Should you take aspirin after you exercise?

I just got back from a beautiful four and a half mile walk around a reservoir behind my house.  I always take 2 adult aspirin after such things like this.  A recent paper implies that perhaps I should not [ Proc. Natl. Acad. Sci. vol. 114 pp. 6675 – 6684 ’17 ].  Here’s why.

Muscle has a set of stem cells all its own.  They are called satellite cells.  After injury they proliferate and make new muscle. One of the triggers for this is a prostaglandin known as PGE2 — https://en.wikipedia.org/wiki/Prostaglandin_E2 — clearly a delightful structure for the organic chemist to make.  It binds to a receptor on the satellite cell (called EP4R) following which all sorts of things happen, which will make sense to you if you know some cellular biochemistry.  Activation of EP4R triggers activation of the cyclic AMP (CAMP) phosphoCREB pathway.  This activates Nurr1, a transcription factor which causes cellular proliferation.

Why no aspirin? Because it inhibits cyclo-oxygenase which forms the 5 membered ring of PGE2.

I think you should still aspirin afterwards, as the injury produced in the paper was pretty severe — muscle toxins, cold injury etc. etc. Probably the weekend warriors among you don’t damage your muscles that much.

A few further points about aspirin and the NSAIDs

Now aspirin is an NSAID (NonSteroid AntiInflammatory Drug) — along with a zillion others (advil, anaprox, ansaid, clinoril, daypro, dolobid, feldene, indocin — etc. etc. a whole alphabet’s worth). It is rather different in that it has an acetyl group on the benzene ring.  Could it be an acetylating agent for things like histones and transcription factors, producing far more widespread effects than those attributable to cyclo-oxygenase inhibition.   I’ve looked at the structures of a few of them — some have CH2-COOH moieties in them, which might be metabolized to an acetyl group –doubt.  Naproxen (Anaprox, Naprosyn) does have an acetyl group — but the other 13 structures I looked at do not.

Another possible negative of aspirin after exercise, is the fact that inhibition of platelet cyclo-oxygenase makes it harder for them to stick together and form clots (this is why it is used to prevent heart attack and stroke). So aspirin might result in more extensive micro-hemorrhages in muscle after exercise (if such things exist).

Gotterdamerung — The Twilight of the GWAS

Life may be like a well, but cellular biochemistry and gene function is like a mattress.  Push on it anywhere and everything changes, because it’s all hooked together.  That’s the only conclusion possible if a review of genome wide association studies (GWAS) is correct [ Cell vol. 169 pp. 1177 – 1186 ’17 ].

 It’s been a scandal for years that GWAS studies as they grow larger and larger are still missing large amounts of the heritability of known very heritable conditions (e.g. schizophrenia, height).  It’s been called the dark matter of the genome (e.g. we know it’s there, but we don’t know what it is).

If you’re a little shaky about how GWAS works have a look at https://luysii.wordpress.com/2014/08/24/tolstoy-rides-again-schizophrenia/ — it will come up again later in this post.

We do know that less than 10% of the SNPs found by GWAS lie in protein coding genes — this means either that they are randomly distributed, or that they are in regions controlling gene expression.  Arguing for randomness — the review states that the heritability contributed by each chromosome tends to be closely proportional to chromosome length.  Schizophrenia is known to be quite heritable, and monozygotic twins have a concordance rate of 40%.  Yet an amazing study (which is quoted but which I have not read) estimates that nearly 100% of all 1 megabase windows in the human genome contribute to schizophrenia heritability (Nature Genet. vol. 47 pp. 1385 – 1392 ’15). Given the 3.2 gigaBase size of our genome that’s 3,200 loci.

Another example is the GIANT study about the heritability of height.  The study was based on 250,000 people and some 697 gene wide significant loci were found.  In aggregate they explain a mere SIXTEEN PERCENT.

So what is going on?

It gets back to the link posted earlier. The title —  “Tolstoy rides again”  isn’t a joke.  It refers to the opening sentence of Anna Karenina — “Happy families are all alike; every unhappy family is unhappy in its own way”.  So there are many routes to schizophrenia (and they are spread all over the genome).

The authors of the review think that larger and larger GWAS studies (some are planned with over a million participants) are not going to help and are probably a waste of money.  Whether the review is Gotterdamerung for GWAS isn’t clear, but the review is provocative.The review is new and it will be interesting to see the response by the GWAS people.

So what do they think is going on?  Namely that everything in organismal and cellular biochemistry, genetics and physiology is related to everything else.  Push on it in one place and like a box spring mattress, everything changes.  The SNPs found outside the DNA coding for proteins are probably changing the control of protein synthesis of all the genes.

The dark matter of the genome is ‘the plan’ which makes the difference between animate and inanimate matter.   For more on this please see — https://luysii.wordpress.com/2015/12/15/it-aint-the-bricks-its-the-plan-take-ii/

Fascinating and enjoyable to be alive at such a time in genetics, biochemistry and molecular biology.

The best laid plans of mice and men

I sent a copy of the previous post (reprinted below) about an idea to diagnose and treat chronic fatigue syndrome to Dr. Norman Sharpless, the author of the Cell review on cellular senescence.  He thought the idea was “great”; and, even better, he ran the lab which did the test I wanted to try.  I also sent a copy to a patient group.  “Solve ME/CFS Initiative”, and they want to use the post on their website.

