Cardiac Amyloid Buildup Correlates with Risk of Atrial Fibrillation

Amyloids are misfolded proteins that become insoluble in their incorrect configuration, forming structures that encourage other molecules of the same protein to also misfold in the same way. These structures spread, grow, and clump together into solid deposits in and around cells. Only a handful of proteins can form amyloid, and many are associated with age-related disease. Consider the amyloid-β characteristic of Alzheimer's disease, for example. The better understood forms of amyloid are known to be accompanied by a surrounding halo of toxic biochemistry that harms cells and cell function.

Setting aside genetic diseases in which proteins are created in a damaged form, more prone to amyloid formation, there are a couple of forms of amyloid that tend to show up in heart tissue with age, light chain amyloid and transthyretin amyloid. The present consensus is that light chain amyloidosis is more common to heart disease in comparison to the presence of transthyretin amyloid, but this may or may not in fact be the case, given (a) the comparatively lack of rigorous data for the subclinical, early stage of amyloidosis, and (b) work of recent years showing a greater prevalence of transthyretin amyloid in patients than previously suspected.

Everyone accumulates transthyretin amyloid as aging progresses, transthyretin amyloidosis is the major cause of death of supercentenarians, but it is far from clear as to how much harm is being done - relative to other issues - in earlier old age. Consider that aging is an accumulation of damage, and amyloids are a form of damage that degrades tissue function, but the clinical community tends to only declare a diagnosis of amyloidosis somewhere well past the point at which meaningful long-term harm is probably resulting.

The study here is an attempt to gain data on just how much damage might be taking pace to heart function in older individuals as a result of subclinical levels of amyloid in the heart. The authors do not distinguish between types of amyloid, but I think that this sort of effort is useful. You might compare it to a paper from recent years in which researchers discovered a significant presence of transthyretin amyloid in a minority of heart failure cases. The more evidence that is obtained, the more support there will be for development programs that can clear transthyretin amyloid, not just slow down its accumulation, such as that undertaken at Covalent Bioscience.

Atrial Fibrillation in the Elderly: The Role of Sub-Clinical Isolated Cardiac Amyloidosis

Atrial fibrillation (AF) is the most common cardiac arrhythmia, and is associated with considerable morbidity and mortality. The prevalence of AF increases with increasing age and is related to the increasing prevalence of comorbidities and structural remodelling of the atria that is believed to occur with aging. Increasing atrial fibrosis is known to be associated with more frequent paroxysms of AF, persistent AF, and refractoriness to medical therapy.

Recently, a considerable proportion of elderly patients with heart failure with preserved ejection fraction (HFpEF) have been found to have isolated amyloid deposits in the heart. Further, a proportion of patients with valvular heart disease have been found to have clinically undetected amyloid deposits on atrial biopsies obtained during cardiac surgery and such deposits have been shown to be associated with an increased risk of AF. The role of such clinically undetected atrial amyloid deposits in the aetiopathogenesis of AF occurring in the absence of valvular heart disease has not been previously evaluated.

In this study, we sought to assess the prevalence of AF in patients with clinically undetected isolated cardiac amyloidosis (ICA) detected at autopsy and identify electrocardiographic (EKG) markers of such amyloid deposits. A total of 1083 patients were included in the study and 3.1% of patients were found to have asymptomatic ICA. Patients with ICA were older and had a higher odds of AF independent of age and CHA2DS2VASc score. Amongst patients with AF, those with ICA were more likely to have persistent forms of AF and had a lower sinus rhythm P-wave amplitude. Further studies are required to further define this entity, identify imaging modalities to aid in antemortem diagnosis of ICA and to establish the optimal management strategies in these patients.

Exercise Reduces Inflammatory Leukocyte Production, Slowing Development of Atherosclerosis

Researchers here report on the investigation of a lesser known mechanism by which exercise lowers risk of cardiovascular mortality. It alters cell signaling that drives the creation of inflammatory immune cells, and in turn thus accelerates the development of atherosclerosis. Atherosclerosis is the buildup of fatty deposits called plaques that narrow and weaken blood vessels, leading to heart attack and stroke. It is a condition resulting from dysfunction in the innate immune cells called macrophages that are responsible for clearing out fats from blood vessel walls. Once atherosclerosis has started, chronic inflammation from any source will accelerate its progression, by making it even harder for macrophages in an atherosclerotic plaque to adopt the set of behaviors required to help clear the damage.

Researchers examined how physical activity affects the activity of bone marrow, specifically hematopoietic stem and progenitor cells (HSPCs). HSPCs can turn into any type of blood cell, including white blood cells called leukocytes, which promote inflammation. The body needs leukocytes to defend against infection and remove foreign bodies. But when these cells become overzealous, they start inflammation in places where they shouldn't, including the walls of arteries.

Researchers studied a group of laboratory mice who were housed in cages with treadmills. Some of the mice ran as much as six miles a night on the spinning wheels. Mice in a second group were housed in cages without treadmills. After six weeks, the running mice had significantly reduced HSPC activity and lower levels of inflammatory leukocytes than other mice who simply sat around their cages all day. Exercising caused the mice to produce less leptin, a hormone made by fat tissue that helps control appetite, but also signals HSPCs to become more active and increase production of leukocytes. In two large studies, the team detected high levels of leptin and leukocytes in sedentary humans who have cardiovascular disease linked to chronic inflammation.

Reassuringly, the study found that lowering leukocyte production levels by exercising didn't make the running mice vulnerable to infection. This study underscores the importance of regular physical activity, but further focuses on how mechanisms by which exercise dampens inflammation could lead to novel strategies for preventing heart attacks and strokes.


Reporting on the Aging Research and Drug Discovery Meeting Held at BASAL Life 2019

Earlier this year the Aging Research and Drug Discovery meeting was organized as a part of the broader BASAL LIFE scientific conference. As is traditional for such events, the organizers put together a paper reviewing the proceedings. A few of the early highlights are noted below, but many more presentations are briefly discussed in the open access paper. It is a representative selection of the present distribution of projects and research goals in the scientific community focused on intervention in the aging process.

Aging poses profound health-related challenges that need to be tackled to reduce the social and economic burden on our aging society. Multidisciplinary perspectives will be of tremendous importance to understand the underlying molecular processes of aging and to accelerate the discovery and development of effective aging interventions. It is therefore indispensable that industry and academia develop deeper cooperation and greater interchange of knowledge and technology. For this purpose, world leading experts from diverse research fields and various sectors came together at the 6th installment of the Aging, Drug Discovery and Artificial Intelligence conference, which was held from the 10th to the 12th September 2019 in Basel as part of the Basel Life Science Week.

Although great progress has been made towards the understanding of aging mechanisms, effective drug interventions are still missing for most age-related disorders. Targeting the aging process contrasts the traditional approach of "one disease-one drug"; thus, multiple challenges need to be overcome, as discussed by Nir Barzilai from the Albert Einstein College of Medicine, NY, USA. In particular, the political attention needs to be further strengthened by highlighting the clinical and economic benefits of aging interventions. However, no party will cover intervention costs without an indication for which simple and reliable biomarkers are still lacking. Towards a resolution of this issue, the Targeting Aging with Metformin (TAME) study driven by Nir Barzilai may represent a proof-of-concept that could pave the way to clinical trials leading to healthy aging.

How can we fill the gap between lab animal research, which has traditionally stopped at murine studies, and human clinical trials? Matt Kaeberlein from the University of Washington, Seattle, USA, and colleagues several years ago initialized the dog aging project to overcome this barrier. Companion animals like dogs as model organisms provide multifarious advantages including a faster aging pace than humans, high genetic diversity, and a shared environment with humans. The dog aging project aims to investigate the influence of genetic and environmental determinants on the life- and healthspan of domestic dogs based on survey, sequencing, blood biochemistry and -omics data collection. Further, the project provides the opportunity to test aging interventions, as already initiated for the mammalian Target Of Rapamycin (mTOR) inhibitor rapamycin. Notably, the completed phase 1 for the rapamycin intervention trial revealed no-side effects and improved cardiac function in treated dogs

Aubrey de Grey from the SENS Research Foundation, Mountain View, California, USA, emphasized that placing the focus on healthspan and not on lifespan will help to rebut societal concerns for longevity investigations. Further, he discussed that human diseases with a higher prevalence at older ages should be treated and explored differentially than communicable diseases. In this regard, he introduced the SENS Research Foundation (SRF) and their concept of maintenance by targeting mechanisms that mitigate cellular damage accumulating during aging. Notably, treatments of age-related diseases directed by spinouts of SRF aim to increase the healthspan of elderly - increased longevity is considered as a positive side-effect.


Werner Syndrome is Strongly Mediated by Mitochondrial Dysfunction

Researchers here report that NAD+ upregulation to improve mitochondrial function, via supplementation with nicotinamide riboside and nicotinamide mononucleotide, does a decent job of rescuing the life span of flies and worms with the genetic mutation that causes Werner syndrome. It is not quite all the way restored to match wild-type animals, but close. Werner syndrome is a DNA repair deficiency condition in which patients exhibit, at the high level, what appears to be accelerated aging: early onset of a range of age-related conditions, early mortality. It is not, however, accelerated aging. Natural aging stems from rising levels of molecular and cellular damage, but damage of a particular blend of varieties. Werner syndrome is one specific class of damage, stochastic nuclear DNA damage, elevated to a very large degree. There are important differences, and it is never all that clear as to whether we can apply lessons learned in DNA deficiency conditions to normal aging - it depends greatly on the fine details in each case.

The most interesting point here is that, in at least short-lived species such as flies and worms, the harm done by this particular DNA repair deficiency is to a large degree mediated by an early collapse in mitochondrial function. It remains to be see whether this is also true in mammals: the literature might lead us to expect that high levels of stochastic mutational damage to nuclear DNA could be causing all sorts of other harms. Mitochondria are the power plants of the cell, responsible for packaging the chemical energy store molecule ATP needed to power cellular operations. NAD+ is vital to mitochondrial operation, and its levels decline with age, alongside mitochondrial function. Artificially boosting NAD+ levels has been shown to restore the ability of mitochondria to function in a more youthful fashion, but as yet there is only the one small clinical trial to show health benefits in older humans.

Can we take this paper as evidence for mitochondrial decline to be very important in normal aging? That would be the question. If I were speculatively joining pieces of the jigsaw puzzle, I would take this study, and put it next to the recent finding that suggests double strand breaks in nuclear DNA will cause epigenetic drift of the sort observed in aging. So the more of this sort of DNA damage, more epigentic change. Problems with mitochondria are perhaps proximately caused by changing levels of specific proteins, such as those necessary for the process of fission. Too little fission results in ever larger mitochondria that are not easily cleared out by the maintenance processes of mitophagy when they become worn and damaged. Protein levels are, of course, under epigenetic control. Perhaps this all fits together, but it still needs a lot of work to shore up the relevant evidence; it should be treated as speculative.

NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome

We report that Werner syndrome (WS) is associated with a significant mitochondrial dysfunction, mainly manifested as defective mitophagy. This is reflected in lower NAD+ levels across species from worms to humans. NAD+ supplementation improves mitochondrial function and other age-related metabolic outcomes. Mitochondrial disease can manifest itself in multiple clinical outcomes amongst which neurodegeneration and impaired metabolism are common. Some features of WS may be explained by genomic instability due to mutation in the gene encoding the Werner protein (WRN), an important DNA helicase/exonuclease involved in DNA repair, telomere and heterochromatin maintenance, and cancer regulation. However, the relationship between WRN mutations and the syndrome's severe dysregulation of energy metabolism is unclear.

Mitochondrial quality and function decline with age, contributing to insulin resistance and metabolic diseases in the elderly. Mitochondrial quality control is regulated by biogenesis and mitophagy. Mitophagy involves the targeting of damaged mitochondria to the lysosomes wherein the mitochondrial constituents are degraded and recycled. Defective mitophagy is prominent in aging and age-predisposed disorders, including metabolic diseases and neurodegeneration. However, the role of mitophagy in WS has not been investigated.

The metabolic molecule nicotinamide adenine dinucleotide (NAD+) is emerging as a fundamental regulator of mitochondrial homeostasis, genome stability, neuroprotection, healthy aging, and longevity. Interestingly, genetic and/or pharmacological upregulation of intracellular NAD+ levels protects against obesity and type 2 diabetes in rodents, and against age-related diseases and neurodegenerative diseases such as Alzheimer's disease.

We therefore examined whether mitochondrial dysfunction and NAD+ depletion occur in WS, and if so, how it contributes to the molecular pathology in WS. We report that NAD+ depletion is a major driver of the severe metabolic dysfunction in WS through dysregulation of mitochondrial homeostasis. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.

SHMT2 in the Age-Related Decline of Mitochondrial Function

Mitochondria are the descendants of ancient symbiotic bacteria, several hundred of them in every cell. Their primary task is to produce the chemical energy store molecule adenosine triphosphate (ATP) to power cellular operations. With aging, mitochondria throughout the body decline in function. They change their morphology, the balance between fission and fusion shifts, the ability of the cell to remove worn and damaged mitochondria is impaired. Researchers have made some inroads into the proximate causes of these global changes, meaning upregulation or downregulation of specific proteins, but the connection to the root causes of aging remains unclear. The research noted here is an example of continued efforts in this direction, and in this specific case offers a hint that mitochondrial decline with aging may be a part of the evolved trade-off between (a) cancer risk due to active cells in a damaged environment and (b) functional decline due to inactive cells that fail to maintain an increasingly damaged environment.

In a previous study, we proposed that age-related mitochondrial respiration defects observed in elderly subjects are partially due to age-associated downregulation of nuclear-encoded genes, including serine hydroxymethyltransferase 2 (SHMT2), which is involved in mitochondrial one-carbon (1C) metabolism. This assertion is supported by evidence that the disruption of mouse Shmt2 induces mitochondrial respiration defects in mouse embryonic fibroblasts generated from Shmt2-knockout E13.5 embryos experiencing anaemia and lethality.

Here, we elucidated the potential mechanisms by which the disruption of this gene induces mitochondrial respiration defects and embryonic anaemia using Shmt2-knockout E13.5 embryos. The livers but not the brains of Shmt2-knockout E13.5 embryos presented mitochondrial respiration defects and growth retardation. Metabolomic profiling revealed that Shmt2 deficiency induced foetal liver-specific downregulation of 1C-metabolic pathways that create taurine and nucleotides required for mitochondrial respiratory function and cell division, respectively, resulting in the manifestation of mitochondrial respiration defects and growth retardation.

The results in this study also suggest that age-associated downregulation of SHMT2 would furthermore control age-related growth retardations, as well as mitochondrial respiration defects, in human fibroblasts from elderly subjects. Therefore, activation of SHMT2 or uptake of certain supplementary 1C sources, such as formate and glycine, might thwart the manifestation of age-related disorders. By contrast, activation of SHMT2 or intake of these supplements might enhance tumour growth due to the fact that SHMT2 is activated in certain human tumour cells, and that its disruption suppresses tumour growth, as well as respiratory function. Therefore, it appears to be controversial whether the activation of SHMT2 or intake of these supplements extends lifespan by restoring mitochondrial respiratory function and cell division or shortens lifespan by the activation of tumour growth and tumour progression. To resolve this controversial issue, further investigation is required.


Towards Small Molecule Drugs that Suppress α-Synuclein Aggregation

Researchers here report on efforts to find small molecules that can interfere in the molecular biochemistry of synucleinopathy. Parkinson's disease is the best known of the synucleinopathies; these are neurodegenerative conditions characterized by the misfolding and consequent aggregation of α-synuclein. This is one of a handful of proteins in the body that can misfold in a way that encourages other molecules to also misfold, forming structures and then solid deposits that cause considerable harm as they spread throughout the aging brain. The best form of therapy would be some form of periodic clearance of these errant molecules, but, absent that, a way to interfere in the processes of misfolding and aggregation would be a step forward.

The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes.

Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn.

Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson's disease.


The Tight Junctions of the Blood-Brain Barrier in Aging and Neurodegeneration

Today's open access paper is a review of the tight junction structures of the blood-brain barrier in aging and neurodegeneration. The blood-brain barrier is a structure of specialized cells that lines the blood vessels that pass through central nervous system tissue. The barrier allows only certain molecules and cells to pass between blood vessels and the central nervous system, thus preserving its separation from the rest of the body. Unfortunately the integrity of the blood-brain barrier breaks down with advancing age, and the entry of unwanted molecules and cells into the brain then contributes to inflammation and tissue dysfunction.

A number of studies have shown links between blood-brain barrier dysfunction and the progression of neurodegenerative conditions. Researchers have identified leakage of fibrinogen into the brain as a cause of inflammation and synaptic damage. The inappropriate passage of molecules across the blood-brain barrier begins when the behavior of endothelial cells changes for the worse, and there is also a progressive loss in the number of pericyte cells. These changes correlates with cognitive decline even in the absence of other signs of pathology, such as protein aggregates. Interestingly, the amyloid-β characteristic of Alzheimer's disease has been shown to cause blood-brain barrier dysfunction.

The Blood-Brain Barrier and Its Intercellular Junctions in Age-Related Brain Disorders

The functional state of the central nervous system (CNS) is greatly dependent on the quality of the vasculature. As the centuries old saying goes: "A man is as old as his arteries". Today, especially for the brain, this concept should be redefined: You are as old as your microvessels and capillaries. There is increasing evidence that the cerebral microvasculature and the neurovascular unit play a critical role in age-related brain dysfunctions. The multitude of brain microvascular changes accompanied by aging includes endothelial dysfunction, blood-brain barrier (BBB) breakdown, decrease in blood flow, microhemorrhages, vessel rarefication, and neurovascular uncoupling.

Cerebral endothelial cells (CECs) lining brain capillaries are considered the principal barrier forming endothelial cells. They are interconnected by a continuous line of tight junctions and characterized by a high number of mitochondria and low number of caveolae. These characteristics contribute to the formation of a paracellular and transcellular barrier.

With aging, the density of brain vasculature is decreased and cerebrovascular dysfunction appears to precede and accompany cognitive dysfunction and neurodegeneration. Cerebrovascular angiogenesis is decreased and cerebral blood flow is inhibited by anomalous blood vessels such as tortuous arterioles and thick collagen deposits in the walls of veins and venules. In most mammals, the capacity of CECs to divide is limited and endothelial cells are prone to be senescent. Aging is associated with endothelial dysfunction, arterial stiffening, and remodeling, impaired angiogenesis, defective vascular repair and with an increasing prevalence of atherosclerosis. In the aging brain cerebral blood flow declines and perfusion pressure either is constant or increases.

The paracellular barrier properties of CECs are determined by the tight junction (TJ), which are composed of transmembrane proteins that control the transport across the intercellular space between adjacent cells and cytoplasmic plaque. Limited data is available on what changes develop in the function of the BBB and the composition and structure of endothelial TJs in the healthy aging human brain. In a meta-analysis of BBB permeability studies, the barrier function was negatively impacted by age. Though there were some discrepancies, paracellular permeability was generally increased in the aged human brain. Permeability changes are likely the result of decreased expression and disorganized localization of TJ proteins.

With the building evidence that dysfunction of the microvasculature is not just coincident but is part of the underlying mechanisms of aging and associated neurovascular and neurological disorders, new therapeutic possibilities are opened. The significant heterogeneity of BBB disruption data in studies using aging postmortem brain tissue suggests that more data is necessary to clearly understand the role of BBB disruption and to see whether it is a symptom or a cause. Thus further comprehensive BBB TJ and permeability studies are needed in the field of aging and aging associated disorders.

Nanotics Aims at Preventing Senescent Cells from Evading Immune Surveillance

Nanotics works on a nanoparticle platform that can modulate cell signaling via depletion of arbitrary target signaling molecules, something that has a great many potential uses, such as altering the behavior of the immune system. Given the present level of interest in clearance of senescent cells as an approach to treating aging, it isn't surprising to see platform companies of this ilk turning their attention to the production of senolytic treatments in addition to their existing pipelines. Here the approach is to deny lingering senescent cells the capacity to protect themselves against immune surveillance, and thus enable the immune system to destroy a greater fraction of these errant cells than would otherwise be the case.

Many cell signals are normally delivered in an intelligent cell-mediated way, meaning that one cell actually moves through the body until it's quite close to a target cell, before releasing the appropriate signal molecules. This greatly increases the likelihood that only the target cell - and not an "off-target" cell - will receive the signal. However, in various diseases, the delivery of the signal molecule is dysregulated in some way, sometimes because a cell is secreting a signal molecule systemically rather than focally to a target cell. The cell that receives this signal molecule - which it would not have received under normal circumstances - may do something it shouldn't do, which can manifest at the tissue level as disease. But the target cell may in fact be responding in a perfectly appropriate way to an incorrect signal. Most medicine still focuses on tissues and organs, rather than the underlying cells and signal molecules.

In cancer, the main problem is not the existence of cancer cells - which the immune system routinely clears out - but rather that cancer cells sometimes persist long enough to erect an inhibitory shield. The secreted molecules inhibit either the immune system's killer cells or the "death signals" those cells produce. There are times when this type of inhibitory signaling is completely appropriate. For instance, the fetus must inhibit the mother's immune system in order to survive, given that's genetically only half the mother and thus looks like an invader. Cancer not only mimics the process used by the fetus/placenta but employs many of the same molecules to do it.

For anti-aging, one would deplete the various known pathogenic signaling molecules that increase with age. Many of these are inflammatory and are responsible for what's termed inflammaging. We are also designing an approach to deplete immune inhibitors secreted by senescent cells for their defense, as a new generation of senolytic therapy. For the vast majority of non-genetic diseases, the drivers or enablers of the disease are signal molecules or molecular signal inhibitors. All of these targets can be depleted by our approach, without drugs and thus without the side effects of drugs.


Common Mechanisms of Blood-Brain Barrier Dysfunction to Underlie Many Forms of Damage to the Brain

Researchers here note a signature of blood-brain barrier dysfunction that is common in many forms of damage and injury to the brain, suggesting it to be more broadly relevant to pathology than suspected. There is already good evidence for dysfunction of the blood-brain barrier to be an early feature of neurodegenerative diseases. The specialized cells of the blood-brain barrier line blood vessels that pass through the central nervous system, managing the passage of molecules and cells. When the barrier fails, unwanted molecules such as fibrogen can enter the brain to cause inflammation - and chronic inflammation in the brain is known to be important in the progression of neurodegeneration.

Whether in the wake of a stroke, seizure, massive neuroinflammation, or a blow to the head, the endothelial cells of the blood-brain barrier (BBB) respond with remarkable similarity, according to a new study. Researchers reported that the endothelial cells that make up the BBB normally express a suite of genes that distinguishes them from the endothelia of other organs. However, BBB cells damaged in various ways lost this specialized signature, changing over to an expression profile more akin to endothelial cells in other parts of the body. The findings suggest that common mechanisms of BBB dysfunction underlie different brain injuries and diseases. "This raises the possibility that successfully preventing (or increasing) endothelial cell gene-expression changes that occur in one disease may lead to a potential therapy for other types of CNS disorders."

Tasked with shielding the precious brain from toxic insults while allowing crucial nutrients to cross, the blood-brain barrier is highly selective. Ergo, the endothelial cells that line the brain's vessels are highly specialized, forming ultra-tight junctions and mobilizing molecular transporters not typically found in vessels supplying other organs. Disruption of the barrier is thought to play a hand in the pathogenesis of multiple injuries and diseases.

How do brain endothelial cells change in the face of injury or disease? The researchers tracked gene-expression changes following four different insults known to disrupt the barrier: seizures, stroke, trauma, and experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. They then tracked the permeability of the barrier at three time points. In the earliest, or "acute" phase of each injury, the researchers found only minimal disruption of the BBB. However, in the so-called "subacute phase," which was one or two days later depending on the model, the leakiness of the BBB reached its peak. About a month later, in the "chronic phase," the barrier had partially or fully regained its integrity.

Each injury induced a bevy of gene-expression changes in brain endothelial cells. They varied substantially among injuries in the acute phase, but shared striking commonalities in the subacute phase, when the barrier was leakiest. By the chronic phase, gene expression had largely returned to normal in the stroke, seizure, and traumatic brain injury models, but remained highly altered in the EAE model. Interestingly, the researchers found that the genetic signature of the healthy BBB endothelium was most downregulated in the acute phase of traumatic brain injury, and the subacute phase of stroke, seizure, and EAE. Conversely, genes expressed predominantly in endothelial cells outside of the brain were turned up in the brain endothelium at these time points. Together, the findings suggest that while brain endothelial cells may initially respond differently to unique insults, they soon converge on a gene-expression profile that resembles those of endothelial cells in other organs of the body. Among other functions, these gene-expression changes likely ramp up interactions between the endothelium and circulating immune cells.


