Tag Archives: proteomics

The Matrix

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You’ve probably heard about the disease called frontotemporal dementia (FTD).  It produces many of the same cognitive and behavioral symptoms as PSP, but more intensely, with much less of PSP’s other symptoms.  Familial forms of FTD are much more common than in PSP, amounting to about 20% of all cases.  Of those, about a quarter have a mutation in their MAPT gene – that’s the one that encodes the tau protein.  (Most of the rest have mutations in two obscure genes called “C9orf72” and “progranulin.”)  As for nearly all neurodegenerative diseases, a protein aggregates in the affected brain cells in FTD and that protein corresponds to the specific mutated gene in that individual.  So, MAPT-associated genetic FTD is a tauopathy very much like PSP, but in a slightly different set of brain cells.

Neurologists at UCSF and the Mayo Clinic are leading a large, multi-institutional observational study of FTD called “ALL-FTD.”  They’re gathering histories, neurological exams, imaging, skin biopsies, blood, and most relevant here, spinal fluid.  They re-gather much of this every year and track the patients’ progression.  The idea is to find diagnostic markers not only to diagnose the disease, but to predict its onset in healthy mutation carriers and to predict the progression of the disease in those who already have symptoms.  All of this can be useful in designing clinical treatment trials and in patient and family counseling.

Using spinal fluid from 116 people with FTD mutations and 39 controls, the ALL-FTD neurologists analyzed the levels of over 4,000 proteins.  They found some of the proteins increased or decreased as functional modules.  That means that subgroups of the affected proteins tended to share a common function in the brain. Then they tested spinal fluid from people with ordinary, non-familial PSP-Richardson syndrome for the same protein disturbances, and found some.  In fact, all 31 of the functional modules disrupted in genetic FTD were disrupted in PSP as well. (This research article is posted on a site for manuscripts not yet peer-reviewed called “Research Square.”)

Mind you, this doesn’t mean that non-familial PSP is actually caused by genetic mutations in the three FTD-related genes.  We still don’t know the initial cause of PSP.  But the study does show that familial FTD and non-familial PSP share some very fundamental similarities. 

The most important modules disrupted in the two conditions relate to the brain cells’ “extracellular matrix.”  That’s the soup of chemicals outside and between the brain cells that serves many protective and nutritive functions.  I suspect that it descended from the coat of slime secreted by our ocean-dwelling, single-celled ancestors.  In ourselves it functions in cell growth, fetal development and injury repair as well as providing physical protection against trauma and a trap for nutrients floating by. It’s easy to see how a genetic or non-genetic defect in the contents our brain’s extracellular matrix could be a problem.

So, let’s add the extracellular matrix to our list of potential drug targets in PSP.

Seeds of a revolution?

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Decades ago, the discovery that specific proteins aggregated in the brain cells of specific neurodegenerative diseases was a major advance.  But like so many other scientific breakthroughs, it created another question: Why are there so many different clinical pictures among different people with the same neurodegenerative disease (like PSP) despite the fact that they all host the same aggregating protein (in this case, tau)? The ability of abnormal tau to “seed” the disease process into previously healthy brain areas is at the root of the disease process, but we’ve had scant clue as to how that works, exactly.

For PSP, the most important clinical variable is the eight subtypes (PSP-Richardson’s syndrome vs PSP-Parkinsonism vs PSP-progressive gait freezing, etc), and slightly less variable features are the onset age and rate of progression.  In the past year or two, it’s become clear that the different subtypes tend to emphasize different areas of the brain, but that doesn’t explain why two people with the same subtype can have different onset ages and rates of progression.

This mystery became even more mysterious recently when a new electron microscopy technique called “cryo-EM” proved able to visualize individual protein molecules. It showed that for everyone with a given disease, the protein for that disease had the same misfolded shape.  In other words, the tau molecule assumes the same rigid squiggle in everyone with PSP, a different rigid squiggle in everyone with Alzheimer’s, yet another in everyone with corticobasal degeneration, and so on.  But that raised the question as to the reason for the variability among patients of the PSP onset age and rate of progression.

Now, researchers at the University of Toronto’s Rossy Centre, an institution dedicated solely to PSP research at the , have found new evidence supporting the old idea that the key may be in the “oligomers” or “high-molecular weight tau” or “HMW tau.”  These are stacks of tau protein molecules small enough to remain dissolved in the brain’s fluids, as opposed to single molecules or the large, insoluble neurofibrillary tangles visible through a conventional microscope. 

The top-line result was that the patients with more rapidly-progressive PSP and brain regions with the worst damage had higher levels of HMW tau.  In a tour-de-force of lab experiments, the Toronto researchers also showed that:

  • HMW tau was more resistant to the brain’s mechanism for breaking down such protein clusters.
  • The study’s 25 PSP patients could be divided into high-, medium- and low-seeders based on the speed with which their tau converted healthy tau to their own misfolded form.
  • Tau with phosphate groups attached to amino acids 202 and 205 were least likely to form the HMW tau clusters.
  • The pattern of production of proteins (i.e., the “proteomics”) in the brain areas rich in HMW tau showed disruption of the brain’s adaptive immune system and two other cellular systems previously known to be related to neurodegeneration.

The importance of all this is that we now have a more specific idea of the structure of the most toxic form of tau aggregates and that boosting the brain’s adaptive immune system with medication could discourage the seeding of misfolded tau into healthy cells.

The study’s first author, Dr. Ivan Martinez-Valbuena, published an editorial in the journal Brain Pathology explaining all this in language that non-specialist scientists can understand.

