Band Camp for Adults — 2015

This is not a scientific post, although it contains a lot of scientific types. Adult amateur chamber musicians are an interesting lot. The festival just concluded contained two people with books coming out, two organic chemists, two math profs, an english prof, multiple MDs, a retired foreign service officer and those were just the people I played with. Not everyone attending these things is so fancy and one of the best amateur cellists I ever played with was a moving man and probably the best violist ever was a 300 pound jail matron.

I’d not been to this one for 11 years or so, and it was amazing how people remembered the things I’d done back then. One classic neurotic back then was quite worried she was crazy. Her friend said she’d repeat over and over what I told her — I know crazy and you’re not crazy. Unfortunately she died an awful death of metastatic ovarian carcinoma, far too young in her 40s, as did another good friend, an RN.

Someone else brought up what I’d done for her at her first time at the festival. I’d totally forgotten about it. The first time my wife an I went there, I wasn’t assigned a group the first day and we didn’t know a soul. Everyone else appeared to know everyone else and had play dates arranged for the rest of the week. So the evening of the first day my wife and I were moping about in a local bar, when a gregarious participant came up to us, found out what was going on and set up the Dvorak piano quintet for me the next day.

The following year after the general initial assembly was over, I and the gregarious one got up and announced that we wanted 7 first timers for an immediate session — the Schumann piano quintet for me and a Mozart string quartet for her. The process has since been institutionalized.

I even met a reader of the blog, an excellent young violinist and organic chemist who I tried to steer into drug design. Probably not a good idea given the employment upheavals appearing nearly daily in Derek’s blog. One of the things we played was Vaughn Williams 6 studies on English Folk song. They have been scored for piano and violin, piano and viola, piano and clarinet and piano and cello. Few seem to know of them. They are each two piano pages long, extremely interesting musically and just not that hard to sight read.

Another great thing about the site, is that there is a whole piano and percussion building and many rooms have two pianos. This means two pianists can get together and play without squeezing onto the same bench. I strongly recommend trying two transcriptions of Bach concerti in C major and C minor. Both parts are quite interesting and well done musically, and you can switch so you’ll get to play each part. It’s  Peters edition #s 2200a, 2200b (BWV 1061, no BWV # given for 2200b). Start with the slow movements of each, the back and forth of the voices is great. I heard it today scored as a concerto for oboe and violin.

Unfortunately many of the people I played with 11 years ago had passed on, including Edwin Gould a violinist. Organic chemists of a certain age know him has the author of the ‘bible’ of physical organic chemistry back in the 60s.

Just by chance, two of the MDs were at places I’d trained — Colorado General Hospital and Children’s Hospital of Philadelphia, and it was fascinating to hear how they’d changed.

Also just by chance there were two graduates from Brown who would have been near classmates with my son, had he chosen to go there. Fascinating to hear about paths not taken.

One of the math profs has a book coming out and the other explained what a Toric variety is (David Cox, from whom I audited a course, wrote a 600 page book on the subject).

All in all an intellectually and musically stimulating week.

How ‘simple’ can a protein be and still have a significant biological effect

Words only have meaning in the context of the much larger collection of words we call language. So it is with proteins. Their only ‘meaning’ is the biologic effects they produce in the much larger collection of proteins, lipids, sugars, metabolites, cells and tissues of an organism.

So how ‘simple’ can a protein be and still produce a meaningful effect? As Bill Clinton would say, that depends on what you mean by simple. Well one way a protein can be simple is by only having a few amino acids. Met-enkephalin, an endogenous opiate, contains only 5 amino acids. Now many wouldn’t consider met-enkehalin a protein, calling it a polypeptide instead. But the boundary between polypeptide and protein is as fluid and ill-defined as a few grains of sand and a pile of it.

Another way to define simple, is by having most of the protein made up by just a few of the 20 amino acids. Collagen is a good example. Nearly half of it is glycine and proline (and a modified proline called hydroxyProline), leaving the other 18 amino acids to make up the rest. Collagen is big despite being simple — a single molecule has a mass of 285 kiloDaltons.

This brings us to [ Proc. Natl. Acad. Sci. vol 112 pp. E4717 – E4727 ’15 ] They constructed a protein/polypeptide of 26 amino acids of which 25 are either leucine or isoleucine. The 26th amino acid is methionine (which is found at the very amino terminal end of all proteins — remember methionine is always the initiator codon).

