Role of Pur Alpha in Neuronal Cells

Pur Alpha 在神经元细胞中的作用

• 批准号: 6877735
• 负责人: Jennifer Gordon
• 金额: $ 28.6万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6877735
• 关键词: PC12 cells; SDS polyacrylamide gel electrophoresis; apoptosis; autoradiography; cell differentiation; cell migration; confocal scanning microscopy; cyclin dependent kinase; developmental neurobiology; enzyme activity; fluorescence microscopy; gene targeting; genetically modified animals; genotype; immunocytochemistry; immunoprecipitation; laboratory mouse; neurogenesis; phosphorylation; polymerase chain reaction; protein structure function; tissue /cell culture; western blottings

CNS disorders are a major sequelae of HIV-1 infection occurring in more than 60% of HIV-1 infected individuals. Neuronal cells are particularly vulnerable and their injury is related to the development of cognitive and motor impairment seen in AIDS patients. A brain derived nucleic acid binding protein, Pura has been implicated in diverse biological events including control of the HIV-1 genome and several cellular genes whose expression are affected upon HIV-1 infection of the CNS. The association of Pur(alpha), which is highly expressed in neurons several key regulatory proteins including cyclins and cyclin dependent kinases coupled with its fluctuating levels throughout the cell cycle, position Pur(alpha) as a crucial factor in the control of cell growth and differentiation. Recent studies in our laboratory have revealed the ability of Pur(alpha) to associate with cdk5, a brain- specific isoform of cdc2, which upon interaction with the neuron- specific p35 protein gains catalytic activity in differentiated neurons. A complex of protein containing cdk5 and p35 with kinase activity is essential for neurite outgrowth during neuronal differentiation. Evidently, the cdk5:p35 complex phosphorylates several axonal cytoskeletal proteins including neurofilaments , tau, and the beta- amyloid precursor protein, and plays an important role in the regulation of N-cadherin-mediated cell adhesion during cortical development. Mice with targeted disruptions of either cdk5 or p35 genes exhibit abnormalities in the laminar structure of the cerebral cortex and contain neurofilaments which are hypophoshorylated at their critical C-terminal domains. These mutant mice show neurological defects including spontaneous seizures. Our effort to determine the biological role of Pur(alpha) in mice through gene knockout has yielded a surprising discovery as mice with no (Pur(alpha) -/-) or reduced (Pur(alpha) -/+) level of Pur(alpha) demonstrated similar abnormalities in the cortical lamination, hypophosphorylation of neurofilaments, and spontaneous seizures as the cdk5, p35 mice. In light of earlier data showing the ability of Pur(alpha) to associate with the cdk5 complex, we hypothesize that Pur(alpha) may play a critical role in regulating the activity of cdk5 and its associated factor(s) in neurons and thus may be important for neuronal cell development and the formation of cortical layers. To examine this hypothesis, we will: (i) examine the developmental expression and association of Pur(alpha) with the cdk5 complex, and the ability of Pur(alpha) to modulate cdk5 kinase activity during brain development; (ii) utilize biochemical assays t o map the region within Pur(alpha) which is important for cdk5 binding and its kinase activity, and examine the function of Pur(alpha) mutants in neuronal cell differentiation in a cell culture system; (iii) examine the importance of Pur(alpha) in neuronal cell differentiation and the development of stratification of cortical neurons at various stages of brain development by generating transgenic mice that conditionally inactivate Pur(alpha) function. The results of these studies should provide insight into molecular events involved in neuronal differentiation and further assist us in understanding the neuronal injury and neuropathology seen in several debilitating CNS disorders and NeuroAIDS.

中枢神经系统疾病是HIV-1感染的主要后遗症，在超过60%的HIV-1感染者中发生。神经细胞特别脆弱，它们的损伤与艾滋病患者的认知和运动障碍的发展有关。PURA是一种脑源性核酸结合蛋白，参与了多种生物学事件，包括控制HIV-1基因组和几个细胞基因的表达，这些基因的表达受到HIV-1感染中枢神经系统的影响。PUR(α)在神经元中高表达几种关键调控蛋白，包括细胞周期蛋白和细胞周期蛋白依赖蛋白依赖蛋白，其在细胞周期中的水平波动，使PUR(α)成为控制细胞生长和分化的关键因子。我们实验室最近的研究表明，PUR(α)能够与cdc2的大脑特异性亚型CDK5结合，当与神经元特异性p35蛋白相互作用时，它在分化的神经元中获得催化活性。在神经元分化过程中，含有CDK5和p35的蛋白与激酶活性的复合体对于轴突的生长是必不可少的。显然，CDK5：P35复合体可磷酸化几种轴突细胞骨架蛋白，包括神经丝、tau和β-淀粉样前体蛋白，并在调节N-钙粘附素介导的细胞黏附中发挥重要作用。靶向突变CDK5或p35基因的小鼠表现出大脑皮层层状结构的异常，并含有神经细丝，这些神经丝在其关键的C-末端区域被低磷短化。这些突变小鼠表现出神经缺陷，包括自发性癫痫发作。我们通过基因敲除来确定PUR(α)在小鼠中的生物学作用的努力取得了一个令人惊讶的发现，因为PUR(α)水平没有(PUR(α)-/-)或水平降低(PUR(α)-/+)的小鼠表现出与CDK5，p35小鼠相似的皮质层异常、神经细丝低磷酸化和自发性癫痫发作。根据早期数据显示PUR(α)与CDK5复合体结合的能力，我们推测PUR(α)可能在调节神经元中CDK5及其相关因子(S)的活性方面发挥关键作用，因此可能对神经细胞的发育和皮质层的形成起重要作用。为了验证这一假设，我们将：(I)检测PUR(α)与CDK5复合体的发育表达和联系，以及PUR(α)在脑发育过程中调节CDK5激酶活性的能力；(Ii)利用生化分析来定位PUR(α)内对CDK5结合及其激酶活性重要的区域，并检测PUR(α)突变体在细胞培养系统中神经细胞分化中的作用；(Iii)通过产生条件失活Pur(Alpha)功能的转基因小鼠，研究Pur(Alpha)在神经细胞分化和大脑发育不同阶段皮质神经元层化发展中的重要性。这些研究的结果将有助于我们深入了解神经元分化所涉及的分子事件，并进一步帮助我们理解在几种衰弱的中枢神经系统疾病和神经艾滋病中所见的神经元损伤和神经病理。