Induced Pluripotency as a Tool to Enable Rejuvenation of Blood Production

It has been a decade or so since the first induced pluripotent stem (iPS) cells were produced. Researchers discovered a recipe by which ordinary, limited, adult somatic cells could be reprogrammed into a state near identical to that of embryonic stem cells, meaning they are pluripotent and can then in principle be used to produce any of the cell types in the body. Doing so in practice requires researchers to establish a suitable methodology to guide cellular differentiation in the right direction, only accomplished at this point for a fraction of all possible cell types. The early attempts at induced pluripotency worked, and were easy to set up, but were also comparatively inefficient. Since then researchers have produced considerable improvement in the methodologies used, and along the way have explored other facets of this reprogramming process. One of the most intriguing aspects of induced pluripotency is that it appears to produce a form of cellular rejuvenation, a sweeping reset and repair of many forms of damage.

There are many open questions regarding this incompletely explored cellular rejuvenation achieved through induced pluripotency: how it works at the detail level; exactly which types of damage are repaired and which are not; how it relates to the equivalent process that occurs in the early development of the embryo. How do old gamete cells, laden with the molecular damage of aging, produce young offspring who lack that damage? Somewhere in there, rejuvenation happens. Is there any way to adapt this process of rejuvenation for use in therapies? It seems unwise to, for example, apply pluripotency reprogramming methods directly to a patient. This sounds a lot like opening the door to a high risk of uncontrolled cellular replication, or cancer in other words. Nonetheless, that experiment was recently carried out in mice, more or less, so we'll likely hear more about the risks in the years ahead. It is possible that such an approach will in the end fall into the same ballpark as stem cell therapies when it comes to overall degree of risk, though it is worth noting that, when performed improperly, stem cell therapies can also result in cancer, and considerable amount of work has gone into minimizing that outcome in those therapies that have made it to widespread clinical availability.

There are other possibilities when it comes to using the rejuvenation that occurs during the induced pluripotency process, however. Take a population of cells that are damaged and dysfunctional in an old individual, for example. Obtain a sample, create an induced pluripotent lineage from that sample, and then apply a suitable recipe to differentiate the pluripotent cells back into the original cell type. Do these recreated cells now behave as though they are younger, and can thus form the basis for a cell therapy to replace the old cells in the patient? Researchers here demonstrate that this is in fact the case for the stem cells responsible for generating blood:

How blood can be rejuvenated

When we are young, our blood stem cells produce an even and well-balanced number of red and white blood cells according to need. As we age, however, the capacity of the blood stem cells to produce the number of blood cells we need declines. "This type of age-related change can have major consequences as it can lead to an imbalance in stem cell production. For example, a reduced production of immune cells or excessive production of other types of cells can be a precursor to leukaemia."

A fundamental question was whether blood stem cells age differently within a single individual or whether all blood stem cells are equally affected by advancing age. In an initial stage, it was therefore important to genetically mark old blood stem cells, to enable the identification and tracking of those most affected by age. In the next step, these traceable cells were reprogrammed to another type of stem cell - known as iPS cells, which can generate all cells in an individual and not only blood cells. When the cells are reprogrammed, their identity is "re-set"; when these reprogrammed iPS cells formed new blood stem cells, the researchers observed that the re-set had entailed a rejuvenation of the cells. "We found that there was no difference in blood-generating capacity when we compared the reprogrammed blood stem cells with healthy blood stem cells from a young mouse. This is, as far as we know, the first time someone has directly succeeded in proving that it is possible to recreate the function of young stem cells from a functionally old cell.ˮ

The research team's studies have also thereby shown that many age-related changes in the blood system cannot be explained by mutations in the cells' DNA. If the changes depended on permanent damage at the DNA level, the damage would still be present after the re-set. Instead, epigenetic changes appear to underlie the decline in function associated with advancing age.

Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate

While age-related diseases evidently can arise due to changes that compromise or alter the function of mature effector cells, this is harder to reconcile with organs such as the blood, that rely on inherently short-lived effector cells in need of continuous replenishment. Rather, accumulating data have suggested that the de novo production of subclasses of haematopoietic cells shifts in an age-dependent manner, akin to that seen during more narrow time windows in early development. These findings have to a large extent also challenged the classically defining criteria of haematopoietic stem cells (HSCs) as a homogenous population of cells with differentiation capacity for all haematopoietic lineages. Rather, the differentiation capacity of HSCs might be more appropriately defined by a continuous multilineage haematopoietic output, but which might not necessarily include the production of all types of blood cells at all points in time.

The mechanisms that drive ageing at both the organismal and cellular level have attracted significant attention as they represent prime targets for intervention. An increased function of aged cells by 'young'-associated systemic factors has been proposed. Whether such approaches indeed reflect rejuvenation at a cellular level or rather stimulate cells less affected by age is mostly unclear. This concern applies also to previous studies approaching the prospects of reversing cellular ageing by somatic cell reprogramming, which have typically failed to distinguish between functionally versus merely chronologically aged cells.

Here we approach these issues by genetic barcoding of young and aged HSCs that allows for evaluations, at a clonal level, of their regenerative capacities following transplantation. This allows us to establish that ageing associates with a decrease of HSC clones with lymphoid potential and an increase of clones with myeloid potential. We generate induced pluripotent stem (iPS) lines from functionally defined aged HSC clones, which we next evaluate from the perspective of their blood-forming capacity following re-differentiation into HSCs by blastocyst/morula complementation. Our experiments reveal that all tested iPS clones, including such that were originally completely devoid of T-cell and/or B-cell potential, perform similar to young HSCs both in steady-state and when forced to regenerate lymphomyeloid haematopoiesis in secondary transplantations. This regain in function coincides with transcriptional features shared with young rather than aged HSCs. Thereby, we provide direct support to the notion that several functional aspects of HSC ageing can be reversed to a young-like state.

Working on a Drug to Stimulate Regeneration of Lost Hair Cells in the Inner Ear

One class of the numerous forms of age-related deafness is caused by loss of hair cells in the inner ear. These cells are a necessary part of the chain of systems that leads from sound outside the body to signals passing along nerves into the brain for interpretation. As these hair cells are lost, so is hearing capacity. A range of efforts to reverse this loss are underway at various stages of development, such as reprogramming a cell sample into patient-matched hair cells, or, as in this case, finding ways to provoke regeneration in situ, changing cellular behavior so that they rebuild where they would normally not do so.

Within the inner ear, thousands of hair cells detect sound waves and translate them into nerve signals. Each of us is born with about 15,000 hair cells per ear, and once damaged, these cells cannot regrow. Noise exposure, aging, and some antibiotics and chemotherapy drugs can lead to hair cell death. In some animals, those cells naturally regenerate, but not in humans. However, researchers have now discovered a combination of drugs that expands the population of progenitor cells (also called supporting cells) in the ear and induces them to become hair cells, offering a potential new way to treat hearing loss.

The research team began investigating the possibility of regenerating hair cells during an earlier study on cells of the intestinal lining. In that study, researchers reported that they could generate large quantities of immature intestinal cells and then stimulate them to differentiate, by exposing them to certain molecules. During that study, the team became aware that cells that provide structural support in the cochlea of the ear express some of the same surface proteins as intestinal stem cells. The researchers decided to explore whether the same approach would work in those supporting cells.

They exposed cells from a mouse cochlea, grown in a lab dish, to molecules that stimulate the Wnt pathway, which makes the cells multiply rapidly. At the same time, to prevent the cells from differentiating too soon, the researchers also exposed the cells to molecules that activate another signaling pathway known as Notch. Once they had a large pool of immature progenitor cells, the researchers added another set of molecules that provoked the cells to differentiate into mature hair cells. This procedure generates about 60 times more mature hair cells than the technique that had previously worked the best, which uses growth factors to induce the supporting cochlea cells to become hair cells without first expanding the population.

The researchers found that their new approach also worked in an intact mouse cochlea removed from the body. In that experiment, the researchers did not need to add the second set of drugs because once the progenitor cells were formed, they were naturally exposed to signals that stimulated them to become mature hair cells. "We only need to promote the proliferation of these supporting cells, and then the natural signaling cascade that exists in the body will drive a portion of those cells to become hair cells." Because this treatment involves a simple drug exposure, the researchers believe it could be easy to administer it to human patients. They envision that the drugs could be injected into the middle ear, from which they would diffuse across a membrane into the inner ear.

Link: http://news.mit.edu/2017/drug-treatment-combat-hearing-loss-0221

Senescent Cells Implicated as a Cause of Idiopathic Pulmonary Fibrosis

The number of senescent cells in tissues grows with age, and these cells cause harm through forms of signaling that induce inflammation, destructively remodel the extracellular matrix, and alter the behavior of other cells for the worse. Now that clearance of senescent cells has been shown to robustly extend healthy life span in mice, there is a lot more interest in the research community in joining the dots between cellular senescence and specific age-related diseases. The past year has seen a range of publications that directly implicate senescent cells in various age-related diseases, or attempt to quantify exactly how much of the detrimental alterations in aged tissues are caused by these cells. In this particular case, researchers are looking at the lung condition known as idiopathic pulmonary fibrosis, and you might compare these results with another promising study of senescent cells in the lungs carried out last year. We can hope that the various companies developing clearance therapies will bring them to the clinic sooner rather than later.

A study has shown evidence linking the biology of aging with idiopathic pulmonary fibrosis, a disease that impairs lung function and causes shortness of breath, fatigue, declining quality of life, and, ultimately, death. Researchers believe that these findings are the next step toward a possible therapy for individuals suffering from idiopathic pulmonary fibrosis. "Idiopathic pulmonary fibrosis is a poorly understood disease, and its effects are devastating. Individuals with idiopathic pulmonary fibrosis express difficulty completing routine activities. There are currently no effective treatment options, and the disease leads to a dramatic decrease in health span and life span, with life expectancy after diagnosis between three to five years."

Researchers studied the lung tissue of healthy individuals and of persons with mild, moderate and severe idiopathic pulmonary fibrosis. Researchers found that the markers of cellular senescence, a process triggered by damage to cells and linked to aging, were higher in individuals with idiopathic pulmonary fibrosis, and senescent cell burden increased with the progression of the disease. Then, they demonstrated that factors secreted by senescent cells could drive inflammation and aberrant tissue remodeling and fibrosis, which are hallmarks of idiopathic pulmonary fibrosis. "Up to this point, research efforts have largely focused on understanding the unique elements that contribute to idiopathic pulmonary fibrosis. Here, we are considering whether the biology of aging is accelerated in this aggressive disease. What we've found is that senescent cells are prevalent, secreting toxic molecules that affect healthy cells in that environment and are essentially promoting tissue fibrosis."

