Category Archives: Medicine in general

Bad news on the AIDs front

Bad news for those hoping for an AIDs cure. As you know, the active virus (HIV1) has a genome made of RNA. However, thanks to an enzyme it possesses called reverse transcriptase (which has led to Nobel prizes), it copies itself into DNA and integrates into the genome of lymphocytes. There it sits presumably doing nothing, but it’s always capable of activating and producing more infectious virus.

We seem to have fought the virus to a draw, using a cocktail of drugs which attack different aspects — HAART (Highly Active Antiretroviral Therapy). Success is usually considered being unable to detect viral RNA in the blood (see later). However blood cells are short-lived. What about the longer living lymphocytes found in the lymph nodes and spleen.

That’s what was studied in a current paper [ Nature vol. 530 pp. 5` – 45 ’16 ] but in only 3 people. All had no detectable virus in the blood (under 48 copies/milliLiter — an incredibly tiny amount — see later). What they did was to biopsy lymph nodes in the groin on study entry and at 3 and 6 months.

Then they sequenced the genomes of the lymphocytes from the nodes, to study the HIV1 DNA integrated into the genome. They found that the genome changed with time. This is very bad. Why?

Because it implies that, even though you the virus in the blood, the virus was not staying latent in the lymph nodes, but coming out of the lymphocytes and forming infectious virus which then mutated. Subsequently the mutated virus integrated into the genome of another lymphocyte. So even with what we consider excellent control, the virus is not purely latent. Drug resistance could arise from mutations (although they didn’t see it in this study).

Clearly, more people need to be studied this way (but serial biopsies? It will probably be done in prisoners, if such things are still done).

It’s worthwhile thinking about how incredibly selective and accurate our methods of analysis are. 48 copies of the viral RNA per milliLiter of blood is the lower limit of detection. Remember that water has a molecular weight of 18, so a liter of distilled water is 1000 grams / 18 grams = 55.5 Molar. A mole has 6 x 10^23 molecules. A milliLiter is 10^-3 liters. So 1 milliLiter of distilled water has 55 * 6 * 10^23 * 10^-3 == 3 * 10^22 molecules of water in it so the assay is finding 48 or more molecules of HIV1 RNA in the water haystack. Even figuring that the concentration of water in blood is 1/10 that of distilled water, this is still impressive.

The checklistization of medicine

Today at the ophthalmologists the assistant who prepped me by putting in eyedrops and checking my visual acuity, had to put some information in her computer. One of the questions was how long I’d been taking eye drops. I told her to look at the chart, since the information was already there. She asked me to guess, so I did, and she duly entered the guess, which is (probably) now in the (electronic) chart and certainly less accurate than what is already there. Such is the checklistization of medicine today. Thank God I’m retired. The ophthalmologist said it’s part of the software that insurance companies require.

My brother, who is still practicing internal medicine, now gets 20 (paper) sheets of mostly useless information for every ER visit of one of his patients. This includes
l. a sheet saying the patient did not fall off the gurney
2. attestation that the patient was treated in a culturally appropriate manner
3. attestation that the patient was given the opportunity to ask questions.
I’m not making this up, and neither is he.

He says the residents are complaining that less time is available for the patient due to all this.

I guarantee you that this malarkey was not put in at the request of physicians and nurses actually attempting to take care of people.

It ain’t the bricks it’s the plan — take II

A recent review in Neuron (vol. 88 pp. 681 – 677 ’15) gives a possible new explanation of how our brains came to be so different from apes (if not our behavior of late).

You’ve all heard that our proteins are only 2% different than the chimp, so we are 98% chimpanzee. The facts are correct, the interpretation wrong. We are far more than the protein ‘bricks’ that make us up, and two current papers in Cell [ vol. 163 pp. 24 – 26, 66 – 83 ’15 ] essentially prove this.

This is like saying Monticello and Independence Hall are just the same because they’re both made out of bricks. One could chemically identify Monticello bricks as coming from the Virginia piedmont, and Independence Hall bricks coming from the red clay of New Jersey, but the real difference between the buildings is the plan.

