DECODING LOAD-GWAS DISCOVERIES: GENE EXPRESSION CHANGES IN SINGLE NEURONS THROUGHOUT LOAD PATHOLOGICAL PROGRESSION

Abstract

In the post genome-wide-association-studies (GWAS) era we are shifting gears toward translation of genetic disease loci to molecular mechanisms of pathogenesis. Large multi-center GWAS have found associations between >20 genomic loci and late-onset Alzheimer's disease (LOAD), and candidate genes were inferred by the proximity to the associated-SNP. However, the precise target genes within the LOAD-associated genomic regions and the causal variants effecting LOAD-risk are yet to be uncovered. Determining alterations in gene expression profiles on neuronal, astrocyte- and microglia- specific levels between normal and LOAD-pathological stages will advance the identification of the target genes within the LOAD-associated loci. We developed a method to quantify cell-type specific (neuronal and glial) gene expression levels. Archive human frozen brain tissues were used to prepare slides for rapid immunostaining, and single-cells, neurons or astrocytes, were isolated by Laser Capture Microdissection (LCM). Following RNA extraction, gene expression was determined digitally using nCounter Single Cell gene expression assay (NanoString). We have been analyzing the expression profiles of genes within LOAD-risk regions in neurons and astrocytes, isolated from brain tissues of severe-AD, mild-AD, mild cognitive impairment (MCI) and normal controls. We then evaluated the association between expression changes and LOAD-pathological stages and found differential expression for several genes. In the neurons, APOE- and TOMM40-mRNAs were higher in MCI compared to normal, while the SORL1-mRNA levels were lower in MCI. These results suggest that alterations in the expression of these genes occur in neurons early in the development of the disease. APOE-mRNA levels in the isolated neurons were further increased with disease progression, mild and severe AD, while APP expression levels decreased throughout disease progression. The expression levels of several other genes also showed variation between LOAD-pathological stages. Analyses of gene expression changes in the isolated astrocytes are underway. We present a state-of-the-art method for detection of cell type-specific gene expression changes using archive frozen human brain samples. Our results showed changes in expression of critical genes in early stages of the disease course, and therefore suggest that the expression regulation of these genes possibly has a causative role in the etiology of LOAD.