Category Archives: Neurology & Psychiatry

A research idea yours for the taking

Why would the gene for a protein contain a part which could form amyloid (the major component of the senile plaque of Alzheimer’s disease) and another part to prevent its formation. Therein lies a research idea, requiring no grant money, and free for you to pursue since I’ll be 80 this month and have no academic affiliation.

Bri2 (aka Integral TransMembrane protein 2B — ITM2B) is such a protein.  It is described in [ Proc. Natl. Acad. Sci. vol. 115 pp. E2752 – E2761 ’18 ] http://www.pnas.org/content/pnas/115/12/E2752.full.pdf.

As a former neurologist I was interested in the paper because two different mutations in the stop codon for Bri2 cause 2 familial forms of Alzheimer’s disease  Familial British Dementia (FBD) and Familial Danish Dementia (FDD).   So the mutated protein is longer at the carboxy terminal end.  And it is the extra amino acids which form the amyloid.

Lots of our proteins form amyloid when mutated, mutations in transthyretin cause familial amyloidotic polyneuropathy.  Amylin (Islet Amyloid Polypeptide — IAPP) is one of the most proficient amyloid formers.  Yet amylin is a protein found in the beta cell of the pancreas which releases insulin (actually in the same secretory granule containing insulin).

This is where Bri2 is thought to come in. It is also found in the pancreas.   Bri2 contains a 100 amino acid motif called BRICHOS  in its 266 amino acids which acts as a chaperone to prevent IAPP from forming amyloid (as it does in the pancreas of 90% of type II diabetics).

Even more interesting is the fact that the BRICHOS domain is found in 300 human genes, grouped into 12 distinct protein families.

Do these proteins also have segments which can form amyloid?  Are they like the amyloid in Bri2, in segments of the gene which can only be expressed if a stop codon is read through.  Nothing in the cell is perfect and how often readthrough occurs at stop codons isn’t known completely, but work is being done — Nucleic Acids Res. 2014 Aug 18; 42(14): 8928–8938.

I find it remarkable that the cause and the cure of a disease is found in the same protein.

Here’s the research proposal for you.  Look at the other 300 human genes containing the BRICHOS motif (itself just a beta sheet with alpha helices on either side) and see how many have sequences which can form amyloid.  There should be programs which predict the likelihood of an amino acid sequence forming amyloid.

It’s very hard to avoid teleology when thinking about cellular biochemistry and physiology.  It’s back to Aristotle where everything has a purpose and a design.  Clearly BRICHOS is being used for something or evolution/nature/natural selection/the creator would have long ago gotten rid of it.  Things that aren’t used tend to disappear in evolutionary time — witness the blind fish living in caves in Mexico that have essentially lost their eyes. The BRICHOS domain clearly hasn’t disappeared being present in over 1% of our proteins.

Suppose that many of the BRICHOS containing proteins have potential amyloid segments.  That would imply (to me at least) that the amyloid isn’t just junk that causes disease, but something with a cellular function. Finding out just what the function is would occupy several research groups for a long time.   This is also where you come in.  It may not pan out, but pathbreaking research is always a gamble when it isn’t stamp collecting.

 

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Amyloid again, again . . .

Big pharma has spent (and lost) several fortunes trying to attack the amyloid deposits of Alzheimer’s.  But like my late med school classmate’s book — “Why God Won’t Go Away” ==https://www.amazon.com/Why-God-Wont-Go-Away/dp/034544034X, amyloid won’t go away either.   It’s a bit oblique but some 300 of our proteins contain a 100 amino acid stretch called BRICHOS.  Why? Because it acts as a chaperone protein preventing proteins with a tendency to form amyloid from aggregating into fibrils.   The amino acids form a beta sheet surrounded front and back by a single alpha helix.

[ Proc. Natl. Acad. Sci. vol. 115 pp. E2752 – E2761 ’18 ] Discusses Bri2 (aka Integral Transmembrane protein 2B (ITM2B), a 266 amino acid type II transmembrane protein. Bri2 contains a carboxy terminal domain Bri23 released by proteolytic processing between amino acids #243 #244 by furinlike proteases. Different missense mutations at the stop codon of Bri2 cause extended carboxy terminal peptides called  Abri or Adan to be released by the proteases. Abri produces Familial British Dementia (FBD) and Adan produces Familial Danish Dementia (FDD). Both are associated with amyloid deposition in blood vessels, and amyloid plaques throughout the brain along with neurofibrillary tangles.

What is fascinating (to me) is that the cause and cure are both present in the same molecule Bri2 also contains a BRICHOS domain.  This implies (to me) that possibly the segment possibly forming amyloid is being used by the cell in some other fashion.

Bri2 is found in the beta cell of the pancreas (produces insulin).  The beta cell also produces Islet Amyloid PolyPeptide (IAPP  aka amylin ) one of the most potent amyloid forming proteins known.  Nonetheless the pancreas makes tons of it, and like insulin, is secreted by the beta cell in response to elevated blood glucose.  The present work shows that Bri2 is what keeps IAPP from forming amyloid.  The BRICHOS segment (amino acids #130 – #231) is released from Bri2 by ADAM10 (you don’t want to know what the acronym stands for).