Equipped with the findings from their studies of human lung tissue, researchers then replicated the process in mice. They found that, much like in humans, mice with clinical features of idiopathic pulmonary fibrosis also demonstrated increased amounts of senescent cells. Researchers used a genetic model programmed to make senescent cells self-destruct and a drug combination of dasatinib and quercetin which, in previous studies, was shown to eliminate senescent cells. Results showed that clearing senescent cells from unhealthy mice improved measures of lung function and physical health, such as exercise capacity on a treadmill. "We are exploring whether senolytic drugs, or drugs that can selectively kill senescent cells, can be used for the treatment of aging-associated conditions, including idiopathic pulmonary fibrosis. More research is needed to validate this, and our goal is to move quickly from discovery to translation to application, and, ultimately, meet the unmet needs of our patients."

Link: http://newsnetwork.mayoclinic.org/discussion/mayo-clinic-researchers-discover-link-between-aging-devastating-lung-disease/

Towards Therapies Capable of Reversing the Progression of Fibrosis

Fibrosis is a significant component of many age-related conditions, a failure of the normal regenerative process that leads to the formation of increasing amounts of scar-like, fibrous connective tissue in organs. This disrupts normal tissue structure and degrades proper function. It features prominently in common forms of heart disease, kidney failure, and liver disease, among others. As is the case for many specific aspects of aging, there is no good treatment for fibrosis, if by this we mean a reliable way to turn back its progression and restore failing tissues to their former state.

The causes of fibrosis lie somewhere downstream of the fundamental forms of cell and tissue damage outlined in the SENS view of aging. Insofar as it is cells that work to produce fibrotic structures, built from the same materials as the normal extracellular matrix, the proximate causes of fibrosis are thus altered cell signaling and behavior, such as that related to the increased chronic inflammation that accompanies aging. The nature of these signals is much debated, and likely varies considerably from tissue to tissue.

Given the importance of fibrosis to the progression of age-related disease, there is considerable interest in finding ways to reverse its progression, not just slow it down. Most such research, as is the case in the paper linked below, is focused on the proximate causes of fibrosis, the altered cellular signaling and behavior. Researchers hope that by forcing a change here, through the use of small molecule drugs and the like, they can change cellular behavior for the better despite the continued existence of underlying damage that causes dysfunction, and set cells to removing fibrosis and correctly regenerating tissue. Or at last tilt the balance somewhat in that direction.

Peptide reverses cardiac fibrosis in a preclinical model of congestive heart failure

Cardiac fibrosis, an abnormal thickening of the heart wall leading to congestive heart failure, was not only halted but also reversed by a caveolin-1 surrogate peptide (CSD) in a preclinical model, report researchers. CSD was able to decrease the fibrotic ventricular wall thickness and improve heart function, all with apparently no toxicity and minimal off-target effects. More than a decade ago, researchers noted that the skin and lung cells producing excess collagen in scleroderma, leading to fibrosis, were deficient in caveolin-1. Supplementing these cells with a caveolin-1 surrogate peptide (CSD; caveolin-1 scaffolding domain peptide), a truncated version of the original compound, showed a reversal of fibrosis.

Hypertrophic overgrowth and profibrogenic signaling of the cardiac muscle occurs under pressure overload. Fibrosis that develops under these conditions is detrimental to the heart's pumping efficiency as it causes a stiffer and less compliant cardiac muscle, leading to the progression of congestive heart failure. To mimic the cardiac fibrosis typical of heart failure, researchers used a transverse aortic constriction mouse model to create pressure overload hypertrophy that then led to the development of fibrosis. Treatment with CSD not only halted the progression of the cardiac fibrosis but also led to its reversal with improved ventricular function.

Although promising, these findings are preliminary - only reflecting outcomes in mice. The researchers plan to run larger preclinical studies using the same approach to generate more definitive data, and if all goes as expected, to move forward to the large-animal studies necessary to take a compound forward into clinical trial. They also note that they are testing CSD in a different congestive heart failure model, the angiotensin II infusion model, which also affects the kidneys. CSD is showing promising anti-fibrotic effects on both the heart and the kidneys in this model. "Fibrotic diseases are related to each other no matter the affected organ. In our case, we were studying lung and skin fibrosis. We had the opportunity to test the same reagent in heart fibrosis and, lo and behold, it worked even better than in lung and skin fibrosis models. And there are plenty of other diseases with a fibrotic element to them where we think the CSD peptide might be useful."

Reversal of maladaptive fibrosis and compromised ventricular function in the pressure overloaded heart by a caveolin-1 surrogate peptide

Chronic ventricular pressure overload (PO) results in congestive heart failure (CHF) in which myocardial fibrosis develops in concert with ventricular dysfunction. Caveolin-1 is important in fibrosis in various tissues due to its decreased expression in fibroblasts and monocytes. The profibrotic effects of low caveolin-1 can be blocked with the caveolin-1 scaffolding domain peptide (CSD, a caveolin-1 surrogate) using both mouse models and human cells.

We have studied the beneficial effects of CSD on mice in which PO was induced by trans-aortic constriction (TAC). Beneficial effects observed in TAC mice receiving CSD injections daily included: improved ventricular function (increased ejection fraction, stroke volume, and cardiac output; reduced wall thickness); decreased collagen I, collagen chaperone HSP47, fibronectin, and CTGF levels; decreased activation of non-receptor tyrosine kinases Pyk2 and Src; and decreased activation of eNOS. To determine the source of cells that contribute to fibrosis in CHF, flow cytometric studies were performed that suggested that myofibroblasts in the heart are in large part bone marrow-derived. Two CD45+ cell populations were observed. One (Zone 1) contained CD45+/HSP47-/macrophage marker+ cells (macrophages). The second (Zone 2) contained CD45moderate/HSP47+/macrophage marker- cells often defined as fibrocytes. TAC increased the number of cells in Zones 1 and 2 and the level of HSP47 in Zone 2. These studies are a first step in elucidating the mechanism of action of CSD in heart fibrosis and promoting the development of CSD as a novel treatment to reduce fibrosis and improve ventricular function in CHF patients.

Projecting out Current Life Expectancy Trends to 2030

I think it a given that trend projection at the present time is going to greatly underestimate gains in life expectancy over the next few decades. This present decade and the next encompass a transition from palliative and compensatory medicine that inadequately patches over the causes of aging, and a research community that has no interest in treating aging itself as a medical condition, to a field of rejuvenation treatments that do actually address the forms of cell and tissue damage that cause degenerative aging, and a research community that is now very interested in working towards therapies for aging. Past gains have occurred despite the fact that research and development efforts made no attempt to treat root causes in aging. Future gains, produced by those actually trying to address aging, will be larger and occur more rapidly.

A new study analysed long-term data on mortality and longevity trends to predict how life expectancy will change in 35 industrialised countries by 2030. Nations in the study included both high-income countries, such as the USA, Canada, UK, Germany, Australia, and emerging economies such as Poland, Mexico and the Czech Republic. The study revealed all nations in the study can expect to see an increase in life expectancy by 2030. The results also found that South Koreans may have the highest life expectancy in the world in 2030. "The increase in average life expectancy in high income countries is due to the over-65s living longer than ever before. In middle-income countries, the number of premature deaths - i.e. people dying in their forties and fifties, will also decline by 2030."

The team calculated life expectancy at birth, and predicted a baby girl born in South Korea in 2030 will expect to live 90.8 years. Life expectancy at birth for South Korean men will be 84.1 years. The researchers also calculated how long a 65-year-old person may expect to live in 2030. The results revealed that the average 65-year-old woman in South Korea in 2030 may live an additional 27.5 years. Scientists once thought an average life expectancy of over 90 was impossible. "We repeatedly hear that improvements in human longevity are about to come to an end. Many people used to believe that 90 years is the upper limit for life expectancy, but this research suggests we will break the 90-year-barrier. I don't believe we're anywhere near the upper limit of life expectancy - if there even is one."

French women and Swiss men were predicted to have the highest life expectancies at birth in Europe in 2030, with an average life expectancy of 88.6 years for French women and nearly 84 years for Swiss men. The results also revealed that the USA is likely to have the lowest life expectancy at birth in 2030 among high-income countries. The nation's average life expectancy at birth of men and women in 2030 (79.5 years and 83.3 years), will be similar to that of middle-income countries like Croatia and Mexico. The team also predicted a 65-year-old UK man in 2030 could expect to live an additional 20.9 years (12th in the table of countries), while a 65-year-old woman in the UK could expect to live an additional 22.7 years, up (22nd in the table of countries). The research also suggested the gap in life expectancy between women and men is closing. "Men traditionally had unhealthier lifestyles, and so shorter life expectancies. They smoked and drank more, and had more road traffic accidents and homicides. However as lifestyles become more similar between men and women, so does their longevity."

Link: http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_21-2-2017-15-33-52

Is a Clone Born at Age Zero?

In the overlap between research into aging and research into regeneration there is some interest in what exactly it is that happens between fertilization and later development of a zygote that enables old reproductive cells to produce young children. Some form of reset takes place, a clearing out of damage. This is also seen in induced pluripotency, whereby ordinary somatic cells are reprogrammed into a state very similar to that of embryonic stem cells. It is an open question as to whether any part of this natural rejuvenation mechanism can be safely harnessed and turned into a therapy, though it is worth noting that induction of induced pluripotency in the tissues of adult mice has been tried recently. Animal cloning is another line of research that might help to shed light on what happens in early development, a topic that was covered in some depth last year. Do clones age normally, and are they born with a similar level of molecular damage as their natural peers? Why, if so?

In 1997, Dolly the sheep was introduced to the world. The implications of cloning animals in our society were self-evident from the start. Our advancing ability to reprogram adult, already-specialized cells and start them over as something new may one day be the key to creating cells and organs that match the immune system of each individual patient in need of replacements. But what somehow got lost was the fact that a clone was born - at day zero - created from the cell of another animal that was 6 years old. Researchers have spent the past 20 years trying to untangle the mysteries of how clones age. How old, biologically, are these animals born from other adult animals' cells?

When Dolly was cloned, she was created using a cell from a 6-year-old sheep. And she died at age 6-and-a-half, a premature death for a breed that lives an average of nine years or more. People assumed that an offspring cloned from an adult was starting at an age disadvantage. Rather than truly being a "newborn," it seemed like a clone's internal age would be more advanced than the length of its own life would suggest. Thus the notion that clones' biological ages and their chronological ones were out of sync, and that "cloned animals will die young."

Some of us were convinced that if the cloning procedure was done properly, the biological clock should be reset - a newborn clone would truly start at age zero. We worked very hard to prove our point. We were not convinced by a single DNA analysis done in Dolly showing slightly shorter telomeres - the repetitive DNA sequences at the end of chromosomes that "count" how many times a cell divides. We presented strong scientific evidence showing that cloned cows had all the same molecular signs of aging as a nonclone, predicting a normal lifespan. Others showed the same in cloned mice. But we couldn't ignore reports from colleagues interpreting biological signs in cloned animals that they attributed to incomplete resetting of the biological clock. So the jury was out.

