We hypothesize that microRNAs (miRNAs) play important roles in the human brain during healthy aging and in Alzheimer's disease (AD). MiRNAs are small RNAs that regulate gene expression. Most miRNAs act through hybridization with "target" mRNAs. We and others have observed an altered pattern of miRNA expression in AD brain tissue. In various models where miRNAs are reduced, neurodegeneration ensues quickly. The mechanisms of miRNA-mediated neuroprotection and neurodegeneration are poorly understood, partly because most of the mRNA targets of miRNAs are still unknown. Unfortunately, there has been no suitable technique to indicate experimentally which mRNAs are miRNA targets. We developed a high-throughput assay to identify miRNA targets in human brain tissue. Here we propose to use this novel assay to better understand the neurochemistry of AD. Our research program has the following Specific Aims: 1. Obtain brain tissue from the University of Kentucky Alzheimer's Disease Center Brain Bank that is thoroughly characterized including clinical evaluations near death, short post-mortem intervals, and state-of-the-art neuropathology. Clinical cohorts will include brain tissue of non- demented controls, mild cognitive impairment controls, AD, and non-AD dementia controls. 2. Optimize a novel biochemical assay for accurate, specific, and direct miRNA target identification. This assay, the Co-immunoprecipitation MicroRNA Assay Procedure (CoMAP), has been optimized in cell culture and preliminarily in brain tissue. CoMAP will be performed on the brain tissue described in Specific Aim #1. 3. Analyses of CoMAP data will incorporate data from mRNA microarray, miRNA microarray, clinical data, and pathological data referent to the same specimens. These analyses will focus on discovering miRNA targets relevant to AD treatment and diagnosis, and on elucidating the mRNA targets that subserve the neuroprotective functions of miRNA. The raw and analyzed datasets, and the CoMAP assay itself, will be shared freely with other investigators. These Specific Aims are intended to answer the following fundamental questions: 7 What mRNAs are miRNA targets in healthy brain aging? 7 What mRNAs are miRNA targets in Alzheimer's disease and other altered brain states? 7 How do changes in mRNA targets correlate with changes in expression of miRNAs? PUBLIC HEALTH RELEVANCE: The objective of this project is to use a novel technique to better understand the causes of Alzheimer's disease and why some people remain Alzheimer's disease-free during aging. We will characterize in human brains a newly-discovered high impact level of gene regulation, which are called microRNAs, using a method called a Co-immunoprecipitation MicroRNA Assay Procedure (CoMAP). We think that we can produce information that directly or indirectly contributes to diagnostics and therapeutics for Alzheimer's disease patients.