Tubulin needs a lot of help from its friends

Our neurons (and us) would be the size of amoebas if weren’t for tubulin which forms the superhighways (microtubules) along which cargo is shipped to the end of axons.   Your average NBA player has axons over 3 feet long going from his sacral spinal cord to his calf muscles.   Split the difference and call it a meter.  Diffusion is way too slow to get anything that far. The trucks schlepping things back and forth on the microtubular highway are called Kinesin and dynein. I think in terms of nanoMeters (10^-9 meters).  Each tubulin dimer is 80 nanoMeters long, and K & D essentially jump from one to the other in 80 nanoMeter steps.

How many jumps do Kinesin and Dynein have to make to go a meter? Just 10^9/80 — call it 10,000,000. Kinesin and Dynein also have to jump from one microtubule to another, as the longest microtubule in our division is at most 100 microns (.1 milliMeter).  So even in the best of cases they have to make at least 10,000 transfers between microtubules.  It’s a miracle they get the job done at all.

To put this in perspective, consider a tractor trailer (not a truck — the part with the motor is the tractor, and the part pulled is the trailer — the distinction can be important, just like the difference between rifle and gun as anyone who’s been through basic training knows quite well).  Say the trailer is 48 feet long, and let that be comparable to the 80 nanoMeters Kinesin and Dynein have to jump. That’s 10,000,000 jumps of 48 feet or 90,909 miles.  It’s amazing they get the job done.

Now that you’re sufficiently impressed with tubulin’s importance, it’s time to see why it needs help.  First a bit of history.  Christian Anfinsen was a Swarthmore football player who happened to win the Nobel prize 50 years ago for his work on the protein ribonuclease, an enzyme.  If you heat it, enzymatic activity is lost (the protein is said to be denatured).  This is because the exact 3 dimensional path of the protein backbone forming the catalytic site of ribonuclease was lost. However if you leave the denatured protein alone (under the proper conditions) it folds back up to the correct 3 dimensional shape.  His point was that the amino acid sequence of the protein was all that was needed to determine ‘the’ three dimensional shape of the protein.  This was at a time when we didn’t know that most proteins have a variety of shapes not just one.

Unfortunately tubulin does not fold up to the shape found in microtubules.  It needs significant help from two friends, prefoldin and TRiC.  TRiC is a monster conglomerate of 2 copies each of 8 different proteins with a molecular mass over 1,000,000 Daltons (e.g. a megaDalton).  What is one Dalton — it’s the mass of a hydrogen atom.   TRiC is made of two back to back rings (with built in lids) each ring consisting of 8 different but related proteins).  Each of the proteins has a domain which binds ATP and a domain which binds the protein to be folded.  There is a central cavity 90 x 90 x 50 Angstroms in size.  Since each hydrogen atom is about 1 Angstrom in diameter, it can fit 405,000 hydrogen atoms inside, or about 200,000 carbons, hydrogens, oxygens and nitrogens — enough room for most proteins.

Prefoldin is equally amazing.  It basically looks like a Portuguese man o’ war — https://en.wikipedia.org/wiki/Portuguese_man_o%27_war.  It is made of 2 copes of one protein and 4 of another.  The tentacles are long alpha helices projecting down from the body.

The tentacles interact with tubulin, carrying it in an unstructured form, thrusting one of its tentacles into the central chamber of TRiC carrying unstructured tubulin with it.   ATP addition leads to lid closure and tubulin encapsulation in the chamber.

A magnificent paper [ Cell vol. 185 pp. 4770 – 4787 ’22 ] describes what happens to tubulin in the TRiC chamber at near atomic resolution.  They are literally watching tubulin fold as it passes from one of the 8 different proteins making up the TRiC ring to another.  The disordered carboxy terminal chains of TRiC are postulated to function as a tethered solvent allowing the intially disordered amino acid sequence of tubulin, to slither into their correct positions more easily.

I’m sure it’s behind a paywall, but if you can look at the figures in the paper, you’ll be bound to be impressed.

So Anfinsen turned out to be wrong, and some 10%  of newly translated proteins turn our to need TRiC’s help.  And yet he wasn’t, because AlphaFold uses only the amino acid sequence of proteins to predict their three dimensional structure.