How many of the 300 or so human proteins containing the BRICHOS domain also have amyloid forming segments.  If they do, this implies that the amyloid forming segments are doing something physiologically useful.

 

 

Stephen Hawking R. I. P.

Stephen Hawking, brilliant mathematician and physicist has died.  Forget all that. He did something for my patients with motor neuron disease that I, as a neurologist, could not do.  He gave them hope.

What has chemistry done for them?  Quite a bit, but there’s so much left.

Chemistry, when successful, just becomes part of the wallpaper and ignored. All genome sequencing depends on what some chemist did.

For one spectacular example of what, without chemistry, would be impossible is Infantile Spinal Muscular Atrophy (Werdnig Hoffmann disease).  For the actual molecular biology behind it — please see — https://luysii.wordpress.com/2016/12/25/tidings-of-great-joy/.   Knowing the cause has led to not one but two specific therapies — an antisense oligonucleotide and a virus which infects neurons and actually changes the gene.

So knowing what the cause of a disease is should lead to a treatment, shouldn’t it?  Hold that thought.  Sometimes one form of motor neuron disease (amyotrophic lateral sclerosis or ALS) can be hereditary.  Find out what is being inherited to find how ALS is caused.

Well, the first protein in which a mutation is associated with familial ALS (FALS) was found exactly 25 years ago.  It is called superoxide dismutase (SOD1).  Over 150 mutations have been found in the protein associated with FALS, and yet despite literally thousands of papers on the subject we don’t know if the mutations cause a loss of function, a gain of function (and if so what that function is), an increased tendency to fold incorrectly, and on and on and on.  It’s a fascinating puzzle for the protein chemist and over the years my notes on the papers I’ve read about SOD1 have ballooned to some 25,000 words.

If you’re tired of working on SOD1, try a few of the other proteins in which mutations have been associated with FALS — Alsin, TAF15, Ubiquilin, Optineurin, TBK1 etc. etc.  The list is long.

Now it’s biology’s turn.  Motor neurons go from the spinal cord (mostly) and brain to produce muscle contraction.  Why should only this tiny (but crucial) minority of cells be affected.  The nerve fibers leave the spinal cord and travel to muscle in nerves which contain sensory nerve fibers making the same long trip, yet somehow these nerves are spared.

More than that, why should these mutations affect only these neurons, and that often after decades.  Also why should great athletes (Lou Gehrig, Ezzard Charles, etc. etc. ) get the disease.

One closing point.  Hawking shows why, in any disease median survival (when 50% of those afflicted die) is much a more meaningful statistic than average duration of survival.  Although he gave my patients great hope, they all died within a few years even as he mightily extended average survival.

 

Hillary Clinton’s latest health event

On a recent trip to India Hillary clearly had difficulty placing her left foot and nearly fell down a set of stairs twice.  You can watch the video on the following website http://dailycaller.com/2018/03/12/hillary-falls-down-stairs-india/.  Please ignore all the snarkiness of the commentary and just look at the video over and over.  She comes out of an old building and starts going down some worn stone steps linking her left arm into that of a large man.  Stop the video when she begins to fall and notice how she placed her left foot.  Fortunately you can go back and forth as many times as you wish.  It clearly wasn’t where it should have been. The same thing happened with her second near fall.  Then watch the way she places her left leg as she walks to the car.  It’s as though she doesn’t really know where it is.

This all fits with my opinion that she suffered a stroke in December of 2012.  The press bought what I thought was a rather hokey explanation that it was traumatic in origin.  At any rate we do know that she had a blood clot in a vein and had double vision lasting for several weeks.  You can read the reasoning behind this here — https://luysii.wordpress.com/2012/12/31/medical-tribulations-of-politicians-degrees-of-transparency/

Then during the campaign in 2016 at an event to commemorate 9/11 she fainted.  The press cast this as a stumble, but I don’t think it was. Once again you have a video of the event with a link to it in a post about the event — https://luysii.wordpress.com/2016/09/13/hillarys-fainting-spell/.  As Richard Pryor famously said when his wife caught him with another woman. He denies anything is going on, and asks his wife, “Who you gonna believe, me or your lying eyes?”

So what does this retired neurologist and former board examiner think is going on?  Given the little released about her health there are many possibilities.  Statistically people who have had one stroke have around a 6% chance of another one in a given year (each and every year).  Given the way she didn’t seem to know where her left foot was, a stroke in the right parietal region is a possibility.

It is clear that the original area of neurologic deficit in 2012 – 13 was in the brainstem, as it affect the nerves to her eyes.  This is an area intimately involved in coordination, but (fortunately) not in thinking.  So she may have suffered a further stroke in this area.  We don’t know if she’s still taking a blood thinner.

She did look pretty frail, and it’s fortunate for her health that she doesn’t have the stresses of the presidency to deal with.

