Neuronal Activity-Dependent Regulation of MeCP2

描述（由申请人提供）：MeCP 2（一种甲基-CpG结合蛋白，作为整体转录抑制因子）中的突变是Rett综合征（RTT）（一种X连锁进行性神经系统疾病）的主要原因。虽然MeCP 2在神经元中的选择性失活足以在小鼠中赋予Rett样表型，但MeCP 2在有丝分裂后神经元中的具体功能尚不清楚。我们已经发现MeCP 2结合到BDNF启动子III中转录起始位点的3'端的位点上，并起到抑制BDNF基因表达的作用。膜去极化触发钙依赖性磷酸化和MeCP 2从BDNF启动子的释放，从而促进BDNF启动子III依赖性转录。这些发现表明，MeCP 2在控制活性依赖性基因表达中起着关键作用，并表明该过程的失调可能是RTT病理学的基础。为了开始检验这一假设，我们提出了以下具体目标：1）表征膜去极化/钙依赖性MeCP 2磷酸化的位点。 我们将利用各种方法来识别MeCP 2上活性调节磷酸化的位点，并开发磷酸化位点特异性抗体来研究培养的神经元和脑切片中这些修饰对各种刺激方案的调节。2)评估磷酸化对MeCP 2活性的影响。将在神经元培养物中产生和表达MeCP 2的不可磷酸化的突变形式，以测试活性诱导的MeCP 2磷酸化对于BDNF启动子活性的适当调节以及对于神经元过程如突触发育和维持是必需的这一假设。3)确定MeCP 2的其他活性调节神经元靶点。我们的研究结果提高了MeCP 2可能是活性依赖性基因表达的一般调节剂的可能性。我们将采用多种技术，包括基因表达谱分析，RT-PCR和染色质免疫沉淀，以确定其他活动调节的目标MeCP 2在有丝分裂后的神经元。我们希望，拟议的实验将提供一个更好地了解MeCP 2功能，深入了解活性依赖性基因表达的机制，并提供新的机会，为发展治疗策略，以减轻RTT病理。

DESCRIPTION (provided by applicant): Mutations in MeCP2, a methyI-CpG-binding protein that functions as a global transcriptional repressor, are a major cause of Rett Syndrome (RTT), an X-linked progressive neurological disorder. While the selective inactivation of MeCP2 in neurons is sufficient to confer a Rett-like phenotype in mice, the specific functions of MeCP2 in post-mitotic neurons are not known. We have found that MeCP2 binds to a site in BDNF promoter III just 3' to the site of transcriptional initiation and functions to repress expression of the BDNF gene. Membrane depolarization triggers the calcium-dependent phosphorylation and release of MeCP2 from the BDNF promoter, thereby facilitating BDNF promoter Ill-dependent transcription. These findings indicate that MeCP2 plays a key role in the control of activity-dependent gene expression and suggest that the deregulation of this process may underlie the pathology of RTT. To begin to test this hypothesis, we propose the following specific aims: 1) To characterize the sites of membrane depolarization/calcium-dependent MeCP2 phosphorylation. We will utilize a variety of methods to identify sites of activity-regulated phosphorylation on MeCP2 and develop phosphorylation site-specific antibodies to investigate the regulation of these modifications in cultured neurons and brain sections in response to a variety of stimulation protocols. 2) To assess the effect of phosphorylation on MeCP2 activity. Non-phosphorylatable mutant forms of MeCP2 will be generated and expressed in neuronal cultures to test the hypothesis that activity-induced MeCP2 phosphorylation is required for proper regulation of BDNF promoter activity as well as for neuronal processes such as synaptic development and maintenance. 3) To identify additional activity-regulated neuronal targets of MeCP2. Our findings raise the possibility that MeCP2 may be a general regulator of activity-dependent gene expression. We will employ a variety of techniques including gene expression profiling, RT-PCR, and chromatin immunoprecipitation to identify other activity-regulated targets of MeCP2 in post mitotic neurons. It is our hope that the proposed experiments will provide a better understanding of MeCP2 function, give insight into the mechanisms of activity-dependent gene expression, and provide new opportunities for the development of therapeutic strategies to alleviate RTT pathology.