It’s not the proteins, but where and when and how much of them are made. The control for this (plan if you will) lies outside the genes for the proteins themselves, in the rest of the genome (remember only 2% of the genome codes for the amino acids making up our 20,000 or so protein genes). The control elements have as much right to be called genes, as the parts of the genome coding for amino acids. Granted, it’s easier to study genes coding for proteins, because we’ve identified them and know so much about them. It’s like the drunk looking for his keys under the lamppost because that’s where the light is.

We are far more than the protein ‘bricks’ that make us up, and two current papers in Cell [ vol. 163 pp. 24 – 26, 66 – 83 ’15 ] essentially prove this.

All the molecular biology you need to understand what follows is in the following post — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure.

The neuron paper is detailed and fascinating to a neurologist, but toward the end it begins to fry far bigger fish.

Until about 10 years ago, molecular biology was incredibly protein-centric. Consider the following terms — nonsense codon, noncoding DNA, junk DNA. All are pejorative and arose from the view that all the genome does is code for protein. Nonsense codon means one of the 3 termination codons, which tells the ribosome to stop making protein. Noncoding DNA means not coding for protein (with the implication that DNA not coding for protein isn’t coding for anything).

Well all that has changed. The ENCODE Consortium showed that well over half (and probably all) our DNA is transcribed into RNA — for details see https://en.wikipedia.org/wiki/ENCODE. This takes energy, and it is doubtful (to me at least) that organisms would waste this much energy if the products were not doing something useful.

I’ve discussed microRNAs elsewhere — for details please see — https://luysii.wordpress.com/2010/07/14/junk-dna-that-isnt-and-why-chemistry-isnt-enough/. They don’t code for protein either, but control how much of a given protein is made.

The Neuron paper concerns lncRNAs (long nonCoding RNAs). They don’t code for protein either and contain over 200 nucleotides. There are a lot of them (10,000 – 50,000 are known to be expressed in man. Amazingly 40% of them are expressed in the brain, and not just in adult life, but during embryonic development. Expression of some of them is restricted to specific brain areas. It is easier for an embryologist to tell what type a cell is during brain cortical development by looking at the lncRNAs expressed than by the proteins a given cell is making. The paper contains multiple examples of the lncRNAs controlling when and where a protein is made in the brain.

lncRNAs can contain multiple domains, each of which has a different affinity for a particular RNA (such as the mRNA for a protein), or DNA, or protein. In the nucleus they influence the DNA binding sites of transcription factors, RNA polymerase II, the polycomb repressor complex. The review goes on with many specific examples of lncRNA function — synaptic plasticity, neurotic extension.

Getting back to proteins, the vast majority are nearly the same in all mammals (this is where the 2% Chimpanzee argument comes from). Here is where it gets interesting. Roughly 1/3 of lncRNAs found in man are primate specific. This includes hundreds of lncRNAs found only in man. The paper gives evidence that hundreds of them have shown evidence of positive selection in humans.

So the paper provides yet another mechanism (with far more detail than I’ve been able to provide here) for why our brains are so much larger, and different in many ways than our nearest evolutionary ancestor, the chimpanzee. This is the largest molecular biological difference found so far for the human brain as opposed to every other brain. Fascinating stuff. Stay tuned. I think this is a watershed paper.

Man’s best friend

I usually pay little attention to animal models of neurologic disease. After all, our brain is what separates us from animals (recent human behavior excepted). Neuromuscular disease is different because our peripheral nerves and muscles work the same way as animals. An astounding paper from Harvard and Brazil, gives us an entirely new angle to treat muscular dystrophy, particularly the Duchenne form. I ran a muscular dystrophy clinic for 15 years in the 70s and 80s and haplessly watched young boys deteriorate and die from Duchenne. The major therapeutic advance during that time was — hold your breath — lighter weight braces, allowing the boys to stay out of wheelchairs a bit longer.

