Fight Aging! Newsletter, March 12th 2018

Fight Aging! provides a weekly digest of news and commentary for thousands of subscribers interested in the latest longevity science: progress towards the medical control of aging in order to prevent age-related frailty, suffering, and disease, as well as improvements in the present understanding of what works and what doesn't work when it comes to extending healthy life. Expect to see summaries of recent advances in medical research, news from the scientific community, advocacy and fundraising initiatives to help speed work on the repair and reversal of aging, links to online resources, and much more.

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  • How to Plan and Carry Out a Simple Self-Experiment, a Single Person Trial of Senolytic Peptide FOXO4-DRI
  • Undoing Aging: an Interview with Michael Greve of the Forever Healthy Foundation
  • New Findings in the Biochemistry of Parkinson's Disease
  • High Levels of Physical Activity Prevent Several Aspects of Immunosenescence
  • A Future of Gene Therapies to Greatly Reduce the Incidence of Cardiovascular Disease
  • Loss of Ribosomal DNA is Associated with Aging in Flies
  • The Prospects for Enhancing Repair Systems in the Brain to Treat Stroke Patients
  • Funding More Work on Deep Learning for Drug Discovery to Treat Aging
  • Greater Levels of Dbx2 Appear Connected to Neural Stem Cell Decline in Aging
  • ERRγ as a Target for the Development of Exercise Mimetics
  • Senolytic Drugs Fail to Kill Cancerous Cells with Senescent Gene Expression Signatures, but a Gene Therapy Succeeds
  • Why Do Some Mitochondrial Mutations Expand to Overtake All Mitochondria in a Cell?
  • Evidence Against Adult Human Neurogenesis
  • Visible Light Influences the Longevity of Nematodes
  • Aspirin as a Calorie Restriction Mimetic that Enhances Autophagy

How to Plan and Carry Out a Simple Self-Experiment, a Single Person Trial of Senolytic Peptide FOXO4-DRI

This lengthy post covers the topic of setting up and running a self-experiment, a human trial of a single individual, to assess the senolytic effects of the peptide FOXO4 D-Retro-Inverso (FOXO4-DRI). This is the protein produced from the FOXO4 gene, with D-amino acids substituted for L-amino acids, reversing the chirality of the molecule. This means it cannot be processed in the usual way by cellular metabolism, and the consequence of interest is that this sabotages the survival efforts of lingering senescent cells in old tissues, causing them to self-destruct. This peptide was evaluated in aged mice in 2017, showing destruction of senescent cells without side-effects, and producing the usual array of benefits to measures of health and age-related decline as a result. This is quite interesting when compared to the various chemotherapeutic senolytic drug candidates that exibit similar degrees of senescent cell destruction in mice, but an array of unpleasant side-effects.

Of course, the chemotherapeutics have extensive human data that catalogs the side-effects at various doses in our species, while at the time of writing there is no human data for FOXO4-DRI whatsoever. This is a very important point! It is perhaps the most important consideration here.

The purpose in publishing this outline is not to encourage people to immediately set forth to follow it. If you come away thinking that you should do exactly that, and as soon as possible, then you have failed at reading comprehension. This post is intended to illustrate how to think about self-experimentation in this field: set your constraints; identify likely approaches; do the research to fill in the necessary details; establish a plan of action; perhaps try out some parts of it in advance, such as the measurement portions, as they never quite work as expected; and most importantly identify whether or not the whole plan is worth actually trying, given all that is known of the risks involved. Ultimately that must be a personal choice.


  • Why Self-Experiment with Senolytics?
  • Caveats in More Detail
  • Summarizing FOXO4-DRI
  • Establishing Dosage
  • An Introduction to Injections
  • Considering Autoinjectors
  • Obtaining a Needle-Free Injection System
  • Obtaining Vials of the Correct Size
  • Preparing FOXO4-DRI for Injection
  • Obtaining FOXO4-DRI
  • Storing FOXO4-DRI
  • Validating the Purchased FOXO4-DRI
  • Establishing Tests and Measures
  • Guesstimated Costs
  • Practice Before Working with FOXO4-DRI
  • Schedule for the Self-Experiment
  • Where to Publish?
  • Final Thoughts: Why Not Wait?

Why Self-Experiment with Senolytics?

Senolytic therapies are those that selectively destroy senescent cells. The build up of senescent cells is one of the causes of aging. So obviously, one hope is to benefit personally from such a therapy sooner than would otherwise be the case, balancing that against incurring some unknown degree of risk of failure or harm. The first human trials, those that establish numbers for that risk, will take another few years to wind through to robust conclusions, and further years beyond that will be required for the medical community to become willing to prescribe senolytics generally. Further, those trials will almost all test only a single candidate therapy, and the evidence to date in mice suggests that different senolytics with different mechanisms are tissue-specific in their effects on senescent cells. Multiple different compounds may be more effective than one - but that won't be discovered in the formal trial process. Lastly, well run self-experimentation carried out by a number of people, where the results are published, can help to guide the direction of later, formal studies.

All of these reasons must be balanced against a sober assessment of the risks involved in obtaining and using an injected peptide that has no published human data whatsoever, and an acceptance of personal responsibility for consequences should one choose to run those risks.

Caveats in More Detail

There are two areas of personal responsibility to consider here. Firstly, this involves injecting a peptide that has no published data on human use at all. The anecdotal data from people claiming to have used FOXO4-DRI should be ignored, as it only covers the most serious short-term consequences, and few if any of these individuals are verifying that they are indeed obtaining the right compound, and nor are they carefully checking their own biochemistry for outcomes. Anything is possible in the long-term, from cardiovascular failure to cancer to subtle increases in disease or organ failure risk, no matter how compelling the animal data might appear to be. The absence of any human data should be far more concerning to any rational individual than the case of a compound with extensive human data showing serious side-effects. The latter can be planned and accounted for. The former cannot: it is a leap into the unknown.

Secondly, obtaining and using arbitrary novel peptides such as FOXO4-DRI in the manner described here is potentially illegal: not yet being a formally registered medical treatment, it falls into a nebulous area of regulatory and prosecutorial discretion as to which of the overly broad rules and laws might apply. In effect it is illegal if one of the representatives of the powers that be chooses to say it is illegal in any specific case, and there are few good guidelines as to how those decisions will be made. The clearest of the murky dividing lines is that it is legal to sell peptides that are not defined as a therapy for research use, but illegal to market and sell them for personal use in most circumstances. This is very selectively enforced, however, and reputable sellers simply declare that their products are not for personal use, while knowing full well that this is exactly what their customers are doing in many cases.

Choosing to purchase and use FOXO4-DRI would therefore likely be a matter of civil disobedience, as is the case for anyone obtaining medicines or potential medicines outside the established national system of prescription and regulation. People are rarely prosected for doing so for personal use in the US - consider the legions of those who obtain medicines overseas for reasons of cost, despite the fact that doing so is illegal - but "rarely" is not "never." If you believe that the law is unjust, then by all means stand up against it, but accept that doing so carries the obvious risks of arrest, conviction, loss of livelihood, and all the other ways in which the cogs of modern society crush those who disagree with the powers that be.

Lastly, senolytics is a fast-moving field. This post will become outdated quite rapidly in its specifics, as new FOXO4-DRI research arrives on the scene, and FOXO4-DRI may well be obsoleted by better options. Nonetheless, the general outline should still be a useful basis for designing new self-experiments involving later and hopefully better compounds, as well as tests involving more logistical effort.

Summarizing FOXO4-DRI

You might look at an earlier post for a high level overview of how FOXO4-DRI works to selectively destroy senescent cells. The short version of the story is that functional FOXO4 interacts with p53 in order to suppress the cell self-destruction mechanisms that are primed for activation in all senescent cells. This appears to be the primary method by which a small fraction of senescent cells manage to linger in order to cause age-related dysfunction in tissues. Since FOXO4-DRI does not function correctly in cellular metabolism, the FOXO4-p53 interation fails when FOXO4-DRI is present in sufficient amounts to replace enough of the native FOXO4, and the cell destroys itself.

From studies in mice, FOXO4-DRI appears to cause no issues in other cell types; it is simply ignored - at least in the short-term, and in mice. In the longer term, high levels of non-functioning FOXO4 in calls might indeed cause problems, but the treatment are intended to last only a short time, with the FOXO4-DRI being broken down in a matter of a day or so. There is no human data to show that any of this also applies to our species, but given a few years for the research community to make progress, that will change.

Establishing Dosage

The only definitive way to establish a dosage for a pharmaceutical in order to achieve a given effect is to run a lot of tests in humans. Testing in mice can only pin down a likely starting point for experiments to determine a human dose, but the way in which you calculate that starting point is fairly well established for most cases. That established algorithm is essentially the same for most ingested and intravenously (or intraperitoneally in small animals) injected medicines, but doesn't necessarily apply to other injection routes. More on that in the next section of this post. Some compounds - as always - are exceptions to the rule, and the only way that scientists discover that any specific compound is an exception is through testing at various doses in various species.

When considering dosage of any substance, it is important to emphasise that more is not better; this cannot be approached in the way people tend to naively approach the (over)use of dietary supplements. The primary goal, if self-experimenting, is to take as little as necessary of any senolytic compound. The first look at FOXO4-DRI by the research community suggests that higher doses should do nothing more than is achieved by the therapeutic doses, and with the same absence of immediate side-effects - but that is only the result of an initial examination. No long-term assessment has taken place, even in mice. If FOXO4-DRI does turn out to produce lasting or subtle side-effects, then following the maxim of using as little of it as possible should help to lower the impact. That the dose makes the poison is an ancient adage, but no less true today.

The steps to figure out a suitable starting point for a human test of an injected senolytic pharmaceutical are as follows: firstly read the mouse studies for the senolytic compound in question, in order to find out how much was given to the mice and for how long. Doses for most pharmaceuticals are expressed in mg/kg. Secondly apply a standard multiplier to scale this up to human doses, which you can find in the open access paper "A simple practice guide for dose conversion between animals and human". Do not just multiply by the weight of the human in kilograms - that is not how this works. The relative surface area of the two species is the more relevant scaling parameter. Read the paper and its references in order to understand why this is the case. Again, note that the result is only a ballpark guess at a starting point in size of dose. The duration of treatment translates fairly directly, however. For the period of treatment, start with the same number of doses, spacing of doses, and duration as takes place in senolytic studies in mice.

In the case of FOXO4-DRI, the mice in the single study were injected intraperitoneally with 5 mg/kg of FOXO4-DRI dissolved in phosphate buffered saline, three times, taking place every other day. For a 60kg human, that translates to a 25mg dose via intravenous injection, carried out three times on alternate days.

An Introduction to Injections

The relationship between different forms of injection, dosage, and effects is actually a complicated and surprisingly poorly mapped topic. There are four type of injection to consider, here listed in descending order of difficulty to carry out safely: (a) intraperitoneal, through the stomach muscle into the abdominal body cavity, which is rare in human medicine but common in studies using small animals; (b) intravenous, into a vein, which requires some practice to get right; (c) intramuscular, into the muscle beneath the skin; and (d) subcutanous, into the lower levels of the skin.

The amount of fluid that can be easily injected varies by type. In humans, effectively unlimited amounts of fluid can be introduced via intraperitoneal or intravenous injection. The subcutaneous route is limited to something less than 1ml, and intramuscular is limited to 2-3ml depending on location. These are all very fuzzy numbers - some sources, for example, give an upper limit of 5ml for intramuscular injections, but I can't say as I would be lining up to be on the receiving end of that. Measure out 5ml and take a look at it. Ouch.

