Category Archives: Molecular Biology

Science fiction for the cognoscenti – III — not all the background you need will be explained

Now that every team in the NFL has its own molecular biologist and antiVirologist, you might be interested in knowing how it all started.  Like most technologies affecting our lives it had a military origin.

The escape of the Taiwanese pacifist virus started it all –https://luysii.wordpress.com/2018/11/19/a-science-fiction-story-for-the-cognoscenti-answer-to-the-puzzle-and-a-bit-more.

The technology of infectious gene transfer by recombinant adeno-associated virus  (AAV) was well advanced long before there were garage molecular biologists.

The NFL wars began with the New England Patriots, (who else?), they of deflategate and other nefarious ways to win.

Tom Brady was getting all set to win superbowl LVII in 2023 at age 45 when the first counterattack was successful.

His wife, the beautiful Gisele, hated the idea of him playing so long, being very worried about dementia pugilista from all the head trauma.  Tom had agreed to yearly PET scans with Pittsburgh compound B, an uncharged derivative of thioflavin T which gets through the blood brain barrier and which stains senile plaques.  They showed no evidence of plaques (although plenty of demented people don’t have them) so he kept on playing behind Belichick’s not so secret weapon — 400 pound linemen.  Even though he’d lost a step or two, his eye and arm were still good and the linemen gave him plenty of time to throw.

Football players have always been bulking up.  Even the early experience with extra testosterone (which causes testicular atrophy in high doses) didn’t dissuade them.  Newer anabolic steroids had somewhat fewer testicular effects.  Eventually players took to using HCG to help normalize things, but some testicular atrophy was a price they were willing to pay.  The cheerleaders felt a lot safer around those using them.

So how did the Patriots have 400 pound linemen when no one else did?  The answer goes back to Piedmontese and Belgian Blue cattle which were bred for their large muscles.  They turned out to have inactivating mutations in the gene for myostatin, a protein which causes muscles to stop growing.

Boston isn’t known as the home of biotech for nothing, and Belichick contracted with an as yet un-named biotech firm (their depositions having been sealed by the court) to come up with a small molecule (compound M) absorbable through the skin which inhibited myostatin.

No one caught on why Belicheck had separate showers installed for the lineman and defensive backs, but they had to use them and got  dosed that way.  Testing for performance enhancing drugs was always negative. The linemen loved it, as their testicles grew back to normal size.  The cheerleaders didn’t.

So there the Patriots were, about to play the Arizona Cardinals, a team only winning 3 games in 2018 in superbowl LVII. No one understood how the Cardinals turned around and how they got those very slippery running backs.

No one, except the molecular biologist they hired.  But that’s for next time.

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Another way to study Alzheimer’s

Until I read the paper PLOS Genet. 14, e1007791 (2018)., I thought that this was a sure way to win Nobel prize.  It’s still pretty interesting.  The abstract in Science was misleading, implying that there was an APOE4 variant which was actually protective against Alzheimer’s disease. That would have been fantastic, as it would provide a clue as to just what the APOE4 allele was doing to increase the risk of Alzheimer’s disease.

A huge amount of work has been done on APOE4.   Googling produced 433,000 results (0.46 seconds).  Theories abound but we still don’t know.

The authors studied Blacks and Puerto Ricans and found that if you inherited the APOE4 allele from an African source (rather than a European source), your chance of developing Alzheimer’s disease was significantly less.  A total of 1,766 African American and 220 Puerto Rican individuals with late-onset Alzheimer disease, and 3,730 African American and 169 Puerto Rican cognitively healthy individuals (> 65 years) participated in the study.

The numbers: ApoE ε4 alleles on an African background conferred a lower risk than those with a European ancestral background, regardless of population (Puerto Rican: OR = 1.26 on African background, OR = 4.49 on European; African American: OR = 2.34 on African background, OR = 3.05 on European background).

Note that the ORs are still up for Alzheimer’s if you have APOE4, but the differences are significant and certainly real given the size of the study.

The authors think it’s the area around the APOE  gene, rather than the total genetic background (African vs. European etc. etc.)

It still might be worth doing the following.  Take skin fibroblasts from all four types of people (Puerto Ricans with APOE4 on African background, Puerto Ricans with APOE4 on European background, Blacks with APOE4 on African background, APOE4 on a European background).

