Thursday, July 20, 2017

Senolytics and allotopic expression

Aging and Cell Senescence

The pursuit of an indefinite healthy lifespan, or healthspan, is the most noble challenge put before mankind. To get there, many bold technologies have been proposed and explored. Among them are two approaches that have already achieved some preliminary measure of success. In particular, they are the elimination of cellular senescence by drugs or immune activation, and the so-called ‘allotopic expression’ of mitochondrial genes into the nuclear DNA. Regrettably, neither of these methods stand a chance, for reasons I will detail in this essay. Fortunately, at the confluence of these seemingly isolate approaches there is a way forward. This interface -- namely, the mitochondrial control of cellular senescence -- offers deep promise.

Cellular senescence refers to cells that can no longer divide. Neurons and heart cells are senescent, but they are not a threat. Rather, they are the most important cells in the body. In terms of the standard cell cycle, these quiescent cells exist in the resting or ‘G0  growth phase’. By contrast, there are many other senescent cells in the body that have outlived their useful lifespans and linger on in a crippled limbo. When things are working properly, such cells have built in programs to bow out gracefully -- they safely self-destruct in a process called apoptosis.

However it is often the case, that cells are damaged to the extent that they lose this self-regulating capability and become a liability. Sometimes, cells end up senescent as a result of too much replicative power, as in cancer. Here, the cell (often with highly damaged DNA) manages to halt its runaway replication, but fails to deploy the full apoptotic program. The rapidly exploding new field of ‘senolytics’ aims to eliminate these cells with senolytic drugs.

Allotopic expression

The other approach to strike at the heart of the aging process is take the genes normally expressed in mitochondria and put them into the nucleus. The logic behind this is that here they would be immune to corruption from the free radicals that saturate the mitochondrial interior. The success of this strategy depends on the ability to re-target the proteins made by these genes back to the mitochondria where they are assembled into various subunits of the essential respiratory complexes. Proponents of this philosophy fancy this endeavor as a natural completion of a long evolutionary process of naturally off-loading the genes of our mitochondrial endosymbionts to the nucleus for safe keeping.

High-tech billionaires
These two anti-aging schools of thought have drawn ample attention both from established corporate biotech, and from those individuals most naturally disposed to command them -- namely, the high tech billionaires. One place that has figured prominently in the development of both plans is the SENS (Strategies for Engineered Negligible Senescence) Foundation. Its primary founder, Aubrey de Grey, has been the most influential proponent of these technologies for the last 15 years. Multi million dollar care packages have been donated to SENS by the likes of Peter Thiel, Larry Ellison, and many other high tech luminaries. In turn, SENS oversees both in-house experiments as well spinning these funds off to other outside researchers that apply to them for grants. See recent profile on Aubrey by the editor in chief of Billionaire Magazine;

Some successes

Research in senolytics has so far hinged on several assumptions. The main one is that senescent cells possess something in common which can be used both to identify them, and also target some therapy towards them. Typically this would be expression of a specific protein that marks them, or a defined pathway of protein operations would render them uniquely susceptible.


The first problem with senolytics is that the assumptions made have in the fullness of time proven to be largely invalid. There simply is no unique marker only expressed by senescent cells, and there is no pathway that can be uniquely targeted. The second problem is that the drugs that have been promoted are actually highly toxic. The only reason that any of them have managed to gain any traction in the anti-aging community is by virtue of the fact that they have been previously approved by the FDA as chemotherapy or anti-viral drugs. That is no small feat in today’s regulatory pipeline. The kicker though, is that these drugs are now being promoted as senolytics simply because were too toxic to continue to justify their use in killing tumors and have therefore become unemployed. But change a small functional group here or there and you have a different drug as far as historical baggage -- albeit one with largely the same functionality in the body. This is madness.

With allotopic expression, the main problem is that the field has failed to distinguish in any tangible way between what I would call ‘soft’ and ‘hard’ allotopic expression as it pertains to aging. Soft expression would be simple putting a copy of single gene into the nucleus. There is ample evidence from nature that this might in theory be made to work. There are also recent results from companies like GenSight and Foundations like SENS indicating that individuals who have massive functional deficits in these particular mitochondrial genes might be improved.

