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（ALS）涉及神经元的病理变化，其导致神经元通过细胞凋亡而丧失。我们实验室这项研究的长期目标是了解信号控制的细胞内机制如何在发育和神经变性过程中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（Cdk 5）在成熟神经元凋亡中起关键作用。我们最近的研究结果表明，Cdk 5在细胞核中的功能，以调节细胞凋亡。在本申请中，我们提出鉴定Cdk 5诱导神经元凋亡的新的核机制。我们的具体目标是：1.鉴定和表征Cdk 5在神经元核中的新调控靶点; 2.探讨Cdk 5对神经元p38 MAPK信号通路的调节作用; 3.鉴定和表征MEF 2（肌细胞增强因子2）中的其他Cdk 5调控位点。为了鉴定Cdk 5的新的核底物，我们将使用Cdk 5结合试验首先从原代神经元的核制备物中分离蛋白质，并通过诱变的大量磷酸化来揭示它们的鉴定。我们将评估Cdk 5介导的p38 MAPK磷酸化如何影响其亚细胞定位和光谱分析。然后，我们将在生化和生物测定中确认它们作为Cdk 5底物。为了探讨Cdk 5是否直接调节p38 MAPK通路，我们将通过体外激酶试验和体内磷酸化研究来测试p38 MAPK是否是Cdk 5的底物。我们将确定其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Cdk 5介导的p38 MAPK磷酸化。我们还将探讨Cdk 5是否直接调节p38 MAPK上游激活剂。将通过诱变鉴定MEF 2各种亚型中的其他调控位点。将在报告基因激活试验和神经元存活试验中使用MEF 2C突变体评估Cdk 5介导的选择性剪接MEF 2C变体磷酸化的潜在影响。确定这些新的Cdk 5核调控靶点将使我们能够探索Cdk 5调节细胞核中生存机制的机制。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