Adopt an orphan

If PSP is an orphan disease, corticobasal degeneration (CBD) can’t even get into the orphanage.  Like PSP, it’s a “pure 4R tauopathy”; it can resemble PSP in many cases; it leads to disability and death after a similar span of time; and it’s no more treatable.  But its prevalence is about 10-20% that of PSP and it’s very difficult to diagnose in a living person.  People fulfilling the accepted, published diagnostic criteria for the most common type of PSP (PSP-Richardson syndrome) actually have that disease at autopsy in over 90% of cases, but for CBD, the figure is less than 50%.  That makes it hard to recruit a group of subjects for a drug trial — or any research — without other diseases influencing the result.  That has put quite a damper on CBD research.

To add injury to injury, googling “CBD” reveals a lot more about cannabidiol than about corticobasal degeneration.

So, an objective diagnostic test for CBD would be great.  Now, researchers mostly at Washington University in St. Louis (WUStL) and University of California, San Francisco (UCSF) have shown that two tiny fragments of the tau protein are less abundant in the spinal fluid of people CBD than in healthy people or those with PSP or three other rare tau disorders called argyrophilic grain disease, Pick’s disease and frontotemporal lobar degeneration associated with aggregation of TDP-43.  They found no difference between CBD and Alzheimer’s disease or frontotemporal lobar degeneration with mutations in the tau (MAPT) gene, but in practice, those two disorders can be readily distinguished from CBD by other means.

The paper appears in the prestigious journal Nature Medicine and it’s open access, so I can provide you this file to download.  The first author is Kanta Horie, PhD and the senior authors are Chihiro Sato, PhD and Randall Bateman, PhD, all of WUStL. 

Panel “a” shows the tau protein. The four microtubule-binding domains are R1 to R4. The one whose inclusion or exclusion makes the difference between the 4R and 3R tauopathies is R2, which is encoded by the gene’s exon 10. The amino acids are numbered starting at the N terminus on the left. Two short stretches of amino acids, numbers 275 to 280 and 282 to 290, were the object of this paper’s analysis. N1 and N2 are two other sections, encoded by exons 2 and 3, respectively, that can be included or excluded in the finished tau protein.

Panel “b” shows the analysis of the 275-280 fragment of tau in the spinal fluid (CSF). The vertical axis is the ratio of the concentration of the 275-280 fragment divided by the concentration of total tau. The horizontal axis lists the tauopathies analyzed in this project. Each circle is one patient. The “box-and-whisker” plot shows, from top to bottom, the maximum value, the 75th percentile, the median, the 25th percentile, and the minimum value. The asterisks indicate the statistical significance of the comparison between the two groups at the ends of each horizontal line segment. One asterisk is a weak difference and four is the strongest. Pairs of groups without a horizontal line connecting them did not differ (i.e. the p value was greater than 0.05, meaning that any difference between them could have occurred by chance with a likelihood of more than 1 in 20).

Panel “c” shows the same thing, but for the 282-290 fragment of tau. The results are essentially the same as for the 275-280 fragment.

The odd thing is that the same analysis using autopsy brain tissue rather than spinal fluid gave a very different result: The values (i.e., the ratio of the fragment to total tau) was actually higher for CBD than for the other groups. The authors present various theories to explain this, but in any case, it does not detract from the diagnostic value of the spinal fluid results. Take a look and the brain tissue results:

So, what does this mean for people diagnosed with CBD, present and future?  It means that if someone like a drug company has an experimental treatment that might help CBD, they could recruit a group of patients with a high level of confidence that they have excluded other diseases that could confound their results.  That level of confidence is expressed as the “area under the receiver operating curve” or AUC.  A previous post on this blog explains that statistic, which varies from 0.5 for a diagnostic test no better than a throw of the dice to 1.0 for a test that’s perfectly accurate every time.  The AUC for this test to distinguish CBD from those other disorders (other than AD and FTLD-MAPT) is 0.800 to 0.889.  That’s close to the figure for PSP using the neurological history and exam.

If this diagnostic test is confirmed (a big “if”) and enters use by researchers and drug companies, and if a drug company sees a route to profitability in so rare a disease, the only problem is finding enough patients with CBD for a trial.  If CBD is 20% as common as PSP, and the new test for CBD is just as good as the present clinical diagnosis of PSP, then it will require five times the number of participating clinical test sites to fill a trial.  But with international collaboration, it’s do-able. 

Now, let’s hope that this test is adopted and that CBD is adopted. 

A step forward or backward? Let’s vote.

I’m interested in your opinions on this.

An important paper just appeared in the prestigious British journal Brain from researchers in Bordeaux, France and Lausanne, Switzerland led by Dr. Morgane Darricau, a junior scientist working with eight other scientists under senior researchers Dr. Erwan Bezard and Dr. Vincent Planche. 

