Global Approaches to the Role of miRNAs in Drug-Induced Changes in Gene Expression

Neurons modulate gene expression with subcellular precision through excitation-coupled local protein synthesis, a process that is regulated in part through the involvement of microRNAs (miRNAs), a class of small non-coding RNAs. The biosynthesis of miRNAs is reviewed, with special emphasis on miRNA families, the subcellular localization of specific miRNAs in neurons, and their potential roles in the response to drugs of abuse. For over a decade, DNA microarrays have dominated genome-wide gene expression studies, revealing widespread effects of drug exposure on neuronal gene expression. We review a number of recent studies that explore the emerging role of miRNAs in the biochemical and behavioral responses to cocaine. The more powerful next-generation sequencing technology offers certain advantages and is supplanting microarrays for the analysis of complex transcriptomes. Next-generation sequencing is unparalleled in its ability to identify and quantify low-abundance transcripts without prior sequence knowledge, facilitating the accurate detection and quantification of miRNAs expressed in total tissue and miRNAs localized to postsynaptic densities (PSDs). We previously identified cocaine-responsive miRNAs, synaptically enriched and depleted miRNA families, and confirmed cocaine-induced changes in protein expression for several bioinformatically predicted target genes. The miR-8 family was found to be highly enriched and cocaine-regulated at the PSD, where its members may modulate expression of cell adhesion molecules. An integrative approach that combines mRNA, miRNA, and protein expression profiling in combination with focused single gene studies and innovative behavioral paradigms should facilitate the development of more effective therapeutic approaches to treat addiction

Global Approaches to the Analysis of Cocaine-Induced Gene Expression

For over a decade, DNA microarrays have dominated genome-wide gene expression studies, revealing widespread effects of drug exposure on neuronal gene expression. More targeted single gene approaches have added invaluable insights into their behavioral, biochemical, and molecular effects. The more powerful next-generation sequencing technology may soon supplant microarrays for the analysis of complex transcriptomes. RNA-Seq is unparalleled in its ability to identify and quantify low-abundance transcripts without prior sequence knowledge, facilitating detection and investigation of known and novel alternative splicing events, imprinting, and RNA editing, among others. We first used RNA-Seq technology to catalog microRNA (miRNA) expression in total tissue and at postsynaptic densities (PSDs) from control and cocaine-treated mice. Neurons modulate gene expression with subcellular precision through excitation-coupled local protein synthesis, a process that may be regulated in part through the involvement of miRNAs. We identified cocaine-responsive miRNAs, synaptically-enriched and depleted miRNA families, and confirmed cocaine-induced changes in protein expression for several predicted target genes. The miR-8 family was found to be highly enriched and cocaine-regulated at the PSD, where its members may modulate expression of cell adhesion molecules. We then used next-generation sequencing of mRNAs purified from the same brain regions to gain a more complete picture of the molecular response to chronic cocaine exposure and withdrawal. Bioinformatic analysis revealed paradigm-specific regulation of multiple components of a limited number of signaling pathways. We identified signaling pathways that utilize distinctly different sets of receptors and signaling molecules under control conditions, after chronic exposure to cocaine, and during withdrawal. The Rho-GEFs expressed in the nucleus accumbens and localized to the postsynaptic density were reviewed because they are strategically positioned to effect rapid alterations in dendritic spine structure. Structural changes mediated by synaptic Rho guanine nucleotide exchange factors (GEFs) control actin cytoskeletal rearrangements and contribute to the long-lasting effects of cocaine. This review is then followed by a brief look at several ongoing and completed collaborative bioinformatics projects. Future studies will employ integrative approaches that combine mRNA, miRNA, and protein expression profiling with focused single gene studies and innovative behavioral paradigms and will facilitate development of integrated approaches to treat addiction

Kalrn Promoter Usage And Isoform Expression Respond To Chronic Cocaine Exposure

Background The long-term effects of cocaine on behavior are accompanied by structural changes in excitatory glutamatergic synapses onto the medium spiny neurons of the striatum. The Kalrn gene encodes several functionally distinct isoforms; these multidomain guanine nucleotide exchange factors (GEFs) contain additional domains known to interact with phosphatidylinositides as well as with a number of different proteins. Through their activation of Rho proteins and their interactions with other proteins, the different Kalirin isoforms affect cytoskeletal organization. Chronic exposure of adult male rodents to cocaine increases levels of Kalirin 7 in the striatum. When exposed chronically to cocaine, mice lacking Kalirin 7, the major adult isoform, fail to show an increase in dendritic spine density in the nucleus accumbens, show diminished place preference for cocaine, and exhibit increased locomotor activity in response to cocaine. Results The use of alternate promoters and 3\u27-terminal exons of the mouse Kalrn gene were investigated using real-time quantitative polymerase chain reaction. While the two most distal full-length Kalrn promoters are used equally in the prefrontal cortex, the more proximal of these promoters accounts for most of the transcripts expressed in the nucleus accumbens. The 3\u27-terminal exon unique to the Kalirin 7 isoform accounts for a greater percentage of the Kalrn transcripts in prefrontal cortex than in nucleus accumbens. Western blot analyses confirmed these differences. Chronic cocaine treatment increases usage of the promoter encoding the Δ-Kalirin isoforms but does not alter full-length Kalirin promoter usage. Usage of the 3\u27-terminal exon unique to Kalirin 7 increases following chronic cocaine exposure. Conclusions Kalrn promoter and 3\u27-terminal exon utilization are region-specific. In the nucleus accumbens, cocaine-mediated alterations in promoter usage and 3\u27-terminal exon usage favor expression of Kalirin 7 and Δ-Kalirin 7. The Δ-isoform, which lacks a Sec14p domain and four of the nine spectrin-like repeats found in full-length Kalirin isoforms, increases spine headsize without increasing dendritic spine numbers. Thus cocaine-mediated changes in alternative splicing of the Kalrn gene may contribute importantly to the behavioral, morphological and biochemical responses observed