The research paper itself is posted by the authors in bioRxiv (“bio-archive”) an on-line, open-access website for articles awaiting word from the peer-review process at a conventional journal. Its senior author is Dr. Gabor Kovacs, one of the world’s leading neuropathologists in the field of neurodegenerative diseases.

Proteomics hits paydirt

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If you know anything about PSP at its molecular level, you know that the tau protein in the neurofibrillary tangles is almost entirely of the “4-repeat” or 4R variety.  The other kind is “3-repeat” or 3R.  Normal adult human brain has equal amounts of 3R and 4R.  So do the tangles of Alzheimer’s disease.  But the tangles of Pick’s disease are 3R. 

The thing that’s repeating is the section of the protein that binds it to microtubules, the brain cells’ internal skeleton and monorail system for transporting chemicals along axons.  The gene encoding tau, called the “microtubule-associated protein tau” (MAPT) gene, has four sections, called exons, each encoding one microtubule-binding repeat.  MAPT has 16 exons and the four in question are exons 9, 10, 11 and 12.  4R tau includes the repeat encoded by exon 10 and 3R tau doesn’t.

There’s pretty good evidence that in PSP, the extreme imbalance of 3R and 4R tau is a major factor in making the tau toxic to brain cells.  But why can’t the brain cells in someone with PSP make enough 3R tau?  In other words, what prevents the brain cells in PSP from excluding the repeat from exon 10 half of the time, as normal brain cells do?

In the latest issue of the journal RNA Biology, a group mostly from McMaster University in Hamilton, Ontario, Canada report at least part of the answer.  They have found that the protein called “heterogeneous nuclear ribonuclear protein C” (hnRNPC) prevents the normal process from happening by binding to messenger RNA.  hsRNPC has been known for years in relation to certain types of cancer, but its role in the brain or in neurodegenerative disease was not previously well studied.  To accomplish this work, the team developed a new technique called “RNA antisense purification by mass spectroscopy” (RAP-MS).  They also found, critically, that in PSP brain, the level of hnRNPC is abnormally elevated, an important confirmatory observation.  (Why is it elevated?  I can’t wait for the next installment in this story!)

The authors point out that hnRNPC can now be considered a target for drugs to slow or halt the progression of PSP.  Pharmaceutical companies, take note.

The senior author of the study and lab head at McMaster was Yu Lu, PhD, a specialist in proteomics.  His grad student Sansi Xing was first author.  The team included others from McMaster, the University of Iowa in Iowa City and Mount Sinai in New York. 

A clue from proteomics

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The annual conference of the International Parkinson and Movement Disorders Society (“MDS”) is in progress this week on line.  The location of this meeting normally migrates from city to city world-wide and this year was supposed to be Philadelphia.  Nice city to visit – great history, great art, great restaurants (both fancy and ethnic).  Oh, well.  One of our many sacrifices to the pandemic and all things considered, not a serious one.

Of the 1,000 posters reporting new research, 17 were on PSP.  One that sounds very interesting is from Hiroshi Takigawa and colleagues at Tottori University in Yonago, Japan.  They did a proteomic survey of cerebrospinal fluid (CSF) from people with PSP, Parkinson’s, corticobasal syndrome and some healthy, age-matched volunteers.  Proteomics is a generic term for big-data studies of all the proteins in a biological samples, just as genomics is the study of all the genes.  In this case, they compared the collection of thousands of CSF proteins among the four groups listed and found that the only one that’s higher, on average, in PSP relative to the other three to a statistically significant degree is something called chromogranin B.  They also found that a small fragment of the 657-amino acid chromogranin B protein was the only protein (or fragment thereof) that was less abundant in CSF in PSP, on average, than in the other conditions. The fragment, which is only 31 amino acids long, is called bCHGB-6255.

For neither of these findings was the magnitude or consistency of the difference enough for use as a diagnostic test at the individual level.  (Statistical digression: For the biostatisticians among you, the area under the ROC for bCHGB-6255 was only 0.67.  For the rest of you, the receiver operating characteristic is a graph comparing the likelihood of true positives with that of false positives for the full range of possible definitions of an abnormal level.  The area under the ROC, if the each axis of the graph goes up to 1.00, has a theoretical maximum of 1.00, in which case there’s no risk of false positives in exchange for full identification of the true positives.  A result of 0.80 is barely acceptable for a test to be useful at the individual level and 0.90 is preferred.)

The value of the finding is the demonstration that chromogranin B might have something to do with the degenerative process underlying PSP but none of the related diseases.  Furthermore, the inverse relationship of the full chromogranin B molecule and its bCHGB-6255 fragment suggests that there’s something about the fragmentation process that may be uniquely important to PSP.  Maybe an enzyme that cleaves chromogranin B is deficient, damaged or suppressed in PSP.  Only further research will work that out.

What does chromogranin B normally do?  We don’t know.  It’s present in a wide variety of brain cells that use norepinephrine as their neurotransmitter and also in many cells in other organs.  It’s somehow associated with the secretion of norepinephrine and its blood levels are known to be elevated by certain tumors.  Tests for it are available from commercial medical labs.  But as I emphasized above, the test would be diagnostically useless at the individual level.

Most of the presentations at important meetings like the MDS are research that has not yet passed peer review, or at least not yet published.  So you have to take it with a grain of salt.  Of course, the same thing can be said for any research that has not been confirmed by other labs using other methods.  And even then . . . I’ll tell you about other interesting MDS posters in the next few days.