The different injection routes can alter the character of the injected medicine; how much is required to gain a given effect, how long it takes to get into the system and how fast it does it. A rare few types of medication cannot be injected subcutaneously, because the metabolism of the skin will degrade them, while some are better given subcutaneously. If you root through the literature looking for comparisons between performance and dosage for different injection types, you'll find a very ragged collection of examples showing that there are few coherent rules. Some compounds have no discernible differences between injection route, some see altered peaks of concentration, some require higher doses when subcutaneous, some require lower doses when subcutenous. Oil-based solutions can produce a very slow uptake of medication when injected into muscle or skin in comparison to an intraveous injection, while water-based solutions result in just as rapid an uptake into the bloodstream.

Do we know how FOXO4-DRI will be affected by different injection routes? No. That data has yet to be established. So it seems acceptable to say that a self-experimenter should try to use the much easier paths of subcutaneous and intramuscular injection, rather than attempting intravenous administration, and just keep the same dose as was established for intravenous injection. For most people, intraveous injections require a helper or a lot of painful practice. For subcutaneous and intramuscular injections, there is a market of autoinjection tools that can remove many of the challenges inherent in managing injections.

Considering Autoinjectors

Sticking a needle into one's own flesh is not an easy thing to do, and this is the rationale for the range of autoinjection systems that have been developed by the medical community. They are most easily available for subcutaneous injections; spring-based devices that accept a standard needle and syringe, and that are trigged by a button push. Intramuscular autoinjectors do exist, but unfortunately not in a general or easily available way. All of the needle-based intramuscular autoinjectors are regulated devices that come preloaded with a particular medicine, and are not otherwise sold in a more generally useful way. Unfortunately, there is no automation that can help with intravenous injections. You are on your own there.

Option 1: Subcutaneous Autoinjection with Needle and Syringe

If intending to carry out subcutaneous injections it is easy enough to order up a supply of disposible needles and syringes, an autoinjector device that accepts the standard needle and syringe arrangement, and other necessary items such as sterilization equipment from the sizable diabetes-focused marketplace. Such injections are relatively easy to carry out, a wide range of vendors sell the materials, and there is a lot of documentation, including videos, available on how to carry out subcutaneous injections. All of the equipment is cheap. Buying these materials will probably put you on a list in this era of the drug war, but there are many people out there doing it.

Option 2: Subcutaneous or Intramuscular Needle-Free Autoinjection

Are there viable alternatives to needles? As it turns out, yes, and some can solve the problem of missing general intramuscular autoinjectors as well. Needle-free autoinjectors that use a thin, high-pressure fluid jet to punch medication through the skin are a growing area of development. These systems have numerous advantages over needles, but they are more expensive, most can only manage subcutanous injections, and all are limited in the amount of fluid they can inject in comparison to the traditional needle and syringe. Nonetheless, for the purposes of this outline, I'll focus on needle-free systems. The biggest, primary, and most attractive advantage of a needle-free system is in the name: it means not having to deal with needles in any way, shape, or form.

Obtaining a Needle-Free Injection System

There are a fair number of needle-free injectors on the market, but most are hard to obtain unless you happen to be a regulated medical facility running through the standard regulated purchase model, and are looking for large numbers of units in a bulk purchase. Some systems use compressed gas, others use springs. The spring-based systems tend to be less complicated and more reliable. From my survey of the marketplace, the two systems worth looking at are (a) PharmaJet, which can be purchased in the US via intermediary suppliers such as Moore Medical, and (b) Comfort-in, which is sold directly to consumers in most countries by an Australian group. So far as I can tell, PharmaJet is the only available needle-free system that is capable of intramuscular rather than subcutaneous injection.

PharmaJet is the better engineered and more expensive of these two systems, and its specialized syringes are very definitely built to be one-use only. Further, loading fluid into the syringes requires the use of vials and a vial adaptor. First the vial is loaded with the fluid to be injected, then the vial is connected to the syringe via the adaptor to transfer the fluid. Comfort-in has a similar setup, but is more flexible, and on the whole more consumer-friendly when considering the entire package of injector and accessories. It is has a wider range of vial and other adaptors. Further, the Comfort-in syringes can in principle be reused given sterilization, though of course that is not recommended.

The instructions for both of these systems are extensive, and include videos. They are fairly easy to use.

Obtaining Vials of the Correct Size

If using the insulin needle and subcutaneous injection approach, then any variety of capped glass vial will do when it comes to mixing and temporarily holding liquids for injection. It does, however help greatly to either use preassembled sterile vials or assemble your own vials with rubber stoppers and crimped caps, as described below, as that sort of setup makes it easier to take up small amounts of a liquid into a syringe. If using the needle-free systems, then vials of a specific type and size are necessary in order to fit the adaptors. The rest of this discussion focuses on that scenario.

There are many, many different types of vial manufactured for various specialized uses in the laboratory. The type needed here is (a) crimp-top vial, also called serum vials by some manufacturers, with (b) a 13mm (for PharmaJet and Comfort-in) or 20mm (for Comfort-in only) diameter open top aluminium cap, one that has a central hole to allow needles and adaptor spikes through, and (c) a rubber or rubber-like stopper that is thin enough in the center to let a needle or adaptor spike past. The cap is crimped on over the rubber seal to keep everything in place - this requires a crimping tool, and removing it requires the use of another tool.

There are two options here. The first option is to purchase preassembled empty sterile vials of the right size and a set of disposable needles and syringes to transfer liquid into the vials. In order to continue to bypass the whole business of needles, however, the other alternative is to purchase vials, stoppers, and aluminium caps separately, or in a kit, and assemble your own vials. A crimping tool is also needed in order to seal the cap. That tool, like the vials and the caps, must be of the right size. Be careful when purchasing online. Vials are categorized by many different dimensions, and descriptions tend to mix and match which dimensions of the vial they are discussing, or omit the important ones. For sterile vials, it is usually only the cap diameter that is mentioned. For crimp-top vials, there are any number of dimensions that might be discussed; the one that needs to match the cap diameter is the outer diameter of the mouth or crimp.

It is usually a good idea to buy a kit where possible, rather than assembling the pieces from different orders, but if taking the assembly path, it is best to buy all the pieces from the same company. Wheaton is a decent manufacturer, and it is usally possible to find much of their equipment for sale via numeous vendors. One can match, say, the crimp-top 3ml vials #223684 with 7mm inner mouth and 13mm outer mouth with snap-on rubber stoppers #224100-080 of the appropriate dimensions and 13mm open top caps #224177-01. Then add a 13mm crimping device #W225302 and pliers #224372 to remove 13mm crimped caps.

Preparing FOXO4-DRI for Injection

The objective is to wind up with the right amount of FOXO4-DRI dissolved in phosphate buffered saline in a sealed vial, ready to be used with the injection system, and with as little contamination as possible from the environment. Depending on the size of the vial, it might contain doses for multiple injections - in fact it is much easier to set things up this way. FOXO4-DRI dissolves very readily in saline, so placing a single human dose into 0.5ml or 1ml is quite feasible. A 3ml vial can hold three doses for the treatment without issue.

One approach is to measure out FOXO4-DRI by weight using a suitable microscale, then transfer that dose to a mixing container, such as larger glass vial. Pipette in the desired amount of saline and stir with a rod to ensure it is fully dissolved. Then either inject the mix into a sterile vial using a standard needle and syringe, or pipette the mix into an clean open vial that is then sealed, capped, and crimped. Keeping hands, tools, vials, and surfaces clean and sterile is important: wash everything carefully and wipe down surfaces with an alcohol solution before and after use. There are many best practices guides and summaries available online.

Obtaining FOXO4-DRI

There are a small number of peptide synthesis companies worldwide that advertise their willingness to sell FOXO4-DRI, as an established recipe for synthesis rather than a special order. Any other reputable peptide synthesis company will be able to manufacture FOXO4-DRI to order from the description provided in the 2017 paper.

H-ltlrkepaseiaqsileaysqngwanrrsggkrppprrrqrrkkrg-OH. Molecular weight: 5358.2 It was manufactured by Pepscan (Lelystad, the Netherlands) at more than 95% purity and stored at -20°C in 1mg powder aliquots until used to avoid freeze-thawing artifacts. For in vitro experiments FOXO4-DRI was dissolved in Phosphate Buffered Saline to generate a 2mM stock. For in vivo use, FOXO4-DRI was dissolved in Phosphate Buffered Saline to generate a 5mg/ml stock solution, which was kept on ice until injection. Before injection the solution was brought to room temperature.

An advantage of the companies that advertise FOXO4-DRI in their catalog is that they will have established mass spectra and other data sheets for the compound that can be compared with one another, or used as a basis for evaluating the quality of the product. The disadvantage, in at least the case of NovoPro Bioscience, is that the price charged is outrageous - possibly a rational choice made to discourage amateur purchasers. Chinese companies, on the other hand have a comparatively low price for their offerings, significant lower than that of even the most aggressively competitive companies in the US and Europe. Among European and US synthesis companies out there, Pepscan and Genscript are options, with Genscript coming recommended by a number of sources.

Without focusing on any of the specific vendors mentioned above, the easiest way to obtain cost-effective synthesis of peptides is to follow the process outlined in the last post on self-experimentation, which is to use Alibaba to find and connect with smaller-sized suppliers in China. There are a good many reputable peptide synthesis concerns in that part of the world, even if only one or two explicitly advertise FOXO4-DRI as a product at this time.

As noted at the outset of this post, all of these efforts to obtain, ship, and use a peptide are to some degree illegal - it would be an act of civil disobedience carried out because the laws regarding these matters are unjust, albeit very unevenly enforced. Many people regularly order pharmaceuticals from overseas, with and without prescriptions, for a variety of economic and medical reasons, and all of this is illegal. The usual worst outcome for individual users is intermittent confiscation of goods by customs, though in the US, the FDA is actually responsible for this enforcement rather than the customs authorities. Worse things can and have happened to individuals, however, even though enforcement is usually targeted at bigger fish, those who want to resell sizable amounts of medication on the gray market, or who are trafficking in controlled substances. While the situation with an arbitrary peptide isn't the same from a regulatory perspective, there is a fair amount written on the broader topic online, and I encourage reading around the subject.

Open a Business Mailbox

A mailbox capable of receiving signature-required packages from internal shipping concerns such as DHL and Fedex will be needed. Having a business name and address is a good idea. Do not use a residential address.

Use Alibaba to Find Manufacturers

Alibaba is the primary means for non-Chinese-language purchasers to connect to Chinese manufacturers. The company has done a lot of work to incorporate automatic translation, to reduce risk, to garden a competitive bazaar, and to make the reputation of companies visible, but it is by now quite a complicated site to use. It is a culture in and of itself, with its own terms and shorthand. There are a lot of guides to Alibaba out there that certainly help, even if primarily aimed at retailers in search of a manufacturer, but many of the specific details become obsolete quickly. The Alibaba international payment systems in particular are a moving target at all times: this year's names, user interfaces, and restrictions will not be the same as next year's names, user interfaces, and restrictions.

Start by searching Alibaba for peptide synthesis companies. There are scores of biotech companies in China for any given specialty. Filter the list for small companies, as larger companies will tend to (a) ignore individual purchasers in search of small amounts of a peptide, for all the obvious economic reasons, and (b) in any case require proof of all of the necessary importation licenses and paperwork. Shop around for prices - they may vary widely, and it isn't necessarily the case that very low prices indicate a scam of some sort. Some items and services are genuinely very cheap to obtain via some Chinese sources. Remember to ask the manufacturer for mass spectra and liquid chromatography data if they have it.

