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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

    350.10: MicroRNA-495 competes with the RNA-binding protein HuD for control of addiction related genes

    Location: Halls B-H

    Neurosciences, Univ. of New Mexico HSC, ALBUQUERQUE, NM

    Abstract Body: RNA-binding proteins and microRNAs (miRNAs) serve as master switches controlling gene expression, with mRNA stability estimated to control approximately 20% of brain-expressed genes. HuD is a well characterized mRNA stabilizer that binds specific U-rich elements in the 3’ untranslated regions (UTRs) of target mRNAs. In contrast, miRNAs silence gene expression via translational repression and mRNA degradation. Our research in animal models of addiction suggests that HuD and miR-495 have opposing roles in the regulation of gene expression and behavior. In addition, HuD and miR-495 target significantly more addiction-related genes (ARGs, http://karg.cbi.pku.edu.cn) compared to the whole genome. Given that one of the binding sites of HuD (GUUUGUUU) is complementary to the seed region of miR-495, we proposed that this miRNA could compete with HuD for the binding of common target mRNAs. To examine the competition between HuD and miR-495, we engineered two luciferase-3’ UTR constructs: one containing only the GUUUGUUUG binding motif that is shared by both HuD and miR-495 (pLuc-m2) and one containing a shared site and a separate HuD binding site (pLuc-m2/3). We also engineered a miR-495-sponge, containing six copies of miR-495 binding sites. As expected, we found that for both constructs reporter activity was: a) reduced in the presence of miR-495, b) rescued in the presence of the sponge and c) increased in the presence of HuD. When HuD and miR-495 were co-transfected, we found that miR-495 outcompetes HuD for binding of shared sites (pLuc-m2), but HuD can compensate to stabilize the mRNA when an additional HuD binding site is available in the 3’ UTR (pLuc-m2/3). We also constructed luciferase reporters containing the short or long 3’UTRs of BDNF, a well-known addiction-related gene. The short 3’UTR (BDNF-S) contains a putative miR-495 binding site, while the long 3’UTR (BDNF-L) contains shared binding sites for HuD and miR-495 and an additional binding site for HuD, similar to pLuc-m2/3. As expected, BDNF-S was not greatly affected by miR-495 or HuD, but BDNF-L was regulated by both. Furthermore, deletion of the shared binding sites in BDNF-L decreased its response to these post-transcriptional regulators. Finally, in neuronal cultures and in vivo, miR-495 decreased the expression of BDNF-L (see Oliver et al, SfN 2013) while HuD increased mRNA levels. Our results suggest that the effects of HuD and miR-495 on ARG gene expression depend not only on the levels of these regulatory molecules but also on 3’ UTR sequences present in the target transcripts. These findings have broad implications for the regulation of addiction-related genes and gene expression in general.

    Lay Language Summary: Our team discovered a novel molecular mechanism underlying changes in brain function during the development of drug addiction. We identified two molecules that compete for the control of addiction-related genes via binding to a specific messenger ribonucleic acid (mRNA), which is the intermediate between DNA and protein. By stabilizing or destabilizing the mRNA, these factors can increase or dampen production of addiction-related proteins. Thus, the balance between these two competing factors may decide whether an animal or a patient becomes addicted to cocaine.
    Addiction is considered a chronic relapsing disorder. According to the 2008 National Survey on Drug Use and Health (NSDUH), about 26.6 million people in the United States meet the clinical criteria for drug addiction, including 1.4 million Americans with a history of cocaine abuse or dependence. The societal cost of drug addiction through decreased productivity and increased health care costs is a staggering $524 billion/year, of which $193 billion is due to illicit drug use. This burden pales in comparison to the emotional devastation brought about by addiction to the patient and his/her family as current treatments are mostly ineffective.
    When humans or animals are exposed to cocaine, the drug induces not only acute changes in brain activity but also long-lasting changes in gene expression, which may lead to compulsive drug seeking. Controlling the motivation for cocaine, which may require reversal of drug-induced changes in gene expression, is a prerequisite for the successful long-term treatment of cocaine addiction. Gene expression, or how much of a gene is turned “on” or “off,” can be controlled by various mechanisms, one of which includes stabilizing or destabilizing mRNA. Stabilizing RNA-binding proteins such as HuD usually increases mRNA and protein levels. In contrast, small RNAs such as microRNAs bind to mRNAs to prevent protein production. One particular microRNA that is expressed in regions associated with drug use, miR-495, binds to the same mRNA sequence that is recognized by HuD, suggesting that these molecules could compete for control of addiction-related gene expression and behavior.
    To test the hypothesis, we first devised experiments using the firefly luciferase gene to examine the competition between HuD and miR-495 for binding to sequences attached to the gene. In this assay, light emitted from cells is directly proportional to the levels of gene expression. As expected, we found that when HuD bound and stabilized the gene, the output was greater light. Conversely, when miR-495 bound and destabilized the gene, the output will be less light. Interestingly, in the presence of both HuD and miR-495, we found that the microRNA could outcompete HuD for binding to mRNAs with only shared sequences. However, HuD could compensate by binding to additional sequences that are present in some mRNAs such as that for brain-derived neurotrophic factor (BDNF), a well-known addiction-related protein.
    Our research also demonstrated that cocaine causes an imbalance in the levels of HuD and miR-495 in the nucleus accumbens, one of the regions associated with drug use. Furthermore we found that mice with increased HuD levels in this region show increase motivation for cocaine. In contrast, as shown by Oliver et al., 2013 (this series) increasing miR-495 levels in the same brain region decreases both drug seeking and addiction-related gene expression.
    Altogether our results suggest that the effects of HuD and miR-495 on drug seeking behavior depend not only of the levels of these regulatory molecules but also in the type of target mRNA they are competing for. Given that in animal models HuD increases while miR-495 decreases cocaine seeking, our studies provide a new molecular framework in which to develop and test novel drug treatments for cocaine addiction.