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.

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