Many manufacturers will state that they require a large (often ridiculously large) minimum order; that can be ignored. Only communicate with gold badge, trade assurance suppliers with several years or more of reputation and a decent response rate. Make sure the companies exist outside Alibaba, though for many entirely reliable Chinese businesses there are often sizable differences between storefronts on Alibaba, real world presence, and the names of owners and bank accounts. Use your best judgement; it will become easier with practice.

Arrange Purchase and Shipping via Alibaba

Given the names of a few suppliers, reach out via the Alibaba messaging system and ask for a quote for a given amount of FOXO4-DRI; you will have to provide the sequence and a reference to the paper in which it is described. Buy more than you'll think you need, as a small amount of it will be used to validate the identity and quality of the compound batch. Payment will most likely have to be carried out via a wire transfer, which in Alibaba is called telegraphic transfer (TT). Alibaba offers a series of quite slick internal payment options that can be hooked up to a credit card or bank account, but it is hit and miss whether or not those methods will be permitted for any given transaction. Asking the seller for a pro-forma invoice (PI), then heading to the bank to send a wire, and trusting to their honesty should work just fine when dealing with companies that have a long-standing gold badge.

To enable shipping with tracking via carriers such as DHL, the preferred method of delivery for Chinese suppliers shipping to the US or Europe, you will need to provide a shipping address, email address, and phone number. Those details will find their way into spam databases if you are dealing with more than a few companies, and will be, of course, sold on by Alibaba itself as well. Expect to see an uptick of spam after dealing with suppliers via Alibaba, so consider using throwaway credentials where possible.

Chinese manufacturers active on Alibaba are familiar with international shipping practices. On their own initiative may or may not decide to declare the true cost and contents of the shipped package. This is another form of widely practiced civil disobedience, but is much more common in the shipping of pharmaceuticals than in the shipping of synthesized peptides such as FOXO4-DRI. The former are likely to be confiscated by customs officials, while the latter are not. If the true cost is declared, then expect to pay customs duty on that cost; payment is typically handled via the carrier.

Storing FOXO4-DRI

Peptides are usually shipped in powder form, and while in this form are easily stored in a refrigerator for the short-term, or in a freezer for the long term. It has a much shorter life span once it has been mixed with liquid for injection, however, and should be kept on ice, and used within days or or at most weeks.

Validating the Purchased FOXO4-DRI

A peptide of a given sequence may have been ordered, but that doesn't mean that what turns up at the door is either the right nondescript powder or free from impurities or otherwise of good quality. Even when not ordering from distant, infrequent suppliers, regular testing of batches is good practice in any industry. How to determine whether a peptide is what it says it is on the label? Run it through a process of liquid chromatography and mass spectrometry, and compare the results against the standard data for a high purity sample of that compound. Or rather pay a small lab company to do that.

Note that there isn't a cost-effective way to validate that the peptide in question does in fact have D-amino acids substituted for L-amino acids. They look the same in mass spectrometry, and largely the same in liquid chromatography. Lacking that configuration, it will have no effect on senescent cells - but if a vendor can otherwise provide the right sequence, it seems unlikely that they would fail on the D versus L matter. It isn't any harder or more expensive for a synthesis group to build the D form versus the L form, and the instructions are quite clear in the sequence provided.

Obtain the Necessary Equipment

Since this process will involve weighing, dividing up, and shipping powders in milligram amounts, a few items will be necessary: spatulas or scoops for small amounts of a substance; a reliable jeweler's scale such as the Gemini-20; sealable vials; small ziplock bags; labels; and shipping and packing materials. All of these are easily purchased online. The recommended shipping protocol is to triple wrap: a labelled vial, secured within a ziplock bag and tape, and then enclosed within a padded envelope.

Use Science Exchange to Find Lab Companies

Science Exchange is a fairly robust way to identify providers of specific lab services, request quotes, and make payments. Here again, pick a small lab company to work with after searching for LC-MS (liquid chromatography and mass spectrometry) services. Large companies will want all of the boilerplate registrations and legalities dotted and crossed, and are generally a pain to deal with in most other ways as well. Companies registered with Science Exchange largely don't provide their rates without some discussion, but a little over 100 per sample is a fair price for LC-MS to check the identity and purity of the compound.

Work with the Company to Arrange the Service

The process of request, bid, acceptance, and payment is managed through the Science Exchange website, with questions and answers posted to a discussion board for the task. Certainly ask if you have questions; most providers are happy to answer questions for someone less familiar with the technologies used. Service providers will typically want a description of the compounds to be tested and their standard data sheets, as a matter of best practice and safety. Here provide the mass spectra and other data sheets from the vendor, or use those published by NovoPro or other sources.

Ship the Samples

Measure out 1-5mg or so of FOXO4-DRI as a distinct sample, label it carefully, and package it up. More in the sample is better than less, as several attempts might be needed to get a good result out of the machines used, but each attempt really only needs a very tiny amount of the compound. Ship the sample via a carrier service such as DHL, UPS, or FedEx. Some LC-MS service companies may provide shipping instructions or recommendations. These are usually some variety of common sense: add a description and invoice to the package; reference the order ID, sender, and receiver; clearly label sample containers; and package defensively with three layers of packing; and so forth.

Examine the Results

Once the LC-MS process runs, the lab company should provide a short summary regarding whether or not the compound is in fact the correct one and numbers for the estimated purity. Also provided are the mass spectra, which can be compared with the existing spectra from the vendor, and from other sources such as NovoPro.

Establishing Tests and Measures

Unfortunately there is no available, established, proven, useful test that can directly assess senescent cell level in humans or human biopsies. It is possible to use immunohistochemistry to assess cellular senesence in tissue samples, which is a standard approach in animal studies, but no-one appears to have yet validated that in humans, given biopsies taken from a living individual. Since senescent cells are generated temporarily by wounding, it is quite possible that anything that starts with a biopsy will prove to be unhelpful as a before and after comparison measure for senolytic trials - the levels measured may not bear any resemblance to the normal levels absent a wound.

Without a direct measure, we must fall back on indirect assessments of the detrimental effects of senescent cells. The objective here is a set of tests that anyone can run without the need to involve a physician, as that always adds significant time and expense. Since we are really only interested in the identification of large and reliable effects as the result of an intervention, we can plausibly expect a collection of cheaper and easier measures known to correlate with age to be useful. Once that hill has been climbed, then decide whether or not to go further - don't bite off more than is easy to chew for a first outing.

From an earlier exploration of likely tests, I picked the following items on the basis of a likely connection to the actions of senescent cells, reasonable cost and effort, and ability to carry out the test without a physician's office being involved. Note that this does rule out, to pick one example, the interesting and relevant examination of kidney and liver function, as it would have to be carried out via the radioactive tracer methods of nuclear medicine to obtain decent results. That leaves the tests below quite focused on (a) the cardiovascular system, particularly measures influenced by vascular stiffness, and (b) inflammatory and other markers in the bloodstream:

  • A standard blood test, with inflammatory markers.
  • Resting heart rate and blood pressure.
  • Heart rate variability.
  • Pulse wave velocity.
  • Biological age assessment via DNA methylation patterns.

The cardiovascular health measures in that list are those that are impacted by changes in the elasticity or functional capacity of blood vessels, such as would be expected to occur to some degree following any rejuvenation therapy that addresses senescent cells, chronic inflammation, or other factors that stiffen blood vessels, such as calcification or cross-linking. Positive change of the average values in most of these metrics are achievable with significant time and effort spent in physical training, so movement in the numbers in a short period of time as the result of a treatment should be an interesting data point.


There exist online services such as WellnessFX where one can order up a blood test and then head off the next day to have it carried out by one of the widely available clinical service companies. Of the set of test packages offered by WellnessFX, the Baseline is probably all that is needed for present purposes. But shop around; this isn't the only provider.

Resting Heart Rate and Blood Pressure

A simple but reliable tool such as the Omron 10 is all you need to measure heart rate and blood pressure. It is worth noting here a couple of general principles for cardiovascular measures. Firstly, the further away from the center of the body that the measurement is taken, the less reliable it is - the more influenced by any number of circumstances, such as position, mood, stress, time of day, and so forth. Fingertip devices are convenient, but nowhere near as useful as something like the Omron 10 that uses pressure on the upper arm. Secondly, all of the above-mentioned line items also influence every cardiovascular measure, so when you are creating a baseline or measuring changes against that baseline, carry out each measure in the same position, at the same time of day, and make multiple measurements over a week to gain a more accurate view of the state of your physiology. The Omron 10 is solid: it just works, and seems quite reliable.

Heart Rate Variability

Surprisingly few of the numerous consumer tools for measuring heart rate variability actually deliver the underlying values used in research papers rather than some form of aggregate rating derived by the vendor; the former is required for any serious testing, and the latter is useless. Caveat emptor, and read the reviews carefully. As an alternative to consumer products, some of the regulated medical devices are quite easy to manage, but good luck in navigating the system to obtain one. The easiest way is to buy second hand medical devices via one of the major marketplaces open to resellers, but that requires a fair-sized investment in time and effort - which comes back to the rule about keeping things simple at the outset.

After some reading around the subject, I settled on the combination of the Polar H10 device coupled with the SelfLoops HRV Android application. I also gave the EliteHRV application a try. Despite the many recommendations for Polar equipment, I could not convince either setup to produce sensible numbers for heart rate variability data: all I obtained during increasingly careful and controlled testing was a very noisy set of clearly unrealistic results, nowhere near the values reported in papers on the subject. However, plenty of people in the quantified self community claim that these systems work reasonably well, so perhaps others will have better luck than I. Take my experience as a caution, and compare data against that reported in the literature before investing a lot of time in measurement.

Pulse Wave Velocity

For pulse wave velocity, choice in consumer tools is considerably more limited than the sitation for pulse wave velocity. Again, carefully note whether or not a device and matching application will deliver the actual underlying data used in research papers rather than a made-up vendor aggregate rating. I was reduced to trying a fingertip device, the iHeart, picked as being more reliable and easier to use than the line of scales that measure pulse wave velocity. Numerous sources suggest that decently reliable pulse wave velocity data from non-invasive devices is only going to be obtained by measures at the aorta and other core locations, or when using more complicated regulated medical devices that use cuffs and sensors at several places on the body.

Still, less reliable data can be smoothed out to some degree by taking the average of measures over time, and being consistent about position, finger used for a fingertip device, time of day, and so forth when the measurement is taken. It is fairly easy to demonstrate the degree to which these items can vary the output - just use the fingertip device on different fingers in succession and observe the result. All of this is a trade-off. A good approach is to take two measures at one time, using the same finger of left and right hand, as a way to demonstrate consistency. While testing an iHeart device in this way, I did indeed manage to obtain consistent and sensible data, though there is a large variation from day to day even when striving to keep as many of the variables as consistent as possible. That large variation means that only sizable effects could be detected.

DNA Methylation

DNA methylation tests can be ordered from either Osiris Green or Epimorphy / Zymo Research - note that it takes a fair few weeks for delivery in the latter case. From talking to people at the two companis, the normal level of variability for repeat tests from the same sample is something like 1.7 years for the Zymo Research test and 4.8 years for the Osiris Green tests. The level of day to day or intraday variation between different samples from the same individual remains more of a question mark at this point in time, though I am told they are very consistent over measures separated by months. Nonetheless, as for the cardiovascular measures, it is wise to try to make everything as similar as possible when taking the test before and after a treatment: time of day, recency of eating or exercise, recent diet, and so forth.