Aging studies are very hard to do because there are only two data points that really count: date of birth and date of death. If you want to know the lifespan of an individual you have to wait until its natural death. By 2012 that was in fact being accomplished: there were several cloned Dollies, all much older than Dolly at the time she had died, and they looked terrific. This work was finally published last year. "For those clones that survive beyond the perinatal period, the emerging consensus, supported by the current data, is that they are healthy and seem to age normally."

The new Dollies are now telling us that if we take a cell from an animal of any age, and we introduce its nucleus into a nonfertilized mature egg, we can have an individual born with its lifespan fully restored. They confirmed that all signs of biological and chronological age matched between cloned and noncloned sheep. There seems to be a natural built-in mechanism in the eggs that can rejuvenate a cell. We don't know what it is yet, but it is there. Our group as well as others are hard at work, and as soon as someone finds it, the most astonishing legacy of Dolly will be realized.

Link: http://www.the-scientist.com/?articles.view/articleNo/48571/title/Opinion--Is-a-Clone-Really-Born-at-Age-Zero-/

An Interview with Aschwin de Wolf on Cryonics at LongeCity

Aschwin de Wolf of Advanced Neural Biosciences and the Institute for Evidence-Based Cryonics (IEBC) is a noted advocate for cryonics as an industry and area of research. He was recently interviewed by the folk over at LongeCity, and as usual it makes for interesting reading. You might also look at a 2013 interview for more of the same, and in addition you'll find many articles at the IEBC site covering a mix of technical and non-technical topics in the the cryonics field. This is one slice of a great deal of technical writing and advocacy for cryonics published over the course of the past few decades, a fair portion of it by people who are now themselves cryopreserved at Alcor or the Cryonics Institute.

The term cryonics covers the technology, community, and practice of placing people into a vitrified state as soon as possible following clinical death. Tissues are perfused with cryoprotectant and cooled to liquid nitrogen temperatures in stages, leading to a glass-like state of minimal ice-crystal formation. Under good conditions, this preserves the fine structures of neural tissue, the synapses, dendrites, and dendritic spines within which the data of the mind is thought to be stored. For so long as that data remains intact, and the vitrified individual in low-temperature storage, there is the possibility of future restoration in an era with more proficient technology than our own. In this age of progress, cryonics is a necessary backup plan for those of who may not live long enough to benefit from the near future of rejuvenation therapies after the SENS model. It is a great pity that it remains a small and marginal undertaking, largely non-profit, and unknown to many who would benefit, even as tens of millions march towards their own personal oblivion each and every year.

While higher animals cannot yet be thawed, cleared of cryoprotectant, and brought back to life, that outcome can be achieved with lower animals such as nematode worms. Thawing and transplantation has also been demonstrated in prototype for mammalian organs in recent years. At present there is the makings of a small industry working on reversible cryopreservation for tissue engineering and organ transplantation, where such a technology would greatly reduce costs and simplify logistics. So when we talk about preserving people for the chance at a future restoration, this isn't done in a vacuum, and isn't a flight of fancy; there is good reason to think that there is a chance of success in this endeavor. It certainly beats the odds of revival from the grave, which is to say zero.

Interview with Aschwin de Wolf (February, 2017)

How has the cryopreservation procedure evolved since the first human was placed in cryostasis?

The most important element in the progress of cryopreservation procedures in cryonics is the progressive elimination of ice formation. When cryonics started, patients were often cryopreserved without any cryoprotection or very low concentrations of cryoprotectant. In the 1980's and 1990's organizations such as Alcor started adapting mainstream perfusion technologies to introduce high concentrations of cryoprotectants (such as glycerol) to mitigate ice formation. In 2000 Alcor formally introduced vitrification with the aim of eliminating freezing altogether.

The elimination of ice formation, which can be achieved in good cases, removes one major form of mechanical damage in the cryopreserved brain. One very attractive feature of a low-toxicity vitrification agent like M22 is that it does not require rapid cooling to prevent ice formation. Under good circumstances (no prior ischemia) it can also be used in whole-body patients without edema - a problem that seemed to plague prior DMSO-based cryoprotectants in cryonics. Elimination of ice formation and reduced toxicity has substantially reduced the degree of damage associated with cryopreservation.

Which foreseeable advances in the field of cryobiology do you believe will lead to improvements in cryonics?

I foresee further advances in two areas; a more detailed understanding of the nature of cryoprotectant toxicity and the design of brain-optimized cryoprotectants. Cryoprotectant toxicity is currently the most formidable obstacle preventing reversible cryopreservation of complex mammalian organs. With the exception of the work of Dr. Greg Fahy and his colleagues at 21st Century Medicine, it is rather surprising how little theoretical and experimental research has been done to illuminate the mechanisms of cryoprotectant toxicity. It is also increasingly recognized that the poor penetration of cryoprotectants across the blood-brain barrier causes dehydration of the brain. We need to develop brain-optimized vitrification solutions and/or identify better methods to deliver cryoprotectants to the brain without such significant changes in brain volume. Resolving these two issues will bring us much closer to reversible brain cryopreservation.

What evidence is there that the brain is not damaged by the cryopreservation process to such an extent that the information in it may be lost forever?

To start with, if we can eliminate ice formation in the brain, the damage associated with cryoprotectant toxicity is assumed to be mostly of a biochemical nature (i.e. denatured proteins) and does not alter the ultrastructure of the brain in a way that precludes inferring the original state. Cryoprotectant-induced dehydration of the brain is a little more of a wild card because we do not have much detailed information about the kind of ultrastructural changes associated with it. Hence, the priority to avoid the brain shrinking that is routinely observed in "good" cases. Ultimately, our incomplete knowledge of the neuroanatomical basis of identity, and about the exact capabilities and limits of future medicine, prompt us to be agnostic about the degree of damage that is still compatible with meaningful revival. Advocates of cryonics are sometimes accused of being too optimistic about future science, but perhaps skeptics are too pessimistic.

To our knowledge (which is based on cryobiological studies and theoretical calculations), deterioration of patients stored at cryogenic temperatures should be non-existent or negligible. Things get a little bit more complicated when we store patients at intermediate temperatures instead of liquid nitrogen temperatures. It has been suggested that nucleation may still occur slightly below the temperature where the vitrification solution turns into a glass (-123 degrees Celsius). At that temperature, however, nucleation does not translate into ice formation but it might create more challenging repair and revival scenarios.

Do you have any hypotheses on how the cryoprotectant could be removed from the body during the reanimation procedure and how hypoxic injury during this removal procedure could be prevented?

In the vision of researchers such as Robert Freitas and Ralph Merkle, a mature form of mechanical nanotechnology will be used to conduct the initial stages of repair and cryoprotectant removal at cryogenic temperatures. If this vision of nanotechnology is plausible, cryoprotectant can be removed while providing (local) metabolic and structural support to prevent damage or freezing. An alternative vision of nanomedicine will involve the use of biological repair machines such as modified viruses or modified white blood cells that operate using conventional diffusion-driven chemistry rather than molecular mechanical nanotechnology. Repair is more challenging in this biological scenario because tissue first needs to be warmed to temperatures at which the cryoprotectant solution inside cells and tissue becomes liquid. This risks movement of damaged structures, possible growth of ice, and cryoprotectant toxicity accumulation occurring at the same time as repairs are being made.

Cryogenic storage of genetic mutants is already a common procedure in the roundworm C. elegans. Are you aware of any research taking place that tries to expand cryogenic storage to other model organisms?

Natasha Vita-Moore, who conducted recent studies on the effects of vitrification on memory in C. elegans, has suggested that the next step would be a slightly more complex organism such as the Greenland Woolly Bear Caterpillar or the ozobranchid leech. One of the most common suggestions I get is to attempt suspended animation on a mouse or rat. This would definitely provide powerful proof of principle for the feasibility of human suspended animation, but I do not think that the challenges in achieving reversible biostasis in a small mammal are that much smaller than in humans. We would need to overcome the same obstacles: minimizing cryoprotectant toxicity, chilling injury, dehydration of the brain, ischemia during cooling, and cryoprotective perfusion, etc. The majority opinion in cryonics is to solve these individual problems more thoroughly before attempting reversible cryopreservation of a complete animal.

The Risks of Current Approaches to Rebooting the Immune System

The present approaches to rebooting the immune system have shown considerable promise in treatment of autoimmune diseases such as multiple sclerosis. Unfortunately the current methods of immune destruction involve chemotherapy, which is a damaging process in and of itself, and there is as yet too little attention being given to protection against infection in the period while the immune system is absent or near-completely suppressed. The risks are significant, and until addressed mean that this remains useful only for patients who will suffer worse absent the therapy.

Both of the major risks noted above could be addressed in the near future, however. Firstly through the development of targeted cell destruction methods with minimal side-effects, such as that currently pioneered by Oisin Biotechnologies, and secondly through delivery of new immune cells generated from the patient's own cells. It is in all our interests to see a broadening of immune reboot work, as this class of therapy could help clear out the malfunctioning and misconfigured cells from an age-damaged immune system, producing a partial rejuvenation of immune function in the elderly.

A type of treatment for multiple sclerosis that 'resets' the immune system may stop progression of the disease in nearly half of patients. In a new study the treatment prevented symptoms of severe disease from worsening for five years, in 46 per cent of patients. However, as the treatment involves aggressive chemotherapy, the researchers stress the procedure carries significant risk. The treatment in the current study, called autologous hematopoietic stem cell transplantation (AHSCT), was given to patients with advanced forms of the disease that had failed to respond to other medications.

The one-off treatment aims to prevent the immune system from attacking the nerve cells. All immune system cells are made from stem cells in the bone marrow. In the treatment, a patient is given a drug that encourages stem cells to move from the bone marrow into the blood stream, and these cells are then removed from the body. The patient then receives high-dose chemotherapy that kills any remaining immune cells. The patient's stem cells are then transfused back into their body to re-grow their immune system. Previous studies have suggested this 'resets' the immune system, and stops it from attacking the nerve cells.

However, because the treatment involves aggressive chemotherapy that inactivates the immune system for a short period of time, some patients died from infections. Out of the 281 patients who received the treatment in the study, eight died in the 100 days following the treatment. Older patients, and those with the most severe forms of the disease, were found to have a higher risk of death. "In this study, which is the largest long-term follow-up study of this procedure, we've shown we can 'freeze' a patient's disease - and stop it from becoming worse, for up to five years. However, we must take into account that the treatment carries a small risk of death, and this is a disease that is not immediately life-threatening."

