Category Archives: Medicine in general

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– — 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) — 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 -

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 – 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 —

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 — 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 —

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.

Play the (genetic) hand you’ve been dealt but don’t spindle, fold or mutilate your cards

Back in the day, computers were programmed by inserting multiple punch cards, each containing a machine instruction. At the bottom of the card it said “do not fold, spindle, or mutilate”. My wife used them back then when she expected to be a widow if and when I got sent to Vietnam.

So it is with you and the genetic hand of coronary artery disease risk you’ve been dealt. [ Cell vol. 167 p. 1431 ’16 ] refers to a recent New England Journal of Medicine article –2016;DOI:

It’s a very good study, with large numbers of participants in three prospective cohorts — 7814 participants in the Atherosclerosis Risk in Communities (ARIC) study, 21,222 in the Women’s Genome Health Study (WGHS), and 22,389 in the Malmö Diet and Cancer Study (MDCS) — plus 4260 participants in the cross-sectional BioImage Study for whom genotype and covariate data were available. Adherence to a healthy lifestyle among the participants was also determined using a scoring system consisting of four factors: no current smoking, no obesity, regular physical activity, and a healthy diet (hardly complicated).

As you probably know, Genome Wide Association Studies have identified over 50 places in our genomes in which slight variations (the technical term is single nucleotide polymorphisms — SNPs ) are associated with increased risk of coronary artery disease. Since vascular disease is a generalized problem, these SNPs also increase the risk of other vascular problems, notably stroke. None of them increases the risk very much, and even together they don’t explain much of the genetic risk of vascular disease (which we know is there). However, they were all determined (at least in the 4260) and a genetic risk score was calculated. So there were people with high, low and medium degrees of risk.

In all risk groups, high, low, whatever, a simple healthy lifestyle (no smoking, not fat, some exercise, healthy diet) decreased the coronary event rate (heart attack, death) by nearly half. So how bad was high risk? Bad indeed, the event rate in the high risk group was nearly twice that of the low risk group.

Even better, healthy lifestyle decreased risk the most just where you’d want it — in the highest risk group. You can reduce your risk of being eaten by a bear by not going to Yellowstone by 99% or more but so what.

This work is to be believed, because the number of events is high enough –1230 coronary events were observed in the ARIC cohort (median follow-up, 18.8 years), 971 coronary events in the WGHS cohort (median follow-up, 20.5 years), and 2902 coronary events in the MDCS cohort (median follow-up, 19.4 years).

So as my late father said (who lived to 100) when asked what his secret was “I chose my parents very carefully”. Well, we can’t do that, but don’t spindle the cards.

Very sad

The failure of Lilly’s antibody against the aBeta protein is very sad on several levels. My year started out going to a memorial service for a college classmate, fellow doc and friend who died of Alzheimer’s disease. He had some 50 papers to his credit mostly involving clinical evaluation of drugs such as captopril. Even so it was an uplifting experience — here’s a link –

There is a large body of theory that says it should have worked. Derek Lowe’s blog “In the Pipeline” has much more — and the 80 or so comments on his post will expose you to many different points of view on Abeta — here’s the link.

It’s time to ‘let 100 flowers bloom’ in Alzheimer’s research — E. g. it’s time to look at some far out possibilities — we know that most will be wrong that they will be crushed, as Mao did to all the flowers. Even so it’s worth doing.

So to buck up your spirits, here’s an old post (not a link) raising the possibility that Alzheimer’s might be a problem in physics rather than chemistry. If that isn’t enough another post follows that one on Lopid (Gemfibrozil).

Could Alzheimer’s disease be a problem in physics rather than chemistry?

Two seemingly unrelated recent papers could turn our attention away from chemistry and toward physics as the basic problem in Alzheimer’s disease. God knows we could use better therapy for Alzheimer’s disease than we have now. Any new way of looking at Alzheimer’s, no matter how bizarre,should be welcome. The approaches via the aBeta peptide, and the enzymes producing it just haven’t worked, and they’ve really been tried — hard.