Addendum 14 March: Apparently she tripped/fell/passed out while on a tour in England breaking a toe 6 months ago http://www.foxnews.com/politics/2017/10/16/hillary-clinton-book-tour-stumbles-after-ex-candidate-falls-and-hurts-foot.html

You don’t have to go to medical school or take a neurology residency to know that a 70 year old woman with 4 neurological events in the past 5 years and 3 months is not in good shape.

Addendum 15 March: Unfortunately she’s had another fall, resulting in a fractured wrist since the episode on the stairs. Here’s the report — https://timesofindia.indiatimes.com/india/hillary-clinton-injured-during-rajasthan-visit/articleshow/63290246.cms

It all adds up to a significant neurological problem with balance.

The death of the pure percept — otoacoustic division

Rooming with 2 philosophy majors warps the mind even if it was 60 years ago.  Conundrums raised back then still hang around.  It was the heyday of Bertrand Russell before he became a crank.  One idea being bandied about back then was the ‘pure percept’ — a sensation produced by the periphery  before the brain got to mucking about with it.   My memory about the concept was a bit foggy so who better to ask than two philosophers I knew.

The first was my nephew, a Rhodes in philosophy, now an attorney with a Yale degree.  I got this back when I asked —

I would be delighted to be able to tell you that my two bachelors’ degrees in philosophy — from the leading faculties on either side of the Atlantic — leave me more than prepared to answer your question. Unfortunately, it would appear I wasn’t that diligent. I focused on moral and political philosophy, and although the idea of a “pure precept” rings a bell, I can’t claim to have a concrete grasp on what that phrase means, much less a commanding one.

 Just shows what a Yale degree does to the mind.

So I asked a classmate, now an emeritus prof. of philosophy and got this back
This pp nonsense was concocted because Empiricists [Es]–inc. Russell, in his more empiricistic moods–believed that the existence of pp was a necessary condition for empirical knowledge. /Why? –>
1. From Plato to Descartes, philosophers often held that genuine Knowledge [K] requires beliefs that are “indubitable” [=beyond any possible doubt]; that is, a belief counts as K only if it [or at least its ultimate source] is beyond doubt. If there were no such indubitable source for belief, skepticism would win: no genuine K, because no beliefs are beyond doubt. “Pure percepts” were supposed to provide the indubitable source for empirical K.
2. Empirical K must originate in sensory data [=percepts] that can’t be wrong, because they simply copy external reality w/o any cognitive “shopping” [as in Photoshop]. In order to avoid any possible ‘error’, percepts must be pure in that they involve no interpretation [= error-prone cognitive manipulation].
{Those Es who contend  that all K derives from our senses tend to ignore mathematical and other allegedly a priori K, which does not “copy” the sensible world.} In sum, pp are sensory data prior to [=unmediated by] any cognitive processing.

So it seems as though the concept is no longer taken seriously.

I’ve written about this before — as it applies to the retina — https://luysii.wordpress.com/2013/02/11/retinal-physiology-and-the-demise-of-the-pure-percept/

This time it involves the ear and eye movements.  Time for some anatomy.  Behind the eardrum are 3 tiny little bones (malleus, incus and stapes — the latter looking just like a stirrup with the foot plate pressed against an opening in the bone to communicate movement of the eardrum produced by sound waves to the delicate mechanisms of the inner ear).  There is a a tiny muscle just 1 millimeter long called the stapedius which stabilizes the stapes making it vibrate less protecting the inner ear against loud sounds.  There is another muscle called the tensor tympani which tenses the eardrum meaning that external sounds vibrate it less.  It protects us against loud sounds.

An article in PNAS (vol. 115 pp. 1309 – E1318 ’18) shows that just moving your eyes to a target causes the eardrum to oscillate.  Even more interesting, the eardrum movements occur 10 milliSeconds before you move your eye.  The oscillations last throughout the eye movement and will into subsequent periods of steady fixation.

It is well recognized in addition to the brain receiving nerve input from the inner ear, it sends nerves to the inner ear to control it.  So ‘the brain’ is controlling the sense organs proving input to it.  Of course the whole question of control in a situation with feedback is up in the air — see https://luysii.wordpress.com/2011/11/20/life-may-not-be-like-a-well-but-control-of-events-in-the-cell-is-like-a-box-spring-mattress/

As soon as feedback (or simultaneous influence) enters the picture it becomes like the three body problem in physics, where 3 objects influence each other’s motion at the same time by the gravitational force. As John Gribbin (former science writer at Natureand now prolific author) said in his book ‘Deep Simplicity’, “It’s important to appreciate, though, that the lack of solutions to the three-body problem is not caused by our human deficiencies as mathematicians; it is built into the laws of mathematics.” As John Gribbin (former science writer at Natureand now prolific author) said in his book ‘Deep Simplicity’, “It’s important to appreciate, though, that the lack of solutions to the three-body problem is not caused by our human deficiencies as mathematicians; it is built into the laws of mathematics.” The physics problem is actually much easier than the brain because we know the exact strength and form of the gravitational force. We aren’t even close to this for a single synapse.