Sharpless noted that the problem with ideas like this is accumulating patients, something the patient group could probably provide.  So all went well until 8 days ago when Dr. Sharpless was named to be the head of the National Cancer Institute, with its 4.5 billion dollar  budget by President Trump.  Being a full prof at the University of North Carolina Medical School, he would have been the ideal individual to run the study (or find someone to do it), but he now has far bigger fish to fry.

After I wrote to congratulate him, he wrote back reiterating that the idea was good, but he said he had to sever all connections with the lab he founded due to conflict of interest considerations.  He did give me the name of someone to contact there, which is where the matter stands presently.

Since the idea is based on the correlation between the amount of fatigue after chemotherapy with the level of a white cell protein (p16^INK4a), he would have had no problem accumulating chemotherapy patients as head of NCI, but again the spectre of conflict of interest rears its ugly head.  Repeating the chemotherapy study to make sure the results are in fact real is the first order of business.

So there you have a research idea, endorsed by the new head of the NCI.  I am a retired neurologist, who no longer has a license to practice medicine (but who doesn’t need a license to think).

If you’re an academic out there, looking for something to do, write up a grant proposal.  The current treatments do help people live with chronic fatigue syndrome, but they are in no sense treatments of the underlying problem.

Here is the original post

How to (possibly) diagnose and treat chronic fatigue syndrome (myalgic encephalomyelitis)

As a neurologist I saw a lot of people who were chronically tired and fatigued, because neurologists deal with muscle weakness and diseases like myasthenia gravis which are associated with fatigue.  Once I ruled out neuromuscular disease as a cause, I had nothing to offer then (nor did medicine).  Some were undoubtedly neurotic, but there was little question in my mind that some of them had something wrong that medicine just hadn’t figured out.  Not it hasn’t been trying.

Infections of almost any sort are associated with fatigue, probably because components of the inflammatory response cause it.  Anyone who’s gone through mononucleosis knows this.    The long search for an infectious cause of chronic fatigue syndrome (CFS) has had its ups and downs — particularly downs — see https://luysii.wordpress.com/2011/03/25/evil-scientists-create-virus-causing-chronic-fatigue-syndrome-in-lab/

At worst many people with these symptoms are written off as crazy; at best, depressed  and given antidepressants.  The fact that many of those given antidepressants feel better is far from conclusive, since most patients with chronic illnesses are somewhat depressed.

Even if we didn’t have a treatment, just having a test which separated sufferers from normal people would at least be of some psychological help, by telling them that they weren’t nuts.

Two recent papers may actually have the answer. Although neither paper dealt with chronic fatigue syndrome directly, and I can find no studies in the literature linking what I’m about to describe to CFS they at least imply that there could be a diagnostic test for CFS, and a possible treatment as well.

Because I expect that many people with minimal biological background will be reading this, I’ll start by describing the basic biology of cellular senescence and death

Background:  Most cells in our bodies are destined to die long before we do. Neurons are the longest lasting (essentially as long as we do).  The lining of the intestines is renewed weekly.  No circulating blood cell lasts more than half a year.

Cells die in a variety of ways.  Some are killed (by infections, heat, toxins).  This is called necrosis. Others voluntarily commit suicide (this is called apoptosis).   Sometimes a cell under stress undergoes cellular senescence, a state in which it doesn’t die, but doesn’t reproduce either.  Such cells have a variety of biochemical characteristics — they are resistant to apoptosis, they express molecules which prevent them from proliferating and most importantly, they secrete proinflammatory molecules (this is called the Senescence Associated Secretory Phenotype — SASP).

At first the very existence of the senescent state was questioned, but exist it does.  What is it good for?  Theories abound, one being that mutation is one cause of stress, and stopping mutated cells from proliferating prevents cancer. However, senescent cells are found during fetal life; and they are almost certainly important in wound healing.  They are known to accumulate the older you get and some think they cause aging.

Many stresses induce cellular senescence.  The one of interest to us is chemotherapy for cancer, something obviously good as a cancer cell turned senescent has stopped proliferating.   If you know anyone who has undergone chemotherapy, you know that fatigue is almost invariable.

One biochemical characteristic of the senescent cell is increased levels of a protein called p16^INK4a, which helps stop cellular proliferation.  While p16^INK4a can easily be measured in tissue biopsies, tissue biopsies are inherently not easy. Fortunately it can also be measured in circulating blood cells.

The following study — Cancer Discov. vol. 7 pp. 165 – 176 ’17 looked at 89 women with breast cancer undergoing chemotherapy. They correlated the amount of fatigue experienced with the levels of p16^INK4a in a type of circulating white blood cell (T lymphocyte).  There was a 44% incidence of fatigue in the highest quartile of  p16^INK4a levels, vs. a 5% incidence of fatigue in the lowest. The cited paper didn’t mention CFS nor did the highly technical but excellent review on which much of the above is based [ Cell vol. 169 pp. 1000 -1011 ’17 ]

But it is definitely time to measure p16^INK4a levels in patients with chronic fatigue and compare them to people without it.  This may be the definitive diagnostic test, if people with CFS show higher levels of p16^INK4a.

If this turns out to be the case, then there is a logical therapy for chronic fatigue syndrome.  As mentioned above, senescent cells are resistant to apoptosis (voluntary suicide).  What stops these cells from suicide? Naturally occurring cellular suicide inhibitors (with names like BCL2, BCL-XL, BCL-W) do so .  Drugs called sensolytics already exist to target the inhibitors causing senescent cells to commit suicide.