An Example Set of Daily Measures

An example of one approach to the daily cardiovascular measures is as follows, adding extra measures as a way to demonstrate the level of consistency in the tools:

  • Put on the Polar H10; this is involved enough to increase heart rate a little for a short period of time, so get it out of the way first.
  • Sit down in a comfortable position and relax for a few minutes.
  • Measure blood pressure and pulse on the left arm using the Omron 10.
  • Measure blood pressure and pulse on the right arm using the Omron 10.
  • Measure pulse wave velocity on the left index fingertip over a 30 second period using the iHeart system.
  • Measure pulse wave velocity on the right index fingertip over a 30 second period using the iHeart system.
  • Measure heart rate variability for a ten minute period using the Polar 10 and Selfloops.

Consistency is Very Important

Over the course of an experiment, from first measurement to last measurement, it is important to maintain a consistent weight, diet, and level of exercise. Sizable changes in lifestyle can produce results that may well prevent the detection of any outcome resulting from a first generation senolytic therapy using the simple tests outlined here. Further, when taking any measurement, be consistent in time of day, distance in time from last exercise or meal, and position of the body. Experimentation with measurement devices will quickly demonstrate just how great an impact these line items can have.

Guesstimated Costs

The costs given here are rounded up for the sake of convenience, and in some cases are blurred median values standing in for the range of observed prices in the wild. The choice to use needles for subcutaneous injection is obviously much cheaper than exploring the world of needle-free injections and vial assembly.

  • Business mailbox, such as from UPS: 250 / year
  • Baseline tests from WellnessFX: 220 / test
  • MyDNAage kits: 310 / kit
  • Osiris Green sample kits: 70 / kit
  • Omron 10 blood pressure monitor: 80
  • Polar H10 heart monitor: 100
  • iHeart monitor: 210
  • American Weigh Gemini-20 microscale: 90
  • Miscellenous equipment: spatulas, labels, vials, a vial rack, etc: 60
  • Subcutaneous autoinjector for use with needle and syringe: 45
  • Needles and syringes: 40
  • Small pack of 13mm sterile serum vials: 35
  • PharmaJet Needle-free Injection Kit: 1020
  • Comfort-in Needle-free Injection Kit: 470
  • Bulk 13mm serum vial parts and capping tools: 750
  • 200mg of FOXO4-DRI via Alibaba: 2000
  • Customs import duty on FOXO4-DRI: 150
  • Shipping and LC-MS analysis of a sample: 200

Practice Before Working with FOXO4-DRI

Do you think you can measure and move milligrams of powder and crystals around between containers without dropping it or otherwise losing a sizable fraction of it? Or reliably pipette fluid in 0.5ml amounts between small vials? Or cap vials or connect adaptors or fill syringes or carry out an injection without messing it up somewhere along the way? Perhaps you can. But it is a very good idea to practice first with salt and saline solution rather than finding out that your manual dexterity and methods are lacking while handling the expensive peptide. You will doubtless come to the conclusion that more tools or different tools are needed than was expected to be the case. It is possible to get by with a spatula small enough to fit into vial mouths, vials, labels, a vial rack to keep vials in place while hands are doing something else, and pipettes sized for moving small amounts of fluid. Other items may be helpful, such as suitably sized powder funnels, though there is considerable utility in a small, singly folded piece of paper for moving non-sticky powders from a measuring device to a small-mouthed container.

Schedule for the Self-Experiment

One might expect the process of discovery, reading around the topic, ordering materials, and validating an order of FOXO4-DRI to take a couple of months. Once all of the decisions are made and the materials are in hand, pick a start date. The schedule for the self-experiment is as follows:

  • Day 1-10: Once or twice a day, take measures for blood pressure, pulse wave velocity, and heart rate variability.
  • Day 10: Bloodwork and DNA methylation test.
  • Day 11: Test a 1/10 dose of FOXO4-DRI and abandon the effort if issues are experienced.
  • Day 12: Inject FOXO4-DRI.
  • Day 14: Inject FOXO4-DRI.
  • Day 16: Inject FOXO4-DRI.
  • Day 46-55: Repeat the blood pressure, pulse wave velocity, and heart rate variability measures.
  • Day 55: Repeat the bloodwork and DNA methylation test.

The exact timing is not really important, but it is a good idea to allow enough time following the end of the dosage for things to settle down. In animal studies, senolytic effects occurred fairly rapidly, as did the benefits, but allowing a few weeks of time in a human self-experiment still sounds like a good idea. Certainly it costs nothing to take that step.

Where to Publish?

If you run a self-experiment and keep the results to yourself, then you helped only yourself. The true benefit of rational, considered self-experimentation only begins to emerge when many members of community share their data, to an extent that can help to inform formal trials and direction of research and development. There are numerous communities of people whose members self-experiment with various compounds and interventions, with varying degrees of rigor. One can be found at the LongeCity forums, for example, and that is a fair place to post the details and results of a personal trial with senolytics. Equally if you run your own website or blog, why not there?

When publishing, include all of the measured data, the doses taken, duration of treatment, and age, weight, and gender. Fuzzing age to a less distinct five year range (e.g. late 40s, early 50s) is fine. If you wish to publish anonymously, it should be fairly safe to do so, as none of that data can be traced back to you without access to the bloodwork provider. None of the usual suspects will be interested in going that far. Negative results are just as important as positive results. For example, given the measures proposed in this post it is entirely plausible that positive changes as a result of present senolytic treatments in a basically healthy late 40s or early 50s individual will be too small to identify - they will be within the same range as random noise and measurement error. Data that confirms this expectation is still important and useful for the community, as it will help to steer future, better efforts.

Final Thoughts: Why Not Wait?

Given all of the cautions above, why not wait? Waiting can be a very sensible strategy. The state of senolytic therapies is progressing rapidly. New senolytics are emerging, and more data is being published for existing senolytics. At some point in the next few years, reliable direct tests for senescence will arrive on the scene, allowing a much better view of whether or not these treatments are actually achieving the claimed results. That said, it doesn't hurt to plan, and it doesn't hurt to tinker with some of the component parts of a plan. That is how we can determine whether or not it is worthwhile to experiment now versus waiting to experiment later with better tools.

Undoing Aging: an Interview with Michael Greve of the Forever Healthy Foundation

The Undoing Aging conference is coming up later this month in Berlin, the latest in a long series of conferences focused initially on the science and later on commercial development of SENS rejuvenation biotechnology. In addition to prominent researchers from many parts of the field, this year we'll see a much greater presence of startup companies and investors. The first legitimate rejuvenation therapies, those based on the SENS model of periodic repair of the cell and tissue damage that causes aging, have reached the stage of clinical development. Some of the companies are a few years old now. More will arrive in the years ahead, as support for this cause grows.

The Undoing Aging conference is co-hosted by the SENS Research Foundation and the Forever Healthy Foundation. The former we're all familiar with by now, I'd hope, as an umbrella organization that coordinates and funds a wide range of scientific programs to unblock important lines of rejuvenation research. The latter is investor and philanthropist Michael Greve's non-profit organization. You might recall his support for the SENS Research Foundation in past years of fundraising; in 2016 he pledged 10 million to SENS rejuvenation research programs and the startups that will emerge from those programs. Hosting Undoing Aging is a part of his continued material support for SENS research and the development of practical near-future rejuvenation therapies. Here the Life Extension Advocacy Foundation volunteers interview Michael Greve on his work and his views on the field.

Undoing Aging With Michael Greve

You have spoken about your own advocacy efforts. How you think the public's perception of the subject has changed over the years?

In general, public opinion has already changed significantly over the past two or three years. Nowadays, you read much more often and positively on extending the healthy human lifespan. I firmly believe that once the first working rejuvenation therapy is out there, the whole discussion will immediately change. It will turn from abstract arguments about over-population and such to a very personal one. Do I want to live twenty more years in good health or not? At that point, I guess nobody will say, "Well no, I won't use that treatment and rather get cancer because of, you know, overpopulation." So, the best thing we can all work on is to make this very first therapy happen and then really promote it.

Many people in our community are hoping to see more wealthy people engaged because they have more resources at their disposal and could have a greater impact by donating even a small share of their wealth. However, we don't see that happening much. What messages might be more convincing to these wealthy people? Are they any different from what we usually say when we are trying to convince someone?

Large-scale philanthropy in a very early market such as rejuvenation biotech is hard and only for a few very forward-thinking individuals. I think the most straightforward and effective message to rich people, in general, is to show them a way to become even richer. I see the acceleration of the development of actual rejuvenation therapies as a three-stage process. First, motivate scientists to enter the field and work on the underlying science, then spin out promising research results as early as possible into fundable startups and finally bring in private capital to fuel development of the actual therapies. This last step will allow those high net-worth individuals to both put their money to good use and benefit from it at the same time.

That is why we are organizing the Undoing Aging conference, funding basic research and working hard to move promising research into fundable startups, allowing private capital to fuel the journey from there on. In terms of startups, we have done this already a few times and are seeing a lot of positive effects there.

What do you expect out of UA2018? What do you expect it to impact the most? Public awareness, investors' interest, or networking among scientists?

First and foremost, we are focussed on the science itself. We want to provide a platform for the existing scientific community that already works on damage repair and strengthens the community itself. At the same time, Undoing Aging offers interested scientists and students a first-hand understanding of the current state of affairs to attract new scientists to our exciting field. Apart from that, we have invited the broader longevity community to enable extended networking and support all advocates that do public work. Since we have a lot of interest from journalists, bloggers and several TV stations, there is going to be a public aspect as well. So, yes, you could say it's networking on all levels to advance our cause.

Are there any plans to make Undoing Aging into an annual event?

Yes, we are in this for the long run. This year, we have already received so much positive feedback and even more registrations than we expected. That is very encouraging.

Many people are concerned about affordability. Do you imagine that governments will necessarily have to step in and subsidise rejuvenation therapies that are otherwise difficult to afford?

There is no need to worry about that. We are talking about a market with billions of customers, numerous possible approaches to each aspect of aging, such as clearance of senescent cells. And you can't patent an approach in general, e.g. clearing senescent cells, just the particular implementation. In such a market, the fundamental economic forces as in any other industry will apply, and healthy competition and a multitude of products in combination with a massive customer base will force quality up and prices down as products quickly mature and become a general commodity.

You are currently supporting several biotech startups that are taking rejuvenation treatments into clinical trials. Is there an estimate of the baseline cost of these therapies, and do you know what the companies are planning to do to make them more affordable?

At the end of the day, these therapies are going to be an extremely affordable commodity.

New Findings in the Biochemistry of Parkinson's Disease

Today I'll point out a couple of recently published research results that add to the understanding of Parkinson's disease and its progression. Parkinson's disease is comparatively straightforward as neurodegenerative diseases go - which is to say that its biochemistry is still enormously complex in detail, but it hasn't proven as hard to identify the important aspects as is the case for Alzheimer's disease. At root, this is a synucleinopathy, a condition caused by the accumulation of α-synuclein deposits. This results in mitochondrial dysfunction and cell death in a small but important population of dopamine-generating neurons connected to motor function, but also a more widespread disruption of normal function in the brain. The challenge in Parkinson's is less a matter of knowing where to intervene, meaning the targeted removal of α-synuclein, but rather the construction of an effective methodology. You might look at one of the SENS Research Foundation reviews on the topic to get a sense of just how difficult it is to safely clear a specific form of metabolic waste from the brain.

Why do only some people develop Parkinson's disease? In a small number of cases, it is due to mutated genes, particularly those like parkin that are important in the processes of cellular maintenance. Impairment of autophagy directed at quality control of mitochondria appears to be an important facet of Parkinson's disease, but in those patients without evidence of mutation, the path to Parkinson's may be a random one of small differences in age-related damage and declining cellular maintenance that snowball and accelerate over time. A little less removal of metabolic waste leads to a little more α-synuclein and a little more mitochondrial dysfunction, which in turn further impacts maintenance systems, and so the feedback loop progresses, ever faster over time. Given enough time, everyone would suffer Parkinson's disease eventually - but as things stand, other processes of aging kill most people before that can happen.

