NEUROTROPHIC FACTOR DEPRIVATION AND NEURONAL CELL DEATH

描述（由申请人提供）：本项目的主要目的是揭示共同的机制，管理发育和病理性神经元死亡，并应用这些知识来制定治疗中风，创伤和神经退行性疾病。拟议的研究将集中在“凋亡细胞周期途径”深入研究，在各种发育和病理性神经元死亡范例中发挥所需的作用。在该途径中，细胞周期蛋白依赖性激酶4（Cdk 4）在神经元中响应于凋亡刺激而被激活。这又使Rb口袋蛋白家族成员磷酸化，使它们与E2 F转录因子的基因阻遏物复合物解离。Rb-E2 F阻遏物复合物的解离导致Myb转录因子的去阻遏和诱导，所述Myb转录因子结合并激活包括Bim的凋亡蛋白的启动子。Bim的诱导促进线粒体蛋白的释放和半胱天冬酶的激活，导致神经元死亡。该建议侧重于凋亡刺激激活Cdk 4，特别是cdc 25 a磷酸酶的机制。在细胞周期中，cdc 25 a通过去除抑制性磷酸基团来激活Cdk 4。我们将评估cdc 25 a活性升高的神经元凋亡刺激，这是一个顶端事件在触发凋亡细胞周期途径的假设。初步的支持性数据包括发现cdc 25 a蛋白和转录本在神经元细胞中被几种不同的凋亡刺激物升高，并且cdc 25 a的敲低或化学抑制具有神经保护作用。有两个具体目标。1）。为了在体外模型中测试cdc 25 a在细胞凋亡中起近端作用的假设， 发育和病理性神经元死亡，并剖析其参与的凋亡细胞周期级联的上游和下游元件。这将涉及各种细胞培养系统和模型的发展（神经生长因子剥夺）和病理（AD，PD）神经元死亡。我们将探讨a）在我们的各种模型中cdc 25 a在神经元死亡中所需的作用，B）通过凋亡刺激调节cdc 25 a的程度，c）位于cdc 25 a下游的死亡级联组件，以及d）调节cdc 25 a表达和活性的上游机制以及这些机制如何通过凋亡刺激参与。我们将特别热衷于识别涉及cdc 25 a的前馈死亡环。2）。验证cdc 25 a是体内发育神经元死亡所必需的假设。使用上级颈神经节中发育神经元死亡的范例，我们将询问a）cdc 25 a水平是否在垂死的神经元中被诱导，B）所选下游细胞周期途径组分是否在这些神经元中共同升高，c）生物耐受的化学cdc 25 a抑制剂是否抑制该系统中的神经元死亡，以及d）这种死亡是否通过交感神经节中cdc 25 a的选择性遗传消融而被抑制。目前的研究结果表明cdc 25 a是一个药物靶点，其在体内的抑制作用几乎没有副作用。因此，当前目标的成功具有导致病理性神经元死亡的疗法的发展的明显潜力。

DESCRIPTION (provided by applicant): The over-arching aim of this project is to uncover shared mechanisms that govern developmental and pathologic neuron death and to apply this knowledge to formulate treatments for stroke, trauma and neurodegenerative disorders. The proposed studies will focus on the "apoptotic cell cycle pathway" studied in depth under this grant that plays a required role in a variety of developmental and pathological neuron death paradigms. In this pathway, the cyclin-dependent kinase-4 (Cdk4) is activated in neurons in response to apoptotic stimuli. This is turn phosphorylates Rb pocket protein family members causing them to dissociate from gene-repressor complexes with the E2F transcription factors. Dissociation of Rb-E2F repressor complexes leads to derepression and induction of Myb transcription factors that bind to and activate the promoter for apoptotic proteins including Bim. Induction of Bim promotes release of mitochondrial proteins and activation of caspases, resulting in neuron death. This proposal focuses on the mechanism by which apoptotic stimuli activate Cdk4 and in particular on the cdc25a phosphatase. During the cell cycle, cdc25a activates Cdk4 by removing an inhibitory phosphate group. We will assess the hypothesis that cdc25a activity is elevated by apoptotic stimuli in neurons and that this is an apical event in triggering the apoptotic cell cycle pathway. Preliminary supportive data include findings that cdc25a protein and transcripts are elevated in neuronal cells by several different apoptotic stimuli, and that knockdown or chemical inhibition of cdc25a is neuroprotective. There are two specific aims. 1). To test, in in vitro models, the hypothesis that cdc25a plays a proximal role in developmental and pathological neuron death and to dissect the up- and downstream elements of the apoptotic cell cycle cascade in which it partakes. This will involve a variety of cell cultue systems and models for developmental (NGF deprivation) and pathological (AD, PD) neuron death. We will explore a) the required role of cdc25a in neuron death in our various models, b) the extent of cdc25a regulation by apoptotic stimuli, c) the death cascade components that lie downstream of cdc25a, and d) the upstream mechanisms by which cdc25a expression and activity are regulated and how these are engaged by apoptotic stimuli. We will be particularly keen to identify feed-forward death loops involving cdc25a. 2). To test the hypothesis that cdc25a is required for developmental neuron death in vivo. Using the paradigm of developmental neuron death in superior cervical ganglia, we will query whether a) cdc25a levels are induced in dying neurons, b) selected downstream cell cycle pathway components are co-elevated in such neurons, c) biologically-tolerated chemical cdc25a inhibitors suppress neuron death in this system and d) whether such death is inhibited by selective genetic ablation of cdc25a in sympathetic ganglia. Current findings indicate that cdc25a is a druggable target whose in vivo inhibition has little side effects. Thus success of the current aims has a clear potential to lead to development of therapies for pathologic neuron death.