Slower DNA Damage Accumulation in Immune Cells Correlates with Species Life Span

Today's open access research is an assessment of DNA damage accumulation in a variety of species, showing the pace of mutational damage correlates with species life span, at least as assessed here in immune cells from blood samples, and using a marker that identifies the response to short telomeres as well as forms of DNA damage. The DNA of the cell nucleus, the genetic blueprint for near all of the proteins produced in a cell, accumulates damage over time due to the normal haphazard chemical reactions that take place constantly inside cells. These mutational changes are largely irrelevant to cellular operation, but some can cause disruption in metabolism, or, worse, make a cell cancerous, by causing certain proteins to be produced in a broken or altered state. Near all mutational damage to DNA is quickly repaired by the highly efficient array of DNA repair mechanisms that a cell is equipped with. But some inevitably slips past.

The way in which mutation leads to cancer is fairly straightforward, but it is less obvious as to how random mutation in single cells can contribute meaningfully to other aspects of aging, such as widespread tissue dysfunction. The present consensus is that the important mutations are those that occur in stem cells and progenitor cells, able to spread widely throughout a tissue via the replication of daughter somatic cells created by those stem cells and progenitor cells. It was also recently suggested that DNA damage, even when repaired, and more or less regardless of what is damaged in DNA, leads to epigenetic changes characteristic of aging, and these epigenetic changes are what causes cell function to decline. In this view, a larger amount of unrepaired DNA damage, the marker usually measured, is indicative of the true cause of harm, which is more frequent DNA repair and thus epigenetic change.

The authors here are primarily focused on DNA damage markers that occur due to critically short telomeres in additional to mutational damage. Average telomere length in tissues, and the fraction of cells with critically short telomeres, is most likely downstream of stem cell function. Telomeres shorten inexorably with each cell division in somatic cells, and cells eventually self-destruct, or become senescent and are destroyed by the immune system. Stem cells use telomerase to maintain long telomeres, and deliver daughter somatic cells with long telomeres into tissues to make up the losses. So telomere length in tissues is a function of how rapidly cells divide and how frequently replacement cells are delivered by the supporting stem cell population - the pace of the latter is well known to decline with age.

Slower rates of accumulation of DNA damage in leukocytes correlate with longer lifespans across several species of birds and mammals

Different species have very different lifespans ranging from less than 1 day for mayflies to more than 400 years for the Greenland shark. However, the exact cause of these differences in longevity are still largely unknown. Our group recently showed that the rate of telomere shortening with age correlates with lifespan in a variety of species from birds to mammals. Species with very fast telomere shortening rates such as mice have very short lifespans, and species with very slow telomere shortening rates such as humans have very long lifespans. It is interesting to note that species that share a similar longevity in spite of being evolutionarily distant like flamingos and elephants, also show a similar rate of telomere shortening, while evolutionarily closer species like mice and elephants, show very different longevities and also have very different rates of telomere shortening.

These findings suggest that longevity can be determined, at least in part, by epigenetic traits, such as the rate of telomere shortening. Furthermore, these findings pose the interesting question of which is the molecular determinant by which higher telomere shortening rates lead to shorter longevities. An obvious answer is that higher rates of telomere shortening will be associated to faster accumulation of critically short/dysfunctional telomeres, which are known to contribute to activation of a persistent DNA damage response stemming from telomeres, which leads to loss of cell viability and aging phenotypes. Thus, species that shorten telomeres at faster rates will reach telomere exhaustion and trigger a persistent DNA damage response earlier than those species that are able to maintain telomeres protected for a longer period of time. A short/dysfunctional telomere is recognized by the cell as an irreparable DNA double strand break (DSB), triggering a persistent DNA damage response which results in phosphorylation of γH2AX, and which eventually leads to cell death and/or senescence. In turn, induction of cellular senescence either owing to critically short telomeres or to other insults is also associated with increased γH2AX levels, involving in some instances the mTOR pathway. Thus, accumulation of cells with DNA damage throughout lifespan should also correlate with species longevity.

Here, we find that increased global rates of DNA damage, as determined by the DNA damage marker γH2AX which detects occurrence of double stranded DNA breaks in the genome, inversely correlates with species longevity. In particular, we determined here the rates of increase of the DNA damage marker γH2AX in leukocytes of phylogenetically distant species of birds and mammals in parallel and using the same experimental method. Previous studies have also shown a correlation between certain types of DNA damage and aging. Indeed, DNA damage accumulation with aging and telomere shortening may be related processes. Critically short telomeres as the result of cell proliferation throughout life to repair damaged tissues trigger a DNA damage signal specifically at telomeres.

We also measured the percentage of short telomeres of the species in this study, and we found that all of the species showed an increase in the percentage of short telomeres with age. This result is concomitant with the fact that average telomere length shortens with age in many species. Several studies have suggested that the percentage of short telomeres is more indicative of health and senescence than average telomere length. The percentage of short telomeres is an important metric since it is the length of the shortest telomere in a cell that induces a DNA damage response and cell senescence rather than the average telomere length of the telomeres on all of the chromosomes. Here we also noticed a mild trend for species with longer maximum lifespans to have a lower rate of increase of percent short telomeres, thus accumulating short telomeres more slowly with age. We also observed that species with the highest rates of γH2AX increase have the highest rates of increase of percent short telomeres with age. These results make a connection between γH2AX DNA damage, short telomeres, and lifespan. As cells accumulate DNA damage and short telomeres, they will enter into a state of senescence, thus accelerating the aging process and shortening lifespan.

Topical Rapamycin Evaluated as a Treatment for Skin Aging

Given the attention that descends upon any prospect of reversing skin aging, I should probably open by saying that much of the data here for extended low dose topical treatment with rapamycin over eight months, that regarding visible skin aging and collagen production, is no more exciting than that obtained by any number of other approaches, such as topical application of keratinocyte growth factor (KGF). Effect sizes are the only thing that matters, and also the one thing that all too many observers fail to consider. Looking at the paper, I would say that the primary point of interest is the 50% reduction in markers of cellular senescence in skin. Given what is known of rapamycin this seems unlikely to be a senolytic effect, so not destruction of existing senescent cells, but rather a reduction in the number of cells becoming senescent. This in turn suggests that there remains some meaningful level of ongoing natural clearance of lingering senescent cells at older ages.

This study demonstrates a clear impact of rapamycin treatment on both the molecular signature associated with senescence and the clinical signs of aging in the skin. These data support the idea that a reduction in the burden of senescent cells underlies these improvements. The results could reflect a modification of the senescent cells present in the skin or a reduction in the number of senescent cells. Although rapamycin has been shown to reduce pro-inflammatory secretions produced by senescent cells, the fact that p16INK4A is reduced suggests that the absolute number of senescent cells in the epidermis is reduced. This implies that rather than simply modifying senescent cells present in the tissue, rapamycin treatment is either reducing the number of cells entering senescence or increasing the clearance of senescent cells. Based on our studies demonstrating that rapamycin prevents the senescence transition and improves functionality in vitro, we favor the concept that rapamycin reduces entry into senescence, but we cannot rule out an additional role for clearance of senescent cells. Whether the reduction in senescent cells is due to reduced entry or increased clearance, a reduction in the burden of senescent cells would be expected to improve functionality.

Senescent cells produce pro-inflammatory cytokines, matrix metalloproteins, and reduced levels of anti-angiogenic factors, creating a secretory profile known as the Senescence-Associated Secretory Phenotype (SASP). Thus, we anticipate that rapamycin treatment reduces inflammatory cytokines in the skin, although the verification of this change represents a technical challenge due to the fact that such cytokines are present in picomolar amounts. One quantifiable aspect of skin biology that is improved by the rapamycin treatment is the incorporation of collagen VII into the basement membrane, which represents a functional measure of skin quality that is improved upon treatment with rapamycin. Collagen VII is essential for a functional skin barrier, and the levels of collagen VII decrease with age and specifically beneath wrinkles. Although the mechanism whereby rapamycin may increase collagen VII protein levels is not clear at this time, the known effects of rapamycin on autophagy and intracellular trafficking of vesicles may allow for intracellular processing of misfolded collagen and increase proper localization at the cell periphery and basement membrane.

A notable aspect of this study is the use of such a low dose of rapamycin (10 μM, or 0.001%) for topical application. Topical treatment with higher concentrations (0.1-1%) has been employed for the treatment of tuberous sclerosis complex (TSC) in adults and children and has shown efficacy. We chose to use rapamycin at a ten-fold lower dose because the concentrations used in TSC patients are intended to inhibit cell growth, while our aim was to improve cell function while maintaining proliferative potential and preventing senescence. The positive impact of our treatment regimen suggests that age-related therapy with rapamycin may be feasible at doses far below those associated with side effects; however, this possibility will require careful evaluation in each specific clinical setting.


In Search of Genes that Were Lost in Longer-Lived Mammals

Researchers here describe an interesting approach to improving the understanding of how differences in species longevity arise from differences in the operation of cellular metabolism. They report on a search for genes in short-lived mammals, mice in this case, that have been lost in long-lived mammals such as our species. Finding such genes can then lead to an investigation of specific aspects of cell and tissue function relevant to life span. As is often the case in this field, the work is of scientific interest, but not really all that relevant to near future efforts to produce rejuvenation. A complete understanding of how exactly aging progresses in detail and which mechanisms are more or less important would be helpful, but it is by no means necessary. The research and development community can forge ahead to repair the known causes of aging without a full understanding of aging - indeed, this is already progressing quite well in the matter of stem cells and senescent cells.

The genetic propensity of certain species for longevity and anti-aging is a challenging problem in vertebrate biology. Of particular interest are the genes that influence life expectancy differences among species. These genes are expected to be the real longevity genes of interest and should explain the wide differences in the rate of aging among diverse species and why similarly sized rodents or primates sometimes have anomalous life expectancies - such as naked mole-rats or humans.

No such genes have been unequivocally identified. We performed a computer-aided analysis of data relevant to lifespan and made a bioinformatic search for the genes, the loss of which might modulate lifespan. This search is based on the general idea that such genes are lost in a predefined set of species but are present in another predefined set of species. Examples of such pairs of sets include long-lived vs short-lived, homeothermic vs poikilothermic, among others. Species are included in one of two sets depending on the property of interest, such as longevity or homeothermy. A bioinformatics method and software relevant to the idea are universal towards these sets and the property that defines them.

Here, the proposed method was applied to study the longevity of Euarchontoglires species. It largely predicted genes that are highly expressed in the testis, epididymis, uterus, mammary glands, and the vomeronasal and other reproduction-related organs. In conclusion, the developed method and its software allowed us to identify a short list of presumably lost genes associated with a long lifespan in Euarchontoglires. The predicted lost genes largely demonstrate specific expressions in reproductive organs, which agrees with Williams' hypothesis concerning the reallocation of the physiological resources of the body between self-maintenance and reproduction (transition from r-strategy to К-strategy in the species evolution). The loss of some predicted vomeronasal and olfactory receptor genes in human and naked mole-rat conforms to their specific anatomical features. We suggest that the loss of certain genes in evolution is one of the essential determinants of lifespan. Overall, it is a likely driving force for many aspects of species evolution in vertebrates.


Notes on the 1st Alcor New York Science Symposium

This past weekend, I was in New York City for a meeting organized by Alcor New York, a cryonics community group that is presently seeking to set up a more robust Biostasis Society of New York complete with well-organized standby capacity to help people achieve a successful cryopreservation at the end of life. Setting aside technical issues, the greatest challenge in cryopreservation is the fact the euthanasia, and thus the ability to arrange time of death, remains largely illegal. Hence there must be expensive standby operations, suboptimal deaths that cause significant damage to the brain, and a scramble to ensure rapid cooldown and preservation when death does occur. Since there are only two reputable cryonics providers in the US, local organizations capable of coordinating standby and transport are essential.

A number of folk in the cryonics community can be found in and around New York of late; Aschwin de Wolf and Chana Phaedra of Advanced Neural Biosciences, for example. In the introduction to the meeting, it was noted that early cryonics of the 60s and 70s started as much in New York as in California - there was a Cryonics Society of New York, and restoring that entity seems a worthy goal. There were a few noteworthy visitors from elsewhere, such as one of the Nectome folk, and a representative of the European Biostasis Foundation in Switzerland - this is not CryoSuisse, interestingly enough, but a distinct initiative with some overlapping members.

The talks at the meeting were divided between discussions of progress towards slowing aging or attaining rejuvenation, and discussions of cryonics itself. In general, cryonicists have a strong interest in not dying if all possible, and thus most are quite interested in what is going on in the newly formed longevity industry. The weight given to cryonics is tempered by expectations as to how soon rejuvenation therapies will arrive, and how effective they will be over time.

Joao de Magelhaes presented remotely from the UK, and gave his view on where things stand in working towards therapies to treat aging and thereby slow or reverse age-related degeneration and mortality. He is fairly conservative and pessimistic; he doesn't think that there will be enough progress in our lifetimes to achieve actuarial escape velocity, but he does think that we will see a slowing of aging in our later lives. Therefore cryonics is very important, and it is particularly important to achieve for cryonics the same that has already been achieved for work on the treatment of aging - to move it from a small, comparatively poorly regarded fringe concern to a field with notable technical successes and greater financial support. In this, there is little substitute for the hard work of bootstrapping, advocacy, research in resource constrained environment, and so forth. On the cryonics side of the house, de Magelhaes is involved in setting up the UK Cryonics and Cryopreservation Research Network to spur more academic research into relevant technologies.

Ben Best spoke about NAD+ upregulation and senolytics; he works at the Life Extension Foundation, and the principals there have recently started to heavily promote these approaches to treating aging. To the extent that they work, this is an example of what will happen to the "anti-aging" industry of fraud and hope and supplements that do little good: many of the people involved are motivated to do something about aging, and thus the good should chase out the bad, given time and therapies that actually work. Ben Best has experimented with these approaches to therapy, as one of the physicians connected with the LEF is willing to prescribe the senolytic dasatinib and NAD+ infusions, but committed the cardinal sin of not assessing metrics before and after. This is sadly prevalent in the self-experimentation community. If you don't measure, nothing happened. Still, there is evidence for both NAD+ upregulation and senolytics to be beneficial in older people, and sooner or later ever more physicians will become comfortable enough with the evidene to prescribe these therapies the many who might benefit.

Mike Perry gave a fascinating talk on the early history of cryonics, starting in the mid-1960s. It is eye-opening just how much information can be lost even at a distance of a mere fifty to sixty years. For example, James Bedford was the second preserved individual; the first may have been a woman called Sarah Gilbert, but this is uncertain. Of the fifteen people cryopreserved from 1966 to 1973, only Bedford remains. All of the others were lost to the haphazard, unprofessional nature of the early initiatives. Perry exhibited a short film made in 1968 by members of the New York Cryonics Society, showing the process of cryopreservation in one of the dewars of the time. It is quite the artifact of its era.

Chana Phaedra gave a presentation on paths towards optimization of cryopreservation. The success of cryopreservation depends upon delivery of cryoprotectant to the brain, efficiently and rapidly. At present, even in the best of circumstances the perfusion of cryoprotectant isn't optimal. This is challenging on a number of fronts: the skull is in the way; you can't just push fluid through tissue at high pressures; the blood-brain barrier blocks all cryoprotectants to some degree. The present conclusion based on work at Advanced Neural Biosciences is that the low-hanging fruit here is finding ways to bypass or open the blood-brain barrier. That may mean new cryoprotectants, or some chemical way of disrupting the blood-brain barrier rapidly and selectively. Other options to improve the situation: faster perfusion, less ischemia, and better assays that can be used in animal studies or on preserved human brains to reliably establish the quality of the preservation.

Aschwin de Wolf discussed the prospects for revival of patients who were frozen rather than vitrified, in part or in whole. The present wisdom is that straight freezing - which can and does occur in sections of the brain given a suboptimal perfusion of cryoprotectant - is highly destructive and causes large amounts of ice crystal formation. Can people with this sort of damage be repaired? De Wolf argued that the best approach, conceptually, is some form of low-temperature repair, via nanomachinery capable of operating in a preserved tissue at liquid nitrogen temperatures. The more interesting part of the discussion was a presentation of straight frozen and then thawed brain tissue that doesn't appear to have anywhere near as much damage as we might expect. The state of the tissue is worse than the same case for vitrification, but perhaps not as much worse as thought. More work is needed to assess this conjecture, however.

Since one of the presenters was ill, I filled in and gave an impromptu talk on self-experimentation: how to do it responsibly and effectively. We might consider four classes of self-experimentation at increasing levels of sophistication. Class 1: the sort of thing that everyone does with dieting for weight loss or eating foods and supplements for benefits. Class 2: compounds that are easy to obtain, easy to use, have great human safety data, and that may have effects on aging, such as metformin (a poor idea, I think) or senolytics (a better prospect). Class 3: treatments that are logistically challenging, and that may need a personal lab. Few people would be able to safety inject themselves with myostatin antibodies, for example. Get that wrong, and you die. But it is technically plausible, and helpful in terms of spurring muscle growth, given the evidence. Class 4: treatments that require a company or other significant effort to create. Liz Parrish's efforts with Bioviva , in order to self-experiment with telomerase gene therapy, for example. Or cryonics, for that matter. In near all cases, from dieting to quite sophisticated efforts, people tend self-experiment poorly. They do not do the one fundamental thing, which is to measure the effects.

Researchers in the fields of neurobiology and cryobiology gave a couple of technical presentations. One was an interesting outline of methods that could be used to evaluate the quality of brain preservation protocols, not limited to cryonics. It essentially boils down to examining labelled dendritic spines in neural tissue, which can be done before and after an experimental preservation to see how well the fine structures survived. It is even in principle possible to do this in a brain, rather than just in sections of brain tissue. The second presentation was on the use of computer modelling and machine learning to optimize cryopreservation procedures. There are many variables that can be tweaked, from cooldown trajectory to type and mix of cryoprotectants. Modelling could be used to find optimal parts of this large state space more effectively than other forms of experimentation.

The European Biostasis Foundation (EBF) representative outlined their efforts to build a professional cryonics provider in Switzerland, which would be the first in Europe if they are successful. I liked a lot of what he had to say, particularly that customer focus and scalability are the weak points of the present cryonics industry, given its non-profit roots. Thus one of the initial projects is to ramp up the professionalization of signup and standby. They are launching a brand called Tomorrow, which streamlines the process of signing up for cryopreservation, making it an entirely online process that runs more smoothly and requires less work on the part of the individual. They are also looking into how to make a for-profit cryonics organization viable through the path of long-term asset management, meaning partnership with life insurance companies. As you may know, most cryopreservations are funded by life insurance policies, making it quite cost-effective, particularly if started at a younger age. Middlemen in the the life insurance industry are a well established business model, and so this might be a path towards for-profit cryonics. Beyond these early stage efforts, EBF supports research efforts to improve the quality and reliability of cryopreservation, and is planning a storage facility, but this will be contingent on success in the initial for-profit path, opening the door to capital investment.

Unfortunately I had to leave before the final keynote by Robin Hanson, but it was an interesting event. The cryonics community needs to grow and find success: we live in a strange world, in which there is an alternative to oblivion and the grave, but it is poorly capitalized, poorly supported, and rarely used. Cryonics, as happened for the treatment of aging as a medical condition, must find its way to success and growth. I think that this will be achieved in part by the slow process of building technologies that work, such as reversible vitrification of donor organs, carried out in research communities that presently have little funding for rapid progress, and in part by efforts such as those of the EBF, the process of discovery in business models and persuasion.

To What Degree Does Loss of Skeletal Muscle with Age Contribute to Immunosenescence?

Sarcopenia, the progressive loss of muscle mass and strength, is characteristic of aging. A perhaps surprisingly large fraction of the losses can be averted by strength training, but there are nonetheless inexorable processes of aging that, until therapies exist to repair this damage, will cause decline in muscle tissue over time even for those who maintain their fitness as best as possible. Researchers here consider the evidence for skeletal muscle tissue to do more than just move us around, but also to be an active participant in many aspects of metabolism. The focus in this open access paper is on the immune system: to what degree does sarcopenia contribute to the loss of immune function that also occurs with age?

In the last two decades, the perception of skeletal muscle as a pure locomotors unit has shifted. Muscle is increasingly recognized as an organ with immune regulatory properties. As such, skeletal muscle cells modulate immune function by signalling through different soluble factors, cell surface molecules, or cell-to-cell interactions. Although our knowledge of the muscle-immune system interplay has advanced considerably, the impact of age is relatively unknown. Sarcopenia may severely disturb this interaction, providing a potential explanation for the observed clinical outcomes of sarcopenic patients

Muscle is increasingly recognized as an endocrine organ producing and releasing cytokines and other peptides, which exert autocrine, paracrine, and endocrine activity on numerous tissues. Consequently, these soluble factors are commonly termed myokines. Proteomic profiling has been applied to the secretome of skeletal muscle and identified more than 300 potential myokines. Myokines such as IL-6, IL-7, IL-15, or LIF have been shown to modulate the immune system. Remarkably, serum concentrations of myokines such as IL-7 and IL-15 are inversely correlated with age, suggesting a link between skeletal muscle and age-dependent loss of immune system function.

As humans age, the immune system undergoes drastic changes. The umbrella term immune senescence is used to encompass these changes. Moreover, ageing is associated with increased serum levels of pro-inflammatory molecules. Skeletal muscle exhibits immune regulatory properties and that chronic, low-grade inflammation may induce muscle wasting. The concept of skeletal muscle as a regulator of immune function is relatively new and adds a new layer of complexity to the muscle-immune system link. Consequently, the muscle-immune system connection might be bidirectional: chronic, low-grade inflammation induces muscle catabolism via pleiotropic mechanisms mediated by the inflammatory secretome. Concurrently, homeostasis of skeletal muscle is, in part, responsible for healthy immune function. However, when dysregulated, insufficient myokine signalling, alteration of membrane bound factors towards a pro-inflammatory profile and impaired regenerative capacities of immune cells might result in disruption of immune system function.

We propose that biological aging may disturb the equilibrium of muscle-immune system homeostasis with skeletal muscle acting as a potential central link between sarcopenia and immune senescence. Healthy muscle function is gradually lost in an aging biological system due to physical inactivity, metabolic changes and the accumulation of chronic, low-grade inflammation. In turn, impaired muscle function curtails skeletal muscle cell signalling needed for immune regulation and maintenance, culminating in a vicious cycle in which immune and muscle system dysfunction sustain each other.


Upregulation of Autophagy Improves Vascular Function in an Animal Model of Type 2 Diabetes

Autophagy is the name given to a collection of cellular maintenance processes that recycle damaged structures, unwanted protein, and other metabolic waste. Many forms of stress, such as heat, lack of nutrients, and so forth spur greater autophagy, and this is thought to be a large part of why mild, temporary stress can produce lasting benefits to health - a process known as hormesis. Cell function is improved, and thus tissue function is improved. Many of the methods shown to modestly slow aging in laboratory species involve increased autophagy, and at least some, such as the practice of calorie restriction, have been shown to depend on functional autophagy for their benefits.

Rather than applying stress to generate autophagy, development programs focus on the use of small molecule drugs to influence the gene networks that regulate stress responses - such as targeting mTOR through rapamycin and analogous mTOR inhibitors. The research results here are an example of the type, showing that forcing a greater pace of autophagy helps to resist some of the metabolic consequences of type 2 diabetes - though of course this compares unfavorably with low calorie diets and consequent reduction in excess visceral fat tissue, the cause of the condition, as an approach to therapy in this specific case.

Vascular dysfunction is a major complication in type 2 diabetes (T2D). It has been suggested dysregulation of autophagy is associated with various cardiovascular diseases. However, the relationship between autophagy and vascular dysfunction in T2D remains unclear. Thus, we examined whether reduced autophagy is involved in vascular dysfunction and stimulation of autophagy could improve vascular function in diabetes.

Ten to 12-week old male type 2 diabetic (db-/db-) mice and their control (db-/db+) mice were treated with rapamycin or trehalose. Mesenteric arteries (MAs) were mounted in the arteriography and diameter was measured. Western blot analysis and immunofluorescence staining were assessed. Myogenic response (MR) was significantly increased, whereas endothelium-dependent relaxation (EDR) was significantly attenuated in the MAs of diabetic mice. These results were associated with increased expressions of LC3II, p62, and beclin-1 in diabetic mice.

Treatment of autophagy stimulators significantly reduced the potentiation of MR and improved EDR in the diabetic mice. Furthermore, autophagy stimulation normalized expressions of LC3II, p62, and beclin-1 in the diabetic mice. In addition, phosphorylation level of eNOS was decreased in diabetic mice, which was restored by rapamycin and trehalose. In conclusion, T2D impairs vascular function by dysregulated autophagy. Therefore, autophagy could be a potential target for overcoming diabetic microvascular complications.