Some background for those who don’t know, the molecular defect in Duchenne was found in ’87. Interestingly Kunkel, one of the authors on the original paper [ Cell vol. 51 pp.; 919 – 928 ’87 ] is an author on the present one [ Cell vol. 163 pp. 1204 – 1213 ’15 ]. Duchenne dystrophy affects only males, as the gene for the protein (dystrophin) is found on the X chromosome, so women with a normal X and a mutant X escape. To show how pathetic things were back then, we tried to find out if a sister of a patient was a carrier. How did we do it. By measuring an enzyme released by damaged muscle (CPK) on several occasion. Carriers often showed an elevation.

The mutated protein is called dystrophin. It hooks the contractile apparatus of a muscle cell to the membrane. Failure of this makes muscle cells more fragile when they contract resulting in eventual loss. From a molecular biological point of view the protein is fascinating. The gene is one of largest known, stretching over 2,220,233 positions (nucleotides) on the X chromosome and containing 79 exons. Figuring a transcription rate of 100 nucleotides a second, it takes 6 hours to make the messenger RNA (mRNA) for it. The protein has 3,685 amino acids and figuring a translation rate of 3 – 6 amino acids/second it takes 10 minutes for the ribosome to make it. Given that it takes only 3 nucleotides to code for an amino acid, the protein coding part of the gene takes up only .5% of the gene. Correctly splicing out the introns is a huge task, which we all perform well. This size and complexity of the gene explains why mutations are so common, making it the most common form of hereditary muscular dystrophy (most are).

There are currently all sorts of efforts underway to correct the mutation, particularly in a milder form called Becker dystrophy. Derek has covered them and they constitute a logical direct attack on the pathology.

What is so remarkable about the current Cell paper is that it gives us an entirely new and different way to attack Duchenne (and possible all forms of muscular dystrophy). It involves a colony of dogs in Brazil. They have GRMD (Golden Retriever Muscular Dystrophy) with a mutation in one of the many splice sites in dystrophin (it has 79 exons in man) leading to a premature stop codon and no functional dystrophin in the dogs’ muscles. The animals weaken and become non ambulatory with a shortened lifespan. However, a few of the dogs in the colony seemed pretty normal. So they went to work. The obvious reason was that gene was in some way repaired so the animals had normal amounts of dystrophin. Not so, even though ambulatory, the animals’ muscles had no dystrophin. So the whole genome was sequenced. What they found was that a mutation at an upstream site of a protein called Jagged1 lead to increased transcription of the gene and increased levels of the protein.

Jagged1 is a protein ligand for the Notch system of receptors. The Notch system is important in muscle regeneration. The myoblasts of the animals had more proliferative capacity. The Notch system is far too complicated to go into here — https://en.wikipedia.org/wiki/Notch_signaling_pathway, but expect to see a lot more research money pumped into it.

What I find so fabulous about this paper, is that it gives us an entirely new way of thinking about Duchenne, totally unrelated to the genetic defect, which had been our focus up to now. It also rubs our noses in how little we understand about our molecular biology and cell physiology. If we really understood things, we’d have been focused on Notch years ago. Yet another reason drug discovery is so hard. We are trying to alter a system we only dimly understand.

Les fleurs du PTEN

Les fleurs du Mal is a volume of poetry by Baudelaire about the beauty of evil and depravity. I have the same esthetic appreciation for the horrible things a mutant of PTEN does. It’s awful, but incredibly elegant chemically.

Back in the day med students used to be told ‘know syphylis and you’ll know medicine’ because of its varied clinical manifestations. PTEN is like that for cellular and molecular biology.

PTEN (Phosphatase and TENsin homolog) is a gene mutated in many forms of cancer. So it was regarded as a tumor suppressor, keeping our cells on the straight and narrow. Naturally cancer cells ‘try’ (note the anthropomorphism) to neutralize it. PI3K is a universal tumor driver, integrating growth factor signaling with downstream circuitries of cell proliferation, metabolism and survival.

