NEUROTROPHIC FACTOR DEPRIVATION AND NEURONAL CELL DEATH

描述（由申请人提供）：该项目的首要目标是揭示支配发育和病理性神经元死亡的共同机制，并将这些知识应用于制定中风、创伤和神经退行性疾病的治疗方法。拟议的研究将集中在“凋亡细胞周期途径”的深入研究，在各种发育和病理神经元死亡范式中发挥必要的作用。在这一途径中，细胞周期蛋白依赖性激酶-4 （Cdk4）在神经元中响应凋亡刺激而被激活。这又使Rb口袋蛋白家族成员磷酸化，使它们与E2F转录因子的基因抑制复合物分离。Rb-E2F抑制因子复合物的解离导致Myb转录因子的抑制和诱导，Myb转录因子结合并激活凋亡蛋白（包括Bim）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。本研究的重点是凋亡刺激激活Cdk4的机制，特别是cdc25a磷酸酶。在细胞周期中，cdc25a通过去除一个抑制性磷酸基团激活Cdk4。我们将评估cdc25a活性通过神经元凋亡刺激而升高的假设，并且这是触发凋亡细胞周期途径的一个顶端事件。初步的支持数据包括发现cdc25a蛋白和转录本在几种不同的凋亡刺激下在神经元细胞中升高，并且cdc25a的敲除或化学抑制具有神经保护作用。有两个具体目标。1）. 为了验证，在体外模型中，cdc25a在

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.