Libella Gene Therapeutics to Run a Patient Paid Trial of Telomerase Gene Therapy

After Bioviva Science, Libella Gene Therapeutics is the second company to take a run at commercializing telomerase gene therapy treatments for human use. Telomerase is the enzyme responsible for lengthening telomeres, repeated DNA sequences at the ends of chromosomes, though it may have other roles. Telomeres are a part of the mechanism that limits the number of times that a somatic cell can replicate. Telomeres shorten with each cell division, and when too short they trigger programmed cell death or cellular senescence followed by destruction by the immune system. Ordinary somatic cells in humans do not express telomerase; it is only present in stem cells, which can replicate indefinitely to supply tissues with new somatic cells with long telomeres. This split between a small privileged stem cell population and the vast majority of restricted somatic cells is how higher forms of animal life keep cancer risk low enough for evolutionary success. Obviously not low enough for comfort, but evolution was never about individual happiness.

Telomerase gene therapies have been demonstrated to extend life span and reduce cancer risk in mice. The former outcome is likely largely due to increased stem cell activity, while the latter outcome is somewhat counterintuitive: if damaged cells are pushed into more activity and replication that they would not normally have undertaken, won't this raise the risk of cancerous mutations arising? It may be that improvements in immune function act to more than offset this risk - a primary task of the immune system is to destroy potentially cancerous cells before they have the chance to form a tumor. There is still some concern, in that mice have very different telomere and telomerase dynamics in comparison to humans. Will the balance of risk and improved function be the same in our species? The way we will find out is via brave volunteers trying the therapies, most likely, rather than any of the other, much slower options.

Neither Bioviva nor Libella took the standard regulatory path forward, opting for some combination of regulatory arbitrage and medical tourism to bring their therapies to patients. This sort of effort, carried out responsibly, is, I think, necessary and must spread if the present excesses of the FDA are to be reined in. The FDA sees its role as reducing risk to zero, at any and all cost, including the cost of slowing medical development to a crawl. Analyses have long shown that the cost in lives of this regulatory burden of slowed development far outweighs the benefits - but absent therapies are invisible and arouse no media outrage. Bureaucracies inevitably optimize to minimize visible problems. The only way to combat this issue effectively, given that working to change the system from within, and political advocacy to change the system from the outside, have been ongoing energetically for the past few decades, a time over which the financial burden imposed by the FDA has more than doubled, is to prove out a viable, responsible, cost-effective path to market outside the FDA system of regulation.

Libella Gene Therapeutics recently announced a patient paid trial to be held outside the US. Patient paid trials are unfairly excoriated by the research and regulatory establishment. As I have remarked upon in the past, they are an entirely legitimate approach to obtaining data. The chief objection is the lack of a control group in most such trials - but if we are only interested in large, reliable effect sizes, then the control group is the rest of the patient population, and that works just fine. In general, good therapies for aging, those that target relevant mechanisms in ways that will truly move the needle on life span, will indeed have large and reliable effects.

A second objection, more valid, is the sort of marketing that tends to accompany these trials. That is very much in evidence here, sadly. Libella should rein that in; in the long term it only harms the very necessary development of reliable, well-defined pathways for regulatory arbitrage. Telomerase gene therapies are not a cure for aging. They are a compensatory or enhancement therapy that addresses one of the downstream consequences of aging, while having little to no effect on a wide range of other important issues, such as accumulation of persistent metabolic waste in long-lived cells. No amount of telomerase will enable the body to break down harmful compounds that it cannot break down even in youth. Further, no-one has yet demonstrated that you can reverse, say, even an epigenetic clock measure by 20 years in humans using telomerase gene therapy. Even if you could, one can't say that this corresponds to 20 years of rejuvenation, given how little is known of what exactly these clocks measure. These sorts of claims are just aggravating. I understand the need for marketing, but one can carry out good marketing without having to resort to this sort of thing.

The Libella Gene Therapeutics trial will likely make waves because of the cost, at $1 million per patient. This is, however, a systemically administered gene therapy using AAV as the vector, stacked with one of the new biotechnologies that can reduce the ability of neutralizing antibodies to destroy the viral particles. If Libella is manufacturing to one of the usual Good Manufacturing Practice (GMP) standards, it is likely that the overwhelming majority of that $1 million cost is the cost of manufacture. AAV, while the most popular vector in the gene therapy development community, remains enormously expensive to manufacture. For a point of comparison, there is a systemically administered viral gene therapy for an inherited disease that is used in newborns, where it requires a 100,000 square foot facility 40 days to produce one dose. That costs more than $2 million. Everyone in the industry agrees that this situation must change and will change, that there will be disruptive advances in cost and efficiency, just as happened for monoclonal antibodies - but it hasn't happened yet.

Breakthrough Gene Therapy Clinical Trial is the World's First That Aims to Reverse 20 Years of Aging in Humans

Libella Gene Therapeutics, LLC ("Libella") announces an institutional review board (IRB)-approved pay-to-play clinical trial in Colombia (South America) using gene therapy that aims to treat and ultimately cure aging. This could lead to Libella offering the world's only treatment to cure and reverse aging by 20 years. Under Libella's pay-to-play model, trial participants will be enrolled in their country of origin after paying $1 million. Participants will travel to Colombia to sign their informed consent and to receive the Libella gene therapy under a strictly controlled hospital environment.

Bill Andrews, Ph.D., Libella's Chief Scientific Officer, has developed an AAV gene therapy that aims to lengthen telomeres. The AAV gene therapy delivery system has been demonstrated as safe with minimal adverse reactions in about 200 clinical trials. Dr. Andrews led the research at Geron Corporation over 20 years ago that initially discovered human telomerase and was part of the team that led the initial experiments related to telomerase induction and cancer.

Telomerase gene therapy in mice delays aging and increases longevity. Libella's clinical trial involves a new gene-therapy using a proprietary AAV Reverse (hTERT) Transcriptase enzyme and aims to lengthen telomeres. Libella believes that lengthening telomeres is the key to treating and possibly curing aging. On why they decided to conduct its project outside the United States, Libella's President, Dr. Jeff Mathis, said, "Traditional clinical trials in the U.S. can take years and millions, or even billions, of dollars. The research and techniques that have been proven to work are ready now. We believe we have the scientist, the technology, the physicians, and the lab partners that are necessary to get this trial done faster and at a lower cost in Colombia."

The Dog Aging Project Forges Ahead with a Large Study

As noted here by the Life Extension Advocacy Foundation, the Dog Aging Project researchers are moving ahead with a large study of companion animals. While much of the study is observational, a sizable cohort will be treated with the mTOR inhibitor rapamycin. Dogs are much closer to humans than mice, so it will be interesting to see what results. Given what is known of the way in which stress response upregulation behaves in different species, we would expect to see similar effects on cellular biochemistry - such as upregulation of autophagy - but smaller relative gains in life span in dogs versus mice. Short-lived species have a much greater plasticity of life span in response to environmental circumstances than longer-lived species, something that probably has its roots in adaptation to seasonal famine. A mouse must extend its reproductive life span by a larger proportion than a dog or a human in order to pass through a famine and carry on its lineage on the other side.

The Dog Aging Project has kicked into high gear and is recruiting 10,000 of our furry friends in what will be the largest dog aging study in history. The researchers hope that the study will also reveal more about human aging and longevity. The National Institute on Aging is funding the $23 million project, which will see a vast amount of data being collected during the five years that the project will run for. The research team will be collecting data such as vet records, DNA samples, gut microbiome samples, and information on diet and exercise.

The study chose to use dogs as they share many things with us humans, including living in the same environment and similar biology, and they even develop many age-related diseases that we do. The dogs in the study will continue to live at home and enjoy their usual daily lives, and the study will include dogs of all ages, sizes, and breeds, including mutts. To be part of the study, owners will have to complete periodic surveys, take their dogs to a vet once a year for examination, and possibly have to make extra visits for additional tests. A panel of animal welfare advisors will be involved in the study to ensure that the participants are treated well. The data from the study will be made available publicly, which is great news for open science and knowledge sharing.

Five hundred lucky pooches will also be given rapamycin, which appears to slow down aging according to various mouse studies; the hope is those results will translate to the dogs in this study. Rapamycin is an immune system suppressant and is currently used in humans to prevent organ rejection during transplants. However, in smaller doses in mouse studies, it has been shown to increase lifespan. A pilot safety study in dogs found no serious side effects.


Evidence for Inflammation to Drive Tau Pathology in Alzheimer's Disease

Researchers here provide evidence for the aggregation of altered forms of tau protein in the aging brain, and the resulting death of neurons, to be driven by chronic inflammation. This is good news if true, given recent work carried out in animal models of tauopathy, in which clearance of inflammatory, senescent glial cells in the brain was achieved via the use of senolytic drugs. The result was a marked reduction in both inflammation and tau pathology. To the degree that senescent cells in the brain prove to be the major cause of the chronic inflammation of aging and neurodegenerative conditions, it may well turn out that senolytic drugs will do a great deal for Alzheimer's patients. Since the senolytic drug dasatinib is off-patent, crosses the blood brain barrier, and is well tested for human use, trials could in principle begin just as soon as a sponsoring organization emerges and chooses to start.

Tau proteins usually stabilize a neuron's cytoskeleton. However, in Alzheimer's disease, frontotemporal dementia (FTD), and other tauopathies these proteins are chemically altered, they detach from the cytoskeleton and stick together. As a consequence, the cell's mechanical stability is compromised to such an extent that it dies off. In essence, tau pathology gives neurons the deathblow. The current study provides new insights into why tau proteins are transformed. As it turns out, inflammatory processes triggered by the brain's immune system are a driving force.

A particular protein complex, the NLRP3 inflammasome, plays a central role for these processes. It is a molecular switch that can trigger the release of inflammatory substances. For the current study, the researchers examined tissue samples from the brains of deceased FTD patients, cultured brain cells, and mice that exhibited hallmarks of Alzheimer's and FTD. In particular, the researchers discovered that the inflammasome influences enzymes that induce a hyperphosphorylation of tau proteins. This chemical change ultimately causes them to separate from the scaffold of neurons and clump together. "It appears that inflammatory processes mediated by the inflammasome are of central importance for most, if not all, neurodegenerative diseases with tau pathology."

This especially applies to Alzheimer's disease. Here another molecule comes into play: amyloid beta (Aβ). In Alzheimer's, this protein also accumulates in the brain. In contrast to tau proteins, this does not happen within the neurons but between them. In addition, deposition of Aβ starts in early phases of the disease, while aggregation of tau proteins occurs later. The results of the current study support the amyloid cascade hypothesis for the development of Alzheimer's. According to this hypothesis, deposits of Aβ ultimately lead to the development of tau pathology and thus to cell death. The study shows that the inflammasome is the decisive and hitherto missing link in this chain of events, because it bridges the development from Aβ pathology to tau pathology. Thus, deposits of Aβ activate the inflammasome. As a result, formation of further deposits of Aβ is promoted. On the other hand, chemical changes occur to the tau proteins resulting into their aggregation.


Dogs as a Model of Human Aging

Dogs are an interesting species when it comes to the study of aging. Firstly they are much closer to human metabolism and cellular biochemistry than mice, and secondly selective breeding has generated lineages with a very wide range of sizes and life spans. Thirdly, they occupy a good compromise position in the range of life spans, study cost, and similarity to humans. Mice live short lives, so studies are rapid and comparatively cheap, but there are sizable, important differences between mouse and human biochemistry. Humans live so long that most studies of aging are simply out of the question. Even in non-human primates that live half or less as long as we do, a study of aging and calorie restriction has lasted for decades, and few organizations can or will commit to that sort of effort.

Interest has picked up in recent years in the dog as a model of aging, to be used in the development of therapies to slow or reverse progression of aging. This is illustrated by the activities of the Dog Aging Project, for example, which seeks to obtain data on mTOR inhibitor therapies via their use in companion animals. Given this increased interest, researchers have started to catalog the holes in present knowledge. Even though dogs are very well studied, there is plenty to room to improve the understanding of how the mechanisms of aging progress and are influenced by genetics in this species.

Genetic Pathways of Aging and Their Relevance in the Dog as a Natural Model of Human Aging

Several genes have been shown to affect the body size variability of dogs, which is unmatched by any other mammalian species. Importantly, dogs also show marked differences in their expected lifespan in connection with body mass. On average, giant sized breeds (above 50 kg) have an expected lifespan of 6-8 years, while small sized breeds (below 10 kg) can live up to 14-16 years. This wide range of expected lifespans, together with other aspects, has made dogs promising as model organisms for aging research. Despite the huge progress in understanding the genetic basis of morphological variability of dogs, still very little is known about the functional relevance of canine homologs of conserved longevity genes. Currently, this may stand as an obstacle in the way of effectively utilizing dogs as aging models. As dogs can provide unique insights into many aspects of human aging, the current lack of detailed information about the canine genetic pathways of aging should be overcome by future research approaches. In this review, we provide an overview of the evolutionary conserved biological mechanisms that contribute to aging, following the Hallmarks of Aging classification, and we summarize current knowledge about these pathways in dogs.

Genomic Instability

The DNA repair machinery involves divergent pathways, each aimed to correct certain forms of DNA damage. These protective mechanisms have been in the focus of cancer and aging research for a long time. Polymorphisms in several genes of the DNA damage response machinery have been linked to longevity in humans. Intriguingly, no canine progeria syndrome, resulting from DNA repair deficiency, has been documented in the scientific literature. On the other hand, several studies that investigated various forms of canine cancer revealed alterations in the DNA repair machinery, which corresponded to findings in human cancers. While these findings clearly promote the dog as a natural model of human cancers, it is still unclear how exactly variations in DNA repair capacity contribute to the expected lifespan of dogs.

Telomere Attrition

Telomere shortening is a characteristic only of somatic cells, while in germ line cells, telomere sequences are constantly restored by telomerase enzymes. The limited proliferative potential of somatic cells may seem disadvantageous for an individual, yet it may increase fitness by limiting the growth of malignant cells. Contrary to mice, dogs were reported to have low or no telomerase expression in normal somatic tissues, a pattern similar to that in humans. Tumors in dogs often showed high levels of telomerase expression, similarly to human malignancies. Although very little is known about the molecular mechanisms regulating telomere maintenance and cell cycle arrest in dogs, such findings indicate that dogs may also share basic telomere biology with humans. Importantly, telomere length was shown to be variable across different dog breeds and was in correlation with expected lifespan. Also, telomere length in individual dogs was found to decrease with age, similarly as described in humans.

Epigenetic Alterations

Although age associated changes in chromatin structure and DNA methylation patterns have been reported in several model animals, there can be major differences between species. For example, epigenetic regulation in C. elegans seems to be limited to chromatin remodeling by histone modifications, limiting its utilization as a model to study epigenetic changes in aging. In dogs, an increasing body of evidence has suggested epigenetic regulation is behind species and breed-specific traits. Importantly, a recent study demonstrated that changes in methylation status in DNA regions, which were homologous to regions with known age-sensitive methylation patterns in humans, were in strong correlation with chronological age in dogs and wolves. This finding supported the applicability of the dog as a model of age-related epigenetic changes, while it also provided a molecular approach to determine the biological age of individual canines.

Disruption of Proteostasis

Chaperone proteins play an important role in the post-translational maturation of nascent proteins by facilitating their folding. They also function as protectors of mature proteins under various stressful conditions, by helping to maintain their natural conformation and by preventing aggregation. In dogs, the few studies that investigated chaperone proteins in relation to aging reported similar age-related changes as in humans. For example, blood levels of the Hsp70 chaperone were shown to decrease with age in dogs, similarly to what had been previously reported in humans.

Deregulation of Nutrient Sensing

Cellular metabolism, protein synthesis, and autophagy are strictly regulated by various signaling pathways. Most of these have evolved to synchronize cell growth and metabolism with nutrient availability; hence, they are often referred to as nutrient sensing pathways. Many of them converge on the target of rapamycin (TOR) kinase. Importantly, the function of mTOR can be efficiently inhibited by rapamycin, which is an already approved immunosuppressant in human medicine, and therefore has been proposed as a promising anti-aging compound to be used in humans. However, it was reported to cause severe side effects in medical dosages. Therefore, optimal dosages, which do not cause undesirable syndromes, yet still exert longevity promoting effects should be carefully determined in preclinical studies. Actually, pharmaceutical studies have already been initiated to investigate the effects of rapamycin on the lifespan of dogs.

Mitochondrial Dysfunction

Nutrient sensing pathways converge on the regulation of mitochondrial activity, as these organelles are the main sources of energy (in the form of adenosine triphosphate, ATP) in eukaryotic cells under normal circumstances, when enough oxygen is present. The availability of nutrients determines the rate of mitochondrial respiration, which, however, generates not only ATP but also chemical by-products, including reactive oxygen species. The oxidative burden created by mitochondria may be especially high in neurons, which solely depend on aerobic mitochondrial respiration as energy source. The role of mitochondrial dysfunction and increased oxidative burden in neural aging has been investigated in dogs. In general, dog brains were shown to accumulate oxidative damage with age. Several mitochondrial diseases are known in dogs, which have human homologs, such as the sensory ataxic neuropathy found in Golden Retriever dogs or the familial dilated cardiomyopathy in Doberman Pinschers. As several promising anti-aging drugs are likely to be tested in dogs in preclinical studies, looking into their effects on mitochondrial function and testing their possible interactions with mitochondrial genotypes can be highly relevant for humans.

Cellular Senescence

A marked elevation of senescent cell numbers was reported in old mice, although not in all tissues. Importantly, this accumulation process can result from both the increased generation of senescent cells and a decreased activity of macrophages that are able to eliminate aged or apoptotic cells from tissues. Little is known about the accumulation of senescent cells in canine tissues, although this phenomenon is also likely to show fundamental similarities with other mammalian species. As there is a growing interest toward pharmacological approaches to deplete senescent cells in tissues by specific apoptosis inducing agents (senolytic drugs), dogs may eventually be involved in testing these types of anti-aging interventions.

Stem Cell Exhaustion

Tissue renewal depends on the abundance and replicative capacity of tissue-specific stem cells. Hematopoietic stem cells (HSCs) were reported to have reduced replicative capacity in both aged mice and humans, mainly because of accumulating DNA damage. This reduction can explain the old age anemia of elderly people. Importantly, similar forms of age-associated changes in blood parameters, including anemia, were reported in dogs. Besides pharmacological interventions, stem cell therapy has also been suggested as a possible anti-aging intervention, with highlighted promises to treat certain forms of neurodegeneration. In this regard, stem cell therapy trials conducted on dogs affected by forms of neurodegeneration could represent a crucial step before progressing to human trials. In the case of the Golden Retriever model for Duchenne muscular dystrophy, successful stem cell-based interventions had actually preceded human clinical trials

Altered Intercellular Communication

In addition to hormones and metabolites, extracellular vesicles released by cells into the blood, called exosomes and ectosomes, have emerged as important transducers of various cellular signals. Consequently, exosomes may also modulate aging and neurodegeneration. Exosome research in dogs have been limited until recently. However, blood miRNA levels - which were hypothesized to be mainly found in exosomes - were reported to correlate with disease phenotypes in canine Duchenne muscular dystrophy. Similarly, miRNA content in circulating exosomes was shown to correlate with progression of secondary heart failure in cases of myxomatous mitral valve disease in dogs. Altogether, investigations about the connections between exosome content and aging or age-related pathologies in dogs may lead to the identification of diagnostic markers with potential translational prospects into human studies.

The Aged Adaptive Immune System is Strange

The adaptive immune system of an older person is a very different beast in comparison to that of the younger self. It has lost the supply of new T cells due to atrophy of the thymus, and the remaining population of T cells becomes ever more damaged, misconfigured, strange, and different. The immune system as a whole is complex enough to still be hiding many unexplored details, even in this era of biotechnology. Here, researchers outline a novel discovery in the immune function of supercentenarians. It seems that at very advanced ages, some T cells start to undertake radical shifts in function in order to compensate somewhat for the growing lack of capacity. It remains to be seen whether or not this only occurs to a significant degree in a minority of the population, and is thus a feature of supercentenarians because it increases the odds of survival.

Supercentenarians, people who have reached 110 years of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from 7 supercentenarians and 5 younger controls. We identified a marked increase of cytotoxic CD4 T cells as a signature of supercentenarians. This characteristic is very unique to supercentenarians, because generally CD4 T cells have helper, but not cytotoxic, functions under physiological conditions. Furthermore, single-cell T cell receptor sequencing of two supercentenarians revealed that cytotoxic CD4 T cells had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15-35% of the entire CD4 T cell population.

The cytotoxic CD4 T cells exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of cytotoxic CD8 T cells. This indicates that cytotoxic CD4 T cells utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Indeed, cytotoxic CD4 T cells extracted from supercentenarians produced IFN-γ and TNF-α upon ex vivo stimulation. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.


7-Ketocholesterol as a Contributing Cause of Multiple Age-Related Diseases

One noteworthy difference between the biochemistry of young and old individuals is a greater presence of oxidative molecules, resulting from dysfunctional cells, inflammatory processes, and other issues. As a consequence, there are also many more oxidized molecules, changed from their original structure and now either broken or actively harmful. Cells clear out this sort of oxidative damage constantly, and are quite efficient at this sort of maintenance until levels of oxidization become high, but they nonetheless struggle with some particularly toxic or resilient oxidized molecules, even in smaller amounts. A good example of the type is 7-ketocholesterol, a form of oxidized cholesterol. It is primarily understood as an important contributing cause of atherosclerosis via its detrimental effects on the macrophages responsible for clearing lipids from blood vessel walls, but there is evidence for it to contribute to other age-related conditions as well.

Cholesterols exist both inside and outside of the cell, as they are important components of all cellular membranes, but these and other nonpolar substances are transported in the plasma via lipoprotein particles. Low density lipoprotein (LDL) is the principle carrier of cholesterol to peripheral tissue. All of the components of LDL are susceptible to oxidation to produce an oxidized form of LDL (OxLDL). OxLDL has been linked to a variety of pathologies. Oxidation of the cholesterol in LDL produces several oxidation products including 7-ketocholesterol (7KC), which is the most abundant oxysterol present in OxLDL. We believe that it is important to distinguish between the effects of OxLDL and that of unsequestered 7KC, as many studies fail to account for this important difference in how 7KC interacts with the cell.

OxLDL is not the only source of 7KC within the body. 7KC can be produced endogenously by a series of oxidation or, much less commonly, enzymatic reactions. It can also be ingested directly in food, however the liver is well equipped to process and rid the body of exogenous toxins, so 7KC is not acutely poisonous to ingest. However, endogenously produced, unsequestered 7KC can wreak havoc inside of most cells. Unesterified 7KC can be found within membranes of organelles where it disrupts fluidity and signaling pathways, causing cellular damage via multiple stress-response pathways. These stress-response pathways induce a vicious cycle by increasing the population of reactive oxygen species, which in turn increases the oxidation of cholesterol and production of 7KC. Particularly in people with already-compromised cholesterol pathways, 7KC buildup can be overwhelming and cause significant damage to membranes, pathways, and overall cell function.

7KC is the most abundant oxysterol in both oxLDL particles and atherosclerotic plaques, indicating the significant role 7KC plays in the progression of atherosclerosis. 7KC has been shown to induce macrophage reprogramming, foam cell formation, and oxiapoptophagy in a multitude of cell types. In atherosclerotic plaques, this results in the deposition of calcium-laden apoptotic bodies, leading to subsequent calcification of the blood vessel.

It has been shown that oxysterols are likely a cause of altered brain cholesterol metabolism which is an integral part of Alzheimer's disease, Parkinson's disease, and other aspects of neurological aging. It is not yet fully understood whether 7KC can cross the blood-brain barrier, but 7KC is highly toxic to neuronal cells and should certainly form spontaneously inside of them with age. Additionally, 7KC is implicated in macular degeneration as it is a major component of the drusen within the retina. 7KC can also damage the liver by disrupting membrane rafts and fenestrations. Lastly, 7KC is also characterized in congenital disorders such as sickle cell, Niemann Pick, and other lysosomal storage disorders. Ambiguous links between many of these diseases, particularly atherosclerosis and neurodegeneration, further implicates 7KC as an unexplored target in many diseases.