Make induced pluripotent stem cells (iPSCs) from them (the technology to do so is quite advanced). Differentiate these iPSCs into neurons  and others into glia (technology quite available).  Study protein and mRNA expression, epigenetic modifications in neurons and glia from all 4 groups.  This might tell you just what APOE4 was doing in high and lower risk people, and possibly might give a clue as to how it was increasing Alzheimer’s risk.

My hopes were really up, because the abstract in Science implied that APOE4 in Blacks and Puerto Ricans was actually absolutely rather than relatively protective, which would have given us some serious clues to Alzheimer pathogenesis, when APOE4 protective cells were contrasted with APOE4 increased risk cells.

Oh well.

Bye bye stoichiometry

Until recently, developments in physics basically followed earlier work by mathematicians Think relativity following Riemannian geometry by 40 years.  However in the past few decades, physicists have developed mathematical concepts before the mathematicians — think mirror symmetry which came out of string theory — https://en.wikipedia.org/wiki/Mirror_symmetry_(string_theory). You may skip the following paragraph, but here is what it meant to mathematics — from a description of a 400+ page book by Amherst College’s own David A. Cox

Mirror symmetry began when theoretical physicists made some astonishing predictions about rational curves on quintic hypersurfaces in four-dimensional projective space. Understanding the mathematics behind these predictions has been a substantial challenge. This book is the first completely comprehensive monograph on mirror symmetry, covering the original observations by the physicists through the most recent progress made to date. Subjects discussed include toric varieties, Hodge theory, Kahler geometry, moduli of stable maps, Calabi-Yau manifolds, quantum cohomology, Gromov-Witten invariants, and the mirror theorem. This title features: numerous examples worked out in detail; an appendix on mathematical physics; an exposition of the algebraic theory of Gromov-Witten invariants and quantum cohomology; and, a proof of the mirror theorem for the quintic threefold.

Similarly, advances in cellular biology have come from chemistry.  Think DNA and protein structure, enzyme analysis.  However, cell biology is now beginning to return the favor and instruct chemistry by giving it new objects to study. Think phase transitions in the cell, liquid liquid phase separation, liquid droplets, and many other names (the field is in flux) as chemists begin to explore them.  Unlike most chemical objects, they are big, or they wouldn’t have been visible microscopically, so they contain many, many more molecules than chemists are used to dealing with.

These objects do not have any sort of definite stiochiometry and are made of RNA and the proteins which bind them (and sometimes DNA).  They go by any number of names (processing bodies, stress granules, nuclear speckles, Cajal bodies, Promyelocytic leukemia bodies, germline P granules.  Recent work has shown that DNA may be compacted similarly using the linker histone [ PNAS vol.  115 pp.11964 – 11969 ’18 ]

The objects are defined essentially by looking at them.  By golly they look like liquid drops, and they fuse and separate just like drops of water.  Once this is done they are analyzed chemically to see what’s in them.  I don’t think theory can predict them now, and they were never predicted a priori as far as I know.

No chemist in their right mind would have made them to study.  For one thing they contain tens to hundreds of molecules.  Imagine trying to get a grant to see what would happen if you threw that many different RNAs and proteins together in varying concentrations.  Physicists have worked for years on phase transitions (but usually with a single molecule — think water).  So have chemists — think crystallization.

Proteins move in and out of these bodies in seconds.  Proteins found in them do have low complexity of amino acids (mostly made of only a few of the 20), and unlike enzymes, their sequences are intrinsically disordered, so forget the key and lock and induced fit concepts for enzymes.

Are they a new form of matter?  Is there any limit to how big they can be?  Are the pathologic precipitates of neurologic disease (neurofibrillary tangles, senile plaques, Lewy bodies) similar.  There certainly are plenty of distinct proteins in the senile plaque, but they don’t look like liquid droplets.

It’s a fascinating field to study.  Although made of organic molecules, there seems to be little for the organic chemist to say, since the interactions aren’t covalent.  Time for physical chemists and polymer chemists to step up to the plate.

Were the initial native Americans inbred?

Usually when I eMail the author(s) of a paper or a math book with a question or a comment I get a quick response.  My cynical wife says thing this is because mathematicians don’t have much to do.  Not so in this case. Hence the hopefully attention getting title of this post.

