Executive dysfunction, as it were

A major announcement: There’s now evidence that an important factor in the cause of PSP are variants in two “regulatory genes” called PLEKHM1 and KANSL1, both of which are very near the MAPT (tau) gene on chromosome 17. 

Regulatory genes are not encoded into protein.  Rather, they affect the rate at which other genes are transcribed into protein, kind of like business executives or military officers directing the activities of others.  Such proteins are called “transcription factors.” The project identified the gene being regulated in this case as SP1, which encodes the protein called Sp1 (note the lowercase p; the fewer abbreviations in science, the better, I always say). SP1 is located on chromosome 12 and works in tandem with a group of other genes to regulate many cellular processes. 

As a comparator, the researchers used samples from people with Alzheimer’s disease, finding that it involved a different set of regulatory genes that did not interact with SP1.  This result could allow drugs that regulate the Sp1 protein or gene-directed therapy for the PLEKHM1 or KANSL1 genes to be developed as treatment or prevention for PSP. The new finding’s significance is underscored by its publication in Science, one of a small handful of top general scientific journals. 

The research was a collaboration between the highly productive neurodegenerative genetics groups at UCLA and UCSF, with Drs. Daniel Geschwind and Martin Kampmann, respectively, as senior members.  The first author was Yonatan Cooper, an MD/PhD student at UCLA.  In response to my email, Yonatan commented, “A key challenge in modern medicine has been interpreting genetic risk factors [for diseases of complex causation] and translating them to understand disease mechanisms. This is especially true for the 17q tau locus, which is the major genetic risk factor for PSP.  The approach that we used was crucial to allowing us to identify these new genes in that region.” 

The “approach that we used” in Yonatan’s quote is called a “massively parallel reporter assay” (MRPA).  It assesses thousands of single-nucleotide DNA variants for their ability to regulate other genes’ protein expression levels.  For this study, the researchers chose the region close to the two variants in or near the MAPT gene found in 2011 by the PSP Genetics Consortium and funded by CurePSP.  That region comprises hundreds of genes, any of which could be actual source(s) of the PSP risk in that region of the genome.

That 2011 study used “single-nucleotide polymorphisms (SNPs)” in DNA from autopsy-confirmed PSP brain tissue and control subjects.  Even now, 11 years later it remains the most informative single study on the genetics of PSP.  (Disclosure: I was a minor co-author.)  It identified a handful of regions across the genome where a marker differs between the two groups, implying that a gene near each marker contributes to the cause of PSP.  Two of those markers were in or near MAPT.  But that technique can’t positively incriminate which of the hundreds of genes in that region of chromosome 17 actually cause(s) the disease.  The current study did that by testing all of the thousands of variants in those genes for their ability to alter protein production in a way that could help cause PSP. 

One next step is to look for drugs or other treatments to counter the effect of these two regulatory abnormalities on the function of the SP1 protein and/or a protein with which it interacts.  Another next step is to use the same technique to look for other regulatory genes near the other PSP-related markers identified in the 2011 SNP study and more recently and to look for drug targets in the protein products of the genes they regulate.

I don’t know about you, but I’m nerdy enough to be very excited about this!

Is PSP genetic? An update

Last week, someone wrote to CurePSP asking if PSP was genetic. I took a look at what I had previously provided CurePSP on that topic to post on its website, and decided it wasn’t nearly detailed enough. So I decided to write up the following. A version of it appears, or will soon appear, at http://www.curepsp.org.

PSP only very rarely runs in families.  Fewer than one in 20 people with PSP knows of even one other family member with PSP, even counting distant cousins. 

But when multiple genetic variants confer only small risks of developing a disease and some sort of non-genetic factor is also necessary, it will be rare for more than one member of a family to have the unlucky co-occurrence of enough of those factors to produce outward signs of the disease. 

That’s basically how PSP works, but then things get a little more complicated:

The gene on chromosome 17 that encodes the tau protein is called MAPT, for “microtubule-associated protein tau.”  The MAPT gene has two variants that are more common in PSP than in the rest of the population.  One of them is called the H1 haplotype and actually consists of a section of the chromosome that is reversed relative to adjacent sections.  About 95 percent of people with PSP have this variant on both of their copies of chromosome 17, while this is true for only about 60 percent of the rest of the population.  So the H1 haplotype is (nearly) necessary but far from sufficient to cause the disease. 