We propose that 7KC could be an effective therapeutic target due to its implication in a wide variety of diseases. Although the abundance of 7KC has not yet been strongly correlated to aging or the severity of different pathologies, there is clear evidence to show its destructiveness in biological systems. As more studies are conducted on toxic oxysterols in aging and disease, we hope that more will become known about 7KC abundance in different cells and tissues. This would increase the potential of 7KC as a therapeutic target for various diseases, especially those specifically associated with aging. Considering nonenzymatic oxysterol accumulation, particularly 7KC, as an integral factor in disease progression could change the way we identify and treat these diseases, offering new and possibly broadly effective therapeutics.


Vaccination and Antiviral Therapies Targeting CMV as an Approach to Reducing Immunosenescence

Today's open access paper discusses possible approaches to the treatment of immunosenescence, the age-related decline in effectiveness of the immune system. Unfortunately it is largely a tour of compensatory treatments, ways to force the cells of the immune system into greater or more useful activity without addressing any of the underlying causes of immunosenescence. Many of these methodologies have serious side-effects, are disruptive of normal immune function and overall health, and cannot be applied for the long term. Checkpoint inhibition, or the delivery of recombinant IL-7, for example, both of which are used as short term interventions to treat cancer.

The path to actually fixing the aged immune system by addressing causes is quite different. It would involve restoring the thymus from atrophy in order to restore a more youthful pace of production of T cells. Replacing the hematopoietic stem cell population to ensure that the right balance of immune cells are produced in the bone marrow. Reversing the degeneration of lymph nodes, where immune cells coordinate. Clearing out the populations of worn, malfunctioning, and misconfigured immune cells in tissues and bloodstream. This is a lot of work, but it is an oversight to omit these active lines of research and development from any review of ways to treat immunosenescence.

The one approach outlined at length in this open access paper that does address a plausible cause of immunosenescence is vaccination against cytomegalovirus (CMV). Near everyone is silently infected by late life, and the adaptive immune system becomes ever more devoted to trying and failing to clear this persistent viral infection. Ever more T cells are specialized to CMV, leaving ever fewer available for other tasks. As the supply of new T cells diminishes with age, this overspecialization becomes a serious issue, contributing greatly to the decline in immune function.

The authors here make the point that all of the necessary knowledge and technology already exists to put together a viable, widely used vaccine for CMV, but the will to do so is absent. We live in a world in which HPV vaccination became a reality, however, and CMV is arguably far worse when it comes to costs and suffering. Perhaps, at some point in the years ahead, the slow machineries of regulation will come to the point at which people are regularly vaccinated against CMV in order to reduce the impact of aging on immune function. I think it likely that selective destruction of CMV-specialized immune cells is more likely to emerge as a branch of therapy before that happens, however.

Immunosenescence and Its Hallmarks: How to Oppose Aging Strategically? A Review of Potential Options for Therapeutic Intervention

Until a few decades ago, a very small fraction of the population would reach 80 years of age. Now this is a frequent event, with the average life expectancy for a newborn to have risen to 80 years in most Western European countries. However, the increase in lifespan does not coincide with increase in healthspan. The link between aging and disease is in part a reflection of the functional changes in the immune system of older people. Different factors contribute to the development of age-related immune dysfunction, but the epilog of an aged immune system is an increased propensity toward a reduced resistance to infection, poorer responses to vaccination, and the development of age-related diseases.

The analysis of the contributing factors to this profound immune remodeling has revealed a complex network of alterations that influence both innate and adaptive arms of the immune system. The diversity of cells, molecules and pathways involved in this remodeling, and their ability to influence each other, including the intra- and inter-individual variability of the immune response, make it hard to identify interventions that can be predicted to improve or, at least, to maintain the immune function in older adults. Within the past few years, numerous studies of the underlying mechanisms of age-related immune decline have laid the groundwork for the identification of targeted approaches, focusing on interventions able to target the hallmarks of immunosenescence.

Taking into account the role of HCMV in the decrease of naïve T cells and increase of memory T cells, the reduction of the latent/lytic viral load, by vaccination and/or antiviral drugs, should be beneficial to diminish HCMV-associated immunosenescence. As a result of 40 years of work, there are many candidate HCMV vaccines. Therefore, we know the antigens needed in a HCMV vaccine, and that vaccination can be protective. To reach the goal of an effective HCMV vaccine, now we need a concentrated effort to combine the important antigens and to generate durable responses that will protect for a significant period.

Further, Letermovir is an antiviral agent that inhibits HCMV replication by binding to components of the terminase complex. In patients undergoing hematopoietic stem cell transplantation, Letermovir daily prophylaxis is effective in preventing clinically significant HCMV infection when used through day 100 after transplantation, with only mild toxic effects and with lower all-cause mortality than placebo. However, there is no suggestion yet for the use of antiviral therapy as a strategy for prophylactic mitigation of immunosenescence.

Greater Physical Fitness Correlates with Lower Risk of Dementia

It is well established that exercise and physical fitness correlate well with reduced incidence of all of the common age-related diseases, and reduced mortality risk. It is hard to establish causation from the contents of human epidemiological databases, but the analogous animal studies convincingly demonstrate that exercise improves health. There is no reason to expect humans to be all that different in this matter. Here, researchers show that, much as expected, greater fitness correlates with reduced risk of dementia. Of note, patients that improved their fitness over the years of later life exhibited reduced disease risk and improved life expectancy.

Cardiorespiratory fitness is associated with risk of dementia, but whether temporal changes in cardiorespiratory fitness influence the risk of dementia incidence and mortality is still unknown. We aimed to study whether change in estimated cardiorespiratory fitness over time is associated with change in risk of incident dementia, dementia-related mortality, time of onset dementia, and longevity after diagnosis in healthy men and women at baseline. We linked data from the prospective Nord-Trøndelag Health Study (HUNT) with dementia data from the Health and Memory Study and cause of death registries (n=30,375). Included participants were apparently healthy individuals for whom data were available on estimated cardiorespiratory fitness and important confounding factors.

Cardiorespiratory fitness was estimated on two occasions 10 years apart, during HUNT1 (1984-86) and HUNT2 (1995-97). HUNT2 was used as the baseline for follow-up. Participants were classified into two sex-specific estimated cardiorespiratory fitness groups according to their age (10-year categories): unfit (least fit 20% of participants) and fit (most fit 80% of participants). To assess the association between change in estimated cardiorespiratory fitness and dementia, we used four categories of change: unfit at both HUNT1 and HUNT2, unfit at HUNT1 and fit at HUNT2, fit at HUNT1 and unfit at HUNT2, fit at both HUNT1 and HUNT2. Using Cox proportional hazard analyses, we estimated adjusted hazard ratios (AHR) for dementia incidence and mortality related to temporal changes in estimated cardiorespiratory fitness.

During a median follow-up of 19.6 years for mortality, and 7.6 years for incidence, there were 814 dementia-related deaths, and 320 incident dementia cases. Compared with participants who were unfit at both assessments, participants who sustained high estimated cardiorespiratory fitness had a reduced risk of incident dementia (AHR 0.60) and a reduced risk of dementia mortality (AHR 0.56). Participants who had an increased estimated cardiorespiratory fitness over time had a reduced risk of incident dementia (adjusted hazard ratio 0.52) and dementia mortality (adjusted hazard ratio 0.72) when compared with those who remained unfit at both assessments. Each metabolic equivalent of task increase in estimated cardiorespiratory fitness was associated with a risk reduction of incident dementia (AHR 0.84) and dementia mortality (AHR 0.90). Participants who increased their estimated cardiorespiratory fitness over time gained 2.2 dementia-free years, and 2.7 years of life when compared with those who remained unfit at both assessments.


Targeting α-Synuclein in the Gut to Turn Back the Progression of Parkinson's Disease

Like most neurodegenerative conditions, Parkinson's disease is driven in large part by the pathological aggregation of misfolded proteins, in this case α-synuclein. These solid deposits of protein spread from cell to cell, and are accompanied by a surrounding halo of harmful biochemical interactions. There is evidence for the protein aggregation of Parkinson's disease to start in the gut and then spread to the brain. You might look at a recent paper that discusses whether or not we should consider Parkinson's to be two diseases with a similar outcome, one in which the α-synuclein aggregation originates in the gut, and the other in which it originates in the brain. In the research noted here, scientists are following the gut origin hypothesis and targeting α-synuclein there in order to slow or reverse the progression of Parkinson's disease.

Aggregates of the protein alpha-synuclein arising in the gut may play a key role in the development of Parkinson's disease (PD). Investigators are testing the hypothesis that by targeting the enteric nervous system with a compound that can inhibit the intracellular aggregation of alpha-synuclein, they can restore enteric functioning in the short term, and possibly slow the progressive deterioration of the central nervous system in the long term. "The concept is that aggregates of the protein alpha-synuclein, thought to play a key role in the disease, arise within the enteric nervous system (ENS) and travel up the peripheral nerves to the central nervous system (CNS) where they ultimately cause inflammation and destruction of parts of the brain. Targeting the formation of alpha-synuclein aggregates in the ENS may therefore slow the progression of the disease."

Alpha-synuclein is one of the defensive proteins produced by enteric nerves when they encounter infections. In children with acute bacterial gastrointestinal (GI) infections, for example, intestinal nerves produce alpha-synuclein. In children who have undergone intestinal transplants and who are prone to GI infections, investigators have shown that enteric neurons start making alpha-synuclein at the time of acute viral infections, and this outlasts the infection by many months, protecting nerve cells for prolonged periods of time. Within a nerve cell, alpha-synuclein could envelop invading viruses and disrupt their replication. It could also attach itself to small vesicles containing neurotransmitters and be released from the nerve cell hitching a ride with them. Once on the outside, it can attract protective immune cells from surrounding tissues.

To determine whether targeting alpha-synuclein within enteric neurons might help patients with PD, researchers are currently conducting clinical trials with a compound called ENT-01, a synthetic derivative of squalamine, a compound originally isolated from dogfish bile. It displaces alpha-synuclein from nerve cell membranes and restores the normal electrical activity of enteric neurons. Investigators completed a 50-patient Phase 2a study (RASMET) in patients with PD in 2018, which corrected constipation, a common symptom of PD, in more than 80% of participants, with the dose titrated up for each patient until a response was obtained. "The RASMET study demonstrated that the ENS is not irreversibly damaged in patients with PD. We believe that this is the first demonstration of the reversal of a neurodegenerative process in humans." Possible benefits were also observed in motor and non-motor symptoms such as hallucinations, depression, and cognitive function. A 110-patient double-blind, placebo-controlled Phase 2b trial (KARMET) evaluating the effect of oral ENT-01 tablets on constipation and neurologic symptoms is currently being conducted.


Heat Shock Proteins as a Basis for Tackling Protein Aggregation in Neurodegenerative Diseases

Neurodegenerative conditions are largely characterized by the aggregation of a few altered proteins that are prone to forming solid deposits in and around neurons. Tissues, such as the brain, made up of long-lived cells, such as neurons, are particularly vulnerable to this sort of dysfunction, as they cannot dilute harmful protein aggregates by cell division, and dysfunctional cells are not readily destroyed and replaced. Cells must rely upon internal quality control mechanisms such as the presence of chaperone proteins responsible for chasing down misfolded or otherwise problematic proteins, and ensuring they are refolded correctly or recycled via autophagy.

The quality control mechanisms of chaperone mediated autophagy are known to be important in aging. Increased autophagic activity is associated with many of the means of modestly slowing aging demonstrated in laboratory animals in past decades. Autophagy declines with age, and this is thought to be important in the development of neurodegenerative conditions precisely because neurons are heavily reliant on quality control to maintain function. Researchers are interested in finding ways to build therapies for age-related conditions based on upregulation of autophagic activity, and, as noted in today's open access paper, the class of chaperone proteins called heat shock proteins are one prominent area of investigation.

Small Heat Shock Proteins, Big Impact on Protein Aggregation in Neurodegenerative Disease

Maintenance of cellular protein homeostasis (proteostasis) is crucial for cell function and survival. Neurons are particularly sensitive to dysregulated proteostasis as evidenced by the accumulation and aggregation of amyloidogenic proteins, which are a hallmark of neurodegenerative disease. Cellular molecular chaperone systems modulate proteostasis, and, therefore, are primed to influence aberrant protein-induced neurotoxicity and disease progression. Molecular chaperones have a wide range of functions from facilitating proper nascent folding and refolding to degradation or sequestration of misfolded substrates.

ATP-dependent chaperones, like the 70 kDa heat shock protein (Hsp70) and the 90 kDa heat shock protein (Hsp90), facilitate refolding, degradation, or sequestration of these misfolded proteins. Small heat shock proteins (sHsps) that lack an ATPase domain and are between 12 and 43 kDa are a class of molecular chaperones that typically associate early with misfolded proteins. These interactions hold proteins in a reversible state that helps facilitate refolding or degradation by other chaperones and co-factors.

Potential therapeutic strategies that aim to modulate endogenous sHsp expression or phosphorylation generally suffer from a lack of specificity for the sHsp family, let alone for discrete sHsps. Heat stress-responsive sHsps can be activated by drugs that generate a challenge to proteostasis, which includes proteasome inhibitors (e.g. Bortezomib), Hsp90 inhibitors (e.g. 17-AAG), and oxidative stress inducers (e.g. terrecyclic acid). However, these treatments also induce expression of other molecular chaperone families (e.g. Hsp70 and Hsp40) and are not specific for sHsp activation. Efforts to identify Hsp co-inducers, substances that potentiate stress responses without inducing a primary stress response on their own, may offer improved selectivity.

Small molecules that interact with sHsps may be a promising strategy for therapeutics, but the nature of this family of chaperones makes drugability difficult. There are no known small molecule ligands to use as a scaffold to start from. The dynamic nature of these proteins taunt the idea of engineering a high affinity binding drug; indeed, these promiscuous proteins likely have many client binding sites with a variety of conformations.

The diversity of sHsps from different organisms, from bacteria to humans, provides a rich set of proteins to explore for aggregation prevention activity. For example, a sHsp from a parasite was shown to be a potent inhibitor of amyloid-β fibrillation and reduced associated toxicity in a neuroblastoma cell model. Specific mutant or engineered sHsp variants, with altered oligomeric structure or client interactions, may prove to have increased chaperone activity towards amyloidogenic proteins. Small peptides derived from human HspB4 and HspB5 sequences, termed mini-chaperones, display chaperone-like activity. One of these constructs reduced cellular toxicity of amyloid-β.

Cellular Senescence May Contribute to Rheumatoid Arthritis in Younger Patients

Senescent cells are a cause of aging, and much of the present focus in the study of cellular senescence is thus on targeting and destroying these unwanted cells in order to treat aging. However, a comparatively recent and intriguing finding is that at least some autoimmune diseases, such as type 1 diabetes, involve cellular senescence. The question at present is whether or not this true for all forms of autoimmunity.

An autoimmune condition must have a trigger, something that prompts the immune system to attack healthy tissues, and it is possible that many different triggers converge on the generation of senescent cells, with their ability to rouse the immune system to action via inflammatory secretions. Here, researchers provide evidence for cellular senescence to be involved in rheumatoid arthritis, but only in younger patients. Rheumatoid arthritis is one of the less well understood autoimmune conditions: it remains unclear as to why it occurs. It may well turn out to be several similar conditions with quite different causes, given the wide variety of patient experiences.

Tissue accumulation of senescent cells has been identified as a deleterious factor that promotes inflammation and tissue damage in different human diseases and animal models of aging related diseases. Regarding joint diseases, evidence of this concept has been only provided in human and experimental osteoarthritis (OA), with the main focus on chondrocytes and cartilage damage. Human cartilage and chondrocyte cultures from OA patients have shown increased number of senescent cells that contribute to cartilage degradation by increased IL-1, IL-6, and MMP-3 expression.

The expression of the senescence marker p16INK4a (p16) was analyzed by immunohistochemistry in rheumatoid arthritis (RA), osteoarthritis (OA), and normal synovial tissues from variably aged donors. The proportion of p16(+) senescent cells in normal synovial tissues from older donors was higher than from younger ones. Although older RA and OA synovial tissues showed proportions of senescent cells similar to older normal synovial tissues, senescence was increased in younger RA synovial tissues compared to age-matched normal synovial tissues. The percentage of senescent SA-β-gal(+) synovial fibroblasts after 14 days in culture positively correlated with donor's age.

Accumulation of senescent cells in synovial tissues increases in normal aging and prematurely in RA patients. Senescence of cultured synovial fibroblasts is accelerated upon exposure to TNFα or oxidative stress and may contribute to the pathogenesis of synovitis by increasing the production of pro-inflammatory mediators.


Against Senolytics

There is no consensus in science that is so strong as to have no heretics. So here we have an interview with a naysayer on the matter of senolytic treatments, who argues that the loss of senescent cells in aged tissues will cause more harm to long-term health than the damage they will do by remaining. To be clear, I think this to be a ridiculous argument given the present evidence. To make it one has to declare the existing results showing extension of healthy life span in mice to be something other than credible data, which just isn't the case. Further, it seems shaky on theoretical grounds to suggest that removal of something like 1% of cells will put onerous stress on the remaining 99%, particularly given that the 1% were contributing to declining stem cell activity via inflammatory signaling. All told, it is hard to take seriously the idea that loss of senescent cells can possibly produce greater degrees of dysfunction in tissue than is caused by the inflammatory signaling of senescent cells.

Your new review on senolytics suggests that senolytics may cause more harm than good. Can you summarize your objections and concerns?

Here is the argument: 1) theoretically, senolytics should make things worse and 2) the available data support this theoretical concern. To use an analogy, imagine that you have a factory in which 10 of the 100 factory workers are feeling overworked and tired. Furthermore, their complaints are disrupting the other workers. You have two possible interventions. You can: (a) Fire the 10 workers, thereby removing the complainers. The result is that the remaining 90 workers are now overworked, and they, too, begin to complain. You end up with 30 workers who are now complaining and disrupting your factory. This is the senolytic approach. (b) Improve the health and conditions of the 10 workers who are overworked and complaining. You now have 100 workers who are doing an excellent job. This is the telomerase therapy approach.

In the first case, your factory has a problem and you make it worse. In the second case, your factory has a problem and you solve the problem. This figure from my new paper illustrates the same point in terms of nine cells subjected to senolytics, with the result being temporary short-term improvement followed by decline and a worse situation than we started with.

This does not take into account the idea of replacing that pool of "workers" by bringing in fresh stem cells.

You have to keep a few points in mind. 1) Will the stem cells populate as desired? 2) If you do get a stem cell population, that requires cell division, which shortens telomeres, which accelerates cell senescence, and once again you have accelerated pathology. 3) Why would you bother recruiting stem cells when you can much more easily reset cell senescence in the resident cells of the tissue? 4) The long-term data (what there is of it) supports the failure of senolytics. Again: remember where those "new cells" come from: you are accelerating senescence in the stem cell pool. The only way to "replace them with healthy working cells" is to simply and effectively reset gene expression, taking senescing cells and turning them into functionally young cells.

It seems that we can only speculate on these issues, as these long-term follow-ups have not yet been done. However, senolytics have been shown to increase median lifespan and healthspan in murine models.

I don't see any credible data that supports the contention that "senolytics have been shown to increase median lifespan and healthspan in murine models".


The Strategy of mTORC1 Inhibition Fails a Phase III Trial

The worst possible outcome when developing a clinical therapy is not an early failure. It is a late failure, in the final and most expensive phase III clinical trial, in which the therapy interacts with a sizable patient population, and after a great deal of time and funding have been devoted to the program. This result is far more likely for therapies based on mechanisms that have smaller rather than larger effect sizes, and where that smaller effect size varies from individual to individual for reasons that are not well understood - something that describes all too much of the past few decades of efforts to treat age-related disease. Unfortunately this worst case phase III failure just happened to resTORbio's mTORC1 inhibitor RTB101, in tests of its ability to improve immune function and reduce the burden of infection in later life.

The inhibition of mTOR, and specifically only the mTORC1 protein complex in order to reduce side-effects resulting from inhibition of mTORC2, is one of a range of potential approaches demonstrated in animal models to modestly slow aging via upregulation of cellular stress response mechanisms. It affects some of the same processes as calorie restriction and exercise. Another way of looking at it is that it pushes metabolism into a state that makes it incrementally more resilient to the accumulated damage of aging. However, all such strategies examined to date perform far better in short-lived species than in long-lived species, a situation that may occur at root because calorie restriction evolved to increase the odds of survival through seasonal famine. A season is a long time for a mouse, a short time for a human, and so only the mouse evolves to demonstrate a sizable relative gain in healthspan and life span due to a restricted calorie intake.

Nonetheless, the clinical evidence to date suggested that mTORC1 inhibition would produce enough benefits in human patients to be worth it from the patient perspective: a low cost pill that produces incremental improvement in the experience of late life medical conditions. I don't think that this outcome is worth it from the point of view of the enormous funding required for development and regulatory approval, however, not when there are far better options on the table, such as senolytic therapies to clear senescent cells and the rest of the SENS rejuvenation research program. The resTORbio team may have made a poor choice of indication to apply their therapy to - though given the promising results to date, I don't think that could have been known in advance. It may be that incremental gains through mTORC1 inhibition can still be obtained for patients with other age-related conditions, but nonetheless, this present failure should dampen our expectations to some degree for any and all other approaches based on stress response upregulation.

Failure in a late stage trial doesn't go unnoticed, and nor should it. It sends ripples through the biotech industry, since there are always networks of companies working on conceptually similar approaches to the therapy that failed at the final hurdle. The best outcome of such events would be for investors and entrepreneurs and researchers to gravitate towards better approaches to the treatment of aging - those with larger and more reliable effect sizes, by virtue of actually repairing the underlying damage of aging. Senolytic therapies are a great example of the type. As two decades of relentless fixation on anti-amyloid immunotherapy in the Alzheimer's industry demonstrates, this can take some time, however. The worst outcome would be for investment in the whole longevity industry to be damaged by failures in its first large trials, because naive investors have little to no insight into the technical and scientific differences between poor strategies and good strategies. This puts greater pressure on the senolytic companies to succeed in their initial trials, as they are up next.

resTORbio Announces That the Phase 3 PROTECTOR 1 Trial of RTB101 in Clinically Symptomatic Respiratory Illness Did Not Meet the Primary Endpoint

resTORbio, Inc., a clinical-stage biopharmaceutical company developing innovative medicines that target the biology of aging to prevent or treat aging-related diseases, today announced that top line data from the PROTECTOR 1 Phase 3 study, evaluating the safety and efficacy of RTB101 in preventing clinically symptomatic respiratory illness (CSRI) in adults age 65 and older, did not meet its primary endpoint, and that it has stopped the development of RTB101 in this indication. RTB101 is an oral, selective, and potent TORC1 inhibitor.

"While we are disappointed in these results, there are extensive preclinical data supporting the potential therapeutic benefit of TORC1 inhibition in multiple aging-related diseases, including Parkinson's disease, for which we have an active Phase 1b/2a trial of RTB101 alone or in combination with sirolimus. Multiple pre-clinical models have demonstrated that inhibition of TORC1 decreases protein and lipid synthesis, increases lysosomal biogenesis and stimulates the clearance of misfolded protein aggregates, such as toxic synucleins, that cause neuronal toxicity in Parkinson's disease. We remain committed to exploring the potential benefits of TORC1 inhibition in patients, and we look forward to the data from our Parkinson's disease trial, which we expect in mid-2020."

The PROTECTOR 1 Phase 3 trial was a randomized, double-blind, placebo-controlled clinical trial that evaluated the safety and efficacy of RTB101 10mg given once daily for 16 weeks during winter cold and flu season to subjects 65 years of age and older, excluding current smokers and individuals with chronic obstructive pulmonary disease. The primary endpoint of the trial was the reduction in the percentage of subjects with clinically symptomatic respiratory illness, defined as illness associated with a respiratory tract infection, or RTI, based on prespecified diagnostic criteria, with or without laboratory confirmation of a pathogen.

The PROTECTOR 1 trial included 1024 patients who were randomized 1:1 to receive RTB101 or placebo administered once daily for 16 weeks. In an analysis of the primary endpoint, the odds of experiencing a CSRI were 0.44 in the placebo cohort and 0.46 in the RTB101 cohort. The Company plans to conduct detailed analyses of the PROTECTOR 1 study, including additional data on safety and secondary and exploratory endpoints, which are not available at this time, with the goal of gaining insights that may explain the difference in RTB101 activity observed in PROTECTOR 1 as compared to prior Phase 2 studies.