While most mammals and other higher-ups typically retain around 13 mitochondrial genes, nature doesn’t always use that mold. Other species like drosophila and many single celled protists can get by with less. Some have even offloaded all their tRNAs or mRNAs to the nucleus and import them as well.They do this however, only by making qualitative sacrifices in those particular genes jettisoned off the nucleus. Typically for proteins, this involves making them less hydrophobic so that they manage to get imported back to mitochondria.

If we want to stretch things a bit, one could even include expressing a few copies from a small set of genes from a particular respiratory complex into the nucleus under the umbrella of soft expression. However, things would rapidly go downhill from there. Hard expression -- putting copies of all the mitochondrial genes into the nucleus -- would be a PR disaster for mitochondria. Their reason to be would vanish.

What the SENS supporters have thus far failed to appreciate is that in the decade or so since the major tenets of their anti-aging strategy were formed researchers learned a lot about how respiratory complexes are actually assembled and deployed. Regarding allotopic expression, my sentiment is simple -- mitochondria aren’t built like that. As genetic hybrids, mitochondria are organelles that exploit two separate transcription and translation systems in what we might call a meta-genetic cooperative. In other words, the seemingly massive and disproportionate overhead of carrying nearly twice as many tRNAs as proteins is actually their primary feature rather than a bug.

Two uniquely optimized translation systems, one on the outside membrane of the mitochondria facing the cytosol, and one on the inside membrane simultaneously working in tandem in the mitochondrial matrix, painstakingly construct the electron transport complexes with precision clockwork. These are in turn positioned with particularly stoichiometries into larger supercomplex geometries within the cristae. What that means for us is that although one might have some success in rescuing gross genetic deficits by allotopically saturating ailing mitochondria with a missing protein, one is not going to meaningfully improve normally aging individuals with these nuclear enticements.

A curious thing has unfolded in aging research that seems to have caught many off guard. That thing is the realization that free radicals (like dual genetic systems) are actually features rather than just bugs. What was the response of the SENS Foundation to this newsflash as it would pertain to the rationale underlying allotopic expression? I have not heard.

I would argue that rather than trying to allotopically diminish our mitochondria, what we should be doing is trying to enhance them. In other words, give them more genes and more powers. This is something we can already do. For example, one group has reported the ability to optically control membrane potential in mitochondria by giving them special proteins.

Charlie Gard was made famous this past July after Donald Trump and Pope Francis made compassionate pleas to an English hospital which was for all intents and purposes holding him as a medical prisoner. When the parents wanted Charlie to see an American doctor and try a seemingly rational (if a long shot) therapy to replace the nucleosides his mitochondria could not make, a court had to intervene to stop the English hospital from pulling the plug on his respirator. Both copies of Charlie’s RRM2B genes were already expressed from the nucleus but both copies he inherited were mutated. What Charlie really needed was an infusion of good mitochondria.

Unfortunately, you can’t really just inject mitochondria into the blood like you would in a transfusion. For one thing, mitochondria are highly immunogenic by virtue of their bacteria-style formylated peptides and undermethylated CpG islands (regions of their genome that frequently given extra methyl group tags via special epigenetic regulatory enzymes). In fact our immune system exploits mitochondria by ejecting them to self-activate. For the all the recent hype regarding the special functions of the so-called microbiome -- the sum total of gut, skin, or other bacteria that lives on and in us -- it should probably just be said that the ‘real’ microbiome is our mitochondria. The mitochondriome being all the unique variants of mitochondria that heterogeneously unfold and exchange within our differentiated tissues.

Notwithstanding this unassailable conventional wisdom, a Chinese group testing this out in mice and found evidence that brute force injection can have some success in the acute phase. They reported no immune activation for in the first 2 hrs after injection, however when I asked if they found any later on, they said they will need to look further. Intriguingly the researchers found that cells from many different organs had taken up apparently functional mitochondria.  