So if excessive senescent cells are the cause of CFS, then killing them should make things better. Sensolytics do exist but there are problems; one couldn’t be used because of side effects.  Others do exist (one such is Venetoclax) and have been approved by the FDA for leukemia — but it isn’t as potent .

So there is a potentially both a diagnostic test and a treatment for CFS.

The initial experiment should be fairly easy for research to do — just corral some CSF patients and controls and run a test for p16^INK4a levels in their blood cells. Also easy on the patients as only a blood draw is involved.

This, in itself, would be great, but there is far more to think about.

If CFS patients have too many senescent cells, getting rid of them — although (hopefully) symptomatically beneficial — will not get rid of what caused the senescent cells to accumulate in the first place. In addition, getting rid of all of them at once would probably cause huge problems causing something similar to the tumor lysis syndrome – https://en.wikipedia.org/wiki/Tumor_lysis_syndrome.

But these are problems CFS patients and

How to (possibly) diagnose and treat chronic fatigue syndrome (myalgic encephalomyelitis)

As a neurologist I saw a lot of people who were chronically tired and fatigued, because neurologists deal with muscle weakness and diseases like myasthenia gravis which are associated with fatigue.  Once I ruled out neuromuscular disease as a cause, I had nothing to offer then (nor did medicine).  Some were undoubtedly neurotic, but there was little question in my mind that some of them had something wrong that medicine just hadn’t figured out.  Not it hasn’t been trying.

Infections of almost any sort are associated with fatigue, probably because components of the inflammatory response cause it.  Anyone who’s gone through mononucleosis knows this.    The long search for an infectious cause of chronic fatigue syndrome (CFS) has had its ups and downs — particularly downs — see https://luysii.wordpress.com/2011/03/25/evil-scientists-create-virus-causing-chronic-fatigue-syndrome-in-lab/

At worst many people with these symptoms are written off as crazy; at best, depressed  and given antidepressants.  The fact that many of those given antidepressants feel better is far from conclusive, since most patients with chronic illnesses are somewhat depressed.

Even if we didn’t have a treatment, just having a test which separated sufferers from normal people would at least be of some psychological help, by telling them that they weren’t nuts.

Two recent papers may actually have the answer. Although neither paper dealt with chronic fatigue syndrome directly, and I can find no studies in the literature linking what I’m about to describe to CFS they at least imply that there could be a diagnostic test for CFS, and a possible treatment as well.

Because I expect that many people with minimal biological background will be reading this, I’ll start by describing the basic biology of cellular senescence and death

Background:  Most cells in our bodies are destined to die long before we do. Neurons are the longest lasting (essentially as long as we do).  The lining of the intestines is renewed weekly.  No circulating blood cell lasts more than half a year.

Cells die in a variety of ways.  Some are killed (by infections, heat, toxins).  This is called necrosis. Others voluntarily commit suicide (this is called apoptosis).   Sometimes a cell under stress undergoes cellular senescence, a state in which it doesn’t die, but doesn’t reproduce either.  Such cells have a variety of biochemical characteristics — they are resistant to apoptosis, they express molecules which prevent them from proliferating and most importantly, they secrete proinflammatory molecules (this is called the Senescence Associated Secretory Phenotype — SASP).

At first the very existence of the senescent state was questioned, but exist it does.  What is it good for?  Theories abound, one being that mutation is one cause of stress, and stopping mutated cells from proliferating prevents cancer. However, senescent cells are found during fetal life; and they are almost certainly important in wound healing.  They are known to accumulate the older you get and some think they cause aging.

Many stresses induce cellular senescence.  The one of interest to us is chemotherapy for cancer, something obviously good as a cancer cell turned senescent has stopped proliferating.   If you know anyone who has undergone chemotherapy, you know that fatigue is almost invariable.

One biochemical characteristic of the senescent cell is increased levels of a protein called p16^INK4a, which helps stop cellular proliferation.  While p16^INK4a can easily be measured in tissue biopsies, tissue biopsies are inherently not easy. Fortunately it can also be measured in circulating blood cells.

The following study — Cancer Discov. vol. 7 pp. 165 – 176 ’17 looked at 89 women with breast cancer undergoing chemotherapy. They correlated the amount of fatigue experienced with the levels of p16^INK4a in a type of circulating white blood cell (T lymphocyte).  There was a 44% incidence of fatigue in the highest quartile of  p16^INK4a levels, vs. a 5% incidence of fatigue in the lowest. The cited paper didn’t mention CFS nor did the highly technical but excellent review on which much of the above is based [ Cell vol. 169 pp. 1000 -1011 ’17 ]

But it is definitely time to measure p16^INK4a levels in patients with chronic fatigue and compare them to people without it.  This may be the definitive diagnostic test, if people with CFS show higher levels of p16^INK4a.

If this turns out to be the case, then there is a logical therapy for chronic fatigue syndrome.  As mentioned above, senescent cells are resistant to apoptosis (voluntary suicide).  What stops these cells from suicide? Naturally occurring cellular suicide inhibitors (with names like BCL2, BCL-XL, BCL-W) do so .  Drugs called sensolytics already exist to target the inhibitors causing senescent cells to commit suicide.

