Date of Completion

5-11-2013

Embargo Period

5-11-2013

Keywords

cocaine, sequencing, miRNA, gene expression

Major Advisor

Brenton R Graveley

Associate Advisor

Marc E Lalande

Associate Advisor

Gordon G Carmichael

Associate Advisor

Blanka Rogina

Field of Study

Genetics and Genomics

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

For over a decade, DNA microarrays have dominated genome-wide gene expression studies, revealing widespread effects of drug exposure on neuronal gene expression. More targeted single gene approaches have added invaluable insights into their behavioral, biochemical, and molecular effects. The more powerful next-generation sequencing technology may soon supplant microarrays for the analysis of complex transcriptomes. RNA-Seq is unparalleled in its ability to identify and quantify low-abundance transcripts without prior sequence knowledge, facilitating detection and investigation of known and novel alternative splicing events, imprinting, and RNA editing, among others. We first used RNA-Seq technology to catalog microRNA (miRNA) expression in total tissue and at postsynaptic densities (PSDs) from control and cocaine-treated mice. Neurons modulate gene expression with subcellular precision through excitation-coupled local protein synthesis, a process that may be regulated in part through the involvement of miRNAs. We identified cocaine-responsive miRNAs, synaptically-enriched and depleted miRNA families, and confirmed cocaine-induced changes in protein expression for several predicted target genes. The miR-8 family was found to be highly enriched and cocaine-regulated at the PSD, where its members may modulate expression of cell adhesion molecules. We then used next-generation sequencing of mRNAs purified from the same brain regions to gain a more complete picture of the molecular response to chronic cocaine exposure and withdrawal. Bioinformatic analysis revealed paradigm-specific regulation of multiple components of a limited number of signaling pathways. We identified signaling pathways that utilize distinctly different sets of receptors and signaling molecules under control conditions, after chronic exposure to cocaine, and during withdrawal. The Rho-GEFs expressed in the nucleus accumbens and localized to the postsynaptic density were reviewed because they are strategically positioned to effect rapid alterations in dendritic spine structure. Structural changes mediated by synaptic Rho guanine nucleotide exchange factors (GEFs) control actin cytoskeletal rearrangements and contribute to the long-lasting effects of cocaine. This review is then followed by a brief look at several ongoing and completed collaborative bioinformatics projects. Future studies will employ integrative approaches that combine mRNA, miRNA, and protein expression profiling with focused single gene studies and innovative behavioral paradigms and will facilitate development of integrated approaches to treat addiction.

Download
COinS