I suggested that they might try injecting mitochondria directly into the ventricular system of the brain and this is something they hope to investigate. If the mitochondria can pass the blood brain barrier, or alternatively can be supplied more directly to different nerve access points, then many new possibilities open up. For example, it is now understood that many cancers are directly controlled by not only by their own mitochondria, but also are transformed by donation of mitochondria from other cells.

It is also known that the many cancers are controlled by the nervous system (gastric cancers, skin cancers, etc). Similarly, various stem cell populations, cell senescence, and regeneration of tissues, organs, and limbs are also all under tight control of the nervous system. The one other key fact we need to tie together these elements is that nerves take the donation and absorption of mitochondria to an extreme. It is their bread and butter. Perhaps even the reason nervous systems first evolved and then later rectified themselves into polarized circuits transmitting from dendrite to axon. (It is not too difficult to imagine mitochondria seeking refuge in the small proto-appendages of cells where they could escape predation and degradation by the lysosomes in the locale of the nucleus, with the fully arborized neuron later evolving as a selector of mitochondria).

The full Monty is now laid bare;
The nervous system controls the body, the many cell niche populations in various states of senescent and proliferation by apportioning mitochondria.

I first published some of the evidence supporting these claims here
And many related comments regarding the nervous system earlier in a previous article for Inference.

In this light cell senescence is not quite so irreversible as one might assume.

The immediate task ahead would be to better identify the best access points to add or siphon off new or obsolete mitochondria, and even modified super mitochondria, as needed. To do this we would essentially need some kind of circuit diagram or principles which reflects how mitochondria are transmitted throughout the body. Much of this diagram is already quite inferable from existing literature.

Thursday, July 13, 2017

Caloric restriction for anti-aging

A recent paper published in GeroScience by researchers at the University of Oklahoma provides some much needed food for thought for the field of caloric restriction. The title, ‘Role of DNA methylation in the dietary restriction mediated cellular memory’ suggests some underlying mechanisms have been uncovered which can explain the presumed healthful benefits of dietary restriction (DR). If so, then what exactly is the nature of these apparent relationships and what are their benefits?

Somewhere in the nebulous aether filling the void between correlation and causality lies the frequently employed dictum of ‘plays a role in’. The way I read things here there are  at least 3 or 4 variables at play upon which we might imposed a natural sequence or order. Namely we have in the following scheme (1 >2 > 3 >4):

Dietary Restriction  >  Decreased Methylation Of Certain Promoters  >  Increased Expression Of The Genes Under Control Of Those Promoters  >  Cell Memory In The Form Persistent Changes To Parts 2 And 3.

While several genes (Pomc, Hsph1, and Nts1) and their inclusive islands of methylation were clearly operated on by dietary restriction,  the most important findings were found within the Nts1 gene. Nts1 encodes the Neurotensin Receptor 1. This G-protein transduces whatever message it is that the 13 amino acid long neuropeptide neurotensin signal locally supplies. This message intimately depends on cell type, and where the cells are found.

Centrally, neurotensin functions in the hypothalamus, a place where each nuclei contains unique neuronal subtypes specializing in the production of a few eclectic signal molecules. Perception of hunger and satiety are believed to be integrated there, as is the nutrient-dependent control of subsequent food- sensing and food-seeking behaviors. It is therefore a logical place to further explore the many established links between nutrients and epigenetic changes like methylation.

