Controlling motivation for cocaine is critical for the successful long-term treatment of cocaine addiction, which may require reversal of drug-induced changes in gene expression. Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in drug abuse has received little attention. RNA binding proteins and microRNAs serve as master switches controlling gene expression, with mRNA stability estimated to control about 20% of brain-expressed genes. Our research suggests that the RNA-binding protein HuD and the microRNA miR-495 play opposite roles in the control of addiction-related gene expression and behavior: 1) they are predicted to bind the same GU-rich sequence in mRNAs;2) their binding sites are overrepresented in transcripts from an addiction-related gene (ARG) database;3) they show differential regulation by cocaine, with miR-495 downregulated and HuD upregulated in the nucleus accumbens;4) in vitro manipulations of these molecules result in opposite effects on the expression of two of their target genes, BDNF and arc;and 5) most importantly, in vivo manipulations of these molecules show contrasting effects on the motivation for cocaine. Based upon these results, we hypothesize that HuD and miR-495 compete for binding to the same sequences to control the expression of ARGs and motivation for drug in opposing directions. To test this hypothesis, in the CEBRA application we propose the following two specific aims:
Aim 1 : To identify mRNA targets of miR-495 and HuD and to map their specific binding sites using a novel UV- crosslinking and RNA immunoprecipitation and high throughput sequencing (CLIP-seq) protocol on the new Ion Torrent PGMTM semiconductor sequencers. These studies will use in vitro and in vivo validation methods as well as unique bioinformatics tools and novel competition studies to define how HuD and miR-495 are able to regulate the same targets in an opposite manner.
Aim 2 : To test the functional consequences of the competitive interactions of HuD vs. miR495 with their targets in vivo both on the motivation of animals for cocaine (Aim 2a) and the post-transcriptional control of shared target mRNAs (Aim 2b). These studies will use HuD overexpressor mice and stereotaxic injections of lentiviral constructs to increase or decrease miR-495 levels in the nucleus accumbens shell. We consider our application to be appropriate for the CEBRA initiative as the cutting-edge sequencing method proposed in Aim 1 to simultaneously identify the common targets and binding sites of HuD and miR-495 is highly innovative and potentially of high risk. Our application is also markedly significant and potentialy of high-impact as these studies will uncover novel molecular targets and unexplored molecular mechanisms involving the competition between RNA-binding proteins and microRNAs in drug addiction. A better understanding of these regulatory mechanisms is a pre-requisite for the application of these new tools in addiction research and ultimately in the treatment of this disorder.

Public Health Relevance

Although post-transcriptional mechanisms play a vital role in the control of gene expression, their role in the establishment of addictive behaviors has received very little attention. Therefore, the identification of the molecular targets of mR-495 an HuD, two post-transcriptional regulators that interact with the same target sequences with opposing effects in gene expression and motivation for cocaine will uncover new mechanisms underlying the maladaptive changes in synaptic plasticity during drug addiction and provide potential new targets for intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21DA034452-02
Application #
8475575
Study Section
Special Emphasis Panel (ZDA1-SXC-E (11))
Program Officer
Satterlee, John S
Project Start
2012-06-01
Project End
2014-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
2
Fiscal Year
2013
Total Cost
$181,200
Indirect Cost
$61,200
Name
University of New Mexico Health Sciences Center
Department
Neurosciences
Type
Schools of Medicine
DUNS #
829868723
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Bastle, R M; Oliver, R J; Gardiner, A S et al. (2018) In silico identification and in vivo validation of miR-495 as a novel regulator of motivation for cocaine that targets multiple addiction-related networks in the nucleus accumbens. Mol Psychiatry 23:434-443
Kim, Hak Hee; Lee, Seung Joon; Gardiner, Amy S et al. (2015) Different motif requirements for the localization zipcode element of ?-actin mRNA binding by HuD and ZBP1. Nucleic Acids Res 43:7432-46
Wright, Carrie; Calhoun, Vince D; Ehrlich, Stefan et al. (2015) Meta gene set enrichment analyses link miR-137-regulated pathways with schizophrenia risk. Front Genet 6:147
Gardiner, Amy S; Twiss, Jeffery L; Perrone-Bizzozero, Nora I (2015) Competing Interactions of RNA-Binding Proteins, MicroRNAs, and Their Targets Control Neuronal Development and Function. Biomolecules 5:2903-18
Wang, Feifei; Tidei, Joseph J; Polich, Eric D et al. (2015) Positive feedback between RNA-binding protein HuD and transcription factor SATB1 promotes neurogenesis. Proc Natl Acad Sci U S A 112:E4995-5004
Wright, Carrie; Turner, Jessica A; Calhoun, Vince D et al. (2013) Potential Impact of miR-137 and Its Targets in Schizophrenia. Front Genet 4:58
Allen, Megan; Bird, Clark; Feng, Wei et al. (2013) HuD promotes BDNF expression in brain neurons via selective stabilization of the BDNF long 3'UTR mRNA. PLoS One 8:e55718