Category Archives: Neurology & Psychiatry

Two disconcerting papers

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

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

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

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

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

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

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

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

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

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

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

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

Book Review — The Kingdom of Speech — Part III

The last half of Wolfe’s book is concerned with Chomsky and Linguistics. Neurologists still think they have something to say about how the brain produces language, something roundly ignored by the professional linguistics field. Almost at the beginning of the specialty, various types of loss of speech (aphasias) were catalogued and correlated with where in the brain the problem was. Some people could understand but not speak (motor aphasia). Most turned out to have lesions in the left frontal lobe. Others could speak but not understand what was said to them (receptive aphasia). They usually had lesions in the left temporal lobe (e.g. just behind the ear amazingly enough).

Back in the day this approach was justifiably criticized as follows — yes you can turn off a lightbulb by flicking a switch, but the switch isn’t producing the light, but is just something necessary for its production. Nowadays not so much, because we see these areas lighting up with increased blood  flow (by functional MRI) when speech is produced or listened to.

I first met Chomsky’s ideas, not about linguistics, but when I was trying to understand how a compiler of a high level computer language worked. This was so long ago that Basic and Pascal were considered high level languages. Compilers worked with formal rules, and Chomsky categorized them into a hierarchy which you can read about here —

The book describes the rise of Chomsky as the enfant terrible, the adult terrible, then the eminence grise of linguistics. Wolfe has great fun skewering him, particularly for his left wing posturing (something he did at length in “Radical Chic”). I think most of the description is accurate, but if you have the time and the interest, there’s a much better book — “The Linguistics Wars” by Randy Allen Harris — although it’s old (1993), Chomsky and linguistics had enough history even then that the book contains 356 pages (including index).

Chomsky actually did use the term language organ meaning a facility of the human brain responsible for our production of language of speech. Neuroscience never uses such a term, and Chomsky never tried to localize it in the brain, but work on the aphasias made this at least plausible. If you’ve never heard of ‘universal grammar, language acquisition device, deep structure of language, the book is a reasonably accurate (and very snarky) introduction.

As the years passed, for everything that Chomsky claimed was a universal of all languages, a language was found that didn’t have it. The last universal left standing was recursion (e.g. the ability the pack phrase within phrase — the example given “He assumed that now that her bulbs had burned out, he could shine and achieve the celebrity he had always longed for” — thought within thought within thought.

Then a missionary turned linguist (Daniel Everett) found a tribe in the Amazon (the Piraha) with a language which not only lacked recursion, but tenses as well. It makes fascinating reading, including the linguist W. Tecumseh Fitch (yes Tecumseh) who travelled up the Amazon to prove that they did have recursion (especially as he had collaborated with Chomsky and the (now disgraced) Marc Hauser on an article in 2002 saying that recursion was the true essence of human language — how’s this horrible sentence for recursion ?

The book ends with a discussion of the quote Wolfe began the book with — “Understanding the evolution of language requires evidence regarding origins and processes that led to change. In the last 40 years, there has been an explosion of research on this problem as well as a sense that considerable progress has been made. We argue instead that the richness of ideas is accompanied by a poverty of evidence, with essentially no explanation of how and why our linguistic computations and representations evolved. We show that, to date, (1) studies of nonhuman animals provide virtually no relevant parallels to human linguistic communication, and none to the underlying biological capacity; (2) the fossil and archaeological evidence does not inform our understanding of the computations and representations of our earliest ancestors, leaving details of origins and selective pressure unresolved; (3) our understanding of the genetics of language is so impoverished that there is little hope of connecting genes to linguistic processes any time soon; (4) all modeling attempts have made unfounded assumptions, and have provided no empirical tests, thus leaving any insights into language’s origins unverifiable. Based on the current state of evidence, we submit that the most fundamental questions about the origins and evolution of our linguistic capacity remain as mysterious as ever, with considerable uncertainty about the discovery of either relevant or conclusive evidence that can adjudicate among the many open hypotheses. We conclude by presenting some suggestions about possible paths forward.”

One of the authors is Chomsky himself.

You can read the whole article at

I think, that Wolfe is right — language is just a tool (like the wheel or the axe) which humans developed to help them. That our brain size is at least 3 times the size of our nearest evolutionary cousin (the Chimpanzee) probably had something to do with it. If language is a tool, then, like the axe, it didn’t have to evolve from anything.

All in all a fascinating and enjoyable book. There’s much more in it than I’ve had time to cover.  The prose will pick you up and carry you along.

Book Review — The Kingdom of Speech — Part II

Although Darwin held off writing up his ideas for 20 years, fearing the reaction he knew would come from the church, the criticisms that really bothered him the most were those of fellow intellectuals about the evolution of language. They began immediately after the Origin of Species came out in 1859, by linguists and later by Wallace himself. Even worse, one critic mocked him. The idea that language evolved from animal sounds was called the bow wow theory, or language arose from sounds that things made (the ding dong theory).

This is all detailed in pp. 54 – 87 of The Kingdom of Speech, about which I knew very little. If any real experts on the early history of evolutionary theory are out there and reading this and disagree, please post a comment. I am assuming that the facts as given by Wolfe are correct (I’ve already disagreed with him about his interpretation of some of them —

The real attack on Darwin’s ideas is that man’s mental capacities were so far above those of animals, that there was no missing link (particularly since there were lots or primates still around). By this critique man was so special, that a special act of creation (not evolution) was called for.  It’s theology getting in the back door, but of course this is essentially the claim of all theologies — special creation by a superior being(s).

In his later book “The Origin of Species and the Descent of Man” – 1871 (which I’ve not read), according to Wolfe Darwin made up all stories (many involving his beloved dog) to show the antecedents of all sorts of things in animal behavior — Darwin actually said that language originated with the songs birds sang during mating. Female protolanguage persists today in mothers cooing to their babies. Darwin spent a lot of time discussing his dog — how it recognized other dogs as a sign of intelligence. Religion came from the love of a dog for his master (Wolfe claims that Darwin said this in the book– I haven’t read the Descent of Man).

