Nuclear mechanisms of Cdk5-mediated neuronal apoptosis

描述（由申请人提供）：许多成人疾病，包括阿尔茨海默病（AD）、帕金森病和肌萎缩侧索硬化症（ALS），都涉及神经元的病理改变，通过细胞凋亡导致神经元的损失。我们实验室的长期研究目标是了解在发育和神经退行性变过程中，信号控制的细胞内机制如何调节神经元的存活和凋亡。研究表明，细胞周期蛋白依赖性激酶5 （cyclin dependent kinase 5, Cdk5）在成熟神经元的凋亡中起关键作用。我们最近的研究结果表明，Cdk5在细胞核中起调节细胞凋亡的作用。我们提出在目前的应用程序，以确定新的核机制，其中Cdk5诱导神经元凋亡。我们的具体目标是：1。识别和表征神经元核中Cdk5的新调控靶点；2. 探讨Cdk5对神经元p38 MAPK信号通路的调控作用；和3。鉴定和表征MEF2（肌细胞增强因子2）中额外的Cdk5调控位点。为了鉴定新的Cdk5核底物，我们将使用Cdk5结合试验首先从原代神经元的核制剂中分离蛋白质，并通过诱变大规模磷酸化揭示它们的鉴定。我们将评估cdk5介导的p38 MAPK磷酸化如何影响其亚细胞定位和光谱仪分析。然后我们将在生化和生物分析中确认它们是Cdk5底物。为了探究Cdk5是否直接调控p38 MAPK通路，我们将通过体外激酶测定和体内磷酸化研究来检验p38 MAPK是否为Cdk5的底物。我们将确定下游目标的调控位点。我们将在神经毒素诱导的细胞凋亡模型中检测cdk5介导的p38 MAPK磷酸化。我们还将探讨Cdk5是否直接调控p38 MAPK上游激活因子。MEF2各种异构体中的其他调节位点将通过诱变确定。在报告基因激活试验和神经元存活试验中，将使用MEF2C突变体来评估cdk5介导的可选剪接MEF2C变异体磷酸化的潜在影响。确定Cdk5的这些新的核调控靶点将使我们能够探索Cdk5调节细胞核存活机制的机制。这将进一步加深我们对神经毒素诱导的细胞凋亡的分子过程的理解，这是许多神经退行性疾病发病机制的基础

DESCRIPTION (provided by applicant): 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 signal-controlled intracellular mechanisms regulate neuronal survival and apoptosis during development and neurodegeneration. Studies have shown that cyclin dependent kinase 5 (Cdk5) plays a key role in the apoptosis of mature neurons. Our recent findings suggest that Cdk5 functions in the nucleus to regulate apoptosis. We propose in the present application to identify novel nuclear mechanisms by which Cdk5 induces neuronal apoptosis. Our specific aims are: 1. to identify and characterize novel regulatory targets of Cdk5 in neuronal nucleus; 2. to explore regulation of p38 MAPK signaling pathway by Cdk5 in neurons; and 3. to identify and characterize additional Cdk5 regulatory sites in MEF2 (myocyte enhancer factor 2). To identify novel nuclear substrates of Cdk5, we will use Cdk5 binding assays to first isolate proteins from nuclear preparations of primary neurons and reveal their identify by Mass phosphorylation by mutagenesis. We will assess how Cdk5-mediated phosphorylation of p38 MAPK affects its subcellular localization and Spectrometer analysis. We will then confirm them as Cdk5 substrates in biochemical and biological assays. To explore whether Cdk5 directly regulates p38 MAPK pathway, we will test if p38 MAPK is a substrate of Cdk5 by in vitro kinase assays and in vivo phosphorylation studies. We will determine sites of regulation of its downstream targets. We will test Cdk5-mediated phosphorylation of p38 MAPK in models of neurotoxin-induced apoptosis. We will also explore whether Cdk5 directly regulates p38 MAPK upstream activators. Additional regulatory sites in various isoforms of MEF2 will be identified by mutagenesis. The potential effects of Cdk5-mediated phosphorylation of alternatively spliced MEF2C variants will be assessed using MEF2C mutants in reporter gene activation assays and neuronal survival assays. Identifying these novel nuclear regulatory targets of Cdk5 will allow us to explore the mechanisms by which Cdk5 regulates the survival machinery in the nucleus. This should further our understanding of the molecular process of neurotoxin-induced apoptosis, which underlies the pathogenesis of many neurodegenerative diseases