So if excessive senescent cells are the cause of CFS, then killing them should make things better. Sensolytics do exist but there are problems; one couldn’t be used because of side effects.  Others do exist (one such is Venetoclax) and have been approved by the FDA for leukemia — but it isn’t as potent .

So there is a potentially both a diagnostic test and a treatment for CFS.

The initial experiment should be fairly easy for research to do — just corral some CSF patients and controls and run a test for p16^INK4a levels in their blood cells. Also easy on the patients as only a blood draw is involved.

This, in itself, would be great, but there is far more to think about. 

If CFS patients have too many senescent cells, getting rid of them — although (hopefully) symptomatically beneficial — will not get rid of what caused the senescent cells to accumulate in the first place. In addition, getting rid of all of them at once would probably cause huge problems causing something similar to the tumor lysis syndrome – https://en.wikipedia.org/wiki/Tumor_lysis_syndrome.

But these are problems CFS patients and their physicians would love to have.

What is docosahexenoic acid and why should you care?

Why should drug chemists care about docosahexenoic acid — it’s a fairly trivial organic structure as these things go – a 22 carbon straight chain carboxylic acid with 6 double bonds — https://en.wikipedia.org/wiki/Docosahexaenoic_acid. However the structure is decidedly non-random (see later)

Docosahexenoic acid turns out to be crucial for the function of the blood brain barrier (BBB), something that makes it very difficult to get drugs into the brain. Years of work have shown that the only drugs able to get through the BBB are small lipid soluble molecules of mass under 400 kiloDaltons with fewer than 9 hydrogen bonds. Certainly not a large group of drugs. The more we know about the BBB, the more likely we’ll be able to figure out something to circumvent it.

The BBB was known to exist more than 100 years ago. Ehrlich found that dyes injected into the circulation were rapidly taken up by all organs except the brain. His student E. Goldmann found that dye injected into the CSF stained the brain but not other organs.

The barrier has at least two components — (1) a very tight seal between the cells lining brain blood vessels (e.g. the endothelium) — see the end of the post — (2)very low transfer across the endothelial cell from the vessel lumen. The latter is called transcytosis and involves formation of small vesicles at the lumenal surface of the endothelium, migration across the endothelial cell with release of vesicle content on the other side.

In general there are two mechanisms of transcytosis — clathrin coated pits, and caveolae. Brain endothelium shows very low rates of transcytosis. There aren’t any coated pits (no explanation I can find) and the rate of caveolar transcytosis is very low.

Dococsahexaenoic acid is the reason for the low rate of caveolar transcytosis. Here is why.

[ Nature vol. 509 pp. 432 – 433, 503 – 506, 507 – 511 ’14 Neuron vol. 82 pp. 728 – 730 ’14 ] An orphan transporter, MFSD2a (Major Facilitator Superfamily Domain containing 2a) is selectively expressed in the BBB endothelium. It is REQUIRED for formation and maintenance of BBB integrity. Animals lacking MFSD2a show uninhibited bulk transcytosis across the endothelium. The animals show no obvious defects in the junctions between the endothelial cells. Pericytes (cells in the brain layer after the endothelium) are important in keeping the levels of MFSD2a at normal levels as animals lacking them show the same defects in the BBB as those lacking MFSD2a. Even though knockouts don’t have much of a BBB, they have normal patterning of vascular networks.

MFSD2a is the major transporter of docohexaenoic acid (DHA), an omega3 fatty acid (more later). DHA isn’t made in the brain and must be transported into it. Knockouts have reduced levels of DHA in the brain accompanied by neuronal loss in the hippocampus and cerebellum and microcephaly. Human cases due to mutation are now known (11/15). Transport of DHA and fatty acids into the brain across the BBB occurs only in the form of esters with lysophosphatidylcholines (LPCs) but not as free fatty acids in a sodium dependent manner. The phospho-zwitterionic headgroup of of LPC is essential for transport. MFSD2a ‘prefers’ long chain fatty acids (oleic, palmitic), failing to transport fatty acids with chain lengths under 14.

So MFSD2a inhibits transcytosis at the same time it promotes fatty acid transport into the brain. Major Facilitator Superfamily (MFS) proteins use the electrochemical potential of the cell to transport substrates. The best known MFSs are the glucose transporters (GLUT1 – 4).

So the blood brain barrier is due in part to the lipid transport activity of MFSD2a which gives BBB endothelium a different lipid composition (with lots of docosahexenoic acid) ) than others, inhibiting caveolar transport. Increased DHA levels are associated with membrane cholesterol depletion, as well as displacement of caveolin1 (the major protein involved in this form of transcytosis) from caveolae.

It is likely that MFSD2A acts as a lipid flippase, transporting phospholipids, including DHA containing species from the outer to the inner plasma membrane leaflet (where caveolin1 binds).

What is so hot about docosahexenoic acid — 22 carbons all in a row, a carboxyl group and 6 double bonds. We’re not talking fused ring systems, alkaloids, bizarre functional groups etc. etc.

Half the answer is that the double bonds are NOT randomly arranged. The 6 occur all in a row (but with methylene groups between them). This tells the chemist that they are not conjugated, hence the chain is probably not straight. Think how unlikely the arrangement is considering the way 6 double bonds and 9 methylenes COULD be arranged in a chain (2^15). Answer 5 ways depending on where the arrangement starts relative to the end of the chain.