We’re still not quite sure how the H1 haplotype increases PSP risk.  It may simply increase the amount of tau produced, which causes that protein to stick together, even if it’s structurally normal.  But more recent work shows that it causes too many methyl groups to stick to the MAPT gene, altering its function. This is exciting because drugs can be developed to alter DNA methylation. Other recent evidence supports the idea that the H1 haplotype reduces the fraction of tau molecules that include the fragment encoded by the MAPT gene’s exon 2. 

The other MAPT variant associated with PSP is statistically independent of the H1 haplotype and its function is unknown.

Over the past two decades a handful of other gene variants not on chromosome 17 have been found to be slightly more common in people with PSP than in those without PSP.  These genes help control a variety of critical processes such as disposal of damaged proteins, inflammatory mechanisms, operation of synapses, and integrity of the brain cells’ insulating sheaths.  However, the effect of these genes, individually or together, is too small to serve as a diagnostic test for the disease or to produce more than one case in a family.   

A gene called LRRK2 has been found to influence (in a rough way) not the likelihood of PSP, but the age at which it starts.  CurePSP is presently supporting a project to pursue this clue to try to find a blood test that might predict the individual’s rate of progression.  As it happens, mutations in LRRK2 are the most common cause of familial Parkinson’s disease and the occasional person with that mutation will have the pathology of PSP at autopsy despite having had the outward appearance of PD during life.  Wonders never cease.  Drugs that suppress the action of abnormal (and normal) LRRK2 are in trials for Parkinson’s.

Despite all I’ve said about the genetic component of PSP being subtle, a small fraction of people with PSP do have a relative with the same diagnosis, raising questions about the risk to their siblings and children.  A few points of advice about that:

  • When a disease occurs in several members of a family in a pattern consistent with either a dominant or a recessive mechanism, it’s easy nowadays to identify that gene.  Despite the dozens of families alleging multiple members with PSP, such a gene has never been reported in the literature. 
  • False-positive diagnoses of PSP are common.  This may account for most of the reports of multiply-affected families, even if one of them had autopsy confirmation.  However, in most situations where two or more relatives have been diagnosed with PSP, there have been no autopsies.
  • A strongly familial disorder called frontotemporal dementia with parkinsonism (FTDP) can mimic PSP, even at autopsy, but the special features of PSP such as balance loss and trouble with downgaze are mild or absent.  Many of the mutations causing this disorder are in the MAPT gene, but those mutations do not occur in non-familial PSP.  Furthermore, FTDP is associated with the MAPT’s H2 rather than H1 haplotype.  Both of these points cast additional doubt on FTDP being real PSP.  The FTDP-associated mutations can be detected by a commercially available blood test with a doctor’s prescription, but they are very rare, with only about 100 such families having been reported in the medical literature world-wide.
  • Despite those caveats, there actually are two or three families world-wide having several members with ordinary PSP (i.e., not FTDP) both during life and at autopsy, with no mutations in the MAPT gene.  Such families can be highly valuable for PSP research, as the gene causing their disease could be encoding a protein that might be key to all PSP.

Bottom line: 

Familial PSP is so rare that people with that condition need not be concerned for their children or siblings.  This advice even accounts for the possibility that what has been diagnosed as PSP may in fact be its rare, familial imitator, FTD with parkinsonism.  Most PSP experts advise their patients’ healthy relatives to make no changes to plans for career, children or finances because of one person with PSP in the family. 

However, when there is a clear indication of two or more close relatives with PSP, one should consider testing one affected person for FTDP by sequencing either the MAPT gene or a battery of genes associated with various dementing neurodegenerative diseases.  This should be done only with the guidance and participation of a genetics counselor or neurologist well-versed in interpreting genetic testing.  If the affected patient has one of those mutations, then another affected relative can be tested as confirmation and healthy relatives can be tested for the same specific mutation if they so choose.  However, a positive result would not predict the age of symptom onset, so there is little or no actionable information to be gained through testing healthy relatives.

Further research results in the near term could change these recommendations, so keep an eye on http://www.curepsp.org for updates.  But if you want me to speculate right now, take a look at the next post.

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.

A family matter

Want to know what’s hot in lab research on PSP?  Or, more accurately, want to know what will be hot in a year or two?  This week, CurePSP will announce its four newest research grant awardees.  Most of the 20 competing applications, a very large crop for CurePSP, were of excellent quality and in a less competitive cycle many of them would have been funded.   A fifth and possibly a sixth application may be funded next month after CurePSP’s leaders have had a chance to discuss the use of a new, unexpected, seven-figure donation.