Beyond clearance of α-synuclein, cell therapy is the other major area of effort in the production of therapies for Parkinson's disease. The goal there is to replace lost dopamine-generating neurons with new cells capable of taking over the same function in the brain. Since the process of loss is gradual, this should provide lasting benefit, even though it doesn't address the causes of cell loss - the new cells will be destroyed in time, just like the old ones. This situation is similar in near any proposed use of cell replacement therapy in older individuals: the tissue environment is typically hostile and damaged, and the details of how and why existing cell populations are no longer working matter greatly when it comes to the potential effectiveness of introducing new cells. Will they function correctly at all, or will they quickly succumb?

Researchers uncover culprit in Parkinson's brain cell die-off

If we could peer into the brains of Parkinson's patients, we'd see two hallmarks of the disease. First, we'd see a die-off of the brain cells that produce a chemical called dopamine. We'd also see protein clumps called Lewy bodies inside the neurons. Researchers believe a key to treating Parkinson's is to study possible links between these two phenomena. "This study identifies the missing link between Lewy bodies and the type of damage that's been observed in neurons affected by Parkinson's. Parkinson's is a disorder of the mitochondria, and we discovered how Lewy bodies are releasing a partial break-down product that has a high tropism for the mitochondria and destroys their ability to produce energy."

Lewy bodies were described a century ago, but it was not until 1997 that scientists discovered they were made of clumps of a misfolded protein called α-synuclein. When it's not misfolded, α-synuclein is believed to carry out functions related to the transmission of signals between neurons. Researchers looked at cell cultures of neurons that were induced to accumulate fibrils made of misfolded α-synuclein, mimicking Lewy bodies in patients with Parkinson's. They discovered that when α-synuclein fibrils are broken down, it often creates a smaller protein clump, which they named pα-syn* (pronounced "P-alpha-syn-star").

It turns out that the result of that partial degradation, pα-syn*, is toxic. Researchers made this discovery by labeling the pα-syn* with an antibody so they could follow it throughout the cell after it was created. They observed that pα-syn* traveled and attached itself to the mitochondria. Further investigation revealed that once the pα-syn* attached, the mitochondria started to break down. These fragmented mitochondria lose their ability to carry an electrochemical signal and produce energy. "The Lewy bodies are big aggregates and they're sitting in the cell, but they don't come into direct contact with the mitochondria in the way pα-syn* does. With this discovery, we've made a direct connection between the protein α-synuclein and the downstream effects that are observed when brain cells become damaged in Parkinson's."

Study Uncovers New Insights Into Cause of Cell Death in Parkinson's

Researchers have found that cardiolipin, a molecule inside nerve cells, helps ensure that a protein called alpha-synuclein folds properly. Misfolding of this protein leads to protein deposits that are the hallmark of Parkinson's disease. These deposits are toxic to nerve cells that control voluntary movement. When too many of these deposits accumulate, nerve cells die. "Identifying the crucial role cardiolipin plays in keeping these proteins functional means cardiolipin may represent a new target for development of therapies against Parkinson's disease."

The study revealed that inside cells, alpha-synuclein binds to mitochondria, where cardiolipin resides. Cells use mitochondria to generate energy and drive metabolism. Normally, cardiolipin in mitochondria pulls synuclein out of toxic protein deposits and refolds it into a non-toxic shape. The researchers found that in people with Parkinson's disease, this process is overwhelmed over time and mitochondria are ultimately destroyed. Understanding cardiolipin's role in protein refolding may help in creating a drug or therapy to slow progression of the disease.

High Levels of Physical Activity Prevent Several Aspects of Immunosenescence

This age of biotechnology is also an age of comparative indolence and comfort. As the research community measures specific biochemical aspects of aging, such as the decline of the cardiovascular system, or metrics relating to immunosenescence in the immune system, we might question the degree to which the results are peculiar to our era. How much of aging is the result of our choices - to eat more and exercise less than our ancestors - rather than the result of inexorable processes of biochemical damage that we, as yet, have little influence over? (Conversely, how much of past aging was due to infectious disease, malnutrition, and other adverse external circumstances that are controlled to a much greater degree today?) This topic crops up fairly often in research into the effects of exercise on health, and the research noted here is a particularly striking example of the type.

The study authors find that the age-related decline of new T cells maturing in the thymus is negligible in some people, those who exercise much more than the rest of us. This diminished supply of new T cells is thought to be an important component of immune system aging, and the failure of the immune system is very influential over many other aspects of aging: senescent cell accumulation, frailty, loss of regenerative capacity, chronic inflammation, cancer risk, and so on. Yet when we look at the demographic evidence for spread of life span based on exercise, it appears to be, at most, 6 or 7 years (with a much larger divergence when it comes to state of health over time). What does this tell us about the likely gains resulting from rejuvenation therapies seeking to regenerate the thymus? Less than we would like, I suspect, and not just because it is hard to evaluate any one contribution to aging in isolation of all of the others.

The thymus atrophies over adult life, with active tissue necessary for the production of T cells being replaced by fat. The first major loss of active thymic tissue occurs at the end of childhood, however, in a process known as involution. Immune cells are generated at a tremendous rate in children in comparison to young adults; evolution selected for a system that would be highly effective at the outset, at the cost of later issues. When it is observed that old people in their 60s and 70s who maintained a high level of fitness throughout life exhibit much the same thymic output as young people in their 20s, that tells us little regarding the outcome were the thymus restored to the same level of active tissue as is present in children. Only a mild restoration, to move thymic activity from typical aged to typical young adult, would be comparable - and why would we stop there?

Exercise can slow the ageing process

Prehistoric hunter-gatherer tribes were highly active, spending a lot of time and energy sourcing their food. If they weren't successful, they would also spend days with or little or no food. By contrast, today we are a highly sedentary society. As we get older, our physical activity levels decline even further. In our research, we have tried to determine how much this low level of physical activity contributes to the ageing of many body systems, including muscle, bone and the immune system.

We examined 125 male and female cyclists, aged 55 to 79, who had maintained a high level of cycling throughout most of their adult lives. These were not Olympians, but very keen cyclists who were able to cycle 100km in under 6.5 hours for the men, and 60km in under 5.5 hours for the women. At mid-life, people start losing muscle mass and strength at a rate of 1% to 2% per year, making it harder to carry out their normal activities such as climbing stairs. Our bones also become thinner with age and this can eventually lead to diseases such as osteoporosis. We showed that the cyclists did not lose muscle mass or strength as they aged, and their bones only became slightly thinner. We then went on to examine a body system that was not so obviously influenced by physical activity - the immune system.

When we compared the immune system of the cyclists to older adults who had not done regular exercise, and to young people in their twenties, we found that their immune systems looked most like the young persons'. In particular, we found that the cyclists still made lots of new immune T cells, produced by an organ called the thymus, which normally starts to shrink after we reach puberty. The older cyclists seemed to have a thymus that was making as many new T cells as the young people's. We investigated why this happened and found that the cyclists had high levels of a hormone called interleukin 7 in their blood, which helps to stop the thymus shrinking. Interleukin 7 is made by many cells in the body, including muscle cells, so we think that active muscles will make more of this hormone and keep the immune system, and especially the thymus, young.

Major features of immunesenescence, including reduced thymic output, are ameliorated by high levels of physical activity in adulthood

What confounds human studies of immunosenescence is that physical activity is not taken into account in either cross-sectional or longitudinal studies of immune aging. The majority of older adults are largely sedentary and fail to meet the recommended guidelines for physical activity of 150 min of aerobic exercise per week. Regular physical activity in older adults has been associated with lower levels of pro-inflammatory cytokines such as IL-6, TNFα, improved neutrophil chemotaxis and NK cell cytotoxicity, increased T-cell proliferation and improved vaccination responses. Thus, the current literature on immunesenescence is not able to determine which aspects of age-related immune change are driven by extrinsic factors and which may be the consequence of a constitutive aging programme.

Here, we studied several aspects of the adaptive immune system in highly physically active older individuals (master cyclists) in which we have shown the maintenance of a range of physiological functions previously reported to decline with age. We show that compared with more sedentary older adults, the cyclists show reduced evidence of a decline in thymic output, inflammaging and increased Th17 cell responses, although accumulation of senescent T cells still occurred. We reveal high serum levels of IL-7 and IL-15 and low IL-6, which would together provide a environment protective of the thymus and also help to maintain naïve T cells in the periphery. We conclude that maintained physical activity into middle and old age protects against many aspects of immune aging which are in large part lifestyle driven.

A Future of Gene Therapies to Greatly Reduce the Incidence of Cardiovascular Disease

It seems plausible that one of the first major mainstream areas of development for human gene therapy will involve disabling genes that sustain levels of lipids in the bloodstream. There are a number of credible targets, including those with sizable numbers of human loss of function mutants who seem to do quite well as a result of their mutation: PCSK9, ANGPTL3, ANGPTL4, and ASGR1 for example. Reducing lipid levels in the bloodstream has the effect of slowing the development of cardiovascular disease, reducing the risk of heart attack, stroke, and other related issues. The success of statin drugs is based on exactly this effect, and gene therapies would be much more effective than statins - a one time treatment producing a larger and permenant benefit.

How does this work under the hood? Why does lowering blood lipids - cholesterol, triglycerides, and so forth - have this beneficial result? The primary mechanism of interest relates to the development of atherosclerosis through damaged lipid molecules. The normal operation of metabolism produces reactive molecules that can oxidize lipids, so some small fraction of the lipids in the bloodstream are damaged in this way. With the progression of aging, various forms of cell and tissue damage and their consequences lead to the generation of many more reactive molecules, and thus greater numbers of damaged lipid molecules entering the bloodstream - the problem becomes worse over time. You might look at the progression of cause and effect that starts with mitochondrial DNA damage, for example, but there are also more systemic issues such as chronic inflammation, which goes hand in hand with greater levels of oxidative damage.

Oxidized lipids can irritate blood vessel walls, giving rise to a feedback loop of inappropriate cellular reactions that produce inflammation and draw in ever more immune cells to try to clean up the mess. Many of these cells die, overwhelmed by forms of oxidized lipid that mammalian biochemistry is not well equipped to handle. The result is a growing, fatty plaque of dead cells and harmful lipids, the signature of atherosclerosis. These plaques weaken and narrow blood vessels, and eventually something ruptures or a blood vessel is blocked - an occurrence that is often fatal, and at best disabling. Interfering in this feedback loop at any point can slow it down: cut down the amount of all lipids entering the bloodstream, make immune cells more resilient or capable, or remove just the problem lipids through some other mechanism.

I think that the latter two are better than the former, as they can in principle be made close to 100% efficient without altering the way in which cellular metabolism functions in ways that are yet to be fully understood over the long term, as is the case for dramatic reductions in blood lipid levels. It isn't the case that all lipids can be removed from the bloodstream, and it isn't the case that all oxidative damage can be prevented. In general, periodic repair is a good deal more useful than partial prevention, as repair can help those already damaged and in late stages of disease. Still, we'll be getting a more effective implementation of the worse option in the near future, it seems, as that is where the bulk of the attention is focused.

A CRISPR edit for heart disease

Consider this scenario: it's 2037, and a middle-aged person can walk into a health centre to get a vaccination against cardiovascular disease. The injection targets cells in the liver, tweaking a gene that is involved in regulating cholesterol in the blood. The simple procedure trims cholesterol levels and dramatically reduces the person's risk of a heart attack. Although antibody-based therapies have been launched to help those most at risk, the cost and complexity of the treatments means that a simpler, one-off fix such as a vaccine would be of benefit to many more people around the world.