I refer to the following papers [ Cell vol. 175 pp. 1173 – 1174, 1185 – 1197 ’18 ]  Nature vol. 563 pp. 303 – 304 ’18,Science vol. 362 pp. 1128 eaav2621  1 –> 11 ’18 ] I’ve sent a bunch letters to the authors and have heard nothing back in a week.

So what is all this about?  It’s about population bottlenecks and founder effects in the ancestors of what are now called ‘native Americans’ — although while living in Montana from ’72 – ’87, if you called an Indian, a Native American, you would have received some strange looks.

I am not a population geneticist, so I wonder just how many people made it over the Bering land bridge during the last ice age, and just how genetically diverse they were.  Northern Siberia today is a rather forbidding place, and I doubt that hordes of genetically different people lived here.  I’m not sure how long the land bridge was open and how many people crossed it.

So modern native Americans may be quite genetically homogeneous.  How to tell?  This is where the papers come in.  They sequenced genomes from a variety of locations in the western hemisphere, all dying over a thousand years ago (before the Europeans came and interbred with them).  It seems that they have around 100 such genomes.

I wrote to ask how similar these genomes are.  No response.  Is it because the answer might be politically incorrect?

I don’t think the question is idiotic.  Possibly we don’t have enough genomes to make a sensible statement, but if they’re all really close (however defined) we could say something.

Anybody out there have any thoughts (or even better)  knowledge about these matters?

Is a little infection good for you? If so what about radiation?

Could a little infection be good for you?  Well how about the immune stimulation it produces?  We’re talking about trained immunity here, and evidence for it started nearly 100 years ago in northern Sweden.  TB was a much bigger problem back then (Hell, my grandmother died of undiagnosed tuberculosis in a university hospital in 1967).  Bacille Calmette Guerin (BCG) is an attenuated form of mycobacterium tuberculosis (TB to you). Immunizing with BCG was thought to be protective against TB.  100 years later people are still arguing about it.

What no one is arguing about is the fact the unvaccinated infants had a 10% mortality in the first year of life (the good old days weren’t that good), while the vaccinated ones had a 3% one year mortality.  The 10% that did die, didn’t die of TB either.

It’s pretty technical, but basically BCG vaccination jazzes up the immune system, making it more responsive to the zillions of critters infesting us. [ Proc. Natl. Acad. Sci. vol. 109 pp. 17537 – 17542 ’12 ] has the details  — inferferon gamma, monocyte derived cytokines, the NOD2 receptor, histone 3 lysine 4 trimethylation etc. etc.

These are rather nonspecific features of innate immunity, and not specifically directed at anything in particular (which is why it is called trained immunity).

Well infection jazzes up the immune system too.  Cell vol, 175 pp. 1634 – 1650 ’18 showed that white cells of mice found in the lungs (alveolar macrophages) developed it after a viral infection, making them more resistant to a subsequent bacterial infection.  Could this be general?

Which brings us to a much larger fish to fry –hormesis.

Toxicology basically had two models of how we deal with toxic agents

l. Threshold model — below a certain dose, no harm results.  Arguably true — can one molecule of anything kill you?  Used by the EPA for nonCarcinogens

2. Linear no-threshold model — now matter how lot the dose, damage is seen. This is obviously crazy (see #1 above) but apparently is used by the EPA for carcinogens.

Enter model #3 — Hormesis

The best example is the famous J curve for alcohol in which small amounts are beneficial  at low doses for the heart and huge amounts are horrible (although a recent meta-analysis has challenged this  [ Lancet vol. 392 pp. 1015 – 1035 ’18 ], but I don’t trust them — for why see http://science.sciencemag.org/content/sci/361/6408/1184.full.pdf.

Hormesis says that other toxic agents (radiation, cadmium, dioxin, saccharin, polychlorinated biphenyls) all have J curves like alcohol.   Articles explaining hormesis can be found in — Nature vol. 421 pp. 691 -692 2003, Scientific American 9/2003.  The reaction to it — Science vol,. 302 pp. 376 – 379, 2003.

So the moral might be don’t be an immunological or a toxicological snowflake — that which doesn’t kill you makes you stronger.

The uses of disorder in the cell

We know that many proteins have disordered segments, and an older (2004) estimate says that over 30% of all eukaryotic proteins have disordered stretches of more than 30 amino acids.  Here is another example where the disordered conformation(s) of a protein is the form used by the cell.

