Fight Aging! Newsletter, March 7th 2016

The neurobiologists treated Alzheimer's disease model mice with a small-molecule inhibitor compound called pexidartinib, or PLX3397, which is currently being used in several cancer studies. The inhibitor works by selectively blocking signaling of microglial surface receptors, known as colony-stimulating factor 1 receptors, which are necessary for microglial survival and proliferation in response to various stimuli, including beta-amyloid. This led to a dramatic reduction of these inflammatory cells, allowing for analysis of their role in Alzheimer's. The researchers noted a lack of neuron death and improved memory and cognition in the pexidartinib-treated mice, along with renewed growth of dendritic spines that enable brain neurons to communicate. Although the compound swept away microglia, the beta-amyloid remained, raising new questions about the part these plaques play in Alzheimer's neurodegenerative process.

In this study, we set out to determine if chronically activated microglia in the Alzheimer's disease brain are also dependent on CSF1R signalling, and if so, how these cells contribute to disease pathogenesis. Ten-month-old 5xfAD mice were treated with a selective CSF1R inhibitor for 1 month, resulting in the elimination of ∼80% of microglia. Chronic microglial elimination does not alter amyloid-β levels or plaque load; however, it does rescue dendritic spine loss and prevent neuronal loss in 5xfAD mice, as well as reduce overall neuroinflammation. Importantly, behavioural testing revealed improvements in contextual memory. Collectively, these results demonstrate that microglia contribute to neuronal loss, as well as memory impairments in 5xfAD mice, but do not mediate or protect from amyloid pathology.

We will not cover all of the compounds that have been studied to extend lifespan, but those that I find most promising or most interesting. We will also discuss compounds that possibly work through different mechanisms. This could mean that we are neglecting some potentially important compounds, so we will try to explain what we think constitutes good evidence for a compound being promising. Extending lifespan in a model organism is certainly an important factor.

Combinatorial interventions are used for treating different diseases in the clinic. In cancer therapies, combinatorial treatments are used with success. The cancer can be attacked from different angles with multiple targeted therapies and cytotoxic agents. So what is the difference between testing one compound and testing a combination of compounds? At some level a single compound may act as a combination of compounds. Single compounds can bind to many targets. But aren't those off-target effects just creating negative side effects? Often that may be true, but some compounds might work well, precisely because they have multiple targets.

If two compounds can extend lifespan separately, together they must be even better, right? Well, it might not be that simple. The only published combination of compounds that has successfully extended lifespan to my knowledge is valproic acid and trimethadione in C. elegans. On their own, valproic acid extended the mean lifespan by 35% and trimethadione extended it by 45% at optimal dose. Together they extended the mean lifespan by 61%. This same study from the Kornfeld lab also shows that combining two compounds that on their own extend lifespan, can produce toxic effects. This was the case when mixing ethosuximide with trimethadione. So why is this? One explanation can be that they are both acting on the same pathway. Too much of a good thing is not always good.

So how do we know which compounds will work well together? The truth is we don't really know. We can try to make educated guesses though. Perhaps targeting different pathways is the way to go. However, sometimes targeting the same pathway can yield very potent effects with genetics. The insulin/IGF1 signaling pathway can be targeted from different angles in C. elegans yielding very long lived worms. There is also the possibility that two compounds targeting the same pathway can maximize the beneficial outcome while minimizing the side effects. Targeting different pathways can also work very well. In C. elegans the TOR and insulin signaling pathway were targeted genetically to produce a worm with nearly 5 times its normal lifespan.

Rapamycin and metformin are two drugs that could potentially work well together to extend longevity. A side effect of rapamycin is insulin resistance but metformin improves this condition. A combination of rapamycin and metformin treatment was started by the NIA Interventions Testing Program in 2011. No results are yet published, but rumor has it, it's working. One combination of compounds that was reported to be working in mice to combat one aspect of aging is quercetin and dasatinib. The combination is for killing senescent cells. They were each effective in killing senescent cells in different tissues but worked better together. No lifespan was measured, but health was improved. The mice were able to run farther on a treadmill.