What does it do? It causes tumors. How so? It binds to the transmembrane domain of the beta variant for the receptor for Platelet Derived Growth factor (PDGFRbeta). The receptor when turned on causes cells to proliferate.

What is the smallest known oncoprotein? It is the E5 protein of Bovine PapillomaVirus (BPV), which is an essentially a free standing transmembrane domain (which also binds to PDGFRbeta). It has only 44 amino acids.

Well we have 26 letters + a space. I leave it to you to choose 3 of them, use one of them once, the other two 25 times, with as many spaces as you want and construct a meaningful sequence from them (in any language using the English alphabet).

Just back from an Adult Chamber Music Festival (aka Band Camp for Adults).  More about that in a future post

The elegance of metabolism control in the cell.

The current two pronged research effort on the possible use of Gemfibrozil (Lopid) to treat Alzheimer’s disease now has far wider implications than Alzheimer’s disease alone. As far as I’m aware, the combination of mechanisms described below to control a cellular pathway as never been reported before.

A previous post has the story up to 3 August — https://luysii.wordpress.com/2015/08/03/takes-me-right-back-to-grad-school/ — you can read it for the details, but here’s some background and the rest of the story.

Background: 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. A paper in the 7 July PNAS (vol. 112 pp. 8445 – 8450 ’15 7 July ’15) 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.

I wrote the author (Dr. Pahan) to ask how they came up with Gemfibrozil (Lopid). He told me that a transcription factor (PPARalpha) helps transcribe the ADAM10 gene into mRNA, and that Gemfibrozil makes PPARalpha a better transcription factor.

I told him to datamine from HMOs to find out if people on Lopid had less Alzheimer’s, he said it would be hard to get such as grant to do this as a basic researcher.

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. So on 3 August, 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, actually using Gemfibrozil.

Both researchers (Dr. Jicha and Nelson) were extremely helpful and cooperative. What is so fascinating is that they got to Gemfibrozil by an entirely different route. There are degrees of Alzheimer’s disease, and there is a pathologic grading scheme for it. They studied postmortem brain of 4 classes of individuals — normal nondemented elderly with minimal plaque, non demented elderly with incipient plaque, mild cognitive impairment and full flown Alzheimer’s. They had studied the microRNA #107 (miR-107) in another context. Why this one of the thousand or so microRNAs in the human genome? Because it binds to the mRNA of BACE1 and prevents it from being made. Why is this good? Because BACE1 chops up APP at a different site so the Abeta peptide is formed.

How did Gemfibrozil get into the act? Just as Dr. Pahan did, they looked to see what transcription factors were involved in making miR-107, and found PPARalpha. So to make less BACE1 they give people Gemfibrozil which turns on PPARalpha which turns on miR-107, which causes the mRNA for BACE1 to be destroyed, hopefully making less Abeta. The study is in progress and will last a year, far too short with far too few people to see a meaningful cognitive effect, but not so short that they won’t see changes in the biologic markers  they are studying in the spinal fluids (yes 72 plucky individuals have agreed to take Gemfibrozil (or not) and have two spinal taps one year apart.

The elegance of all this is simply astounding. One transcription factor turns on a gene for a chopper which inhibits Abeta formation, and turns on a microRNA which stops an APP chopper producing Abeta from being made.

So there’s a whole research program for you. Take a given transcription factor, look at the protein genes it turns on. Then look at the microRNA genes it turns on and then see what protein mRNAs they turn off. Then see they affect the same biochemical pathway as do ADAM10 and BACE1.

The mechanism is so elegant (although hardly simple) that I’ll bet the cell uses it again, in completely different pathways.

One problem with PPARalpha is that it is said to affect HUNDREDS of genes (Mol. Metab vol. 3 pp. 354 371 ’14).  So Gemfibrozil is a nice story, but even if it works, we won’t really be sure it’s doing so by ADAM10 and microRNA-107.

Hillary’s stroke – II

On 31 July The Clinton campaign released a letter from Hillary’s personal physician Lisa Bardack, MD, a board certified Internist,  basically saying that her health was excellent.

Well it isn’t and her letter essentially proves that she had a stroke in December of 2012. Here’s why.

First: a timeline.

