Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cellular Biology
Beverly L. Davidson
Huntington's Disease (HD) is an inherited neurodegenerative disorders caused by CAG repeat expansions in exon 1 of the huntingtin gene (htt). Patients with HD experience profound region specific neural degeneration for reasons that remain incompletely understood. Early studies in HD brain suggest that transcriptional misregulation occurs early in disease, before significant tissue loss and degeneration has occurred. However, a comprehensive understanding of the events that contribute to this remain poorly understood.
In this study, we investigate a functional role for small RNA or miRNAs in the central nervous system (CNS) of patients with HD. Our work identifies subsets of miRNAs misregulated in HD. A functional role for these miRNAs was investigated by identifying their predicted targets. We identify a subset of differentially detected miRNAs which are inversely correlated with predicted downstream predicted 3'UTR target genes. We also identify targets of these differentially detected miRNAs including transcriptional regulators REST, CoREST and cFOS.
The transcription factor REST silences neuronal gene expression in non-neuronal cells. Polyglutamine expansions in Huntingtin, which cause HD, abrogate REST-mediated Huntingtin binding, and as a result REST translocates to the nucleus, occupies RE1 consensus sites and represses the expression of both coding and non-coding RNAs. In this work, we identify miRNAs (miRNAs) with upstream REST consensus sites that are decreased in HD patient primary motor cortices (BA4). One of these miRNAs, miRNA-9/miRNA-9* is capable of regulating the expression of two components of the REST complex: miRNA-9 targets REST and miRNA-9* targets CoREST. These data provide evidence for a double negative feedback loop between the REST silencing complex and the miRNAs it regulates.
In addition to these studies, we identify CNS enriched miRNAs which may differentially regulate human versus non-human primate gene expression. We computationally identified a single nucleotide change from G to A in the 3'UTR of human cFOS 3'UTR which is predicted to be regulated by the brain enriched miRNA-7. A regulatory role for the single nucleotide change in humans (G->A) was assessed by mutating the single nucleotide in the human cFOS 3'UTR (from A->G), as well as by introducing the corresponding human mutation (G->A) into the rhesus and chimpanzee cFOS 3'UTRs. The presence of the A nucleotide in the predicted MRE for miRNA-7 was sufficient to partially abrogate miRNA-7 activity in reporter plasmids. Finally, overexpression of artificial precursor miRNAs in human HEK293 and mouse N2A cell lines confirmed differential targeting of cFOS in human versus mouse cell lines. These data provide evidence for the potential contribution of a single nucleotide change in humans as regards changes in cFOS regulated gene expression. Since cFOS is a transcription factor, downstream affects from altered expression could be significant.
Together, this work provides new support for the role of brain enriched miRNAs in the CNS and identifies functional support that their misregulation or altered expression can impact expression of protein coding transcripts in disease brain, and may be relevant to primate brain evolution.
CNS, Huntington's Disease, microRNA
viii, 139 pages
Includes bibliographical references (pages 131-139).
Copyright 2011 Amy N. Dubinsky