Link: http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_20-2-2017-17-40-27

Identification of a Potential Autophagy Enhancement Drug

Researchers here note the identification of a drug candidate to enhance autophagy, a process of cellular housekeeping responsible for removing damaged proteins and structures in the cell. Enhanced autophagy is associated with many of the interventions known to slow aging in laboratory species, and in at least some cases, such as for calorie restriction, the correct operation of autophagy has been shown to be necessary for extension of life span to take place. Consequently, the research community has for some time shown interest in the development of therapies based on the enhancement of autophagy, but there has been surprisingly little progress on this front to date.

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation.

Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging.

Link: https://dx.doi.org/10.1038/srep42014

The Media Meanders on the Topic of Enhanced Longevity

For various reasons, such as people promoting their books, the mainstream media has been giving more attention than usual these past few weeks to the topic of healthy life extension. The quality of the resulting articles is fairly low, as is usually the case. When given marching orders to cover any particular topic, the average journalist grabs the first few specific items that show up in a search of recent articles, wraps them with some pretty words, and launches the result without any attempt at achieving or conveying real understanding of the subject. When it comes aging and efforts to treat aging as a medical condition, just like any other quite complex topic in science and medicine, that real understanding is absolutely vital in order to distinguish between arrant nonsense, legitimate but poor approaches, and efforts that might do very well indeed if given sufficient support. The media is not the place to search for comprehension, on this or any other subject, sadly. So we see articles in which supplements, calorie restriction mimetic research, senescent cell clearance, and spa treatments are all ranked equally, without judgement or insight - options spanning the gamut of the aforementioned arrant nonsense through to potentially viable rejuvenation therapies.

Does it do the cause of human rejuvenation any good to have the press talk more rather than less, when nine-tenths of what is published is wrong, useless, or outright disinformation? It can be argued that there is no such thing as bad publicity. If these bland articles spur some people into moving from the class of those who do nothing into the class of those who head off to find out more, then some of the more active of those folk will eventually make their way into our community. There are many roads to learning about SENS-like rejuvenation research: from the personal health and fitness world; from time spent in other areas of the life sciences; from a passing interest in living longer acquired via supplement sellers; because it is talked about among members of an otherwise unrelated community, such as in the Bay Area technology circles; and so forth. So long as people arrive and help with meaningful progress in research and development, help to grow the community, I don't think the road taken matters all that much. Even if it starts with a few eye-rollingly terrible articles in the press.

Only Human: Meet the hackers trying to solve the problem of death

It is tempting to see transhumanism as merely the latest rebranding of a very old desire, for immortality. Aubrey de Grey is a biomedical gerontologist who sees death as a disease to be cured. Anders Sandberg, a neuroscientist working on mind uploading, wishes literally to become an "emotional machine." Zoltan Istvan ran a presidential campaign that saw him travel across the country in a coffin-shaped bus to raise awareness for transhumanism. He campaigned on a pro-technology platform that called for a universal basic income, and promoted a Transhumanist Bill of Rights that would assure, among other things, that "human beings, sentient artificial intelligences, cyborgs, and other advanced sapient life forms" be "entitled to universal rights of ending involuntary suffering."

Then there's Max More, a co-founder of Extropianism, who runs the Alcor Life Extension Foundation in Scottsdale, Arizona. Alcor is a cryopreservation facility that houses the bodies of those hoping to be reanimated as soon as the technology exists. The bodies, "are considered to be suspended, rather than deceased: detained in some liminal stasis between this world and whatever follows it, or does not." Alcor is the largest of the world's four cryopreservation facilities, and houses 149 "patients," nearly 70 percent of whom are male.

Those working on immortality are long-term thinkers and fall, broadly, into two camps: those who want to free the human from the body, and those who aim to keep the body in a healthy condition for as long as possible. Randal Koene, like Max More, is in the first group. Instead of cryonics, he is working toward "mind uploading," the construction of a mind that can exist independent of the body. His nonprofit organization, Carboncopies, aims for "the effective immortality of the digitally duplicated self. Maybe it wouldn't be that much of a shock to the system to be uploaded, because we already exist in this prosthetic relationship to the physical world anyway, where so many things are experienced as extensions of our bodies."

Aubrey de Grey is in the second, body preservationist group, whose efforts tend to be slightly more modest: Rather than solving death, they focus on extending life. His nonprofit, SENS Research Foundation, focuses on research in heart disease and Alzheimer's, and other common illnesses and diseases. (SENS, like many organizations the transhumanists are involved with, has received funding from Peter Thiel.) De Grey's most mainstream contribution is the popularization of the concept of "longevity escape velocity," which is explained as follows: "For every year that passes, the progress of longevity research is such that average human life expectancy increases by more than a year-a situation that would, in theory, lead to our effectively outrunning death." One might dismiss such transhumanist visions as too extreme: so many men, so much hubris. And yet, at a time of great cynicism about humanity - and the future we're all barreling toward - there is something irresistible about transhumanism. Call it magical thinking; call it radical optimism.

Why Do People Want to Live So Long, Anyway?

Dr. Ezekiel Emanuel is famous for a lot of reasons. He's an acclaimed bioethicist and oncologist and has two very well known brothers, but another thing people always seem to remember about him is that article he wrote in 2014: "Why I Hope to Die at 75." Emanuel's embrace of an early end - one that's only a few years shy of the U.S. life expectancy of 78.8 -is the exact opposite of how most people in America feel about dying. In a survey from the Pew Research Center, nearly 70% of American adults said they wanted to live to be up to 100 years old. But why?

"The quest to live forever, or to live for great expanses of time, has always been part of the human spirit," says Paul Root Wolpe, director of the Emory Center for Ethics. People now seem to have particular reason to be optimistic: in the past century, science and medicine have extended life expectancy, and longevity researchers (not to mention Silicon Valley types) are pushing for a life that lasts at least a couple decades more.

How Silicon Valley Is Trying to Hack Its Way Into a Longer Life

The titans of the tech industry are known for their confidence that they can solve any problem - even, as it turns out, the one that's defeated every other attempt so far. That's why the most far-out strategies to cheat death are being tested in America's playground for the young, deep-pocketed and brilliant: Silicon Valley. Larry Ellison, the co-founder of Oracle, has given more than $330 million to research about aging and age-related diseases. Alphabet CEO and co-founder Larry Page launched Calico, a research company that targets ways to improve the human lifespan. Peter Thiel, co-founder of PayPal, has also invested millions in the cause, including over $7 million to the Methuselah Foundation, a nonprofit focused on life-extension therapies.

Rather than wait years for treatments to be approved by federal officials, many of them are testing ways to modify human biology that fall somewhere on the spectrum between science and entrepreneurialism. It's called biohacking, and it's one of the biggest things happening in the Bay Area. "My goal is to live beyond 180 years," says Dave Asprey, CEO of the supplement company Bulletproof. "I am doing every single thing I can to make it happen for myself."

Should We Die?

"So, you don't want to die?" I asked Zoltan Istvan, then the Transhumanist candidate for president, as we sat in the lobby of the University of Baltimore one day last fall. "No," he said, assuredly. "Never." Istvan, an atheist who physically resembles the pure-hearted hero of a Soviet children's book, explained that his life is awesome. In the future, it will grow awesomer still, and he wants to be the one to decide when it ends. Defying aging was the point of his presidential campaign. He knew he'd lose, of course, but he wanted his candidacy to promote the cause of transhumanism - the idea that technology will allow humans to break free of their physical and mental limitations. His platform included, in part, declaring aging a disease.

But his central goal-pushing the human lifespan far beyond the record 122 years and possibly into eternity - is one shared by many futurists in Silicon Valley and beyond. Investor Peter Thiel, who sees death as "the great enemy" of man, is writing checks to researchers like Cynthia Kenyon, who doubled the life-spans of worms through gene-hacking. Oracle founder Larry Ellison has thrown hundreds of millions toward anti-aging research, according to Inc magazine, and Google founders Larry Page and Sergey Brin launched the Google subsidiary Calico specifically with the goal of "curing death."

But let's assume, for the sake of argument, that it can be. Let's say human lives will soon get radically longer - or even become unending. The billionaires will get their way, and death will become optional. If we really are on the doorstep of radical longevity, it's worth considering how it will change human society. With no deadline, will we still be motivated to finish things? Or will we while away our endless days, amusing ourselves to - well, the Process Formerly Known as Death - while we overpopulate the planet? Will Earth become a paradise of eternally youthful artists, or a hellish, depleted nursing home? The answers depend on, well, one's opinion about the meaning of life.

SkQ1 Slows Accelerated Aging in Mitochondrial Mutator Mice

Mice engineered to generate a high level of deletion mutations in mitochondrial DNA exhibit accelerated aging. As in most cases of accelerated aging, we can debate whether or not it is correct to call it accelerated aging. The important point is whether or not the type of cellular damage involved provides a significant contribution to the normal aging process, which in this case it does. The normal lower levels of mitochondrial DNA damage are implicated as a cause of aging and age-related disease. Then the question becomes whether or not it is acceptable to continue to call it aging given a vastly greater presence of just one of the types of age-related damage, or is it now some other form of pathology?

Putting this to one side, here researchers show that SkQ1, a mitochondrially targeted antioxidant shown to modestly slow aging in laboratory species, helps to ameliorate the harm done by high levels of mitochondrial deletion mutations. To my eyes, at least, it would have been unexpected to find another outcome, given what is known of the mechanisms involved here. This class of targeted antioxidant compounds come with a good deal of evidence backing their impact on mitochondrial metabolism; to the degree that deletions occur due to oxidative damage, and to the degree that they in turn cause greater levels of oxidative damage throughout the cell through disarrayed mitochondrial function, the presence of antioxidants in the mitochondria should reduce the harmful outcomes. This class of compound is currently being developed as a treatment for inflammatory eye conditions, as this is one of the areas in which the benefits are both reliable and large, and the regulatory path to market is comparatively smooth.

As a cause for the decreasing health status that accompanies aging, mitochondrial deterioration has been repeatedly suggested. Particularly, it has been discussed that an accumulation of errors in mitochondrial DNA (mtDNA) replication would lead to mitochondrial dysfunction, including increased production of reactive oxygen species (ROS) that may both further deteriorate the mitochondria and affect the function of the rest of the cell. However, the significance of ROS for the aging process has been doubted, particularly based on observations in the mtDNA mutator mice. These mice accumulate errors in their mtDNA and demonstrate subsequent alterations in their respiratory chain composition. They also demonstrate an early occurrence of characteristics normally associated with aging, and they die at a young age. However, there has been no convincing evidence that oxidative damage causes these problems.