Histone H1 (aka the linker histone) binds to DNA between nucleosomes.  It is thought to be important in the 10,000 or so compaction of the 3 meters or so of DNA each cell has so it fits into a 10 micron nucleus.  Histone H1 has a disordered carboxy terminal tail of 100 amino acids.  Unsurprisingly it is strongly positively charged (so it binds to the negatively charged phosphates holding DNA together).

H1 was studied in an interesting paper [ Proc. Natl. Acad. Sci. vol. 115 pp. 11964 – 11969 ’18 ].  The tail was added to short (36 basepairs) double stranded segment of DNA, under various stoichiometries and ionic compositions.  They found regions where the complex formed liquid droplets the size of microns.

We know DNA is compacted and people have looked for the 30 nanoMeter DNA fiber of DNA bound to nucleosomes for years without success.  It is possible that the compaction in DNA is due to phase separation (which is basically unstructured) rather than the rather specific structures proposed.  H1 may be acting as a likquidlike glue.  Fascinating.

In other work H1 was complexed with another protein (Prothymosin alpha) which is another intrinsically disordered protein which actually serves as a histone H1 chaperone.  Prothymosin is is polyAnionic, so it binds to polyCationic H1.  What is fascinating is that the binding is quite tight (picoMolar) and yet even when so tightly bound H1 remains disordered, something to confound drug chemists who are always looking for specific binding conformations.

The paper also describes Psi DNA, which is formed in solutions of cationic polymers. Here DNA condenses into a compact solvent excluded state.  It is an ordered assembly of B-DNA arranged in parallel twisted helical segments with a well define spacing.  It produces an anomalously large scattering signal in circular dichroism spectra.

Here is an older post in which the functional form of a protein is the unstructured one

When the active form of a protein is intrinsically disordered

Back in the day, biochemists talked about the shape of a protein, influenced by the spectacular pictures produced by Xray crystallography. Now, of course, we know that a protein has multiple conformations in the cell. I still find it miraculous that the proteins making us up have only relatively few. For details see — https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/.

Presently, we also know that many proteins contain segments which are intrinsically disordered (e.g. no single shape).The pendulum has swung the other way — “estimations that contiguous regions longer than 50 amino acids ‘may be present” in ‘up to’ 50% of proteins coded in eukaryotic genomes [ Proc. Natl. Acad. Sci. vol. 102 pp. 17002 – 17007 ’05 ]

[ Science vol. 325 pp. 1635 – 1636 ’09 ] Compared to ordered regions, disordered regions of proteins have evolved rapidly, contain many short linear motifs that mediate protein/protein interactions, and have numerous phosphorylation sites compared to ordered regions. Disordered regions are enriched in serine and threonine residues, while ordered sequences are enriched in tyrosines — this highlights functional differences in the types of phosphorylation. Interestingly tyrosines have been lost during evolution.

What are unstructured protein segments good for? One theory is that the disordered segment can adopt different conformations to bind to different partners — this is the moonlighting effect. Then there is the fly casting mechanism — by being disordered (hence extended rather than compact) such proteins can flail about and find partners more easily.

Given what we know about enzyme function (and by inference protein function), it is logical to assume that the structured form of a protein which can be unstructured is the functional form.

Not so according to this recent example [ Nature vol. 519 pp. 106 – 109 ’15 ]. 4EBP2 is a protein involved in the control of protein synthesis. It binds to another protein also involved in synthesis (eIF4E) to suppress a form of translation of mRNA into protein (cap dependent translation if you must know). 4EBP2 is intrinsically disordered. When it binds to its target it undergoes a disorder to ordered transition. However eIF4E binding only occurs from the intrinsically disordered form.

Control of 4EBP2 activity is due, in part, to phosphorylation on multiple sites. This induces folding of amino acids #18 – #62 into a 4 stranded beta domain which sequesters the canonical YXXXLphi motif with which 4EBP2 binds eIF4E (Y stands for tyrosine, X for any amino acid, L for leucine and phi for any bulky hydrophobic amino acid). So here we have an inactive (e.g. nonbonding) form of a protein being the structured rather than the unstructured form. The unstructured form of 4EBP2 is therefore the physiologically active form of the protein.