The first paper [ Proc. Natl. Acad. Sci. vol. 111 pp. 16124 – 16129 ’14 ] made surfaces with arbitrary degrees of roughness, using the microfabrication technology for making computer chips. We’re talking roughness that’s almost smooth — bumps ranging from 320 Angstroms to 800. Surfaces could be made quite regular (as in a diffraction grating) or irregular. Scanning electron microscopic pictures were given of the various degrees of roughness.

Then they plated cultured primitive neuronal cells (PC12 cells) on surfaces of varying degrees of roughness. The optimal roughness for PC12 to act more like neurons was an Rq of 320 Angstroms.. Interestingly, this degree of roughness is identical to that found on healthy astrocytes (assuming that culturing them or getting them out of the brain doesn’t radically change them). Hippocampal neurons in contact with astrocytes of this degree of roughness also began extending neurites. It’s important to note that the roughness was made with something neurons and astrocytes never see — silica colloids of varying sizes and shapes.

Now is when it gets interesting. The plaques of Alzheimer’s disease have surface roughness of around 800 Angstroms. Roughness of the artificial surface of this degree was toxic to hippocampal neurons (lower degrees of roughness were not). Normal brain has a roughness with a median at 340 Angstroms.

So in some way neurons and astrocytes can sense the amount of roughness in surfaces they are in contact with. How do they do this — chemically it comes down to Piezo1 ion channels, a story in themselves [ Science vol. 330 pp. 55 – 60 ’10 ] These are membrane proteins with between 24 and 36 transmembrane segments. Then they form tetramers with a huge molecular mass (1.2 megaDaltons) and 120 or more transmembrane segments. They are huge (2,100 – 4,700 amino acids). They can sense mechanical stress, and are used by endothelial cells to sense how fast blood is flowing (or not flowing) past them. Expression of these genes in mechanically insensitive cells makes them sensitive to mechanical stimuli.

The paper is somewhat ambiguous on whether expressing piezo1 is a function of neuronal health or sickness. The last paragraph appears to have it both ways.

So as we leave paper #1, we note that that neurons can sense the physical characteristics of their environment, even when it’s something as un-natural as a silica colloid. Inhibiting Piezo1 activity by a spider venom toxin (GsMTx4) destroys this ability. The right degree of roughness is healthy for neurons, the wrong degree kills them. Clearly the work should be repeated with other colloids of a different chemical composition.

The next paper [ Science vol. 342 pp. 301, 316 – 317, 373 – 377 ’13 ] Talks about the plumbing system of the brain, which is far more active than I’d ever imaged. The glymphatic system is a network of microscopic fluid filled channels. Cerebrospinal fluid (CSF) bathes the brain. It flows into the substance of the brain (the parenchyma) along arteries, and the fluid between the cellular elements (interstitial fluid) it exchanges with flows out of the brain along the draining veins.

This work was able to measure the amount of flow through the lymphatics by injected tracer into the CSF and/or the brain parenchyma. The important point about this is that during sleep these channels expand by 60%, and beta amyloid is cleared twice as quickly. Arousal of a sleeping mouse decreases the influx of tracer by 95%. So this amazing paper finally comes up with an explanation of why we spend 1/3 of our lives asleep — to flush toxins from the brain.

If you wish to read (a lot) more about this system — see an older post from when this paper first came out —

So what is the implication of these two papers for Alzheimer’s disease?

The surface roughness of the plaques (800 Angstroms roughness) may physically hurt neurons. The plaques are much larger or Alzheimer would never have seen them with the light microscopy at his disposal.

The size of the plaques themselves may gum up the brain’s plumbing system.

The tracer work should certainly be repeated with mouse models of Alzheimer’s, far removed from human pathology though they may be.

I find this extremely appealing because it gives us a new way of thinking about this terrible disorder. In addition it might explain why cognitive decline almost invariably accompanies aging, and why Alzheimer’s disease is a disorder of the elderly.

Next, assume this is true? What would be the therapy? Getting rid of the senile plaques in and of itself might be therapeutic. It is nearly impossible for me to imagine a way that this could be done without harming the surrounding brain.