Experimentally, an alternative avenue to examine the possible involvement of ROS in the development of aging characteristics would be to examine the ability of mitochondrially targeted antioxidants to ameliorate the health problems associated with experimentally induced aging. In this paper, we find that the mitochondrially targeted antioxidant 10-(6'-plastoquinonyl)decyltri-phenylphosphonium cation (SkQ1) substantially counteracts the acquisition of aging characteristics in the mtDNA mutator mice. We also find that parameters for oxidative damage not earlier examined (cardiolipin depletion and accumulation of hydroxynonenal protein adducts) are diminished by SkQ1 treatment. These data clearly indicate that ROS production and oxidative damage are substantial factors in the development of aging characteristics in the mtDNA mutator mice.

As the presently reluctance to associate mitochondrial dysfunction with aging through ROS and oxidative damage are largely based on the notion that these phenomena were apparently not involved in aging in mtDNA mutator mice, and as our present data indicate the opposite to be the case, our observations may also be of significance for discussions of the nature of aging and the possibility to ameliorate the aging process therapeutically.

Link: http://dx.doi.org/10.18632/aging.101174

A Diet of Old Tissues Modestly Shortens Life Span

In an interesting series of experiments, researchers found evidence for a diet of old tissues to modestly reduce life span in flies and mice. If speculating on specific mechanisms, we might look to the various forms of metabolic waste and damaged proteins that accumulate with age; some of that might find its way past the digestive process to be incorporated into tissues and thereby accelerate the aging process. This sort of dietary influence on aging is already a much-debated topic regarding advanced glycation end-products, for example. The results of the studies here offer reinforcement for the SENS approach of damage repair to create rejuvenation, but sadly that is not the conclusion reached by the researchers involved. They instead look ahead to a much harder task with the prospect of only marginal benefits, which is to say safely altering cellular metabolism in order to slow down damage accumulation. This is an inferior approach to periodic damage repair, requiring far more research to realize, and capable of producing only lesser gains in health and life span.

A study offers evidence bolstering one long-held theory: that aging is caused, at least in part, by molecular damage accumulating in the cells. This damage is generated by nearly every cellular process by the work of enzymes and proteins and the life-sustaining metabolic processes that occur at every level of complexity, from simple molecules and cell components to whole cells and entire organs. Over time we have many, many damage forms, byproducts of enzyme function, for example, or of protein-to-protein interactions, errors in DNA transcription or translation. As a function of age, they accumulate, and eventually, it's more than the body can cope with.

Researchers found that feeding a diet of "old" organisms to yeast, fruit flies, and mice shortened their lifespans by roughly 10 percent. Here's how it worked: for yeast, the researchers formulated one cell-culture medium composed of extracts from young yeast cells and another of extracts from old ones. They then grew new yeast cells on each medium and watched to see which set would live longer. "Our hypothesis was that as yeast ages, it accumulates certain damage forms, and we wanted to test that specific damage and find out if it is deleterious for yeast."­­­­ The team replicated the same basic procedure in fruit flies and mice: they collected 5,000 freshly dead flies that had lived an average of 45 days, and sacrificed 5,000 others that were three to five days old. Then they prepared two homogenized diets, one composed of young flies and the other using the old ones. They fed these diets to young female fruit flies. The mice were fed diets of skeletal muscle from young and old farmed red deer (three years old versus 25) that replaced the animal-product components (insects, carrion, worms, etc.) of a normal mouse diet. Using mouse tissue was not feasible because of the large quantities needed for the experiment; deer meat was a suitably close match.

The experiments raise new questions - in a field that's full of them - and some of the results were a little unexpected. The researchers had expected to see larger differences in the test organisms' relative lifespans. The effect was consistent, however, across all three species. In the study, the authors interpret the minor-but-consistent effect to mean that damage accumulation may be only one contributing factor in aging, and also that damage caused by internal molecular changes may have a stronger effect than damage introduced through the diet. It's also likely that the damage arises from many processes. "And they all work together in a deleterious way. So the question is, how do we slow down this process? How do we restructure cellular metabolism so that this damage accumulates at a slower rate?"

Link: http://harvardmagazine.com/2017/02/old-food-reduces-lifespan

Discussions of Stem Cell Rejuvenation

Earlier this week I noticed a couple of very readable open access papers in which the authors discuss the potential for rejuvenation of stem cells as a means to address some aspects of aging. Reversing age-related stem cell decline has long been a topic of considerable interest in the broader longevity science and advocacy communities, ever since the stem cell medicine industry started up in earnest. Indeed, back in the early days of SENS rejuvenation research advocacy, when stem cells were in the news every other week, it was frequently necessary to emphasize that stem cell repair and replacement was just one of a range of necessary approaches to the treatment of aging. Even if an individual's stem cells were somehow perpetually kept in pristine condition, the other forms of cell and tissue damage that lie at the root of aging would still result in degeneration and death. The degree of benefit achieved from fixing just one type of damage is an open question - we will most likely only find out some years after the relevant therapies become widely available, as is about to happen for senescent cell clearance.

Stem cells and their supporting structures are, of course, important in the aging process. Stem cells are responsible for generating replacement somatic cells needed to keep tissues functioning, but with advancing age the supply of new cells dwindles. This decline is one of the causes of frailty and organ failure. At present it looks likely that the changes in stem cell activity are as much a matter of altered cell signaling as of damage to the stem cells themselves. Temporarily restored signaling may be one of the means by which cell therapies produce benefits, by putting native cells back to work. Why does signaling change with aging, however? From an evolutionary perspective this reaction to rising levels of damage may exist because it serves to reduce cancer risk and thereby lengthen life, at the cost of a slower demise through organ failure, though programmed aging advocates would argue that stem cell decline is selected to promote aging as a fitness strategy. From a purely mechanical perspective, it is still up for debate as to the degree to which stem cell declines are secondary to the other forms of molecular damage and waste accumulation outlined in the SENS view of aging. It isn't unreasonable to think that comprehensive repair elsewhere would lead to some degree of renewed stem cell activity as the signaling environment becomes more youthful.

Rejuvenating stem cells to restore muscle regeneration in aging

Adult muscle stem cells, originally called satellite cells (SCs), are essential for muscle repair and regeneration throughout life. Besides a gradual loss of mass and function, muscle aging is characterized by a decline in the repair capacity, which blunts muscle recovery after injury in elderly individuals. A major effort has been dedicated in recent years to deciphering the causes of SC dysfunction in aging animals, with the ultimate goal of rejuvenating old SCs and improving muscle function in elderly people. The emerging evidence indicates that the functional and numerical loss of SCs is a progressive process occurring throughout the lifetime of the organism. The long-lived quiescent SC accumulates many lesions caused by loss of homeostasis, metabolic alterations, and the aging environment. Although this process is gradual, it is accelerated in advanced old age to the extent that SCs become practically non-functional owing to senescence or apoptosis. In this context, disputes about which factors, intrinsic or extrinsic, are more dominant in dictating the fate of old SCs seem misplaced, and it is likely that both make important contributions to SC functional decline with aging.

A degree of success has been obtained in restoring the regenerative capacity of old muscle with both parabiosis experiments (extrinsic effect) and transplantation of ex vivo-rejuvenated SCs into old animals (intrinsic effect). The simplest explanation for these effects is the heterogeneous nature of SCs. Even in old age, the SC population includes a small percentage of functional SCs, with only limited accumulated damage that can be reversed still by extrinsic signaling factors or by ex vivo pharmacological inhibition of stress pathways such as p38 MAPK or JAK/STAT3. It is thus likely that the success of biochemical or genetic strategies applied to old SCs in transplantation experiments results from the proliferative amplification of a subset of highly regenerative cells. Alternatively, the health and fitness of old SCs could be increased by refueling "clean up" activities such as autophagy (which declines with aging) to eliminate damage, thus improving SC regenerative capacity after muscle injury and in transplantation procedures. Future interventions that could also be considered for combating age-related muscle regenerative decline may utilize the restoration of SC-niche interactions via the delivery of bioengineered molecules.

The key finding that the SC pool enters a state of irreversible senescence at a geriatric age implies that any treatment to rejuvenate endogenous stem cells should be implemented before this point of no return. It is also important to consider the link between SC regenerative potential and quiescence. It is generally well accepted that the more quiescent a stem cell is, the more regenerative capacity it has. It has also become clear that somatic stem cell populations are heterogeneous, with cells showing differing levels of quiescence. Highly quiescent subpopulations probably change with aging to become less quiescent and therefore of reduced regenerative capacity. SC heterogeneity should therefore be further investigated, with the aim of deciphering the molecular basis of quiescence. Understanding the quiescent state will allow early intervention aimed at preserving the highly regenerative quiescent subpopulations throughout life.

Likewise, strategies directed towards the expansion of relevant subpopulations of resident progenitor cells in the SC niche may be envisioned for reversing the age-associated muscle regenerative loss. Another unresolved issue is the interaction among the various events contributing to the loss of SC regenerative potential with aging. Research needs to focus on determining which events are causative and which are consequential. For example, DNA damage may induce the loss of baseline autophagy flux in old SCs, or alternatively DNA damage may be the consequence of oxidative stress resulting from the loss of autophagy flux. Defining the hierarchy of events leading to SC deterioration will enable the targeting of upstream events in order to achieve more efficient rejuvenation of SCs. Last but not least, in a low-turnover tissue like muscle, much of the damage to the quiescent SC is the result of the gradual decline (aging) of the niche composition and the systemic system. Future efforts to rejuvenate the regenerative potential of SCs should thus adopt a holistic view of the SC and its supportive environment.

Preventing aging with stem cell rejuvenation: Feasible or infeasible?

Preventing pathological conditions caused by aging, including cancer, osteoporosis, sarcopenia, and cognitive disorders, is one of the most important issues for human health, especially in societies with large aging populations. Although aging, defined by functional decline of cells/organs or accumulation of cell/organ damage, is one of the most recognizable biological characteristics in all creatures, our understanding of mechanisms underlying the aging process remains incomplete. The primary cause of functional declines occurring along with aging is considered to be the exhaustion of stem cell functions in their corresponding tissues. Stem cell exhaustion is induced by several mechanisms, including accumulation of DNA damage and increased expression of cell cycle inhibitory factors, such as p16 and p21.

Meanwhile, aging at cellular, tissue, organ and organismic levels has been reversed by exposing tissues from old animals to a young environment. Recent studies have suggested that stem cell rejuvenation could reverse organismal aging phenotypes, and that this could be achieved by inhibiting fibroblast growth factor 2, mammalian target of rapamycin (mTOR) complex 1, guanosine triphosphatase and cell division control protein 42. Several additional experiments, such as cross-age transplantation and heterochronic parabiosis, have revealed that some factors in the young systemic milieu can rejuvenate declined thymus gland function, as well as neural and muscle stem cell functions, in samples derived from elderly donors. Furthermore, heterochronic parabiosis experiments have also shown strong inhibition of young tissue stem cells by the aged systemic milieu or old serum.