Cell biological porn

Could there actually be cell biological porn?  Yes indeedy, and hopefully the following is not behind a paywall.  https://www.cell.com/cell/fulltext/S0092-8674(18)31308-4 — [ Cell vol. 175 pp. 1430 – 1442 ’18 ]

For why I find the pictures (and videos) in the article sexy, we have to go back the bad old days of 1962 when I entered medical school and saw my first electron micrograph.  Possessed of an immense ego and a newly minted masters of chemistry, I thought I could look at the pictures and figure out what what going on chemically to produce what was seen, namely Robertson’s unit membrane.  We know what’s going on in cell membranes now, but here’s what I had to deal with back then.

Membranes fixed with osmium tetroxide revealed a characteristic tri-laminar appearance con­sisting of two parallel outer dark (osmiophilic) layers and a central light (osmiophobic) layer.

The osmiophilic layers typically measured 20-25 Å (2.0-2.5nm) in thickness and the osmiophobic layers measured 25-35 Å (2.5-3.5 nm), yielding a total thickness of 65-85 Å (6.5-8.5 nm). This value com­pared favorably with the thickness predicted on the basis of chemical studies.

According to Robertson, the unit membrane consisted of a bimolecular lipid leaflet sandwiched between outer and inner layers of protein organized in the pleated sheet con­figuration. Such an arrangement was presumed to be basically the same in all cell membranes.

Well that was the state of the art back then.  I figured I could do better, particularly since I’d used osmium tetroxide as a chemist to convert olefins to vic-diols.  Little did I know that the osmium was being used because of its high atomic weight (76 protons and over 100 neutrons) making it relatively impenetrable to the electrons of the electron microscope.

But then I looked at what was done to prepare tissue for electron microscopy — fix with glutaraldehyde, then osmium.  Dehydrate the (dead) dissue, and embed it in a monomeric resin which polymerizes to form a solid block of plastic, then cut the block, into a very thin section, place it on a copper grid covered with carbon, pump the air out so the electrons could get through, and take a picture (prayer optional).

As soon as I read this, any hope of chemical analysis disappeared.  It also taught me that it was a very large leap to assume the electron micrographs reflected what was going on in living tissue.

Which is why the above paper is so spectacular.  It uses two types of living cells (COS-7 a fibroblast like cell line from kidney and U2OS, an osteosarcoma cell line). The technique (Grazing Incidence Structured Illumination Microscopy — GI-SIM) is incredibly complicated (but well described in the paper).  It allows you to image events near the part of the cell resting on the microscope stage at 970 Angstrom resolution at rates of ‘up to’ 266 frames/second over thousands of time points.  Recall that the lowest wavelength of visible light is 3,800 Angstroms.

Various dyes are used to differentially stain microtubules, the membranes of the endoplasmic reticulumn (ER), late endosomes (LEs), mitochondria and lysosomes.  To my amazements the pictures look the electron micrographs of yore.

You can watch mitochondria touching the ER and then splitting, ER tubules growing and shrinking and being pulled along LEs riding on microtubules etc. etc. The pictures show the same cell over a period of 4 minutes.

Then to make a neurologist’s day complete, they watch dendritic spines form and unform in cultured hippocampal neurons.

So look at the paper if you can.  You don’t even have to read it th,e pictures are explanatory.

An extraordinarily impressive work, considering where we’ve been.

A science fiction story (for the cognoscenti) — answer to the puzzle and a bit more

Comrade Chen we have a serious problem.

Don’t tell me one of our bugs escaped confinement.

Worse.  One of theirs did.  And it’s affecting the PLA (People’s Liberation Army).  Some are turning into pacifists.

It doesn’t kill them?

No. But for our purposes it might as well.

It’s a typical adenoassociated virus (AAV) like we use.

Well, what does the genome look like?

We’ve sequenced it and among other things, it codes for a protein which enters the brain and alters behavior.

What?

Well, the enemy has some excellent biologists, one of whom works on Wolbachia.

What’s that?

It’s a rickettsial organism which changes the sex life of some insects.

I don’t believe that.

Do you have a cat?

Yes.

Well many cats contain another organism (toxoplasma gondi).

So what.

Rats infected by the organism become less afraid of cats.

Another example please.

A fungus infecting carpenter ants causes the ant to leave its colony, climb a tree, chomp down on the underside of a leaf and die, freeing fungal spores to fall on the ground where they can reinfect new ants.

Well what is the genome of the virus?

It has some very unusual sequences, and one which proves that the Wolbachia biologist on the other side has a very large ego.