So much work, so little progress

Two years ago, I found going to a memorial service for a friend and classmate who died of Alzheimer’s curiously uplifting  (see the link at the end). The disease is far from ignored. A monster review in Neuron vol. 97 pp. 32 – 58 ’18 — http://www.cell.com/neuron/fulltext/S0896-6273(17)31081-4  contained references to over 400 research articles half of them published since January 2013.

Still I found it quite depressing.  Tons of work and tons findings, and yet no coherent path to the cause (or causes); something absolutely necessary for a rational treatment, unless we somehow stumble into a therapy.

In a way it’s like cancer.  The cancer genome atlas intensively studied the genome of various cancers, looking for ‘the’ or ‘the set of’ causative mutations.  They found way too much.  The average colon and breast cancer had an average of 93 mutated genes, of which 11 were thought to be cancer promoting.  Not only that, but the same 11 were not consistent from tumor to tumor.

So it is with this epic review.  Which of the myriad findings described are causative of the disease and which are responses of the nervous system to the ’cause’ (or causes).

In the review the authors posit that Alzheimer’s disease is due to failure of ‘homeostatic systems’ that maintain a ‘set point’ of neuronal firing.  Unfortunately what is measured to determine the set point isn’t known. This seems to be an example of redefining a question into an answer.  Clearly if you juice up neuronal firing rates by stimulation they come back down, or if you inhibit them, they come back up.  So you can operationally define set point without defining it mechanistically.   It must be due to some sort of feedback on whatever it is that is sensing ‘the set point’ , but what is it that is being sensed?

The following is from an earlier post but is quite relevant to homeostasis and set points.

The whole notion of control in the presence of feedback is far from clear cut.  Here’s the story of the first inklings of feedback in endocrinology.  I watched it happen.

Endocrinology was pretty simple in med school back in the 60s. All the target endocrine glands (ovary, adrenal, thyroid, etc.) were controlled by the pituitary; a gland about the size of a marble sitting an inch or so directly behind the bridge of your nose. The pituitary released a variety of hormones into the blood (one or more for each target gland) telling the target glands to secrete, and secrete they did. That’s why the pituitary was called the master gland back then.  The master gland ruled.

Things became a bit more complicated when it was found that a small (4 grams or so out of 1500) part of the brain called the hypothalamus sitting just above the pituitary was really in control, telling the pituitary what and when to secrete. Subsequently it was found that the hormones secreted by the target glands (thyroid, ovary, etc.) were getting into the hypothalamus and altering its effects on the pituitary. Estrogen is one example. Any notion of simple control vanished into an ambiguous miasma of setpoints, influences and equilibria. Goodbye linearity and simple notions of causation.

As soon as feedback (or simultaneous influence) enters the picture it becomes like the three body problem in physics, where 3 objects influence each other’s motion at the same time by the gravitational force. As John Gribbin (former science writer at Natureand now prolific author) said in his book ‘Deep Simplicity’, “It’s important to appreciate, though, that the lack of solutions to the three-body problem is not caused by our human deficiencies as mathematicians; it is built into the laws of mathematics.” The physics problem is actually much easier than endocrinology, because we know the exact strength and form of the gravitational force.

https://luysii.wordpress.com/2016/01/05/an-uplifting-way-to-start-the-new-year/

Why drug development is hard #31: retroviruses at the synapse

What if I told you that a very important neuronal synaptic protein Arc (Arg3.1) is acting like like a virus, sending copies of itself (and its messenger RNA) across the synapse?  Would a team of shrinks, who’ve never examined me, tell you that I was crazy and unfit to blog?  Well there is very good evidence that exactly this occurs in one situation and probably many more [ Cell vol. 172 pp. 8 – 10, 262 – 274, 275 – 288 ’18] — http://www.cell.com/cell/fulltext/S0092-8674(17)31509-X.

Arc stands for Activity Regulated Cytoskeleton associated protein.  It’s messenger RNA (mRNA) is transcribed from the gene in response to neuronal activity.  More importantly, the mRNA for  Arc is rapidly distributed to active synapses through the cell body and dendrites, where it is translated into protein. It is locally and rapidly stimulated during the induction of long term depression and plays a critical role in removing a class of glutamic acid receptors (AMPA receptors) from the synapse.  To whet the interest of drug developers, Arc regulates the activity dependent cleavage of the Amyloid Precursor Protein (APP) and beta amyloid production by its interaction with presenilin

Several posts could easily be filled with what Arc does, but that’s not what is so amazing about these papers.  Parts of the Arc protein arose from one of the many transcriptionally dead retroviruses found in our genome.  Our species literally wouldn’t exist without other retroviral gifts.  For instance syncytin1 is a protein expressed a high levels in the placenta.  It is produced from the envelope gene of an endogenous retrovirus (HERV-W) which has undergon inactivating mutations in its other major genes (gag and pol).  Mutant mice in which the gene has been knocked out die in utero due to failure of placenta formation.