Testing different compounds in combination is certainly a daunting task. How many compounds do you want in your longevity cocktail? Lets say we want to test 100 promising geroprotectors in combination. Perhaps we are assaying lifespan of C. elegans in a 96-well format. If we are just doing pairwise combinations it is almost manageable. 10,000 combinations performed in triplicate make 30,000 wells to be assayed. Only very large effects will be noticed in these types of screens if we are to eliminate false positives. If we are combining 3 or 4 compounds together in our screen we need to assess 3,000,000 or 300,000,000 wells respectively. In this setup, we are only using one concentration per compound. We have seen that sometimes concentrations of drugs need to be lowered when used together with another drug. A way to get around some of the problem is to first do a pairwise screen and use your best combination as the base for the next screen. This way you can keep adding compounds to your cocktail with a manageable amount of screening. This will of course miss some combinations that would be in the massive 300,000,000 well screen but some compromises always have to be made.

One of the main problems with research in general is the reliability of the data. Many times a finding is not repeatable when tried by another lab. This is usually not due to fraud or anything as nefarious as that. It is mainly that research is hard and there are many ways to fool yourself. One way to easily fool yourself is to use too small a sample size. This is quite common. Using a small sample size when looking at lifespan increases the chance that a long-lived or short-lived population was picked by chance. Generally around 100 animals should give around 80% chance of detecting a 10% difference in lifespan. By detecting I mean the difference being statistically significant (if they indeed are). But not only large sample sizes and statistical significance are important. Large effects are generally more reproducible than small effects. What I like to see is a large effect with a large sample size with a good statistically significant result. It is always nice to see if can be replicated by other groups. However, many small badly designed studies do not equal one large well-designed study.

Besides Weismann's evolutionary theory, many additional theories have been proposed to explain the complexity of aging. These include the antagonistic pleiotropy theory, the free radical theory, age-associated shortening of telomeres, development of insulin resistance, decreased immune function, the mitochondrial theory, as well as deregulation of the circadian clock. While these theories indicate functional diversity in the etiology of aging, it must be stressed that each one relies on the concept of internal alterations in individual cells, and does not explain how the microscopic cellular damage manifests as macroscopic aging and tissue breakdown in the organism (with a few exceptions, such as changes in hormone function and declines in immune function). Theories of mutation accumulation and antagonistic pleiotropy address the genetic causes of aging, and environmental stress or lack of it contributes to modulation of the epigenome as well as physiological alterations in different tissues of the whole organism, but each theory revolves around the functional competence of different components of cells and again does not explain how this manifests as macroscopic organismal aging. Experimental evidence unifying the interactions of some components has started to emerge, but we propose that all of the changes described by diverse theories ultimately converge on the cellular senescence theory.

Since aging is a progressive condition that steadily advances from invisible to visible and localized to ubiquitous, the central question as to the direct cause of the entire process is key. The answer has been elusive due to its complex nature. Our model proposes that the process of aging results from a sequential passage through three distinct phases and can be described by the following blueprint: (1) molecular damage which results in (2) cessation of proliferation leading to cellular senescence followed by (3) body-wide aging of the organism. The first step occurs when localized, microscopic damage accumulates to a point where the burden to repair overwhelms the system. Despite the tissue source or broad input of molecular damage, crossing of this threshold results in the second phase, the crux of the entire process - arrest of cellular proliferation, acquisition of the senescence-associated secretory phenotype (SASP), and imminent cellular senescence. Once this occurs, the third phase of aging begins. This final phase is marked by tissue dysfunction and breakdown that results in the visible signs of comprehensive organismal aging.

The incremental advance proposed by our model is that while there are many undisputed factors that trigger the onset of cellular senescence and result in cessation of proliferation and SASP, the first phase in the model (cumulative molecular damage) is a precursor, rather than a final cause of aging. The complexity normally imposed by countless variables (i.e., age of onset, site of damage, affected cell type, mechanism of damage, and even species) that need to be overcome is rendered manageable by eliminating the first phase in the aging schematic. And since organismal aging can be artificially and reversibly induced by blocking and restarting cellular proliferation, this indicates that the second phase in the model - cessation of proliferation followed by cellular senescence - clearly represents the essential cause of aging. Placing cellular senescence in the pivotal junction between cause and effect, the causal nexus, to yield an integrated model of aging will serve to advance identification of crucial targets for future therapeutic investigation. By identifying cellular senescence as the causal nexus of aging, the process of treating, reversing and possibly even eventually eliminating this once inevitable outcome draws closer to reality.

