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"POST-TRANSCRIPTIONAL REGULATION OF
D1 DOPAMINE RECEPTOR EXPRESSION
IN COCAINE ADDICTION:
INVESTIGATING THE ROLE OF MICRO-RNAs"

by
Krishna Eugenia Tobón
Pharmacology & Physiology Program
B.S. 2003, William Paterson University



Thesis Advisor: Eldo Kuzhikandathil, Ph.D.
Associate Professor
Department of Pharmacology & Physiology

Tuesday, September 18, 2012
11:30 A.M., MSB -H609b


Abstract

In 2009, the National Institute on Drug Abuse (NIDA) reported that 4.8 million Americans age 12 and older had abused cocaine. The effects of cocaine use on the juvenile brain and behavior are poorly understood. Currently there are no FDA medications approved to treat cocaine addiction. In adult animal models, it is well understood that intermittent repeated cocaine administration results in behavioral sensitization wherein there is an exaggerated locomotor response to a challenge dose of cocaine. This behavioral phenomenon reportedly models several aspects of cocaine addiction in humans. Data from pharmacological studies and knock-out animals, suggests that the D1 dopamine receptor (D1R) is necessary for the manifestation of cocaine-induced behavioral sensitization. Data from the literature, while incomplete and inconsistent, suggested that repeated cocaine administration results in increased D1R mRNA expression but no concomitant increase in D1R protein expression, suggesting that repeated cocaine administration might induce D1R post-transcriptional regulation. In this thesis project we tested the hypothesis that D1R post-transcriptional regulation (PTR) is centrally involved in cocaine-induced behavioral sensitization and that D1R PTR is mediated by a microRNA mechanism. The objective of my thesis project was to identify and characterize D1R PTR in physiological and pathophysiological conditions, and identify the molecular mechanism mediating D1R PTR.
PTR is an important regulatory mechanism in which the expression of a gene is controlled at the level of the mRNA, commonly by modulating stability and/or translation of the mRNA. The molecular mechanisms that regulate the expression of D1R, particularly at the post-transcriptional level, are not well understood. Here we report that the D1R is post-transcriptionally regulated during postnatal mouse brain development, repeated cocaine administration in juvenile mice, and in the mouse CAD catecholaminergic neuronal cell line. We show that the 3’untranslated region (3’UTR) of D1R mRNA is necessary and sufficient for mediating the PTR and that in CAD cells the D1R PTR is specifically mediated by microRNA 142-3p (miR-142-3p). Inhibiting miR-142-3p in CAD cells not only increases D1R protein expression levels but also enhances D1R mediated signaling. We also show that in the mouse brain, during postnatal development, there is an inverse correlation between miR-142-3p and D1R receptor protein expression.
The effects of pro-dopamine drugs such as cocaine are rarely reported in juvenile mouse models. Here we report that juvenile mice of two different strains, C57/Bl6 and Drd1-EGFP, develop and manifest behavioral sensitization in response to binge cocaine administration. In adult mouse models, the role of the D1R in the activation of extracellular signal-regulated kinase (ERK) in the nucleus accumbens and dorsal striatum has been well studied as a necessary pathway for cocaine-mediated behaviors such as behavioral sensitization. In this study, we show that repeated cocaine administration to juvenile mice also leads to increased basal ERK activation in the nucleus accumbens. We observed that the Drd1-EGFP mouse model has higher locomotor activity when compared to the C57/Bl6 mouse model. This could be in part due to the Drd1-EGFP mice having higher levels of D1R and D2R mRNA in the dorsal striatum. We also demonstrate that the behavioral sensitization is abolished after a two week cocaine-free period, suggesting that while juvenile mice manifest cocaine-induced behavioral sensitization, the cocaine-induced neuronal modifications are reversible.
The studies from juvenile cocaine-sensitized mice demonstrate that the D1R also exhibits PTR wherein the sensitization induces an increase in D1R mRNA but not D1R protein, specifically in the caudate brain region. We propose that elevated levels of D1R mRNA would provide a pool that could be rapidly translated into D1R protein following new cocaine stimulation. Indeed, we demonstrate that in cocaine-sensitized mice, de novo D1R protein synthesis occurs within 30 minutes of a challenge dose of cocaine. As D1R expression levels are elevated 30 minute post cocaine challenge, miR-142-3p expression levels are decreased. The results suggest that D1R PTR in the juvenile cocaine model might be mediated by a mechanism that involves microRNAs.
In summary, the results of this thesis project suggest that PTR of D1R expression is important for the manifestation of cocaine-induced behavioral sensitization. We determined that the D1R PTR was mediated by microRNA miR-142-3p and showed that the levels of this microRNA inversely correlated with D1R protein expression during postnatal mouse brain development and in cocaine-sensitized mice. Modulation of miR-142-3p levels might provide a novel therapeutic approach to treat cocaine addiction.


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