The work was performed using rhesus monkeys, also called “macaques,” which have been productively and frequently used in research for over a century. The researchers injected abnormal tau protein from patients with PSP into the midbrain of two macaques. As controls, they injected normal tau from the brains of two people whose autopsies showed no neurological disease into the midbrain of two other macaques.  The result was that starting six months later, the first group started to show abnormal control of walking and loss of performance on a cognitive task requiring opening a box containing a treat. 

The deficits progressed, and after another 12 months, the animals were euthanized.  Brain tissue of the two recipients of the abnormal tau showed the same sort of tau aggregation seen in human PSP. Also, crucially, the tau abnormality had spread to several areas known to be connected to the original injection. Those areas — the putamen, caudate, globus pallidus and thalamus — are among the main sites of involvement in human PSP.  They must have received the abnormality from the injection site through axons and across synapses, not by mere proximity. The two control macaques had neither symptoms nor brain abnormalities at autopsy.

Similar experiments have been done with mice over the past decade with similar results, but:

  • The mice did not display the full range of PSP-related brain changes that occurred in the monkeys.
  • The mouse brain’s simpler circuitry and much smaller size do not closely mimic the “environment” in which the abnormal tau spreads in human PSP. 
  • The types of normal tau in the brain, a mix of 3R and 4R, is like that of humans, while normal mice produce only the 3R type.  (“R” is a stretch of amino acids in the tau protein that allows it to attach to the brain cells’ microtubules.  The number is how many such stretches exist in the tau molecule.)  This suggests that macaques and humans share a similar genetic control of tau production.
  • The complexity of monkeys’ normal movements and cognitive processes more closely resemble those of humans, allowing more valid extension of the experimental observations to humans and their diseases.  This complexity also allows a finer-grained evaluation of the effects of the experimental intervention.

The authors point out that while only four macaques were necessary to demonstrate this result, larger numbers would be needed to confirm the findings and to turn this model into a practical research tool.  Once that happens, many research labs the world over could use this technique in studying PSP and testing drugs designed to slow, stop or reverse its progression.

Now here’s the issue at hand:  The last line of the paper is:

“ . . . our results support the use of PSP-tau inoculated macaques as relevant animal models to accelerate drug development targeting this rare and fatal neurodegenerative disease.”

At one level, they are probably right: using macaques in research would bring a cure for PSP faster than using mice.  But some people oppose the use of animals of this level of intelligence in scientific research, no matter the benefit to humans.  I’m interested in your opinion: should macaques be used in PSP research? 

No, I don’t know how many macaques might ultimately be needed.  Nor do I know how much sooner a cure would be found compared to the present practice of using only rodents.  So, try to provide an opinion that transcends those important specifications. 

Please use the “reply” or “leave a comment” feature (whichever your browser shows) below.  Thanks.

Bad for the goose, good for the gander?

A disturbing piece of news this week about an influential 2006 paper in Nature about Alzheimer’s disease.  Turns out it was likely that some of the data in the published version were deliberately faked.  The paper was about beta amyloid, which is not an issue in PSP.  In fact, this could actually be good news for PSP research.  Here’s why:

In the experiments reported in the 2006 paper, researchers at the University of Minnesota Twin Cities used mice carrying a copy of the human amyloid precursor protein (APP) gene with a mutational variant known to cause AD in humans.  (In the normal human brain, the protein product of the APP gene is cut to form beta-amyloid, abbreviated, “A-beta.”)  The researchers allowed the mice to develop cognitive deficits, analyzed their brains, and found a type of small A-beta aggregates never before seen, dubbing them “A-beta*56.”  They extracted the small aggregates, called “oligomers,” and injected them into the brains of genetically normal (“wild-type”) rats, which proceeded to develop AD-like cognitive disabilities. 

Ever since A-beta was identified as a critical player in AD in 1984, researchers had been trying to nail down just what form that protein takes in the process of causing, or contributing to, the disease.  The 2006 paper seemed finally to answer that question and formed the basis for innumerable subsequent experiments world-wide and hundreds of millions of dollars spent by the NIH, philanthropies and drug companies to build upon it in pursuit of an AD treatment. 

The 2006 Nature paper used a commonplace lab technique called Western blot to separate out different proteins from a mixture.  A bit of the mixture is placed on a flat layer of absorptive material and subjected to an electrical field.  The heavier proteins move more slowly than the lighter ones.  The resulting array is exposed to an antibody-based stain that allows it to be seen.  The positions and sizes of the individual protein spots are then analyzed. 

A sample Western blot unrelated to the research discussed here. The numbers on the left are the molecular weights of the proteins in kiloDaltons (one Dalton is the weight of one proton). The leftmost column shows the separation of a standard mixture of proteins of known weight as a benchmark. The other two columns show components of a protein mixture before (middle) and after (right) the addition of an enzyme that cuts proteins.