There are no comparable human studies with CR. Type 2 diabetes mellitus in humans is currently described as a progressive disease with a pathophysiology that involves over eight different organ systems. However, this understanding of disease does not really give a valid explanation to the reversibility and induction of normal glucose tolerance in patients with type 2 diabetes who undergo bariatric surgery. The improvements in glucose control happen within a few days after surgery much before there is any significant reduction in body weight. There are many explanations offered for early improvement in glucose tolerance like changes in gut hormone profile, changes in gut bacteria, etc., Both these overlook the most logical explanation for the phenomenon which is an acute profound decrease in calorie intake.

However, considering the difficulties in getting healthy adults to limit food intake science has focused on understanding the biochemical processes that accompany calorie restriction (CR) to formulate drugs that would mimic the effects of CR without the need to actually restrict calories. Drugs in this emerging therapeutic field are called CR mimetics. Some of the currently used anti-diabetic agents may have some CR mimetic like effects. This review focuses on the CR mimetic properties of the currently available anti-diabetic agents.

Overall, our study has demonstrated that increasing levels of CR lead to a graded expression of genes involved in both hunger signaling and circadian rhythms. The expression of genes in these pathways wwere correlated with circulating levels of leptin, insulin, TNF-α and IGF-1, but not resistin or IL-6. We also demonstrated the phenotypic responses to CR (body temperature and physical activity) were significantly associated with the key hunger and core clock genes. Our results suggest that under CR modulation of the hunger and circadian signaling pathways, in response to altered levels of circulating hormones, drive some of the key phenotypic outcomes, such as activity and body temperature, which are probably important components of the longevity effects of CR.

Recently evidence was provided that the integration of cell-free nucleic acid with host cells occurs in vivo as well as in vitro. Mice were injected intravenously with human cfDNA and Cfs and analysis of heart, lung, liver, and brain of the mice sacrificed 7 days after injection revealed genomic localization of nucleic acids, with Cfs localizing more efficiently than cfDNA. Of note, genomic integration of Cfs in the mouse brain indicated that chromatin particles are able to cross the blood-brain barrier. This recent work offers a fascinating new mechanism of age-related mutagenesis, highlighting the fate and effects of free nucleic acids within our body. However, many questions remain. Probably the most interesting question is whether cfDNA truly behaves as mobile genetic elements under normal conditions. That is, rather than extracting concentrated cfDNA from heterogenic serum samples and intravenously injecting that in the mouse, integration of its own cfDNA should be studied, for example, as a function of age. Because integrated DNA fragments can then no longer be uniquely aligned as foreign DNA to a reference sequence, single cells or clones should be studied for insertion events as compared to the germline sequence, which is considerably more difficult than screening for reads containing human sequences.

While still lacking in important details, this recent work opens up the intriguing prospect of a new, endogenous source of genome instability that could well contribute to increased genome mosaicism with age. In this respect, cfDNA could act similarly to the previously described age-related derepression of endogenous retrotransposons in the somatic genome during aging. In this respect, there is evidence that cfDNA becomes increasingly frequent in the circulation as a function of age, for example, due to increased vulnerability of aged and damaged cells to cell death. Its activation of the DNA damage response could increase the level of genome instability considerably, contributing to aging-related degenerative processes, such as cellular senescence, cancer, and inflammation. Further research on the biological and pathological roles of cell-free nucleic acids will help to elucidate its importance as an intrinsic mechanism of aging.

"We can explain a lot of the disagreement in the C. elegans aging field by realizing that FuDR can dramatically change the answer. There were very different effects in published papers that had different doses of FUdR in them. Sometimes it's a very profound disagreement." Moreover, some other studies may involve FUdR-related discrepancies but insufficient documentation of the methods prevented researchers from being sure.