Darwin’s second book didn’t get much response. Postive reviews avoided his reasoning, and negative reviews said it was thin. In 1872 the Philological Society of London gave up on trying to find out the origin of language, and wouldn’t accept patpers about it. The Linguistic Society of Paris did this even earlier (1866).

Evolutionists basically stopped talking about language from 1872 to 1949.

As soon as Mendel’s work on genetics was discovered, evolution went into scientific eclipse. Here was something that wasn’t just armchair speculation about things happening in the remote past, something on which experiments could be done.
Mendel’s experiments with green peas took 9 years and involved 28,000 plants.

In a fascinating aside, Wolfe notes that Mendel actually sent his work to Darwin. Tragically it was found unread with its pages uncut in Darwin’s papers after his death. In all fairness to Darwin, he and his peers had no idea how heredity worked and there are parts in The Origin of Species in which Darwin appears to accept the inheritance of acquired characteristics (the blacksmith’s large muscles passed on to his son etc. etc.). I don’t think you can read the Origin without being impressed by the tremendous power of Darwin’s mind, and how much work he put in and how far he got with how little he had to go on.

Wolfe says Darwin’s ideas about the origin or language were mocked by Gould  one hundred years later (1972) as “Just So Stories”, fantastic bizarre explanations for why animals are the way they are — see I’m not so sure, the citation for this gives an article  Sociobiology which Gould and Lewontin (see later) relentlessly attacked. Gould himself saw what he wanted to see in his book “The Mismeasure of Man” — for details see —

As you can see,The Kingdom of Speech is full of all sorts of interesting stuff, and I’m not even halfway through talking about it.

Next up, linguistics, to include Noam  Chomsky and his admission that he doesn’t understand language or where it came from.

Hillary’s health — you can see a lot by looking

Last night’s debates should put two suggestions about Hillary’s health to rest and gives some evidence for two others. First, she does not have Parkinson’s disease. Second, she does not have epilepsy. Third, her eye movements still show some residua from the stroke of December 2012. Fourth, she may have a mild proximal myopathy.

Now to elaborate.

Parkinson’s disease: Two great things happened in September 1970 — I finished my two years in the Air Force and L – DOPA was released for use in the USA. American neurologists had been reading about the great things it was doing for the disease in Europe for almost 10 years. So when I went back to complete the last two years of my residency, the chief put me in charge of the L – DOPA clinic he’d just set up. So until retirement in 2000, I treated lots of people with it.

As the chief said — Parkinson’s disease is a Yellow Cab disease. If you see a Yellow Cab on the street, you don’t write down the license number, go down to city hall and find that it was registered as a Yellow Cab. You look at it and say “that’s a Yellow Cab”.

Parkinsonians have a rather immobile face (masklike) — Hillary’s face is quite mobile. Their speech lacks the normal musicality of speech (prosody), Hillary’s speech has normal inflection. Parkinsonians have a slow, stiff gait with difficulty initiating it. Hillary has none of this. Finally there is no sign of any tremor.

Epilepsy: Videos of purported seizures are out and about on the internet, particularly one during an interview. I thought that the ones I saw looked rather edited, as though some individual frames had been deleted from the videos. Fortunately last night we had an opportunity to see for ourselves. Toward the end of the debate, she had another episode, during which she shook her head and her shoulders for a few seconds. This happened in real time, so we could run the video recording backwards and forwards. At no time did she appear to be out of contact, and she then continued on with what she was saying without pause. So it’s just something she voluntarily does. It isn’t epilepsy.

Eye movements: Recall that after the stroke in December 2012, Hillary had double vision and had to wear Fresnel lenses to correct it for a few weeks afterwards — pictures of her testifying in congress January 2013 show this. So last night there was a 90 minute opportunity to watch the way her eyes move. They aren’t quite normal – on looking to her left the right eye lags and doesn’t bury the white. Even though Trump was to her right, she turned her head rather than her eyes to look at him, so I only saw her look to her right on a few occasions, but when she did her eyes appeared to move together. No other residua of a brainstem stroke were present such as slurred speech (dysarthria), facial weakness, facial asymmetry.

Proximal muscle weakness: The internist referred to in a previous post noted the following:

“There were shots a month or so ago of her needing help to get up outdoor stairs and also needing a small step-stool to get up into a Secret Service Suburban. My wife and I hop in and out of a Yukon and do not need any step device (they are of comparable age). After a photo of her doing that was published, she started getting in and out of vehicles on the side away from cameras and was also switched to a taller van with a step mounted on the vehicle. In February, press was forbidden by her staff from filming her climbing the stairs to board her private jet.”

He wondered if she could have something like limb girdle dystrophy.

Well, such a dystrophy is certainly possible. Although Hillary  had no difficulty standing for 90 minutes, at the end, she appeared to waddle as she walked toward the moderator.. There wasn’t really enough time to definitely say that she waddled.  It’s worth carefully watching the way she walks in the future.

Why is waddling a sign of mild weakness of the muscles of the pelvic girdle? Believe it or not the buttocks are not a secondary sexual characteristic. The main buttock muscle (gluteus maximus) is so big because it has so much work to do.

Think about what you do when you take a step forward with your right foot. To remain stable, your entire upper body weight must  be strongly plastered to your left hip. You need a strong, large muscle to do this (the gluteus maximus). What happens if the muscle is weak? Your upper body would fall to the right. How would you prevent this? By throwing your upper body to the left, putting its center of gravity there, so it presses on the left hip with greater force. A similar thing happens when stepping forward with the left foot. The net effect is that you waddle, which is what Hillary appeared to do.

It’s worth watching her walk in the future.

Stamina: she was under 90 minutes of stress, and showed no sign of fatigue.

Now, hopefully, back to the science, with a very long (over 1,000 Angstroms) allosteric effect.

Hillary’s fainting spell

And I thought I’d retired as a neurologist. What is there to say about the video that shows Hillary Clinton being held up by a woman on her left, later by others, and then collapsing sufficiently that her head is at most 3 feet from the pavement in one frame. You don’t have to go to medical school to call this a fainting spell.