One further point.  The ancestral bacterial protein for tubulin is called FtsZ.  It happily folds to the correct structure by itself.  However tubulin developed new domains, some of which are for the motor proteins Dynein and Kinesin, and others are for microtubule associated proteins such as tau, the major component of the neurofibrillary tangle of Alzheimer’s disease. These domains are on the surface of the protein, making it harder to fold by itself.

All this information would have been impossible to get 10 years ago, and it’s all due to the sharpening of our technological tools.

Kinetic traps and life

“It is well known that the thermodynamically stable state of proteins in a crowded environment is insoluble fibrils” [ Proc. Natl. Acad. Sci. vol. 119 pp. e2122078119 ’22 ].  However even under ideal conditions the time scale for their formation is hours to days [ Nat. Rev. Mol. Cell Biol. 15, 384–396 (2014) ].  Hopefully it’s even longer (decades) for senile plaques (abeta) neurofibrils (tau) and Lewy bodies (alpha-synuclein) to form.  The fact that equilibrium takes such a long time to reach, allows rapid synthesis and degradation of proteins to avoid their aggregation.  So we live because our proteins are trapped in a less the equilibrium (metastable) state by kinetics — e.g. a kinetic trap.

Will flickering light treat Alzheimer’s disease ? — Take II

30 months ago, a fascinating paper appeared in which flickering light improved a mouse model of Alzheimer’s disease.  The authors (MIT mostly) have continued to extend their work.   Here is a copy of the post back then.  Their new work is summarized after the ****

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/

****

The new work appears in two papers.

First [ Cell vol. 1777 pp. 256 – 271 ’19 ] 7 days of auditory tone stimuli at 40 cycles/second (40 Hertz) for just one hour a day reduced amyloid in the auditory cortex of the same pathetic mice described above (the 5XFAD mice).  They call this GENUS (Gamma ENtrainment Using sensory Stimuli).  Neurologists love to name frequencies in the EEG, and the 40 Hertz is in the gamma range.

The second paper [ Neuron vol. 102 pp. 929 – 943 ’19 ] is even better.  Alzheimer’s disease is characterized by two types of pathology — neurofibrillary tangles inside the remaining neurons and the senile plaque outside them.  The tangles are made of the tau protein, the plaques mostly of fragments of the amyloid precursor protein (APP).  The 5XFAD mouse had 3 separate mutations in the APP and two more in the enzyme that chops it up.

The present work looked at the other half of Alzheimer’s the neurofibrillary tangle.  They had mice with the P301S mutation in the tau protein found in a hereditary form of dementia (not Alzheimer’s) and also with excessive levels of CK-p25 which also results in tangles.

Again chronic visual GENUS worked in this (completely different) model of neurodegeneration.

This is very exciting stuff, but I’d love to see a different group of researchers reproduce it.  Also billions have been spent and lost on promising treatments of Alzheimer’s (all based on animal work).

Probably someone is trying it out on themselves or their spouse.  A EE friend notes that engineers have been trying homebrew transcranial magnetic and current stimulation using themselves or someone close as guineapigs for years.

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.

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

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

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

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

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

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

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

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

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

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

Research on Alzheimer’s disease: the bad news, the good news

The past few months have seen a flurry of work on Alzheimer’s disease.  The news has been both good and bad.  First, some background for the nonMds.

Just as the gray hair on the head of an 80 year old looks the same under the microscope as one from a prematurely gray 30 year old, the brain changes of Alzheimer’s disease are the same regardless of the age of onset.  Alois Alzheimer described the microscopic changes in a youngish person so initially the disorder was called presenile dementia.

There are basically two distinctive changes (1) senile plaques outside neurons (2) neurofibrillary tangles inside neurons.  Note that common to all dementias there is a severe loss of neurons, so something is killing them.   The major protein component of the senile plaque is a 40+ amino acid peptide called the Abeta peptide.  It is a fragment of a much larger protein (the amyloid precursor protein which comes in 3 forms containing 770, 751 or 695 amino acids).  The major protein component of the neurofibrillary tangle is hyperphosphorylated tau protein.