The good news is that a combination of gene discovery and the blossoming of genome-editing technologies such as CRISPR-Cas9 has given this vision of a vaccine-led future for tackling heart disease a strong chance of becoming reality. The breakthrough came in 2003, when researchers investigated three French families with members who had potentially lethal levels of low-density lipoprotein (LDL) cholesterol and who harboured a mutation in the gene PCSK9. PCSK9 encodes an enzyme that regulates levels of LDL - or 'bad' - cholesterol. Sensing the possibilities, investigators sought to determine whether naturally occurring mutations in PCSK9 could also have the effect of lowering LDL cholesterol. After combing the data from about 3,600 individuals who provided a blood sample, the researchers sequenced DNA from the 128 participants with the lowest levels of LDL cholesterol. They discovered that about 2% of African-American participants had one broken copy of PCSK9. A follow-up study of a different, larger population similarly found mutations in almost 3% of African Americans, which was associated with an 88% reduction in the risk of ischaemic heart disease.

The liver is a preferred target organ of gene therapy for companies such as Editas Medicine, Sangamo Therapeutics, and CRISPR Therapeutics; it is straightforward to deliver genes to the liver, and the CRISPR-Cas9 tool is especially efficient in the organ, editing a greater proportion of cells than it does in most other tissues. The liver is also an excellent place from which to tackle cholesterol - it clears LDL cholesterol from the blood and is also a main engine of lipid synthesis. Researchers have shown that more than half of Pcsk9 genes in the mouse liver could be silenced with a single injection of an adenovirus containing a CRISPR-Cas9 system directed against Pcsk9. This led to a roughly 90% decrease in the level of Pcsk9 in the blood and a 35-40% fall in blood LDL cholesterol.

The approach is "absolutely plausible, even feasible", from a technological point of view. But there is also a philosophical barrier to negotiate. "You don't necessarily want to treat people who haven't got a disease yet." Others go further. "Changing lifestyle may be much more effective for a population than focusing on high-cost interventions." They worry that a gene therapy for individuals at high risk would hinder efforts to help people to help themselves. "It is the way the human mind works. Take a pill and we think we are protected."

There is certainly a reluctance to follow through in permanent gene therapies for prevention and enhancement at the moment - the work could be proceeding much more rapidly than it is, given the rapidly falling costs of genetic biotechnology. It will probably require more adventurous groups such as BioViva Sciences or Ascendance Biomedical to break down that door by simply going ahead and offering the gene therapies that are technologically plausible outside the mainstream regulatory system. These treatments will initially have a low effectiveness, in terms of the proportion of cells transfected by the therapy, but that is a challenge that will be solved with increasing efficiency over the next decade. Someone has to get started, go first, show the way. If regulatory systems as they presently exist make that hard, then the start will occur outside the regulatory framework, just as it did for stem cell therapies - and a good thing too, as that is pretty much the only circumstance that might help to make current medical regulation less oppressively terrible.

Loss of Ribosomal DNA is Associated with Aging in Flies

Researchers here make an interesting discovery in the genetics of fly aging. Old flies lose repeated DNA sequences in the genome that encode for RNA related to the ribosome, a cellular structure important in the intricate, multi-stage process by which proteins are created from their genetic blueprints. Protein creation changes in numerous ways in later life, better ribosomal function is associated with greater species longevity, and it is known that ribosomal RNA genes acquire epigenetic markers in a characteristic way with age. How exactly this all links together is yet to be determined in detail.

The more interesting part of the report here is that young flies regain lost ribosomal DNA, if they were the offspring of old parents and thus inherited a genome with few repeats of ribosomal DNA. This suggests that, whatever is going under the hood, the loss of ribosomal RNA genes is a secondary aspect of aging, driven by some other process. We might ask whether this observation in flies is relevant to mammals. It may not be, judging from the results of an older study in aged mice that examined this part of the genome and found no great losses - but that was sufficiently long ago that revisiting the topic is certainly on the agenda.

Studies in fruit flies have shown how cells in the offspring of older fathers can replace copies of genes that have been lost due to aging. The findings provide clues as to how some cells could overcome genomic shrinkage that appears to occur as an organism ages. If the same results can be confirmed in humans, they could offer a new level of understanding about how cells deteriorate with time.

The team looked specifically at ribosomal DNA (rDNA) loci that contain the genes for ribosomal RNA (rRNA). These loci are repeated at multiple sites on different chromosomes. For example, five human chromosomes contain regions in which rDNA genes are repeated hundreds of times. However rDNA is very unstable. "rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. The end result is that some copies are lost every cycle. They are popping out of the chromosome."

Studies have confirmed that in yeast cells, at least, this rDNA instability and gene copy loss underlies aging, via a process known as replicative senescence. What isn't yet known, however, is whether rDNA instability contributes to aging in multicellular organisms. To investigate this further, the researchers turned to the fruit fly, Drosophila melanogaster. Their studies looks more closely at the dynamics of rDNA loci and rDNA loss during aging in male Drosophila germline stem cells (GSCs), which continue to divide throughout adulthood. The results of their cell analyses showed that in comparison with younger male fruit flies, older males had fewer copies of the rDNA genes on the Y chromosome in their GSCs-in effect their Y chromosomes had shrunk. These older fathers then passed the reduced amount of rDNA on to their male offspring.

However, rather make do with fewer rDNA genes, the offspring were able to rebuild the number of rDNA copies in the Y chromosomes of their GSCs. By the time they had reached about 10 days of age, the sons of aged fathers had comparable amounts of rDNA to those male offspring of younger fathers that had passed on less depleted Y chromosomal rDNA. Interestingly, recovery of of rDNA copy number was limited to young adults, suggesting that the mechanisms at work might only occur under certain conditions. The results indicate that rejuvenation of rDNA in sons plays a key role in the persistence of stem cells from father to son. What isn't known yet is whether the same rebuilding of lost rDNA can also occur in female stem cells in the ovary.

Further analysis indicated that the process of rDNA copy number recovery uses the same factors that are needed for a phenomenon known as rDNA magnification, in which DNA copy number is rapidly expanded in the male germline of animals that are deficient in rDNA due to large rDNA deletions. "Our study also indicates that the phenomenon classically regarded as 'rDNA magnification' might be a manifestation of a general 'maintenance' mechanism that operates in the population that experiences normal fluctuations in rDNA copy number." The researchers suspect that mechanisms allowing cells to reset gene copy number may also be present in some types of human cells, but this has yet to be demonstrated.

The Prospects for Enhancing Repair Systems in the Brain to Treat Stroke Patients

A sizable fraction of the regenerative medicine community is interested in finding ways to improve existing repair systems in the body, and particularly in the central nervous system, which exhibits little ability to recover from injury in mammals. Initiatives in progress include efforts to increase the rate at which new neurons are created and integrated into the brain, work on ways to encourage more glial cells to adopt a pro-regenerative state, and the usual range of approaches based on delivering signal molecules found to be significant in stem cell therapies or heterochronic parabiosis studies. This open access review paper looks over some of the areas of present research. One of the more interesting points made by the authors is that the window of time for a successful regenerative intervention to restore function is very long, years or more. Any significant advance in the field will bring benefits to a large number of existing patients.

A stroke is caused by a sudden interruption of cerebral blood supply to a specific region of the brain, resulting in regional brain tissue death. Once a stroke occurs, brain tissue that is located inside and outside the infarct/lesion area undergoes significant changes over time. The major pathological cascades include primary neuron loss, secondary neuron loss, brain edema, neuroinflammation, dead cell removal, neuron functional reorganization, blood vessel regeneration, and neural network rewiring. Stroke represents a very serious medical condition and causes huge medical and financial burdens throughout the world. It remains the leading cause of long-term disability and the second leading cause of death worldwide.

Over the past few decades, major advances have been made in understanding of the pathophysiology of stroke, while there has not been much progress in the development of stroke treatment, especially for stroke recovery. Extensive efforts have been devoted to developing neuroprotective therapies to rescue dying neurons within the limited hours post-stroke, and this approach has been shown effective in animal models; unfortunately, the neuroprotective agents have all failed in clinical trials.

Despite the permanent brain tissue damage, spontaneous recovery occurs days, weeks, and months after stroke onset. This type of recovery occurs during the first 3-6 months after stroke with the most dramatic recovery from neurological impairments in the first 30 days. The mechanism underlying the spontaneous recovery after stroke has not been fully understood. Early recovery post-stroke is associated with resolution of edema and reperfusion of the ischemic tissue. Later recovery is related to brain plasticity. Brain plasticity is an intrinsic ability of the brain to reorganize its function and structure in response to stimuli and injuries from both internal and external sources. Brain plasticity is centered on neuronal plasticity, which is coupled with the changes of other types of cells in the brain such as astrocytes, microglia, and blood vascular cells. Convincing evidence shows that brain plasticity exists throughout a person's lifespan.

The involvement of astrocytes and microglial cells in neuroinflammatory responses during the early stage of stroke has been intensively studied. Microglia, the brain tissue resident macrophages, are classified into pro-inflammatory phenotype (M1 type) and anti-inflammatory phenotype (M2 type) based on their responses to local environment. The pro-inflammatory phenotype microglia release destructive pro-inflammatory cytokines, whereas the anti-inflammatory phenotype microglia produce molecules and trophic factors that participate in anti-inflammatory and tissue repair. M2 type microglia have shown beneficial effects in neurogenesis, axonal regeneration, angiogenesis, and vascular repair.

Neural stem cells (NSCs) or neural progenitor/precursor cells (NPCs) are multipotent cells that have the capacity for self-renewal and differentiation into neurons, astrocytes, and oligodendrocytes. Although extensive research has been done over the past decade, understanding the role of endogenous NSCs/NPCs in brain self-repair and spontaneous functional recovery after stroke still remains incomplete. The original hypothesis has been proposed that the NSC/NPC-generated new neurons may replace the stroke-damaged neurons, leading to brain self-repair and functional recovery after stroke. The vast majority of studies have been directed by this hypothesis and are searching for evidence that stroke-induced NSC/NPC proliferation, migration, differentiation, and survival/integration are linked to spontaneously functional recovery.

In total, convincing evidence supports that the brain has the intrinsic ability to repair itself, which is the foundation of spontaneous functional recovery after stroke. However, the capability of brain self-repair post-stroke is limited, especially in severe stroke, as spontaneous recovery is often incomplete in most stroke patients. Clearly, the brain needs more help for reinforcing the repair process. Can we provide extrinsic interventions or treatments to enhance the intrinsic ability of brain self-repair for further strengthening stroke recovery? Emerging evidence has renewed our knowledge on the time window for stroke recovery, which is much longer than previously thought. By contrast to the limited several-hour-effective window of thrombolytic/thrombectomy treatment, the therapeutic window of restorative/rehabilitative interventions is much broader, from years after stroke to lifelong applicability. Recognizing this unique feature of restorative approach will direct stroke research into a fruitful direction and provide great opportunities to develop more treatments for maximizing stroke recovery.

Funding More Work on Deep Learning for Drug Discovery to Treat Aging

Recently Y Combinator announced their intent to fund companies working on treatments for aging. It is one of the many signs of a growing interest in this area of development in the venture community. One of the early results appears to be more funding for computational methods of improving drug discovery, with therapies for aging as the rallying cry, after the established Insilico Medicine model. It makes sense that a primarily software-focused part of the venture community would move into a new area, biotechnology, by funding ventures that apply computational technology to the space. That says nothing about the effectiveness of the approach, of course, just that it is a natural evolution of established knowledge and interests.