As to what caused the episode, we can only speculate. I see no reason to trust what the campaign is putting out, that she had ‘pneumonia’ for the previous two days. Since I’ve already gone on record that she had a stroke in December 2012 ( ), not due to head trauma sustained in what was said to another fainting spell, people have asked me what the event could be neurologically.

But I’m a neurologist not an internist, so I talked to a very smart one for his take.

“Somewhat oddly, her campaign now reports that pneumonia had been “diagnosed” as of two days before her collapse. However, she was not acting as if she is infectious, going out into crowds and getting close to small children. The Clintons are known for lawyerly parsing of phrases carefully, so it may matter what the meaning of pneumonia “is.” Therein may lie a clue which puts the chronic non-productive cough of many months duration, along with apparent decreased stamina and a carefully tuned and truncated schedule over a similar period into perspective.

Chronic lung disease, particularly a mildly progressive idiopathic pulmonary fibrosis/interstitial pneumonia could fit that picture. It would also be technically true as a diagnosis. Whatever pulmonary condition she has does not appear to be acute.”

He also had an interesting observation on the way the faint was handled. “There must be some chronic known condition, as she has two attendants with her now at all times–large black male and heavyset white woman. Her collapse was handled as if it were familiar territory. Hustling a woman of her age into a van and driving to her daughter’s apartment is a highly unusual way to handle such a loss of consciousness in a 68-year old woman, particularly when there had to be a number of emergency vehicles loaded with EMT’s on the scene and well as several hospitals at least as close as her daughter’s apartment.” To which a friend noted that the secret service is trained to react immediately to situations like this, going through dry runs of all sorts of eventualities etc. etc.

Taking her to her daughter’s apartment is quite strange, given the way the secret service was acting 40 or so years ago. Back then, a neurosurgeon in Billings Montana told me that the secret service had called him up and asked him to be available in the coming weekend as the president would be visiting Yellowstone, a mere 140 miles away by the nearest road. It seems likely that some hospital close by was on alert that Hillary was in the neighborhood.

The internist has been watching her a lot more closely than I have and noted the following “There were shots a month or so ago of her needing help to get up outdoor stairs and also needing a small step-stool to get up into a Secret Service Suburban. My wife and I hop in and out of a Yukon and do not need any step device (they are of comparable age). After a photo of her doing that was published, she started getting in and out of vehicles on the side away from cameras and was also switched to a taller van with a step mounted on the vehicle. In February, press was forbidden by her staff from filming her climbing the stairs to board her private jet.” He wondered if she could have something like limb girdle dystrophy — watching her walk and stand during the upcoming debates will be helpful for determining that.

Finally he noted — “There are also a number of cardiovascular causes (transient arrhythmia for starters) as well as pulmonary microemboli which can cause collapse like that.”

Now for the neurologic possibilities.

There are peculiar videos purporting to show Hillary having a ‘seizure’ during a press conference. They look doctored to me. She appears to be compulsively laughing. Such seizures are called gelastic epilepsy. They are rare but I’ve seen them. They arise from the hypothalamus and the temporal lobes. Nothing in the current video is suggestive of a seizure. Loss of consciousness at this age rarely is due to a seizure. Cardiovascular causes are far more likely.

Another possible cause is a brainstem transient ischemic attack (TIA), since we have been told that the clot of 2012 was in a draining sinus of the posterior fossa (we have no pictures of any sort from that episode). Recovery in 90 minutes is consistent with either syncope or TIA.

The final possibility is that the event is a warning of an impending second stroke. If you look again at the post about the events of 2012, you’ll see that I speculated that the ‘faint’ occuring in the week of 9 December could have been a transient warning of the cerebral venous thrombosis she suffered that month. I don’t think this likely, but when I examined for the Neurology boards, fellow examiners always wanted to see how many possibilities for diagnoses the candidates can muster.

So what do I think it was? A fainting spell (syncope if you want to be impressive). Her blood pressure dropped for some reason or other, the brainstem which maintains alertness didn’t get enough fresh blood and she passed out nearly. The people with her did NOT help by keeping her erect, which kept the brainstem from getting the blood (and the oxygen it delivers) it needed. In fact holding someone up who has fainted is the perfect crime, as the brain deprived of oxygen long enough begins to die, and no marks will be found on the body.

Why out of the thousands there, on a warm but not excessively hot day, she was the only one to pass out can only be the subject of speculation until more details are forthcoming. The health of a possible future president is simply too important not to speculate about.

What neuropharmacology can’t tell us about opiates and addiction

A friend’s wife had some painful surgery and is trying to get by with as little opiates as possible, being very worried about becoming an addict, something quite reasonable if all she had to go on was the popular press with lurid stories of hapless innocents being turned into addicts by evil physicians overprescribing opiates (it’s the current day Reefer Madness story). Fortunately her surgeon wisely told her that her chances of this happening were quite low, since she’d made it past 50 with no dependency problems whatsoever. Here’s why he’s right and why neuropharmacology can’t tell us everything we want to know about opiates and addiction.

Back in the day, disc surgery required general anesthesia, dissection of the back muscles down to the spine, sometimes chipping away at the bones of the spine to remove a bone spur (osteophyte) and/or removal of the offending herniated intervertebral disc. This meant a hospital stay (unlike my ophthalmologist who had a microdiscectomy as an outpatient a few years ago). This was the era of the discovery of the protein receptor for morphine and other opiates, and we were all hopeful that this would lead to the development of a nonAddicting opiate (narcotic). Spoiler alert — it hasn’t happened and likely won’t.

Often, I was the neurologist who diagnosed the disc and told the surgeon where it was likely to be found (this was in the preCT and later the preMRI era). I’d developed a relationship with most of those I’d referred for surgery (since it was never recommended, without a trial of rest — unless there were compelling reasons not to — trouble controlling bowels and bladder, progressive weakness etc. etc.). I was their doc while they tried to heal on their own.

So post-operatively I’d always stop by to see how the surgery had worked for them. All were on a narcotic (usually Demerol back then) as even if the source of their preoperative pain had been relieved, just getting to the problem had to cause significant pain (see above).

