Mechanisms of MEF2-dependent synapse elimination

描述（由申请人提供）：正常突触形成、消除和维持的缺陷被假设为许多神经认知障碍（包括精神发育迟滞和自闭症）的基础，并且也被认为有助于与药物成瘾相关的行为。神经元活动促进突触消除以修剪在发育期间形成的多余突触，并稳态地维持突触连接的稳态数量。转录因子肌细胞增强因子2（MEF2）是发育和成人大脑中兴奋性突触消除的关键活性依赖性调节因子。然而，MEF2控制突触数量的分子和细胞机制还不清楚。我们的实验室最近发现，RNA结合蛋白，脆性X智力迟钝蛋白（FMRP）是MEF2诱导的突触消除所必需的。脆性X综合征（FXS）是最普遍的自闭症遗传形式，由编码FMRP的Fmr1基因失活引起。患有FXS的个体和该疾病的小鼠模型，Fmr1基因敲除小鼠，显示皮质树突棘密度增加，表明FXS的缺陷， 突触消除可能是FXS的基础。我们认为MEF2和FMRP共同作用调节共同的转录物，诱导兴奋性突触的消除，并提出具体目标1来研究这一假设。在这个目标中，我们将利用CLIP-seq，一种配对交联免疫沉淀（CLIP）技术，然后进行高通量测序，以确定MEF2调节的基因靶点，其转录本与FMRP相关。然后，我们将通过进行mEPSC的双光子活细胞树突棘成像和全细胞膜片钳记录来测试MEF2调节和FMRP相关候选物在结构和功能突触消除中的需求。在具体目标2中，我们将表征MEF2依赖性突触消除的过程，通过进行双光子显微镜和全细胞电生理记录来检查MEF2诱导的结构和功能突触消除的动力学，以及MEF2介导的结构突触消除的形态学过程。由于突触消除缺陷可能导致自闭症，脆性X综合征和与药物滥用相关的行为适应，我们相信我们的研究将为理解这些疾病和相关疾病提供重要意义。 公共卫生相关性：转录因子肌细胞增强因子2（MEF2）在突触连接的正常发育和维持、学习和记忆以及与药物成瘾相关的行为中起着关键作用;然而，MEF2介导这些作用的分子和细胞机制尚未完全了解。在这里，我们建议研究MEF2调节大脑中正确连接形成的机制，这项研究将对理解自闭症，脆性X综合征，药物成瘾和相关疾病产生广泛的影响。

DESCRIPTION (provided by applicant): Deficits in proper synapse formation, elimination, and maintenance, are hypothesized to underlie numerous neurocognitive disorders, including mental retardation and autism, and are also thought to contribute to behaviors associated with drug addiction. Neuronal activity promotes synapse elimination to prune excess synapses formed during development, and to homeostatically maintain a steady-state number of synaptic connections. The transcription factor myocyte enhancer factor 2 (MEF2) is a critical activity-dependent regulator of excitatory synapse elimination in developing and adult brains. However, the molecular and cellular mechanisms by which MEF2 controls synapse number are not well understood. Our lab recently discovered that the RNA-binding protein, Fragile X Mental Retardation Protein (FMRP) is required for MEF2-induced elimination of synapses. Fragile X Syndrome (FXS), the most prevalent inherited form of autism, results from inactivation of the Fmr1 gene, which codes for FMRP. Individuals with FXS and a mouse model for the disorder, Fmr1 knockout mice, display increased cortical dendritic spine density, indicating that deficits in synapse elimination may underlie FXS. We propose that MEF2 and FMRP function together to regulate common transcripts to induce elimination of excitatory synapses and propose Specific Aim 1 to investigate this hypothesis. In this aim, we will utilize CLIP-seq, a technique pairing cross-linking immunoprecipitation (CLIP) followed by high throughput sequencing to identify MEF2-regulated gene targets whose transcripts associate with FMRP. We will then test a requirement for MEF2-regulated and FMRP-associated candidates in structural and functional synapse elimination by performing 2-photon live-cell dendritic spine imaging and whole-cell patch clamp recordings of mEPSCs. In Specific Aim 2, we will characterize the process of MEF2-dependent synapse elimination, examining the kinetics of MEF2-induced structural and functional synapse elimination, as well as the morphological process of MEF2-mediated structural synapse elimination by performing 2-photon microscopy and whole-cell electrophysiology recordings. As synapse elimination defects may contribute to autism, Fragile X Syndrome, and behavioral adaptations associated with drug abuse, we believe that our studies will provide significant implications towards understanding these and related disorders. PUBLIC HEALTH RELEVANCE: The transcription factor myocyte enhancer factor 2 (MEF2) has critical roles in the proper development and maintenance of synaptic connections, in learning and memory, and in behaviors associated with drug addiction; however, the molecular and cellular mechanisms by which MEF2 mediates these effects are not well understood. Here, we propose to investigate the mechanisms by which MEF2 regulates the formation of proper connections in the brain, a study which will have broad implications for understanding autism, Fragile X Syndrome, drug addiction, and related disorders.