At some time in the week of 9 December 2012 Mrs. Clinton is said to have fainted suffering a concussion. The New York Times reported on this 13 December.

She remained at home until 31 December at which point she was admitted to New York-Presbyterian Hospital when a blood clot was found in a vein draining the brain. (12 August — correction.  She was admitted 30 December).

Subsequently she had double vision due to her eye muscles not working together for a month or so and had to wear special glasses to correct this.

Second: The following explanation for these events was given by Dr. Bardach. You may read the entire letter at http://online.wsj.com/public/resources/documents/clintonhealth2015.pdf but the relevant paragraph is directly quoted below.

“In December of 2012, Mrs. Clinton suffered a stomach virus after traveling, became dehydrated, fainted and sustained a concussion. During follow up evaluations, Mrs. Clinton was found to have a transverse sinus venous thrombosis and began anticoagulation therapy to dissolve the clot. As a result of the concussion, Mrs. Clinton also experienced double vision for a period of time and benefited from wearing glasses with a Fresnel Prism. Her concussion including the double vision, resolved within two months and she discontinued the use of the prism. She had followup testing in 2013, which revealed complete resolution of the effects of the concussion as well as total dissolution of the thrombosis. Mrs. Clinton also tested negative for all clotting disorders. As a precaution, however, it was decided to continue her on daily anticoagulation.”

Third: Why should you believe what yours truly, a neurologist and not a neurosurgeon says about the minimal likelihood of this clot being due to the head trauma she sustained when she fainted? Neurologists rarely deal with acute head trauma although when the smoke clears we see plenty of its long term side effects (post-traumatic epilepsy, cognitive and coordination problems etc. etc.). I saw plenty of it in soldiers when I was in the service ’68 – ’70, but this was after they’d been stabilized and shipped stateside. However, I had an intense 42 month experience managing acute head injuries.

To get my kids through college, I took a job working for two busy neurosurgeons. When I got there, I was informed that I’d be on call every other night and weekend, taking first call with one of the neurosurgeons backing me up.  Fortunately, my neurosurgical backup was excellent, and I learned and now know far more about acute head trauma than any neurologist should. We admitted some of the head trauma cases to our service, but most cases had trauma to other parts of the body, so a general surgeon would run the show with our group as consultants. I was the initial consultant in half the cases. When I saw them initially, I followed the patients until discharge. On weekends I covered all our patients and all our consults, usually well over 20 people.

We are told that Hillary had a clot in one of the large draining veins in the back of her head (the transverse dural venous sinus). I’d guess that I saw over 300 cases of head trauma,but I never saw a clot develop in a dural sinus due to the trauma. I’ve spoken to two neuroradiologists still in practice, and they can’t recall seeing such a clot without a skull fracture over the sinus. Such a fracture has never been mentioned  at any time about Hillary.

Hillary’s neurologic deficit involved a nerve going to the muscles of her left eye. These start in the brainstem, a part of the brain quite near the site where she is said to have had the clot in her vein. The brainstem is crucial in maintaining consciousness, and it is more likely that the faint earlier in December was a warning sign of the stroke she had subsequently.

Why does the letter essentially prove Hillary had a stroke back then ?

I find it impossible to believe that the double vision occurred when she fainted. No MD in their right mind would not immediately hospitalize a case of head trauma with a neurologic deficit such as double vision. This is just as true for the most indigent patient as for the Secretary of State.

My guess was that the double vision came up later — probably after Christmas. Who gets admitted to the hospital the day of New Year’s Eve? Only those with symptoms requiring immediate attention.  Update 9 August — an alert reader asked how I knew she was admitted during the day and not in the evening.  It made me Google this point further — finding this http://news.yahoo.com/blogs/ticket/clinton-admitted-hospital-blood-clot-015548623–politics.html — showing that she was admitted 30 December.  Thanks Joe.

Dr. Bardack’s letter states, “As a precaution,however, it was decided to continue her on daily anticoagulation.” I couldn’t agree more. However, this is essentially an admission that she is at significant risk to have more blood clots. While anticoagulation is not without its own risks, it’s a lot safer now than it used to be. Chronic anticoagulation is no walk in the park for the patient (or for the doctor).   The most difficult cases of head trauma we had to treat were those on anticoagulants. They always bled more.

