Alzheimer's disease-linked microRNA Exploration of UTR Polymorphisms (AdmiRE-UP)

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Effective treatments are urgently needed to cure or delay Alzheimer’s disease (AD). Major hallmarks of AD are extracellular plaque deposits of amyloid-β (Aβ) peptide, which is derived from the Aβ precursor protein (APP), and intracellular tangles of hyperphosphorylated tau protein. Further, myeloid cells may be involved, such as implied by connections between AD and the TREM2 protein. APP, tau, and TREM2 expression are regulated by non-coding microRNA (miRNA), via targets in their mRNAs. Several single-nucleotide polymorphisms (SNP) associate with AD. We hypothesize that naturally occurring SNPs within the UTR sequences by altering miRNA recognition sites can alter risk and/or progression of AD. We propose to test miRNAs that 1) are experimentally confirmed to alter AD-related protein levels, 2) have experimentally verified targets in these mRNAs, and 3) have perturbed levels in human AD brain. We propose a systematic Alzheimer’s disease-linked microRNA exploration of UTR polymorphisms (AdmiRe-Up) to elucidate effects of these variations. Our innovation is to assess naturally-occurring polymorphisms (SNPs) in miRNA target sites for functional activity in relation to disease. Novel AdmiRe-Up platform rationally winnows candidate polymorphisms through successive stages. Our outcome is to functionally validate known naturally-occurring polymorphisms in confirmed miRNA sequences in the UTRs, employing three specific aims (SA). SA1: Test hypothesis—naturally occurring SNPs alter reporter response to miRNA. SA2: Test hypothesis—naturally occurring SNPs alter target protein levels in response to miRNA treatment. SA3: Test hypothesis—naturally, occurring SNPs alter target protein levels in response to miRNA treatment in human induced pluripotent stem (iPS) cells. The AdmiRe-Up platform requires a SNP be within an miRNA target sequence that 1) has been experimentally confirmed to alter target protein levels; 2) was experimentally validated for sequence; and 3) has perturbed levels in association with AD. Variants will then be cross-indexed with disease genomic variation databases to prioritize already-reported associations. SNPs that pass these criteria will be used for dual-reporter based functional assays. SNPs activity that differ from wildtype will be used as templates to engineer chromosomal mutants in cell cultures. SNPs that pass these stages would then be used in disease-specific iPS cells from clinical sources. We expect each successive step of the process to produce a tightening circle of “active” variants. Those variants that still show significant difference from wildtype APP will then be searched for in the ADNI database for potential associations with AD-associated phenotypes. Within the limits of an R21, proof of the concept will lay a path for rigorous and systematic exploration of functional effects of SNPs in the UTR sequences of uncounted other disease-associated genes. A systematic method, AdmiRe-Up, of testing polymorphism effects on such regulation will accelerate higher-throughput functional analysis and greater understanding of miRNA modulation of disease effects or resistance.