Part of the arc gene arose from the Gag gene (Group specific antigen gene) of a retrovirus.  Recall most viruses have proteins coating their genetic material when they’re on the move (e. g. a capsid).  In the case of retroviruses, the genetic material is RNA rather than DNA.  Well the gag elements of the Arc protein form a capsid containing the mRNA for Arc (just like a virus).  In some way or other the capsid containing mRNA gets outside the neuron at the nerve muscle junction and gets into muscle.  The evidence is good that this happens, but in a system somewhat removed from us — the fruitfly (Drosophila).  Fruitfly neuromuscular junctions lacking this mechanism are weaker.

Well that’s pretty far from us.  However one of the papers (275 – 288) showed that the Arc protein and its mRNA was found in extracellular vesicles released from mouse neurons cultured from their cerebral cortex.  Could viral-like particles be crossing the synapses in our brains (which are already pretty chockfull of stuff — see https://luysii.wordpress.com/2017/11/15/the-bouillabaisse-of-the-synaptic-cleft/).  It’s very early times (in fact the Cell issue came out 3 days ago) but people are sure to look.  There are at least 100 Gag derived genes in the human genome (Campillos, M., Doerks, T., Shah, P.K., and Bork, P. (2006). Computational characterization of multiple Gag-like human proteins. Trends Genet. 22, 585–589.).

Remarkable.  Remember CRISPR was hiding in plain sight for half a century.  We have a lot to learn.  No wonder drugs have unexpected side effects.

Are the inclusions found in neurologic disease attempts at defense rather then the cause?

Thinking about pathologic changes in neurologic disease has been simplistic in the extreme.  Intially both senile plaques and neurofibrillary tangles were assumed to be causative for Alzheimer’s.  However there are 3 possible explanations for any microscopic change seen in any disease.  The first is that they are causative (the initial assumption).  The second is that they are a pile of spent bullets, which the neuron uses to defend itself against the real killer.  The third is they are tombstones, the final emanations of a dying cell.

A fascinating recent paper [ Neuron vol. 97 pp. 3 – 4, 108 – 124 ’18 ] http://www.cell.com/neuron/pdf/S0896-6273(17)31089-9.pdf gives strong evidence that some inclusions can be defensive rather than toxic.  It contains the following;

“In these studies, we found that formation of large inclusions was correlated with protection from a-synuclein toxicity”

The paper is likely to be a landmark because it ties two neurologic diseases (Parkinsonism and Alzheimer’s) together by showing that they may due to toxicity produced by single mechanism — inhibition of mitochondrial function.

Basically, the paper says that overproduction of alpha synuclein (the major component of the Lewy body inclusion of Parkinsonism) and tau (the major component of the neurofibrillary tangle of Alzheimer’s disease) produce death and destruction by interfering with mitochondria.  The mechanism is mislocalization of a protein called Drp1 which is important in mitochondrial function (it’s required for mitochondrial fission).

Actin isn’t just found in muscle, but is part of the cytoskeleton of every cell.  Alpha-synuclein is held to alter actin dynamics by binding to another protein called spectrin (which also binds to actin).  The net effect is to mislocalize Drp1 so it doesn’t bind to mitochondria where it is needed.  It isn’t clear to me from reading the paper, just where the Drp1 actually goes.

In any event overexpressing spectrin causes the alpha-synuclein to bind to it forming inclusions and protecting the cells.

There is a similar mechanism proposed for tau, and co-expressing alpha synuclein with Tau significantly enhances the toxicity of both models of tau toxicity which implies that they work by a common mechanism.

Grains of salt are required because the organism used for the model is the humble fruitfly (Drosophila).

Not a great way to end 2017

Not a great way to end 2017

2017 ended with a rejection of the following letter to PNAS.

As a clinical neurologist with a long standing interest in muscular dystrophy(1), I was referred many patients who turned out to have chronic fatigue syndrome (CFS) . Medicine, then and now, has no effective treatment for CFS.

A paper (2) cited In an excellent review of cellular senescence (3) was able to correlate an intracellular marker of senescence (p16^INK4a) with the degree of fatigue experienced by patients undergoing chemotherapy for breast cancer. Chemotherapy induces cellular senescence, and the fatigue was thought to come from the various cytokines secreted by senescent cells (Senescence Associated Secretory Phenotype—SASP) It seems logical to me to test CFS patients for p16^INK4a (4).
I suggested this to the senior author; however, he was nominated as head of the National Cancer Institute just 9 days later. There the matter rested until the paper of Montoya et al. (5) appeared in July. I looked up the 74 individual elements of the SASP and found that 9 were among the 17 cytokines whose levels correlated with the degree of fatigue in CFS. However, this is not statistically significant as Montoya looked at 51 cytokines altogether.

In October, an article(6) on the possibility of killing senescent cells to prevent aging contained a statement that Judith Campisi’s group (which has done much of the work on SASP) had identified “hundreds of proteins involved in SASPs”. (These results have not yet been published.) It is certainly possible that many more of Montoya’s 17 cytokines are among them.

