Date of Award

Spring 1-1-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Integrative Physiology

First Advisor

Marissa A. Ehringer

Second Advisor

Jerry A. Stitzel

Third Advisor

Robert L. Spencer

Fourth Advisor

Michael C. Stallings

Fifth Advisor

Christopher A. Lowry

Abstract

Modern large-scale genetic approaches like GWAS have allowed the identification of common genetic variations that contribute to the risk architecture of psychiatric disorders. Majority of such susceptibility variants are located in non-coding genomic regions spanning multiple genes. Multiple GWAS have linked certain polymorphisms in the CHRNA5-CHRNA3-CHRNB4 gene cluster on chromosome 15q21, encoding for the alpha5, alpha3 and beta4 subunits of the nicotinic acetylcholine receptors (nAChRs) respectively, with an increased risk for a variety of smoking and drug-related behaviors, lung cancer, COPD, and reduced levels of cognitive performance (in domains such as attention, response inhibition, and discriminative abilities). One of the strongest impacts on risk has been associated with non-coding functional variations in the CHRNA5 and CHRNB4 genes known to modulate CHRNA5 and CHRNB4 mRNAs expression. Here we studied the effects of these genetic polymorphisms on gene expression, nicotine and learning and memory-related behaviors applying in vitro (cell cultures) and in vivo (live organism, C57BL/6J mice) models.

The first series of analyses focused on non-coding SNPs within CHRNA5 distal upstream enhancer/repressor and the core promoter regions, shown previously to regulate CHRNA5 mRNA expression. The results of the latter pointed towards a 22bp-indel polymorphism (rs3841324) in the core CHRNA5 promoter region as a main modulator of the luciferase expression activity in vitro in two human neuroblastoma and one human-derived small cell lung carcinoma cell lines. The subsequent goal was to characterize if/how the Sp1 transcription factor interacts with the 22bp-indel DNA polymorphism (that has a predicted Sp1 binding site) to modulate CHRNA5 gene expression.

In addition, in vivo (live organism) models (C57BL/6J mice) were used to study whether the effects of miR-138 (known to silence CHRNB4 gene expression), observed in vitro (in cell cultures), also replicate in vivo and how this affects nicotine- and learning and memory-related behaviors. Adeno-Associated Viral (AAV) vectors were utilized for the in vivo delivery of the miR-138 in a specific brain region (dorsal hippocampus) of C57BL/6J mice and investigated miR-138 role on nicotine consumption (Nicotine preference/2-bottle free choice) and fear conditioning and extinction tests. The results suggest that the targeted silencing of CHRNB4 in dorsal hippocampus does not change nicotine preference/consumption and that the CHRNB4 was most likely not independently involved/associated with dorsal hippocampal-dependent memory processes in C57BL/6J mice.

Overall, these studies have the potential to provide vital information on future research directions with clinical implications for nicotine addiction, lung cancer, PTSD; that may advance new avenues for a targeted treatment of these devastating disorders.

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