But now, a whistleblower has reported evidence that some of the Western blot images in the publication and many others from the same lab were placed where they didn’t belong, citing faint lines between blots that could result from cutting-and-pasting.  There was also an instance of two blots with identical size and shape, something with a likelihood approaching zero absent a copy-and-paste operation.  The journal Science hired two scientists unconnected to the Minnesota team to take a look.  They confirmed that deliberate falsification is highly likely, though there’s no smoking gun, which would require access to the original Western blot images or to the original data readouts. Nor, so far, has there been a confession. 

Meanwhile, what’s the upshot?  For the AD field, it means that the treatment trials of anti-A-beta drugs were based on much less laboratory evidence than was thought, possibly explaining why they all failed.  (Aducanumab, the antibody approved in 2021 by the FDA, targets A-beta, but its clinical benefit is highly controversial, Medicare refuses to cover the treatment, and most neurologists opt not to prescribe it.)  That means that by default, anti-AD treatments addressing tau, the other protein aggregating in AD, deserve more attention.

Some experts have questioned the importance of A-beta in AD for decades, but only in the last 15 years or so has AD research into tau as the alternative received serious support.  In PSP, tau is the only protein that consistently aggregates and there’s no evidence of A-beta misbehavior at all.  PSP is therefore considered by many scientists to be a good test bed for anti-tau treatments for AD.  That’s why I think that if these new doubts about A-beta in AD direct attention to tau, an intensification of tau-based PSP research could result, and that could, by extension, benefit AD as well. 

While two anti-tau antibodies have failed to slow the progression of PSP in clinical trials, there are many other ways to address tau in PSP, including one trial currently recruiting and at least two more set to start in the next year. 

So, let’s hope that this week’s revelation gives PSP research a boost and AD research a long-overdue redirection.

Here’s a detailed editorial in Science explaining all of this (without my own speculation about the possible benefit for PSP research). But it’s behind the journal’s paywall and I didn’t want to post the pdf I have access to through my university. That would be another form of dishonesty.

A paradigm shift?

You already know that PSP and CBD are “pure tauopathies,” meaning that tau is the only protein consistently aggregating in the degenerating brain cells.  You also know that Alzheimer’s disease has two such aggregating proteins, beta-amyloid and tau, and that problems in the former seem to induce the problems in the latter.  But now there’s evidence that in PSP there’s a second protein causing the tau problem. It’s called “filamin-A” and if the evidence is correct, it’s a very big deal. 

A research group from several centers in Japan led by Dr. Koyo Tsujikawa of Nagoya University encountered a pair of identical twins with PSP.  They found that each man had multiple copies of a normal region of their X chromosome where 16 different genes reside.  One of those 16, called “FLNA” because it encoded the protein filamin-A, was previously known to play a role in the brain cell’s internal skeleton.  Of course, tau is also involved with the cytoskeleton, so the scientists focused on filamin-A before the other 15 proteins. 

The paper lists 31 authors.  I know two of the senior guys and can vouch that they have produced consistently excellent work for decades. 

Their lab experiments showed that this mutation in the twins and their PSP are cause-and-effect rather than just coincidental. Sorry, but this gets a little tech-y:

  • Autopsied brain cells from the twins with PSP showed not only the excess tau expected in PSP, but also excess filamin-A, and the two proteins aggregated into insoluble clumps in the same brain cells.
  • The twins had tufted astrocytes, a tau-laden feature of PSP brain tissue found in no other disease, and those same cells had abundant filamin-A. This means that this is real PSP, as best we can define it, and not some imitator.
  • Filamin-A levels were normal in autopsied brain samples from people with no brain disease and from brains of patients with CBD, AD, Parkinson’s and dementia with Lewy bodies.
  • In cultured human cells, excessive filamin-A produced by adding an extra copy of the FLNA gene increases the production of tau; and reducing filamin-A production with “silencing RNA” directed at FLNA prevented excessive tau production.
  • Mice engineered to over-produce tau (called “MAPT knock-ins”) did not develop high filamin-A levels, showing that in the direction of causality goes from filamin-A to tau, not the reverse.
  • FLMA knock-in mice produced tau that was not only over-abundant, but qualitatively abnormal as well, with excessive attachment of phosphate groups (“hyperphosphorylation”), an important known driver of neurodegeneration in PSP and the other tauopathies.
  • The genetic abnormalities in FLNA appear to damage tau by interacting with a third protein called F-actin. Genetic abnormalities in F-actin have not been found in PSP, but the function of that protein is impaired by mutations in the gene LRRK2 (“lark-two”), which are over-represented in PSP.  (It was previously known that lab-induced abnormalities in F-actin can cause tau to malfunction in a way that damages brain cells, but there was no reason to think this was relevant to human tauopathies until now.)
  • Among 312 patients with non-familial PSP analyzed in the new paper, none had the same mutation found in the twins (i.e., extra copies of FLNA) but there were 12 patients (4%) with other kinds of mutations in FLNA.  Much lower percentages of FLNA mutations were present in patients with CBD, AD and healthy individuals. 

So, what does this mean?