Before we all get too excited it should be noted that the correlation between senile plaque burden and cognitive function is far from perfect. Some people have a lot of plaque (there are ways to detect them antemortem) and normal cognitive function. The work also leaves out the second pathologic change seen in Alzheimer’s disease, the neurofibrillary tangle which is intracellular, not extracellular. I suppose if it caused the parts of the cell containing them to swell, it too could gum up the plumbing.

As far as I can tell, putting the two papers together conceptually might even be original. Prasad Shastri, the author of the first paper, was very helpful discussing some points about his paper by Email, but had not heard of the second and is looking at it this weekend.

Also a trial of Lopid (Gemfibrozil) as something which might be beneficial is in progress — there is some interesting theory behind this. The trial has about another year to go. Here’s that post and happy hunting

Takes me right back to grad school

How many times in grad school did you or your friends come up with a good idea, only to see it appear in the literature a few months later by someone who’d been working on it for much longer. We’d console ourselves with the knowledge that at least we were thinking well and move on.

Exactly that happened to what I thought was an original idea in my last post — e.g. that Gemfibrozil (Lopid) might slow down (or even treat) Alzheimer’s disease. I considered the post the most significant one I’d ever written, and didn’t post anything else for a week or two, so anyone coming to the blog for any reason would see it first.

A commenter on the first post gave me a name to contact to try out the idea, but I’ve been unable to reach her. Derek Lowe was quite helpful in letting me link to the post, so presently the post has had over 200 hits. Today I wrote an Alzheimer’s researcher at Yale about it. He responded nearly immediately with a link to an ongoing clinical study in progress in Kentucky

On Aug 3, 2015, at 3:04 PM, Christopher van Dyck wrote:

Dear Dr. xxxxx

Thanks for your email. I agree that this is a promising mechanism.
My colleague Greg Jicha at U.Kentucky is already working on this:

Our current efforts at Yale are on other mechanisms:

We can’t all test every mechanism, but hopefully we can collectively test the important ones.

-best regards,
Christopher H. van Dyck, MD
Professor of Psychiatry, Neurology, and Neurobiology
Director, Alzheimers Disease Research Unit

Am I unhappy about losing fame and glory being the first to think of it? Not in the slightest. Alzheimer’s is a terrible disease and it’s great to see the idea being tested.

Even more interestingly, a look at the website for the study shows, that somehow they got to Gemfibrozil by a different mechanism — microRNAs rather than PPARalpha.

I plan to get in touch with Dr. Jicha to see how he found his way to Gemfibrozil. The study is only 1 year in duration, and hopefully is well enough powered to find an effect. These studies are incredibly expensive (and an excellent use of my taxes). I never been involved in anything like this, but data mining existing HMO data simply has to be cheaper. How much cheaper I don’t know.

Here’s the previous post —

Could Gemfibrozil (Lopid) be used to slow down (or even treat) Alzheimer’s disease?

Is a treatment of Alzheimer’s disease at hand with a drug in clinical use for nearly 40 years? A paper in this week’s PNAS implies that it might (vol. 112 pp. 8445 – 8450 ’15 7 July ’15). First a lot more background than I usually provide, because some family members of the afflicted read everything they can get their hands on, and few of them have medical or biochemical training. The cognoscenti can skip past this to the text marked ***

One of the two pathologic hallmarks of Alzheimer’s disease is the senile plaque (the other is the neurofibrillary tangle). The major component of the plaque is a fragment of a protein called APP (Amyloid Precursor Protein). Normally it sits in the cellular membrane of nerve cells (neurons) with part sticking outside the cell and another part sticking inside. The protein as made by the cell contains anywhere from 563 to 770 amino acids linked together in a long chain. The fragment destined to make up the senile plaque (called the Abeta peptide) is much smaller (39 to 42 amino acids) and is found in the parts of APP embedded in the membrane and sticking outside the cell.

No protein lives forever in the cell, and APP is no exception. There are a variety of ways to chop it up, so its amino acids can be used for other things. One such chopper is called ADAM10 (aka Kuzbanian). ADAM10breaks down APP in such a way that Abeta isn’t formed. The paper essentially found that Gemfibrozil (commercial name Lopid) increases the amount of ADAM10 around. If you take a mouse genetically modified so that it will get senile plaques and decrease ADAM10 you get a lot more plaques.

