Debomoy K. Lahiri
Laboratory of Molecular Neurogenetics, Department of Psychiatry and of Medical & Molecular Genetics, 791 Union Drive, Indiana University School of Medicine, Indianapolis, IN, USA

Abstract:

Alzheimer’s disease (AD) accounts for 50 to 80 percent of dementia cases. Other types of dementia include vascular dementia, mixed dementia, Lewy bodies and frontotemporal dementia. AD is characterized by amyloid plaques, neurofibrillary tangles, synaptic dysfunction, neuronal loss and cognitive decline. These aberrations are believed to result, in part, from the over-production of amyloid-β peptide (Aβ), a product of Aβ precursor protein (APP). Expression studies suggest that, dysregulation of proteins involved in Aβ production (e.g. APP, BACE1) and Aβ clearance (e.g. neprilysin) 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).  These are short, non-coding RNAs that act as post-transcriptional regulators of gene expression through site-specific interactions mostly with 3’UTRs of target mRNAs. Here we present data demonstrating miRNA-mediated regulation of APP.

Using multiple bioinformatic tools, a set of miRNA predicted to target the APP 3’UTRmRNA was identified.  Corresponding miRNA mimics were co-transfected into HeLa cells along with a reporter construct containing the 1.2kb APP 3’UTRmRNA inserted into the 3’UTR of Renilla luciferase.  Reporter assays identified multiple MiRNAs that mediate inhibitory effects on luciferase reporter expression, with the strongest effect produced by miR-101.  miR-101 putatively targets two sites in the APP 3’UTR.  The seed sequences in these two sites were mutated by site-directed mutagenesis.  Reporter assays indicated that mutation of one site eliminated the inhibitory effect of miR-101, while mutation of the second site had little to no effect on this inhibition.  To confirm the effects of miR-101 on native protein expression, miR-101 was transfected into HeLa and neuronal cells and changes in APP levels were measured by Western blot, and miRNA levels by qRT-PCR.  APP level was significantly inhibited relative to the cells transfected with a negative control miRNA.  Thus, miR-101 appears capable of regulating APP expression.  Taken together, our results reveal a novel regulatory interaction between important AD-related genes and specific endogenously expressed miRNA species. Ongoing experiments with specific miRNAs targeting different APP pathways genes would lead to other potential therapeutic agents for AD.

This work is supported by NIH grants to Dr. D.K. Lahiri