At the superficial level, it means that some sort of abnormalities in filamen-A could explain tau misbehavior in PSP, just as abnormalities in beta-amyloid abnormalities explain tau misbehavior in AD.  Only a small minority of people (in Japan) with PSP actually have a mutation in the gene for filamen-A, but like any protein, its function may be impaired by many other things such as toxins, trauma, inflammation, and genetic or non-genetic defects in proteins with which it interacts. 

At a more profound level, this new insight could mean that finding the ultimate cause of PSP should start with filamin-A or F-actin even though effective treatments for the diseases could act elsewhere, like with tau itself.  Attacking a disease “upstream,” where the problem starts, is theoretically better than downstream, though the latter is closer to the actual loss of brain cells.

There are a couple of caveats:

  • Mutations in FLNA have long been known to cause a developmental brain abnormality with cognitive delay.  Both twins’ brains had subtle forms of that.  So their PSP may not be a good model for ordinary, non-familial PSP occurring in developmentally normal individuals. 
  • The frequency of FLNA mutations in the 312 Japanese patients with non-familial PSP may not apply to other populations.  The genetic studies of PSP in non-Japanese populations to date have not found a relationship with FLNA, but there are technical reasons for false negatives in that sort of study. 

But these caveats aren’t dealbreakers at all:  Regarding the second issue, remember that rare, atypical, genetic forms of neurodegenerative diseases have in the past provided very valuable insights into the cause of the common, typical, non-familial form of a disease.  For example, in Parkinson’s, 20 members of an extended Italian-American family kindred with young-onset, rapid-progressive PD were found to harbor a mutation in the gene for alpha-synuclein.  On further scrutiny, that protein proved to be central to all PD and trials of anti-alpha-synuclein treatments are under way.  A similarly huge advance in understanding Alzheimer’s disease arose from analyzing the extra chromosome 21 in individuals with Down syndrome (trisomy 21).  A search of that chromosome pointed to the amyloid precursor protein, the source of beta-amyloid, critical to all AD.  In neither PD nor AD does more than a tiny fraction of patients have a mutation in their genes for alpha-synuclein or amyloid precursor protein.

Could we be at the threshold of a similarly radical advance in our understanding of PSP?  Could such a paradigm shift provide targets for a drug to prevent, slow or halt PSP?  We’ll find out — and I hope soon.

Skin is now in the game

Researchers led by Dr. Elena Vacchi of Lugano, Switzerland report new data on the utility of skin biopsies in the diagnosis of PSP and CBS.  This diagnostic approach is further along for Parkinson’s, where the fibers of alpha-synuclein are not difficult to detect in the tiny nerves in skin. The same technique, but for tau, has not been particularly successful for PSP so far, but these researchers did more sophisticated molecular tests. 

They recruited 11 patients with PSP and 4 with CBS, along with 31 with PD, 14 with MSA and 24 healthy controls.  They obtained two cylindrical plugs of skin 3 mm in diameter from the back of the neck and another two from just above one ankle.  They measured the amount of normal and abnormal tau protein and the forms of RNA encoding the most common abnormal tau forms found in PSP and CBD (the 2N4R isoform). 

Comparing the group with PSP or CBS with the group with PD, there was a 90% sensitivity (i.e., the fraction of the patients with PSP or CBS whose biopsy showed an excess of abnormal tau) but only 69% specificity (the fraction of those without PSP or CBS with a normal result) and 0.812 AUC (see the note below).  For the comparison of PSP/CBS versus MSA, the results were better: 90% sensitivity, 86% specificity and 0.900 AUC.  I assume that that’s because of PD’s known tendency to have a little tau aggregation along with its alpha-synuclein, while that happens little in MSA.  For some reason the comparison of PSP/CBS with healthy controls was only moderate, with an AUC of 0.774.  The neck skin proved more informative than the ankle skin. 

The authors point out that their patients’ diagnoses were not autopsy-confirmed.  One solution might be to obtain the skin samples from deceased patients undergoing brain autopsy.  They also point out that the pattern of excessive phosphorylation of tau, which is known to be critical to disease causation, was not considered at all in their otherwise-thorough lab procedures.  So that might improve their results.

An interesting upshot is the authors’ observation that in PD and MSA, there was a reduction of nerve fibers in the skin, while this did not occur in PSP/CBS.  Together with evidence from many other sources, this suggests to them that in PD and MSA, the disease starts in the peripheral tissue (i.e., in non-brain organs such as the skin) and spreads to the brain, while in PSP and CBD, the problem starts in the brain and spreads outwards.

Note: Per Wikipedia, “A receiver operating characteristic curve, or ROC curve, is a graphical plot that illustrates the diagnostic ability of a binary classifier system as its discrimination threshold is varied.”  In other words, how well does a simple positive/negative diagnostic test do in distinguishing true positives from true negatives?  This allows you to optimize the definition of “positive” and “negative” test.  The curve’s vertical axis is the sensitivity or the true positive rate (0 to 1.0) and the horizontal axis is 1 minus specificity or the false positive rate.  The area under that curve (AUC) has a theoretical maximum of 1.0.  Excellent diagnostic tests have an AUC of 0.90 or more, and moderate tests, 0.80 to 0.89.  A coin flip’s AUC is 0.50.