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

Large battles have occurred about the causative roles of tau and Abeta in the Alzheimer’s disease.  As usual, the best evidence is genetics, as there are mutations in these proteins associated with dementia running in families.  Please note that most Alzheimer’s and most dementias are NOT hereditary.  The evidence for Alzheimer causation is strongest for the Abeta peptide and its parent the amyloid precursor protein.  25 of 30 known dominant mutations in the amyloid precursor protein (APP) are associated with Alzheimer’s disease. Over 200 mutations are known in APP and the proteins which process it to Abeta [ Neuron vol. 68 pp. 270 – 281 ’10 — this has a link to an updated database of mutations ].

So an obvious attack on Alzheimer’s disease is to reduce the amount of Abeta present in the brain.   This is unlikely to be curative, as human neurons (as opposed to animal neurons) don’t regrow — the evidence for this is quite good despite publicity to the contrary — See Neuron vol. 74 pp. 595 – 596, 634 – 639 ’12 and references therein.  However, to stop the disease in its tracks wouldn’t be bad at all.

One approach would be to get rid of the accumulated Abeta peptide in the brain. Here’s some bad news.  Unfortunately a trial of antibodies against Abeta, just reported didn’t work.

For details see http://pipeline.corante.com/archives/2012/07/24/bapineuzumab_does_not_work_against_alzheimers.php.  Be sure to read the comments as they are interesting.  There is a lot of discussion of the fact that antibodies are large proteins, which don’t get into the brain  very well (if they get in at all). Also they didn’t see how antibodies would mobilize what is basically the insoluble gunk of the senile plaque.  So this doesn’t damn the idea of lowering Abeta as a way to slow the disease, just the way they did it.

In fact there is an interesting animal model based on a Ayurvedic medicine preparation (yes Ayurvedic medicine !), which DID reduce Abeta peptide in mouse brain.  The animals were said to be getting smarter (but mouse smartness has never impressed me).  The intriguing point is that the reduction occurred by chewing up Abeta in the liver, implying that to work the drug doesn’t have to get into the brain.  Presumably, by le Chatelier’s principle even the most insoluble gunk is in equilibrium with a small amount of soluble material.  For details see

https://luysii.wordpress.com/2012/03/04/could-le-chateliers-principle-be-the-answer-to-alzheimers-disease/

A second approach would be to stop the Abeta peptide from forming.  Since it is a 40  – 42 amino acid fragment of the much larger amyloid precursor protein, an enzyme (a protease) must be breaking APP down to form it.  Actually there are 3 enzymes known which break down APP.  They are called secretases, because most of APP lies outside the cell, and when it is cleaved, part of the protein is secreted.  It would be great if all we had was alpha secretase, as this breaks down APP  right in the middle of Abeta, so it is never formed.  Actually it wouldn’t great at all, because one of the other enzymes — gamma secretase, breaks APP in the middle of the membrane, and the part remaining in the cell (called AICD) has important work to do.  At any rate lots of work is in progress on beta and gamma secretase inhibitors, but impressive results aren’t to be found as yet.

The failure of trials to lower Abeta peptide has led to some doubt as to whether Abeta peptide is really the killer of neurons.  This is where the good news comes in.

Here’s a summary, but the print editorial and paper can be found in the 2 August ’12 Nature (vol.  388 pp.  38 – 39, 96 – 99 ’12).  The quick and dirty is that a mutation has been found in the amyloid precursor protein which PROTECTS against Alzheimer’s disease.  The authors sequenced the APP gene of 1,795 Icelanders, just to look for low frequency variants.  A mutation was found 1 amino acid away from the site cleaved by beta secretase (it changes amino acid #673 from alanine to threonine (written A673T).  When the protein is cleaved this becomes amino acid #2 of the Abeta peptide.

The mutation is far from common — around 1/200 in Scandinavian populations, and even lower in a more heterogeneous North American population.

Then they looked at two groups of people — those with and those without Alzheimer’s disease.  5 times fewer people with Alzheimer’s disease 1/1000 had the mutation than those without, so the mutation in some way is associated with protection against the disease.

What’s going on?  A study in isolated cells shows that the mutation is associated with a 40% reduction in the formation of Abeta peptide from APP.  This makes sense. A different variant at this position (alanine to valine) INCREASES Abeta formation, and is associated with Alzheimer’s.  So this is excellent evidence that APP and Abeta are involved in Alzheimer’s disease.

The news gets better and better, the (rare) variant increased the odds of reaching 85% by 50%.  Then they studied people over 85 living in nursing homes.  41 carriers of A673T had better cognitive function that 3,673 non carriers.