Inositol is a 6 membered ring (all carbons) with one OH group attached to each carbon, which are numbered 1 through 6. PI3K puts phosphate on the 3 position, PTEN takes it off. Since this is how PI3K signaling begins, cells lacking PTEN grow faster and migrate aberrantly (e.g. spread).

Enter Proc. Natl. Acad. Sci. vol. 112 pp. 13976 – 13981 ’15 which carefully studied a PTEN mutant found in an unfortunate man with aggressive prostate cancer. It just changed one of the 403 amino acids (#126) from alanine to glycine. Not a big deal you say,it’s just a change of CH3 (alanine) to H (glycine). #126 is near the active site of the enzyme. One might expect that the mutation inhibits PTEN’s phosphatase activity (e.g. its enzymatic activity). Not so — the mutations shifts the activity so the enzyme. Instead of removing phosphate from the 3 position of inositol, the phosphate at the 5 position is removed (leaving the 3 position alone). This shifts inositol phosphate levels in the cell with hyperactivation of PI3K signaling (which requires inositol phospholipids containing phosphate at the 3 position).

What happens is that inositol phosphates fit into the mutant active site with the 5 position near the catalytic amino acid (cysteine). Essentially the 6 membered ring rotates the 3 position away from cysteine and puts the 5 position there instead. This changes PTEN from a tumor suppressor (anti-oncogene) to an oncogene.

To a chemist this is elegant and beautiful (apologies Baudelaire).

PTEN has taught us a huge amount about the control of protein levels, pseudogenes, competitive endogenous RNA (ceRNA). You can read all about this in https://luysii.wordpress.com/2014/01/20/why-drug-discovery-is-so-hard-reason-24-is-the-3-untranslated-region-of-every-protein-a-cerna/

That’s fairly grim, so here’s a link to one of the great comedians of years past — Jonathan Winters

http://biggeekdad.com/2013/04/jonathan-winters-stick/

It’s politically incorrect and sure to offend the humorless pompous prigs. Enjoy ! ! !

Carly’s cancer

Carly Florina had breast cancer surgery (bilateral mastectomy) 2 March 2009 at Stanford University Hospital followed by chemotherapy and radiation. She was given an excellent prognosis for full recovery — https://en.wikipedia.org/wiki/Carly_Fiorina.

So far so good and it’s coming up on 7 years. But it is reasonable to ask just what her prognosis really is, particularly as she may be our next president. I asked an old friend and colleague who has been involved in research on breast cancer and in many of the clinical trials of therapy over the past 35 years.

So I wrote the following — I’m writing you for some idea what the chances of someone with breast cancer being free for 6+ years (Carly’s surgery was 2/09) will be free for the next 5+? I know that there are all sorts of statistics on survival in breast cancer (because the cohort is so large). If anyone would know them, it would be you.

and got this back

Impossible to answer your question. Too many variables and NO DATA or info. Many people, docs and patients alike call ductal carcinoma in situ,” cancer” but cure rate is 99%. If she was one of those then, of course, she’s likely to be cured . Stage 1 ,luminal A tumors (even though real cancers) have excellent prognoses—probably > 90% cured. For other real cancers Lots depend on stage, hormone receptors ad infinitum. On thin ice lumping anyone into a broad statement without lots more info

just what you’d expect from an circumspect intelligent expert

So I dug a bit more and sent him this

I tried to find out just what type of breast cancer Carly had. No luck, but various newspaper articles show that she did receive postop chemo causing her hair to fall out as well as radiation. Would ductal carcinoma in situ (Dcis) be treated this way? Would stage 1 luminal A tumors be treated this way?