If the original pain was much improved (as it usually was), I’d ask them how they liked the way the demerol made them feel. There were two types of responses.

#1 I hate feeling like this. I don’t care about anything. I’m just floating, and feel rather dopey. I’m used to being in control.

#2 I love it ! ! ! ! I don’t have a care in the world. All my troubles are a million miles away as I just float along.

Love it or hate it, both groups are describing the same feeling. Neuropharmacology can help to tell us why opiates produce this feeling, but it can’t tell us why some like it (about 5%) and the majority (95%) do not. This clearly is the province of psychology and psychiatry. It’s the Cartesian dualism between flesh (opiate receptor) and spirit (whether you like what it does). It also shows the limitation of purely physical reductionism of the way we react to physical events.

The phenomenon of a small percentage of people becoming addicted to a mind altering substance is general and is not confined to one class of drug. We were told never to prescribe chronic benzodiazepines (valium, etc. etc.) to a recovered alcoholic. People who get hooked on one thing are very likely to get hooked on another.

I realize that some of this could be criticized as blaming the victim, but so be it. Medical facts are just that, like what they say or not.

Addendum 11 Sep ’16 — I’m not saying that you won’t become physically dependent on opiates if you get them long enough and at high enough doses. We all would. Even if this happened to you. When you no longer needed them for pain and went through medically supervised withdrawal, you wouldn’t crave them, and do crazy things to get them (e.g. you were physically dependent but never addicted to them — it is important to make the distinction).

Example — when I was in the service ’68 – ’70, we had half a million men in Vietnam. Everyone I’ve talked to who was over there says that heroin use among the troops was 25 – 50% (high grade stuff from Thailand was readily available). As soon as they got back to the states, the vast majority gave them up (and with minimal withdrawal requiring my attention – I think I saw one convulsion due to withdrawal).

Baudelaire comes to Chemistry

Could an evil molecule be beautiful? In Les Fleurs du Mal, a collection of poems, Baudelaire argued that there was a certain beauty in evil. Well, if there ever was an evil molecule, it’s the Abeta42 peptide, the main component of the senile plaque of Alzheimer’s disease, a molecule whose effects I spent my entire professional career as a neurologist ineffectually fighting. And yet, in a recent paper on the way it forms the fibrils constituting the plaque I found the structure compellingly beautiful.

The papers are Proc. Natl. Acad. Sci. vol. 113 pp. 9398 – 9400, E4976 – E4984 ’16. People have been working on the structure of the amyloid fibril of Alzheimer’s for decades, consistently stymied by its insolubility. The authors solved it not by Xray crystallography, not by cryoEM, but by solid state NMR. They basically looked at the distance constraints between pairs of isotopically labeled atoms, and built their model that way. Actually they built a bouquet of models using computer aided energy minimization of the peptide backbone. Another independent study produced nearly the same set.

The root mean square deviation of backbone atoms of the 10 lowest energy models of the bouquets in the two studies was small (.89 and .71 Angstroms). Even better the model bouquets of the two papers resemble each other.

There are two chains of Abeta42, EACH shaped like a double horseshoe (similar to the letter S). The two S’s meet around a twofold axis. The interface between the two S’s is form by two noncontiguous areas on each monomer (#15 – #17) and (#34 – #37).

The hydrophilic amino terminal residues (#1 – #14) are poorly ordered, but amino acids #15 – #42 are arranged into 4 short beta strands (I only see 3 obvious ones) that stack up and down the fibril into parallel in register beta-sheets. Each stack of double horseshoes forms a thread and the two threads twist around each other to form a two stranded protofilament.

Glycines allow sharp turns at the corners of the horseshoes. Hydrogen bonds between amides link the two layers of the fibrils. Asparagine side chains form ladders of hydrogen bonds up and down the fibrils. Water isn’t present between the layers because the beta sheets are so close together (counterintuitively this decreases the entropy, because water molecules don’t have to align themselves just so to solvate the side chains).

Each of the horseshoes is stabilized by hydrophobic interactions among the hydrophobic side chains buried in the core. Charged residues are solvent exposed. The interface between the two horsehoes is a hydrophobic interface.

Many of the famlial mutations are on the outer edges of double S structure — they are K16N, A21G, D23N, E22A, E22K, E22G, E22Q.

The surface hydrophobic patch formed by V40 and A42 may explain the greater rate of secondary nucleation by Abeta42 vs. Abeta40.

The cryoEM structures we have of Abeta42 are different showing the phenomenon of amyloid polymorphism.

The PNAS paper used reombinant Abeta and prepared homogenous fibrils by repeated seeding of dissolved Abeta42 with preformed fibrils. The other study used chemically synthesized Abeta and got fibrils without seeding. Details of pH, peptide concentration, salt concentration differed, and yet the results are the same, making both structures more secure.

The new structure doesn’t immediately suggest the toxic mechanism of Abeta.

To indulge in a bit of teleology — the structure is so beautiful and so intricately designed, that the aBeta42 peptide has probably been evolutionarily optimized to perform an (as yet unknown) function in our bodies. Animals lacking Abeta42’s parent (the amyloid precursor protein) don’t form neuromuscular synapses correctly, but they are viable.

Hillary Clinton’s stroke in 2012

Now that Hillary Clinton is the Democratic Party nominee and the campaign has less than 3 months to go, it is time to republish the post of April 2016 so that people can think it over. I am a retired board certified neurologist and former examiner for the American Board of Psychiatry and Neurology.

First: a timeline.

At some point 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 30 December at which point she was admitted to New York-Presbyterian Hospital when a blood clot was found in a vein draining her brain.

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

Second: The following explanation for these events was given by Lisa Bardach M. D, a board certified internist in a letter released by the Clinton campaign 31 July 2015 (as of 24 August 2016 nothing more has been forthcoming).

You may read the entire letter at 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.”

In my opinion this letter essentially proves that Mrs. Clinton had a stroke.

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.

Fourth: 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 for observation in 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. I suppose it’s possible that the double vision came up right away, and Dr. Bardach was talked into following her at home. Docs can be bent to the whims of the rich and powerful. Witness Michael Jackson talking his doc to giving him Diprivan at home, something that should never be given outside the OR or the ICU due to the need for minute to minute monitoring.