A frozen treat

I wish I could report on some breakthrough in the treatment or prevention of PSP, but that hasn’t happened yet. But what I can do is to report on a major advance in understanding an “upstream” step in the process that’s killing brain cells in PSP. The abnormal tau molecule in PSP has now been imaged.

The prevailing, and probably correct, theory is that brain cells are damaged somehow by tau protein that has folded upon itself, causes its healthy sibling tau molecules to similarly misfold via a templating process. The tau then forms stacks like checkers, called “fibrils,” which are toxic to the cells. Tau doesn’t normally have any folding pattern at all – if it’s not attached to the cells’ internal skeleton, doing it’s normal job, it’s floating around in the cytoplasm (the cells’ internal fluid) like overcooked spaghetti in boiling water. We’ve known for decades that through the electron microscope, the fibrils of PSP look different from those of Alzheimer’s, which in turn are different from those of corticobasal degeneration, which are different from those of Pick’s disease, and so on. We’ve hypothesized that this is because the pattern of misfolding determines the geometry of the stacks of tau, and those stacks determine which cells are attacked, and that determines the patient’s symptoms. But we had no details.

Well, now we do, thanks to a new lab technique called “cryo-electron microscopy” or “cryo-EM.” That’s where the sample is imaged at a temperature cold enough to keep it from wiggling around as much, allowing a far greater visual resolution than was possible using other methods. It still does wiggle a bit, which is why the technique starts by making a video of a tau molecule and then computer-averages the image. The resulting still photo shows the protein molecule’s pattern of loops and folds; even the individual amino acids are visible as fuzzy bumps on the chain. The resolution of the images was 2.7 Angstroms, about the same as the diameter of a water molecule.

Over the past couple of years, researchers applied cryo-EM to tau in a few tauopathies including AD, CBD and Pick’s disease. Only now has PSP been studied by this technology, along with a bunch of much less common tauopathies. The new paper is from an international team led by Michel Goedert, Sjors H. W. Scheres, Yang Shi and Wenjuan Zhang, all of Cambridge University. It was published in Nature, probably the world’s most prestigious and selective biomedical journal.

Misfolded tau from a patient with PSP (Shi Y., et al. Nature 2021)

They imaged tau from 11 diseases, a number large enough to justify a classification system using a few different features such as the number of layered folds and hairpin turns. They found that the folding patterns of the various PSP subtypes were identical except for one case of a rare type, PSP-F, a variant with disproproportionate frontal lobe behavior and cognitive problems. Under the new classification system based on tau folding, the tauopathy most similar to PSP was globular glial tauopathy, a rare cause of dementia diagnosable only at autopsy.

They found the folding pattern of Alzheimer’s tau to be identical to that of primary age-related tauopathy (PART), familial British dementia (FBD) and familial Danish dementia (FDD). The pattern in chronic traumatic encephalopathy (CTE) was similar but not identical to that. Corticobasal degeneration (CBD) was similar to argyrophilic grain disease (AGD) and aging-related tau astrogliopathy (ARTAG). Finally there was Pick’s disease, which was similar to none of the other ten.


Why is all this important? I’ll let the authors themselves explain:

“The presence of a specific tau fold in a given disease is consistent with its formation in a small number of brain cells, followed by the prion-like like spreading of tau inclusions. This may underlie the temporo-spatial staging of disease. Knowledge of the tau folds in the different diseases provides a framework for studying tauopathies that will lead to a better understanding of disease pathogenesis. At a diagnostic level, our findings will inform ongoing efforts to develop more specific and sensitive tau biomarkers.”


By “temporo-spatial spreading,” they mean where in the brain the damage spreads and how fast. So the next big step is to figure out just how the molecular structure presented on the surface of each kind of misfolded tau interacts with healthy brain structures. Once we do that, we can find a monkey wrench to throw into that process – one monkey wrench for PSP and globular glial tauopathy; another for AD and its pals PART, FBD and FDD and possibly CTE; one for Pick’s; and yet another for CBD, AGD and ARTAG.

I’ll try to keep you updated on this, and soon I’ll opine on the collateral question of whether this new work casts doubt on our long-held position that PSP, as a “pure tauopathy,” is a good test-bed for all of the tauopathies, including AD.

Drilling into a gene

So here’s the second of five installments in this fall’s series on CurePSP’s newly funded grants. 

