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，从而控制神经元的总数。许多成人疾病包括阿尔茨海默病（Alzheimer's disease，AD）、帕金森病（Parkinson's disease）和肌萎缩侧索硬化症（amyotrophic lateral sclerosis，ALS）都涉及神经元的病理变化，导致神经元凋亡。我们实验室这项研究的长期目标是了解在发育和神经变性过程中，转录依赖机制如何调节神经元的存活和凋亡。利用MEF 2在发育和成熟神经元的存活中的关键作用，我们在本申请中提出探索细胞周期蛋白依赖性激酶5（Cdk 5）抑制MEF 2功能的关键机制。我们的具体目标是：1.探讨CDK 5介导的磷酸化调节MEF 2稳定性的分子机制; 2.研究Cdk 5介导的磷酸化对MEF 2的协同调节作用及其机制; 3.研究Cdk 5介导的磷酸化对MEF 2亚细胞和亚核分布的影响及其机制。神经毒性诱导的小脑颗粒神经元凋亡将被用作我们研究的模型。结合生物化学，分子生物学和细胞生物学的方法将被用来确定MEF 2去稳定谷氨酸，Cdk 5介导的磷酸化在这一过程中的作用。将通过胱天蛋白酶切割测定来评估胱天蛋白酶在MEF 2不稳定性中的作用。控制MEF 2稳定性的独特调控机制将通过结构域映射进行研究。Cdk 5依赖性调节MEF 2及其共调节因子（激活剂和抑制剂）之间的相互作用将通过体外下拉测定、共免疫沉淀和染色质免疫沉淀来研究。将研究可能拮抗Cdk 5功能的信号通路。最后，MEF 2的亚核和亚细胞分布将通过免疫细胞化学分析和亚细胞分级来研究。通过DNA结合试验和启动子募集试验研究Cdk 5对MEF 2 DNA结合的影响。 这些实验将定义神经毒素和Cdk 5诱导的磷酸化调节MEF 2功能和神经元死亡的具体机制，这对我们理解病理条件下神经元凋亡的过程至关重要。

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.