My guess was that the double vision came up later — probably after Christmas. Who gets admitted to the hospital the day before New Year’s Eve? Only those with symptoms requiring immediate attention.

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).

This is not a criticism of Dr. Bardach’s use of anticoagulation, spontaneous clots tend to recur and anticoagulation is standard treatment. 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.

Fifth: Isn’t this really speculation? Yes, of course it is and this is absolutely typical of medical practice where docs do the best they can with the information they have while always wishing for more. The Clinton campaign has chosen to release precious little.

So what information that we don’t currently have would be useful? First Dr. Bardach’s office notes. I’m sure Mrs. Clinton was seen the day she fainted, and subsequently. The notes would tell us when the double vision arose. Second the admission history and physical and discharge summary from NY Pres. Her radiologic studies (not just the reports) — plain skull film, CT (if done), MRI (if done) should be available.

Sixth: why is this important? 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 decisions they made in that state were not happy for the USA or the world.

Seventh (new): I’ve seen the videos of the ‘seizure’ during a press conference. I find them unconvincing and possibly doctored. The idea that Mrs. Clinton suffers from post-traumatic syndrome seems far fetched to me. She wouldn’t be on anticoagulants if all she did was fall and hit her head. Stay tuned. Mrs. Clinton has not had a press conference in 300 days.
Actually, 264 days. Washington Post keeps a counter on this, which is running as you view the following
The debates should be watched closely As Joe Louis (almost) said in another context “[s]he can run but [s]he can’t hide”.

Addendum 11 Sep ’16 — Lest you think that my concern about Mrs. Clinton’s health is something new, or politically driven, have a look at the following post written the last day of 2012. She was but one of 3 politicians I blogged about that day.The initial story about Hillary’s medical problems made no sense to me back then, nor does it now.

The plural of anecdote is NOT data (in medicine at least)

The previous post ( showed that collecting a bunch of small studies (anecdotes) was extremely helpful in seeing the larger picture.

In medicine exactly the opposite occurs. The only way to find out if something works is to do a controlled study. [ Science vol. 297 p 325 ’02 ] There were over 50 observational studies showing benefits for hormone replacement in menopausal women.. Observational studies are basically anecdotes. During the planning study for the Women’s Health Initiative (WHI), some argued that it was unethical to deny some women hormones and give them a placebo. The reason HERS (Heart and Estrogen/Progesterone Replacement Study) was even done was that Wyeth couldn’t get the FDA to approve hormone replacement therapy as a treatment to prevent cardiovascular disease, so they funded HERS to prove their case. Most readers of this have probably read all sorts of bitching about the slowness of the FDA in approving drugs but in this case they did the female populace a huge favor.

As you probably know, the results of hormone replacement in both studies were a disaster (the HERS trial was stopped at 5.2 years after because of increased breast cancer in the treated group). There was also an increased risk of coronary heart disease by 30%, stroke by 41%. At least hip fracture was reduced. Fortunately, even though these were bad outcomes, they were infrequent,(but more frequent in the treated group).

These weren’t lab animals, but someone’s wife and/or mother.

How could they have been so far off? Before all this started, estrogen users were different from nonUsers in several respects — first they were doing something about their health, and clearly had more medical supervision. In addition they were better educated, smoked less and of a higher social class, all of which tend to diminish morbidity and mortality.

Something very similar happened in my field of neurology (not that vascular disease doesn’t severely impact the nervous system). There was a very logical operation to improve cerebral circulation — the pulse just in front of your ear is the superficial temporal artery, a branch of the common carotid after it splits in the internal carotid which goes into the skull and supplies blood to the brain, and the external carotid. If the internal carotid is blocked and the common carotid artery is open, then open the skull and hook (anastomose) the superficial temporal artery to a vessel on the surface of the brain, bypassing the blockage. If you want to know how it is done see —

There was all sorts of anecdotal evidence of miraculous recovery from stroke. The neurosurgeons and vascular surgeons mounted a wonderful controlled study of the surgery even though many thought it was unnecessary — so 1377 patients were prospectively randomized to have the surgery or medical management. The surgery wasn’t better than medical management N Engl J Med 1985; 313:1191-1200November 7, 1985DOI: 10.1056/NEJM198511073131904, so the procedure was abandoned.

What reading the literature is like when things are barely understood

There is a very exciting paper to be described in a post to appear shortly. I ran a muscular dystrophy clinic for 15 years, and saw lots of Amyotrophic Lateral Sclerosis (ALS) — even though, strictly speaking it is not a muscular dystrophy. The muscular Dystrophy Association was founded by parents of weak children, before we could actually separate motor neuron disease from myopathy. In retirement, I’ve kept up an interest in ALS (particularly since all I could do for patients as a doc was — (drumroll) — basically nothing).

The fact that a fair amount of even sporadic ALS has a problem with a protein called C9ORF72 was particularly fascinating. All this came out less than five years ago (October 2011). Everything is far from clearcut even now.

That being the case, it might be of interest to look at the notes I accumulated as scientists began to explore what was wrong with C9ORF72, how the protein normally does whatever it does (we still don’t know really) and how the mutated product of the gene causes trouble (there are 3 main theories).

What you’ll see in what follows is the heat of scientific battle (warts and all), where things are far from clear. Enjoy. This is basically what used to be called a core-dump (back in the day when computer memory was made of metallic cores). Things are far from cut and dried even now so it might be of interest to see the many angles of attack on the problem, the confusion, the conflicting theories, as things became a bit more clear. It’s the scientific enterprise in action against a very horrible disease (trust me).

I’ll try and clear up the typos. I’ll also try to put the notes on the papers in semi-chronological order, but I make no guarantees. The notes may be incomprehensible, as they include only what I didn’t know rather than all the background needed to understand what’s in them .

First a bit of background — FTD stands for FrontoTemporal Dementia.