If this is the case, a rational therapy for CFS is immediately apparent; namely, the senolytics, a class of drugs which kills senescent cells. A few senolytics are currently available clinically and many more are under development as a way to attack the aging process (6).

If Montoya still has cells from the patients in the study, measuring p16^INK4a could prove or disprove the idea. However, any oncology service could do the test. If the idea proves correct, then there would be a way to treat the debilitating fatigue of both chemotherapy and CFS—not to mention the many more medical conditions in which severe fatigue is found.
Chemotherapy is a systemic process, producing senescent cells everywhere, which is why DeMaria (2) was able to use circulating blood cells to measure p16^INK4a. It is possible that the senescent cells producing SASP in CFS are confined to one tissue; in which case testing blood for p16^INK4a would fail. (That would be similar to pheochromocytoma cells, in which a few localized cells produce major systemic effects.)

Although senolytics might provide symptomatic treatment (something worthwhile having since medicine presently has nothing for the CFS patient), we’d still be in the dark about what initially caused the cells to become senescent. But this would be research well worth pursuing.

Anyone intrigued by the idea should feel free to go ahead and test it. I am a retired neurologist with no academic affiliation, lacking the means to test it.
References

1 Robinson, L (1979) Split genes and musclar dystrophy. Muscle Nerve 2: 458 – 464

2. He S, Sharpless N (2017) Senescence in Health and Disease. Cell 170: 1000 – 1011

3. Demaria M, et al. (2014) Cellular senescence promotes adverse effects of chemotherapy and cancer relapse. Cancer Discov. 7: 165 – 176

4. https://luysii.wordpress.com/2017/09/04/is-the-era-of-precision-medicine-for-chronic-fatigue-syndrome-at-hand/

5. Montoya JG, et al., (2017) Cytokine signature associated with disease severity in chronic fatigue syndrome patients, Proc Natl Acad Sci USA 114: E7150-E7158

6. Scudellari M, (2017) To stay young, kill zombie cells Nature 551: 448 – 450

The flying Wallendas of the synapse

Is anything similar to the flying Wallendas ( https://en.wikipedia.org/wiki/The_Flying_Wallendas) going on in the synapse? The first electron micrographs of the synaptic cleft back in the day showed a clear space about 400 Angstroms (40 nanoMeters) thick.  Well we now know that there are tons of proteins occupying this space — a copy of a previous post

The bouillabaisse of the synaptic cleft

appears after the **** at the end of this post.  It shows just how many proteins occupy that clear space. Could a presynaptic protein directly bond to a postsynaptic protein across the cleft (perhaps with the help of a third or fourth Wallenda protein between the two?  A nice review [ Neuron vol. 96 pp. 680 – 696 ’17b ] http://www.cell.com/neuron/fulltext/S0896-6273(17)30935-2 sets out what is known.

We know that neurexins (presynaptic) bind to neuroligins (postsynaptic) across the cleft.  This is the best studied pair, and most of the earlier post discusses what is known about them.

Figure 1c p. 682 is particularly fascinating as it shows that there are many more molecules which shake hands across the cleft.  Even more interesting is the fact that just where they are relative to the center/periphery of  the synapses isn’t shown for the neurexin/neuroligin pair and the LAR/Strk pair (e.g. one of the best studied pairs) because apparently this isn’t known.   The ephrins/ephrin pair and the syncam pair are in the center, while N-cadherin is shown at the edge.

One of the crucial elements of the post-synaptic membrane, the AMPAR receptor for glutamic protrudes its amino terminal domain 1/3 of the way across the cleft (assuming it is 40 nanoMeters thick).

Postsynaptic receptors are said to be clustered in nanoDomains 80 – 100 nanoMeters in diameter, Similarly, presynaptic RIM nanoClusters are the same size and are said to be aligned with postSynaptic nanoClusters of PSD95 as measured by 3D-STORM, the current most cutting edge technique we have for visualizing these things [ Nature vol. 536 pp. 210 – 214 ’17 ].

So, all in all, the paper is fascinating and shows how much more there is to know.

Unfortunately the paper contains one statement which raises my chemical hackles;  “A consistent prediction across models is that the glutamate concentration profile reaches a very high peak (over 1 milliMolar), but only for a brief time period (100 microSeconds) and over a small distance (100 nanoMeters).” Glutamate is the major excitatory neurotransmitter in brain and is what binds to AMPAR.

Models are lovely, but how many molecules of glutamic acid are they talking about?  It’s easy (but tedious) to figure this out.

We know the volume they are talking about: a cylinder 100 nanoMeters in diameter and 40 nanoMeters tall (the width of the synaptic cleft).   So it contains pi * 100 * 40 = 12,566 cubic nanometers –round this down to 10^4 cubic nanoMeters. A liter is a cube .1 meters (10 centimeters) on a side. So 10 centimeters is 10^8 nanoMeters, meaning that a liter contains (10^8)^3 = 10^24 cubic nanoMeters.

