Tag Archives: Alzheimer’s disease

RIPK1

The innate immune system is intrinsically fascinating, dealing with invaders long before antibodies or cytotoxic cells are on the scene.  It is even more fascinating to a chemist because it works in part by forming amyloid inside the cell.  And you thought amyloid was bad.

The system becomes even more fascinating because blocking one part of it (RIPK1) may be a way to treat a variety of neurologic diseases (ALS, MS,Alzheimer’s, Parkinsonism) whose treatment could be improved to put it mildly.

One way to deal with an invader which has made it inside the cell, is for the cell to purposely die.  More and more it appears that many forms of cell death are elaborately programmed (like taking down a stage set).

Necroptosis is one such, distinct from the better known and studied apoptosis.   It is programmed and occurs when a cytokine such as tumor necrosis factor binds to its receptor, or when an invader binds to members of the innate immune system (TLR3, TLR4).

The system is insanely complicated.  Here is a taste from a superb review — unfortunately probably behind a paywall — https://www.pnas.org/content/116/20/9714 — PNAS vol. 116 pp. 9714 – 9722 ’19.

“RIPK1 is a multidomain protein comprising an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The intermediate domain of RIPK1 contains an RHIM [receptor interacting protein (rip) homotypic interaction motif] domain which is important for interacting with other RHIM-containing proteins such as RIPK3, TRIF, and ZBP1. The C-terminal DD mediates its recruitment by interacting with other DD-containing proteins, such as TNFR1 and FADD, and its homodimerization to promote the activation of the N-terminal kinase domain. In the case of TNF-α signaling, ligand-induced TNFR1 trimerization leads to the assembly of a large receptor-bound signaling complex, termed Complex I, which includes multiple adaptors (TRADD, TRAF2, and RIPK1), and E3 ubiquitin ligases (cIAP1/2, LUBAC complex).”

Got that?  Here’s a bit more

“RIPK1 is regulated by multiple posttranslational modifications, but one of the most critical regulatory mechanisms is via ubiquitination. The E3 ubiquitin ligases cIAP1/2 are recruited into Complex I with the help of TRAF2 to mediate RIPK1 K63 ubiquitination. K63 ubiquitination of RIPK1 by cIAP1/2 promotes the recruitment and activation of TAK1 kinase through the polyubiquitin binding adaptors TAB2/TAB3. K63 ubiquitination also facilitates the recruitment of the LUBAC complex, which in turn performs M1- type ubiquitination of RIPK1 and TNFR1. M1 ubiquitination of Complex I is important for the recruitment of the trimeric IκB kinase complex (IKK) through a polyubuiquitin-binding adaptor subunit IKKγ/NEMO . The activation of RIPK1 is inhibited by direct phosphorylation by TAK1, IKKα/β, MK2, and TBK1. cIAP1 was also found to mediate K48 ubiquitination of RIPK1, inhibiting its catalytic activity and promoting degradation.”

So why should you plow through all this?  Because inhibiting RIPK1 reduces oxygen/glucose deprivation induced cell death in neurons, and reduced infarct size in experimental middle cerebral artery occlusion.

RIPK1 is elevated in MS brain, and inhibition of it helps an animal model (EAE).  Mutations in optineurin, and TBK1 leading to familial ALS promote the onset of RIPK1 necroptosis

Inflammation is seen in a variety of neurologic diseases (Alzheimer’s, MS) and RIPK1 is elevated in them.

Inhibitors of RIPK1 are available and do get into the brain.  As of now two RIPK1 inhibitors have made it through phase I human safety trials.

So it’s time to try RIPK1 inhibitors in these diseases.  It is an entirely new approach to them.  Even if it works only in one disease it would be worth it.

Now a dose of cynicism.  Diseased cells have to die one way or another.  RIPK1 may help this along, but it tells us nothing about what caused RIPK1 to become activated.  It may be a biomarker of a diseased cell.  The animal models are suggestive (as they always are) but few of them have panned out when applied to man.

