Another neuropharmacologic surprise.

Our genome contains 826 different genes for G Protein Coupled Receptors (GPCRs) which are targeted by at least 475 FDA approved drugs (Nature vol. 587 p. 553 ’20 ). Yet part of the fascination of reading the current literature is the surprises it brings.

Our basic understanding was that the GPCRs sit on the surface of the cell waiting for ligands outside the cell to bind to it, which produces a conformational change on the cytoplasmic side of the cell membrane, changing the way the GPCR binds to the G protein, triggering all sorts of effects inside the cell.

As far as I recall, we never thought that different GPCRs would bind to each other in the cell membrane, even though a single cell can express ‘up to’ 100 different GPCRs [ Mol. Pharm. vol. 88 pp. 181 – 187 ’15 ].  Neurons express GPCRs and some are thought to be involved in neuropathic pain

But that’s exactly what Proc. Natl. Acad. Sci. vol. 119 e2123511119  ’22  is saying.

First a few definitions, if you’re as rusty about them as I was

A cytokine is an extracellular protein or peptide  helping cells to communicate with each other.  A chemokine is an extracellular protein which attracts cells.

Our genome has over 50 chemokines.  Most are  proteins with about 70 amino acids. The are classified by where the cysteines lie in them.  We have 23 receptors for chemokines, 18 of which are GPCRs.   Binding is promiscuous — a given chemokine binds to multiple receptors, and a given receptor binds to multiple chemokines.

Clearly the chemokines and their receptors are intimately involved in inflammation which always involves cell migration.  Neurons express chemokine receptors GPCRs and some are thought to be involved in neuropathic pain.

We also know that the nervous system is involved in immune function, particularly inflammation.  One prominent neurotransmitter is norepinephrine, and a variety of receptors bind to it.  There are 3 alpha1 norepinephrine receptors (a, b and d), all of which are GPCRs.

What is so shocking is that alpha1 GPCRs bind to chemokine receptors (forming heteromers), and that this binding is required for chemokines to have any effect on cell migration.  Even more interesting is that binding of norepinephrine to the alpha1 component of the heteromer INHIBITs cell migration.

So how many of our 826 GPCRs bind to each other, and what effects do they have?

Reading the literature is like opening presents, you find new fascinating toys to play with, some of which may actually benefit humanity

 

How can a cytokine still act after it’s gone

Here is yet another new wrinkle in how drugs act.

Type I interferons are  very well studied cytokines, important in the defense against viruses.  There are 16 of them, and in response to infection, they induce the expression of over 300 antiviral genes (ISGs — Interferon Stimulated Genes).

Type I interferon is gone from the plasma after intramuscular injection in a few hours, yet its effects last days to weeks.  Well maybe the proteins it induces hang around a long time.

Not so says Proc. Natl. Acad. Sci. vol. 117 pp. 17510 – 17512 ’20.  It hangs around inside the cell in little vesicles formed from pinching off part of the cell membrane containing type I interferon bound to its receptor.  These are called endosomes.  The authors call this siloing.  The silos persist for days and can actually be seen if you use fluorescently labeled type I interferon.  The endosomes with type I interferon inside persist for days.

I’m sure people are actively studying other cytokines of which there are many, many — e.g. there are more than 50 interleukins that we know about.

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.