Although cumulative cellular "intrinsic changes", such as DNA damage, oxidative damage, increased expression of cell cycle inhibitors and mitochondria dysfunction, have been considered likely culprits for the tissue decline observed with aging, cellular rejuvenation induced by young systemic milieu would have been impossible if "intrinsic changes" were the only cause of cellular aging. Therefore, these so-called "causes of aging" should be more properly regarded as effects of aging (i.e., these processes are not causes, but rather consequences of aging), the result of cellular decisions often defined by responses to "extrinsic stimuli". Here some questions arise: If aging at the cellular level were reversed, would it lead to the rejuvenation of the animal at an organismic level? Would it result in prevention of aging and, eventually, life extension?

Examining Changes in Fat Tissue Metabolism with Aging and Calorie Restriction

Here researchers look at some of the changes wrought in the metabolism of fat tissue, both over the course of aging, and under conditions of calorie restriction. Calorie restriction is the practice of eating fewer calories while still obtaining optimal levels of micronutrients. It has been shown to extend life in near all species and lineages tested to date. In the short term in humans it considerably improves measures of health, and over the long term is expected to greatly reduce incidence of age-related disease.

Understanding exactly how calorie restriction produces these benefits is a challenge, since it changes near every aspect of metabolism. Wading through the complexity of cellular biology in search of definitive proof and root causes has proven to be a sizable undertaking. Just look at the much-hyped investigation of sirtuins over the past decade or so, for example, and that is just one tiny slice of the molecular biochemistry relevant to calorie restriction. My prediction is that attempts to understand the calorie restriction response and other common altered states of metabolism in mammals will still be ongoing well into the era of widespread availability of rejuvenation therapies based on the SENS vision, as implementing treatments that repair known forms of cell and tissue damage is a much simpler undertaking than trying to recreate or improve upon the changes created by calorie restriction.

It has been long established that aging is the greatest risk factor for a range of diseases. Caloric restriction (CR) is a dietary intervention that delays aging and extends the period of health in diverse species. One of the hallmarks of caloric restriction is the marked reduction in adiposity, a consequence that may be important in the mechanisms of CR given the endocrine function of adipose tissue. Adipokines and lipokines secreted from white adipose tissue impact peripheral tissue fuel utilization and the balance of energy generation from lipid or carbohydrate sources. However, it is unclear what effect aging has on adipose tissue metabolic integrity and how that relates to secretion of systemic regulatory factors. Prior studies of gene expression in adipose tissues from old rats and adult mice show that CR induces expression of genes involved in multiple aspects of metabolism. A further difference includes the increased circulating levels of the adipose tissue-derived peptide hormone adiponectin with long-term stringent (40%) CR.

In order to understand whether age-related changes in adiposity are associated with a change in adipose tissue function, we undertook a cross-sectional mouse study focusing on adipose tissue metabolism and circulating levels of adipose tissue-derived signaling molecules. To capture the trajectory of aging, the study involved adult, late-middle-aged, and advanced-aged C3B6F1 hybrid mice. Parallel groups of mice on modest (16%) CR taken at each age served to uncover aspects of adipose tissue aging that were responsive to delayed aging. We investigated the relationship between adiposity, adipocyte size, and adiponectin levels at three age groups of mice on control or CR diets. We determined whether differences with age and diet were associated with changes in factors downstream of adiponectin and factors that connect with adiponectin signaling including NAD metabolism. To investigate differences in adipose tissue lipid metabolism, we profiled serum lipids including free fatty acids that are derived from adipose tissue. The goal of these studies was to determine how age and CR impacted adipose tissue function beyond simple differences in adiposity and whether relationships between adipocyte size and secretory profiles were sustained with age or altered with CR.

Adiposity and the relationship between adiposity and circulating levels of the adipose-derived peptide hormone adiponectin were age-sensitive. CR impacted adiposity but only levels of the high molecular weight isoform of adiponectin responded to CR. Activators of metabolism including PGC-1a, SIRT1, and NAMPT were differentially expressed with CR in adipose tissues. Although age had a significant impact on NAD metabolism, the impact of CR was subtle and related to differences in reliance on oxidative metabolism. The impact of age on circulating lipids was limited to composition of circulating phospholipids. In contrast, the impact of CR was detected in all lipid classes regardless of age, suggesting a profound difference in lipid metabolism. These data demonstrate that aspects of adipose tissue metabolism are life phase specific and that CR is associated with a distinct metabolic state, suggesting that adipose tissue signaling presents a suitable target for interventions to delay aging.

Link: http://onlinelibrary.wiley.com/doi/10.1111/acel.12575/full

Continued Trials to Quantify the Benefits of a Fasting Mimicking Diet

Beyond the actual science, researcher Valter Longo's innovation in calorie restriction studies was to find a way to commercialize the undertaking of eating less, thereby pulling more money and attention into the field. With commercial backing comes the funding needed for larger, more rigorous trials and monitoring of outcomes. Moving beyond the earlier studies of human calorie restriction, such as CALERIE, researchers are now attempting to reliably quantify the degree to which one needs to eat less to achieve meaningful benefits: how little and how long. The suggestion resulting from the more recent studies is that intermittent periods of low calorie intake may capture a sizable portion of the benefits realized from fasting or full time calorie restriction. As always it is worth noting that there is nothing special about the product under discussion here; a fasting mimicking diet is easy enough to put together on your own given the calorie and nutrient targets.

A new study finds that providing the body with a temporary, specifically formulated fasting mimicking diet (FMD) called ProLon causes cellular changes normally generated by several days of consecutive water-only fasting and may increase health and lifespan by partially turning back the aging clock. After animal results showing that this FMD reduces incidence of cancer and inflammatory diseases and extends lifespan, researchers have now published the results of a 100-participant randomized Phase II clinical trial demonstrating that ProLon targets the aging process and reduces risk factors for age related diseases such as diabetes, cancer, and cardiovascular disease in humans. These effects are believed to be caused by an increase in stem cell number and regeneration.

Pre-clinical studies demonstrated that ProLon provides the body with the necessary macro and micronutrients while keeping it in a fasting mode and activates stem cell-based regeneration in multiple organs and systems. ProLon is perhaps the first success story in a new but rapidly developing nutri-technology field. The understanding of the molecular connections between specific food components and genes that regulate aging and regeneration allows food to be used to promote cellular changes that are safe but more coordinated than those caused by drugs.

Researchers tested the effects of three monthly ProLon cycles on metabolic markers and risk factors associated with aging and age-related diseases. Each ProLon cycle lasts five consecutive days and does not require alteration to lifestyle during the remaining days of the month. Findings in humans were consistent with mouse studies showing a spike in circulating stem cells and delay in biological aging by promoting regeneration in multiple systems. Body weight, BMI, total body fat, trunk fat, waist circumference, systolic and diastolic blood pressure, cholesterol, insulin-like growth factor 1 (IGF-1), and C-Reactive Protein (a marker of inflammation) were significantly reduced, particularly in participants at risk for diseases, while relative lean body mass (muscle and bone mass) was increased. Low levels of IGF-1 are associated with a lower risk of cancer and diabetes. No serious adverse effects were reported.

Link: https://www.eurekalert.org/pub_releases/2017-02/cci-fm020717.php

A Sample of Recent Work on New Means of Detecting and Targeting Senescent Cells

Senescent cells are receiving a great deal more attention from the research community these days, as illustrated by the two papers on methods of senescent cell identification I'll point out today. How things have changed; it wasn't only a few short years ago that scientists struggled to raising funding for animal studies of senescent cell removal, in an environment of little interest in this aspect of cellular biology. That was the state of the field despite the weight of evidence, gathered over decades, for increased cellular senescence in old tissues to be a root cause of aging and age-related disease. Now that studies have demonstrated that targeted clearance of senescent cells improves health and extends healthy life span in mice, and now that the methods of clearance are being used to produce stronger direct evidence for specific age-related disease and loss of function to involve senescent cells, it seems that every other gerontologist is either revising existing views of aging to incorporate cellular senescence or adding studies of cellular senescence to their portfolio.

Most cells fall into a senescent state when they reach the end of their replicative life span, at which point they either self-destruct or are removed by the immune system. Damage from random mutation or a toxic tissue environment can also result in senescence, and should in theory lead to cell death in the same way as for replicative senescence. Complicating the picture somewhat, short-term localized increases in senescent cell presence also appear to be involved in the wound healing process. There may also be numerous multiple distinct forms of senescence with somewhat different behaviors - this is one of many blank spots remaining on the map of cellular biochemistry, presently under active investigation. Regardless, at the end of the day the ideal situation is that all cells that become senescent should self-destruct or be destroyed fairly soon thereafter. Unfortunately that is not the case in practice, and a fraction of these cells linger on, their numbers growing over the years. These cells cause harm primarily through the signals they generate, producing a potent mix of molecules know as the senescence-associated secretory phenotype (SASP) that degrades nearby extracellular matrix structures necessary for tissue function, spurs increased inflammation, and alters the behavior of neighboring cells for the worse. By the time that 1% or more of cells in a tissue have become senescent the SASP and its downstream consequences become a serious threat to health and organ function.

All of this amounts to a very good reason to support research into identification and removal of senescent cells. Therapies capable of clearing senescent cells should produce a form of limited, narrowly focused rejuvenation, improving health at any point in old age. Those therapies will have to be accompanied by improved assays in order to determine exactly how well they remove senescent cells, as well as to definitively establish links between senescence and specific aspects of age-related degeneration. Below find linked a couple of interesting open access papers in which the authors explore potential new approaches to assessing levels of cellular senescence in tissues and tissue samples. The more of this sort of thing the better, to my eyes. Competition tends to result in better solutions at the end of the day.

Detecting senescence: a new method for an old pigment

Senescent cells have been recently shown to contribute causally to the aging process. Elimination of senescent cells by suicide gene-meditated ablation of p16Ink4a-expressing senescent cells in INK-ATTAC mice has led to improvements in healthspan and lifespan suggesting that senescent cells are drivers of aging. This has prompted the scientific community to identify new interventions to target senescence as a therapy against aging and age-related diseases. However, despite remarkable advances, the detection of senescent cells, particularly in tissues, is still a major challenge. There are several reasons, both of a biological and methodological nature, which have hindered the identification of specific markers able to determine whether a cell is senescent or not.