 

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How to treat Alzheimer’s disease

Let’s say you’re an engineer whose wife has early Alzheimer’s disease.  Would you build the following noninvasive device to remove her plaques?  [ Cell vol. 177 pp. 256 – 271 ’19 ] showed that it worked in mice.

Addendum 18 April — A reader requested a better way to get to the paper — Here is the title — “Multisensory Gamma Stimulation Ameliorates Alzheimer’s Associated Pathology and Improves Cognition”.  It is from MIT — here is the person to correspond to  —Correspondence — lhtsai@mit.edu

The device emits sound and light 40 times a second.  Exposing mice  to this 1 hour a day for a week decreased the number of senile plaques all over the brain (not just in the auditory and visual cortex) and improved their cognition as well.

With apologies to Steinbeck, mice are not men (particularly these mice which carry 5 different mutations which cause Alzheimer’s disease in man).  Animal cognition is not human cognition.  How well do you think Einstein would have done running a maze looking for food?

I had written about the authors’ earlier work and a copy of that post will be found after the ****.

What makes this work exciting is that plaque reduction was seen not only  in the visual cortex (which is pretty much unaffected in Alzheimer’s) but in the hippocampus (which is devastated) and the frontal lobes (also severely affected).  Interestingly, to be effective, both sound and light had to be given simultaneously

Here are the details about the stimuli  —

“Animals were presented with 10 s stimulation blocks interleaved with 10 s baseline periods. Stimulation blocks rotated between auditory-only or auditory and visual stimulation at 20 Hz, 40 Hz, 80 Hz, or with random stimulation (pulses were delivered with randomized inter-pulse intervals determined from a uniform distribution with an average interval of 25 ms). Stimuli blocks were interleaved to ensure the results observed were not due to changes over time in the neuronal response. 10 s long stimulus blocks were used to reduce the influence of onset effects, and to examine neural responses to prolonged rhythmic stimulation. All auditory pulses were 1 ms-long 10 kHz tones. All visual pulses were 50% duty cycle of the stimulation frequency (25 ms, 12.5 ms, or 6.25 ms in length). For combined stimulation, auditory and visual pulses were aligned to the onset of each pulse.”

The device should not require approval by the FDA unless a therapeutic claim is made, and it’s about as noninvasive as it could be.

What could go wrong?  Well a flickering light could trigger seizures in people subject to photic epilepsy (under 1/1,000).

Certainly Claude Shannon who died of Alzheimer’s disease, would have had one built, as would Fields medal winner Daniel Quillen had he not passed away 8 years ago.

Here is the post of 12/16 which has more detail

 

*****

Will flickering light treat Alzheimer’s disease ?

Big pharma has spent zillions trying to rid the brain of senile plaques, to no avail. A recent paper shows that light flickering at 40 cycles/second (40 Hertz) can do it — this is not a misprint [ Nature vol. 540 pp. 207 – 208, 230 – 235 ’16 ]. As most know the main component of the senile plaque of Alzheimer’s disease is a fragment (called the aBeta peptide) of the amyloid precursor protein (APP).

The most interesting part of the paper showed that just an hour or so of light flickering at 40 Hertz temporarily reduced the amount of Abeta peptide in visual cortex of aged mice. Nothing invasive about that.

Should we try this in people? How harmful could it be? Unfortunately the visual cortex is relatively unaffected in Alzheimer’s disease — the disease starts deep inside the head in the medial temporal lobe, particularly the hippocampus — the link shows just how deep it is -https://en.wikipedia.org/wiki/Hippocampus#/media/File:MRI_Location_Hippocampus_up..png

You might be able to do this through the squamous portion of the temporal bone which is just in front of and above the ear. It’s very thin, and ultrasound probes placed here can ‘see’ blood flowing in arteries in this region. Another way to do it might be a light source placed in the mouth.

The technical aspects of the paper are fascinating and will be described later.

First, what could go wrong?

The work shows that the flickering light activates the scavenger cells of the brain (microglia) and then eat the extracellular plaques. However that may not be a good thing as microglia could attack normal cells. In particular they are important in the remodeling of the dendritic tree (notably dendritic spines) that occurs during experience and learning.