There is certainly a lot of room for improvement when it comes to the cost and effort required to find and prove out small molecule and other drugs to treat specific conditions or target specific biological mechanisms with minimal side-effects. It is reasonable to think that established deep learning approaches can be fruitfully applied here, to focus attention on molecules in the standard libraries that might otherwise be overlooked, and to design new therapeutic molecules based on existing data and desired characteristics. There is, however, a sizable difference between, say, applying this technology to the search for senolytics and cross-link breakers, approaches that can in principle produce rejuvenation, or applying it to the search for more geroprotectors like metformin, rapamycin, and so forth. The latter can only marginally slow the progression of aging, and the research community is struggling to produce anything in that part of the field that can do any better than exercise and calorie restriction. It remains to be seen as to the direction taken by this venture.

Over the past few decades, an unignorable amount of evidence has piled up that we are able to slow the biological processes of aging in animals. This evidence has been accumulating along multiple lines of research covering many different therapies. We're left with the same conclusion: by understanding and directly treating the biological damage accumulated while aging, we can find powerful new therapies for fighting disease and living healthier, longer lives.

At Spring Discovery, we're accelerating the discovery of these therapies with our machine learning-based drug discovery platform. And we're proud to announce that we've raised a 4.25M seed round from a team of biotech funders who support our long-term vision. Why do therapies focused on aging present such a profound opportunity? Because aging is the single greatest risk factor for the most detrimental diseases on Earth - cardiovascular disease, neurodegenerative disease, pulmonary disease, cancer, muscle wasting, and more - and drugs that slow the biological damage accumulated while aging have the potential to reduce the incidences of these diseases, possibly simultaneously.

Combined, aging represent A) one of the most important problems facing humanity and B) a problem that looks increasingly possible to tackle. The diseases of old age don't discriminate, but they can be fought. We believe that in the not-too-distant future the discovery of therapies for aging will provide some of the most effective tools in history for reducing our burden of disease and extending our healthy lifespan. Spring Discovery's mission is to dramatically accelerate the realization of that future. And we're bringing a new set of machine learning tools to bear on this challenge.

Greater Levels of Dbx2 Appear Connected to Neural Stem Cell Decline in Aging

Stem cells are responsible for tissue maintenance, delivering replacement somatic cells and a variety of signals that help to keep organs and other biological systems running. There are many varieties of stem cell, at least one for every tissue type, and all have significant differences in their biochemistry. Unfortunately, one of the shared behaviors in all stem cell populations is a slowing of activity with advancing age, an evolved response to rising levels of damage in cells and tissues that probably serves to reduce the risk of cancer, but at the cost of a decline into organ failure, as essential maintenance shuts down.

The research here is characteristic of a wide range of initiatives that seek to find signal and regulator proteins that can override the evolved reduction in stem cell activity. The aim is to increase activity to youthful levels, and thus avoid the slowdown. Evidence from stem cell therapies and a variety of other approaches to regenerative medicine suggest that this will not cause as great a risk of cancer as feared, even though it doesn't address the underlying damage in cells. Forcing damaged cells in a damaged environment into greater activity must have adverse consequences at some point, but it seems there is nonetheless some leeway to do just that within the present natural state of stem cell aging.

Cells in the brain are constantly dying and being replaced with new ones produced by brain stem cells. As we age, it becomes harder for these stem cells to produce new brain cells and so the brain slowly deteriorates. By comparing the genetic activity in brain cells from old and young mice, the scientists identified over 250 genes that changed their level of activity with age. Older cells turn on some genes, including Dbx2, and they turn off other genes.

By increasing the activity of Dbx2 in young brain stem cells, the team were able to make them behave more like older cells. Changes to the activity of this one gene slowed the growth of brain stem cells. These prematurely aged stem cells are not the same as old stem cells but have many key similarities. This means that many of the genes identified in this study are likely to have important roles in brain ageing.

The research also identified changes in several epigenetic marks - a type of genetic switch - in the older stem cells that might contribute to their deterioration with age. Epigenetic marks are chemical tags attached to the genome that affect the activity of certain genes. The placement of these marks in the genome change as we age and this alters how the cells behave. The researchers think that some of these changes that happen in the brain may alter causing brain stem cells to grow more slowly. "We hope this research will lead to benefits for human health. We have succeeded in accelerating parts of the ageing process in neural stem cells. By studying these genes more closely, we now plan to try turning back the clock for older cells. If we can do this in mice, then the same thing could also be possible for humans."

ERRγ as a Target for the Development of Exercise Mimetics

The research noted here is a representative example of efforts to reverse engineer the mechanisms by which exercise produces benefits, with an eye to achieving the same result with pharmaceutical compounds rather than exertion. Exercise works to grow muscle, improve endurance, and maintain long-term cardiovascular health through some set of mechanisms, as yet far from fully explored. The future of efforts to develop exercise mimetic drugs will no doubt be as laborious and difficult as the past fifteen years of work on calorie restriction mimetics, and for all the same reasons. Both are enormously broad and complex swathe of cellular biochemistry, poorly mapped, and expensive to explore.

In this area of research, even incremental advances in understanding have required years and a great deal of funding to achieve - just look at ongoing work on sirtuins, for example. As yet none of these programs have delivered meaningful approaches to therapy, treatments that might capture a sizable fraction of the effects of either exercise or calorie restriction. This will all change at some point, as biotechnology becomes ever more capable, but it seems foolish to imagine that it will happen in the next few years, given the past record. Even when it does, "so what?" we might ask. Exercise and calorie restriction cannot add decades to healthy life spans. We need a different approach to produce far longer healthy life.

If you've ever wondered how strenuous exercise translates into better endurance, researchers may have your answer. "ERRγ helps make endurance exercise possible. It gears up the energy-creating cellular power plants known as mitochondria, creating more blood vessels to bring in oxygen, take away toxins, and help repair damage associated with muscle use. This makes ERRγ a really interesting potential therapeutic target for conditions with weakened muscles."

The story starts with the PGC1α and PGC1β proteins, which stimulate 20 other proteins associated with skeletal muscle energy and endurance exercise, including ERRγ. In turn, ERRγ, a hormone receptor, acts to turn on genes. Researchers wanted to precisely understand ERRγ's role in skeletal muscle energy production and how that impacts physical endurance. To unravel this relationship, the team studied mice without PGC1α/β. In some, they increased ERRγ selectively in skeletal muscle cells. This approach allowed them to measure how ERRγ and PGC1 act independently, as well as how they function in combination.

Losing PGC1 had a negative impact on muscle energy and endurance. However, boosting ERRγ restored function. The team found ERRγ is essential to energy production, activating genes that create more mitochondria. In other words, they found the power switch for skeletal muscles. The researchers also showed that increased ERRγ in PGC1-deficient mice boosted their exercise performance. By measuring voluntary wheel running, they found that increasing ERRγ produced a five-fold increase in time spent exercising compared to mice with no PGC1 and normal ERRγ levels. "Now that we have detected this direct target (ERRγ) for exercise-induced changes, we could potentially activate ERRγ and create the same changes that are being induced by exercise training."

Senolytic Drugs Fail to Kill Cancerous Cells with Senescent Gene Expression Signatures, but a Gene Therapy Succeeds

Some cancerous cells express signatures normally associated with senescent cells, so why not try senolytic compounds against them? This is something of a full circle, given that most of the current senolytic drug candidates were originally characterized and tested as potential chemotherapeutics. The open access paper here is interesting for two points: firstly, that senolytic drugs didn't kill cancerous cells with a senescent signature, and secondly that a suicide gene therapy targeting that signature does work against both normal senescent cells and cancerous cells with a senescent signature. The gene therapy approach reported here is conceptually similar (at a very high level) to the Oisin Biotechnologies gene therapy used to destroy senescent cells, but less flexible. The Oisin Biotechnologies founders have shown that targeting p53, a cancer suppressor, rather than p16 / p16Ink4a, a signature of senescence, is highly effective against cancer, but it appears that p16 is also a viable trigger for cell killing gene therapy mechanisms in many cancers.

p16Ink4a arrests cell cycle progression by inhibiting the S phase. Cellular senescence, a tumor suppressive mechanism defined as irreversible growth arrest and induced by accumulation of DNA damage, is often associated to induction of p16Ink4a. Consequently, p16Ink4a is considered a strong tumor suppressor. Loss-of-function mutations affecting p16Ink4a are a common mark of various human tumors, and considered an essential step towards tumor progression. However, in the presence of mutations affecting RB or CDK4/CDK6, p16Ink4a activity is not sufficient to arrest cell cycle progression. Moreover, p16Ink4a overexpression has been observed at the invasive front of endometrial, colorectal and basal cell carcinoma and correlated with high aggressiveness. Thus, under these conditions targeting p16Ink4a-overexpressing cells could be a potent anti-cancer intervention.

Despite the mutation-enabled bypass of the senescence program, sarcoma cells overexpressing RAS and with inactive p53 induced high level of p16Ink4a. We then hypothesized that treatment with compounds shown to selectively eliminate senescent p16Ink4a-overexpressing cells could be an efficient strategy. Two of the most effective compounds with senolytic properties (i.e. selectively toxic against senescent cells) are ABT-263 and ABT-737, well-known anti-cancer agents inhibiting the BCL2 family of anti-apoptotic proteins. However, neither treatment was toxic for these cancerous cells. This suggests that p16Ink4a overexpressing tumor cells are resistant to currently available compounds with specificity against p16Ink4a+ cells.

We then reasoned that an alternative strategy for elimination of p16Ink4a-overexpressing tumor cells could make use of gene targeting therapy. Suicide gene therapy has been investigated in various types of cancers because of its superior specificity compared to standard genotoxic therapies. A previous effort in testing a suicide gene therapy under the regulation of the p16Ink4a promoter - the so-called INK-ATTAC system - failed to kill p16Ink4a+ cancer cells, despite being effective in eliminating p16Ink4a+ senescent cells. We have recently developed a similar suicide system, called p16-3MR. The major difference is that the p16-3MR gene is under the regulation of the full p16Ink4a promoter, while the INK-ATTAC is regulated by a small portion proximal to the transcription starting site of the INK4a locus.

Our strategy, which we have shown being highly effective in non-proliferating cells, showed high toxicity for cancerous cells both in cell culture and in vivo. Additionally, since it has been shown that in some instances p16Ink4a+ cells are precursor of malignant cells, the 3MR system could allow reduction of tumor incidence via removal of p16Ink4a+ pre-malignant cells. At this stage, extensive research should to be done to test the toxicity of a p16Ink4a-driven suicide gene therapy strategy against additional tumor types.

Why Do Some Mitochondrial Mutations Expand to Overtake All Mitochondria in a Cell?

There is a constantly replicating herd of mitochondria in every cell, the evolved descendants of ancient symbiotic bacteria now well integrated into cellular mechanisms. They still bear a small remnant of the original bacterial DNA, however, and this is prone to mutational damage. Some forms of this damage cause mitochondria to both malfunction and become more resilient or more able to replicate than their peers. As a result, the cell is quickly overtaken by broken mitochondria and becomes broken itself, exporting damaging reactive molecules into surrounding tissues, the bloodstream, and the body at large.

This process is one of the root causes of aging, so it is a matter of considerable interest to the research community to understand exactly how it is that these damaged mitochondria can so quickly replicate to fill a cell with their descendants. That said, the beauty of the SENS rejuvenation research approach to the problem is that it really doesn't depend on how the damage occurs or spreads. It aims to place backup copies of mitochondrial genes into the cell nucleus, thus ensuring that there is always a supply of the proteins encoded in mitochondrial DNA. So if mitochondrial DNA does become damaged, then there are no further consequences, and mitochondria will nonetheless continue to function correctly.