In the absence of any stress, FUdR makes no difference to lifespan in normal worms, they confirmed. But when worms were exposed to a modest concentration of salt, animals who were not exposed to FUdR had only half the lifespan of those who were exposed to the drug. Meanwhile, adding even more FUdR caused even longer lifespan under salt stress. A tenfold increase in FUdR concentration extended lifespan by a factor of three. Other experiments suggested that FUdR causes better stress resistance in hot or low-oxygen conditions. Further research revealed details of how FUdR protects the worms from stress. They found evidence that the drug turns on the gene that produces the protein FOXO, a master regulator of stress resistance in many organisms that is often central in longevity studies. They also found that exposure to FUdR forced DNA mutations that then activated a DNA-repair process. That process, once activated, also fixes a lot of DNA damage caused by environmental stresses, including dreaded double-strand breaks, a clean severing of the DNA molecule.

Most heart muscle diseases involve problems with contraction. Many strategies increase the calcium signal to improve heart contraction. However, they do so at the expense of other functions. This can cause negative side effects, such as arrhythmias and cell death, and ultimately increase mortality. The team evaluated TnC L48Q in a common heart pathology - myocardial infarction, or a heart attack. Compared to the infarcted control group, TnC L48Q mice had better heart function and cardiovascular performance. There were also no signs of congestive heart failure or increased mortality, both of which were observed in the control group. When assessing the long-term effects and therapeutic potential of TnC L48Q, the researchers observed steady and significant improvement in heart function, cardiovascular performance, and significantly less detrimental remodeling compared to the control group. This resulted in better survival.

"It's long been presumed that altering the receptor would be ineffective, that it was better to change the calcium signal. We're seeing strong evidence that's not the case. Changing the calcium receptor does have a significant and safer impact." The scientists report these results were achieved by replacing only a modest amount of the original TnC receptor through gene therapy. This makes it more likely that this strategy will be a viable and personalized treatment option in the future. The researchers believe these findings could open the door for new treatments against cardiac diseases. In previous in vitro work, the team has customized several TnC receptors designed to combat various cardiac disorders. The team is also working on engineering other calcium receptors for a variety of diseases, such as high blood pressure and heart arrhythmias.

It's hard for anyone to put a number on it. Depending on whom you talk to, those estimated ages of longevity - again, 81.4 years for men and 84.3 for women, according to 2011 actuarial tables - may be just so much hokum. As the science of human longevity continues to accelerate, this will likely translate into improved life expectancy across the board.

Heart disease, cancer and other terminal illnesses can all be traced back to the natural wear and tear that comes with living. As we age, our bodies break down: Tissues and organs become defective and the likelihood increases that our cells will fail us. "Ninety percent of us will die of diseases that do not kill 20-year-olds," says George Church, a professor of genetics at Harvard Medical School. Advances in medicine - which over the past century raised the average lifespan in the United States from 47 to 77 - simply delay the inevitable certainty that we will grow old and, eventually, die. But what if we could reverse aging? According to Church, "That's already happening." Remember those stem cells? Other body parts are figuring into the mix, too. To be clear, Church is talking about mice. Still, the research is compelling.

Such experiments, along with advancements in regenerative medicine, stem cell therapy and gene editing, mean that in a hazy but potentially not-too-distant future, we will no longer die from natural causes. Death, in other words, will be relegated to the realms outside age and disease. Plan to live longer than you expect to. After all, the anecdotal evidence out there supports it.

Some prominent biogerontologists have provided comprehensive weighted responses explaining the dangers of separation of aging from disease and benefits of proactive preventative approaches that are likely to result from recognizing the pathological nature of aging. In spite of the many breakthroughs providing proof of concept for successful interventions in aging in model organisms, human progress has been surprisingly slow. One major cause of inaction is a widely held, but flawed, conceptual framework concerning the relationship between aging and disease that categorizes the former as "natural" and the latter as "abnormal". One comprehensive review of the many arguments for and against classifying aging as a disease with a definite and eloquent recommendation that calls for a complete quote: "We must draw aside the rosy veil of tradition and face aging for what it is, and in all its horror: the greatest disease of them all."

Bulterijs et al. explained the many benefits of classifying aging a disease, while Stambler provided a historical perspective arguing that acknowledging the possibility of successful intervention into the aging process, in other words treating aging as a curable disease, has been a long and highly respected tradition of biomedical thought. Dubnikov and Cohen provided an overview of multiple theories of aging and recommended further research to understand the relationship between aging and disease. Advocates for longevity research provided new survey data indicating that the majority (74.4%) of Americans are interested to live to 120 or longer if health was guaranteed, but only 57.4% wished to live that long if it wasn't, contradicting previous surveys that used different approaches to surveying the general population and generally indicated negative attitudes toward increased longevity and longevity-boosting interventions.