He replied

Dcis definitely no. luminal a probably shouldn’t be. Sounds like a significant cancer. Next issue is did she get antihormonal therapy. Estrogen receptor tumors are the ones that tend to relapse after 5 years. ER neg. tumors while more aggressive overall seldom recur >5 yrs after dx. The radiation part doesn’t mean much unless she had a mastectomy since all lumpectomy patients get radiation. – If she had mastectomy and chemo and radiation it was probably a poorer risk tumor. Even chemo might not be so bad—–we give chemo to node neg tumors which could end up with very good long term prognosis.AMONG RELAPSES in ER pos pts. 15% recur before year 5 and 17% recur after year 5. However overall likelihood of relapse depends on whether or not she had positive or negative nodes and was ER + or Neg. Sorry to be so wordy but prognosis has been improving steadily. I would guestimate that we’re curing about 70% of women with newly diagnosed breast cancer—excluding dcis who are virtually all cured.

I realized that I’d neglected to tell him that she’d had a bilateral mastectomy as well and got the following back after I did.

If she indeed had radiation after a mastectomy as well as chemo it speaks for a more aggressive presentation. Rule of thumb—-post mastectomy xrt reserved for patients with > 4 positive nodes or tumors >5 cm in size. Today, many are giving post mastectomy xrt to 1-3 positive nodes although that was very controversial for years . newer data impies benefit. So, just guessing, but she probably had positive nodes—a poorer prognostic sign for long term—but only if she was estrogen receptor pos. as noted in prior email.

So there you have it — she’s fortunately well presently, but the tumor and prognosis doesn’t sound that good. Still unknown are histologic type of the tumor, presence or absence of spread to lymph nodes (and if so how many), estrogen receptor positivity, which would certainly give us a better idea of her ultimate prognosis (and the country’s should she become president).

I take no pleasure in any of these posts. See https://luysii.wordpress.com/2015/08/06/hillarys-stroke-ii/ Both Carly and Hillary are brilliant women it would be an honor to know and I wish them both the best. FYI Hillary was valedictorian of her class at Wellesley.

So why write about their potential health problems? Look at the sad saga of Hugo Chavez who claimed he was cured in July elected in the fall with death before he could take office in March of the following year — see https://luysii.wordpress.com/2013/03/05/q-e-d/. Also consider the last months in office of Woodrow Wilson and Franklin Roosevelt and the results of the League of Nations and the Yalta conference when they were both impaired.

My wife asked my why I pick on female candidates, and I’ll address Christie’s massive obesity should he rise in the polls.

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).

How little we know

Well it’s basic biochem 101, but enzymes only allow equilibrium to be reached faster (by lowering activation energy), they never change it. This came as a shock to the authors of [ Proc. Natl. Acad. Sci. vol. 112 pp. 6601 – 6606 ’15 ] when Cytosolic Nonspecific DiPeptidase 2 (CNDP2), a proteolytic enzyme, was found to tack the carboxyl group of lactic acid onto the amino group of a variety of amino acids, essentially running the proteolytic reaction in reverse. Why? Because intracellular levels of lactic acid and amino acids are in the high microMolar to milliMolar range. It’s Le Chatelier’s principle in action.

The compounds are called N-Lactoyl amino acids. No one had ever seen them before. They are part of the ‘metabolome’ — small molecules found in our bodies. God knows what they do. The paper was really shocking to me for another reason, because I had no idea how many members the metabolome has.

How large is the metabolome? Make a guess.

Well METLIN (https://metlin.scripps.edu/index.php has 240,000, and Human Metabolome DataBase http://www.hmdb.ca/metabolites?c=hmdb_id&d=up&page=1676 has 42,000. I doubt that we know what they are all doing. Undoubtedly some of them are binding to proteins producing physiologic effects. Drug chemists may be mimicking some of them unknowingly, producing untoward and unexpected side effects.

What’s even more shocking to me is the following statement from the paper. State of the art untargeted metabolomics studies still report ‘up to’ 40% unidentified, but potentially important metabolitcs which can be detected reproducibly. The unknown metabolites are only rarely characterized because of the extensive work required for de novo structure determination..

So we really don’t know everything that’s out there in our bodies, and even if we did, we don’t know what they are doing. Drug discovery is hard because we only dimly understand the system we are trying to manipulate. Until I read this paper, I had no idea just how dim this is.

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