So this gives a lot of hope to the decrease Abeta and slow down or prevent Alzheimer’s disease hypothesis and therapies aiming to do so.  Whether or not doing this in people who’ve already begun to decline from Alzheimer’s disease will be helpful isn’t known.  

Nonetheless, hope in this awful disorder is always welcome.

Now for a social note:  When a study shows a particular therapy doesn’t work, the approach is abandoned.  Not so for therapies targeting society at large.  The war on poverty is now nearly 50 years old, and a recent story said that the incidence of poverty (as currently defined) is approaching a level not seen since the 60’s– http://www.google.com/hostednews/ap/article/ALeqM5gnvKZsBhqzm6Z_4QmbymuHsa2UTw?docId=c2d37e75d61549f382b8b200bf54848c.

As far as I can tell, there have been no calls to abandon the current approach, and try something else.  Changing it will be difficult, I have several family members that poverty has been very good to.  They work in various social agencies to help the poor.  They live quite comfortably.

Could le Chatelier’s principle be the answer to Alzheimer’s disease ?

A recent paper [ Proc. Natl. Acad. Sci. vol. 109 pp. 3199 – 3200, 3510 – 3515 ’12 ] found a way to dissolve the senile plaques in a mouse model of Alzheimer’s disease.  Essentially it uses le Chatelier’s principle to do so, although it is doubtful that Ayurvedic praticioners were thinking along these lines when they first gave an extract of Ashwagandha (Indian ginseng) to improve memory.  As the examples of digitalis and curare show, you ignore folk pharmacology at your peril.  Whole Foods and health food stores certainly don’t and make piles of money as a result, whether or not their nostrums do any good.  The FDA stands by gnashing its teeth as by law it cant’ touch this sort of thing.

First, a bit of background for the nonMDs.  Just as the gray hair on the head of an 80 year old looks the same under the microscope as one from a prematurely gray 30 year old, the brain changes of Alzheimer’s disease are the same regardless of the age of onset.  Alois Alzheimer described the microscopic changes in a youngish person and the disorder firstt came to be known as presenile dementia.

There are basically two distinctive changes (1) senile plaques outside neurons (2) neurofibrillary tangles inside neurons.  Like all dementias there is a severe loss of neurons in addition.  The major protein component of the senile plaque is a 40+ amino acid peptide called the Abeta peptide.  It is a fragment of a much larger protein (the amyloid precursor protein).  The major protein component of the neurofibrillary tangle is hyperphosphorylated tau.

Thinking about pathologic changes and neurologic disease has been simplistic in the extreme.  Both plaques and tangles were assumed to be causative.  However there are 3 possible explanations any microscopic change seen in disease.  The first is that they are causative (which is what everyone had assumed for years).  The second is that they are a pile of spent bullets, that the cell used to defend itself against the real killer.  The third is they are tombstones, the final emanations of a dying cell.

Large battles have occurred about the causative roles of tau and Abeta in the disease.  As usual, the best evidence genetics, as there are mutations these proteins associated with dementia.  The evidence for Alzheimer causation is strongest for Abeta and its parent the amyloid precursor protein.

Now to the paper itself.  Mice were created with mutations known to cause Alzheimer’s disease in man.  They developed senile plaques and were then given Ashwagandha extract.  The plaques got smaller.  This is surprising in itself, as huge attempts have been made to solublilize the aggregated Abeta in plaques and determine its structure without notable success  Lots more work needs to be done, but they think the liver begins chewing up Abeta, forcing the insoluble Abeta in the plaque to solublilze and move to the liver.  Le Chatelier’s principle in action !

The mice got smarter on various tests, but I take all this stuff with a grain of salt.  Animals are smart at doing what they need to do to survive, and running mazes is not one of them.  For that matter, how good would with Newton or Einstein have been running a maze.

The crucial point is the plaques got smaller.  This sort of thing is exactly what the field needs — a slightly different approach.  Immune attacks on the plaques have been a disaster, and inhibiting the enzyme complex which fragments the amyloid precursor protein into Abeta is likely to have other effects, as the complex is responsible for processing many different proteins.

At this point, they aren’t using pure compounds, but extracts of the plant which sure to a mixture.  Stay tuned. First the work needs to be replicated.  Nice to see that most of the work is from India.