P2‐132: EXPRESSION STUDY ON THE EFFECT OF DELETING A MAPT ENHANCER CONTAINING RS242557 IN MICE BRAIN

Abstract

Microtubule associated protein tau encoded by the MAPT gene aggregates in the brain in phenotypically diverse neurodegenerative diseases including Alzheimer's disease (AD), Progressive supranuclear palsy (PSP) and Corticobasal degeneration. rs242557, in MAPT exon 0, is in a FANTOM5 enhancer that correlates with MAPT, STH and CRHR1 promoter expression. This polymorphism is also strongly associated with increased risk for AD, PSP and Parkinson's disease. We were interested in determining whether this enhancer regulates expression of Mapt and its neighboring genes in mice. Established protocols for CRISPR-Cas9 editing were used to delete this enhancer in mice. Whole brain or region specific (cortex, cerebellum and hippocampus) tissues were dissected from adult mice to extract total protein and RNA. cDNA, synthesized from RNA samples, were used for qRT-PCR to study expression of Mapt isoforms and its neighboring genes viz., Crhr1, Nsf and Kansl1. Western blots were also performed on the corresponding proteins. From the multiple CRISPR-edited pups born only one with a 756 bp deletion encompassing rs242557 and the enhancer was selected. This animal was used to generate wild type, CRISPR-edited heterozygous and homozygous mutated mice for further studies. Whole brain extracts from adult mice did not show any significant differential expression in any of the studied genes or their proteins among wild type, CRISPR-edited heterozygous and homozygous mice (n=6 each). Similar experiments were performed to identify any brain region specific effect on hippocampal, cortical and cerebellar samples from the 3 groups. None showed any significant differential expression in Nsf, Crhr1, Kansl1 and Mapt (including 3R, 4R, ex2+3 and all 4 isoforms). Our results show that deleting rs242557 with its two flanking sequences encompassing the enhancer region neither alter the expression of Mapt nor its neighboring genes both at the mRNA and protein levels in mice. Future studies on neuronal cell lines should be performed to address whether this enhancer can modulate MAPT and its neighboring genes’ expression in humans.