The #9p21 chromosomal region is another locus for ALS/FTD. It contains something called C9orf72, which contains a GGGGCC hexnucleotide repeat in the intron between noncoding exons 1a and 1b. Normal alleles contain less than 24 repeats (range 2 – 23). Those with ALS + FTD contain over 30 (actually they think the repeat length is much higher — 700 to 1,600 ! ! !). ORF probably stands for open reading frame.

The expansion is present in 12% of familial FTD and 22.5% of familial ALS — making it the most common genetic abnormality in both conditions. More importantly it is found in 21% of sporadic ALS and 29% of FTD in the Finnish population. Later they say it is the most common genetic cause of sporadic ALS (but only in 4%).

There are 3 possible mechanisms of toxicity
l. The RNA transcribed from the repeat acts as an RNA sponge, binding all sorts of RNAs it shouldn’t
2. Repeat Assoaicted Non-ATG translation (RAN translation) see later
3. Decreased expression of the mRNA for C9ORF72.

[ Science vol. 338 pp. 1282 – 1283 ’12 ] Now 40% of familial ALS, 21% of familial frontotemporal dementia, and 8% of sporadic ALS, 5% of sporadic frontotemporal dementia have expansions in C9orf72.

Not much is known about C9orf72 — it is conserved across species. It contains no previously known protein domains. The expansion leads to loss of one alternatively spliced C9ORF72 isoform (normally 3 isoforms are expressed), and to the formation of nuclear RNA foci (which appear to be composed mostly of the expansion). [ Neuron vol. 79 pp. 416 – 438 ’13 ] The function of C9ORF72 is unknown (8/13).

The current (12/12) thinking is that the repeats produce a glob of RNA which traps RNA binding proteins which have better things to do. The best analogy is myotonic dystrophy in which an expanded 3 nucleotide repeat sequesters muscleblind, an RNA binding protein involved in splicing.

The expansion is present in 46% of familial ALS in Finland and 21% of sporadic ALS there. But Finns are somewhat different genetically. The expansion is found in 1/3 of European ancestry familial ALS.

Interestingly some of the patients with FTD presented with nonfluent progressive aphasia.

[ Cell vol. 152 pp. 691 – 698 ’13, Neuron vol. 77 pp. 639 – 646 ’13 ] The protein aggregates of C9orf72 mutants contain TDP43 inclusions. But they also show additional p62 and ubiquilin positive pathology (with no TDP43 present). The abnormal proteins are due to translation of the expanded GGGGCC repeats (which should be nonCoding as they are in introns). This is an example of Repeat Associated Non-ATG translation (RAN). This was first shown for expanded CAG repeats, which can be translated in all 3 reading frames giving polyGlutamine, polyLysine and polySerine . A minimum of 58 CAG repeats was required for translation.

This work looked for translation of GGGGCC in all 3 reading frames (poly glycine-proline, poly glycine-alanine, polyglycine-arginine. They found that poly glycine-proline was found and in the protein inclusions which were p62 positive and TDP43 negative. Similar inclusions weren’t present in other neurodegenerative diseases, known to have nucleotide inclusions.

[ Proc. Natl. Acad. Sci. vol. 110 pp 7533 – 7534, 7778 – 7783 ’13 ] The expanded C9orf72 repeat is enough to cause neurodegeneration (mammalian neurons, and D. melanogaster). They placed either 3 or 30 copies of GGGCC into an epidermal growth factor vector between the start of transcription and the first ATG codon. The repeat can sequester the RNA binding protein Pur alpha (and other Pur family members). Interestingly, TDP43 didn’t bind to the repeat RNA, nor did hnRNP A2/B1 which binds to fragile X CGG repeat containing RNA. Overexpression of of Pur alpha is able to abort the neurogeneration in the mammalian neuonal cell line (Neuro-2a). So probably the excessive repeat number is acting as an RNA sponge.

Pur alpha is evolutionarily conserved. It controlls the cell cycle and differentiation. It is also a pomonent of the RNA transport granule. It interacts with Pur beta.

30 was as many repeats as they could manipulate experimentally — normals have 2 – 8 repeats, but patients with disease have from 100s to 1,000s of repeats, so the pathogenesis might be different.

[ Neuron vol. 80 pp. 257 – 258, 415 – 428 ’13 ] Expression of C9orf72’s mRNA in frontotemporal dementia/als (FTD/ALS) patients is reduced by 50%, and the expanded repeat and neighboring CgP islands are hypermethylated consistent with transcriptional silencing. Also the cytoplasmic aggregates staining positively for P62 appear to result from protein translation through the hexanucleotide repeat.

This work used induced pluripotent stem cells (iPSCs) derived from C9ALS/FTD patients. They show decreased C9orf72 mRNA, nuclear and cytoplasmic GGGGCC RNA foci, and expression of one RAN product (Gly Pro dipeptide). Neurons derived from the iPSCs also show enhanced sensitvity to glutamic acid excitotoxicity, and a transcriptional profile that ‘partially’ overlaps with transcriptional changes seen in iPSC neurons derived from mutant SOD1 ALS patients.

In addition, some 19 proteins were found which associate with the GGGGCC repeats in vitro. ADARB2 does this and participates in RNA editing.

ASOs (AntiSense OIigonucleotides ??) were used to suppress C9orf72 RNA expression. This led to reversal in many of the phenotypes of the iPSC neurons (suppression of glutamic acid toxicity, reduction in RNA foci formation). This implies that the GGGGCC repeats trigger toxicity through a gain of function mechanism. [ Proc. Natl. Acad. Sci. vol. 110 pp. E4530 – E4539 ’13 ] Nuclear RNA foci containing GGGCC in patient cells (wbc’s fibroblasts, glia, neurons) were ssen in patients with repeat expansion. The Foci weren’t present in sporadic ALS or ALS/FTD caused by other mutations (SOD1, TDP43, tau), Parkinsonism, or nonNeurological controls. Antisense oligonucleotides reduced the GGGGCC containing nuclear foci without alteraling overall C9orf72 RNA levels. SiNRAS didn’t work.

The Rx was applied to living mice and it was well tolerated.