Do you recall that the 2011 publication by CurePSP’s Genetics Corsortium discovered four new places in the genome where a slight variant is associated with slightly greater chance of developing PSP?  The genes containing those variants are cryptically called MAPT, STX6, EIF2AK3 and MOBP.  The first one stands for “microtubule-associated protein tau” and the protein it codes for is, of course, our old frenemy, the tau protein.  Well, when you break the effect down statistically, it turns out that in the MAPT gene, two different variants are each independently associated with increasing PSP risk.  One has been known since 1998 and remained the only known PSP genetic risk factor until 2011.  Its name is the “H1 haplotype.”  (Click on the link for explanatory details. )  The other one, previously unknown, is called only “rs232557.” That variation specifically is the substitution of one “letter” in the genetic code for another, called a single-nucleotide polymorphism, or SNP, pronounced “snip.”  The substitution is probably not itself be the cause of the PSP risk; it’s only a “marker,” a variation that was already present in the MAPT gene in the individual in which the PSP-causing mutation originally occurred. That person then passed both the innocuous old marker variant and the new disease-causing variant together to subsequent generations.  The two stayed together on the chromosome (in this case, chromosome 17) through all those generations because their very close physical proximity means that a break between them during the reproductive process (where sperm cells or ova are made, called “meiosis”) is statistically unlikely. 

Still with me here?  Back to the new grant, where Rueben G. Das, PhD of the University of Pennsylvania proposes to figure out just how the variant revealed by the rs242557 marker increases PSP risk.  We already know that rs242557 is located in an intron of the MAPT (tau) gene.  Introns are long stretches of DNA between the shorter stretches, the exons, which actually encode the structure of proteins.  Introns can do a variety of things, though many of them seem to be just “junk DNA” deactivated by the evolutionary process somewhere between single-celled creatures and ourselves.  But some introns regulate the “expression” of the exons, i.e., the number of RNA molecules the exons produce, which in turn determines the number of molecules of the corresponding protein that the cell makes. 

Introns may also regulate which specific exons in the gene actually get encoded into RNA and which don’t.  That’s relevant for PSP, where nearly all the tau molecules in the neurofibrillary tangles include the product of MAPT’s exon 10, producing “4-repeat” tau.  This contrasts with normal tau in adult human brain, which includes exon 10’s stretch of amino acids on only half of the copies.  The other half are called “3-repeat” tau.

One of Dr. Das’ experiments will simply excise (“knock out”) the variable nucleotide at the rs242557 site.  Others will knock out or change one of the nucleotides nearby.  These experiments will be tried in both mouse and human brain cells.  One of the outcomes the researchers will look for will be the ratio of messenger RNA for the two forms of tau, 3-repeat tau and 4-repear.  A ratio favoring 4-repeat tau would suggest a PSP-causing effect.  Of course, they will also look at the finished tau proteins corresponding to these MAPT gene variants.

Additional readouts will be the messenger RNAs for other genes located in the same general area of chromosome 17 (and their associated proteins) that have bene associated with PSP or Alzheimer’s disease.  Those genes are called NSF, KANSL1, LRR37A and CRHR1.

Dr. Das completed a postdoctoral fellowship (the final stage of training for a lab scientist) at Penn in 2017 under the mentorship of Gerard Schellenberg, PhD, a world authority in the genetics of neurodegenerative disorders.  They continue to work together now that Dr. Das has graduated to Senior Research Investigator at Penn.  Jerry serves on CurePSP Scientific Advisory Board and of course recused himself from the evaluation of this grant application.

Back to science: To create these tiny, targeted changes in the DNA at the rs242557 site, Dr. Das and colleagues will use the new gene-editing technique called CRISPR-Cas9.  Just a month ago, the two scientists most prominent in the development of that technique, Jennifer Doudna and Emmanuelle Charpentier, received the Nobel Prize for that work, which appeared in 2012 and has been called one of the most important advances in biological science in history.   Like many useful techniques in biology and medicine, this one harnesses something from nature, in this case an anti-viral defensive mechanism present in about half of all species of bacteria, an enzyme called CRISPR (clustered regularly-interspaced short palindromic repeats).  The CRISPR protein is coupled with another bacterial protein, Cas9, which can cut DNA.  (“Cas” means CRISPR-associated, and yes, there are at least eight other Cas enzymes.)  The researcher then adds to the complex of CRISPR and Cas9 a stretch of synthetic RNA custom-designed to complement the stretch of DNA targeted for alteration.  That “guide RNA” allows the complex to recognize the DNA site of interest, where the Cas9 proceeds to make a cut. 

If this project is successful and reveals which nearby genes are up- or down-regulated by variants in rs242557, the next steps would be to try to normalize the function of the resulting protein by other means such as conventional drugs. Another approach might reduce the expression of the offending DNA variant by giving an anti-sense oligonucleotide.

This grant is only a one-year project and I’ll report its results once published or otherwise publicly presented.  Stay tuned now for posts on the other new CurePSP grants.

Of spice and dog breeds

Got some catching-up to do.  Since my last blog post about six months ago, I’ve retired from my professorial job at Rutgers, which means I’ve stopped seeing patients.  But I’ll still do clinical PSP trials as a volunteer, so I’ll still see that kind of patient.  Retiring from Rutgers also means that I retire from teaching.  But I’ll continue my work with PSP, so I’ll continue that kind of teaching.  Still working a little on my own research, which is mostly about that cluster of PSP in France.  Then there are all of my non-neurological retirement activities (see spouse for details).  I’ve been neglecting my PSP blog, but that has just changed.