Firstly, while senescence is characterized by numerous changes in gene expression, very few of these differences are exclusive to senescent cells. Secondly, senescence is a kinetic, multifactorial process, with several phenotypic changes occurring at different time points following the initial cell cycle arrest. This could explain why aged tissues are highly heterogeneous, possibly containing cells at different stages of the senescent programme. Thirdly, senescent cells manifest the phenotype differently depending on the type of inducing stimuli or the cell type. Finally, recent data have highlighted that senescence may play different physiological roles in different contexts. For instance, an 'acute' type of senescence has been shown to play a beneficial role during processes such as development or tissue repair, while a 'chronic' type of senescence may contribute to aging and age-related disease. The recent realization that there may be different types of senescent cells in tissues has created an additional obstacle to the identification of a universal marker.

The detection of senescence-associated β-galactosidase (SA-β-Gal) activity at pH 6 is probably the most widely utilized method for identification of senescent cells. Nevertheless, there are major limitations to this method. Given the growing realization that senescence is a multifactorial process, a multimarker approach is being favoured by many researchers in the field. Examples of currently used markers are as follows: increased expression of cyclin kinase inhibitors p21 and p16 and absence of proliferation markers; telomere-associated DNA damage foci; senescence-associated heterochromatin foci; loss of lamin B1; senescence-associated distension of satellites (SADS); and expression of components of the SASP amongst several others. Nonetheless, there is also growing realization that many of these markers are not exclusive to all types of senescence and may only occur in specific cell types.

Lipofuscin is a nondegradable aggregate of oxidized lipids, covalently cross-linked proteins, oligosaccharides and transition metals which accumulate within lysosomes. Multiple studies indicate that lipofuscin accumulates in various tissues and species with age, particularly postmitotic tissues such as the brain and cardiac and skeletal muscle. However, lipofuscin has also been shown to accumulate during replicative senescence of human fibroblasts. Lipofuscin is autofluorescent and can be visualized using fluorescent microscopy; however, several other histochemical methods have been described based on lipid detection, such as staining using Sudan Black B (SBB) amongst others. Here, a structurally similar compound to SBB has been designed and coupled to biotin. Commercially available SBB contain numerous impurities which impact on staining quality and justified the need to synthesize a new analogue. The chemical coupling with biotin allows its detection using antibiotin antibodies and thereby increases its detection sensitivity. This method is versatile: it can be used in fresh, frozen cells and tissues, but also in fixed material. Furthermore, it can be identified in cells using both microscopy and flow cytometry.

While the authors have convincingly demonstrated that lipofuscin accumulation correlates with senescent markers in cell culture and that lipofuscin increases in tissues with age, future work should investigate more thoroughly whether and to what extent the lipofuscin signal overlaps with other established senescent markers. A separate question which arises from this work is whether lipofuscin accumulation is a mere consequence of the induction of the senescence programme or whether its accumulation contributes causally to the development of senescence.

Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody

Studying the phenomenon of cellular senescence has been hindered by the lack of senescence-specific markers. As such, detection of proteins informally associated with senescence accompanies the use of senescence-associated β-galactosidase as a collection of semiselective markers to monitor the presence of senescent cells. To identify novel biomarkers of senescence, we immunized BALB/c mice with senescent mouse lung fibroblasts and screened for antibodies that recognized senescence-associated cell-surface antigens by FACS analysis and a newly developed cell-based ELISA. The majority of antibodies that we isolated, cloned, and sequenced belonged to the IgM isotype of the innate immune system.

In-depth characterization of one of these monoclonal, polyreactive natural antibodies, the IgM clone 9H4, revealed its ability to recognize the intermediate filament vimentin. By using 9H4, we observed that senescent primary human fibroblasts express vimentin on their cell surface, and mass spectrometry analysis revealed a posttranslational modification on cysteine 328 (C328) by the oxidative adduct malondialdehyde (MDA). Moreover, elevated levels of secreted MDA-modified vimentin were detected in the plasma of aged senescence-accelerated mouse prone 8 mice, which are known to have deregulated reactive oxygen species metabolism and accelerated aging.

Based on these findings, we hypothesize that humoral innate immunity may recognize senescent cells by the presence of membrane-bound MDA-vimentin, presumably as part of a senescence eradication mechanism that may become impaired with age and result in senescent cell accumulation. Given the growing evidence that oxidized proteins are involved in the development of human disease, the detection and monitoring of secreted proteins like oxidized vimentin is certain to become a vital and noninvasive biomarker for monitoring age-related illnesses.

The Benefits of Hormesis Require Autophagy

Hormesis describes the outcome of a little damage inflicted upon an organism or tissue resulting in a net gain in health and function. Exercise, lack of nutrients, heat, and low levels of toxins or radiation all stress cells, damaging proteins and structures, causing the affected cells to boost their repair and maintenance efforts for some time. If the exposure to damaging circumstances is sufficiently mild and short-lived, then the overall result is an improvement, the additional maintenance activities more than compensating for the damage inflicted. Researchers here demonstrate that this beneficial response requires the cellular recycling process of autophagy, responsible for removing structures and proteins that have become damaged or dysfunctional. The research community has for some time shown an interest in building therapies to slow the progression of aging based on enhancement of autophagy, but beyond calorie restriction mimetic research there has been surprisingly little concrete progress on this front.

Biologists have known for decades that enduring a short period of mild stress makes simple organisms and human cells better able to survive additional stress later in life. Now, scientists have found that a cellular process called autophagy is critically involved in providing the benefits of temporary stress. Autophagy is a means of recycling cells' old, broken, or unneeded parts so that their components can be re-used to make new molecules or be burned for energy. The process had previously been linked to longevity. The new results suggest that long life and stress resistance are connected at the cellular level.

The researchers incubated C. elegans worms at 36 °C, significantly above the temperature they are usually kept at in the laboratory, for one hour. After this short heat exposure - a mild form of stress that improves the organism's survival - autophagy rates increased throughout the worms' tissues. When they exposed these heat-primed worms to another, longer heat shock a few days later, worms that were deficient in autophagy failed to benefit from the initial mild heat shock, as observed in heat-primed worms with intact autophagy.

The researchers reasoned that a mild heat stress might also improve the worms' ability to handle another condition that worsens with age - buildup of aggregated proteins, which is stressful for cells. To test this hypothesis, they used worms that model Huntington's disease, a fatal inherited disorder caused by neuronal proteins that start to stick together into big clumps as patients age, leading to degeneration throughout the brain. Exposing worms that make similar sticky proteins in different tissues to a mild heat shock reduced the number of protein aggregates, suggesting that a limited amount of heat stress can reduce toxic protein aggregation. "Our finding that brief heat exposure helps alleviate protein aggregation is exciting because it could lead to new approaches to slow the advance of neurodegenerative diseases such as Huntington's. This research raises many exciting questions. For example, how does induction of autophagy by a mild heat stress early on make cells better able to survive heat later - what's the cellular memory? There's a lot to follow up on."

Link: http://www.newswise.com/articles/what-doesn-t-kill-you-makes-you-stronger

Theorizing on a Mitochondrial Death Spiral

Mitochondria are the power plants of the cell, their activities essential for all energetic processes and actions in the body. They are descendants of symbiotic bacteria, a swarm in every cell, and carry their own DNA. Unfortunately that DNA can become damaged in ways that subvert the normal cellular quality control mechanisms to cause significant dysfunction; that a growing number of cells fall into this state over time is one of the contributing causes of aging and age-related disease. The author of this paper theorizes that mitochondrial DNA damage in aging is an example of antagonistic pleiotropy, meaning that it exists because evolution has guided mitochondrial structure and quality control processes to enhance early life success via mechanisms that also cause later failure and dysfunction.

From an evolutionary perspective, aging has been difficult to understand. Natural selection increases organismal fitness, and yet aging, which clearly decreases fitness, is not only observed, but also appears to be nearly universal within multicellular (and even some single-celled) organisms. To address this dilemma, it was proposed that aging occurs and is fixed in populations because alleles that have deleterious effects in old age benefit growth, survival, and reproduction in youth. This theory is called antagonistic pleiotropy (AP) theory. In this view, aging occurs because alleles that in the short term are beneficial in solving problems in growth and reproduction serve to exacerbate the problem in the long run. Therefore, aging can be viewed as a form of death spiral. A death spiral, also known as a vicious circle, is a specific form of positive feedback in which steps taken to handle a particular problem, while successful in the short term, exacerbate the problem in the long term.

If this premise is accepted, the next step is to identify the alleles that mediate AP, understand the nature of these alleles, how they might exert AP, and finally identify and define the critical cellular processes affected by AP. Although genes of the insulin signaling pathway likely participate in AP, the insulin-regulated cellular correlates of AP have not been identified. The mitochondrial quality control process called mitochondrial autophagy (mitophagy), which is inhibited by insulin signaling, might represent a cellular correlate of AP. In this view, rapidly growing cells are limited by ATP production; these cells thus actively inhibit mitophagy to maximize mitochondrial ATP production and compete successfully for scarce nutrients. This process maximizes early growth and reproduction, but by permitting the persistence of damaged mitochondria with mitochondrial DNA mutations, becomes detrimental in the longer term.

I suggest that as mitochondrial ATP output drops, cells respond by further inhibiting mitophagy, leading to a further decrease in ATP output in a classic death spiral. I suggest that this increasing ATP deficit is communicated by progressive increases in mitochondrial reactive oxygen species (ROS) generation, which signals inhibition of mitophagy via ROS-dependent activation of insulin signaling. This hypothesis clarifies a role for ROS in aging, explains why insulin signaling inhibits autophagy, and why cells become progressively more oxidized during aging with increased levels of insulin signaling and decreased levels of autophagy. I suggest that the mitochondrial death spiral is not an error in cell physiology but rather a rational approach to the problem of enabling successful growth and reproduction in a competitive world of scarce nutrients.

Link: http://onlinelibrary.wiley.com/doi/10.1111/acel.12579/full

Ichor Therapeutics Announces Lysoclear SENS Rejuvenation Therapy and Series A Fundraising for Further Development

As regular readers will be aware, the company Ichor Therapeutics has for the past year or so been actively developing one of the results of the LysoSENS medical bioremediation program in order to produce a viable therapy. Today there is news of progress, and the work is moving on to the next stage of development and funding. This line of research sought to find bacterial enzymes that can degrade forms of metabolic waste that our cellular biochemistry struggles with, particular the constituents of lipofuscin. Lipofuscin compounds, varying in type from tissue to tissue, accumulate in the cellular recycling system known as the lysosome. That is where cellular waste ends up, but what happens when it cannot be effectively broken down? The answer is that lysosomal activity starts to fail, and cells fall into a form of garbage catastrophe as a result, a process of growing damage and functional decline that, as it happens across all cells in a tissue, contributes to degenerative aging. In most cases the process of cause and effect that leads from lipofusin to age-related disease isn't clearly and completely mapped, but for some conditions the contribution of lipofuscin compounds is quite direct, and so better known. Ichor is focused on the metabolic waste compound A2E as it pertains to macular degeneration, a common age-related condition of progressive blindness. Here, researchers have very good evidence for the harms caused fairly directly via A2E accumulation.