A Mechanism by which Cellular Senescence Drives Pulmonary Fibrosis

The lingering senescent cells that accumulate with age are an important contributing cause of degenerative aging. If nothing else, their secretions generate a significant fraction of the chronic inflammation of aging, disrupting tissue function and immune function. Chronic inflammation is in turn well known to accelerate all of the most common age-related conditions. Fibrosis is a consequence of dysfunctional tissue maintenance and regeneration, in which scar-like deposits form, degrading tissue function. There is good evidence for fibrotic diseases, such as those of the lung, kidney, and heart, to be driven in large part by the presence of senescent cells. This is good news for patients, as while there is little that can be done to treat these conditions in the practice of medicine at the present time, senolytic therapies to clear senescent cells may well help to turn back fibrosis.

Accumulation of senescent cells is associated with the progression of pulmonary fibrosis but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM) lipid extracts and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT regardless of the origin of the cells or the modality of senescence induction.

The synthesis of LT was biphasic and followed by anti-fibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts induced pro-fibrotic signaling in naïve fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl leukotriene in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cells removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5+cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16Ink4a+). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs.

This study demonstrates for the first time that senescent cells secrete functional LTs, significantly contributing to the LTs pool known to cause or exacerbate idiopathic pulmonary fibrosis.


A Role for B Cells in the Chronic Inflammation Generated by Visceral Fat Tissue

Much of the long-term harm caused by excess visceral fat tissue is due to raised levels of chronic inflammation, the inappropriate over-activation of the immune system characteristic of both obesity and aging. Chronic inflammation accelerates the progression of near all of the common age-related conditions. There are numerous mechanisms via which fat tissue rouses an immune response: cellular debris that triggers immune cells into action; generation of excessive numbers of senescent cells; inappropriate signaling from fat cells that mimics the response to infection; infiltration of inflammatory macrophages into fat tissue; and so forth. Researchers here investigate some of the details of the way in which the immune system interacts with visceral fat, focusing on a role for B cells in spurring the inflammation that results.

Previous work found that as people age, their body's ability to generate energy by burning belly fat is reduced. Consequently, fat that surrounds the internal organs increases in the elderly. Researchers had found that the immune cells necessary to the fat-burning process, called macrophages, were still active but their overall numbers declined as belly fat increased with aging. This latest study found that something else is happening as well. Adipose B cells in belly fat unexpectedly proliferated as animals aged, contributing to increased inflammation and metabolic decline. "These adipose B cells are a unique source of inflammation. Normally the B cells produce antibodies, and defend against infection. But with aging, the increased adipose B cells become dysfunctional, contributing to metabolic disease."

When they are working correctly, some B cells expand as needed to protect the body from infection, and then contract to baseline. But with aging, they don't contract in belly fat. This predisposes to diabetes and metabolic dysfunction like inability to burn fat. Researchers theorizes that this ongoing expansion may be due to increased human life expectancy - a pushing of the body's cells beyond their evolutionary limits. Researchers discovered that adipose B cells expand by receiving signals from nearby macrophages. Relatedly, they found that by reducing the macrophage signal and by removing adipose B cells, they could reverse the expansion process, and protect against age-induced decline in metabolic health.


Notes on the 2019 Longevity Week Events in London

I was recently in London for the Longevity Week, a collection of single day and evening events organized by investor Jim Mellon of Juvenescence and supporting groups. Varied events focused separately on (a) educating investors in the science of aging, (b) generating a larger investment community for the new longevity industry, and (c) improving the non-profit world and its efforts to explain the merits of treating aging to the public, to bring therapies to the clinic, and to improve the state of older life using presently available tools. Jim Mellon clearly understands that building an industry focused on the medical control of aging, particularly in regions where medical development and clinical practice is so very heavily regulated, requires raising the water level when it comes to understanding of that industry and its potential.

The first event, the Science Summit, was a small and selective gathering that I was kindly invited to. It was the successor to an earlier master class on the science of aging organized for an audience of investors interested in the field, based on the concept that investors, and then the field as a whole, will benefit from a better understanding of the underlying science. I should say that these are largely investors in funds rather than investors in companies, occupying the higher valuation end of the investment community. These are people who, collectively as a class, have a lot of influence over the shape and pace of development of future industries through their choices in what to invest in.

A number of scientists gave presentations on the field, eclectic in topic. Work on bisphosphonates was mentioned early in the day. You might recall that these were shown at the start of the decade to extend life span by five years in one cohort, and in numerous other studies have been shown to reduce mortality, heart attack incidence, and so forth. This was one of the early examples that prompted the question of whether or not it is reasonable to expect there to be any sizable effects on life span hiding in the existing portfolio of medical therapies, unnoticed. This was, of course, well before the advent of senolytics to clear senescent cells, and the discovery that numerous existing drugs and supplements may be senolytic to a degree that will affect life expectancy in patient populations. When it comes to bisphosphonates, it has been challenging for the research community to raise funds to study the mechanisms involved, so little work is ongoing on the possible mechanisms. This is a common theme in scientific research on interventions in aging, sad to say. Effects on life span due to bisphosphonates are thought to depend on improved DNA repair and reduced levels of cellular senescence, but this is far from confirmed, and these drugs do have non-trivial side-effects. The likely future course of development would involve the slow process of identifying the important mechanism and then finding alternative ways of targeting it.

There was a fascinating presentation on the use of 670nm wavelength light, showing that in a number of different species it can improve mitochondrial function in the cells that the light reaches. In small animals this wavelength can penetrate much of the body and brain. The underlying mechanism by which mitochondrial function is improved in cells under this wavelength of light is unclear, but it would be interesting to see researchers comparing the effects of this with, say, NAD+ upregulation, in search of a better understanding of the general malaise that affects mitochondria with aging. Which parts of the problem are the most important? Given quite distinct forms of intervention - light and small molecules - one might have a chance of learning by comparison.

Another interesting point that emerged, watching presentations on a variety of topics related to the biology of aging, specific age-related diseases, and programs of development, is that cellular senescence now appears near everywhere. Scientific programs that wouldn't have mentioned cellular senescence as recently as five years ago are now fitting it into their work, or making it a focus. We might treat this as a leading indicator of what the senolytics industry will look like a few years from now - there will be many, many companies and development programs.

The final presentation of the day was by Joao de Magelhaes, and he spent some time discussing the question of how development of therapies for aging might fail. The point of thinking about this is of course to prevent this from happening - to convince ourselves that either matters are progressing well, or that there is something important that must be done in order to enable matters to progress well. Do we actually understand how and why aging happens? Are animal models too different from humans in ways that matter? While stress responses related to calorie restriction appear very similar across most species examined to date, and these processes influence life span, is that really representative of the degree to which processes of aging are different between species? Can the results of any human trial of at most a few years really tell us anything about what a therapy is doing to aging over the long term of decades? Are we successfully prioritizing and selecting which of the limited number of research projects and trials can be conducted, given the resources to hand?

The second event, Investing in the Age of Longevity, was a part of the investor-focused programs organized by Jim Mellon's Master Investor organization, a way to promote understanding of - and participation in - the longevity industry among the members of the broader investor community. This is of course of great benefit to Juvenescence, cofounded by Jim Mellon, but it is also of great benefit to everyone else. If we are to see this industry thrive and deliver on the promise of treating aging as a medical condition, it absolutely must be promoted and made interesting to the investment community. The day opened with an overview of the state of science to slow or reverse aging, and examples of specific programs, by Aubrey de Grey, Nir Barzilai, and Michael West of AgeX Therapeutics. Their overriding theme was that things have changed since a decade or two ago, that we've reached a tipping point, that now is the time to really work on bringing therapies to the clinic. The scientific and technical capabilities, the arrival of capital, the changing of opinions, all have advanced considerably since the turn of the century, and things are changing ever more rapidly year to year now. Then investors, including Laura Deming of the Longevity Fund said much the same thing from their perspective on the space.

The afternoon saw the showcasing of various companies, biotech startups working on aging in some way, and at different stages of progress. I presented on Repair Biotechnologies, alongside a number of other founders. In our own way, the startup entrepreneurs of the field also demonstrate that now is the time, that things are moving rapidly - if it wasn't, if they weren't, then we wouldn't have each recently chosen to start a company in the space.

The final event was the yearly Longevity Forum, in which the focus is on non-profits, governments, and the process of explaining the promise of treating aging as a medical condition to society at large. While it is quite possible for a small group of people, a small research and investor community, to build the first rejuvenation biotechnologies that will change the world, it remains the case that, on the large scale and over the long term, public support is vital to generating an industry and bringing rejuvenation therapies to the world. We want a world in which the average fellow in the street thinks of aging in the same way as he thinks about cancer: that something should be done about it, and that it is obviously a good idea to fund research into therapies that can treat the condition. This change won't happen by itself. It requires a great deal of work on the part of patient advocates and others, and hence the need for conferences and other community gatherings.

The Longevity Forum started, as on the previous day, with researchers talking about the state of the science, and why this is an exciting time - just to a different audience. From there it moved to discussions relating to the involvement of government and non-profits in the process of deploying means of slowing or reversing aging, and then moved back and forth between the science on one hand and the interactions between government, public, and public health services on the other. The UK has set the goal of extending healthy life span by five years by 2030, and this had a central place in the presentations of the day. It was an interesting mix of (a) those people who want to address aging in only a very minor way, via better diet, exercise, and ordinary preventative healthcare, and who think it will be a struggle to make progress, and (b) those people who are looking at the development of therapies to produce more radical changes in life span. Watching these two factions interact is quite interesting. It is a microcosm of what will happen over the next few years as people start to realize that senolytics will have a major impact on the state of human aging. What will happen to the community of people fixated on diet, exercise, and improving NHS and local health authority practices when it becomes clear that cheap senolytics produce a meaningful degree of rejuvenation?

Alongside the three days of events were evening gatherings of folk in the longevity community, a chance to catch up with people who are working on interesting projects, or funding those interesting projects. Much of the more important networking happens outside the events, as is ever the case. It was an interesting week, all told, and it is clear that we're all going to be very much better off in the years ahead as a result of the efforts of Jim Mellon and his allies and staff. This is a step up in the scale of advocacy for the treatment of aging in comparison to past years.

Proposing Parkinson's Disease to Originate in Either the Brain or the Gut

Parkinson's disease is characterized by the aggregation and spread of misfolded α-synuclein throughout the brain, though, as for all neurodegenerative conditions, there are many layers of cause and effect, and chronic inflammation and cellular dysfunction play noted roles as well. There has been some debate in recent years over whether the α-synuclein aggregation of Parkinson's disease begins in the gut or the brain, with evidence presented for both sides. The authors of this open access paper suggest that both are the case, and Parkinson's can be divided into two subtypes depending on the origin of α-synuclein misfolding.

Parkinson's disease (PD) is a highly heterogeneous disorder, which probably consists of multiple subtypes. Aggregation of misfolded alpha-synuclein and propagation of these proteinacious aggregates through interconnected neural networks is believed to be a crucial pathogenetic factor. It has been hypothesized that the initial pathological alpha-synuclein aggregates originate in the enteric or peripheral nervous system (PNS) and invade the central nervous system (CNS) via retrograde vagal transport. However, evidence from neuropathological studies suggests that not all PD patients can be reconciled with this hypothesis. Importantly, a small fraction of patients do not show pathology in the dorsal motor nucleus of the vagus.

Here, it is hypothesized that PD can be divided into a PNS-first and a CNS-first subtype. The former is tightly associated with REM sleep behavior disorder (RBD) during the prodromal phase and is characterized by marked autonomic damage before involvement of the dopaminergic system. In contrast, the CNS-first phenotype is most often RBD-negative during the prodromal phase and characterized by nigrostriatal dopaminergic dysfunction prior to involvement of the autonomic PNS. The existence of these subtypes is supported by in vivo imaging studies of RBD-positive and RBD-negative patient groups and by histological evidence. The present proposal provides a fresh hypothesis-generating framework for future studies into the etiopathogenesis of PD and seems capable of explaining a number of discrepant findings in the neuropathological literature.


Particulate Air Pollution Correlates with Atherosclerosis Risk

It is known that exposure to airborne particles, such as smoke from cooking fires, correlates with increased mortality due to cardiovascular disease. Setting aside commentary on wealth and its correlation with exposure to particulate air pollution, the obvious candidate mechanism is an increase in chronic inflammation due to the effects of inhaled particles on lung tissue. Raised inflammation then leads to an accelerated progression of atherosclerosis, the fatty deposits that narrow and weaken blood vessels, ultimately leading to heart failure, stroke, and heart attack. Researchers here provide epidemiological data to support this chronic inflammation hypothesis for the harms caused by particulate air pollution.

Cardiovascular diseases are the leading cause of mortality and morbidity worldwide, including in many low- and middle-income countries (LMICs). India has experienced a rapid epidemiological transition, resulting in notable prevalence of hypertension, diabetes, and obesity. India is also affected by high levels of ambient and household air pollution (HAP), resulting in a setting with high baseline cardiovascular risk and widespread exposure to high levels of air pollution.

Long-term exposure to ambient particulate matter (PM) has been associated with risk of acute myocardial infarction, stroke, and cardiovascular mortality. The most plausible pathway by which PM causes cardiovascular diseases is by promoting inflammation and atherosclerosis. Atherosclerosis is a systemic vascular disease representing the aging process and the cumulative adaptive response to cardiovascular risk factors (e.g. hypertension, diabetes). Carotid intima-media thickness (CIMT) is a non-invasive, surrogate marker of subclinical atherosclerosis, associated with cardiovascular risk factors, events, and mortality. There is evidence for a positive association between long-term ambient PM and CIMT. However, the magnitude of associations has been heterogeneous and studies are limited to high-income countries with low or moderate levels of air pollution. Additionally, the evidence for the association between HAP and CIMT is limited.

To our knowledge, there is no previous evidence about the association between outdoor ambient fine particulate matter (PM) and CIMT from populations in low- and-middle income countries. In this population-based study of 3372 participants, with annual mean ambient PM of 32.7 µg/m3, annual mean PM was associated with carotid intima-media thickness among men. 60% of participants used biomass cooking fuel, which was strongly associated with carotid intima-media thickness in women cooking with an unvented stove. Women had higher values of carotid intima-media thickness compared with men, which might be attributed to high cumulative exposure to household air pollution.


An Interview with Matthew O'Connor, as Underdog Pharmaceuticals Secures Seed Funding

Matthew O'Conner presented at Undoing Aging earlier this year on the startup biotech company Underdog Pharmaceuticals. The company is spinning out of the SENS Research Foundation (SRF), based on research conducted by the scientific team there in recent years. The company is focused on a class of molecule known cyclodextrins, and have candidates capable of efficiently binding and sequestering 7-ketocholesterol. This form of oxidized cholesterol is of great importance to the progression of atherosclerosis, and possibly other age-related conditions as well. In the case of atherosclerosis, the presence of oxidized cholesterols, and particularly 7-ketocholesterol, causes the macrophage cells, which are responsible for clearing out cholesterol from blood vessel walls, to become dysfunctional and inflammatory. Remove the 7-ketocholestrol, and the problem should largely go away.

I'm pleased to note that Underdog Pharmaceuticals has secured nearly $4 million in seed funding, and is thus well set to move ahead with the clinical development of this approach over the next few years. I would hope that this will be one of numerous biotech companies focused on rejuvenation research to emerge directly from the SENS Research Foundation in-house programs, joining the many others that have emerged within the broader network of researchers and entrepreneurs interested in tackling causes of aging. To mark the occasion, I recently had the chance to chat with Matthew O'Conner about Underdog Pharmaceuticals; as you can see, the company is a demonstration in and of itself of how the networks built by the foundation over the past decade have matured.

Underdog Pharmaceuticals, Inc. (Underdog), and SENS Research Foundation (SRF) today announced the launch of Underdog and the completion of its seed round, providing $3.95 million to promote Underdog's development of disease-modifying treatments for atherosclerosis and other age-related diseases. SRF also announced two senior appointments. The Underdog round is led by Michael Greve's Kizoo Technology Capital, part of the Forever Healthy Group and one of the premier organizations focusing on accelerating rejuvenation biotechnologies.

Underdog was built from an SRF flagship program that has driven two years of applied development designed to explore and repair the underlying causes of cardiovascular disease. Its co-founders are Matthew O'Connor, Ph.D. and Michael Kope, formerly the V.P. of Research and the founding CEO, respectively, of SRF. "We've taken a well-known and extremely safe compound, and have created novel derivatives that can specifically target the toxic biomolecule that drives the development of atherosclerosis, the cause of most heart attacks and strokes." Underdog's research has combined computational and synthetic chemistry programs to create custom-engineered cyclodextrins (polysaccharides with known industrial and pharmaceutical uses) to capture, and remove from cells, oxidized cholesterol derivatives such as 7-ketocholesterol, which are broadly toxic molecules with no known biological function.

Who are the Underdog Pharmaceuticals team, and how did you all become involved in this business of defeating atherosclerosis?

Underdog is being co-founded by Michael Kope and myself, formerly the founding CEO and VP of Research, respectively, of SRF. We are exiting our decade-long service at SRF to do this. We are building a great team of researchers and partners including our home-grown computational chemist Amelia Anderson and veteran bench industry biologists Daniel Clemens and Tamari Kirtadze. We are partnering with Cyclolab LTD for cyclodextrin chemistry expertise, MD.USE for cyclodextrin computational chemistry, and Biolacuna for regulatory affairs.

Amelia Anderson was one of our elite SRF Summer Scholars two years ago. It was that summer that she conceived of our computational chemistry program and started to build it. Dave Brindley, founder of Biolacuna, was the first SRF-sponsored PhD student and has since become a world-renowned biomedical regulatory expert specializing in developing methods for gaining approval of the new classes of rejuvenation biotechnologies that are going to be needed to revolutionize medicine. SRF's investment in education is really coming home to help us.

Every name has a story, why Underdog Pharmaceuticals?

We chose the name Underdog to connote our core mission - to attack the underlying causes of age-related disease; and also to represent the broader fight as the underdog seeking to overturn the current costly and inefficient paradigm for the treatment of such diseases ... and because Mike and I couldn't otherwise agree on whose dog to name the company after.

You outlined the science behind Underdog at Undoing Aging earlier this year, but if you could give a summary for the audience here?

We are targeting 7-ketocholesterol (7KC) in aging and disease. 7KC is a toxic oxysterol formed by the reaction of a cholesterol molecule with an oxygen free radical. 7KC has no redeeming qualities and is difficult to clear once it has become lodged in a cell. It is highly toxic to cells and as a fundamental damage molecule implicated many diseases of aging including atherosclerosis (and therefor heart attacks and strokes), Alzheimer's disease, and macular degeneration. We are engineering cyclodextrins to bind 7KC with high affinity and specificity. Cyclodextrins are cyclic sugar molecules that are very safe and amenable to engineering. We have created a new class of cyclodextrins that can bind 7KC more than ten times better than any other cyclodextrin currently available. The goal is to remove 7KC from cells and tissues and have it be excreted from the body.

Do you plan to work towards sequestration of other molecular targets via cyclodextrins, now that you've demonstrated success with one?

Yes! The tools that we are developing are quite amenable to targeting other damage molecules with cyclodextrins. Particularly the cyclodextrin computational platform that we are developing can easily be adapted for other targets; as well as our automated cyclodextrin screening tool and safety testing assays. For at least the next few years, however, we'll be spending 99% of our time and energy on getting our first drug to the clinic.

SENS Research Foundation is a non-profit, Underdog Pharmaceuticals is a startup; has it been challenging to make the shift to the for-profit world?

It has not. SRF's mission has always been to bring the promise of rejuvenation biotechnologies to real-life people. The organization has always known that successful technology transfer is inherent and intimate to that mission. SRF has always been looking for opportunities for translation with all of our projects. With respect to Underdog this transition for us has been been super fun. We've been delighted with the response to our research from the scientific community, discussions with financial agents, and with our development collaborators all around the world. We have an exciting message and it's been gratifying to see how enthusiastically it's been received. Our investors are all enthusiastic about our mission which is why we've been able to raise the funds that we need and transition so quickly into this new company.

Is this the starting point for SENS Research Foundation to become an incubator of a series of biotech startups?

SRF won't be developing wholly owned subsidiaries; it will be creating companies using the same rubric as university tech transfer programs. As with Underdog, SRF would have both royalty and equity interests in such companies. It is inherent to the mission that SRF tries to do this to make these technologies publicly available, and indeed we think one sees evidence of that both in the success of this spinout and in the new leadership that's been chosen for SRF. Jim O'Neill is the new interim CEO at SRF. He has an extensive background in both government research funding and private investment and is committed to driving this push towards translating SRF technologies into therapies. Professor Alexandra Stolzing is coming on board to replace me as VP of Research. Her reputation in rejuvenation / regenerative medicine should need no introduction, but she has played both faculty and private industry leadership roles focused on translational medicine in aging. SRF is in the process of reviewing incubator and accelerator programs focused on translating SENS-style damage repair technologies. We'd love to see them do more of that!

Will we see the allotopic expression project spun out in the same way?

The MitoSENS allotopic expression project is plowing ahead. It started as a basic research program but has made great strides and could be ready to make a transition to the treatment of mitochondrial genetic disease quite soon.

If this all works out amazingly well, atherosclerosis is much reduced, and Underdog Pharmaceuticals becomes a billion dollar company, what next?

Well we want to not only reduce atherosclerosis, but eliminate it entirely! But on a broader scale our team genuinely hopes to be able to contribute to a change in paradigm, from both a regulatory perspective and a clinical perspective, on how we treat diseases of aging. This could be a long-term challenge, and if done appropriately, we, together with other companies with similar missions, can work with regulatory agencies to look at age-related disease in a new way. This is a big part of the reason that we want to do this company now.

The Gut Microbiome in Neuroinflammation and Alzheimer's Disease

The microbial populations of the gut influence and are influenced by the state of the immune system. They also have effects on tissue function throughout the body via secreted compounds such as butyrate, mediating some of the effects of diet on long-term health. These microbes change with age, losing beneficial populations and gaining harmful populations that contribute to chronic inflammation. These changes are far from fully explored at the present time, but may have effects on health that rival those resulting from regular exercise. In this open access review, researchers discuss the influence of gut microbes on chronic inflammation of the brain, and the development of neurodegenerative conditions such as Alzheimer's disease.

Alzheimer's disease (AD) is a complex, multi-factorial disease affecting various brain systems. This complexity implies that successful therapies must be directed against several core neuropathological targets rather than single ones. The scientific community has made great efforts to identify the right AD targets beside the historic amyloid-β. Neuroinflammation is re-emerging as determinant in the neuropathological process of AD. A new theory, still in its infancy, highlights the role of gut microbiota in the control of brain development, but also in the onset and progression of neurodegenerative diseases.

Bidirectional communication between the central and the enteric nervous systems, called gut-brain axes, is largely influenced by gut microbiota and the immune system is a potential key mediator of this interaction. Growing evidence points to the role of gut microbiota in the maturation and activation of host microglia and peripheral immune cells. Several recent studies have found abnormalities in gut microbiota (dysbiosis) in AD populations. These observations raise the intriguing question whether and how gut microbiota dysbiosis could contribute to AD development through action on the immune system and whether, in a therapeutic prospective, the development of strategies preserving a healthy gut microbiota might become a valuable approach to prevent AD.


Rapamycin Prevents Deterioration in Brain Circulation in Aged Rats

The mTOR inhibitor rapamycin is well known to slow aging in animal models. As for most of the methods shown to achieve this goal in short-lived species, upregulation of cellular maintenance processes such as autophagy features prominently in the changes produced by the drug. Every one of these approaches that produce sweeping changes in cellular metabolism and a general slowing of age-related decline provides the research community with an essentially unlimited range of projects to undertake when it comes to assessing specific metrics of aging and how they are affected.

Here, researchers look at how rapamycin affects age-related deterioration in blood circulation in the brain. There are many reasons why this might decline: a weakened or failing heart; loss of capillary network density; narrowing of blood vessels due to atherosclerosis; and so forth. The brain is an energy-hungry organ, and any reduction in the supply of oxygen and nutrients will have detrimental effects on tissue function, contributing to the onset of neurodegeneration.

Cerebrovascular dysfunction and cognitive decline are highly prevalent in aging, but the mechanisms underlying these impairments are unclear. Cerebral blood flow decreases with aging and is one of the earliest events in the pathogenesis of Alzheimer's disease (AD). We have previously shown that the mechanistic target of rapamycin (mTOR) drives disease progression in mouse models of AD and in models of cognitive impairment associated with atherosclerosis, closely recapitulating vascular cognitive impairment. In the present studies, we sought to determine whether mTOR plays a role in cerebrovascular dysfunction and cognitive decline during normative aging in rats.