Dr. Bardack’s letter is quite clever.  She never comes out and actually says that the head trauma caused the clot, but by the juxtaposition of the first two sentences, the reader is led to that conclusion.  Suppose, Dr. Bardack was convinced that the trauma did cause the clot.  Then there would be no reason for her to subject Mrs. Clinton to the risks of anticoagulation, given that the causative agent was no longer present.    In all the cases of head trauma we saw, we never prescribed anticoagulants on discharge (unless we had to for non-neurosurgical reasons). I certainly agree with her use of anticoagulation, as I highly doubt that the trauma had anything at all to do with the blood clot in the transverse sinus. It is even possible that the clot was there all the time and caused the faint in early December.

The really important medical data would be Dr. Bardack’s office notes from December and the consultations of the neurosurgeon and admitting physician at Presbyterian 31 December, but I doubt that we’ll ever see them.

Why does this matter? Fortunately, Mrs. Clinton has recovered. However, statistically a person who has had one stroke is far more likely to have another than a person who has never had one. This is particularly true when we don’t know what caused the first (as in this case).

We’ve had two presidents neurologically impaired by stroke in the past century (Woodrow Wilson after World War I and Franklin Delano Roosevelt at Yalta). The decision they made in that state were not happy for the USA or the world.

Lest you regard this as anti-Hillary, concern for the health of future presidents is not confined to Democratic candidates.  Reagan’s age was raised as a legitimate issue by his opponents  as Christie’s near-morbid obesity should be if he gets the Republican nomination.  The resignation of Thomas Eagleton, the first running mate of George McGovern in 1972 because he had a history of electroshock therapy for depression, again shows that these concerns are not limited to any time or  party.

Addendum 14 Aug ’15: Will be away from the net for several weeks.  If you’ve commented after that, you’ve not been ignored or rejected, just held till I get back.

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:
https://www.nia.nih.gov/alzheimers/clinical-trials/gemfibrozil-predementia-alzheimers-disease

Our current efforts at Yale are on other mechanisms:
http://www.adcs.org/studies/Connect.aspx

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

Professor

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).

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

Professor

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).

Why drug discovery is so hard (particularly in the brain): Reason #28: The brain processes its introns very differently

Useful drug discovery for neurologic and psychiatric disease is nearly at a standstill. It isn’t for want of trying by basic researchers and big and small pharma. A recent excellent review [ Neuron vol. 87 pp. 14 – 27 ’15 ] helps explain why. In short, the brain processes its protein coding genes rather differently.

This post assumes you know what introns, exons and alternate splicing are. For pretty much all the needed background see the following.

First: https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/
Second:https://luysii.wordpress.com/2010/07/11/molecular-biology-survival-guide-for-chemists-ii-what-dna-is-transcribed-into/

When splicing first came out I started making a list of proteins which were alternatively spliced. It is now safe to assume that any gene containing introns (95% of all protein coding genes [ Proc. Natl. Acad. Sci. vol. 112 pp. 17985 – 17990 ’08 ]) results in several protein products due to alternative splicing. The products produced vary from tissue to tissue, probably because most tissues express different splicing regulators.

Here are a few. A2BP1 (aka Rbfox1, aka FOX1) is a brain specific RNA splicing factor found only in postmitotic terminally differentiated neurons. It is deleted in 10% of glioblastomas. Another is nSR100 (neural Specific Related protein of 100 kiloDaltons) — see later.

To show how crucial alternative splicing is for the every existence of the brain, consider this. The neuronal splicing regulator PTBP2 is barely expressed in most tissues. It is upregulated in neurons. Both PTBP1 and PTBP2 are repressors of neural alternative splicing (but some genes are actually enhanced). In a given region of the brain either PTPB1 or PTBP2 is expressed (but not both). PTBP1 promotes skiping of a neural specific exon (exon #10) in PTBP2 transcripts. This exposes a premature termination codon in PTBP2 leading to nonsense mediated decay (NMD). PTPB1 is expressed in most nonNeural tissues and neural precursor cells, but is silenced in developing neurons by the microRNA miR-124. The mRNA for PTBP2 contains an alternative exon which triggers nonsense mediated decay (NMD) when skipped. Inclusion of the exon requires positive transacting factors such as nSR100 in neurons. Repression is mediated by PTBP1 in undifferentiation. microRNAs (which ones?) downregulate PTBP1 during neuronal differentiation, relieving the negative regulation of PTBP2. Depletion of PTBP1 in fibroblasts is enough for PTBP2 induction and neuronal transdifferentiation.