The other half is that all the double bonds are cis, making it very unlikely that the 21 carbon chain can straighten out and cross the membrane. Lots of DHA means a very disordered membrane, which may be impossible to caveolin1 (and clathrin) to bind to.

So even though it’s years and years since I left organic chemistry, it permits the enjoying of the biochemical esthetics of the blood brain barrier.

The tight junctions between endothelial cells are primarily responsible for barrier function. These tight junctions are found only in the capillaries and postcapillary venules of the brain. Endothelial cells of the brain have few pinocytotic vesicles and fenestriae. [ Neuron vol. 71 p. 408 ’11 ] The brain vasculature has the thinnest endothelial cells, with the tightest junction and a higher degree of pericyte coverage coverage (‘up to’ 30%). [ Neuron vol. 78 pp. 214 – 232 ’13 ] The tight junctions are made from occludin, claudins and junctional adhesion molecules, and are closer to the lumen than the adherens junctions (which also link endothelial cells to each other) made by the cadherins (E, P and N). (ibid p. 219) TLR2/6 specific stimuli.

Progress has been slow but not for want of trying

Progress in the sense of therapy for Alzheimer’s disease and Glioblastoma multiforme is essentially nonexistent, and we could use better therapy for Parkinsonism. This doesn’t mean that researchers have given up. Far from it. Three papers all in last week’s issue of PNAS came up with new understanding and possibly new therapeutic approaches for all three.

You’ll need some serious molecular biological and cell physiological chops to get through the following.

l. Glioblastoma multiforme — they aren’t living much longer than they were when I started pracice 45 years ago (about 2 years — although of course there are exceptions).

The human ZBTB family of genes consists of 49 members coding for transcription factors. BCL6 is also known as ZBTB27 and is a master regulator of lymph node germinal responses. To execute its transcriptional activity, BCL6 requires homodimerization and formation of a complex with a variety of cofactors including BCL6 corerpressor (BCoR), nuclear receptor corepressor 1 (NCoR) and Silencing Mediator of Retinoic acid and Thyroid hormone receptor (SMRT). BCL6 inhibitors block the interaction between BCL6 and its friends, selectively killing BCL6 addicted cancer cells.

The present paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 3981 – 3986 ’17 ] shows that BCL6 is required for glioblastoma cell viability. One transcriptional target of BCL6 is AXL, a tyrosine kinase. Depletion of AXL also decreases proliferation of glioblastoma cells in vitro and in vivo (in a mouse model of course).

So here are two new lines of attack on a very bad disease.

2. Alzheimer’s disease — the best we can do is slow it down, certainly not improve mental function and not keep mental function from getting worse. ErbB2 is a member of the Epidermal Growth Factor Receptor (EGFR) family. It is tightly associated with neuritic plaques in Alzheimer’s. Ras GTPase activation mediates EGF induced stimulation of gamma secretase to increase the nuclear function of the amyloid precursor protein (APP) intracellular domain (AICD). ErbB2 suppresses the autophagic destruction of AICD, physically dissociating Beclin1 vrom the VPS34/VPS15 complex independently of its kinase activity.

So the following paper [ Proc. Natl. Acad. Sci. vol. 114 pp. E3129 – E3138 ’17 ] Used a compound downregulating ErbB2 function (CL-387,785) in mouse models of Alzheimer’s (which have notoriously NOT led to useful therapy). Levels of AICD declined along with beta amyloid, and the animals appeared smarter (but how smart can a mouse be?).

3.Parkinson’s disease — here we really thought we had a cure back in 1972 when L-DOPA was first released for use in the USA. Some patients looked so good that it was impossible to tell if they had the disease. Unfortunately, the basic problem (death of dopaminergic neurons) continued despite L-DOPA pills supplying what they no longer could.

Nurr1 is a protein which causes the development of dopamine neurons in the embryo. Expression of Nurr1 continues throughout life. Nurr1 appears to be a constitutively active nuclear hormone receptor. Why? Because the place where ligands (such as thyroid hormone, steroid hormones) bind to the protein is closed. A few mutations in the Nurr1 gene have been associated with familial parkinsonism.

Nurr1 functions by forming a heterodimer with the Retinoid X Receptor alpha (RXRalpha), another nuclear hormone receptor, but one which does have an open binding pocket. A compound called BRF110 was shown by the following paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 3795 – 3797, 3999 – 4004 ’17 ] to bind to the ligand pocked of RXRalpha increasing its activity. The net effect is to enhance expression of dopamine neuron specific genes.

More to the point MPP+ is a toxin pretty selective for dopamine neurons (it kills them). BRF110 helps survival against MPP+ (but only if given before toxin administration). This wouldn’t be so bad because something is causing dopamine neurons to die (perhaps its a toxin), so BRF110 may fight the decline in dopamine neuron numbers, rather than treating the symptoms of dopamine deficiency.

So there you have it 3 possible new approaches to therapy for 3 bad disease all in one weeks issue of PNAS. Not easy reading, perhaps, but this is where therapy is going to come from (hopefully soon).

An obvious idea we’ve all missed

In 3+ decades as a clinical neurologist I saw several hundred unfortunate people with primary brain tumors. Not one of them was made of proliferating neurons. Not a single one. Most were tumors derived from glial cells (gliomas, glioblastomas, astrocytomas, oligodendrogliomas) which make up half the cells in the brain. Some came from the coverings of the brain (meningiomas), or the ventricular lining (ependymomas).