The main international agency responsible for disease classification is the World Health Organization (WHO), which maintains and publishes the International Statistical Classification of Diseases and Related Health Problems (ICD) since 1948. The 10th revision of the ICD, referred as ICD-10, was first published in 1992, and the 11th revision (ICD-11) is expected to be released in 2018. WHO classifies aging as a disease in the ICD-10 with the R54 code. However, this code is generally regarded by the Global Burden of Disease (GBD) statisticians as a "garbage code" and cannot be considered to be actionable. Actionable classification of aging as a disease may lead to more efficient allocation of resources by enabling funding bodies and other stakeholders to use quality-adjusted life years (QALYs) and healthy-years equivalent (HYE) as metrics when evaluating both research and clinical programs. In order to classify aging with an actionable code or set of codes linked to specific age-related diseases, we propose an international task force to be organized to develop and communicate proposals to the WHO at the national and international levels.

Previous activity-tracking studies have drawn similar conclusions. But such studies usually ask participants to monitor their own exercise frequency and quantity, numbers they notoriously over-report. Also, the trackers used for NHANES have a higher level of precision than what's typically employed. "Because the device captures the intensity of activity so frequently, every minute, we can actually make a distinction between people who spent two hours a day doing those activities versus people who spent an hour and a half." To account for chronic conditions or illness influencing mortality rates, researchers statistically controlled for factors like diagnosed medical conditions, smoking, age and gender. They also completed a secondary examination from which they entirely excluded participants with chronic conditions. Though the scientists didn't discover any magic threshold for the amount a person needs to move to improve mortality, they did learn that even adding just 10 minutes per day of light activity could make a difference. Replacing 30 minutes of sedentary time with light or moderate-to-vigorous physical activity produced even better results.

The increase in expression of NKG2D ligands, particularly MICA and ULBP2, is likely a general feature of human senescent cells. A number of other studies have demonstrated the expression of MICA and/or ULBP2 in senescent cells derived from different cell types. Furthermore, senescent cells also acquire unique NKG2D ligand expression profiles consisting of several additional NKG2D ligands that result from differences between cell types (or cell-strains) and the mechanism by which senescence was induced. The repertoire of NKG2D ligands in mice is vast and similar to human cells, however based on sequence comparisons, mouse ligands are not homologous to the human ligands. Of note, NKG2D ligands are present on mouse cells that become senescent following p53 reactivation, and participate in the interaction of these cells with NK cells. In addition to their expression in senescent cells, NKG2D ligands are upregulated in other cell contexts related to cellular stress, including cancer, virally infected cells or following DNA damage. Therefore, the expression of these ligands might be part of a general stress response of cells that is utilized by senescent cells.

Our findings add to the emerging conceptual idea that the senescent program might represent a change in cell state that is associated with conversion to an immunogenic phenotype, functioning to remove damaged cells by immune clearance rather than through apoptosis. In addition to the upregulation of NKG2D ligands, the secretion of chemoattractants or the expression of adhesion molecules are further examples by which senescent cells become immunogenic. Immune clearance of senescent cells is likely beneficial in complex organisms where the regenerative capacity is dependent on non-resident stem cell populations and therefore temporal preservation of tissue architecture is necessary. Elimination of senescent cells following short-term insults, mediated by immune clearance, has physiological functions in tumor suppression and wound healing. Moreover, inefficient clearance might lead to the long-term persistence of senescent cells in tissues that has been associated with promotion of cancer development, ageing and age-related disease. Therefore, understanding the normal processes and mechanisms by which senescent cells are eliminated by the immune system will enable the formulation of conjectures concerning the mechanism responsible for impaired senescent cells elimination in later life. Such an understanding could lead to novel therapeutic strategies that enhance elimination of senescent cells by the immune system to improve tissue repair, cancer therapy and prevent deleterious effects of accumulation of senescent cells.

