Novel Specific MicroRNAs in Regulating Neuronal Genes Implicated In Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is mainly characterized by brain depositions of amyloid plaques and neurofibrillary tangles, synaptic dysfunction and cognitive decline. These aberrations are believed to result from the over-production of amyloid-β peptide (Aβ), which is derived from amyloid precursor protein (APP). Overproduction of Aβ is most likey due to dysregulation of genes involved in AD. Dysregulation of proteins involved in APP trafficking (e.g. SORL1), Aβ production (APP, BACE1) and Aβ clearance (neprilysin [NEP]) may contribute to excess Aβ deposition. Elucidating how expression of these proteins is regulated will ultimately reveal new drug targets. We have taken the novel approach of studying the regulation of these gene products by microRNAs (miRNAs), which interact with the 3'UTRs of target mRNA. Here we report the identification of novel miRNAs that are significant for AD. Using bioinformatic tools a set of miRNA predicted to target the human APP, BACE1, NEP and SORL1 mRNA 3'UTR was identified. Chimeric 3'UTR-reporter constructs were then prepared by inserting the APP, BACE1, NEP or SORL1 3'UTR downstream of a reporter Renilla luciferase gene. To identify miRNA-target gene interactions, corresponding miRNA mimics were independently co-transfected into mammalian cells along with a specific reporter construct described above. Sensitive reporter assays identified multiple miRNAs that mediate inhibitory (and some stimulatory) effects on luciferase expression for each 3'UTR. We found that miR-153 and miR-346 inhibited APP 3'UTR reporter expression to a significant degree. To confirm the effects of miR-153 and miR-346 on native protein expression, miRNA mimics were transfected into HeLa cells and changes in APP protein levels were measured. Notably, APP levels were significantly decreased by miR-153 but surprisingly increased by miR-346 relative to controls. Experiments are underway to confirm the site-specific nature of these interactions, to validate endogenous regulation of APP by miR-153 and miR-346. and to test candidate miRNA directed against BACE1, NEP and SORL1 3'UTR. Our results reveal a novel regulatory interaction between important AD-related genes and specific endogenously expressed miRNA species, and open up a novel drug target(s) for AD therapeutics. Further characterization of unique miRNA(s) would have significant therapeutic implication for AD.