microRNAs miR-124, let-7d and miR-181a regulate Cocaine-induced Plasticity

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Abstract

MicroRNAs play key regulatory roles in cellular processes including neurogenesis, synapse development and plasticity in the brain. Psychostimulants induces strong neuroadaptive changes through a surfeit of gene regulatory mechanisms leading to addiction. MicroRNA profiling for identifying miRNAs regulating cocaine-induced, plasticity-related genes revealed significant regulation of a set of miRNAs upon cocaine administration, especially let-7d, miR-181a and the brain-specific miR-124. These miRNAs target many genes involved in cocaine addiction. Precursor and mature miRNA quantification by qRT-PCR showed that miR-124 and let-7d are significantly downregulated, whereas miR-181a is induced in the mesolimbic dopaminergic system under chronic cocaine administration. Results were confirmed by in situ hybridization, Northern blots, FISH analysis and RNase protection assay. Using lentiviral-mediated miRNA expression, we show a significant downregulation of BDNF and D3R both at mRNA and protein levels by miR-124 and let-7d, respectively. Our data suggest that miR-124, let-7d and miR-181a may be involved in a complex feedback loop with cocaine-responsive plasticity genes, highlighting the possibility that some miRNAs are key regulators of the reward circuit and may be implicated in addiction.

Introduction

Addictive drugs (cocaine and amphetamines), depressants (ethanol) and opiate narcotics (heroin and morphine) are very powerful reinforcers and produce their rewarding effects of euphoria or pleasure through an interaction with the mesolimbic dopaminergic system (Nestler, 1997, Koob et al., 1998, Woolverton and Johnson, 1992). Important neuroplastic changes occur in the reward- and memory-related brain centers following drug action. Exposure to cocaine, both acute and chronic, blocks the dopamine transporter, preventing dopamine re-uptake and alters gene expression in midbrain dopaminergic neurons (Nestler, 2001), however the precise molecular machinery involved in this chronically relapsing disorder remains largely unclear.

Recent studies have demonstrated that post-transcriptional regulation of gene expression plays a key role in neurogenesis, synaptic plasticity and in diseases associated with the CNS. MicroRNAs, a class of small non-coding transcripts are involved in coordinating the fine-tuning of gene expression during differentiation and development of the brain (Kosik, 2006). The target mRNAs are either translationally suppressed or subjected to transcriptome degradation by miRNA based on the imperfect or perfect complementarity in the 3′ UTR of the mRNA (Kosik, 2006). The miRNAs in mature neurons display a high degree of diversity and current estimates speak of about 100 miRNAs in postmitotic neurons (Fiore and Schratt, 2007a). Enriched expression of certain miRNAs in the brain suggests that they could be involved in long-term potentiation and in regulating structural and functional aspects of synaptic plasticity, like neuronal morphogenesis and activity dependent translation during synapse formation, memory and addiction (Kosik, 2006, Fiore and Schratt, 2007b, Schratt et al., 2006, Ashraf et al., 2006, Huang and Li, 2008, Pietrzykowski et al., 2008). miRNAs in the vertebrate system predominantly inhibit productive translation of mRNAs (Schratt et al., 2006), a function that could be used to keep dendritic mRNAs dormant during transport and storage near synapses (Ashraf et al., 2006). This is further supported by recent studies demonstrating that aberrant expression of few miRNAs may cause synaptic dysfunction, leading to the etiology of different neurological disorders (Beveridge et al., 2008, Wang et al., 2008a, Wang et al., 2008b, Nelson et al., 2006, Johnson et al., 2008, Niwa et al., 2008). Moreover a role of miRNA in feedback circuit has been demonstrated in midbrain dopaminergic neurons (Kim et al., 2007).

We have previously shown that cocaine treatment induces changes in expression of axon guidance molecules (Bahi and Dreyer, 2005), which may contribute to cognitive deficits associated with drug abuse. Interestingly, a number of axon guidance molecules implicated in cocaine addiction are regulated by miRNAs (John et al., 2004). Therefore expression analysis aimed at identifying miRNAs involved in cocaine-induced alteration in gene expression may provide cues to a better understanding about this reward circuit. We show in this study that miR-124 and let-7d are significantly downregulated, whereas miR-181a is strongly up-regulated after cocaine treatment. Our observation provides insight into a possible miRNA-mediated feedback loop involving miRNAs, transcription factors and other genes involved in signal transduction in cocaine-induced plasticity.

Section snippets

Computational prediction of cocaine-specific miRNAs

Screening for miRNA targeting cocaine-responsive genes was performed on the basis of lists of genes modulated upon chronic cocaine (Brenz Verca et al., 2003). For this purpose, miRNA target prediction softwares were used (http://www.microrna.org) (Betel et al., 2008). The results were later compared with other open access softwares (Griffiths-Jones et al., 2006). This comparison allowed us to narrow down the miRNAs candidates possibly involved in cocaine-induced altered gene expression, based

Discussion

In this study, application of bioinformatic predictions on cocaine-responsive genes revealed that several cocaine-induced genes were targeted by miR-124 and let-7d either individually or in a co-operative manner, whereas miR-181a has putative target sites on at least four cocaine-suppressed genes. Using various quantification methods, we found that cocaine indeed causes a strong induction of miR-181a in different regions of the midbrain whereas the levels of miR-124 and let-7d are significantly

Animal handling

All animal experiments were carried out in accordance with the guidelines and regulations for Animal Experimentation, BAG, Bern, Switzerland. Male Wistar rats weighing 250–300 g (BRL, Fillingsdorf, Switzerland) were used for all the experiments. The animals were housed in groups (four per cage) in clear plastic cages with wire grid lids. The animals were kept on a 12-h light/dark cycle (lights off at 07.00 am), with access to food and water ad libitum.

Cocaine administration

Chronic cocaine administration was

Financial disclosure

This study was supported, by Swiss National Foundation grants 3100-059350 and 3100AO-100686 (JLD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors are grateful to Dr F. Boyer and Dr A. Bahi, for critical comments and for skillful assistance, and Mrs. C. Deforel-Poncet for skillful assistance. We also thank Prof. P. Descombes, Genomics Platform Manager, Geneva, Switzerland, for skillful assistance in screening for miRNAs.

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