It gets more complicated still. PTBP1 inhibits splicing of introns at the 3′ end of some genes involved in presynaptic function. This results in nuclear retention and turnover via components of the nuclear RNA surveillance machinery. As PTBP1 is downregulated during neuronal differentiation, the target introns are spliced out and the mature mRNAs are found.

Now we get to microExons, something unknown until 2014. For more details see — https://luysii.wordpress.com/2015/01/04/microexons-great-new-drugable-targets/.
Briefly, microexons are defined as exons containing 50 nucleotides or less (the paper says 3 – 27 nucleotides). They have been overlooked, partially because their short length makes them computationally difficult to find. Also few bothered to look for them as they were thought to be unfavorable for splicing because they were too short to contain exonic splicing enhancers. They are so short that it was thought that the splicing machinery (which is huge) couldn’t physically assemble at both the 3′ and 5′ splice sites. So much for theory, they’re out there.

The inclusion in the final transcript of most identified neural microExons is regulated by a brain specific factor nSR100 (neural specific SR related protein of 100 kiloDaltons)/SRRM4 which binds to intronic enhancer UGC motifs close to the 3′ splice sites, resulting in their inclusion. They are ‘enhanced’ by tissue specific RBFox proteins. nSR100 is said to be reduced in Autism Spectrum Disorder (really? all? some?). nSR100 is strongly coexpressed in the developing human brain in a gene network module M2 which is enriched for rare de novo ASD assciated mutations.

MicroExons are enriched for lengths which are multiples of 3 nucleotides. Recall that every 3 nucleotides in mRNA codes for an amino acid. This implies strong selection pressure was used to preserve reading frames as 3n+1 and 3n+2 produce a frameshift. The microExons are enriched in charged amino acids. Most microExons show high inclusion at late stages of neuronal differentiation in genes associated with axon formation and synapse function. A neural specific microExon in Protrudin/Zfyve27 increases its interation with Vessicale Associated membrane protein associated Protein VAP) and to promote neurite outgrowth.

[ Proc. Natl. Acad. Sci. vol. 112 pp. 3445 – 3450 ’15 ] Deep mRNA sequencing of mouse cerebral cortex expanded the list of alternative splicing events TENfold and showed that 72% of multiexon genes express multiple splice variants. Among the newly discovered alternatively spliced exon are 1,104 exons involved in nonsense mediated decay (NMD). THey are enriched in RNA binding proteins including splicing factors. Another set of alternatively spliced NMD exons is found in genes coding for chromatin regulators. Conservation of NMD exons is found in lower vertebrates, but those involving chromatin regulators are found later into the mammalian lineage. So the transcriptome in the brain is even more complicated.

A bit more about the actual effects on protein structure of alternate splicing. The sites chosen for this aren’t random. Cell and tissue differentially regulated alternative splicing events are significantly UNDERrepresented in functionally defined folded domains in proteins, they are enriched in regions of protein disorder that typically are surface accessible and embed short linear interaction motifs (with other proteins and ligands). Among a set of analyzed neural specific exons enriched in disordered regions, 1/3 promoted or disrupted interactions with partner proteins. So regulated exon splicing might specify tissue and cell type specific protein interaction networks. They regard their inclusion/exclusion as protein surface microsurgery.

How much can a little microexon do to protein function? Here’s an example of a 6 nucleotide microexon (two amino acids). Insertion of the microExon in the nuclear adaptor protein Apbb1 enhances its interaction with Kat5/Tip60 a histone deacetylase. The microExon adds Arginine and Glutamic acid to a phosphotyrosine binding domain (PTB domain) which binds Kat4. This enhances binding.

Had enough? The complexity is staggering and I haven’t even talked about recursive splicing — that’s for another post, but here’s a reference if you can’t wait — [ Nature vol. 521 pp. 300 – 301, 371 – 375, 376 – 379 ’15 ]. Pity the drug chemist figuring out which alternatively spliced form of a brain protein to attack (particularly if it hasn’t been studied for microExons).