The authors didn’t artificially increase the amount of ADAM10 to see if the animals got fewer plaques (that’s probably their next paper).

So there you have it. Should your loved one get Gemfibrozil? It’s a very long shot and the drug has significant side effects. For just how long a shot and the chain of inferences why this is so look at the text marked @@@@


How does Gemfibrozil increase the amount of ADAM10 around? It binds to a protein called PPARalpha which is a type of nuclear hormone receptor. PPARalpha binds to another protein called RXR, and together they turn on the transcription of a variety of genes, most of which are related to lipid metabolism. One of the genes turned on is ADAM10, which really has never been mentioned in the context of lipid metabolism. In any event Gemfibrozil binds to PPARalpha which binds more effectively to RAR which binds more effectively to the promoter of the ADAM10 gene which makes more ADAM10 which chops of APP in such fashion that Abeta isn’t made.

How in the world the authors got to PPARalpha from ADAM10 is unknown — but I’ve written the following to the lead author just before writing this post.

Dr. Pahan;

Great paper. People have been focused on ADAM10 for years. It isn’t clear to me how you were led to PPARgamma from reading your paper. I’m not sure how many people are still on Gemfibrozil. Probably most of them have some form of vascular disease, which increases the risk of dementia of all sorts (including Alzheimer’s). Nonetheless large HMOs have prescription data which can be mined to see if the incidence of Alzheimer’s is less on Gemfibrozil than those taking other lipid lowering agents, or the population at large. One such example (involving another class of drugs) is JAMA Intern Med. 2015;175(3):401-407, where the prescriptions of 3,434 individuals 65 years or older in Group Health, an integrated health care delivery system in Seattle, Washington. I thought the conclusions were totally unwarranted, but it shows what can be done with data already out there. Did you look at other fibrates (such as Atromid)?

Update: 22 July ’15

I received the following back from the author

Dear Dr.

Wonderful suggestion. However, here, we have focused on the basic science part because the NIH supports basic science discovery. It is very difficult to compete for NIH R01 grants using data mining approach.

It is PPARα, but not PPARγ, that is involved in the regulation of ADAM10. We searched ADAM10 gene promoter and found a site where PPAR can bind. Then using knockout cells and ChIP assay, we confirmed the participation of PPARα, the protein that controls fatty acid metabolism in the liver, suggesting that plaque formation is controlled by a lipid-lowering protein. Therefore, many colleagues are sending kudos for this publication.

Thank you.

Kalipada Pahan, Ph.D.

The Floyd A. Davis, M.D., Endowed Chair of Neurology


Departments of Neurological Sciences, Biochemistry and Pharmacology

So there you have it. An idea worth pursuing according to Dr. Pahan, but one which he can’t (or won’t). So, dear reader, take it upon yourself (if you can) to mine the data on people given Gemfibrozil to see if their risk of Alzheimer’s is lower. I won’t stand in your way or compete with you as I’m a retired clinical neurologist with no academic affiliation. The data is certainly out there, just as it was for the JAMA Intern Med. 2015;175(3):401-407 study. Bon voyage.


There are side effects, one of which is a severe muscle disease, and as a neurologist I saw someone so severely weakened by drugs of this class that they were on a respirator being too weak to breathe (they recovered). The use of Gemfibrozil rests on the assumption that the senile plaque and Abeta peptide are causative of Alzheimer’s. A huge amount of money has been spent and lost on drugs (antibodies mostly) trying to get rid of the plaques. None have helped clinically. It is possible that the plaque is the last gasp of a neuron dying of something else (e.g. a tombstone rather than a smoking gun). It is also possible that the plaque is actually a way the neuron was defending itself against what was trying to kill it (e.g. the plaque as a pile of spent bullets).

A scary paper: Cancer by proxy

Can a good kid growing up in a bad neighborhood turn bad? Most think so. What about a genetically normal cell growing up in a bad neighborhood? Can it turn cancerous if its neighbors have a mutation ? A recent paper [ Nature vol. 539 pp.304 – 308 ’16b] demonstrates how this can happen.