Since I’m driving this bus, I’ll start with the funded grant I consider the most intriguing, though it’s the smallest of the four.  For two decades, researchers at the University of Southern California have been following a Mexican-American family with PSP in 14 members over three generations.  Two of the 14 have had autopsies confirming the diagnosis.  The inheritance pattern is most likely autosomal dominance (look it up).  There are six living, affected members and another 19 who are at 50% risk because they have an affected parent or sibling.  One of the affected members has had sequencing of the gene that encodes tau (called the MAPT gene).  That revealed no mutations.  Now, John M. Ringman, PhD and his USC colleagues plan to sequence the entire genome of four affected and one unaffected family members.

It’s entirely possible that the result will be a mutation in one of the half dozen or so genes besides MAPT that have already been identified by other methods as conferring a slight risk of developing PSP.  That wouldn’t be so exciting, though it would show that one mutation in that gene suffices to cause the disease while other mutation(s) in the same gene only raise PSP risk slightly.  That would shed light on just how that gene works with respect to PSP.

A more exciting result would be if the culprit gene in this family turns out to be one that has not been previously associated with PSP.  Even though this particular mutation would clearly not be the cause of “regular” PSP, perhaps the protein that this gene encodes will prove to be part of a molecular pathway critical to the pathogenesis of PSP but not yet investigated carefully.  That could point to scads of new treatment targets for drug developers and maybe even a diagnostic test.  Very cool.

I led a project like this on Parkinson’s disease back in the 1980s and 1990s, though we didn’t have whole-genome sequencing then.  I won’t get into the details, but you can read about the big family I found and worked with here, my subsequent clinical analysis of the family here, the report of the culprit gene here, the discovery of its significance to PD in general here, the development of a diagnostic test based on the gene’s product here, the efforts to prevent PD in lab models by reducing the gene’s product here and an initial safety report on a Phase 1 human trial here.  Maybe that’s why I find Dr. Ringman’s little project so intriguing.

More on the other new grantees in the next post.

PSP by the Bay

Just returned from the annual CurePSP International Research Symposium, held this year in on the campus of University of California San Francisco on October 27.  About 120 researchers attended, many from Europe and Japan.  The first keynote speaker was Bruce Miller from UCSF, perhaps the country’s leading behavioral neurologist, who gave an overview of PSP/CBD research with an emphasis on activity of the Tau Consortium, the multi-institutional research group that he directs.  The other keynoter was Robert Stern, a neuroscientist at Boston University who directs clinical research at BU’s Chronic Traumatic Encephalopathy Center.

Bruce’s talk touched on many topics — from the nosology and pathology of the various cognitive/behavioral syndromes in the tauopathies to the sleep disturbance in PSP (hyperarousal is common in PSP, while hypoarousal predominates in CBD).  Perhaps most interesting was his up-to-the minute summary of the state of tau PET imaging in PSP diagnosis (not nearly ready for prime time, though potential exists).

Bob’s lecture summarized the story of CTE.  He emphasized that the most important frequent cause isn’t concussions, but the continual sub-concussive blows to the head such as those suffered by football players during routine blocking and tackling.  He was too smart to speculate much about a relationship between the tauopathy of CTE and that of PSP, but I’m not:  I’ll say that in both cases, individuals with a genetic predisposition to tau aggregation are exposed to a precipitating factor – repeated brain tissue stretching for one, some sort of toxin for the other.  If we can find the genetic background for one, we may find it for the other.

Perhaps the genetic answers will emerge from the whole-exome sequencing project that is complete and in the writing phase or the whole-genome sequencing project that is well under way. But as pointed out in another Symposium talk by Jerry Schellenberg, the U Penn geneticist who heads those efforts for the PSP Genetics Consortium, there’s a lot of “genetic dark matter” in the form of genomic deletions, undetectable by mere sequencing.

Maybe in future posts I’ll get more into the other excellent CurePSP Symposium talks – and the 18 concomitant poster presentations.  Or maybe I’ll get distracted by a random shiny object I find somewhere else.

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

Road test for a Roadmap

This is a request for your suggestions to improve/amend CurePSP’s research plan, which is now two years old.