I’m not making this up

Docs hate res ipsa loquitur — a favorite of malpractice lawyers — e.g. the thing speaks for itself — If accepted this means that negligence or malpractice requires no proof. So here I am, using it as a rhetorical device in an important malpractice case.

Below are the 7 priority areas set out by Katherine Archuleta, the late unlamented director of the Office of Personnel management. It’s a marvelously politically correct document, with all the appropriate buzzwords to warm the heart (and numb the mind). Unfortunately security wasn’t one of the seven.

OPM has set out seven priority areas:

Honoring the Workforce: OPM will be the champion of the Federal workforce. Through such programs as the OPM Innovation Lab and the Learning Center, we will provide career training and skill development for Federal employees. OPM will serve as the thought leader in research and data-driven human resource management and policy decision-making.

Build a More Diverse and Engaged Workforce: OPM, which by Executive Order is the lead agency on increasing diversity and inclusion in the Federal workforce, will recruit qualified individuals to serve and expand access to the job pipeline from entry and mid-level positions to leadership posts. OPM will provide leadership in helping agencies create work environments where a diverse Federal workforce is fully engaged and energized.

World Class Customer Service: OPM will respond to the interests of its many and diverse customers throughout the lifecycle of an employee. Whether it’s a recent graduate seeking to start a Federal career, a current employee looking for a training opportunity, or a retiree, OPM will provide timely, accurate and responsive service.

IT Improvement: Under the leadership of a new Chief Information Officer and Chief Technology Officer, OPM will implement its IT Strategic Plan to streamline and update IT systems to better serve Federal employees from resume through retirement.

Background Investigations: In partnership with the Office of the Director of National Intelligence, OPM will implement the revised Federal Investigative Standards and will lead efforts to strengthen the background investigations program across government as we maintain the highest standards of quality and timeliness.

Retirement: OPM is closing in on our goal to process 90 percent of cases within 60 days. The agency will continue to update our systems as we continue to transition to a paperless process.

Health Care: OPM will fully implement the Multi-State Plan provision of the Affordable Care Act, provide coverage to Tribal employees and continue providing high quality health insurance benefits to the Federal workforce.

Here’s a link to the original https://www.opm.gov/about-us/our-director/top-priorities/

Res ipsa loquitur

Why drug discovery is so hard: Reason #27 Moonlighting effects.

Well, we all know what heat shock proteins (Hsps) do — they bind to proteins which have lost their shape due to heat (or other stressors), cuddle them hydrolyze ATP and nurse them back to health. But what  if some of them do other things? The phenomenon is called moonlighting.

The case of Hsp70 is instructive. Some background first. The Hsp70 chaperone transiently associates with its substrates in a manner controlled by its ATPase cycle. ATP binding to the amino terminal nucleotide binding domain (NBD) induces a conformational change in the carboxy terminal substrate binding domain (SBD) which results in low affinity for substrate. Hydrolysis of ATP converts the Hsp70 to the ADP state, which binds substrates with higher affinity. Exchange of ADP for ATP releases substrate completing the cycle. The hydrolysis of ATP is stimulated by J-domain containing cochaperones. These are the nucleotide exchange factors.  Back and forth Hsp70 and the damaged protein go through the cycle until the protein is nursed back to normal or, failing this, is destroyed.

The Hsp70 family acts early in protein synthesis by binding to a small stretch of hydrophobic amino acids on a protein’s surface. Aided by a set of smaller Hsp40 proteins (also known as J proteins), a hsp70 monomer binds to its target protein and then hydrolyzes ATP to ADP, undergoing a conformational change that causes the hsp70 to clamp down very tightly on the target. After the hsp40 dissociates (see below), the dissociation of the hsp70 protein is induced by the rapid rebinding of ATP after ADP release. Repeated cycles of hsp protein binding and release help the target protein to refold.

Enter [ Proc. Natl. Acad. Sci. vol. 112 pp. E3327 – E3336 ’15 ] This work shows Hsp70 is methylated on arginine #469 by Coactivator Associated aRginine Methyltransferase 1/Protein aRginine MethylTransferase 4 (CARM1/PRMT4) and demethylated by JuMonJi Domain containing 6 (JMJD6) — hideous acronyms shortening even more hideous names. Methylated Hsp70 then functions in transcription as a ‘regulator’ of Retinoid Acid Receptor beta 2 (RARbeta2) transcriptional acitivty. R468Mmethylated Hsp70 mediates the interaction between Hsp70 and TFIIH (Transcription Factor IIH).