[ Proc. Natl. Acad. Sci. vol. 110 pp E4968 – E4977 ’13 ] C9orf72 antisense transcripts are elevated in the brains of those with the expansion. Repeat expansion GGCCCC RNAs accumulate in nuclear foci in the brain. Sense and antisense foci accumulate in the blood and are potential biomarkers. RAN translation occurs in BOTH sense and antisense expansion transcripts — so all 6 proteins described above are made. The proteins accumulate in cytoplasmic aggregates in affected brain regions (e.g. frontal and motor cortex, spinal cord neurons).

[ Nature vol. 507 pp. 175 – 177, 195 – 200 ’14 ] C9orf72 has repeated hexanucleotide units (GGGGCC). Two or more G quartets stacked on top of one another form a G-quadruplex. In the expanded repeats of C9orf72 in ALS and frontotemporal dementia, stable quadruplexes form in DNA as well as the RNA transcribed from it.

Sequences which can form G-quadruplexes are conserved during evolution, so they presumably are doing something useful. They are found in transcriptional start sites. This work shows that G-quadruplex assembly in DNA increases transcriptional pauses in the expanded repeat (unsurprising). Also the G-quadruplexes in C9orf72 DNA promote the formation of stable R-loops — triple stranded structures that assemble when a newly form RNA transcript exiting RNA polymerase II invades the double helix and binds to one DNA strand, displacing the other. If the R-loops aren’t resolved, they can halt transcriptional elongation.

Not only that, but abortive GGGGCC containing RNAs accumulate in the spinal cord and motor cortex of patients with the expanded repeats. The RNAs are truncated in the GGGGCC region, and the amount is linearly proportional to the length of the hexanucleotide repeat. This explains how they could accumulate along with decreased level of full length C9orf72 mRNA (and presumably the protein made from it).

A ‘few dozen’ proteins binding the GGGGCC repeats have been found. One of them is nucleolin, involved in the formation of the ribosome within the nucleolus It is mislocalized to RNA foci in neurons of the motor cortex of patients with C9orf72 related disease. The lack of mature ribosomes results in the buildup of untranslated mRNA in the cytoplasm.

[ Science vol. 345 pp. 1118 – 1119, 1139 – 1145, 1192 – 1194 ’14 ] Normally the number of GGGGCC repeats in C9orf72 ranges from 2 to 23, with hundreds or even thousands of copies in the disease range. Possibilities
l. Interference with C9orf72 expression — e. g. loss of function
2. Sponging up RNA binding proteins by the transcript
3. Repeat associated non-ATG translation (RAN translation) in all reading frames (sense and antisense).

A series of stop codons in both the sense and antisense RNAs was engineered every 12 repeats, stopping formation of the dipeptide repeat proteins. The new RNAs still formed the G-quadruplexes, and both RNAs formed RNA foci when expressed in cultured neurons.

Putting them into Drosophila showed that the pure repeats able to form dipeptides causing degeneration in the fly eye, while the interrupted constructs (producing RNA only) did not. The same was true when expressed in the nervous systems of adult flies. Blocking translation of the RNA partially suppressed the phenotype.

There are 5 possible dipeptide products of RAN of GGGGCC (GA, GP, PA, GR, PR — G == Glycine, P == Proline, A == Alanine, R = Arginine). Then RNAs using alternate codons for the dipeptides were used (so GGGGCC wasn’t present). Expressing Glycine Arginine (GR) or Proline Arginine (PR) was toxic, Glycine Alanine showing ‘some’ toxicity later in life.

Some RNA binding proteins containing low complexity sequences (aka prion-like domains) — these are FUS, EWSR1, TAF14, hnRNPA2 — form polymeric assemblies, which incorporate into hydrogels in vitro. The assemblies are similar to RNA granules. Many of the RNA binding proteins associating with hydrogels hare serine arginine (SR) sequences. The SR domain proteins are regulated by phosphorylation on serine, also controlling the association with hydrogels. It is hypothesized that the GR and PR transcripts associate with hydrogels (or similar assemblies such as RNA granules), but are impervious to the regulatory action of the kinases (no serine to phosphorylate), so they might clog up the trafficking of SR domain containing RNA binding proteins moving in an out of the granules to transfer information throughout the cell.

[ Neuron vol 84 pp. 1213 – 1225 ’14 ] Proline Arginine dipeptides are neurotoxic. They form aggregates in nucleoli in experimental systems. Nuclear aggregates were also found in postmortem spinal cord from C9ORF72 ALS and ALS/FTD patients. Intronic GGGGCC transcripts are also toxic. Repeat associated non-ATG translation (RAN translation) is thought to depend on RNA hairpin structures using GC pairing.

[ Cell vol. 158 pp. 967 ’14 (abstract of something to appear in Science) ] Peptide translated from GGGGCC expansions containng arginines (Gly Arg and Pro Arg) are harmful — 3 other dipeptide repeats are harmless. The peptides bind to nucleoi and impede RNA biogenesis. Interestingly Ser-Arg repeats proteins (SR proteins) are important in RNA splicing. The GlyARG and PROARG repeat peptides alter splicing of the amino acid transporter EAAT2, similar to that seen in ALS. Interestingly, the peptides are readily taken up by cells in culture, translocating to the nucleus.

Also a small molecule has been developed which targets GGGGCC RNA expansions. It inhibits translation of the dipeptide repeat proteins from the expansions (see Science vol. 353 pp. 64 ****

GlyPro in CSF is a biomarker of ALS patients with the C9orf7s expansion.

The normal function of C9orf72 isn’t known. It is structurally related to DENN (Differentially Expressed in Normal and Neoplastic cells) proteins, which are GDP/GTP exchange factors for Rab GTPases.

At this point it isn’t known if the proteins generated by RAN are toxic. The protein inclusions are present in unaffected areas of the brain (lateral geniculate) as well as the vulnerable areas (cortex, hippocampus).

The initiation of RNA translation is thought to depend on RNA hairpin structures which use C:G complementary pairing. CAG (but not CAA) repeats undergo RAN translation. Protein aggregates occured only in brain intestes despite the fact that C9orf72 is expressed all over the body (but expression is highest in brain).

