基于lncRNA对mesolimbic多巴胺通路形成的调控机制探究药物成瘾的起源性分子

Previous studies have already demonstarted that mesolimbic dopaminergic pathway is the anatomical basis for drug addiction. However, the mechanisms of the formation of mesolimbic projection are unclear. Our proposal will explore the molecular mechanisms of mesolimbic projection formation and the origin of drug addiction. It’s a great challenge for isolation the transcripts of specific subtye dopaminergic neurons in mesolimbic projection.In our preliminary study, using Ribotag methology and Cre-loxp recombination technique, we labeled these neurons with Adeno-Cre virus injection to make tdtomato fluorescent protein expression, and to make Rpl22 protein expression taged with hemagglutinin (HA). We then immunoprecipitaed the HA associated transcripts from mesolimbic dopaminergic neurons. Subsequently, long non-coding RNA (lncRNA) microarry was employed to analyze lncRNA and mRNA expression profiles (lncRNA microarry contains mRNA probes). Bioinformatics analysis showed that differential expression lncRNAs targeting axon guidance pathway in which lncRNA-MRUC007VKO targeting semaphorin5A. In particular, methamphetamine increased both MRUC007VKO and semaphorin5A expression levels, which was closely related with addiction. The following of this proposal will perform the first experiment to analyze the function of MRUC007VKO and semaphorin5A by gene over-expression and knock-down in cultured dopaminergic neurons, as well as to know how lncRNA regulates semaphorin 5A was determined. The second experiment will confirm the regulation of MRUC007VKO and semaphorin5A in mesolimbic projection with in utero electroporation experiment. The final experiment will determine the expressions of MRUC007VKO and semaphorin5A and to investigate the possible intervention to methamphetamine abuse. These studies will provide important new insights into the molecular mechanisms of mesolimbic projection and should facilitate the understanding of reward and the development of new therapeutic strategies for drug abuse.

已经证明，中脑边缘（mesolimbic）多巴胺通路是药物成瘾的解剖学基础，但目前该通路形成的机制不清。本项目旨在揭示mesolimbic多巴胺通路形成的调控机制，探究药物成瘾的起源性分子。在预实验中，我们创新性的采用Ribotag和Cre-loxp重组技术免疫共沉淀了mesolimbic通路形成期多巴胺亚类神经元转录本。经lncRNA表达谱分析，发现差异lncRNAs作用axon通路。其中MRUC007VKO靶向semaphorin5A。并且MA上调二者表达，初步显示与药物成瘾相关。下一步，拟分析MRUC007VKO对semaphorin5A及轴突生长的调控作用；采用胚胎脑内基因干扰获得lncRNAs及靶基因控制mesolimbic通路发育形成的可靠证据；通过模型鼠证实lncRNAs及靶基因与药物成瘾的相关性和干预性，有望找出药物成瘾的特异性分子靶点。

长链非编码RNA（lncRNA）是一类含量多且不编码蛋白的RNA分子，具有广泛的生物学功能。LncRNA与神经发育、神经细胞分化和突触可塑性变相关。已经证明，中脑边缘（mesolimbic）多巴胺通路是药物成瘾的解剖学基础，但目前该通路发育形成的机制不清。本项目旨在从神经发育的角度，揭示lncRNA对mesolimbic多巴胺通路形成的调控机制，寻找特异性调控多巴胺环路形成的分子，并分析与药物成瘾的关系。首先，构建了含Ribotag的逆行示踪病毒载体，进行了脑内注射和神经环路标记；结合前期实验研究，通过转录组测序，进行lncRNA表达谱分析，寻找MA损伤神经元后的差异表达lncRNAs。通过表达验证后，选定了进一步分析研究的特异表达lncRNA。在随后的实验中，构建了针对特异表达lncRNA的干扰病毒载体，经体外转染神经元和抑制表达验证确认后，进行了功能分析。分析了候选lnc-miR670hg对MA致神经元损伤及关联分子通路的影响。在对多巴胺通路进行示踪的基础上，分析了标记神经元电生理学特性。本项实验从长链非编码RNA调控神经通路功能和神经元损伤的角度，分析了lncRNA在药物成瘾中可能的机制，对理解成瘾机制具有重要意义。