The best thing to do with unwanted metabolic waste is to find a way to safely break it down so that its components can be recycled appropriately. Given that this waste is a cause of aging, successful removal will be a narrow, targeted form of rejuvenation. The path chosen by the SENS Research Foundation in their LysoSENS program was based on the observation that graveyards and similar locations do not appear to be saturated with human metabolic waste. Therefore soil bacteria must be consuming this material. Given the enormous number and variety of bacterial species, somewhere in there is very likely to be found one or more molecules that can form the basis for a drug that can break down metabolic waste compounds without harming cells. Finding such a compound starts with culturing bacteria in order to find those that can thrive on a diet of human lipofuscin, and after some years of work, a range of candidates for various forms of metabolic waste were indeed discovered, including one for A2E. Given a single suitable molecule, it is then possible to build others in the same class, and search for those that are most effective and least likely to harm cells and tissues via unwanted side-effects.

The lipofuscin constituent A2E is peculiar to the very energetic metabolism of retinal cells, so Ichor Therapeutic's work is the production of a very narrowly focused rejuvenation therapy indeed, applicable only to this tissue in the eye. It is nonetheless a rejuvenation therapy and one of a growing number of examples of the work of the SENS Research Foundation moving to the clinic. This may be just one tissue, but there are a great many patients suffering with macular degeneration, no currently effective treatment for the dry form of the condition, and consequently signs of progress in the field tend to attract attention from Big Pharma. The work of the SENS Research Foundation and Ichor Therapeutics here is another article of proof to show that the right way to proceed towards the effective treatment of aging and age-related disease is to repair and reverse the fundamental differences between old and young tissue - such as the accumulation of metabolic waste that our biochemistry cannot effectively break down.

Considering all of this, I'm pleased to note, both as an investor in the company and as someone who wants to see the field of rejuvenation research grow enormously, that the work at Ichor Therapeutics has continued to produce excellent results as it moved into animal studies. The company has accordingly announced the Lysoclear product line, and is now seeking series A funding for further development leading towards a clinical therapy to turn back the progression of macular degeneration by removing one of the root causes of the condition, the A2E accumulation. Everyone who, back in the day, helped out with the early LysoSENS research in one way or another, as researchers, advocates, and donors to the Methuselah Foundation and SENS Research Foundation, should be feeling proud and vindicated today.

Lysoclear

Lysoclear is an enzyme therapy being developed for age-related macular degeneration (AMD) and Stargardt's macular degeneration. Age-related macular degeneration (AMD) is the leading cause of vision loss among people over the age of 50, affecting 20 million Americans. Stargardt's macular degeneration is an inherited conditions that robs children of their sight. Lysoclear shows promise as a highly targeted treatment for both conditions. Lysoclear has been extensively studied in buffer systems, cell culture models, and in vivo, and findings suggest that Lysoclear is safe and effecting at destroying toxic vitamin A aggregates (A2E) that may cause these diseases. Lysoclear is safe and effective at breaking down toxic A2E, removing up to 10% with each dose. Lysoclear selectively localizes to the lysosomes of retinal pigmented epithelial (RPE) cells where A2E accumulates, and destroys it.

Age-related macular degeneration (AMD) and Stargardt's macular degeneration (SMD) are thought to arise from the gradual loss of RPE cells of the macula, the area of the eye responsible for central vision. The accumulation of toxic vitamin A aggregates, including the bis-retinoid A2E, have been implicated in these diseases. Recent research suggests that A2E is capable of binding native lysosomal enzymes, inhibiting their function. As A2E accumulation reaches a critical threshold, lysosomal impairment leads to the accumulation of intracellular lipofuscin, extracellular drusen deposition, and eventually RPE cell death. Lysoclear is a recombinant enzyme product under development by Ichor Therapeutics that is able to selectively localize to the lysosomes of RPE cells where A2E accumulates, and destroy it.

Ichor Therapeutics announces series A offering for LYSOCLEAR to move first SENS therapy into the clinic

Today, Ichor Therapeutics, a biotechnology company that focuses on developing drugs for age-related diseases, announced a series A offering to bring its Lysoclear product for age-related macular degeneration (AMD) and Stargardt's macular degeneration (SMD) through Phase I clinical trials. This product would be the first clinical candidate based on the SENS paradigm, pioneered by biomedical gerontologist Dr. Aubrey de Grey. AMD is the leading cause of vision loss among people over the age of 50. The underlying pathology of AMD is thought to be caused by the death of retinal pigmented epithelial (RPE) cells, which photoreceptors in the macula rely upon to survive. RPE cells assist photoreceptors in various metabolic roles, including the recycling of vitamin A, an essential component of the visual cycle. However, this is a leaky process, and trace by-products are formed that accumulate in the lysosomes of RPE cells. The most well studied of these by-products is A2E, a toxic compound which may play a causative role in AMD and SMD.

Although A2E accumulates gradually over the lifespan, it is generally not until later age that A2E reaches a threshold necessary to promote toxicity. At high concentrations, A2E promotes the formation of intracellular junk termed lipofuscin. RPE cells attempt to handle this accumulation by shuttling the junk out in the form of extracellular drusen. Eventually, the RPE cells choke on the garbage, and cell death accompanies complement activation, inflammation, and hypoxia. Multiple companies have developed drugs that successfully reduce the rate of A2E formation, but such interventions may be too late for symptomatic patients, who have already had the cascade kicked off.

In 2014, Ichor Therapeutics completed a material and technology transfer agreement for rights to concepts and research pioneered by SENS Research Foundation. In 2017 Ichor announced Lysoclear, a recombinant enzyme product that selectively localizes to the lysosomes of RPE cells where A2E accumulates, and destroys it. Ongoing studies suggest that LYSOCLEAR is safe and effective at targeting A2E, eliminating up to 10% with each dose. Ichor has opened a Series A funding round to support pre-clinical Investigational New Drug (IND) enabling studies and phase I human clinical trials for AMD and SMD.

A Different Take on a Cellular Garbage Catastrophe in Neurodegeneration

The garbage catastrophe view of aging in long-lived cell populations with little turnover, such as those of the brain, is fairly well established. Over-simplifying somewhat, it is a downward spiral in which accumulated molecular damage and metabolic waste in cells makes their maintenance processes ever less efficient, which in turn leads to a faster increase in damage and waste. That ultimately leads to cellular senescence, or programmed cell death, or other forms of dysfunction. Here, researchers present a somewhat different take on a garbage catastrophe, one in which cells sabotage one another by ejecting waste and damaged proteins into the surrounding environment:

Neurodegenerative diseases like Alzheimer's and Parkinson's may be linked to defective brain cells disposing toxic proteins that make neighboring cells sick. Researchers found that while healthy neurons should be able to sort out and rid brain cells of toxic proteins and damaged cell structures without causing problems, this does not always occur. These findings could have major implications for neurological disease in humans and could possibly be the way that disease can spread in the brain. "Normally the process of throwing out this trash would be a good thing. We think that there might be a mismanagement of this very important process that is supposed to protect neurons but, instead, is doing harm to neighbor cells."

Scientists have understood how the process of eliminating toxic cellular substances works internally within the cell, comparing it to a garbage disposal getting rid of waste, but they did not know how cells released the garbage externally. "What we found out could be compared to a person collecting trash and putting it outside for garbage day. They actively select and sort the trash from the good stuff, but if it's not picked up, the garbage can cause real problems."

Working with the transparent roundworm C. elegans, which are similar in molecular form, function, and genetics to those of humans, researchers discovered that the worms - which have a lifespan of about three weeks - had an external garbage removal mechanism and were disposing these toxic proteins outside the cell as well. The team realized what was occurring when they observed a small cloud-like, bright blob forming outside of the cell in some of the worms. Over two years, they counted and monitored their production and degradation in single still images until finally they caught one in mid-formation. Roundworms engineered to produce human disease proteins associated with Huntington's disease and Alzheimer's threw out more trash consisting of these neurodegenerative toxic materials. While neighboring cells degraded some of the material, more distant cells scavenged other portions of the diseased proteins.

Link: http://news.rutgers.edu/research-news/alzheimer%E2%80%99s-may-be-linked-defective-brain-cells-spreading-disease/20170212

Calorie Restriction and the Ribosome

Ribosomes are structures within which protein assembly takes place in cells. Many interventions that modestly slow aging, such as calorie restriction, are associated with both a slower rate of protein production and a slower turnover of ribosomes - which, like near all structures in the cell, are periodically replaced as they become damaged or dysfunctional. The direction of causation in this and associated effects is still up for debate, though a consensus is emerging. In this context it is interesting to note that there is some evidence for selective ribosomal dsyfunction to mimic some of the effects of calorie restriction. Further, naked mole-rats, those paragons of mammalian longevity, have been found to have highly efficient ribosomes. Determining how this all fits together into a coherent picture of the effects of calorie restriction on aging, as well as the differences in aging between short-lived versus long-lived species, is still a work in progress.

Recent research offers one glimpse into how cutting calories impacts aging inside a cell. The researchers found that when ribosomes - the cell's protein makers - slow down, the aging process slows too. The decreased speed lowers production but gives ribosomes extra time to repair themselves. "The ribosome is a very complex machine, sort of like your car, and it periodically needs maintenance to replace the parts that wear out the fastest. When tires wear out, you don't throw the whole car away and buy new ones. It's cheaper to replace the tires." So what causes ribosome production to slow down in the first place? At least for mice: reduced calorie consumption.

Researchers observed two groups of mice. One group had unlimited access to food while the other was restricted to consume 35 percent fewer calories, though still receiving all the necessary nutrients for survival. "When you restrict calorie consumption, there's almost a linear increase in lifespan. We inferred that the restriction caused real biochemical changes that slowed down the rate of aging." The team isn't the first to make the connection between cut calories and lifespan, but they were the first to show that general protein synthesis slows down and to recognize the ribosome's role in facilitating those youth-extending biochemical changes. "The calorie-restricted mice are more energetic and suffered fewer diseases. And it's not just that they're living longer, but because they're better at maintaining their bodies, they're younger for longer as well."

Ribosomes, like cars, are expensive and important - they use 10-20 percent of the cell's total energy to build all the proteins necessary for the cell to operate. Because of this, it's impractical to destroy an entire ribosome when it starts to malfunction. But repairing individual parts of the ribosome on a regular basis enables ribosomes to continue producing high-quality proteins for longer than they would otherwise. This top-quality production in turn keeps cells and the entire body functioning well.

Link: http://news.byu.edu/news/how-eating-less-can-slow-aging-process