Second, why wouldn’t it work? So much has been spent on trying to remove abeta, that serious doubt exists as to whether excessive extracellular Abeta causes Alzheimer’s and even if it does, would removing it be helpful.

Now for some fascinating detail on the paper (for the cognoscenti)

They used a mouse model of Alzheimer’s disease (the 5XFAD mouse). This poor creature has 3 different mutations associated with Alzheimer’s disease in the amyloid precursor protein (APP) — these are the Swedish (K670B), Florida (I716V) and London (V717I). If that wasn’t enough there are two Alzheimer associated mutations in one of the enzymes that processes the APP into Abeta (M146L, L286V) — using the single letter amino acid code –http://www.biochem.ucl.ac.uk/bsm/dbbrowser/c32/aacode.html.hy1. Then the whole mess is put under control of a promoter particularly active in mice (the Thy1 promoter). This results in high expression of the two mutant proteins.

So the poor mice get lots of senile plaques (particularly in the hippocampus) at an early age.

The first experiment was even more complicated, as a way was found to put channelrhodopsin into a set of hippocampal interneurons (this is optogenetics and hardly simple). Exposing the channel to light causes it to open the membrane to depolarize and the neuron to fire. Then fiberoptics were used to stimulate these neurons at 40 Hertz and the effects on the plaques were noted. Clearly a lot of work and the authors (and grad students) deserve our thanks.

Light at 8 Hertz did nothing to the plaques. I couldn’t find what other stimulation frequencies were used (assuming they were tried).

It would be wonderful if something so simple could help these people.

For other ideas about Alzheimer’s using physics rather than chemistry please see — https://luysii.wordpress.com/2014/11/30/could-alzheimers-disease-be-a-problem-in-physics-rather-than-chemistry/

Does gamma-secretase have sex with its substrates?

This is a family blog (for the most part), so discretion is advised in reading further.   Billions have been spent trying to inhibit gamma-secretase.  Over 150 different mutations have been associated with familial Alzheimer’s disease.  The more we know about the way it works, the better.

A recent very impressive paper from China did just that [ Science vol. 363 pp. 690- 691, 701 eaaw0930 pp. 1 –> 8 ’19 ].

Gamma secretase is actually a combination of 4 proteins (presenilin1, nicastrin, APH1 (anterior pharynx defect) and PEN-2 (presenilin enhancer 2). It is embedded in membranes and has at least 19 transmembrane segments.  It cleaves a variety of proteins spanning membranes (e.g it hydrolyzes a peptide bond — which is just an amide).  The big deal is that cleavage occurs in the hydrophobic interior of the membrane rather than in the cytoplasm where there is plenty of water around.

Gamma secretase cleaves at least 20 different proteins this way, not just the amyloid precursor protein, one of whose cleavage products is the Abeta peptide making up a large component of the senile plaque of Alzheimer’s disease.

To get near gamma secretase, another enzyme must first cleave APP in another place so one extramembrane fragment is short.  Why so the rest of the protein can fit under a loop between two transmembrane helices of nicastrin.  This is elegance itself, so the gamma secretase doesn’t go around chopping up the myriad of extracellular proteins we have.

The 19 or so transmembrane helices of the 4 gamma secretase proteins form a horseshoe, into which migrates the transmembrane segment of the protein to be cleaved (once it can fit under the nicastrin loop).

So why is discretion advised before reading further?  Because the actual mechanism of cleavage involves intimate coupling of the proteins.    One of the transmembrane helices of presenilin1 unfolds to form two beta strands, and the transmembrane helix of the target protein unfolds to form one beta strand, the two strands pair up forming a beta sheet, and then the aspartic acid at the active site of gamma secretase cleaves the target (deflowers it if you will).  Is this sexual or what?

All in all another tribute to ingenuity (and possibly the prurience) of the blind watchmaker. What an elegant mechanism.

Have a look at the pictures in the Science article, but I think it is under a paywall.

