA mixture following a bout of high-intensity resistance exercise (RE) stimulates mTORC1 and prolongs myofibrillar protein synthesis for up to 24 hours post-RE in the very same cohort of older men we examined in this study whereas, in the absence of EAA this mTORC1 response is blunted in older adults.

A host of evidence has suggested increased satellite cell (SC) activation and content following RE in human skeletal muscle, yet we and others have demonstrated a blunting of or a delayed ability to activate and increase the SC pool in older men compared with a younger cohort. EAA and leucine provision has been shown to upregulate SC activity via mTORC1. Therefore, we hypothesized that EAA ingestion, which we have previously shown to potently activate mTORC1 following an acute bout of leg resistance exercise, would enhance skeletal muscle satellite cell proliferative capacity and content in older men. We demonstrate that older men do not appear to increase skeletal muscle satellite cell content at 24 hours following heavy, high volume resistance exercise in the absence of EAA ingestion. However, when 10g of EAA is ingested one hour postexercise we found that MHC I myofiber satellite cell content displays obvious trends to be greater than when older men are not given postexercise EAA. Although this pattern is visually evident in MHC II myofibers and when all myofibers are pooled, the current data set did not reach statistical significance.

Link: https://doi.org/10.14814/phy2.13269

Numerous research teams are interested in finding ways to enhance muscle regeneration, and below find the publicity materials for a couple of different lines of research along these lines - researchers in search of specific mechanisms that might be amenable to change, and thus the potential foundation for a drug discovery program and therapies in the clinic. Enhanced muscle regeneration encompasses more than just faster and more comprehensive recovery from injury, as much the same set of mechanisms are also involved in the normal maintenance and growth of muscles. As I'm sure the audience here is well aware, muscle tissue weakens and diminishes with age, a condition known as sarcopenia. Researchers hope that enhancements to the processes of muscle repair will be able to at least partially compensate for the losses of age, or delay those losses somewhat, even though they fail to directly address the underlying reasons for this form of age-related decline.

There is considerable debate over the causes of sarcopenia, and as for most aspects of aging, it is not yet possible to draw a consensus line of cause and effect from the sort of root cause forms of cellular damage outlined in the SENS rejuvenation research proposals all the way to well known age-related diseases, passing through the better explored metabolic changes observed in aging along the way. There is a great deal of data for sarcopenia, however: it is a puzzle in which at least some of the pieces are already joined together, even if it isn't quite settled as how these little islands of knowledge relate to one another in the bigger picture.

Changes in processing of amino acids appear important, as does the decline in diet and exercise in later life. Cellular senescence is implicated, as is becoming the case in many parts of the field now that more attention is being given to removal of senescent cells. Loss of mitochondrial function is also thought important, and there are related views involving loss of capillaries and nutrient supply in older tissue. Of late, there has been a fairly compelling argument to point to loss of stem cell activity as the primary lynchpin issue, though everything else just mentioned may well contribute to that loss. Everything is connected to everything else in cellular metabolism, and there are many angles from which the wise men can approach this elephant.

Inflammatory molecule essential to muscle regeneration in mice

Muscle stem cells usually nestle quietly along the muscle fibers. They spring into action when a muscle is damaged by trauma or overuse, dividing rapidly to generate enough muscle cells to repair the injury. But it's not entirely clear what signals present in inflammation activate the stem cells. Prostaglandin E2, or PGE2, is a metabolite produced by immune cells that infiltrate the muscle fiber as well by the muscle tissue itself in response to injury. Anti-inflammatory treatments have been shown to adversely affect muscle recovery, but because they affect many different pathways, it's been tough to identify who the real players are in muscle regeneration.

Researchers discovered a role for PGE2 in muscle repair by noting that its receptor was expressed at higher levels on stem cells shortly after injury. They found that muscle stem cells that had undergone injury displayed an increase in the expression of a gene encoding for a receptor called EP4, which binds to PGE2. Furthermore, they showed that the levels of PGE2 in the muscle tissue increased dramatically within a three-day period after injury, indicating it is a transient, naturally occurring immune modulator. "This transient pulse of PGE2 is a natural response to injury. When we tested the effect of a one-day exposure to PGE2 on muscle stem cells growing in culture, we saw a profound effect on the proliferation of the cells. One week after a single one-day exposure, the number of cells had increased sixfold compared with controls."

After seeing what happened in laboratory-grown cells, researchers tested the effect of a single injection of PGE2 into the legs of the mice after injury. "When we gave mice a single shot of PGE2 directly to the muscle, it robustly affected muscle regeneration and even increased strength. Conversely, if we inhibited the ability of the muscle stem cells to respond to naturally produced PGE2 by blocking the expression of EP4 or by giving them a single dose of a nonsteroidal anti-inflammatory drug to suppress PGE2 production, the acquisition of strength was impeded." The researchers next plan to test the effect of PGE2 on human muscle stem cells in the laboratory, and to study whether and how aging affects the stem cells' response. Because PGE2 is approved by the Food and Drug Administration for use in the induction of labor, a path to the clinic could be relatively speedy.

Researchers Find Key to Muscle Regeneration

Earlier this year, researchers published findings showing that a nuclear receptor called REV-ERB is involved in lowering LDL cholesterol. They previously studied REV-ERB's role in regulating mammals' internal clocks. Now the researchers are uncovering REV-ERB's role in muscle regeneration. Skeletal muscle comprises 40 to 50 percent of our total body mass and is essential for postural support, locomotion and breathing. With a high capacity for regeneration, skeletal muscle normally maintains muscle mass and function in response to minor injuries and normal wear and tear without much trouble. When injuries are severe - with more than 20 percent loss of muscle mass - normal muscle regeneration often cannot keep pace with the regenerative demands. In this scenario, the loss of skeletal muscle mass can trigger widespread fibrosis and loss of muscle function.

"Identifying new means of accelerating muscle regeneration has proved a daunting challenge. Therefore understanding the underlying mechanisms that regulate muscle cell regeneration and coordinate regenerative repair could provide future therapeutic options for stymieing the loss of muscle function in the traumatically injured." A simplified version of muscle cells' life-cycle looks like this: muscle stem cells produce myoblasts that will either reproduce (proliferate) or form muscle tissue (differentiate). Successful regeneration of skeletal muscle after traumatic injury depends on the replenishment of muscle fibers through elevated myoblast proliferation and differentiation.

The research team identified a mechanism through which REV-ERB may regulate gene expression pre and post muscle differentiation. "We demonstrate that REV-ERB can stimulate muscle regeneration upon acute muscle injury in an animal model. Our findings reveal that REV-ERB may be a potent therapeutic target for the treatment of a myriad of muscular disorders."