How so.

Well in addition to the brain infecting protein, there is a very unusual triplet of peptides all in a row.

Methionine Alanine Aspartic Acid Glutamic acid, then a stop codon, then Isoleucine Asparagine, than a stop codon, then Threonine Alanine Isoleucine Tryptophan Alanine Asparagine.  We think that the first two in some way cause readthrough of the stop codons so the protein following the short peptides is made.

Where does the big ego come in?

Sir, proteins can have hundreds and hundreds of amino acids.  People got tired of writing their full names out, so each of the 20 amino acids was given a single letter to stand for it.

M – Methionine

A – Alanine

D – Aspartic acid

What does D have to do with Aspartic acid?

Nothing sir, look on the letters as Chinese characters.

E -Glutamic Acid

I – isoleucine

What about the stop codon between Glutamic acid and Isoleucine

Just regard it as a space.

N – Asparagine

Nooo! ! ! I I’m beginning to get the picture.

Yes sir, it stands for MADE IN TAIWAN

—-

A few years later

Well the Taiwanese biologist outsmarted himself (or herself).   The Taiwanese soldiers wouldn’t fight either as the virus spread.  Most conflicts between nation states pretty much ended (Russia/Ukraine, North Korea/South Korea) etc. etc.  The Taiwanese biologist was nominated for the Nobel Peace Prize, and did receive it in absentia, as every military type in the world was looking for him (or  her), so he (or she) went into hiding, and is believed to be living in an Ashram near Boulder, Colorado.

Unfortunately, the idea of using viruses to change human behavior spread past nation states, and private groups with their own agendas began using it.

The ‘new soviet man’ of the previous century looked rather benign compared to what subsequently happened.

The next story for the scientific cognoscenti will describe the events leading up to the impeachment trial of President Jon Tester in 2028.

 

A science fiction story (for the cognoscenti)

Comrade Chen we have a serious problem.

Don’t tell me one of our bugs escaped confinement.

Worse.  One of theirs did.  And it’s affecting the PLA (People’s Liberation Army).  Some are turning into pacifists.

It doesn’t kill them?

No. But for our purposes it might as well.

It’s a typical adenoassociated virus (AAV) like we use.

Well, what does the genome look like?

We’ve sequenced it and among other things, it codes for a protein which enters the brain and alters behavior.

What?

Well, the enemy has some excellent biologists, one of whom works on Wolbachia.

What’s that?

It’s a rickettsial organism which changes the sex life of some insects.

I don’t believe that.

Do you have a cat?

Yes.

Well many cats contain another organism (toxoplasma gondi).

So what.

Rats infected by the organism become less afraid of cats.

Another example please.

A fungus infecting carpenter ants causes the ant to leave its colony, climb a tree, chomp down on the underside of a leaf and die, freeing fungal spores to fall on the ground where they can reinfect new ants.

Well what is the genome of the virus?

It has some very unusual sequences, and one which proves that the Wolbachia biologist on the other side has a very large ego.

How so.

Well in addition to the brain infecting protein, there is a very unusual triplet of peptides all in a row.

Methionine Alanine Aspartic Acid Glutamic acid, then a stop codon, then Isoleucine Asparagine, than a stop codon, then Threonine Alanine Isoleucine Tryptophan Alanine Asparagine.  We think that the first two in some way cause readthrough of the stop codons so the protein following the short peptides is made.

Where does the big ego come in?

Figure it out.

 

Answer next week.

Cellular senescence (again, again)

As well as being involved in normal cellular function, wound healing, embryology, and warding off cancer, cellular senescence may be involved in one form of neurodegeneration according to [ Nature vol. 562 pp. 503 – 504, 578 – 582 ’18 ]

Alzheimer’s disease is characterized by two findings visible with only a light microscope — the senile plaque which occurs outside neurons, and the neurofibrillary tangle (which occurs inside them).  The latter is due to accumulation of excessively phosphorylated tau protein.  A few mutations in the tau protein are known to cause neurodegeneration.  One such is the substitution of serine (S) for proline (P) at position #301 in tau (e. g. the P301S mutation).