You might as well watch the Kardashians

You might as well watch the Kardashians.  Reading Shakespeare will not protect you against cognitive decline.  Although you can spindle and mutilate the intellectual cards you were dealt, you can’t play them.  That’s the rather depressing result of from  large (over 1,000 subjects) just in [ Proc. Natl. Acad. Sci. vol. 116 pp. 1832 – 1833, 2021 – 2026 ‘ 19 ].  You have doubtless heard that people who have higher educational attainment, who have had intellectually demanding occupations, who stay mentally and physically active have a lower incidence of Alzheimer’s disease.  This is true, but it’s because they were smarter to begin with.

Before describing the paper please do note that high intellectual attainment (due to high intellectual ability) is not absolutely  protective against Alzheimer’s.  Claude Shannon died of it (https://en.wikipedia.org/wiki/Claude_Shannon), as did a Fields medalist who entered college when I did, as did a classmate who wrote 43 papers testing new drugs.   It does lower the odds though.

There were intimations of this years ago [ J. Am. Med. Assoc. vol. 275 pp. 538 – 532 ’96 ] Catholic nuns ages 75 – 95 were studied. All had written an autobiographical essay at age 22 explaining why they wanted to enter the order.  14 died and some had Alzheimer’s.  The essays were read blind and scored for idea density, grammatical complexity etc. etc. Those with the lowest idea density etc. had Alzheimer’s, while those with the most intellectual complexity were free of Alzheimer neuropathology.  Of the 79 living nuns, the smart ones at age 22 remained smart for the most part at 75+ while the less gifted stayed the same.  This was a select and far from average group — all were college educated and were parochial school teachers for most of their lives.  So the group was controlled for education and occupation.

The PNAS study concerned military recruits (average age 20) entering the service between 1965 and 1975.  The people going in at age 20 were not Ivy League types, who had concocted all sorts of reasons they couldn’t serve.  The Ivy league types going in were JAG officers or Docs like myself, but we were educated and long past 20.  89% were white, 80% did not have combat exposure.

The group was part of the Vietnam Era Twin Study of Aging.  Subjects took the Armed Forces Qualification Test (AFQT) which measures cognitive ability.  Then some 1,237 were  retested at ages 51 – 59 and 1,009 were retested at an average age of 62.

Subjects filled out questionnaires concerning education, job complexity, physical and mental activity etc. etc.

So what was the best predictor of General Cognitive Ability (GCA) at 62?  It was not subsequent education, job complexity, intellectual engagement.  Each of them predicted under 1% of the variance of GCA at age 62.  The best predictor (and not that great) was GCA at 20, which accounted for over 10% of the variance.

Pretty depressing.  You can’t even play the hand you were dealt.

Somehow Princeton undergraduates have found this out and p. 15 of the 6 Feb ’19 issue of the Princeton Alumni Weekly describes the” Kardashian Lifestyle Klub, a registered student organization with about 150 members, meetings and University support“.

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.

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.

Will acyclovir be a treatment for Alzheimer’s ?

When I was a first year medical student my aunt died of probable acute herpes simplex encephalitis at Columbia University Hospital in New York City.  That was 55 years ago and her daughters (teenagers at the time) still bear the scars.  Later, as a neurologist I treated it, and after 1977, when acyclovir, which effectively treats herpes encephalitis came out, I would always wonder if acyclovir would have saved her.

The drug is simplicity itself.  It’s just guanosine (https://en.wikipedia.org/wiki/Guanosine) with two of the carbons of the ribose missing.  Herpesviruses have an enzyme which forms the triphosphate incorporating it into its DNA killing the virus.  Well, actually we have the same enzyme, but the virus’s enzyme is 3,000,000 times more efficient than ours, so acyclovir is relatively nontoxic to us.  People with compromised renal function shouldn’t take it.

What does this have to do with Alzheimer’s disease?  The senile plaque of Alzheimers is mostly the aBeta peptide (39 – 43 amino acids) from the amyloid precursor protein (APP).  This has been known for years, and my notes on various papers about over the years contain 150,000 characters or so.