Here’s a link to the first and current version of our Research Roadmap:  Roadmap for Symposium 2013. You can also find it here.

It was designed as a guide to grant applicants and donors who want to know what CurePSP is interested in funding.  That’s not to say that we wouldn’t fund other things, but proposals that fit into the Roadmap’s model are viewed more favorably in our grant review process.

The elevator explanation of the model is that it uses unbiased gene searches to identify new risk genes, then finds drug targets among the proteins in the related gene products or cellular pathways, then tests those drugs in lab models, then turns to Pharma to develop those drugs clinically.  Along the way, it calls for new models and new clinical markers to assist in the process.

Clearly, The Roadmap ignores important things such as symptomatic treatments, toxic etiologies, clinical characterization, epidemiology and neurophysiologic analysis, not to mention serendipitous neuroprotective treatments with unclear mechanism.  But it provides a focus and an orientation.

So please use the Comments function to leave me your suggestions for improvement.  Keep in mind that the Roadmap should remain relatively simple and generic.  We don’t want to direct research from the top down.  On the other hand, we don’t want the document to be so generic as to be useless.

Obviously, feel free to respond to others’ comments; and have fun!

 

A new PSP genetic risk factor screener

Genetic screening is emerging as a routine and necessary step in clinical research in the neurodegenerative diseases. If you’re looking for the cause of a family cluster, for example, you have to rule out the genetic variants already known to be associated with that disease. If you’re working up a geographical cluster of PSP, as my colleagues in France and I are, you have to look for a genetic founder effect before embarking on a difficult search for environmental causes, and the place to start is with gene variants already known to increase disease risk.
Pathological overlap among the various neurodegenerative diseases is another major current theme. For example, LRRK2 mutations can cause any of a number of pathologies, including PSP, and the tau H1 haplotype is associated with PSP, CBD and PD. It would therefore be convenient to have a single genetic screening device would allow different labs studying different diseases to compare or merge results.
Such a gizmo is now here. It’s a superset of Illumina’s Infinium HumanExome BeadChip called NeuroX. It tests for not only the standard 242,901 gene variants usable in studying any condition but also an additional 24,706 variants focusing on Alzheimer’s, Parkinson’s, MSA, ALS, FTD, multiple sclerosis, Charcot-Marie-Tooth disease, myasthenia gravis — and PSP. The chip is designed to allow easy substitution of subsequent versions of both the basic Illumina chip and easy addition of new neurological variants.
The first author of the report in Neurobiology of Aging is Mike Nalls and the senior author is Andrew Singleton, both of the NIH. The genetic variants included in the chip were derived from multiple genome-wide analyses over the past 20 years. Disclosure: I’m listed way down on the list of “authors” because I was a leader of “GenePD,” one of the consortia whose findings were used in constructing the new chip.  But I have no financial interest in the invention.
At a cost of $57 per sample plus the cost of the basic machine and technician time, you won’t have to be a drug company or the NIH to afford a statistically meaningful series; genetics core labs will be able to offer this as a routine procedure.

Is DNA methylation the key?

Very cool paper in PLoS Genetics this week reporting alterations in DNA methylation in PSP.  It’s from Giovanni Coppola’s lab at UCLA, with Yun Li as first author and collaborators from UCSF.  They used Illumina probes to profile DNA methylation genome-wide in WBCs.  The result was that in PSP, MAPT showed more methylation than controls or subjects with FTD.  But the same was true for three other genes near MAPT: KIAA1267, ARHGAP27 and DND1.  All lie within the H1 haplotype block, an inversion spanning 1.8 Mb and 48 genes at 17q21.31.

A new statistical technique called “causal inference” suggested that something in the H1 haplotype caused the differential methylation, which in turn caused the PSP phenotype.  They conclude that a quantitative trait locus for methylation exists within the H1 haplotype, but that differential methylation is a characteristic of H1 independent of the presence of PSP.

A supplemental experiment looking for differences in gene expression correlating with methylation changes came up empty, unfortunately.

So now we have evidence that the pathogenetic mechanism of the H1 haplotype is differential methylation of MAPT and/or nearby genes.  Work by others has suggested that H1 operates, rather, by increasing MAPT expression, but that observation is not consistently replicated.  Either way, we still have to explain what else is necessary to the etiology of PSP.  After all, H1 is present in 95% of subjects with PSP aut also in a majority of the rest of the population.

Do any geneticists out there have any special insights to share?