A gene called PTPN11 is mutated in myelomonocytic leukemia (MML)in humans and mice. Expressing the mutant in blood cells causes leukemia in mice (nothing spectacular there).

However, expressing the mutant in marrow supporting cells, not blood cells or blood stem cells for long enough gives MML in mice which can be transplanted into normal mice producing MML there.

Note that the blood stem cells don’t contain the mutant gene. One theory has it that mutant PTPN11 recruits monocytes, which then produce other stuff (CCL3 also known as MIP1alpha and interleukin1Beta), which then turns on blood stem cells to proliferate madly causing leukemia. Giving a CCL3 receptor antagonist reverses the myeloproliferation (but it isn’t clear to me if it reverses the leukemia once established)

As far as we know the cells developing into MML don’t contain mutant PTPN11. So it’s cancer by proxy. Obviously some changes (mutations, epigenetic changes) have have occurred in the leukemic cells, but at this point we don’t know what they are.

What is ICP27 trying to tell us? One of you could get a PhD if you figure it out !

It wouldn’t be the first time a viral protein led us to an important cellular mechanism. Consider what the polio virus taught us about the translation of mRNA into protein. It cleaves two components of eIF-4F (eukaryotic Initiation (of ribosome translation of mRNA into protein) Factor 4F totally shutting down synthesis of mRNAs with a cap on their 5′ end (which is most of them). Poliovirus proteins don’t have these caps so their proteins continue to be made.

Well this brings us to ICP27 (Infected Cell Protein 27) a product of the Herpes Simplex virus. You can read all about it in [ Proc. Natl. Acad. Sci. vol. 113 pp. 12256 – 12261 ’16 ]. ICP27 is essential for herpes virus infection. This work shows that it inhibits intron splicing (but in under 1% of cellular genes) and also promotes the use of alternative 5′ splice sites.

It also induces the expression of pre-mRNAS prematurely cleaved and polyAdenylated from cryptic polyAdenylation signals located in intron 1 or intron 2 of an amazing 1% of all cellular genes. These prematurely cleaved and polyAdenylated mRNA sometimes contain novel open reading frames (ORFs). They are typically intronless (they should be) and under 2 kiloBases long. They are expressed early during viral infection and efficiently exported to cytoplasm. The ICP27 targeted genes are GC rich (as are all Herpes simplex genes), contain cytosine rich sequences near the 5′ splice site.

The paper also showed that optimization of splice site sequences, or mutation of nearby cytosines eliminated ICP27 mediated splicing inhibition. Introduction of cytosine rich sequences to an ICP27 INsensitive splicing reporter conferred susceptibility to ICP27.

How is this going to help you get a PhD? Ask yourself. What are cryptic polyAdenylation signals doing in the first two introns in so many genes? It seems obvious (to me) that as well as the virus the cell is using them for some purpose. It isn’t hard to mutate something to the signal for polyadenylation AAUAAA. Interestingly cleavage doesn’t occur here, but 30 nucleotides or so downstream. The sequence occurs every 4^6 == 4096 nucleotides (if they’re random). I’m not sure what the total length of introns #1 and #2 are of our 20,000 or so protein coding genes, but someone should be able to find out and see if 200 occurrences of this sequence is more than would be expected by chance.

The plot thickens when the paper notes that “Over 200 genes are affected by ICP27. Over 30 (including PML, STING, TRAF6, PPP6C, MAP3K7, FBXw11, IFNAR2, NKFB1, RELA and CREBP are related to the immune pathway). Do you think the cell doesn’t use this pathway as well?

What about the existence of other viral (and cellular) proteins doing the same sort of thing (but on different introns perhaps). What are those novel open reading frames in the alternatively spliced mRNAs doing?

Fascinating stuff. Time to get busy if you’re an enterprising grad student, or young faculty member.

The proteasome branches out

The surface of a protein is not at all like a ball of yarn, even though they are both one long string. This has profound implications for the immune system. Look at any solved protein structure. The backbone bobs and weaves taking water hating (hydrophobic) amino acids into the center of the protein, and putting water loving (hydrophilic) amino acids on the surface. So even though the peptide backbone is continuous, only discontinuous patches of it are displayed on the protein surface.

