Phosphorylation-mediated inhibition of MEF2 in neuron

描述（由申请人提供）：细胞凋亡在消除缺乏营养支持的神经元中起关键作用，从而控制神经元总数。许多成人疾病包括阿尔茨海默病（AD）、帕金森氏病和肌萎缩侧索硬化症（ALS）都涉及神经元的病理改变，导致神经元通过细胞凋亡而丧失。我们实验室的长期研究目标是了解转录依赖机制在发育和神经退行性变过程中如何调节神经元的存活和凋亡。利用MEF2在发育和成熟神经元存活中的关键作用，我们提出在本应用中探索细胞周期蛋白依赖性激酶5 （Cdk5）抑制MEF2功能的关键机制。我们的具体目标是：1。探索cdk5介导的磷酸化调控MEF2稳定性的分子机制；2. 研究cdk5介导的磷酸化对MEF2的调控作用及其机制；和3。研究cdk5介导的磷酸化对MEF2亚细胞和亚核分布的影响及机制。神经毒性诱导的小脑颗粒神经元凋亡将作为我们研究的模型。将采用生化、分子生物学和细胞生物学相结合的方法来确定谷氨酸对MEF2的去稳定化作用，以及cdk5介导的磷酸化在这一过程中的作用。caspase在MEF2不稳定性中的作用将通过caspase裂解试验来评估。我们将通过结构域映射研究控制MEF2稳定性的独特调控机制。将通过体外拉下实验、共免疫沉淀和染色质免疫沉淀来研究MEF2与其共调节因子（激活剂和抑制剂）之间相互作用的cdk5依赖性调节。可能拮抗Cdk5功能的信号通路将被研究。最后，将通过免疫细胞化学分析和亚细胞分离来研究MEF2的亚核和亚细胞分布。Cdk5对MEF2 DNA结合的影响将通过DNA结合实验和启动子招募实验进行研究。这些实验将明确神经毒素和cdk5诱导的磷酸化调节MEF2功能和神经元死亡的具体机制，这对我们了解病理条件下神经元凋亡过程至关重要。

DESCRIPTION (provided by applicant): Apoptosis plays a key role in eliminating neurons that lack trophic support, thereby controlling the total number of neurons. Many adult human illnesses including Alzheimer's disease (AD), and Parkinson's disease and amyotrophic lateral sclerosis (ALS) involve pathologic change of neurons, which results in their loss through apoptosis. The long-term objective of this research in our laboratory is to understand how transcription-dependent mechanisms regulate neuronal survival and apoptosis during development and neurodegeneration. Taking advantage of the key role of MEF2 in the survival of both developing and mature neurons, we propose in the present application to explore the critical mechanisms by which cyclin-dependent kinase 5 (Cdk5) inhibits MEF2 function. Our specific aims are: 1. to explore the molecular mechanisms by which CDK5-mediated phosphorylation regulate MEF2 stability; 2. to study the effects and mechanisms of Cdk5-mediated phosphorylation on the regulation of MEF2 by its co-regulators; and 3. to study the effects and mechanisms of Cdk5-mediated phosphorylation on the subcellular and subnuclear distribution of MEF2. Neurotoxicity-induced apoptosis of cerebellar granule neurons will be used as a model for our investigation. A combined biochemical, molecular biological, and cell biological approach will be used to determine MEF2 de-stabilization by glutamate, and the role of Cdk5-mediated phosphorylation in this process. The role of caspase in MEF2 instability will be assessed by caspase cleavage assay. The unique regulatory mechanisms that control MEF2 stability will be studied through domain mapping. Cdk5-dependent regulation of the interaction between MEF2 and its co-regulators, both activators and inhibitors, will be investigated by in vitro pull down assays, co-immunoprecipitation, and chromatin immunoprecipitation. Signaling pathways that may antagonize Cdk5 function will be studied. Finally, sub nuclear and subcellular distribution of MEF2 will be investigated by immunocytochemistry analysis and subcellular fractionation. The effect of Cdk5 on MEF2 DNA binding will be studied by DNA binding assay and promoter recruiting assay. These experiments will define specific mechanisms by which neurotoxin and Cdk5-induced phosphorylation regulate MEF2 function and neuronal death, which are essential to our understanding of the process of neuronal apoptosis under pathological conditions.