Today a journal article caught my eye.  It found that a slight modification of a naturally-occurring component of turmeric may slow or halt the progression of the tau-based neurodegenerative disorders like PSP and Alzheimer’s disease. At least in cells growing in a dish.

First a little background: You probably know that the brain cells being damaged in PSP contain abnormal clumps of a normal protein called tau.   You also know that the clumps are called neurofibrillary tangles.  In the course of forming the tangles, tau molecules first form smaller accumulations called oligomers (Greek for “a few parts”).  The oligomers are toxic but they’re still soluble in water, like ordinary, monomeric tau (you got it: “one part”).  But the oligomers have an Achilles heel: They tend to form larger clumps, the neurofibrillary tangles, which are no longer soluble in the brain cells’ fluid.  The tangles’ inability to float around and interact with things renders them harmless and means that they serve the useful purpose of taking the toxic, soluble oligomers out of the brain cells’ internal soup.

More background: You’ve heard of turmeric, a popular spice related to ginger.  It’s a traditional remedy for what ails you and some responsible researchers feel that it may actually help certain inflammatory conditions and the metabolic syndrome (high blood pressure, diabetes, abnormal lipids and obesity).  But despite some success in animal models, it has never been proven by modern standards to help any medical condition in actual humans.  Furthermore, we don’t know which of the dozens of chemical components of turmeric explains its apparent benefits.  One minor component of turmeric is curcumin, which by itself is used as a coloring agent in food and cosmetics.  Its chemical structure renders it very difficult to absorb from the digestive tract into the blood or from the blood into the brain.  Here’s the good news: We’ve known for a few years that curcumin, when directly applied to brain cells in a dish, can actually can induce the water-soluble, toxic tau oligomers to form insoluble, harmless neurofibrillary tangles. 

The new journal paper is from neuroscientists at the University of Texas Medical Branch in Galveston and the University of Palermo, Italy.  The group’s leader is Rakez Kayed, PhD of UTMB, a respected and well-published researcher in tauopathies and other forms of neurodegeneration.  The first-named author is Filipa Lo Cascio, PhD, a young post-doctoral trainee in his lab.  They cultured two off-the-shelf types of neural cells and added abnormal tau extracted from autopsied brain tissue from people with PSP, Alzheimer’s disease and dementia with Lewy bodies.  Ordinarily, the abnormal tau would misfold, cause the normal host’s tau to misfold in the same disease-specific way, form oligomers, cause damage, spread to other cells and eventually be taken out of action by forming neurofibrillary tangles.  The curcumin caused the toxic oligomers to more rapidly form tangles, thereby reducing their cell-to-cell spread.  By making the tau less soluble, the curcumin also limited its ability to damage components of the cells with the result of improving the cells’ survival. 

The researchers didn’t actually use curcumin itself, as that compound has no future as a neurological treatment because, as mentioned, it can’t get into the brain. So they tweaked its structure by replacing some hydroxy (-OH) and methoxy (-O-CH3) groups (don’t worry about it) with fluorine atoms.  The resulting compound, which they dubbed CL3, had the same beneficial effect regardless of whether the abnormal tau introduced into the cells was from people with PSP, Alzheimer’s or dementia with Lewy bodies. 

The next step is for another lab to replicate the results using different methods.  At the same time, researchers could try the experiment in mice that have received an abnormal tau gene that forms aggregates and kills their brain cells.  That’s currently the best lab model for PSP and the other tauopathies.  But the multiple examples of drugs that prevented this sort of mouse tauopathy and then failed to prevent PSP in clinical trials show that the mouse models, albeit based on human tau, are inadequate as surrogates for people with real PSP or other tauopathies.

The experiments of Lo Cascio, Kayed and colleagues also confirmed that the aggregates forming from tau taken from people with different tau disorders differ from one another in some other important ways.  That’s the new, hot idea of tau strains – they’re like dog breeds.  They’re all tau, but they differ in some important details and when they reproduce (i.e., by templating their abnormal folding pattern onto normal copies of tau in a host cell), their specific variation continues in the next generation.  More about tau strains soon.

Now let’s see if I can keep this up.

Express yourself, or better yet, don’t.

An original and interesting observation just appeared that might help explain how the known genetic variants associated with PSP might cause the disease.  It has to do with regulating gene expression.

Mariet Allen, PhD, a junior researcher at Mayo Clinic Jacksonville, and colleagues published the paper in the current issue of Acta Neuropathologica.  The senior author is Nilüfer Ertekin-Taner, PhD, who received a grant from CurePSP for this work.  The general idea of using such “endophenotypes” to assess the role of genetic variants in causing PSP has long been proposed by their mentor, Dennis Dickson, MD, a leading neuropathologist, who was also an author of this paper.