Neurotensin acts to lower blood pressure, raise blood sugar, lower body temperature, and confer antinociception. Another pathway leads to the release of prolactin and luteinizing hormones, ultimately through activity in the arcuate nucleus (where the Pomc neurons are also found) via its own set of  tediously constructed peptides. The hypothalamus in turn is supplied with signals, nutrient, and even mitochondrial product from the rest of the brain via the thick fiber bundle from the hippocampus known as the fornix.
It has been argued that these diverse hypothalamic and pituitary mediators represent so-called reliable or ‘honest’ signals conforming to the ‘handicap principle’ -- otherwise popularly known as the ‘peacock effect’. One example of this would be the arduous steroid and vitamin D synthesis chains whose rate-limiting steps are frequently resident in the mitochondria. These expensive and rare (ie. difficult to replicate and active at very low concentration) signals of metabolic state get funneled to the body at large through individual kingpin neurons residing at apex positions in the brain.
In peripheral regions, neurotensin’s major local effects are in the small intestine where it leads to secretion and smooth muscle contraction via enteroendocrine cells. It also acts to preserve intestinal stem pool which is why the authors make special mention that memory effects in methylation of the Nts gene may be critical there.  
The primary experimental manipulation performed here was to place mice on a DR diet for a few months and then return them to a normal diet for another few months and see what happens. Perhaps not surprisingly significant changes in gene expression were noted within one month of the initial DR. After normal feeding was done the authors found both reduced methylation at three key CG sites in the Nts gene promoter using standard bisulfite amplicon sequencing, and also an overall increase in expression of Nts1 transcripts using RNA-Seq techniques. This confirms their initial hypothesis flowing from DR to a cell memory effect.

While these expression and methylation findings are all well and good, I think at this point we need to ask a question that might be on the minds of any red-blooded American:

How on Earth does Herschel Walker, top mixed-martial arts competitor today at age 55 and still widely regarded as the greatest college running back of all time, manage to crank out over 1500 push-ups, 1500  sit-ups, 1500 pull-ups, followed up by a grueling sprint and long distance workout, each day, every day, while eating nothing but a small dinner of salad, soup, and bread?

I am not  presenting Hershel’s claims above as facts to be swallowed whole by the reader, but rather offering them up for consumption as something we must potentially account for. Putting up those kinds of numbers, ie. physical work produced from calories burned, is simply not possible by any known man-made machine, and is just barely imaginable for one of flesh and blood. So how does he maintain a stout 225 lb frame where mere mortals attempting to subsist on lumpy green shakes would rapidly shrivel and etiolate? In other words, what exactly is his nuclear DNA, mitochondrial DNA, and epigenetic state?

To answer that we would probably need to do more than a quick cheek swab and sequencing from somebody like 23andMe. In fact we’d have to do a lot more to attempt to understand something so fickle as epigenetics, something that can purportedly respond to any given sandwich with a cascading butterfly effect of changes. In many instances these changes are not limited to simple cell memory, but rather can be experimentally extended into heritability and beyond.

So we must dive deep into our evolutionary roots to uncover the origins, and thereby implications of what methylation really is. It is widely held that methylation in its most ancient incarnation first arose in bacteria for purposes of transposon control. Regarding transmission of acquired characteristics, like diet, it should not escape notice that bacteria are essentially full blown Lamarckian creatures -- their immediate daughter fission products directly feel the full metabolic life history of the parent.

The transposon idea may have some substance to it because methylation is a powerful transcriptional repressor in animals plants and protists that at least in CpG environments can ensure permanent silencing of rogue transposable elements. Furthermore, methylation tends to repress genes of multicellular organisms that need to be silenced in most but not all differentiated tissues.
Yet bacteria do not take methylation to the extent that higher mammals like ourselves do. In fact, one of the ways in which our immune system recognizes and deploys our own bacterial endosymbionts, aka the mitochondria, is by the very absence of any methylated mtDNA should their nucleoids get released into blood.

Another major clue we have is that between generations in mammals, DNA methylation patterns, including many imprinting and X-inactivation marks, are largely erased and then re-created to various levels of fidelity at several key stages. The first is a ‘genetic reboot’ consisting of deliberate demethylation and remethylation during gametogenesis. The next two happen first in pre-implantation period of the zygote and then in the blastula stage where the CpG islands are shielding from a bulk methylation wave so that a global repression phase allows housekeeping genes to be deployed through the entire blastula. After that, methylation patterns become tissue and cell type specific to permit stable differentiation.

At the most basic level many aspects of genome-wide methylation are quite predictable. For example, it has been suggested that the use of thymine in DNA as opposed to uracil in RNA evolved for error-control purposes because any deleterious uracils generated by spontaneous deamination of cytosine could be more easily recognized and removed. 5-methylcytosine has its methyl group at the same spot thymine does, which is the only thing that distinguished thymine from uracil. CpG methylation is evolutionarily costly because over time methylated cytosine spontaneously deaminate to thymine.