Transgenic expression of the mutant tau in mice mimics the human illness.  Long before neurofibrillary tangles appear in neurons, glial cells (which don’t express much tau and never have neurofibrillary tangles) develop cellular senescence.  Neurons don’t show this.

p16^INK4a is a transcription factor which turns on cellular senescence, leading to expression of a bunch of proteins known as the Senescence Associated Secretory Phenotype (SASP).  It was elevated in glia.  The authors were able to prevent the neurodegeneration using another genetic tool, which produced cell death in cells expression p16^INK4a.  There was fewer neurofibrillary tangles in the animals.

The nature of the neural signal to glia causing senescence isn’t known at this point.  How glia signal back also isn’t known.

So are drugs killing senescence cells (senolytics) a possible treatment of neurodegeneration?  Stay tuned.

As readers of this blog well know, I’ve been flogging an idea of mine — that excessive cellular senescence with release of SASP products is behind the faatigue of chronic fatigue syndrome.   I’d love it if someone would measure p16^INK4a in these people — it’s so easy to do, and if the idea is correct would lead to a rational treatment for some with the disorder.

Neurodegeneration is a far larger fish to fry than CFS, and I hope people with it don’t get lost in the shuffle.

Here’s the idea again

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

Is a rational treatment for chronic fatigue syndrome at hand?

If an idea of mine is correct, it is possible that some patients with chronic fatigue syndrome (CFS) can be treated with specific medications based on the results of a few blood tests. This is precision medicine at its finest.  The data to test this idea has already been acquired, and nothing further needs to be done except to analyze it.

Athough the initial impetus for the idea happened only 3 months ago, there have been enough twists and turns that the best way explanation is by a timeline.

First some background:

As a neurologist I saw a lot of people who were chronically tired and fatigued, because neurologists deal with muscle weakness and diseases like myasthenia gravis which are associated with fatigue.  Once I ruled out neuromuscular disease as a cause, I had nothing to offer then (nor did medicine).  Some of these patients were undoubtedly neurotic, but there was little question in my mind that many others had something wrong that medicine just hadn’t figured out yet — not that it hasn’t been trying.

Infections of almost any sort are associated with fatigue, most probably caused by components of the inflammatory response.  Anyone who’s gone through mononucleosis knows this.    The long search for an infectious cause of chronic fatigue syndrome (CFS) has had its ups and downs — particularly downs — see https://luysii.wordpress.com/2011/03/25/evil-scientists-create-virus-causing-chronic-fatigue-syndrome-in-lab/

At worst many people with these symptoms are written off as crazy; at best, diagnosed as depressed  and given antidepressants.  The fact that many of those given antidepressants feel better is far from conclusive, since most patients with chronic illnesses are somewhat depressed.

The 1 June 2017 Cell had a long and interesting review of cellular senescence by Norman Sharpless [ vol. 169 pp. 1000 – 1011 ].  Here is some background about the entity.  If you are familiar with senescent cell biology skip to the paragraph marked **** below

Cells die in a variety of ways.  Some are killed (by infections, heat, toxins).  This is called necrosis. Others voluntarily commit suicide (this is called apoptosis).   Sometimes a cell under stress undergoes cellular senescence, a state in which it doesn’t die, but doesn’t reproduce either.  Such cells have a variety of biochemical characteristics — they are resistant to apoptosis, they express molecules which prevent them from proliferating and — most importantly — they secrete a variety of proinflammatory molecules collectively called the Senescence Associated Secretory Phenotype — SASP).

At first the very existence of the senescent state was questioned, but exist it does.  What is it good for?  Theories abound, one being that mutation is one cause of stress, and stopping mutated cells from proliferating prevents cancer. However, senescent cells are found during fetal life; and they are almost certainly important in wound healing.  They are known to accumulate the older you get and some think they cause aging.

Many stresses induce cellular senescence of which mutation is but one.  The one of interest to us is chemotherapy for cancer, something obviously good as a cancer cell turned senescent has stopped proliferating.   If you know anyone who has undergone chemotherapy, you know that fatigue is almost invariable.

****

One biochemical characteristic of the senescent cell is increased levels of a protein called p16^INK4a, which helps stop cellular proliferation.  While p16^INK4a can easily be measured in tissue biopsies, tissue biopsies are inherently invasive. Fortunately, p16^INK4a can also be measured in circulating blood cells.

What caught my eye in the Cell paper was a reference to a paper about cancer [ Cancer Discov. vol. 7 pp. 165 – 176 ’17 ] by M. Demaria, in which the levels of p16^INK4a correlated with the degree of fatigue after chemotherapy.  The more p16^INK4a in the blood cells the greater the fatigue.