A recent paper in Nature (vol. 543 pp.681 – 686 ’17) decided that it might be worthwhile to figure out why some organs rarely if ever develop cancer (brain, heart, skeletal muscle). Obvious isn’t it? But no one did it until now.

Most of these tissues are terminally differentiated (unlike, skin, lung, breast and gut) and don’t undergo cellular division. This means that they don’t have to copy their DNA over and over to replenish old and dying cells, and so they are much less likely to develop mutation.

They also use oxidative phosphorylation (a mitochondrial function) rather than glycolysis to generate energy. So they looked for genes that were upregulated in terminally differentiated muscle (not brain) cells relative to proliferating muscle cell precursors. Not a complicated idea to test once you think of it (but you and I didn’t). They found 5 such, and tested them for their ability to suppress tumor growth. One such (LACTB) decreased the growth rate of a variety of tumor cells in vitro and in vivo (e.g.– when transplanted into immunodeficient animals). Amazingly it seems to have no effect on normal cells.

Showing how little we understand the goings on inside our cells, why don’t you try to guess what LACTB given your (and our) knowledge of cellular biochemistry and physiology.

LACTB changes mitochondrial lipid metabolism, by reducing the rate of decarboxylation of mitochondrial phosphatidyl serine — say what?

Even when you know what LACTB is doing you’d be hard pressed to figure out how this effect slows cancer cell growth (and possibly prevents it from occuring at all).

So given our knowledge we’d have never found LACTB and having found it we still don’t know how it works.

Why antioxidants may be bad for you

Antioxidants (vitamin E, beta carotene, vitamin C etc. etc. ) were very big a while ago. They were held to prevent all sorts of bad things (heart attack, stroke). However one pretty good study done years and years ago (see the bottom) showed that they increased the risk of lung cancer in 29,000 Finnish male smokers by 18%. People still take them however.

Now we are beginning to find out the good things that oxidation does for you. One oxidation product is 8-oxo-guanine–https://en.wikipedia.org/wiki/8-Oxoguanine — and it is estimated that it occurs 100,000 time a day in every cell in our body. This isn’t very often as we have .24 x 3,200,000 = 768,000,000 guanines in our genome.

One good thing 8-oxo-guanine may do for you is turn on gene transcription [ Proc. Natl. Acad. Sci. vol. 114 pp. 2788 – 2790, 2604 – 2609 ’17 ].This occurs when the guanine occurs in an elegant DNA structure called a G-quartet (G quadruplex) — https://en.wikipedia.org/wiki/G-quadruplex. Oxidation recruits an enzyme to remove it (8-oxo-guanine glycosylase — aka OGG1 ) generating a DNA lesion — a sugar in the backbone without a nucleotide attach. This causes the binding of Apurinic/Apyrmidic Endonuclease 1 (APE1) which recruits other things to repair the DNA.

As you know DNA in our cells is compacted 100,000 fold to fit its 1 meter length into a nucleus .00001 meters in size. Compaction involves wrapping the helix around all nucleosomes and then binding the nucleosomes together.

It’s pretty hard for RNA polymerase to even get to a gene to transcribe it into mRNA, and DNA lesions cause opening up of this compaction so repair enzymes can actually get to the double helix.

One such gene is Vascular Endothelial Growth Factor (VEGF), a gene induced by low oxygen (hypoxia). The promoter of VEGF has a potential G quadruplex sequence. If the authors put 8-oxo-guanine at 5 different positions in the G quartet, transcription of the VEGF gene was increased 2 – 3 times over the next few days. Showing the importance of the DNA lesion, if OGG1 levels were decreased this didn’t happen — showing that guanine oxidation and with the subsequent formation of a DNA lesion is required for increased transcription of VEGF.

Aside from being another mechanism for gene activation under oxidative stress, 8-oxo-guanine may actually be another epigenetic DNA modification, like 5 methyl cytosine.

So this may explain the result immediately below.

[ New England J. Med. vol. 330 pp. 1029 – 1035 ’94 ] The Alpha-Tocopherol, Beta-Carotene Trial (ATBC trial) randomized double blind placebo controlled of daily supplementation with alpha-tocopherol (a form of vitamin E), beta carotene or both to see if it reduced the incidence of lung cancer was done in 29,000 Finnish male smokers ages 50 – 69 (when most of the damage had been done). They received either alpha tocopherol 50 mg/day, beta carotene 20 mg/day or both. There was a high incidence of lung cancer (876/29000) during the 5 – 8 year period of followup. Alpha tocopherol didn’t decrease the incidence of lung cancer, and there was a higher incidence among the men receiving beta carotene (by 18%). Alpha tocopherol had no benefit on mortality (although there were more deaths from hemorrhagic stroke among the men receiving the supplement). Total mortality was 8% higher among the participants on beta carotene (more deaths from lung cancer and ischemic heart disease). It is unlikely that the dose was too low, since it was much higher than the estimated intake thought to be protective in the uncontrolled dietaryt studies. The trial organizers were so baffled by the results that they even wondered whether the beta-carotene pills used in the study had become contaminated with some known carcinogen during the manufacturing process. However, tests have ruled out that possibility.