The maxim that hypermethylated CpG in promoters leads to repression of gene expression is not always the case. In a paper published in Science last week
authors looked at 542 human transcription factors and found that for many, particularly homeodomain proteins active during development, methylated promoters enhanced transcription.

Another study revealed that a reliable biomarker of chronological aging can be extracted by looking at methylation at 353 sites in the human genome. This perhaps is the kind of data we might seek to answer the question we posed above. Namely, how Herschel Walker can look like he does by basically running on fumes while many top powerlifters and bodybuilders with comparable physiques claim to require anywhere from 5000-10000 calories a day.

Complete evulsion of right proximal outer bicep tendon head, bug or feature?

Dear Doc,

I wanted to let you know what I decided to do regarding my right proximal outer bicep tendon tear. First of all, I want to thank you for making the time to see me, and for your excellent consultation and advice. Having watched a couple youtube videos now, I am very impressed with the the surgical tools now available to re-attach tendons, and also the skill with which surgeons like yourself can wield them. I have no doubt you are among the best, and would have every confidence in your abilities if you did surgery on my arm.

When I saw you a couple weeks ago I had fairly severe cramping in the bicep, which I hoped would soon subside, and also had  real concerns it was still in the process of some further tearing throughout the muscle because of the way it felt. For the most part the cramping is gone now. Although I have not had any MRI, mainly because I do not like being stuffed in the tunnel (particularly without any chance to do any kind a quick ‘dry run’ ahead of time to get acclimatized and relieve anxiety), I am sure you were right that the proximal tendon tore completely.

Looking back, I think the tendon must have been pretty much gone for some time and was merely hanging on uselessly by a thread that served to do nothing for me save keep the bicep from crumpling up like it is now, and causing me intense pain for two years. I think that because when it finally went, there was absolutely no blood or bruising (like both of us expected) in the arm at large afterwards, and also because for the last two years that I had the shoulder pain, and also mid bicep cramping (like when I took long jogs), I could notice the slightest little droop in the right bicep relative to left.

I must say I am elated that I can now throw footballs, shoot hoops, and lift weights without the pain I had grown accustomed to and that going through a surgery and the associated recovery time is the furthest thing from my mind right now. My expectation now for surgical reattachment to the humerus somewhere at this point would really only be to serve a cosmetic purpose, and that alone cannot justify surgery given my personal aversion to what I see as the endemic and unavoidable risks to general anesthesia (and intubation). I am not an expert in upper arm anatomy but my impression is that the ideal attachment point to give the muscle any actual purpose for generating additional supination force would be high up in an area where real estate is already expensive -- in other words, I  imagine you would have to make space by taking it from somewhere else.

Yes the bicep still looks a little strange, but having tested things now, the arm as a whole somehow seems to have full strength. I know the conventional wisdom is the tendon does not sprout and reattach, but as I mentioned, my experience with other injured parts of my body and eventual recovery suggests I should give natural healing processes a shot and see what my own rehabilitation efforts might accomplish. I must agree with Leonardo Da Vinci who apparently was the first to say that the biceps is only a minor playing in elbow flexion and really acts primarily to assist in supination. My baseline test for that is that I used to do 10 pullups with a 60lb weight vest on; if I can do that again eventually I will be content. As far as supination, I have not tested things yet by trying to torque down a bolt or open a big bottle of tightly corked Belgian beer, but soon will.

I was curious that in your experience the preferred method of reattachment was a titanium screw as opposed to something that would eventually degrade away, but understand that surgery is not an a-la-carte affair where the patient picks what they imagine might work and the surgeon complies. One last comment. In looking at the curious structure of the tendon and the way it winds around to eventually form the labrum, it is not surprising to me that this thing eventually frays in many men who do evolutionarily odd things like throw balls. If I had to guess I would imagine this kind of structure formerly was a good way to stabilize and provide feedback for walking on four limbs, something of little use to me now, and that my own self pruning has essentially acted more as a feature than a bug.