A 1 molar solution of anything contains 6 * 10^23 molecules per liter (Avogadro’s number), so a 1 milliMolar solution (of glutamate in this case) contains 6 * 10^20 molecules/liter or  6 * 10^-4 molecules per cubic nanoMeter. Multiply this by the volume of the cylinder and you get a grand total of 6 molecules of glutamic acid in the cylinder.

If I’ve done the calculations correctly (and I think I have), “a very high peak (over 1 milliMolar)” is basically scientific garbage, the concept of concentration being stretched far beyond its range of meaningful applicability.

I’d love to stand corrected if my calculations are incorrect. Just make a comment.

Addendum 12 Dec — well my calculation is wrong. Here’s the dialog

APAJ — “We know the volume they are talking about: a cylinder 100 nanoMeters in diameter and 40 nanoMeters tall (the width of the synaptic cleft). So it contains pi * 100 * 40 = 12,566 cubic nanometers –round this down to 10^4 cubic nanoMeters.”
Just one err0r in the maths: the volume is r^2*pi*h so it’s closer to 3^5 cubic nm. This leads to ~188 glumate molecules following your further calculations. A more significant number, but I agree concentrations should not be used in these kind of volumes.

APAJ — Thanks — you’re correct and I’m embarrassed — pi * diameter is circumference not volume. so its pi * 50^2 * 40 = 314,259 cubic microns == 25 x more than 12,566 bringing the number of glutamic acids up to 150 (when 12,566 is rounded down to 10^4).

The criticism still stands. Concentration is meaningless in such small volumes.

 

*****

The bouillabaisse of the synaptic cleft

The synaptic cleft is so small ( under 400 Angstroms — 40 nanoMeters ) that it can’t be seen with the light microscope ( the smallest wavelength of visible light 3,900 Angstroms — 390 nanoMeters).  This led to a bruising battle between Cajal and Golgi a just over a century ago over whether the brain was actually made of cells.  Even though Golgi’s work led to the delineation of single neurons he thought the brain was a continuous network.  They both won the Nobel in 1906.

Semifast forward to the mid 60s when I was in medical school.  We finally had the electron microscope, so we could see synapses. They showed up as a small CLEAR spaces (e.g. electrons passed through it easily leaving it white) between neurons.  Neurotransmitters were being discovered at the same time and the synapse was to be the analogy to vacuum tubes, which could pass electricity in just one direction (yes, the transistor although invented hadn’t been used to make anything resembling a computer — the Intel 4004 wasn’t until the 70s).  Of course now we know that information flows back and forth across the synapse, with endocannabinoids (e. g. natural marihuana) being the major retrograde neurotransmitter.

Since there didn’t seem to be anything in the synaptic cleft, neurotransmitters were thought to freely diffuse across it to being to receptors on the other (postsynaptic) side e.g. a free fly zone.

Fast forward to the present to a marvelous (and grueling to read because of the complexity of the subject not the way it’s written) review of just what is in the synaptic cleft [ Cell vol. 171 pp. 745 – 769 ’17 ] http://www.cell.com/cell/fulltext/S0092-8674(17)31246-1 (It is likely behind a paywall).  There are over 120 references, and rather than being just a catalogue, the single author Thomas Sudhof extensively discusseswhich experimental work is to be believed (not that Sudhof  is saying the work is fraudulent, but that it can’t be used to extrapolate to the living human brain).  The review is a staggering piece of work for one individual.

The stuff in the synaptic cleft is so diverse, and so intimately involved with itself and the membranes on either side what what is needed for comprehension is not a chemist but a sociologist.  Probably most of the molecules to be discussed are present in such small numbers that the law of mass action doesn’t apply, nor do binding constants which rely on large numbers of ligands and receptors. Not only that, but the binding constants haven’t been been determined for many of the players.

Now for some anatomic detail and numbers.  It is remarkably hard to find just how far laterally the synaptic cleft extends.  Molecular Biology of the Cell ed. 5 p. 1149 has a fairly typical picture with a size marker and it looks to be about 2 microns (20,000 Angstroms, 2,000 nanoMeters) — that’s 314,159,265 square Angstroms (3.14 square microns).  So let’s assume each protein takes up a square 50 Angstroms on a side (2,500 square Angstroms).  That’s room for 125,600 proteins on each side assuming extremely dense packing.  However the density of acetyl choline receptors at the neuromuscular junction is 8,700/square micron, a packing also thought to be extremely dense which would give only 26,100 such proteins in a similarly distributed CNS synapse. So the numbers are at least in the right ball park (meaning they’re within an order of magnitude e.g. within a power of 10) of being correct.

What’s the point?

When you see how many different proteins and different varieties of the same protein reside in the cleft, the numbers for  each individual element is likely to be small, meaning that you can’t use statistical mechanics but must use sociology instead.