Needless to say investigators in other beta carotene clinical trials (the Women’s Health Study, the Carotene and Retinoid Efficacy Trial) are upset. [ Science vol. 264 pp. 501 – 502 ’94 ] “In our heart of hearts, we don’t believe [ beta carotene is ] toxic” says one researcher. Touching isn’t it. Such faith in a secular age, particularly where other people’s lives are at stake. I love it when ecology, natural vitamins and pseudoscience take it in the ear.

Will flickering light treat Alzheimer’s disease ?

Big pharma has spent zillions trying to rid the brain of senile plaques, to no avail. A recent paper shows that light flickering at 40 cycles/second (40 Hertz) can do it — this is not a misprint [ Nature vol. 540 pp. 207 – 208, 230 – 235 ’16 ]. As most know the main component of the senile plaque of Alzheimer’s disease is a fragment (called the aBeta peptide) of the amyloid precursor protein (APP).

The most interesting part of the paper showed that just an hour or so of light flickering at 40 Hertz temporarily reduced the amount of Abeta peptide in visual cortex of aged mice. Nothing invasive about that.

Should we try this in people? How harmful could it be? Unfortunately the visual cortex is relatively unaffected in Alzheimer’s disease — the disease starts deep inside the head in the medial temporal lobe, particularly the hippocampus — the link shows just how deep it is -https://en.wikipedia.org/wiki/Hippocampus#/media/File:MRI_Location_Hippocampus_up..png

You might be able to do this through the squamous portion of the temporal bone which is just in front of and above the ear. It’s very thin, and ultrasound probes placed here can ‘see’ blood flowing in arteries in this region. Another way to do it might be a light source placed in the mouth.

The technical aspects of the paper are fascinating and will be described later.

First, what could go wrong?

The work shows that the flickering light activates the scavenger cells of the brain (microglia) and then eat the extracellular plaques. However that may not be a good thing as microglia could attack normal cells. In particular they are important in the remodeling of the dendritic tree (notably dendritic spines) that occurs during experience and learning.

Second, why wouldn’t it work? So much has been spent on trying to remove abeta, that serious doubt exists as to whether excessive extracellular Abeta causes Alzheimer’s and even if it does, would removing it be helpful.

Now for some fascinating detail on the paper (for the cognoscenti)

They used a mouse model of Alzheimer’s disease (the 5XFAD mouse). This poor creature has 3 different mutations associated with Alzheimer’s disease in the amyloid precursor protein (APP) — these are the Swedish (K670B), Florida (I716V) and London (V717I). If that wasn’t enough there are two Alzheimer associated mutations in one of the enzymes that processes the APP into Abeta (M146L, L286V) — using the single letter amino acid code –http://www.biochem.ucl.ac.uk/bsm/dbbrowser/c32/aacode.html.hy1. Then the whole mess is put under control of a promoter particularly active in mice (the Thy1 promoter). This results in high expression of the two mutant proteins.

So the poor mice get lots of senile plaques (particularly in the hippocampus) at an early age.

The first experiment was even more complicated, as a way was found to put channelrhodopsin into a set of hippocampal interneurons (this is optogenetics and hardly simple). Exposing the channel to light causes it to open the membrane to depolarize and the neuron to fire. Then fiberoptics were used to stimulate these neurons at 40 Hertz and the effects on the plaques were noted. Clearly a lot of work and the authors (and grad students) deserve our thanks.

Light at 8 Hertz did nothing to the plaques. I couldn’t find what other stimulation frequencies were used (assuming they were tried).

It would be wonderful if something so simple could help these people.

For other ideas about Alzheimer’s using physics rather than chemistry please see — https://luysii.wordpress.com/2014/11/30/could-alzheimers-disease-be-a-problem-in-physics-rather-than-chemistry/

In a gambling mood? Take II

I increased my holdings of ONTX (Onconova) yesterday on the basis of a trial of their drug Rigosertib jsut reported. Here’s the link — https://finance.yahoo.com/news/onconova-presents-phase-2-data-120100889.html. Basically rigosertib improved survival with no increased toxicity when added to standard therapy for myelodysplastic syndrome.

Big deal you say, that’s a relatively uncommon type of cancer. True but Rigosertib attacks the great white whale of oncology – the ras oncogene. If it works here, it may work in the forms of cancer where ras is mutated (conservatively 20 – 40% of all cancer) This is why buying ONTX is a gamble — you are balancing a 90% – 99% chance that it won’t work, with a 10 – 100 fold payoff. Here’s the old post of last May

Has the great white whale of oncology finally been harpooned?

The ras oncogene is the great white whale of oncology. Mutations in 20 – 40% of cancer turn its activity on so that nothing can turn it off, resulting in cellular proliferation. People have been trying to turn mutated ras off for years with no success.

A current paper [ Cell vol. 165 pp. 643 – 655 ’16 ] describes a new and different way to attack it. Once ras is turned on (either naturally or by mutation) many other proteins must bind to it, to produce their effects — they are called RAS effectors, among which are the uneuphoniously named RAF, RalGDS and PI3K. They bind to activated ras by the cleverly named Ras Binding Domain (RBD) which has 78 amino acids.

The paper describes rigosertib, a not that complicated molecule to the chemist, which inhibits the binding (by resembling the site on ras that the RBD binds to). It is a styryl benzyl sulfone and you can see the structure here — https://en.wikipedia.org/wiki/Rigosertib.

What’s good about it? Well it is in phase III trials for a fairly uncommon form of cancer (myelodysplastic syndrome). That means it isn’t horribly toxic or it wouldn’t have made it out of phase I.

