Neuronal Activity-Dependent Regulation of MeCP2

描述(申请人提供)：MeCP2是一种甲基I-CpG结合蛋白，起着全球转录抑制因子的作用，它的突变是Rett综合征(RTT)的主要原因，RTT是一种X-连锁进行性神经疾病。虽然MeCP2在神经元中的选择性失活足以在小鼠中赋予Rett样表型，但MeCP2在有丝分裂后神经元中的特定功能尚不清楚。我们已经发现MeCP2与BDNF启动子III的转录起始部位仅有3‘端结合，并具有抑制BDNF基因表达的功能。膜去极化触发BDNF启动子的钙依赖性磷酸化和MeCP2的释放，从而促进BDNF启动子Ill依赖的转录。这些发现表明，MeCP2在控制依赖活性的基因表达方面发挥了关键作用，并提示这一过程的放松可能是RTT的病理基础。为了开始验证这一假说，我们提出了以下具体目标：1)表征膜去极化/钙依赖的MeCP2磷酸化位点。我们将利用各种方法来确定MeCP2上活性调节的磷酸化位点，并开发磷酸化位点特异性抗体来研究培养的神经元和大脑切片对各种刺激方案的调节。2)评价磷酸化对MeCP2活性的影响。将产生不可磷酸化的MeCP2突变形式并在神经元培养中表达，以检验活性诱导的MeCP2磷酸化是适当调节BDNF启动子活性以及突触发育和维持等神经元过程所必需的假说。3)确定MeCP2的其他活性调节神经元靶点。我们的发现提出了这样一种可能性，即MeCP2可能是活性依赖基因表达的一般调节因子。我们将使用各种技术，包括基因表达谱，RT-PCR和染色质免疫沉淀来确定MeCP2在有丝分裂后神经元中的其他活性调节靶点。我们希望所提出的实验能够更好地了解MeCP2的功能，深入了解活性依赖基因表达的机制，并为开发减轻RTT病理的治疗策略提供新的机会。

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.