Using behavioral tools and MRI-based functional imaging, together with biochemical and immunohistochemical approaches, we demonstrate that chronic mTOR attenuation with rapamycin ameliorates deficits in learning and memory, prevents neurovascular uncoupling, and restores cerebral perfusion in aged rats. Additionally, morphometric and biochemical analyses of hippocampus and cortex revealed that mTOR drives age-related declines in synaptic and vascular density during aging. These data indicate that in addition to mediating AD-like cognitive and cerebrovascular deficits in models of AD and atherosclerosis, mTOR drives cerebrovascular, neuronal, and cognitive deficits associated with normative aging.

Thus, inhibitors of mTOR may have potential to treat age-related cerebrovascular dysfunction and cognitive decline. Since treatment of age-related cerebrovascular dysfunction in older adults is expected to prevent further deterioration of cerebral perfusion, recently identified as a biomarker for the very early (preclinical) stages of AD, mTOR attenuation may potentially block the initiation and progression of AD.


Cellular Senescence is Important in Zebrafish Fin Regrowth

Species such as salamanders and zebrafish are capable of regrowing lost limbs, fins, and organ tissue without scarring, leading to a fully functional replacement. Regeneration from injury is in general a complex dance of different cell types: immune cells, stem cells, somatic cells. Further, senescent cells play an important part in this process. In response to injury some cells enter a senescent state and their inflammatory secretions help to coordinate the process of regrowth. The senescent cells either self-destruct or are destroyed by the immune system shortly thereafter - though, of course, this process of clearance is not completely efficient, and that inefficiency has sizable consequences over the long term. That some senescent cells linger, and in increasing numbers as the immune system falters with age, is a contributing cause of degenerative aging.

Research into the details of proficient regeneration in a variety of species points to significant differences in the behavior of senescent cells and their interactions with other cell types. Salamanders, for example, exhibit highly efficient clearance of senescent cells by immune cells following regeneration. A prehaps similar situation is present in African spiny mice, which are capable of more extensive regeneration than is the case for most mammals. In today's open access research, the focus is on zebrafish, and the authors show that removal of a sizable fraction of senescent cells via senolytic treatment impairs regrowth but doesn't prevent it. It would be interesting to see the outcome of complete clearance of senescent cells.

Cell senescence contributes to tissue regeneration in zebrafish

Cellular senescence is a terminal cell response consisting on the implementation of a permanent cell cycle arrest and the acquisition of a secretory phenotype with cell-to-cell communication properties. Exhaustion of the proliferative capacity of the cell leads to senescence, and the accumulation of these damaged cells in tissues from old individuals is considered a key element in the process of aging. Despite this detrimental effect, the senescence response has a beneficial side protecting damaged cells from proliferating. This is considered the basis of its tumor-suppressive function. The recent identification of developmentally programmed cell senescence during embryogenesis expanded our view of the positive activities of this response. Senescence during development promotes cell turnover, tissue remodeling, and, paradoxically, growth. A similar positive pro-morphogenetic activity for cell senescence has been suggested to operate during skin wound healing in mice and during limb regeneration in salamanders. Senescent cells seem to appear at wound sites after injury to help promote optimal wound healing.

Here, we decided to evaluate the senescence response in the context of tissue injury using an animal model of complex tissue regeneration, the zebrafish. To study senescence after tissue damage, we amputated the pectoral fin of adult fish (around 1 year old) at approximately 50% of its length and followed regeneration with time. We stained fins for senescence-associated beta-galactosidase (SAbetaGal), the most widely used marker of senescence, after 8, 16, or 30 days postamputation (dpa), a time point in which fins were completely regenerated. Fins at 8 dpa showed intense blue staining compared with light blue at 16 dpa and completely absent staining at 30 dpa. We observed that 8 dpa was the time point that produced a stronger SAbetaGal reaction and this activity was restricted to the distal part of the fin, the area where regeneration takes place. These results support the notion of a transient induction of cell senescence during fin regeneration.

To directly assess the role of senescence induction during fin amputation, we decided to induce the removal of these senescent cells from amputated fins. For this, we treated fish for 48 or 72 hr with ABT-263 (Navitoclax), a senolytic compound that by inhibiting the Bcl-2 antiapoptotic family of proteins triggers specifically the death of the senescent cell. ABT-263 treatment caused a reduction in SAbetaGal staining and a concomitant induction of apoptosis in the regenerating area. We determined the regenerative capacity by measuring the length of regenerate at 8 dpa in fish treated with ABT-263. This analysis revealed that the removal of senescent cells by ABT-263 treatment clearly impaired regeneration, with amputated fins in fish treated with ABT-263 showing a clear reduction in the length of regenerate compared with the one reached in control animals.

Senescent Cells Mediate the Incidence of Periodontitis in Diabetic Patients

Insofar as either type 1 diabetes or type 2 diabetes increase the burden of senescent cells, we might say that the condition literally accelerates aging. The accumulation of lingering senescent cells is a contributing cause of aging; these errant cells disrupt tissue function and produce the characteristic profile of chronic inflammation known as inflammaging via a potent mix of secreted molecules and vesicles. Diabetic patients suffer more and worse gum disease, periodontitis, than their healthy peers, and researchers here show that hyperglycemia leads to increased numbers of senescent cells in gum tissue, causing all of the expected downstream consequences resulting from inflamed gums.

Inflammaging was recently affiliated with the progression of diabetic complications. Local cellular senescence together with senescence-associated secretory phenotype (SASP) are the main contributors to inflammaging. However, little is known about their involvement in diabetic periodontitis. Gingiva is the first line of host defense in the periodontium, and macrophages are key SASP-carrying cells. Here, we explored the molecular mechanism by which hyperglycemia drives the inflammaging in the gingival tissue of diabetic mice and macrophages.

We demonstrated that hyperglycemia increased the infiltrated macrophage senescence in gingival tissue of diabetic mice. Simultaneously, hyperglycemia elevated the local burden of senescent cells in gingival tissue and induced the serum secretion of SASP factors in vivo. Moreover, in vitro, high glucose induced macrophage senescence and SASP factors secretion through phosphorylation of NLRC4, which further stimulated the NF-κB/Caspase-1 cascade via IRF8-dependent pathway.

Deletion of NLRC4 or IRF8 abolished hyperglycemia-induced cellular senescence and SASP in macrophages. In addition, we found that treatment with metformin inhibited NLRC4 phosphorylation and remarkably decreased cellular senescence and SASP in the context of hyperglycemia. Our data demonstrated that hyperglycemia induces the development of inflammaging in gingival tissue and suggested that NLRC4 is a potential target for treatment of diabetes-associated complications.


Delivery of MALAT1 in Exosomes as a Treatment for Osteoporosis

Bone is not a static tissue. It is constantly remodeled, broken down by osteoclast cells and built up by osteoblast cells. The loss of bone mass and strength with age, osteoporosis, is the result of an imbalance in the activities of osteoclasts and osteoblasts, too much destruction and too little creation. This imbalance, as for all aspects of aging, is the result of many deeper overlapping layers of cause and effect, not fully mapped and understood. Thus most approaches to therapy tend to involve ways to force greater activity of osteoblasts or suppress the activity of osteoclasts, rather than delving in search of root causes. The open access paper here is an example of this type of work, outlining an approach to stimulate greater osteoblast activity in mice.

In recent years, promising therapeutic approaches to treat osteoporosis are mainly focused on targeting the functions of skeletal stem cells and osteoblasts. A more detailed understanding of bone biology has led to the identification of novel therapeutic targets with enhanced molecular insights into the communication between bone-forming osteoblasts and bone-resorbing osteoclasts as well as the orchestrating signaling network. Thus, it is necessary to develop new approaches to stimulate osteoblast activity. In the present study, we demonstrate that bone marrow stem cell (BMSC) derived exosomes carrying the long non-coding RNA (lncRNA) MALAT1 could effectively stimulate the osteoblast activity. Our results highlighted the potential of exosomal MALAT1 to prevent osteoporosis in mouse models.

A key finding of the current study indicated that BMSCs-derived exosomal MALAT1 could potentially promote osteoblast activity. Exosomes could actively transport and transfer information between miRNAs, proteins, and mRNAs to target cells, thus affecting their behaviors and strongly modifying the entire microenvironment. Consistent with previous reports, we observed the protective role of exosome-mediated delivery of MALAT1 in disease. Furthermore, we detected that the upregulation of MALAT1 could attenuate the symptoms of osteoporosis in mice. Existing literature has suggested that lncRNAs play critical roles in the initiation and pathogenesis of osteoporosis. For instance, a recent study demonstrated that lncRNA MEG3 suppressed the osteogenic differentiation of mesenchymal stem cells in postmenopausal osteoporosis.

Our study provides evidence that BMSC-derived exosomal MALAT1 may contribute to enhanced osteogenic activity and alleviated symptoms of osteoporosis in the mouse model by acting as a miR-34c sponge to upregulate SATB2 expression. These results provide a broader understanding of the pathogenesis of osteoporosis as well as novel therapeutic strategies for its treatment.


How to Start a Biotech Company in the Longevity Industry

Based on discussions with various folk at scientific and industry conferences earlier this year, regarding whether or not our rejuvenation research, development, and advocacy community is challenging to approach and understand as an outsider, I recently put together an introductory document for entrepreneurs entitled How to Start a Biotech Company in the Longevity Industry (PDF). Given my experiences, it is primarily aimed at entrepreneurs with previous experience in other industries, who are now interested in helping to treat aging as a medical condition and there by greatly improve the human condition.

The young and rapidly growing longevity industry encompasses the clinical development of rejuvenation therapies, such as senolytic therapies to clear senescent cells from old tissues, or the thymic regeneration project taking place at Repair Biotechnologies, the company I founded with Bill Cherman last year. It also includes initiatives that can only modestly slow aging, such as mTOR inhibitor and NAD+ upregulation programs. All told there are around 100 companies in the industry as of late 2019, of which perhaps a fifth could be argued to be working on programs relevant to the SENS damage repair view of aging, and which thus might lead to rejuvenation therapies. Clearly we still have some way to go in persuading people that only damage repair and rejuvenation is worth the effort, when looking at the long term and the big picture.

Yes, once the sizable expense of clinical development has been expended, it will be a good deal for older patients to be able to spend $60 a month on a drug that halves the rate of influenza infection - this more or less describes an early use case for an mTOR inhibitor, based on the work taking place at resTORbio. But the cost of clinical development of an mTOR inhibitor and a senolytic are pretty much the same, and the senolytic is vastly, enormously more beneficial, based on the animal data to date. It is transformative, where mTOR inhibitors produce only incremental gains. No-one should be choosing to work on projects that can only produce small gains, when there are many alternatives that have the potential to produce large gains, and yet most people in the industry are doing just that.

This is not why I wrote an introduction to starting a biotech company in the longevity industry. I wrote it because I was having the same conversation with interested entrepreneurs from other industries over and again at conferences. The longevity industry is in an interesting state at the moment: there is far more funding than there are early stage companies to absorb it, there are not enough entrepreneurs, and there are scores (at the very least) of scientific programs relevant to the treatment of aging as a medical condition that are ready for clinical translation, but lacking anyone to carry out the work. That there is so much venture funding and excitement is attracting interest from the broader entrepreneurial community, but not rapidly or robustly enough. It takes time to find out what questions one should even be asking when coming into the longevity industry completely naive.

Thus the need for more introductory documents, and thus this introductory document. Because it comes from me, it is intended not just to help newcomers find their way, but also to point out that working on rejuvenation is far, far more beneficial for all parties concerned than is the case for work on slowing aging. The first draft of the document is available as a PDF. Hopefully it proves useful, and, as always, feedback is welcome.

How to Start a Biotech Company in the Longevity Industry (PDF)

You are an entrepreneur who wishes to start a longevity industry biotech startup, but your experience to date is in a different industry. This document is an initial primer and guide to help you get started. New classes of therapy, targeting the mechanisms of aging, have the potential to prevent and reverse all age-related disease, and greatly extend healthy human lifespan. The first rejuvenation therapies are already under clinical development in numerous startup companies. This new longevity industry is growing exceptionally rapidly. Venture funding for longevity startups is increasing enormously year over year. Yet there are far too few entrepreneurs and new startups in comparison to the available funding. Your arrival will be welcomed: this is a friendly, and close-knit community.

You are entrepreneurial. You have heard the buzz about the new longevity industry: the rapid growth in funding, the numerous billionaires becoming involved, the new approaches to medicine that are targeting the mechanisms of aging to prevent and reverse the diseases and frailty of old age. You want to get involved, to start a company, to do something about aging ... to change the world for the better. But how? Whatever your past industry, here you must be the business co-founder. Life science and its application to biotechnology is a vast, complex, intimidating field. Aging is its own highly specialized portion of that field. You need an understanding sufficient to identify a project to work on; you need a scientific co-founder; you need to know the investors and the movers and shakers. Where to even start?

This document is a starting point. We hope that it helps.

Greater Waist Circumference, Greater Risk of Dementia

In recent years, epidemiologists have found that waist circumference is a better measure of the burden of excess visceral fat tissue than body mass index (BMI). Progress towards making better use of this information has been slow, as is usually the case in the world of epidemiology. Visceral fat tissue generates chronic inflammation through a variety of mechanisms, from DNA debris activating the immune system to inappropriate signaling by fat cells to an accelerated pace of generation of senescent cells. Chronic inflammation disrupts function and accelerates the progression of all of the common age-related conditions. People who are overweight have a shorter life expectancy and higher lifetime medical costs as a result.

A 2015 large-scale retrospective cohort study of nearly 2 million people from the United Kingdom Clinical Practice Research Datalink showed that the incidence of dementia continued to fall for every increasing BMI category. Two Mendelian randomization studies showed no association between obesity and dementia. BMI is not a perfect measure of adiposity because it cannot discriminate between fat and lean body mass. Waist circumference is a more accurate indicator of abdominal visceral fat level than body mass index (BMI) in the elderly. Studies have been limited, however, and focused on the relationship between waist circumference and dementia in older persons. One study showed that central adiposity, represented by waist circumference, predicted an increased risk for cognitive decline during a 2-year follow-up period in older patients with diabetes. Another study reported that waist circumference was correlated with lower overall cognition and executive performance in older women with type 2 diabetes.

To help determine a healthy waist circumference, researchers compared relative risk of dementia associated with waist circumference and BMI categories using the Korea National Health Insurance Service program. The program is a mandatory social health insurance program that enrolls about 98 percent of Koreans who participate in biannual standardized health examinations. The study population comprised 872,082 participants aged 65 years and older who participated in the Korean national health screening examination between January 1, 2009 and December 31, 2009. The study population was observed from baseline until the date of development of dementia, death, or until December 31, 2015, whichever came first.

The results of the study showed participants with a waist circumference of greater than or equal to 90cm for men and 85cm for women had a significantly increased risk of dementia after adjusting for other factors such as age, BMI, blood pressure, cholesterol, liver function tests and various lifestyle factors. As for the association between BMI categories with dementia in older men and women who were underweight, they experienced a significant increased risk of dementia compared with normal weight individuals after factoring in comorbidities and various lifestyle factors. The relationship with BMI and dementia may be a result of the adverse effects of sarcopenia in the elderly.


Selectively Removing Mutant Proteins by Binding them to Autophagy Components

Researchers here demonstrate a proof of principle for an interesting approach to tackling the aggregation of damaged, altered, or misfolded proteins that is a feature of most neurodegenerative conditions. They target the mutant huntingtin protein, which is probably an easier task than targeting, say, a misfolded protein with a normal sequence. The basic idea is to deploy a linking molecule that binds to the problem protein with high specificity, and also binds to an essential component of autophagy - in this case LC3B, involved in the generation of autophagosomes responsible for carrying materials to lysosomes. This ensures that the whole linked set of molecules is dragged into an autophagosome and transported to a lysosome where it is broken down and recycled.

Several neurodegenerative diseases involve the slow accumulation of a misfolded protein in neurons over many years. The proteins involved in these diseases might differ, but the result is similar - eventually, the neurons die from the build-up of toxic misfolded proteins. Scientists have long been searching for ways to reduce the levels of the disease-driving proteins without also clearing their wild-type counterparts, which typically have myriad crucial functions. Researcher snow show that this can be accomplished using compounds that interact specifically with both the misfolded part of the protein and the neuron's protein-clearance machinery.

The researchers chose to focus on Huntington's disease, which is caused by an abnormally long stretch of glutamine amino-acid residues in the huntingtin (HTT) protein. This expanded polyglutamine tract causes HTT to misfold. Cells are able to degrade the mutant huntingtin (mHTT) through autophagy - a clearance mechanism that involves engulfment of proteins by a vesicle called the autophagosome. Researchers hypothesized that compounds that bind to both the mutant polyglutamine tract and the protein LC3B, which resides in the autophagosome, would lead to engulfment and enhanced clearance of mHTT. But no such compounds had been reported. The authors therefore conducted small-molecule screens to identify candidate compounds.

Researchers initially identified two candidates, dubbed 10O5 and 8F20. These compounds had been shown to inhibit, respectively, the activity of the cancer-associated protein c-Raf and kinesin spindle protein (KSP), which has a key role in the cell cycle. The team found that 10O5 and 8F20 were able to clear mHTT independently of their effects on these other proteins. The researchers showed that the regions of 10O5 and 8F20 that interacted with mHTT and LC3B in the screen shared structural similarities. Next, they screened for compounds that shared these structural properties but were structurally distinct. This led them to discover two more compounds, AN1 and AN2, that link mHTT to LC3B and thereby selectively reduce levels of mHTT.

Researchers validated their discovery by showing that the four compounds reduced levels of the full-length mHTT protein (not just the protein fragment used in the screen). The compounds lowered levels of mHTT both in vitro - in mouse neurons and neurons derived from the biopsied skin cells of people with Huntington's disease - and in vivo, in mouse and fly models of the disease.


Poor Results from an Initial Human Trial of Nicotinamide Mononucleotide

Mitochondria are the power plants of the cell, responsible for packaging energy store molecules that power cellular processes. NAD+ is an essential metabolite for mitochondrial function, but levels decline with age. The proximate causes of this decline are fairly well mapped, and involve insufficient resources in a variety of pathways for synthesis or recycling of NAD+. The deeper reasons are poorly understood, however, meaning how these pathway issues emerge from the underlying molecular damage to cells and tissues that causes aging. Ways to force an increase in NAD+ levels have been shown to improve mitochondrial function in old animals, reversing some of the losses that occur with age. Loss of mitochondrial function is implicated in age-related diseases, particularly those in energy-hungry tissues such as the brain and muscles.

There are a number of ways to raise NAD+ levels: delivery of sizable amounts of NAD+ directly via infusion, of which a tiny fraction makes it into cells where it is needed; delivery of various precursor molecules that are used to manufacture NAD+; or delivery of factors known to improve recycling of NAD+. Most present effort is focused on the second of those options, via supplements such as nicotinamide riboside or nicotinamide mononucleotide, though groups like Nuchido are trying to produce better means of raising NAD+ levels that target multiple mechanisms at once.

Nicotinamide riboside has been trialed in humans, in a small number of people, with data showing reductions in age-related increases in blood pressure through improvement in the function of vascular smooth muscle. A similarly small trial of nicotinamide mononucleotide took place in Japan, and in today's open access paper, the researchers involved report on the results. As you can see from their summary, this approach achieved none of the benefits noted in the trial of nicotinamide riboside. At least some of the patients were old enough to expect some positive outcome on blood pressure, but none was observed.

Recent studies have revealed that decline in cellular nicotinamide adenine dinucleotide (NAD+) levels causes aging-related disorders and therapeutic approaches increasing cellular NAD+ prevent these disorders in animal models. The administration of nicotinamide mononucleotide (NMN) has been shown to mitigate aging-related dysfunctions. However, the safety of NMN in humans have remained unclear. We, therefore, conducted a clinical trial to investigate the safety of single NMN administration in 10 healthy men of 40 to 60 years of age.

A single-arm non-randomized intervention was conducted by single oral administration of 100, 250, and 500 mg NMN. Clinical findings and parameters, and the pharmacokinetics of NMN metabolites were investigated for 5 hours after each intervention. Ophthalmic examination and sleep quality assessment were also conducted before and after the intervention.

The single oral administrations of NMN did not cause any significant clinical symptoms or changes in heart rate, blood pressure, oxygen saturation, and body temperature. Laboratory analysis results did not show significant changes, except for increases in serum bilirubin levels and decreases in serum creatinine, chloride, and blood glucose levels within the normal ranges, independent of the dose of NMN. Results of ophthalmic examination and sleep quality score showed no differences before and after the intervention. Plasma concentrations of N-methyl-2-pyridone-5-carboxamide and N-methyl-4-pyridone-5-carboxamide were significantly increased dose-dependently by NMN administration. The single oral administration of NMN was safe and effectively metabolized in healthy men without causing any significant deleterious effects. Thus, the oral administration of NMN was found to be feasible, implicating a potential therapeutic strategy to mitigate aging-related disorders in humans.


Cardiovascular Aging Contributes to Brain Aging

The brain is an energy-hungry organ, and is sensitive to reductions in the blood supply of oxygen and nutrients. Cardiovascular aging can reduce that supply, whether through conditions such as heart failure, or the progressive loss of density in capillary networks that occurs throughout the body with advancing age, or an accelerated pace of rupture of tiny vessels in the brain, or disruption of the blood-brain barrier, allowing unwanted molecules and cells to enter the brain. Thus, as researchers here note, we would expect to see correlations between cardiovascular disease, or risk factors for cardiovascular disease, and damage and dysfunction in the brain.

Age-related changes in the cerebrovascular system include structural reorganization of the vascular beds, reduced vessel elasticity, and disintegration of the blood-brain barrier. Further observations include reduced cerebral perfusion, and increased lesion burden in the cerebral white matter. Lesions can be observed as white-matter hyperintensities (WMHs) upon magnetic resonance imaging (MRI). They arise from ischemia, hypoperfusion, blood-brain-barrier breakage, and inflammation and are considered manifestations of cerebral small-vessel disease. WMHs are highly prevalent in aging and predictive of broad-ranged cognitive decline, dementia, and mortality.

Dopamine (DA) has been identified as an important modulator of cognitive functions. Maladaptive DA signaling typically gives rise to cognitive impairment, whereas increased DA transmission, if not excessive, may improve performance. Numerous positron emission tomography (PET) studies have demonstrated reduced availability of DA constituents in older individuals, with links to reduced cognitive performance. The age sensitivity of the DA system has therefore been suggested to modulate cognitive trajectories in aging. Research suggests relationships among vascular function, DA status, and atrophy in pathological and normal aging. Cognitive impairments in Parkinson's disease (PD) have been related to deficits in perfusion and DA decline, which are exacerbated in presence of WMHs. Increased WMH burden in normal aging is paralleled by decreased grey-matter and white-matter volume, and has been associated with reduced DA transporter and D1 receptor availability.

Thus we evaluated the interrelation among WMH burden, cerebral perfusion, DA D2-receptor (D2DR) availability, grey- and white-matter structure, and cognition in 181 healthy, older adults aged 64-68 years. Higher cardiovascular risk as assessed by treatment for hypertension, systolic blood pressure, overweight, and smoking was associated with lower frontal cortical perfusion, lower putaminal D2DR availability, smaller grey-matter volumes, a larger number of white-matter lesions, and lower episodic memory performance. Taken together, these findings suggest that reduced cardiovascular health is associated with poorer status for brain variables that are central to age-sensitive cognitive functions, with emphasis on DA integrity.


The MicroRNA mir-83 Disrupts Autophagy in Aging Nematode Worms

Researchers here find a proximate cause of age-related impairments in autophagy in nematode worms. The cellular maintenance processes of autophagy, responsible for recycling unwanted and damaged molecules and structures, are well known to decline with age. This dysfunction contributes to numerous age-related conditions, particularly in tissues containing significant populations of very long-lived cells, in which the build up of damaged components becomes disruptive to function. Upregulation of autophagy, on the other hand, is a feature of many interventions shown to slow aging in laboratory species. In some cases, as for calorie restriction, autophagy is required for the beneficial effects on life span.

Macroautophagy, a key player in protein quality control, is proposed to be systematically impaired in distinct tissues and causes coordinated disruption of protein homeostasis and ageing throughout the body. Although tissue-specific changes in autophagy and ageing have been extensively explored, the mechanism underlying the inter-tissue regulation of autophagy with ageing is poorly understood. Here, we show that a secreted microRNA, mir-83, homologous to mammalian miR-29, controls the age-related decrease in macroautophagy across tissues in Caenorhabditis elegans.

Upregulated in the intestine by hsf-1 with age, mir-83 is transported across tissues potentially via extracellular vesicles and disrupts macroautophagy by suppressing CUP-5, a vital autophagy regulator, autonomously in the intestine as well as non-autonomously in body wall muscle. Mutating mir-83 thereby enhances macroautophagy in different tissues, promoting protein homeostasis and longevity.