I may have been the only reader of both papers with clinical experience wth chronic fatigue syndrome.  It is extremely difficult to objectively measure a subjective complaint such as fatigue.

As an example of the difficulty in correlating subjective complaints with objective findings, consider the nearly uniform complaint of difficulty thinking in depression, with how such patients actually perform on cognitive tests — e. g. there is  little if any correlation between complaints and actual performance — here’s a current reference — Scientific Reports 7, Article number: 3901(2017) —  doi:10.1038/s41598-017-04353.

If the results of the Cancer paper could be replicated, p16^INK4 would be the first objective measure of a patient’s individual sense of fatigue.

So I wrote both authors, suggesting that the p16^INK4a test be run on a collection of chronic fatigue syndrome (CFS) patients. Both authors replied quickly, but thought the problem would be acquiring patients.  Demaria said that Sharpless had a lab all set up to do the test.

Then fate (in the form of Donald Trump) supervened.  A mere 9 days after the Cell issue appeared, Sharpless was nominated to be the head of the National Cancer Institute by President Trump.  This meant Dr. Sharpless had far bigger fish to fry, and he would have to sever all connection with his lab because of conflict of interest considerations.

I also contacted a patient organization for chronic fatigue syndrome without much success.  Their science advisor never responded.

There matters stood until 22 August when a paper and an editorial about it came out [ Proc. Natl. Acad. Sci. vol. 114 pp. 8914 – 8916, E7150 – E7158 ’17 ].  The paper represented a tremendous amount of data (and work).  The blood levels of 51 cytokines (measures of inflammation) and adipokines (hormones released by fat) were measured in both 192 patients with CFS (which can only be defined by symptoms) and 293 healthy controls matched for age and gender.

In this paper, levels of 17 of the 51 cytokines correlated with severity of CFS. This is a striking similarity with the way the p16^INK4 levels correlated with the degree of fatigue after chemotherapy).  So I looked up the individual elements of the SASP (which can be found in Annu Rev Pathol. 21010; 5: 99–118.)  There are 74 of them. I wondered how many of the 51 cytokines measured in the PNAS paper were in the SASP.  This is trickier than it sounds as many cytokines have far more than one name.  The bottom line is that 20 SASPs are in the 51 cytokines measured in the paper.

If the fatigue of CFS is due to senescent cells and the SASPs  they release, then they should be over-represented in the 17 of the 51 cytokines correlating with symptom severity.  Well they are; 9 out of the 17 are SASP.  However although suggestive, this increase is not statistically significant (according to my consultants on Math Stack Exchange).

After wrote I him about the new work, Dr. Sharpless noted that CFS is almost certainly a heterogeneous condition. As a clinician with decades of experience, I’ve certainly did see some of the more larcenous members of our society who used any subjective diagnosis to be compensated, as well as a variety of individuals who just wanted to withdraw from society, for whatever reason. They are undoubtedly contaminating the sample in the paper. Dr. Sharpless thought the idea, while interesting, would be very difficult to test.

But it wouldn’t at all.  Not with the immense amount of data in the PNAS paper.

Here’s how. Take each of the 9 SASPs and see how their levels correlate with the other 16 (in each of the 192 CSF patients). If they correlate better with SASPs than with nonSASPs, than this would be evidence for senescent cells being the cause some cases of CFS. In particular, patients with a high level of any of the 9 SASPs should be studied for such correlations.  Doing so should weed out some of the heterogeneity of the 192 patients in the sample.

This is why the idea is testable and, even better, falsifiable, making it a scientific hypothesis (a la Karl Popper).  The data to refute it is in the possession of the authors of the paper.

Suppose the idea turns out to be correct and that some patients with CFS are in fact that way because, for whatever reason, they have a lot of senescent cells releasing SASPs.

This would mean that it would be time to start trials of senolyic drugs which destroy senescent cells on the group with elevated SASPs. Fortunately, a few senolytics are currently inc linical use.  This would be precision medicine at its finest.

Being able to alleviate the symptoms of CFS would be worthwhile in itself, but SASP levels could also be run on all sorts of conditions associated with fatigue, most notably infection. This might lead to symptomatic treatment at least.  Having gone through mono in med school, I would have loved to have been able to take something to keep me from falling asleep all the time.