It is possible that antisense RNA could be formed from the opposite strand (e.g. CCCCGG) giving poly pro-ala, poly pro-gly and poly pro-arg.

[ Science vol. 1106 – 1112 ’15 ] Just expressing 66 GGGGCC repeats without an ATG start codon using an AdenoAssociated Virus (AAV) vector in mice was enough to produce neurodegeneration with RNA foci, inclusoins of poly QP, GA and GR and TDP43 pathology. There was cortical neuron and cerebellar Purkinje cell loss and gliosis.

[ Nature vol. 525 pp. 36 – 37, 56 – 61, 129 – 133 ’15 ] (GGGGCC)30 was expressed in the Drosophila eye. This leads to the rough eye trait and is easily scored, allowing you to look at the effect of other genes on it. Mutations activating RanGAP suppressed rough eyes. RanGAP binds to GGGGCC on the cytoplasmic face of the nuclear pore. Enhancing nuclear import or suppressing nuclear export of proteins also suppressed neurodegeneration. RanGAP physically interacts with the GGGGCC Hexanucleotide Repeat Expansion resulting in its mislocalization. The mislocalization is found in neurons derived from iPSCs from a patient with C9orf72 type ALS, and also in brain tissue from other patients with C9orf72 ALS.

Nuclear import is impaired due to HRE expression (fly and iPSC derived neurons). The defects can be ‘rescued’ by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. This may actually be a way to Rx ALS ! ! ! !

Another paper crossed (GGGGCC)58 flies with missing chromosomal segments. They found a variety of nuclear import factors whose inactivation worsened rough eye.

Expression of constructs of in GGGGCC)8, 28 and 58 lacking an AUG start codon in Drosophila was done. The constructs could only produce Repeat Associated NonAUG translation products (e.g. dipeptides). The dipeptides disrupt nuclear import of fluorescent test substrates and of normal nuclear proteins (notably TDP43). In addition RNA export from the nucleus is also compromised. The deleterious effects could be modified by 18 genetic regions (found by large scale unbiased genetic screening). THey coded for components of the nuclear pore complex, nuclear RNA export machinery and nuclear import.

Dipeptides produced from GGGGCC and GGGGCCn’s disrupt the nucleolus, so this may be an additional cause of repeat toxicity.

[ Neuron vol. 88 pp. 892 – 901 ’15 ] A mouse model containng the full human C9orf72 repeat which was either normal (15 repeats) or expanded (100 – 1,000 repeats) — using bacterial artificial chromosomes (BACs) — thes mice are called C9-BACexpanded. They show widespread RNA foci and RAN translated dipeptides. Nucleolin distribution was altered. However the mice showed normal behavior and there was no neurodegenration. This is surprising.

[ Nature vol. 535 p. 327’16 (abstr. of Sci. Transl. Med ’16) ] Mice with mutations diminishing or eliminating the function of C9ORF72 (unknown as of 8/13) developed autoimmune disease.

[ Science vol. 351 pp. 1324 – 1329 ’16 ] Two independent mouse lines lacking the ortholog of C9orf72 (3110043021Rik) in all tissues developed normally and aged without any motor neuron disease. Instead they developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophagelike cells. There was age related neuroInflammation similar to C9orf72 ALS but not sporadic ALS. There was no evidence of neurodegeneration however.

[ Neuron vol. 90 pp. 427 – 430, 531 -534, 535 – 550 ’16 ] BAC transgenic mice using patient derived gene constructs expressing (some of? all of?) C9ORF72 are reported.

A germline knockout develops blood abnormalities (splenomegaly, lymphadenopathy and premature death). The data conflict on which of the 5 products of RAN (Repeat Associated NonATG) translation are the most toxic (GP, GA, GR, PA, PA, PR).

In this study, mice with increased levels of repeats (up to 450) showed no evidence of motor neuron disease, and the brain was normal. They at least did have some trouble with cognition.

THe second study put in the full C9 gene with 5′ and 3′ flanking sequences. 4 lines of transgenics with repeats ranging from 37 to 500 were characterized. These mice did have peirpheral and central neurodegeneration, with motor deficits. There was a decrease in cortical neurons, Purkinje cells. This is the first time any transgenic has shown neurodegeneration. The deficits are reversible with antisense oligonucleotides. There was a disparity in disease expression between male and female mice.

RNA foci and DPR (DiPeptide Repeat) proteins don’t accumulate in the most affected brain regions.

[ Science vol. 353 pp. 647 – 648, 708 – 712 ’16 ] Spt4 is a highly conserved transcription elongation factor which regulates RNA polymerase II processivity (along with its binding partner Spt5). Spt4 is required to transcribe long trinucleotide repeats found in open reading frames, or in non protein coding regions of DNA templates (in S. cerevisiae). Mutations of Spt4 decrease synthesis of (and restored enzymatic activity to) expanded polyQ proteins (in yeast) without affecting genes lacking the excessive CAG repeats. It might also work in nonCAG repeats.

Targeting Spt4 (with antiSense oligonucleotides) reduces production of the C9orf72 expansion associated RNA and protein, and helps neurodegeneration in model systems. Repeat expansions are transcribed in both the sense and antisense directions. Yeast Spt4 (human homolog SUPT4H) is a small evolutionarily conserved zinc finger protein which forms a complex with Spt5, which then binds to RNA polymerase II regulating transcription elongation (pol II processivity).

DRB is a RNA polymerase II inhibitor. The complex of Spt4 and Spt5 homologs in man (SUPT4H, SUPT5H) is called DSIF (DRB Sensitivity Inducing Factor)

Depletion of Spt4 or its binding partner (Spt5 ) decreases the number of both sense and antisense repeat transcripts and RNA foci. One of the 6 RAN translation products (polyGlyPro) is substantially reduced by Spt4 depletion.

The study was in human c9ALS fibroblasts. However, side effects are certainly possible — in addition to decreasing the expression of C9ORF72, 95% depletion of SUPT4H1 altered (how?) the expression of another 300 genes. In mice deletion of both copies of SUPT4 is embryonic lethal, but deleting one produced no effects up to 18 months of age.