To define the relationship with previously characterized longevity pathways, we analyzed mutant animals. The lifespan extension caused by reducing the activity of acn-1 was additive with caloric restriction and mitochondrial insufficiency, and did not require sir-2.1, hsf-1 or rict-1, suggesting that acn-1 functions by a distinct mechanism. The interactions with the insulin/IGF-1 pathway were complex, since the lifespan extensions caused by captopril and reducing acn-1 activity were additive with daf-2 and age-1 but required daf-16. Captopril treatment and reducing acn-1 activity caused similar effects in a wide range of genetic backgrounds, consistent with the model that they act by the same mechanism. These results identify a new drug and a new gene that can extend the lifespan of worms and suggest new therapeutic strategies for addressing age-related degenerative changes.

ARGK-1 and its mammalian equivalent, creatine kinase, are enzymes that transport energy in the form of phosphoarginine or phosphocreatine to various locations within cells. The research team found that, as in worms, creatine kinase levels are increased in the brains of mice lacking a similar S6K protein. "Our main goal in studying aging is not to find ways to extend human lifespan, but to understand the processes by which our cells and tissues become less functional over time. Such insight might allow us to develop better preventive care that improves overall health at advanced ages, or interventions that can slow or perhaps even prevent the progression of diseases associated with aging. For example, in cancer, some tumors highly activate S6K to feed tumor growth. Further work to understand the relationship between creatine kinase and S6K may lead to new avenues to pursue novel drugs for age-related diseases, including cancer."

"This is a very exciting new step for our company. Our strategy assumes parallel development of a variety of formulations for our lead compound SkQ1 targeting a spectrum of age-related disorders. Visomitin had been the most advanced drug in our pipeline and already reached Phase III stage for dry eye indication in the U.S. Ophthalmic field, where we have been pursuing uveitis in addition to dry eye syndrome, remains the forefront area of development for Mitotech. At the same time, this new milestone brings us to clinical level of development for a variety of indications outside ophthalmic field. Mitotech is now in a great position to tackle critical disorders associated with aging such as neurodegenerative and metabolic diseases. Here at Mitotech we feel that we are approaching a major clinical breakthrough that could help many patients around the world."

"SkQ1 has demonstrated efficacy when administered systemically in a whole spectrum of preclinical studies. The very unique mechanism of action of the molecule has proved its benefit in models of rare genetic diseases as well as in models of more common age-related disorders. We see enormous potential in this novel mode of action and our clinical team worked very diligently on getting Plastomitin to its first clinical trial. Mitotech's goal is to progress with this new clinical program as efficiently as we did with the ophthalmic program and to deliver Plastomitin to patients within the next few years."

With just three patients frozen in its tanks, Trans Time is a scrappy little cryonics competitor. (The last person to enter one of Trans Time's vats was the company's founder, Paul Segall, a Berkeley Ph.D who co-founded the publicly-traded medical company BioTime. He died of a brain aneurism in 2003.) The two largest cryonics facilities are Alcor, in Arizona, and the Cryonics Institute, in Michigan; that's where you'll find most of the 300 or so people who are currently frozen. There's also KrioRus in Russia, which has 45 people on ice. And there are over 2,000 people worldwide who have signed up to be frozen, but haven't died yet.

Greg Fahy is a cryobiologist at 21st Century Medicine, a scientific institution based in southern California that has received funding from the National Institutes of Health to work on cryonically preserving organs. And he thinks the sci-fi fantasy of bringing frozen bodies back to life may not be as far-fetched as we think. "We're getting pretty good at this. We can load a kidney up with cryoprotectant and save it. We now know we can remove a piece of the brain and preserve it with perfection, and then put it back and it will still operate." Fahy, who has experimented successfully with cryonic preservation in rabbits and rats, thinks it may one day work in humans, too. "There's nothing about brain tissue that prevents it from being cryonically preserved."

In fact, Fahy said, the biggest obstacle to successful cryonic reanimation might be the law, not science. Most cryonics experts agree that cryonic preservation would work best on bodies that aren't yet dead, and haven't begun to decompose. But under current law, cryonics facilities are prohibited from freezing their patients while they're alive. (Doing so would be considered assisted suicide, or possibly murder.) "There may need to be legal changes that need to be made to allow cryonic preservation before deterioration begins."

Cryonics, itself, represents a kind of faith - a faith that scientific progress will continue unabated, and will eventually be able to solve even death itself. Cryonicists believe so strongly in our scientific future that many think that people who bury, cremate or compost their bodies instead of freezing them are, essentially, committing suicide.