The regulation of gene transcription is an entirely novel and unsuspected function for a heat shock protein. A classic example of moonlighting.

Drug chemists and pharmacologists are always concerned about off-target effects. For an interesting example please see https://luysii.wordpress.com/2011/02/02/medicinal-chemists-do-you-know-where-your-drug-is-and-what-it-is-doing/.  Off-target effects occur when their drug hits something else in the cell producing an unexpected (and usually untoward) effect.

If you are unaware that your target of choice is doing a little something else on the side (e.g. moonlighting) you can get an off target effect even when you hit your desired target. It’s a tough business. How many more moonlighters are out there that we don’t know about?

Hsp70 is a good example. Here are two more — no background provided, so you’re on your own — except to point out that glucocorticoids are a widely used class of drug.

[ Proc. Natl. Acad. Sci. vol. 112 pp. E1540 – 1549 ’15 ] Amazingly, the glucocorticoid receptor (GR)plays a role in mRNA degradation by acting as an RNA binding protein. When loaded onto the 5′ UnTranslated Region (5′ UTR) of a target mRNA, the GR recruits UPF1 through Proline-rich Nuclear Receptor Coregulatory protein 2 (PNRC2) in a ligand (of itself?) dependent manner to cuase rapid mRNA degradation. They call this GMD (Glurocorticoid receptor Mediated Decay). Along with Staufen Mediated mRNA Decay (SMD) and Nonsense Mediated mRNA Decay (NMD), they share UPF1 (Upstream Frameshift 1) and PNRC2.

[ Science vol. 323 pp. 723 – 724, 793 – 797 ’09 ] Stat3 proteins represent the canonical mediators of signals elicited by cytokines binding to type I cytokine receptors. However, GRIM19 (Gene associated with Retinoid Interferon Mortality 19), a mitochondrial protein, interacts with Stat3 and inhibits its transcriptional activity (where?). This work shows that Stat3 associates with GRIM19 containing complexes I and II (components of the electron transport chain) in mouse liver and muscle mitochondria. Levels of Stat3 in mitochondria are 10% of cytosolic levels.

Cells lacking Stat3 show decreased activity of mitochondrial complexes I and II. Effects on complex I and II don’t require Stat3’s DNA binding domain, the dimerization motif, or the tyrosine phosphorylation site controlling Stat3 nuclear localization and transcriptional activity — so this is a ‘moonlighting’ role for State3 having nothing to do with gene transcription. The serine phosporylation site on Stat3 is important. So Stat3 is required to maintain normal mitochondrial function.

Happy fourth of July

Two encounters in the past 2 days brought home just how fortunate we are to live in the USA, along with the realization that only the immigrants truly appreciate this country. One was with a Greek friend who is a professor of engineering at a local university. His wife is on her way to Greece acting as a money launderer (well not really) bringing US dollars to her family over there. We get his brains for free and probably those of his 15 year old daughter who grew up here.

The other was with our tile man Sergey, a Russian immigrant of 17 years. He describes how his grandfather was sent by Stalin to Siberia, surviving for 14 years. Why? He wasn’t at all active politically, but was a devout Christian. That was all it took. If you find this difficult to accept — look at the following post — https://luysii.wordpress.com/2010/03/01/reification-in-mathematics-and-medicine/. Some of the most brilliant mathematicians of the Soviet Union were persecuted for the same reason.

Not convinced? Over 25 years ago, the local paper where we were living at the time had an interview with a Ukrainian woman newly arrived in the states. She was asked what it was she liked best about this country. She said it was being able to have people over to her house for prayer without having the draw the curtains.

Yes, we complain a lot about how things could be better, probably a genetic heritage, as only those who were unsatisfied with their condition where they were had the gumption to get up and come here.

However, as great as we are, tonight’s fireworks pale in comparison to those of the Chinese New Year (they invented fireworks after all). Earlier this year my wife and I viewed a 30 minute display from 3 ships firing away in Hong Kong harbor. Some of them even spelled out Chinese characters according to our daughter in law. If you like loud, go to a Buddist temple for their new year celebrations.

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