They used tissue from 422 brains from the CurePSP Brain Bank at Mayo that had been confirmed as having PSP.  They made three types of measurements in each brain: gene expression (measured as messenger RNA), epigenetic methylation of DNA (measured as CpG islands), and numbers of the various classic PSP micro-anatomic changes that have been known for decades.  They correlated those measurements with whether each case carried the major or minor allele of markers reported in  the genome-wide analysis (GWAS) of single-nucleotide polymorphisms published in 2011 by Hoglinger et al. (Disclaimer: I was a minor co-author on the 2011 paper.)

Without getting too much into the details, the results were that the genetic variants and increased DNA methylation at MAPT (the gene for tau protein) and/or MOBP (the gene for myelin-associated basic protein) were associated with increased expression levels of some proteins not previously associated with PSP.  One such protein was “leucine-rich repeat-containing protein 37A4” or LRRC37A, which is coded at chromosome 17q21.31-q21.32.  The genetic marker status at that location was also associated with increased expression levels and methylation levels in two other proteins encoded by genes at the same approximate location, ARL17A and ARL17B.  (Adenosine diphosphate ribosylation factor-like GTPases are involved in protein transcription and like LRRC37A, are located next to the MAPT gene on chromosome 17.)

LRRC37A appears to be involved in regulating interactions of proteins with other compounds.  Its upregulation is known to be harmful to cells.  Intriguingly, its gene produces a wide variety of alternatively spliced protein forms (where some exons’ protein products are included, others excluded, from the finished protein product) in different people and in different species.  This may suggest that this gene is unstable and could easily be induced to make an inappropriate variant of its protein by a subtle exposure to a toxin or a toxic effect of another gene.

Furthermore, the marker status at the MAPT locus correlated with more intense tau aggregates in the form of coiled bodies and tufted astrocytes, two of the standard diagnostic features of PSP.  This reinforces the idea that tau overexpression is part of the pathogenesis of PSP and that inhibiting that expression could provide prevention.

So as the authors modestly conclude, “MOBP, LRRC37A4, ARL17A and ARL17B warrant further assessment as candidate PSP risk genes.”  All of these associations may suggest new drug targets, but it’s a long slog from there to the clinic.  However, if someone screens a library of existing drugs for their ability to suppress overexpression of these proteins, the path to a treatment could be much, much shorter

Is PSP the route to not just Alzheimer’s but also Parkinson’s?

We’ve known for many years that Parkinson’s disease, which the textbooks call an α-synucleinopathy, has some aggregated tau as well. It appears that each of the two proteins, once misfolded, not only induces its own normal brethren to misfold, it also induces copies of the other to misfold.
The first demonstration of this synergistic misfolding and resulting aggregation came in a series of three papers between 2002 and 2004 from the lab of John Trojanowski and Virginia Lee at Penn. The first authors were John Duda, Bernard Giasson and Paul Kotzbauer. (Disclaimer: I was one of their co-authors on all three.)

Now, Julia Gerson, a grad student in the lab of Rakez Kayed at the University of Texas Galveston, has presented work at the Society for Neuroscience that harnesses that finding of a PD/tauopathy overlap for therapeutic purposes. (Another disclaimer: Kayed has a related grant from CurePSP, where I chair the grant review.)

Gerson and friends created antibodies directed at oligomeric tau, which is tau in small aggregates of maybe 20 or 30 molecules, which are still small enough to remain in solution in the cytoplasm and therefore invisible to light microscopy, unlike mature neurofibrillary tangles. They didn’t want to target normal, non-aggregated tau for fear of disrupting the normal function of that protein, which is to stabilize microtubules.

They injected those anti-tau antibodies into mice that had a copy of a variant of the human gene encoding α-synuclein. The variation was an G209A mutation, producing an A53T alteration in the resulting protein. This is the mutation that my colleagues and I found in 1997 as the cause of PD in a large Italian-American family with autosomal-dominant PD, a finding that first linked PD with α-synuclein. (That’s Disclaimer #3. You’re starting to see why I’m so interested in this new finding.)

The antibody protected the mice against the loss of dopaminergic neurons that the α-synuclein mutation caused in the untreated mutant mice. Mice that received antibody against normal tau did even more poorly than the controls.

So here’s the take-home: Developing an anti-tau antibody for treatment of PSP may also help Parkinson’s. We already expect that it may help Alzheimer’s because that’s clearly a tau disorder. But now, the synergistic toxicity of tau and α-synuclein could also allow a single anti-tau antibody to protect against both Parkinson’s and dementia with Lewy bodies (which also has aggregation of both proteins).

If I were the drug companies, I’d be sitting up and taking notice. Two companies, Bristol-Myers Squibb and AbbVie (licensing an antibody from C2N) have already started anti-tau antibody trials in people with PSP. Others have anti-tau programs in progress.

This new report, which may extend the utility of those products to Parkinson’s, should give that snowball an extra push.