Our results not only show that a secreted microRNA is an inter-tissue messenger controlling autophagy for protein homeostasis but also indicate that tissues other than the nervous system (e.g., the intestine) broadcast signals for protein homeostasis throughout the body. Similarly, transcellular chaperone signaling from muscle to intestine and neurons is important in the response against proteotoxic stress.


TRIB1 Regulates Uptake of Oxidized Lipids into Macrophages, and thus Drives Atherosclerosis

Atherosclerosis is a condition of dysfunctional macrophages. Macrophages are responsible for clearing out lipids that end up in blood vessel walls, ingesting these misplaced lipid molecules and handing them off to HDL particles to be carried to the liver for excretion. This works just fine in youth, in an environment of low oxidative stress and few oxidized lipids. Aging brings chronic inflammation, oxidative stress, and oxidized lipids, however. Macrophages cannot process oxidized lipids all that well, and so become pathological, turning into inflammatory foam cells packed with lipids, and unable to do more than send signals for help. The plaques that form to narrow and weaken blood vessels in atherosclerosis might start out as lipid deposits, but become macrophage graveyards as they grow, as ever more macrophages arrive to try and fail to clear the damage.

A number of new approaches to atherosclerosis based on interfering in this process are under development. My company, Repair Biotechnologies, works on a way to allow macrophages to break down oxidized cholesterol in situ. Underdog Pharmaceuticals works on sequestration of the worst oxidized lipid, 7-ketocholesterol. And so forth. The work here, in which researchers identify the gene TRIB1 as a regulator of macrophage uptake of oxidized lipids, offers a new avenue of attack. The evidence presented is fairly compelling for some form of inhibition of TRIB1 to be the basis for a therapy. If this could be done for an extended period of time, then in principle atherosclerosis could be reversed, as macrophages become able to go about their duties and meaningfully clean up atherosclerotic lesions.

Sheffield scientists identify new potential treatment pathway for cardiovascular disease

Research has shown for the first time that a protein expressed in a subset of immune cells contributes towards the build-up of fatty deposits in arteries, which leads to cardiovascular disease. These fatty deposits are caused by macrophages, a subset of immune cells known to take up surplus cholesterol. When this is present in excess, they mature into larger cholesterol-laden cells known as foam cells which accumulate and cause blockages inside arteries. The study shows for the first time that levels of a protein called Tribbles-1 (TRIB1) inside macrophages controls the amount of oxidized cholesterol taken up by foam cells. The research shows that higher levels of TRIB1 increased specific cholesterol uptake receptors, promoting arterial disease, whereas decreasing TRIB1 reduced disease. The findings of this study suggest that inhibiting TRIB1 in macrophages could be a viable therapeutic target in treating cardiovascular disease.

Myeloid Tribbles 1 induces early atherosclerosis via enhanced foam cell expansion

Atherosclerosis, a progressive disease of arterial blood vessels and the main underlying cause of stroke, myocardial infarction, and cardiac death, is initiated by the conversion of plaque macrophages to cholesterol-laden foam cells in the arterial intima. In the early-stage atherosclerotic plaque, this transformation is induced by the uptake of both low density lipoprotein-cholesterol (LDL-C) and oxidized LDL (oxLDL), which may serve a beneficial purpose; but unrestrained, the crucial function of plaque macrophages in resolving local inflammation is compromised, and the development of unstable, advanced lesions ensues.

Tribbles 1 (Trib1) has been detected in murine plaque-resident macrophages, and variants at the TRIB1 locus have been associated with increased risk of hyperlipidemia and atherosclerotic disease in multiple populations. However, no study had examined the macrophage-specific cellular processes dependent on myeloid-specific Trib1 expression and how these tally with the assumed atheroprotective properties of this pseudokinase. At the whole-body level, one study has shown that Trib1-deficient mice have markedly reduced numbers of M2-like macrophages in multiple organs, including adipose tissue. Hence, these studies strongly implicated that loss of macrophage-Trib1 expression within the arterial wall would lead to excessive atherosclerotic plaque inflammation and/or impair inflammation resolution and promote atheroma formation.

In the current study, we found that contrary to expectations, myeloid-specific knockout of Trib1 is atheroprotective, while myeloid-specific Trib1 expression is detrimental. In brief, Trib1 increased OLR1 RNA and protein expression, oxLDL uptake, foamy macrophage formation, and atherosclerotic burden in two distinct mouse models of human disease. The expression of these two genes, as well as those of LPL and SCARB1 (which mediates selective HDL-cholesterol uptake), is also tightly linked in human macrophages. Collectively, our studies reveal an unexpected beneficial effect for selectively silencing Trib1 in arterial plaque macrophages.

Reviewing Leucine Supplementation as a Treatment for Sarcopenia

Sarcopenia is the name given to the characteristic loss of muscle mass and strength that takes place with advancing age. A surprisingly large fraction of this loss is self inflicted: few people undertake the necessary exercise and strength training to maintain muscle in later life. But the rest of the losses are to some degree inevitable, a consequence of damage and disarray in muscle stem cells, neuromuscular junctions, and various processes necessary to muscle tissue maintenance. There is evidence for one those issues to be a growing inability to process leucine, an essential amino acid. Leucine supplementation may thus slow the onset of sarcopenia, even while being a compensatory approach that in no way addresses the underlying causes of this form of age-related degeneration.

One of the main ways in which sarcopenia contributes to disease is that it alters muscular turnover and metabolism. Moreover, older adults exhibit a decreased anabolic response to protein feeding, which is a mechanism underpinning the loss of muscle mass in sarcopenic individuals. Compared to younger adults, those aged over 65 years require ∼70% more protein per meal to maximally stimulate muscle protein synthesis. Furthermore, at a global level, only 40% of older adults meet the recommended daily allowance for protein (0.8 g/kg/day) and 10% of older women do not even meet the estimated average requirement of 0.66 g/kg/day.

One strategy to increase the muscle protein synthesis that has been investigated is the supplementation of diets with leucine, an essential branched-chain amino acid with important regulatory actions in muscles, which are at least partially mediated by the mammalian target of the rapamycin pathway. Leucine modifies protein turnover in skeletal muscles, by decreasing proteolysis and by increasing protein synthesis. Physiological research reports have shown that leucine can enhance muscle protein-synthesis. Furthermore, leucine can stimulate insulin release by pancreatic cells, showing that besides its beneficial effect in enhancing skeletal muscle glucose uptake, it is also an important anabolic signal in skeletal muscle.

Based on the above, administration of leucine-containing supplements is therefore a promising approach for treating sarcopenia. We took a systematic approach to analysing the current scientific evidence in this area, and to ascertaining whether the administration of leucine-containing supplements is effective in the treatment of sarcopenia. We also included interventions that used whey protein as a supplement, because these contain large amounts of leucine (approximately 13 g leucine/100 g protein) and the consumption of whey protein appears to be the most effective at increasing muscle protein synthesis.

In overall terms, published study results show that administration of leucine or leucine-enriched proteins (in a range of 1.2 g to 6 g leucine/day) is well-tolerated and significantly improves sarcopenia in elderly individuals, mainly by improving lean muscle-mass content and in this case most protocols also include vitamin D co-administration. The effect of muscular strength showed mixed results, however, and the effect on physical performance has seldom been studied.


3-D Printing of Skin with Embedded Vasculature

Researchers continue to take incremental steps towards the creation of engineered living tissues containing the vascular networks needed to support it. Absent blood vessels, numerous varieties of functional tissue can be generated from cell samples: lung, liver, kidney, and so forth. These organoids are limited in size to a few millimeters, however, the distance the nutrients and oxygen can perfuse. Generating blood vessel networks is a serious technical challenge, and the major obstacle to the production of entire organs for transplantation. Consider that natural capillary networks exhibit a density of hundreds of vessels passing through every square millimeter of tissue cross-section. Even the best of present efforts are distant from that scale, though in laboratory demonstrations they suffice to produce essentially functional larger tissue sections.

Researchers have developed a way to 3D print living skin, complete with blood vessels. The advancement is a significant step toward creating grafts that are more like the skin our bodies produce naturally. "Right now, whatever is available as a clinical product is more like a fancy Band-Aid. It provides some accelerated wound healing, but eventually it just falls off; it never really integrates with the host cells."

A significant barrier to that integration has been the absence of a functioning vascular system in the skin grafts. Researchers have been working on this challenge for several years, previously showing that they could take two types of living human cells, make them into "bio-inks," and print them into a skin-like structure. Researchers now show that if they add key elements - including human endothelial cells, which line the inside of blood vessels, and human pericyte cells, which wrap around the endothelial cells - with animal collagen and other structural cells typically found in a skin graft, the cells start communicating and forming a biologically relevant vascular structure within the span of a few weeks.

"As engineers working to recreate biology, we've always appreciated and been aware of the fact that biology is far more complex than the simple systems we make in the lab. We were pleasantly surprised to find that, once we start approaching that complexity, biology takes over and starts getting closer and closer to what exists in nature." Once the team grafted the engineered tissue onto a mouse, the vessels from the printed skin began to communicate and connect with the mouse's own vessels. In order to make this usable at a clinical level, researchers need to be able to edit the donor cells using something like the CRISPR technology, so that the vessels can integrate and be accepted by the patient's body. "We are still not at that step, but we are one step closer,."


Cardiomyocytes Expressing SOX10 are Vital to Zebrafish Heart Regeneration

A few higher animal species, such as salamanders and zebrafish, are capable of regeneration of limbs and internal organs, regrowing lost and injured tissue without scarring or loss of function. Numerous research groups are engaged in investigating the biochemistry of proficient regeneration, attempting to find the specific differences between species that might explain how it happens and why adult mammals are largely incapable of such feats of regrowth. Today's open access research is an example of the type, in which the authors narrow down on a specific cell population that appear in zebrafish hearts during regeneration, but not in human tissues.

It may be the case that the mechanisms and capacity for adult regeneration do still exist in mammals, but are suppressed in some way, as suggested by the fact that the human ARF gene can shut down zebrafish regeneration. After all, we all managed to undertake the process of growing organ tissue during embryonic development. Alternatively perhaps a single crucial part of the adult regeneration mechanisms was lost over evolutionary time, and thus there is an opportunity to reinsert it into mammalian tissues via gene therapy or some other form of modern biotechnology. It still remains to be seen as to whether there are simple paths towards enabling greater adult mammalian regeneration, or, as seems equally likely, the situation is a complex mess that will take decades to decipher, and offers no easy path to therapy.

Special cells contribute to regenerate the heart in Zebrafish

In mammals, including humans, the heart muscle has a very limited capacity to recover after injury. After an acute myocardial infarction, millions of cardiac muscle cells, named cardiomyocytes, die, and are replaced by a scar. Unlike mammals, other vertebrates can recover much better from a cardiac damage. This is the case of some fish, including the zebrafish, a well-established animal model in biomedical research which shares with humans most of its genes.

Zebrafish are extremely well suited to study organ regeneration. After heart injury, zebrafish cardiomyocytes can divide and the scar is replaced by new cardiac muscle. Now the researchers show that not all cardiomyocytes in the zebrafish heart contribute equally to regenerate the lost muscle, but that there is a specific subset of cardiomyocytes with enhanced regenerative capacity.

A small subset of cardiomyocytes in the zebrafish heart, marked by sox10 gene expression, expanded more than the rest of myocardial cells in response to injury. These cells differed from the rest of the myocardium also in their gene expression profile, suggesting that they represented a particular cell subset. Furthermore, experimental erasure of this small cell population, impaired heart regeneration. The researchers want to find out whether the absence of such a sox10 cell population in mammals could explain why their heart does not regenerate well. If this is the case, the researchers believe that this finding could be of great importance in stimulating the repair process in the human heart.

Adult sox10+ Cardiomyocytes Contribute to Myocardial Regeneration in the Zebrafish

Like mammals, zebrafish cardiomyocytes (CMs) derive from first and second heart field progenitors. However, in the zebrafish, the neural crest represents a third progenitor population that contributes to the developing heart. Cell transplantation and fluorescent dye tracing experiments suggested that cardiac neural crest cells incorporate not only into the areas of the outflow tract, as in mammals and birds, but also into the atrium and ventricle. Moreover, genetic lineage tracing using sox10 as a neural crest cell marker revealed a cellular contribution of sox10+ cells to the zebrafish heart and suggested that sox10-derived CMs are necessary for correct heart development. Noteworthy, it is still unclear if a sox10+ neural crest population differentiates into CMs or if alternatively, a sox10+ CM subset is relevant for heart development.

Here, we assessed the contribution of sox10-derived cells to the adult zebrafish heart. We found that embryonic sox10-derived cells contributed to significant portions of the adult heart. We also identified adult sox10+ CMs that expanded to a higher degree upon injury than other CMs and significantly contributed to cardiac regeneration. Their transcriptome differed from other CMs in the heart, and their genetic ablation impaired recovery from ventricular injury.

Extracellular Vesicles from Embryonic Stem Cells Make Mesenchymal Stem Cells More Effective in Therapy

Mesenchymal stem cell (MSC) is a category so broad as to be near meaningless, but many varieties are widely used for therapeutic purposes. MSCs are taken from any one of a variety of sources, expanded in culture, and introduced to the patient. Researchers here show that applying extracellular vesicles from embryonic stem cells to the cultured MSCs reduces the usual issues that arise when expanding cells in culture, such as senescence, and improves the effectiveness of MSCs as a therapy when tested in mice. On a practical basis, one would imagine that induced pluripotent stem cells would serve just as well as a source of extracellular vesicles with this capability.

Mesenchymal stem cells (MSCs), derived from several kinds of tissues such as placenta, umbilical cord, bone marrow, and adipose tissue, are multipotent stem cells that can differentiate into many cell types. MSCs have been recognized as important candidates for the treatment of many degenerative diseases or injuries. Furthermore, MSCs can be expanded by continuously passage in vitro, to obtain a sufficient number of cells that can be used for clinical applications. Along with the continuous passage in vitro, MSCs exhibit the senescence-associated features, including enlarged morphology, irreversible growth arrest, enhanced SA-β-gal activity, decreased stemness of stem cells, increased cell apoptosis and DNA damage foci, and telomere attrition.

For senescent MSCs, the characteristics of stem cell are lost, and their therapeutic effects are limited. Therefore, researchers attempt to find a better way to block the cellular senescence. Mouse embryonic stem (ES) cells, derived from the blastocyst stage embryos, are distinguished by their ability to self-renew and differentiate into all cell types. The major barriers to the possible transplantation of ES cells into patients are immune rejection and the risk of forming tumors. It has been reported that conditioned medium from ES cells (ES-CM) has beneficial effects on cell proliferation and tissue regeneration via the factors secreted from ES cells.

Recently studies suggested that extracellular vesicles (EVs), which are biological particles released by many cell types, could be considered for therapeutic utility. The EVs transfer proteins and nucleic acids between cells and play an important role in the target cells. Moreover, EVs isolated from various types of stem cells have different properties such as anti-apoptosis, pro-angiogenesis, and anti-fibrosis. In this study, we explored the effects of EVs derived from ES cells (ES-EVs) on the senescent MSCs. Our results indicated that ES-EVs rejuvenated the senescent MSCs and enhanced their therapeutic effects in a mouse cutaneous wound model.


Success for the MitoMouse Crowdfunding Project

The latest mitochondrial rejuvenation research project to be crowdfunded by the and SENS Research Foundation teams focused on proving out allotopic expression of mitochondrial gene ATP8 in mice with a loss of function mutation in that gene. I'm pleased to note that the community rallied around and the project was fully funded, including its stretch goals.

Mitochondria have their own small genome; allotopic expression is the process of placing a copy of a mitochondrial gene into the nuclear genome, suitably altered to enable the proteins produced to find their way back to the mitochondria where they are needed. This backup source of proteins allows mitochondria to function normally even when their own DNA is damaged. The technique, when applied to single genes, allows for the treatment of inherited mitochondrial conditions, as demonstrated by Gensight Biologics. More importantly, however, when applied to all thirteen mitochondrial genes it will prevent mitochondrial DNA damage from contributing to the aging process.

The MitoMouse campaign has ended, and what a final few days it has been! Thanks to the efforts of the community, an amazing total of $77,525 has been raised in support of this mitochondrial repair project of the SENS Research Foundation. There were 319 people who backed the project and helped to make this the most successful fundraiser on to date, even higher than the previous record breaker, the NAD+ mouse project. This is very impressive and shows that support for the field is growing and that the tide has really turned.

We would like to give special thanks to, which generously stepped in once we reached the first stretch goal and agreed to fully fund the project all the way to the second stretch goal! Thanks to LongeCity, the project not only hit the final stretch goal, which greatly expanded the scope of the project, the total funds raised went well over that goal. We are confident that the extra money will be put to good use by the MitoMouse team, and a few more boxes of mouse food and laboratory supplies are sure to come in handy.

Big thanks to everyone who supported the project, including the team at SENS Research Foundation, John Saunders, the Foster Foundation, Patrick Deane, and the volunteers and staff at LEAF for pulling together to make this happen. Hopefully, MitoMouse will enjoy the same success as the previous MitoSENS project, and we will be one step closer to having a solution to mitochondrial damage and potential cures for inherited mitochondrial conditions as well as age-related diseases.


GDF11 as a Calorie Restriction Mimetic

GDF11 was one of the first factors in blood identified as a possible explanation for the outcome of heterochronic parabiosis. When a young and old mouse have their circulatory systems joined, some aspects of aging reverse in the old mouse, and some aspects of aging are accelerated in the young mouse. GDF11 levels decline with age, and it was thought that increased levels of GDF11 provided by the young animal could act to improve function of cells and tissues in the older animal - though it was not well understood as to how GDF11 worked to produce these results.

Since then there has been some debate over whether or not the original GDF11 research was technically correct, as well as some debate over whether or not factors in young blood are in fact responsible for parabiosis effects. Researchers have demonstrated benefits in old mice by delivery of GDF11 as a treatment, however. A company, Elevian, was founded to carry forward the development of GDF11 as basis for clinical therapy.

Meanwhile, research into GDF11 and aging continues elsewhere in the scientific community. Today's very interesting open access research provides evidence for GDF11 to produce benefits in large part through triggering many of the same mechanisms as calorie restriction. If this is the primary mechanism of action, it would make GDF11 much less interesting as a basis for human therapy. Firstly because calorie restriction already exists, and is essentially free, and secondly because the practice of calorie restriction produces much larger effects on life span in short-lived species than it does in long-lived species.

A blood factor involved in weight loss and aging

In a previous study using mouse models, scientists observed that injecting aged mice with blood from young mice rejuvenated blood vessels in the brain, and consequently improved cerebral blood flow, while increasing neurogenesis and cognition. Scientists put forward the theory that, since calorie restriction and supplementation with young blood were effective in rejuvenating organs, they most likely have certain mechanisms in common.

They therefore examined the molecule GDF11, which belongs to the GDF (Growth Differentiation Factor) protein family and is involved in embryonic development. GDF11 was already known to scientists for its ability to rejuvenate the aged brain. By injecting this molecule into aged mouse models, researchers noticed an increase in neurogenesis and blood vessel remodeling. The scientists also observed that the mice administered with GDF11 had lost weight without changing their appetite. This observation led them to believe that GDF11 could be a link between calorie restriction and the regenerating effects of young blood.

The next step was to confirm this theory by studying adiponectin, a hormone secreted by adipose tissue which induces weight loss without affecting appetite. In animals that have undergone calorie restriction, the blood levels of this hormone are high. In animals that were administered GDF11, researchers also observed high levels of adiponectin, and this shows that GDF11 causes metabolic changes similar to those induced by calorie restriction. Until recently, there has been controversy over the role of GDF11 in aging, and its mechanisms were largely unknown. The findings of this study show that by inducing phenomena similar to those reported for calorie restriction leading to the stimulation of adiponectin and neurogenesis, GDF11 contributes to the birth of new neurons in the brain.

Systemic GDF11 stimulates the secretion of adiponectin and induces a calorie restriction-like phenotype in aged mice

Here, we present evidence that GDF11 induces a healthy calorie restriction-like phenotype together with brain rejuvenation in aged mice, and it acts by stimulating the secretion of adiponectin directly on adipocytes. We demonstrate a potent role for GDF11 as a metabolic actor in the aged organism based on the following findings: (a) systemic administration of GDF11 induced healthy weight loss as early as 1 week after treatment, (b) this weight loss reached a plateau throughout the rest of the treatment and was maintained for 3 weeks beyond the end of the treatment, (c) GDF11 levels were increased in aged mice that were subjected to calorie restriction, (d) metabolic changes were independent of GDF15 activation or anorexia, but correlated with changes in adiponectin levels and the insulin/IGF-1 metabolic pathway, (e) GDF11 activated adiponectin secretion directly from adipocytes, and (f) all the above changes correlated with a brain rejuvenation phenotype in aged mice.

Modulating Gut Microbe Populations to Generate More Butyrate, thus Raising BDNF Levels and Improving Cognitive Function

The microbial populations of the gut have an influence on health and the progression of aging via the molecules that they generate, and which our cells react to. It isn't entirely clear as to the ordering of cause and effect in the detrimental changes that take place with aging in intestinal tissue, immune system, diet, and microbial populations. Studies have shown, however, that restoring more youthful populations can influence the function of tissues throughout the body, including the brain. The authors of this open access paper discuss modulating gut microbial populations in rats so as to upregulate butyrate production and BDNF levels, thereby improving some aspects of cognitive function. Similar examples exist in the literature for a range of other organs and tissues; it is an interesting area of research, though ultimately the size of the effects are probably not all that different from those relating to exercise or diet.

Neuroinflammation is correlated with a decline in cognitive function and memory, primarily because inflammation of the hippocampus tends to cause deleterious changes in synaptic transmission and plasticity. Because BDNF helps to sustain and enhance long-term potentiation (LTP) induction, it serves an essential role in cognitive function. Aging is associated with decreased levels of BDNF, suggesting that the maintenance of adequate BDNF concentrations could potentially help to preclude or delay the onset of cognitive impairment.

One convenient way to raise BDNF levels is supplementation of butyrate, a short-chain fatty acid (SCFA) that functions as a histone deacetylase inhibitor. Butyrate maintains the relaxation of chromatin and thereby enhances BDNF expression in the hippocampus. Secretion of pro-inflammatory cytokines may also be inhibited by BDNF, as the latter molecule interferes with activation of nuclear factor-kappa beta (NF-κβ). In addition, the expression of enzymes involved in the production of glutathione (GSH) may also be triggered by butyrate secretion. GSH is an antioxidant enzyme that relieves oxidative stress - another neurodegenerative risk factor.

The intestinal microbiota is responsible for a significant proportion of SCFA production. However, levels of SCFA decline with age due to dysbiosis, a microbial imbalance that often results in a considerable increase in pathological bacteria (Proteobacterium) at the expense of mutualistic ones (Bifidobacterium). Progression of gut dysbiosis has been linked to chronic systemic inflammation, including inflammation of the brain. Supplementation with probiotics and prebiotics may counteract the damaging effects that aging has on the brain by not only lessening inflammation and oxidative stress but also by increasing neurotrophic factors and neuronal plasticity.

A study was conducted to test how probiotic and prebiotic supplementation impacted spatial and associative memory in middle-aged rats. The results showed that rats supplemented with the symbiotic (both probiotic and prebiotic) treatment performed significantly better than other groups in the spatial memory test, though not in that of associative memory. The data also showed that this improvement correlated with increased levels of BDNF, decreased levels of pro-inflammatory cytokines, and better electrophysiological outcomes in the hippocampi of the symbiotic group. Thus, the results indicated that the progression of cognitive impairment is indeed affected by changes in microbiota induced by probiotics and prebiotics.


HDAC9 in Vascular Calcification

Researchers here show that HDAC9 plays a role in the calcification of blood vessel walls, a process that contributes to the stiffening of blood vessels that leads to hypertension and all of the damage that chronic raised blood pressure causes to delicate tissues throughout the body. That mice lacking HDAC9 are more resistant to calcification suggests that there may be a mechanism here that can serve as the basis for a therapy to slow down the progression of calcification in human tissues. That said, it is worth comparing effort such as this with the potential for senolytic drugs to achieve similar results, based on the evidence for senescent cells to contribute to vascular calcification.

Arterial wall calcification is the buildup of calcium in the blood vessel walls, which can often be a predictor of serious cardiovascular events like heart attacks and strokes. A new study looked at more than 11,000 people and found patients with significant blood vessel calcification were more likely to have a specific variant of HDAC9. This high-risk variant of HDAC9 is present in about 25 percent of the population. In follow-up mouse studies, the researchers also found HDAC9 caused abnormal changes in the cells of the vessel walls, resembling that of human bone cells.

