Cocaine Regulation of FosB Splicing

描述(申请人提供)：尽管最近的研究揭示了许多关于药物成瘾的分子基础，但在我们对与慢性药物摄取相关的基因表达变化的理解方面仍然存在许多空白。慢性可卡因暴露时发生的一个显著变化是纹状体中积累了非常稳定的转录因子d FosB亚型，这种亚型是通过FosB转录本的选择性剪接产生的。DeltaFosB亚型可能参与药物成瘾引起的一些神经和行为改变。影响Delta FosB选择性剪接的因素尚未确定。为了开始理解导致稳定dFosB产生的转录后事件，我们将检验顺式和反式作用因子在这种调控中都很重要的假设。FosB的突变分析将用于鉴定前mRNA剪接所必需的序列。将进行交联和RNA结合研究，以确定从组织培养细胞和大脑制备的剪接提取物中，哪些反式作用因子与RNA调节序列结合。这些研究中确定的因子将在剪接试验中进行测试，以确定它们是否在体外调节剪接。调节剪接因子在大脑中的分布将通过原位杂交组织化学来确定。为了确定在体内对FosB的影响，将改变调控因子的表达。这些研究将确定在长期使用可卡因期间调节剪接所需的因素，并导致对所涉及的机制的理解。由于长期服用其他滥用药物，如苯丙胺、尼古丁和鸦片类药物也会诱发dFosB，这项研究的结果也可能有助于更好地理解药物成瘾。

DESCRIPTION (provided by applicant): Although recent studies have revealed a great deal about the molecular basis of drug addiction, numerous gaps remain in our understanding of the changes in gene expression that are associated with chronic drug intake. One prominent alteration that occurs upon chronic cocaine exposure is accumulation in the striatum of very stable isoforms of the transcription factor d FosB that is produced by alternative splicing of the FosB transcript. The delta FosB isoforms may mediate some of the neural and behavioral modifications that occur with drug addiction. The factors that play a role in the alternative splicing of delta FosB have not yet been identified. To begin to understand the posttranscriptional events that lead to the production of stable dFosB we will test the hypothesis that both cis- and trans-acting factors are important in this regulation. Mutation analysis of FosB will be used to identify the sequences essential for pre-mRNA splicing. Cross-linking and RNA-binding studies will be undertaken to determine which trans-acting factors bind to the RNA regulatory sequences in splicing extracts prepared from tissue culture cells and brains. Factors identified in these studies will be tested in splicing assays to determine if they regulate splicing in vitro. The distribution of the regulatory splicing factors within the brain will be determined using in situ hybridization histochemistry. The expression of the regulatory factors will be changed in order to determine the effect on FosB in vivo. These studies will identify the factors required for the regulation of splicing during chronic cocaine use and lead to an understanding of the mechanisms involved. Because dFosB is also induced by chronic administration of other drugs of abuse, such as amphetamine, nicotine and opiates, the results obtained in this study may also lead to a better understanding of drug addiction.