Long Noncoding RNA Dysregulation Alters Ethanol Drinking Behavior and Ethanol-Related Phenotypes

Alcohol use disorder (AUD) is a chronic, relapsing brain disease associated with a myriad of debilitating consequences. Unfortunately, current therapies are limited, and the etiology is incomplete. Chronic alcohol use impacts molecular processes, one of great importance being gene expression. Transcriptomic changes are thought to underly the transition from recreational drinking to AUD, and persistent transcriptional change is a hypothesized mechanism for AUD withdrawal and relapse. Long noncoding RNAs (lncRNAs) have emerged as key regulators of the transcriptome and hold great therapeutic potential. LncRNA dysregulation has been linked to AUD, drug addiction, psychiatric disorders, and immune responses. However, out of thousands of lncRNAs known to exist, only a small percentage have been functionally characterized. Further, the impact of chronic alcohol misuse on lncRNA regulation and function is largely unknown. For important AUD-regulating lncRNAs to be identified and characterized, ethanol-responsive lncRNAs must be screened for AUD-linked behavior. Based on RNA-sequencing and microarray data from brain tissue of chronically exposed subjects, the ethanol-responsive lncRNAs Tx2, Pitt1, Pitt2, Pitt3, Pitt4, and Gas5 were selected for functional interrogation. Cutting-edge CRISPR/Cas9 mutagenesis techniques were applied to generate mutant mouse cohorts of all six genes. The overall goal was to elucidate the involvement of six ethanol-responsive lncRNAs in ethanol action to test the central hypothesis that individual lncRNAs act as determinants of ethanol consumption and ethanol-related behaviors. Each mutant line was functionally investigated using a variety of behavioral and molecular methods. All six mutant lncRNA lines demonstrated significant sex-specific alterations in ethanol drinking behavior when compared to controls. Ethanol consumption was significantly altered in mutant Tx2 males and mutant Pitt1, Pitt3, and Pitt4 females. Ethanol preference was significantly altered in mutant Tx2, Pitt1, and Pitt2 males, and mutant Gas5 females. These findings add to the literature implicating noncoding RNAs in addiction and suggest that lncRNAs play an important regulatory role in AUD. The dissertation presented herein advances our understanding of the molecular impacts of ethanol-responsive lncRNAs and how they relate to addictive behaviors. This project provided substantial training opportunities, a strong research foundation, and a collaborative environment with other researchers in the field