Which is a big problem for the immune system which wants to recognize the surface of the protein (which is all it first gets to see with an invading bug). Now we know that foreign proteins are ingested by the cell, chopped up by the proteasome, and fragments loaded on to immune molecules (class I Major Histocompatibility Complex antigens) and displayed on the cell surface so the immune system can learn what it looks like and react to it. The peptides aren’t very long — under 11 or so amino acids, but they are continuous.

What if the really distinct part of the protein surface (e.g. the immunogen)  is made of two distinct patches from the backbone? A fascinating paper shows how the immune system might still recognize it. Chop the protein up into fragments by the proteasome, and then have the fragments from adjacent patches put back together. You know that any enzyme can be run in reverse, so if the proteasome can split peptide bonds apart it can also join them together.

This is exactly what was found in a recent paper — Science vol. 354 pp. 354 – 358 ’16. The small peptides (containing at most 11 amino acids) finding their way to the cell surface were analyzed in a technical tour de force. In aggregate they go by the fancy name of immunopeptidome. They found that the proteasome IS actually splicing peptide fragments together. This is called Proteasome Catalyzed Peptide Splicing (PCPS). The present work shows that it accounts for 1/3 of the class I immunopeptidome in terms of diversity and 1/4 in terms of abundance. One-third of self antigens are represented on the cell surface of the immune cell line they studied (GR-LCL the GR-lymphoblastoid cell line) ONLY by spliced peptides. The ordering of the spliced peptide was the same as the parent protein in only half. There was no preference for the length of the protein skipped by the splice.

The work has huge implications for immunology, not least autoimmune disease.

So today I wrote the author the following

Dr. Mishto

Terrific paper ! Do you have any evidence for the spliced peptides being spatially contiguous on the surface of the parent protein. Have you looked?

This makes a lot of sense, because the immune system should ‘want’ to recognize protein conformations as they exist in the living cell, rather than stretches of amino acid sequence in the parent protein. Also, with few exceptions the surface of a given protein in vivo is a collection of discontinuous peptide sequences of the parent protein. I’ve always wondered how the immune system did this, and perhaps your paper explains things.


and got this back almost immediately

Dear Luysii

Interesting idea. We shall have a look for few examples where the crystallography structure or the parental protein is disclosed already.



It doesn’t get any better than this. Tomorrow I will be exactly 78 years and 6 months old. It shows I can still think (on occasion).

Addendum 17 Nov ’16;  It looks as though proteins are fed into the central cavity of the proteasome as a completely denatured single strand.  See figure 5 of PNAS 113 pp 12991 -m12996 ’16.  The channel to get in appears quite narrow.

The butterfly effect in embryology

How the snake lost its legs. No, this isn’t a Just So story a la Rudyard Kipling, but a fascinating paper in Cell (vol. 167 pp. 598 – 600, 633 – 642 ’16 ). All it takes is a 17 nucleotide deletion in ZRS (Zone of polarizing activity Regulatory Sequence), an enhancer of gene expression involved in limb development. The enhancer is at least 1,300 nucleotides long (but I can’t find out just how long ZRS is). The deletion removes a binding site for a transcription factor (ETS) which turns on some limb development genes.

ZRS has long been known to be involved in limb development, and mutations distributed over 700 nucleotides are associated with a variety of human limb malformations. So the authors sequenced the enhancer in a variety of species (including many snakes) and found that only snakes had the deletion.

Then they put the snake ZRS into genetically engineered transgenic mice and found markedly shortened limbs. That was all it took. Reintroducing the missing 17 nucleotides into the transgenics restores normal limb development. Staggering what genetic technology is capable of.

Where does the butterfly effect come in? Because the enhancer is 1,000,000 nucleotides away from some of the genes it controls. If you were studying sequences around the genes it controls, you’d never find the deletion (until you’d run through a large number of grad students). Human biology (with limb malformations) told the authors where to look.