"Our research proved HDAC9 is not just associated with cardiovascular disease but can actually cause it by changing the makeup of those vascular cells. We then investigated it at the molecular level and looked at what would happen if we knocked out HDAC9." The researchers found that inhibiting HDAC9 in mice preserved normal function in vascular cells and prevented vascular calcification, therefore identifying HDAC9 as a target for the potential treatment of cardiovascular disease. "Currently, there are no heart drugs available to patients that would prevent this type of hardening of the arteries. These findings are exciting in that they harness genetics to open the door for future pathways to heart disease prevention."


Assessing Gene Therapy to Upregulate Three Longevity-Associated Genes in Mice

Today's open access research materials report on results obtained in mice using gene therapy to upregulates protein production of several longevity-associated genes. As expected from prior research into these genes and their influence on the operation of metabolism, health is improved in mouse models of age-related disease. As might also be expected based on past results, some combinations are not effective for reasons that remain to be explored: metabolism is complicated, and pulling on levers and turning dials rarely does exactly what was expected.

Evolution does not produce optimal organisms, as seen from the perspective of the individual. This is well illustrated in mice, where any number of single gene alterations, even just dialing up or down the amount of protein produced over time, leads to better health, less disease, longer lives. Why haven't any of these small alterations taken place via random mutation and thereafter prospered and spread through the species over the course of evolutionary time? Because evolutionary competition in the wild is a race to the bottom, in which lineages engineered for early life success at the cost of later life collapse tend to outcompete those with a biochemistry more friendly to the individual.

This applies as much to humans as it does to mice. There are variant human genes that offer much reduced risk of cardiovascular disease, found in a small fraction of the population. Why don't we all have those variants? Because evolution doesn't place much emphasis on late life health and survival. Further, many of the alterations known to improve health and longevity in mice should be expected to at least improve health in humans. So at some point in the years ahead, the use of gene therapies to improve human metabolism so as to reduce age-related disease and improve longevity will be a going concern. It will start with therapies for adults that don't integrate with the genome and are not passed on to children, and at some point, once the will is there, the medical community will start to engineer better human lineages.

This class of approach most likely won't be the most important road to increased longevity in the near future, however. The gains that can be achieved through periodic repair of the human biochemistry that we have today should be far greater than those produced by engineering an improved biochemistry that is more resilient to damage. We know that a youthful mammalian biochemistry works pretty well, and the differences between youth and age emerge from forms of cell and tissue damage, such as accumulation of senescent cells and persistent metabolic waste products. To the degree that the research community can repair that damage, rejuvenation will be the outcome. Yes, that repair may take the form of gene therapies, such as to deliver novel enzymes capable of breaking down metabolic waste, but this is a very different approach in comparison to the type of gene therapy tested in the research noted here, which is an attempt to shift the operation of cellular metabolism into a more resilient state, not to repair damage.

Combination gene therapy treats multiple age-related diseases in mice

Researchers honed in on three genes that had previously been shown to confer increased health and lifespan benefits when their expression was modified in genetically engineered mice: FGF21, sTGFβR2, and αKlotho. They hypothesized that providing extra copies of those genes to non-engineered mice via gene therapy would similarly combat age-related diseases and confer health benefits. The team created separate gene therapy constructs for each gene using the AAV8 serotype as a delivery vehicle, and injected them into mouse models of obesity, type II diabetes, heart failure, and renal failure both individually and in combination with the other genes to see if there was a synergistic beneficial effect.

FGF21 alone caused complete reversal of weight gain and type II diabetes in obese, diabetic mice following a single gene therapy administration, and its combination with sTGFβR2 reduced kidney atrophy by 75% in mice with renal fibrosis. Heart function in mice with heart failure improved by 58% when they were given sTGFβR2 alone or in combination with either of the other two genes, showing that a combined therapeutic treatment of FGF21 and sTGFβR2 could successfully treat all four age-related conditions, therefore improving health and survival. Administering all three genes together resulted in slightly worse outcomes, likely from an adverse interaction between FGF21 and αKlotho, which remains to be studied.

A single combination gene therapy treats multiple age-related diseases

Comorbidity is common as age increases, and currently prescribed treatments often ignore the interconnectedness of the involved age-related diseases. The presence of any one such disease usually increases the risk of having others, and new approaches will be more effective at increasing an individual's health span by taking this systems-level view into account. In this study, we developed gene therapies based on 3 longevity associated genes: fibroblast growth factor 21 (FGF21), αKlotho, soluble form of mouse transforming growth factor-β receptor 2 (sTGFβR2). The gene therapies were delivered using adeno-associated viruses, and we explored their ability to mitigate 4 age-related diseases: obesity, type II diabetes, heart failure, and renal failure.

Individually and combinatorially, we applied these therapies to disease-specific mouse models and found that this set of diverse pathologies could be effectively treated and in some cases, even reversed with a single dose. We observed a 58% increase in heart function in ascending aortic constriction ensuing heart failure, a 38% reduction in α-smooth muscle actin (αSMA) expression, and a 75% reduction in renal medullary atrophy in mice subjected to unilateral ureteral obstruction and a complete reversal of obesity and diabetes phenotypes in mice fed a constant high-fat diet. Crucially, we discovered that a single formulation combining 2 separate therapies into 1 was able to treat all 4 diseases. These results emphasize the promise of gene therapy for treating diverse age-related ailments and demonstrate the potential of combination gene therapy that may improve health span and longevity by addressing multiple diseases at once.

Towards a Rigorous Definition of Cellular Senescence

The accumulation of lingering senescent cells is a significant cause of aging, disrupting tissue function and generating chronic inflammation throughout the body. Even while the first senolytic drugs capable of selectively destroying these cells already exist, and while a number of biotech companies are working on the production of rejuvenation therapies based on clearance of senescent cells, it is still the case that much remains to be settled when it comes to the biochemistry of these errant cells. More rigorous consensus definitions relating to the mechanisms, manifestations, and tissue specificity of senescence have yet to be pinned down. This process will proceed in parallel with the development of more effective therapies, as is often the case.

Cellular senescence is a cell state implicated in various physiological processes and a wide spectrum of age-related diseases. Recently, interest in therapeutically targeting senescence to improve healthy aging and age-related disease, otherwise known as senotherapy, has been growing rapidly. Thus, the accurate detection of senescent cells, especially in vivo, is essential. Here, we present a consensus from the International Cell Senescence Association (ICSA), defining and discussing key cellular and molecular features of senescence and offering recommendations on how to use them as biomarkers.

Over the last decade, improved experimental tools have significantly advanced our knowledge about causes and phenotypic consequences of senescent cells. However, specific markers and a consensus on the definition of what constitutes senescent cells are lacking. Further, although a link to organismal aging is clear, aging and senescence are not synonymous. Indeed, cells can undergo senescence, regardless of organismal age, due to myriad signals including those independent of telomere shortening. Consequently, senescent cells are detected at any life stage from embryogenesis, where they contribute to tissue development, to adulthood, where they prevent the propagation of damaged cells and contribute to tissue repair and tumor suppression. Thus, cellular senescence might be an example of evolutionary antagonistic pleiotropy or a cellular program with beneficial and detrimental effects.

Cellular senescence is a cell state triggered by stressful insults and certain physiological processes, characterized by a prolonged and generally irreversible cell-cycle arrest with secretory features, macromolecular damage, and altered metabolism. Senescent cells secrete a plethora of factors, including pro-inflammatory cytokines and chemokines, growth modulators, angiogenic factors, and matrix metalloproteinases (MMPs), collectively termed the senescent associated secretory phenotype (SASP) or senescence messaging secretome (SMS). The SASP constitutes a hallmark of senescent cells and mediates many of their patho-physiological effects. The SASP composition and strength varies substantially, depending on the duration of senescence, origin of the pro-senescence stimulus, and cell type.

The role of senescence in human disease is clear from cellular studies, while in vivo evidence is only now catching up. Evidence linking senescence to other common age-associated human diseases has recently emerged. These diseases include neurodegenerative disorders, glaucoma, cataract, atherosclerosis and cardiovascular disease, diabetes, osteoarthritis, pulmonary, and renal, and liver fibrosis. In most studies, senescence is assessed in ex vivo cultures or fresh samples by SA-β-gal staining or indirect markers in formalin-fixed tissues. Since SA-β-gal is not suitable for fixed tissues, analyzing senescence in human samples is challenging. Despite promising results from other individual markers, no marker is completely senescence specific. We recommend combining cytoplasmic (e.g., SA-β-gal, lipofuscin), nuclear (e.g., p16INK4A, p21WAF1/Cip1, Ki67) and SASP, context, and/or cell-type-specific markers.


An Investigation of Adverse Effects of Nicotinamide Riboside Supplementation

Nicotinamide riboside is so far the only approach to NAD+ upregulation for which there is published human trial data, though other trials for other approaches are underway at the present time. NAD+ declines with age, for reasons that remain comparatively poorly understood, and this has a negative impact on mitochondrial function. Thus there is considerable enthusiasm at the moment for intervening in this known manifestation of aging by tackling the proximate causes, raising NAD+ levels, but without addressing underlying causes.

Researchers here find the potential for adverse effects on glucose metabolism and white adipose tissue function to result from nicotinamide riboside supplementation, but there are a great many details involved: dietary differences and genetic differences in mice appear important as to whether problems arise, and the final sections of the discussion in the paper are worth reading closely. It is hard to say whether or not the discoveries made in mice that are reported in this open access paper will apply to humans, but the specific details suggest that investigation is warranted.

Nicotinamide riboside (NR) is a nicotinamide adenine dinucleotide (NAD+) precursor vitamin. The scarce reports on the adverse effects on metabolic health of supplementation with high-dose NR warrant substantiation. Here, we aimed to examine the physiological responses to high-dose NR supplementation in the context of a mildly obesogenic diet and to substantiate this with molecular data. An 18-week dietary intervention was conducted in male C57BL/6JRccHsd mice, in which a diet with 9000 mg NR per kg diet (high NR) was compared to a diet with NR at the recommended vitamin B3 level (control NR). Both diets were mildly obesogenic (40 en% fat). Metabolic flexibility and glucose tolerance were analyzed and immunoblotting, qRT-PCR, and histology of epididymal white adipose tissue (eWAT) were performed.

Mice fed with high NR showed a reduced metabolic flexibility, a lower glucose clearance rate and aggravated systemic insulin resistance. This was consistent with molecular and morphological changes in eWAT, including sirtuin 1 (SIRT1)-mediated PPARγ (proliferator-activated receptor γ) repression, downregulated AKT/glucose transporter type 4 (GLUT4) signaling, an increased number of crown-like structures and macrophages, and an upregulation of pro-inflammatory gene markers. In conclusion, high-dose NR induces the onset of WAT dysfunction, which may in part explain the deterioration of metabolic health.


Support Rejuvenation Research by Donating to the SENS Research Foundation Winter 2019 Fundraiser

Winter is upon us, and thus the yearly winter SENS Research Foundation fundraiser just recently started a few days ago. For those who don't know, the SENS Research Foundation remains one of the most important and influential organizations working on advancing rejuvenation research to the point at which it can be moved into clinical development and funded by venture capital. Through conferences and advocacy, the SENS Research Foundation staff also played a sizable role in ensuring that there is in fact an active venture capital community eager to back new companies working on the treatment of aging.

All of this work is powered by charitable donations. The SENS Research Foundation is near entirely funded by philanthropy, by the donations made by our community of visionaries, looking forward to a future in which the causes of aging can be treated effectively, and the course of aging turned back. Over the past six years, our community has provided more than $7 million to the SENS Research Foundation to advance the cause of rejuvenation research.

That funding has paid off. Ever more of the rejuvenation research programs funded, advocates, coordinated, and otherwise supported by the SENS Research Foundation have made the leap to commercial development. In 2016 the results of a decade of work on ways to clear out harmful metabolic waste from the lysosome, all funded by philanthropic donations, materialized in the form of LysoClear. That company is developing an enzyme therapy to break down forms of metabolic waste in the retina that cause age-related blindness, and as of this year is close to entering the FDA IND process. Also as of this year, SENS Research Foundation funded research programs have led to companies such as Revel Pharmaceuticals to tackle glucosepane cross-link breaking, Covalent Bioscience to break down transthyretin amyloid via the use of catabodies, and Underdog Pharmaceuticals to prevent and reverse atherosclerosis by removing 7-ketocholesterol from the body.

Consider also that thanks in part to fifteen years of advocacy and support on the part of the SENS Research Foundation and its predecessor, the Methuselah Foundation, clearance of senescent cells as a rejuvenation therapy has finally moved from being ignored by the research community to being a hot area of development in just the past few years. The SENS Research Foundation funded one of the laboratories working on cellular senescence for a number of years, and in 2015 helped to launch Oisin Biotechnologies, a company working on a best of class therapy to remove these unwanted cells.

This is the sort of result we aim for when we support the SENS Research Foundation: to see previously languishing fields relevant to rejuvenation therapies take off in this manner, gaining large-scale funding and widespread support in a short period of time once the tipping point is reached. Further, all of this progress has spurred many other groups to independently work on tackling causes of aging. Take a look at the Aging Biotech Info resource for a list of companies in the longevity industry, a sizable fraction of which are addressing aging in ways that are informed by the SENS viewpoint of rejuvenation achieved via repair of underlying molecular damage. Growth continues.

Yet there are still numerous forms of molecular damage underlying aging for which research programs have yet to fully emerge from the laboratory, or for which the best approaches to rejuvenation are underdeveloped. There is still a huge role for philanthropy and for organizations like the SENS Research Foundation to ensure that the development required for success in the treatment of aging is in fact carried out to completion. We can all see the young and growing senolytics industry as a success story, but there are half a dozen or more other industries needed in order to comprehensively address the causes of aging. These industries do not yet exist in any meaningful way, and cannot exist until the research is carried out.

The future of a truly effective longevity industry still depends on present philanthropy. Support the SENS Research Foundation in their 2019 winter fundraiser, and in doing so you will help to produce the rejuvenation biotechnology success stories of the years ahead.

Researchers Call for Rigorous Classifications of Aging to Assist Development of Therapies to Treat Aging

The first rejuvenation therapies exist, in the form of senolytic drugs that selectively destroy senescent cells, but no regulatory bodies yet recognize aging as a legitimate target for therapy. A variety of efforts are underway to change this state of affairs, many of which focus on the contents of the International Classification of Diseases (ICD) maintained by the World Health Organization (WHO), which is presently in the process of revision to ICD-11. Incorporating aging into the ICD in a rigorous way would lead, in time, to medical service providers and regulatory bodies worldwide adopting the concept of aging as a condition that can be treated.

Beyond updating the ICD to include aging, other initiatives include the TAME metformin trial, which is an attempt to get the FDA to agree upon trial endpoints that are close enough to aging for practical purposes. Other factions, and I fall into this camp, are of the opinion that real progress will emerge from battles with the regulators over widespread off-label use of cheap rejuvenation therapies that are approved for other conditions, with senolytic treatments such as the dasatinib and quercetin combination leading the way there.

An international team of researchers has put forward a call to action to governments, the WHO, and the scientific and medical communities to come together and develop classifications and staging systems utilizing the framework as the basis for diagnosing and treating age-related diseases, including directly treating all aging tissues and organs. Age-related diseases without adequate diagnostic criteria and severity staging limit the ability for prevention or treatment and the ability to develop new drugs and interventions. Ultimately, this impacts the quality of life for older members of society.

Currently the classification and severity staging of age-related diseases is limited because it is inconsistent, incomplete, and non-systematic. Some types of disease that can be found in many organs, such as intrinsic organ aging, or organ atrophy or wasting, are classified in one organ but not others. Experts, scientists, and physicians have created a position statement which lays out a framework for properly and comprehensively classifying and staging the severity of age-related diseases. Importantly the statement includes aging at the tissue and organ level as well as organ atrophy, pathologic remodelling and calcification, and age-related systemic and metabolic diseases.

While aging is classified as a condition within the WHO International Classification of Diseases (ICD-11) in relation to intrinsic skin aging and photoaging, the framework proposes the classification of aging as conditions in all organs, along with the comprehensive classification of all aging-related diseases and syndromes. As part of this work, initial classification submissions related to age-related diseases in line with the framework have already been submitted to the latest version of WHO ICD-11.


Exercise is a Benefit at Any Age

In our modern societies of convenience and luxury, and the overwhelming majority of human societies today are exceedingly convenient and luxurious in comparison to the hunter-gatherer existence of our ancestors, it is the case that most people do not exercise to the degree needed to maintain function with advancing age. If exercise is seen to produce benefits in near all patients of any later age, and it is, that is the case because those patients are not maintaining themselves sufficiently.

Elderly patients are at a higher risk for complications and accelerated physical deconditioning after a cardiovascular event, yet older patients are largely underrepresented in rehabilitation programs. Studies have shown that this might be due to a lack of referral and encouragement to attend cardiac rehabilitation in older patients. Several studies have looked at the effects of cardiac rehabilitation in older adults. However, these data often focus on patients above the age of 65 with no distinction between old and very old patients and examine either physical or psychological outcomes but not both.

The goal of this study was to compare the effects of an exercise-based cardiac rehabilitation program on physical and psychological parameters in young, old, and very old patients. It also aimed to identify the features that best predicted cardiac rehabilitation outcome. Investigators examined 733 patients who completed a 25-session cardiac rehabilitation program. They were divided into three subgroups: less than 65 years old; between 65 and 80 years old; and 80 years or older. Physical and psychological variables such as scores of anxiety and depression were evaluated for all patients before and after cardiac rehabilitation.

Following the intervention, all patients experienced improvements. "We found a few weeks of exercise training not only significantly improved exercise capacity, but also decreased anxiety and depression. Patients with the greatest physical impairments at baseline benefited the most from exercise. Another interesting result was that patients younger than 65 who were very anxious before rehabilitation benefited the most from exercise training. A similar result was found for depressed patients older than 65. These improvements will surely have a great positive impact on patients' independence and quality of life and might help both clinicians and patients to realize how beneficial exercise rehabilitation can be."


Senescent Cells in Blind Mole Rats do not Exhibit the Senescence-Associated Secretory Phenotype

Naked mole-rats live as much as nine times longer than similarly sized rodent species. A short summary of what is known of their biochemistry is that they exhibit many of the molecular signs of aging found in other mammals, such as oxidative damage, presence of senescent cells, and so forth, but few to none of the consequences found in other mammals. Naked mole rats stay fit and healthy and physiologically youthful right up until very late life. The near relative species of blind mole-rat has many of the same characteristics, although it is less well studied than naked mole-rats at the present time.

The accumulation of senescent cells is an important contribution to the aging process, as shown by the ability of senolytic drugs to significantly reverse many aspects of aging and age-related disease via clearance of lingering senescent cells in aged tissues. So it was something of a mystery as to how naked mole-rats and blind mole-rats could simply ignore the presence of senescent cells and carry on regardless. Here, scientists determine that this is because blind mole-rat senescent cells do not secrete the potent mix of inflammatory and damaging molecules known as the senescence-associated secretory phenotype (SASP). It is this process that causes all the harms resulting from cellular senescence in mice and other mammals.

This raises interesting questions as to how senescent cells in naked mole-rats and blind mole-rats carry out their normal, transient, beneficial functions in cancer suppression and wound healing. Perhaps they simply do not participate in the same way. Certainly these species have a whole array of other extraordinarily efficient means of suppressing cancer. One of the reasons why naked mole-rats are so well studied is their near immunity to cancer; whether researchers can find mechanisms that can be turned into human cancer suppression therapies remains to be seen, however.

Downregulation of the inflammatory network in senescent fibroblasts and aging tissues of the long-lived and cancer-resistant subterranean wild rodent, Spalax

The blind mole rat (Spalax) is a wild, long-lived rodent that has evolved mechanisms to tolerate hypoxia and resist cancer. Previously, we demonstrated high DNA repair capacity and low DNA damage in Spalax fibroblasts following genotoxic stress compared with rats. Since the acquisition of senescence-associated secretory phenotype (SASP) is a consequence of persistent DNA damage, we investigated whether cellular senescence in Spalax is accompanied by an inflammatory response.

Spalax fibroblasts undergo replicative senescence and etoposide-induced senescence, evidenced by an increased activity of senescence-associated beta-galactosidase (SA-β-Gal), growth arrest, and overexpression of p21, p16, and p53 mRNAs. Yet, unlike mouse and human fibroblasts, senescent Spalax cells showed undetectable or decreased expression of the well-known SASP factors: interleukin-6 (IL6), IL8, IL1α, growth-related oncogene alpha (GROα), SerpinB2, and intercellular adhesion molecule (ICAM-1). Apparently, due to the efficient DNA repair in Spalax, senescent cells did not accumulate the DNA damage necessary for SASP activation.

Conversely, Spalax can maintain DNA integrity during replicative or moderate genotoxic stress and limit pro-inflammatory secretion. However, exposure to the conditioned medium of breast cancer cells MDA-MB-231 resulted in an increase in DNA damage, activation of the nuclear factor κB (NF-κB) through nuclear translocation, and expression of inflammatory mediators in RS Spalax cells. Evaluation of SASP in aging Spalax brain and intestine confirmed downregulation of inflammatory-related genes. These findings suggest a natural mechanism for alleviating the inflammatory response during cellular senescence and aging in Spalax, which can prevent age-related chronic inflammation supporting healthy aging and longevity.

Microglial Neuroinflammation as a Cause of Tau Aggregation

Chronic inflammation in the brain is an important aspect of all neurodegenerative conditions. In particular, as discussed in this open access paper, there is good evidence for inflammatory and dysfunctional (and senescent) microglia to drive the tau protein aggregation characteristic of late stage Alzheimer's disease. It remains to be seen as to how the research community will build on past years of research on this topic to develop therapies, but one of the best near term possibilities is the use of senolytic therapies such as the dasatinib and quercetin combination to selectively destroy senescent microglia in the aging brain.

Histopathological features of Alzheimer's disease (AD) are extracellular amyloid-β (Aβ) plaques and intracellular aggregation of hyperphosphorylated tau protein in a form of neurofibrillary tangles (NFTs). The disease is also characterized by loss of neurons and synapses and elevated levels of inflammatory factors. Neuroinflammation occurs in many neurodegenerative diseases, including AD. Several studies confirmed elevated levels of pro-inflammatory cytokines and stronger microglial activation during disease progression. Activated microglia have been found near NFT-bearing neurons. There is also a better correlation between numbers of activated microglia and NFT than between microglial cells' activation and amyloid plaques distribution.

Uncontrolled microglial response in the brain contributes to the progression of many neurodegenerative diseases and several lines of evidence suggest that inflammation may even precede the development of tau pathology in AD. Exosomes could be an important link between tau propagation and microglial activation. Reduction of microglial cells number and exosome synthesis inhibition reduces tau propagation. Further, phagocytosed tau seeds induce inflammasome activation inside microglia causing an overactive microglia state. That could be one of the mechanisms that promote the constant inflammatory response in AD.


Senescent Mesenchymal Stem Cells Contribute to Osteoarthritis

Cellular senescence is a significant contributing cause of osteoarthritis in old age, and senolytic therapies capable of selectively destroying senescent cells are presently undergoing clinical trials in osteoarthritic patients. Researchers here investigate a specific population of senescent cells, the supporting mesenchymal stem cells found in and around joint tissue, and establish that they are important in the progression of osteoarthritis.

Tissue accumulation of p16INK4a-positive senescent cells is associated with age-related disorders, such as osteoarthritis (OA). These cell-cycle arrested cells affect tissue function through a specific secretory phenotype. The links between OA onset and senescence remain poorly described. Using experimental OA protocol and transgenic mice, we found that the senescence-driving p16INK4a is a marker of the disease, expressed by the synovial tissue, but is also an actor: its somatic deletion partially protects against cartilage degeneration.

We test whether by becoming senescent, the mesenchymal stromal/stem cells (MSCs), found in the synovial tissue and sub-chondral bone marrow, can contribute to OA development. We established an in vitro p16INK4a-positive senescence model on human MSCs. Upon senescence induction, their intrinsic stem cell properties are altered. When co-cultured with OA chondrocytes, senescent MSC show also an impairment favoring tissue degeneration.

To evaluate in vivo the effects of p16INK4a-senescent MSC on healthy cartilage, we rely on the SAMP8 mouse model of accelerated senescence that develops spontaneous OA. MSCs isolated from these mice expressed p16INK4a. Intra-articular injection in 2-month-old C57BL/6JRj male mice of SAMP8-derived MSCs was sufficient to induce articular cartilage breakdown. Our findings reveal that senescent p16INK4a-positive MSCs contribute to joint alteration.