Given the mechanism described, it is possible that Rigosertib will be useful in 20 – 40% of all cancer. Can you say blockbuster drug?

Do you have a speculative bent? Buy the company testing the drug and owning the patent — Oncova Therapeutics. It’s quite cheap — trading at $.40 (yes 40 cents !). It once traded as high as $30.00 — symbol ONTX. I don’t own any (yet), but for the price of a movie with a beer and some wings afterwards you could be the proud owner of 100 shares. If Rigosertib works, the stock will certainly increase more than a hundredfold.

Enough kidding around. This is serious business. In what follows you will find some hardcore molecular biology and cellular physiology showing just what we’re up against. Some of the following is quite old, and probably out of date (like yours truly), but it does give you the broad outlines of what is involved.

The pathway from Ras to the nucleus

The components of the pathway had been found in isolation (primarily because mutations in them were associated with malignancy). Ras was discovered as an oncogene in various sarcoma viruses. Mutations in ras found in tumors left it in a ‘turned on’ state, but just how ras (and everything else) fit into the chain of binding of a growth factor (such as platelet derived growth factor, epidermal growth factor, insulin, etc. etc.) to its receptor on the cell surface to alterations in gene expression wasn’t clear. It is certain to become more complicated, because anything as important as cellular proliferation is very likely to have a wide variety of control mechanisms superimposed on it. Although all sorts of protein kinases are involved in the pathway it is important to remember that ras is NOT a protein kinase.

l. The first step is binding of a growth factor to its receptor on the cell surface. The receptor is usually a tyrosine kinase. Binding of the factor to the receptor causes ‘activation’ of the receptor. Activation usually means increasing the enzymatic activity of the receptor in the tyrosine kinase reaction (most growth factor receptors are tyrosine kinases). The increase in activity is usually brought about by dimerization of the receptor (so it phosphorylates itself on tyrosine).

2. Most activated growth factor receptors phosphorylate themselves (as well as other proteins) on tyrosine. A variety of other proteins have domains known as SH2 (for src homology 2) which bind to phosphorylated tyrosine.

3. A protein called grb2 binds via its SH2 domain to a phosphorylated tyrosine on the receptor. Grb2 binds to the polyproline domain of another protein called sos1 via its SH3 domain. At this point, the unintiated must find the proceedings pretty hokey, but the pathway is so general (and fundamental) that proteins from yeast may be substituted into the human pathway and still have it work.

4. At last we get to ras. This protein is ‘active’ when it binds GTP, and inactive when it binds GDP. Ras is a GTPase (it can hydrolyze GTP to GDP). Most mutations which make ras an oncogene decrease the GTPase activity of RAS leaving it in a permanently ‘turned on’ state. It is important for the neurologist to know that the defective gene in type I neurofibromatosis activates the GTPase activity of ras, turning ras off. Deficiencies (in ras inactivation) lead to a variety of unusual tumors familiar to neurologists.

Once RAS has hydrolyzed GTP to GDP, the GDP remains bound to RAS inactivating it. This is the function of sos1. It catalyzes the exchange of GDP for GTP on ras, thus activating ras.

5. What does activated ras do? It activates Raf-1 silly. Raf-1 is another oncogene. How does activated ras activate Raf-1 ? Ras appears to activate raf by causing raf to bind to the cell membrane (this doesn’t happen in vitro as there is no membrane). Once ras has done its job of localizing raf to the plasma membrane, it is no longer required. How membrane localization activates raf is less than crystal clear. [ Proc. Natl. Acad. Sci. vol. 93 pp. 6924 – 6928 ’96 ] There is increasing evidence that Ras may mediate its actions by stimulating multiple downstream targets of which Raf-1 is only one.

6. Raf-1 is a protein kinase. Protein kinases work by adding phosphate groups to serine, threonine or tyrosine. In general protein kinases fall into two classes those phosphorylating on serine or threonine and those phosphorylating on tyrosine. Biochemistry has a well documented series of examples of enzymes being activated (or inhibited) by phosphorylation. The best worked out is the pathway from the binding of epinephrine to its cell surface receptor to glycogen breakdown. There is a whole sequence of one enzyme phosphorylating another which then phosphorylates a third. Something similar goes on between Raf-1 and a collection of protein kinases called MAPKs (mitogen activated protein kinases). These were discovered as kinases activated when mitogens bound to their extracellular receptors.There may be a kinase lurking about which activates Raf (it isn’t Ras which has no kinase activity). Removal of phosphate from Raf (by phosphatases) inactivates it.

7. Raf-1 activates members of the MAPK family by phosphorylating them. There may be several kinases in a row phosphorylating each other. [ Science vol. 262 pp. 1065 – 1067 ’93 ] There are at least three kinase reactions at present at this point. It isn’t known if some can be sidestepped. Raf-1 activates mitogen activated protein kinase kinase (MAPK-K) by phosphorylation (it is called MEK in the ras pathway). MAPK-K activates mitogen activation protein kinase (MAPK) by phosphorylation. Thus Raf-1 is actually mitogen activated protein kinase kinase kinase (sort of like the character in Catch-22 named Junior Junior Junior). (1/06 — I think that Raf-1 is now called BRAF)

8. The final step in the pathway is activation of transcription factors (which turn genes off or on) by MAP kinases by (what else) phosphorylation. Thus the pathway from cell surface is complete.