Even so, there’s a lot we don’t understand about APP and the abeta peptide — e.g. what are they doing for us?  You can knockout the APP gene in mice and they appear normal and fertile.  The paper cited below notes that APP has been present in various species for the past 400,000,000 years of evolutionary time remaining pretty much unchanged throughout, so it is probably doing something useful

A recent paper in Neuron (vol. 99 pp. 56 – 63 ’18) noted that aBeta is actually an antimicrobial peptide.  When exposed to herpes simplex it binds to glycoproteins on its surface and then  oligomerizes forming amyloid (just like in the senile plaque) trapping the virus.  Abeta will protect mice against herpes simplex 1 (HSV1) encephalitis.  Even more important — infection of the mice with HSV1 induced abeta production in their brains.

People have been claiming infections as the cause of just about every neurodegeneration since I’ve been a neurologist, and papers have been written about HSV1 and Alzheimer’s.

Which brings me to the second paper (ibid. pp. 64 – 82) that looked for the viral RNAs and DNAs in over 900 or so brains, some with and some without Alzheimer’s.  They didn’t find HSV but they found two other herpes viruses known to infect man (HHV6, HHV7 — which cause roseola infantum).  Humans are subject to infection with 8 different herpes virus (Epstein Barr — mononucleosis, H. Zoster — chickenpox etc. etc.).   Just about everyone of us has herpes virus in latent form in the trigeminal ganglion — which gets sensory information from our faces.

So could some sort of indolent herpesvirus infection be triggering abeta peptide production as a defense with the senile plaque as a byproduct?  That being the case, given the minimal benefits of any therapy we have for Alzheimer’s disease so far, why not try acyclovir (Zovirax) on Alzheimer’s.

I find it remarkable that neither paper mentioned this possibility, or even discussed any of the antivirals active against herpesviruses.

A pile of spent bullets — take II

I can tell you after being in neurology for 50 years that back in the day every microscopic inclusion found in neurologic disease was thought to be causative.  This was certainly true for the senile plaque of Alzheimer’s disease and the Lewy body of Parkinsonism.  Interestingly, the protein inclusions in ALS weren’t noticed for decades.

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 marker for the cause of death rather than the cause itself.

An earlier post concerned work that implied that the visible aggregates of alpha-synuclein in Parkinson’s disease were protective rather than destructive — https://luysii.wordpress.com/2018/01/07/are-the-inclusions-found-in-neurologic-disease-attempts-at-defense-rather-then-the-cause/.

Comes now Proc. Natl. Acad. Sci. vol. 115 pp. 4661 – 4665 ’18 on Superoxide Dismutase 1 (SOD1) and ALS. Familial ALS is fortunately less common than the sporadic form (under 10% in my experience).  Mutations in SOD1 are found in the familial form.  The protein contains 153 amino acids, and as 6/16 160 different mutations in SOD1 have been found.  Since each codon can contain only 3 mutations from the wild type, this implies that, at a minimum,  53/153 codons of the protein have been mutated causing the disease.  Sadly, there is no general agreement on what the mutations actually do — impair SOD1 function, produce a new SOD1 function, cause SOD1 to bind to something else modifying that function etc. etc.  A search on Google Scholar for SOD1 and ALS produced 28,000 hits.

SOD1 exists as a soluble trimer of proteins or the fibrillar aggregate.   Knowing the structure of the trimer, the authors produced mutants which stabilized the trimer (Glycine 147 –> Proline) making aggregate formation less likely and two mutations (Asparagine 53 –> Isoleucine, and Aspartic acid 101 –> Isoleucine) which destabilized the trimer making aggregate formation more likely.  Then they threw the various mutant proteins at neuroblastoma cells and looked for toxicity.

The trimer stabilizing mutant  (Glycine 147 –> Proline) was toxic and the destabilizing mutants  (Asparagine 53 –> Isoleucine, and Aspartic acid 101 –> Isoleucine)  actually improved survival of the cells.  The trimer stabilizing mutant was actually more toxic to the cells than two naturally occurring SOD1 mutants which cause ALS in people (Alanine 4 –> Valine, Glycine 93 –> Alanine).  Clearly with these two something steric is going on.

So, in this experimental system at least, the aggregate is protective and what you can’t see (microscopically) is what kills cells.

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.

 

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/