Straightened out 1,000,000 nucleotides is 3,200,000 Angstroms,or 320 microns (32 times the size of the average 10 micron nucleus). Remarkable how it finds its target. You might be interested in a series of posts which try to imagine these goings on at human scale — blowing up the nucleus so it fits in a football stadium with our double stranded DNA blown up to the size of linguini with a total total length of 2840 miles. Start here –

Two disconcerting papers

We all know that mutations cause cancer and that MRI lesions cause disability in multiple sclerosis. We do, don’t we? Maybe we don’t, say two papers out this October.

First: cancer. The number of mutations in stem cells from 3 organs (liver, colon, small intestine) was determined in biopsy samples from 19 people ranging in age 3 to 87 [ Nature vol. 538 pp. 260 – 264 ’16 ].th How did they get stem cells? An in vitro system was sued to expand single stem cells into epithelial organoids, and then the whole genome was sequenced of each. Some 45 organoids were used. Some 79,790 heterozygous clonal mutations were found. They then plotted the number of mutations vs. the age of the patient. When you have a spread in patient ages (which they did) you can calculate a tissue mutation rate for its stem cells. What is remarkable, is that all 3 tissues had the same mutation rate — about 40 mutations per year. Not bad. That’s only 4,000 if you live to 100 in your 3.2 BILLION nucleotide genome.

This is so  remarkable because the incidence of cancer is wildly different in the 3 tissues, so if mutations occurring randomly cause cancer, all 3 tissues should have the same cancer incidence (and there is much less liver cancer than gut cancer).

Of course there’s a hooker. The numbers are quite small, only 9 organoids from liver with a relatively small age range spanning only 25 years. There were more organoids from colon and small and the age ranges was wider but, clearly, the work needs o be replicated with a lot more samples. However, a look at figure one shows that the slope of the plot of mutation number vs. age is quite similar.

Second: Multiple sclerosis. First, some ancient history. I started in neurology before there were CAT scans and MRIs. All we had to evaluate the MS patient was the neurologic exam. So we’d see if new neurologic signs had developed, or the old ones worsened. There were all sorts of clinical staging scores and indices. Not terribly objective, but at least they measured function which is what physician and patient cared about the most.

The MRI revolutionized both diagnosis and our understanding of MS. We quickly found that even when the exam remained constant, that new lesions appeared and disappeared on the MRI totally silent to both patient and physician. I used to say that prior to MRI neurologists managed patients the way a hematologist would manage leukemics without blood counts, by looking at them to see how pale they were.

In general the more lesions that remained fixed, the worse shape the patient was in. So new drugs against MS could easily be evaluated without waiting years for the clinical exam to change, if a given drug just stopped lesions from appearing — stability was assumed to ensue (or at least it was when I retired almost exactly 4 presidential elections ago).

Enter Laquinimod [ Proc. Natl. Acad. Sci. vol. 113 pp. E6145 – E6152 ’16 ] which has a much greater beneficial effect on disability progression (e.g. less) than it does on clinical relapse rate (also less) and lesion appearance rate on MRI (also less). So again there is a dissociation between the MRI findings and the patient’s clinical status. Here are references to relevant papers — which I’ve not read —
Comi G, et al.; ALLEGRO Study Group (2012) Placebo-controlled trial of oral laquini- mod for multiple sclerosis. N Engl J Med 366(11):1000–1009.

Filippi M, et al.; ALLEGRO Study Group (2014) Placebo-controlled trial of oral laqui- nimod in multiple sclerosis: MRI evidence of an effect on brain tissue damage. J Neurol Neurosurg Psychiatry 85(8):851–858.

Vollmer TL, et al.; BRAVO Study Group (2014) A randomized placebo-controlled phase III trial of oral laquinimod for multiple sclerosis. J Neurol 261(4):773–783.

It is well known that there are different kinds of lesions in MS (some destroying axons, others just stripping off their myelin). Since I’ve left the field, I don’t know if MRI can distinguish the two types, and whether this was looked at.

The disconcerting thing about this paper, is that we may have given up on drugs which would  clinically help patients (rather than a biological marker) because they didn’t help the MRI ! ! !