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  • Addiction, Drugs
  • Information from Lay-Language Summaries is Embargoed Until the Conclusion of the Scientific Presentation

    350—Cocaine: Neural Mechanisms of Addiction II

    Monday, November 11, 2013, 8:00 am - 12:00 noon

    350.11: miR-495, a post-transcriptional link between gene expression and the behavioral effects of cocaine

    Location: Halls B-H

    1Dept. of Neurosciences, Univ. of New Mexico, Albuquerque, NM; 2Sch. of Life Sci., Arizona State Univ., Tempe, AZ

    Abstract Body: Despite the importance of post-transcriptional mechanisms in the control of gene expression, very little is known about the role of these regulatory processes in the etiology and development of substance use disorders (SUDs) such as cocaine addiction. Our previous in silico analysis of miRNAs with targets enriched in addiction related genes (ARGs), such as BDNF, identified one microRNA, miR-495, as a putative regulator of SUD development. Additionally, we found that miR-495 is expressed within the nucleus accumbens (NAc) and prefrontal cortex (PFC), two regions implicated in SUDs and that lentiviral-mediated overexpression of miR-495 in the NAc shell (NAcSh) decreased cocaine intake in a rat model of cocaine self-administration (Bastle et al, SfN 2012, 456.26). To examine the molecular basis for this effect, we used microarrays to identify miR-495-regulated transcripts in vivo. Overexpression of miR-495 in the NAcsh decreased the levels of several ARGs and plasticity associated genes such as Arc, BDNF, Cnr1, Shank2, and other GABA/Glutamatergic receptors that are predicted miR-495 targets. Ingenuity pathway analysis (IPA) of downregulated genes revealed that the top associated networks included nervous system development and function, cell to cell signaling interaction, and neurological disease. Furthermore, qRT-PCR confirmed that the levels of three putative miR-495 targets, the long-3’UTR isoform of BDNF mRNA (L-BDNF), CaMK2a and Arc were significantly decreased by LV-miR-495 infusions vs. control LV-GFP, both in vivo and in cultured neurons. Additionally, luciferase-3’ UTR reporter assays demonstrated that L-BDNF expression is specifically regulated by miR-495 (Gardiner et al, SfN 2013). To analyze how the levels of miR-495 are regulated in the NAc and PFC in response to different types of cocaine exposure paradigms, C57BL/6 mice were either given a single (acute) or 5 daily (subchronic) i.p. injections of 15 mg/kg cocaine. In addition, a third group underwent a sensitization-inducing exposure, where they received a subchronic treatment regimen followed by 7 days of withdrawal and an acute challenge injection of cocaine (15 mg/kg). We found that both the acute and sensitization exposures decreased miR-495 expression (p = 0.05) in NAc. No changes were observed in the NAc after subchronic exposure or in the PFC under any of these conditions. In the mice that underwent sensitization we found a negative correlation between increased locomotor activity and miR-495 levels within the NAc (p = 0.05). In conclusion, our results indicate that miR-495 may serve an important role in the regulation of ARG expression during the development of cocaine addiction.

    Lay Language Summary: Our research group has identified a novel genetic component that silences other genes and may be involved in the development of cocaine addiction. This component is a small ribonucleic acid (RNA), or microRNA. Previously, RNA was thought to play a role only in the transfer of genetic information from DNA to a protein. However, the vast majority of DNA in our genome does not encode proteins, but rather encodes different forms of RNA that are regulatory in nature. Among these, microRNAs primarily prevent protein production, effectively silencing gene expression. In this study we report that one of these microRNAs, miR-495, regulates cocaine-seeking behavior in animals by silencing genes that have been implicated in cocaine addiction. These findings suggest that manipulating the levels of this microRNA in specific brain regions may provide a new strategy to prevent the development of drug dependence.
    Drug addiction is a significant problem in the United States. According to the 2008 National Survey on Drug Use and Health (NSDUH), about 26.6 million people in the United States meet the criteria for substance use disorder, including 1.4 million Americans with cocaine dependence. Furthermore, it has been estimated that cocaine use accounts for about 25% of all drug related emergency room visits. The societal cost of drug addiction through decreased productivity and increased health care costs is a staggering $524 billion/year, of which about $200 billion is due to illicit drug use. This economic burden pales in comparison to the emotional devastation brought about by addiction to the patient and his/her family as treatment of drug abuse is still rather ineffective.
    To evaluate the possible link between microRNAs and drug addiction, we first searched the Knowledgebase of Addiction-Related Genes database for genes that can be targeted by candidate microRNAs and found that a significant proportion of these were potential miR-495 targets. To validate these findings, we used microarray assays that simultaneously measure the expression of a large number of genes and confirmed that indeed miR-495 regulated multiple addiction-related target genes.
    Cocaine incentive-sensitization is a phenomenon in which progressive use of cocaine increases the motivation for the drug by increasing the amount of dopamine release in the nucleus accumbens and other brain regions. This mechanism has been suggested as the basis for development of drug dependence. Interestingly, only about 15-20% of people who have used cocaine become addicted, implying a genetic predisposition. Using an animal model, we found that low expression of miR-495 in mice was associated with increased cocaine-induced sensitization suggesting that the levels of this microRNA could be one of the genetic factors associated with the development of cocaine addiction.
    In order to demonstrate a more conclusive link between miR-495, its targets, and cocaine addiction, we used viral-mediated gene transfer to increase the levels of this microRNA in the nucleus accumbens of rats that were previously trained to self-administer cocaine. We found that increased levels of miR-495 decreased the amount of cocaine rats self-administered and decreased the expression of genes associated with addiction. Overall our findings indicate that increased levels of this microRNA may be a protective mechanism against the development of addiction. Next, we will try to determine if there is a specific gene or set of genes targeted by this microRNA that is necessary for this protective effect. The identification of several addiction-related genes that are regulated by miR-495, as well as some genes that have not yet been extensively studied in addiction, provide exciting new targets for future studies. Ultimately, we hope to find a treatment that increases the expression of this microRNA which may prove valuable in helping patients recover from cocaine addiction.