An intriguing hallmark of aging in mammals is the appearance of cells carrying significant burdens of mitochondrial DNA (mtDNA) mutants. Unlike the mtDNA mutations which cause inherited diseases, those associated with aging appear to be somatically acquired. Within a given tissue, there is often considerable heterogeneity in the burden of mtDNA mutations, such that affected cells co-exist side by side with healthy cells that carry few, if any, mutations. Furthermore, the frequency of affected cells tends to increase with age and there is evidence that within individual cells, the mitochondrial population is commonly overtaken by a single mutant type, very often a deletion in which a part of the normal mtDNA genome has been lost. The precise mutations tend to differ from one affected cell to another, suggesting that individual mtDNA mutations arise at random. How these mtDNA mutations undergo clonal expansion is a question of longstanding interest.

The possibilities that they multiply either because of a so-called vicious cycle such that defective mitochondria simply generate more reactive oxygen species (ROS), which in turn cause more mutations, or because of random drift, have both been considered but found to be unsatisfactory. Instead, it seems most likely that new mtDNA mutations are acted upon by some form of intracellular selection, causing the expansion of a clone of mutant mitochondria that may come to dominate or entirely exclude the wild type population.

Among the various possibilities to account for a selective advantage favoring mtDNA deletions are that: (i) in a cell where wild type and deleted mtDNA molecules co-exist, there may be a selection advantage for deletion mutants since they have a smaller genome size, which might result in a shorter replication time; (ii) if mitochondria that are compromised by a high burden of mutations have a slower rate of metabolism, they may be less damaged by ROS and so relatively spared from deletion by mitophagy, thereby resulting in survival-based selection through a process that has been termed survival of the slowest; (iii) the selection advantage of mtDNA deletions might be based on features relating to some aspect of the machinery for mtDNA replication, of which several possibilities exist, at least hypothetically.

Possibility (i) has been closely examined but found to be implausible, chiefly because the time required for replication of an mtDNA molecule is only a tiny fraction (less than 1%) of the half-life of mtDNA, which drastically diminishes any scope for a size-based replication advantage to be important. Possibility (ii) has also been found to be unlikely, since not only is it incompatible with mitochondrial dynamics, but it also appears that dysfunctional mitochondria are degraded preferentially rather than more slowly than intact ones By a process of elimination, it appears probable, therefore, that the enigma of clonal expansion of mtDNA deletions requires explanation in terms of the machinery for DNA replication.

Recently, we noticed that when the locations of mtDNA deletions, which had been reported from rats, rhesus monkeys, and humans, were compared, there was a stretch of mtDNA that was overlapped in nearly every instance. Based on this observation and noting that the primer required for DNA replication is provided by processing an mRNA transcript, we suggested a novel mechanism based on this intimate connection of transcription and replication in mitochondria. If a product inhibition mechanism exists that downregulates the transcription rate if sufficient components for the respiration chain exist, then deletion events removing a region of the genome involved in this feedback-loop would confer to such deletion mutants a higher rate of replication priming, leading to a substantial selection advantage. In this article, we report additional data from mice that are strongly consistent with our previous analysis of rats, monkeys, and humans, and we further examine the implications of the hypothesis that a shared sequence, falling within the common overlap of these many individual deletions, might throw light on the underlying mechanism for clonal expansion.

Evidence Against Adult Human Neurogenesis

The results here will cause some upheaval in the research community if verified, and will do doubt lead to considerable debate regardless of the outcome. For decades it is has been considered that neurogenesis, the production and integration of new neurons in the brain, continues past childhood, albeit at a lower rate. This is based largely on studies in mice, but also on a range of human evidence. The researchers here suggest that this is wrong, and in fact humans are not like mice in this regard: we do not generate new neurons at any detectable level as adults. This question of adult neurogenesis has great influence on the strategies adopted in the development of therapies that might enhance maintenance of the brain. This is a topic of considerable importance to the future of human rejuvenation: we are our brains, and damage and loss must be repaired in situ. If there are no naturally occurring mechanisms to achieve that goal in some or all of the brain, this suggests that the task will be that much harder to safely achieve.

One of the liveliest debates in neuroscience over the past half century surrounds whether the human brain renews itself by producing new neurons throughout life, and whether it may be possible to rejuvenate the brain by boosting its innate regenerative capacity. Now scientists have shown that in the human hippocampus - a region essential for learning and memory and one of the key places where researchers have been seeking evidence that new neurons continue to be born throughout the lifespan - neurogenesis declines throughout childhood and is undetectable in adults.

The findings present a challenge to a large body of research which has proposed that boosting the birth of new neurons could help to treat brain diseases such as Alzheimer's disease and depression. But the authors said it also opens the door to exciting new questions about how the human brain learns and adapts without a supply of new neurons, as in seen in mice and other animals. It was once neuroscientific dogma that the brain stops producing new neurons before birth. In the 1960s, experiments in rodents first suggested that new neurons could be born in the adult mammalian brain, but these results remained highly controversial until the 1980s, it was shown that new neurons are born and put to use throughout life in several parts of the songbird brain.

These findings launched a whole field of research. Much work has focused on a region of the hippocampus called the dentate gyrus (DG), where rodents produce newborn neurons throughout life that are thought to help them form distinct new memories, among other cognitive functions. Rodent studies have shown that DG neurogenesis declines with age, but is otherwise quite malleable - increasing with exercise, but decreasing with stress, for example - leading to popular claims that we can boost brain regeneration by living a healthy lifestyle. Beginning in the late '90s, a handful of studies reported evidence of adult neurogenesis in the human brain, either by estimating the birth dates of cells present in postmortem brain specimens or by labeling telltale molecular markers of newborn neurons or dividing neural stem cells. However, these findings, some of which were based on small numbers of brain samples, have remained controversial.

In the new study, researchers collected and analyzed samples of the human hippocampus. They analyzed changes in the number of newborn neurons and neural stem cells present in these samples, from before birth to adulthood, using a variety of antibodies to identify cells of different types and states of maturity, including neural stem cells and progenitors, newborn and mature neurons, and non-neuronal glial cells. The researchers also examined the cells they labeled based on their shape and structure - including imaging with high-resolution electron microscopy for a subset of tissue samples - in order to confirm their identity as neurons, neuronal stem cells, or glial cells.

The researchers found plentiful evidence of neurogenesis in the dentate gyrus during prenatal brain development and in newborns, observing an average of 1,618 young neurons per square millimeter of brain tissue at the time of birth. But the number of newborn cells sharply declined in samples obtained during early infancy: dentate gyrus samples from year-old infants contained fivefold fewer new neurons than was seen in samples from newborn infants. The decline continued into childhood, with the number of new neurons declining by 23-fold between one and seven years of age, followed by a further fivefold decrease by 13 years, at which point neurons also appeared more mature than those seen in samples from younger brains. The authors observed only about 2.4 new cells per square millimeter of DG tissue in early adolescence, and found no evidence of newborn neurons in any of the 17 adult post-mortem DG samples or in surgically extracted tissue samples from 12 adult patients with epilepsy.

Visible Light Influences the Longevity of Nematodes

Researchers have found a reason to distrust the results of past nematode life span studies with modest effect sizes, even those that controlled for the effects of dietary intake on longevity, a now well-known issue in animal studies that has caused plenty of problems in the past. The researchers have found that light exposure affects the life span of the commonly used Caenorhabditis elegans species of nematode. Their data shows a sizable difference between conditions of permanent light and permanent darkness, but the problem would arise more subtly in comparison between studies where duration, intensity, and type of lighting varied - say, by season, by employee hours, by building fixtures, by nematode housing, and so forth. By the sound of it, this is bad news for near all past life span studies carried out with nematodes, casting doubt on a large amount of exploratory data in the field of aging research.

Historically, the nematode Caenorhabditis elegans was believed to lack the ability to sense light due to the absence of a bona fide photoreceptor system and its original isolation in soil samples. However, recent work in C. elegans has identified the LITE-1 taste receptor homolog as a UV-specific photoreceptor. Interestingly, high-energy UV and blue wavelength light trigger escape behavior and feeding inhibition in C. elegans. In contrast to animals with external pigmentation, the transparent body of nematodes allows light to penetrate their body, making them particularly vulnerable to the mutagenic effects of UV.

We used C. elegans to test whether the photoperiod (the interval in a 24-h period during which an animal is exposed to light) could impact its physiology and lifespan. This is also of special interest since standard laboratory manuals and practices for C. elegans handling completely ignore random exposure to light versus dark. Here, we demonstrate that daily exposure to white light decreases C. elegans lifespan and alters development. Importantly, these effects are not mediated through known photoreceptor pathways or through a proper disruption of circadian rhythms. Our results indicate that the effect of light on C. elegans lifespan is not specific to a particular wavelength of the visible spectrum, but is photon energy dependent.

We find that light exposure causes oxidative stress and induces canonical stress responses. Several long-lived mutants that ectopically activate these stress-responsive pathways are resistant to light stress. Furthermore, we find that treatment of wild-type worms with antioxidants is sufficient to rescue their short lifespan due to light exposure. Such findings strongly invite a reconsideration of the standard methods of C. elegans handling, especially in the context of aging research and stress biology.

Aspirin as a Calorie Restriction Mimetic that Enhances Autophagy

One of the better ways to dampen down the unhelpful hype generated by one or another new supplement or drug alleged to modestly slow aging on the basis of animal data is to point out that aspirin does just as good a job in animal studies. We all know what aspirin does for human life span, which is to say pretty much nothing, while still managing to be a useful tool in the pharmaceutical toolbox. Chasing marginal outcomes in human longevity will at best achieve marginal outcomes - and that is the major problem with the mainstream focus on trying to recapture the beneficial effects of calorie restriction through any number of candidate calorie restriction mimetic drugs. We need to do better, to aim higher. This means more work focused on the development of therapies after the SENS model, those that repair the molecular damage that causes aging and thus are capable in principle of achieving rejuvenation and significant extension of healthy human life.

The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs). Here, we show that aspirin or its active metabolite salicylate induce autophagy by virtue of their capacity to inhibit the acetyltransferase activity of EP300.

We demonstrate that aspirin fails to modulate autophagic flux in cells lacking EP300 or cells in which EP300 has been engineered to avoid aspirin binding to the enzyme. As a confirmation of the evolutionarily conserved nature of this process, we demonstrate that aspirin failed to further induce autophagy in Caenorhabditis elegans strains deficient for the EP300 homolog CBP-1 or the essential autophagy gene products ATG-7 and BEC-1.

Based on the results described in this paper, aspirin may be classified as a CRM. Indeed, aspirin fulfills all the criteria of a CRM as it (1) reduces protein acetylation by virtue of its ability to inhibit the acetyltransferase activity of EP300, (2) stimulates autophagic flux, and (3) has no cytotoxic activity. At this point, it remains to be determined to which extent EP300 inhibition and autophagy activation may effectively contribute to these aspirin effects that apparently transcend its well-established anti-inflammatory effects.

Pre-clinical evidence suggests that a brain-permeable aspirin derivative can reduce tau-mediated neurodegeneration in an EP300-dependent fashion. However, the role of autophagy has not been explored in this setting. Epidemiological and experimental data indicate that a high nutritional uptake of the EP300 inhibitor spermidine counteracts cardiac aging, both in humans and rodents. In addition, spermidine reduces arteriosclerosis and colon carcinogenesis in mouse models. These spermidine effects hence show a notable overlap with those of aspirin, in accord with the observation that both compounds inhibit EP300.


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