The review focuses on the neurExins (I capitalize the E  to help me remember that they are prEsynaptic).  Why?  Because they are the best studied of all the players.  What a piece of work they are.  Humans have 3 genes for them. One of the 3 contains 1,477 amino acids, spread over 1,112,187 basepairs (1.1 megaBases) along with 74 exons.  This means that just over 1/10 of a percent of the gene is actually coding for for the amino acids making it up.  I think it takes energy for RNA polymerase II to stitch the ribonucleotides into the 1.1 megabase pre-mRNA, but I couldn’t (quickly) find out how much per ribonucleotide.  It seems quite wasteful of energy, unless there is some other function to the process which we haven’t figured out yet.

Most of the molecule resides in the synaptic cleft.  There are 6 LNS domains with 3 interspersed EGFlike repeats, a cysteine loop domain, a transmembrane region and a cytoplasmic sequence of 55 amino acids. There are 6 sites for alternative splicing, and because there are two promoters for each of the 3 genes, there is a shorter form (beta neurexin) with less extracellular stuff than the long form (alpha-neurexin).  When all is said and done there are over 1,000 possible variants of the 3 genes.

Unlike olfactory neurons which only express one or two of the nearly 1,000 olfactory receptors, neurons express mutiple isoforms of each, increasing the complexity.

The LNS regions of the neurexins are like immunoglobulins and fill at 60 x 60 x 60 Angstrom box.  Since the synaptic cleft is at most 400 Angstroms long, the alpha -neurexins (if extended) reach all the way across.

Here the neurexins bind to the neuroligins which are always postsynaptic — sorry no mnemonic.  They are simpler in structure, but they are the product of 4 genes, and only about 40 isoforms (due to alternative splicing) are possible. Neuroligns 1, 3 and 4 are found at excitatory synapses, neuroligin 2 is found at inhibitory synapses.  The intracleft part of the neuroligins resembles an important enzyme (acetylcholinesterase) but which is catalytically inactive.  This is where the neurexins.

This is complex enough, but Sudhof notes that the neurexins are hubs interacting with multiple classes of post-synaptic molecules, in addition to the neuroligins — dystroglycan, GABA[A] receptors, calsystenins, latrophilins (of which there are 4).   There are at least 50 post-synaptic cell adhesion molecules — “Few are well understood, although many are described.”

The neurexins have 3 major sites where other things bind, and all sites may be occupied at once.  Just to give you a taste of he complexity involved (before I go on to  larger issues).

The second LNS domain (LNS2)is found only in the alpha-neurexins, and binds to neuroexophilin (of which there are 4) and dystroglycan .

The 6th LNS domain (LNS6) binds to neuroligins, LRRTMs, GABA[A] receptors, cerebellins and latrophilins (of which there are 4)_

The juxtamembrane sequence of the neurexins binds to CA10, CA11 and C1ql.

The cerebellins (of which there are 4) bind to all the neurexins (of a particular splice variety) and interestingly to some postsynaptic glutamic acid receptors.  So there is a direct chain across the synapse from neurexin to cerebellin to ion channel (GLuD1, GLuD2).

There is far more to the review. But here is something I didn’t see there.  People have talked about proton wires — sites on proteins that allow protons to jump from one site to another, and move much faster than they would if they had to bump into everything in solution.  Remember that molecules are moving quite rapidly — water is moving at 590 meters a second at room temperature. Since the synaptic cleft is 40 nanoMeters (40 x 10^-9 meters, it should take only 40 * 10^-9 meters/ 590 meters/second   60 trillionths of a second (60 picoSeconds) to cross, assuming the synapse is a free fly zone — but it isn’t as the review exhaustively shows.

It it possible that the various neurotransmitters at the synapse (glutamic acid, gamma amino butyric acid, etc) bind to the various proteins crossing the cleft to get their target in the postsynaptic membrane (e.g. neurotransmitter wires).  I didn’t see any mention of neurotransmitter binding to  the various proteins in the review.  This may actually be an original idea.

I’d like to put more numbers on many of these things, but they are devilishly hard to find.  Both the neuroligins and neurexins are said to have stalks pushing them out from the membrane, but I can’t find how many amino acids they contain.  It can’t find how much energy it takes to copy the 1.1 megabase neurexin gene in to mRNA (or even how much energy it takes to add one ribonucleotide to an existing mRNA chain).

Another point– proteins have a finite lifetime.  How are they replenished?  We know that there is some synaptic protein synthesis — does the cell body send packages of mRNAs to the synapse to be translated there.  There are at least 50 different proteins mentioned in the review, and don’t forget the thousands of possible isoforms, each of which requires a separate mRNA.

Old Chinese saying — the mountains are high and the emperor is far away. Protein synthesis at the synaptic cleft is probably local.  How what gets made and when is an entirely different problem.

A large part of the review concerns mutations in all these proteins associated with neurologic disease (particularly autism).  This whole area has a long and checkered history.  A high degree of cynicism is needed before believing that any of these mutations are causative.  As a neurologist dealing with epilepsy I saw the whole idea of ion channel mutations causing epilepsy crash and burn — here’s a link — https://luysii.wordpress.com/2011/07/17/we’ve-found-the-mutation-causing-your-disease-not-so-fast-says-this-paper/

Once again, hats off to Dr. Sudhof for what must have been a tremendous amount of work