Neuronal Activity-Dependent Regulation of MeCP2

描述(由申请人提供)：摘要/摘要：MeCP2是一种甲基CpG结合蛋白，起着基因表达调节的作用，它的突变是Rett综合征(RTT)的主要原因，RTT是一种X连锁的进行性自闭症谱系障碍，是女孩和妇女严重认知障碍的最常见原因之一。虽然神经元中MeCP2的选择性失活足以在小鼠中授予Rett样表型，但刺激后神经元中MeCP2功能的丧失导致RTT表型的具体机制尚不清楚。我们已经确定MeCP2上的丝氨酸421(S421)是神经元活性依赖的磷酸化位点，在大脑中选择性地诱导对生理刺激的反应。值得注意的是，我们发现S421磷酸化控制着MeCP2调节树突图案、脊椎形态发生的能力，以及在培养的神经元和切片中BDNF转录的活性诱导。为了进一步探索这一调控机制在体内神经发育中的作用，我们产生了一个敲入小鼠，在该小鼠中，MeCP2的S421突变为一个丙氨酸残基(S421A Ki)，阻止了MeCP2在该位点的磷酸化。有趣的是，虽然在体内取消MeCP2 S421磷酸化并不会导致MeCP2表达完全丧失的运动和生存表型，但我们的初步研究发现，这些S421A Ki小鼠的皮质抑制性突触发育存在缺陷，表明活性依赖的磷酸化可能参与与RTT中观察到的突触和认知缺陷相关的MeCP2功能的特定子集。为了验证这一假说并确定MeCP2作为神经元活性依赖性基因表达的一般调节因子的作用程度，我们提出了以下具体目标：(1)研究MeCP2 S421磷酸化对活体经验依赖性突触发育的贡献；(2)评估MeCP2 S421磷酸化在调节活性依赖性神经元基因表达中的作用；以及(3)表征其他活性依赖性MeCP2磷酸化位点。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。 公共卫生相关性：项目叙述：雷特综合症是一种进行性自闭症，是女孩和妇女严重认知障碍的最常见原因之一。为了深入了解这种疾病的潜在分子基础，这项拟议的研究将试图探索这样一种假设，即这种自闭症反映了中枢神经系统中基因动态调节的缺陷。

DESCRIPTION (provided by applicant): Summary/Abstract: Mutations in MeCP2, a methyl-CpG-binding protein that functions as a regulator of gene expression, are a major cause of Rett Syndrome (RTT), an X-linked progressive autism spectrum disorder that is among the most common causes of profound cognitive impairment in girls and women. While the selective inactivation of MeCP2 in neurons has been suggested to be sufficient to confer a Rett-like phenotype in mice, the specific mechanisms by which the loss of MeCP2 function in postimitotic neurons contributes to RTT phenotypes remain unclear. We have identified serine 421 (S421) on MeCP2 as a site of neuronal activity-dependent phosphorylation that is induced selectively in the brain in response to physiological stimuli. Significantly, we have found that S421 phosphorylation controls the ability of MeCP2 to regulate dendritic patterning, spine morphogenesis, and the activity-dependent induction of Bdnf transcription in both cultured neurons and slice preparations. To further explore the role of this regulatory mechanism in neural development in vivo, we have generated a knock-in mouse in which S421 of MeCP2 is mutated to an alanine residue (S421A KI), preventing the phosphorylation of MeCP2 at this site. Intriguingly, whereas the abrogation of MeCP2 S421 phosphorylation in vivo does not result in the motor and survival phenotypes seen with complete loss of MeCP2 expression, our preliminary studies have revealed a deficit in cortical inhibitory synaptic development in these S421A KI mice, suggesting that activity-dependent phosphorylation may be involved in a specific subset of MeCP2 functions relevant to the synaptic and cognitive defects observed in RTT. To begin to test this hypothesis and determine the extent to which MeCP2 functions as a general regulator of neuronal activity- dependent gene expression, we propose the following specific aims: (1) to investigate the contribution of MeCP2 S421 phosphorylation to experience-dependent synaptic development in vivo; (2) to assess the role of MeCP2 S421 phosphorylation in the regulation of activity-dependent neuronal gene expression; and (3) to characterize additional sites of activity-dependent MeCP2 phosphorylation. 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. PUBLIC HEALTH RELEVANCE: Project Narrative: Rett Syndrome is a progressive autistic disorder that is among the most common causes of profound cognitive impairment in girls and women. In an effort to gain insight into the underlying molecular basis of the disorder, the proposed study will seek to explore the hypothesis that this autistic disorder reflects a defect in the dynamic regulation of genes in the central nervous system.