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，发生在 60% 以上的 HIV-1 感染者中。神经元细胞特别脆弱，它们的损伤与艾滋病患者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，与多种生物事件有关，包括控制 HIV-1 基因组和一些细胞基因，这些基因的表达会受到中枢神经系统 HIV-1 感染的影响。 Pur(α) 在神经元中高度表达，与包括细胞周期蛋白和细胞周期蛋白依赖性激酶在内的几种关键调节蛋白结合，加上其在整个细胞周期中水平的波动，使 Pur(α) 成为控制细胞生长和分化的关键因素。我们实验室最近的研究揭示了 Pur(α) 与 cdk5（cdc2 的一种大脑特异性亚型）结合的能力，cdk5 在与神经元特异性 p35 蛋白相互作用后在分化的神经元中获得催化活性。含有 cdk5 和 p35 且具有激酶活性的蛋白质复合物对于神经元分化过程中神经突的生长至关重要。显然，cdk5:p35 复合物磷酸化多种轴突细胞骨架蛋白，包括神经丝、tau 和 β-淀粉样前体蛋白，并在皮质发育过程中调节 N-钙粘蛋白介导的细胞粘附中发挥重要作用。 cdk5 或 p35 基因被靶向破坏的小鼠大脑皮层层状结构表现出异常，并且含有在其关键 C 末端结构域低磷酸化的神经丝。这些突变小鼠表现出神经缺陷，包括自发性癫痫发作。我们通过基因敲除确定 Pur(α) 在小鼠中的生物学作用的努力取得了令人惊讶的发现，因为没有 (Pur(α) -/-) 或降低 (Pur(α) -/+) Pur(α) 水平的小鼠表现出与 cdk5、p35 相似的皮质层压、神经丝低磷酸化和自发性癫痫发作的异常。 老鼠。根据早期数据显示 Pur(α) 与 cdk5 复合物结合的能力，我们假设 Pur(α) 可能在调节神经元中 cdk5 及其相关因子的活性中发挥关键作用，因此可能对神经元细胞发育和皮质层的形成很重要。为了检验这一假设，我们将：（i）检验Pur（α）与cdk5复合物的发育表达和关联，以及Pur（α）在大脑发育过程中调节cdk5激酶活性的能力； (ii) 利用生化测定来绘制 Pur(α) 内对于 cdk5 结合及其激酶活性很重要的区域，并检查 Pur(α) 突变体在细胞培养系统中神经元细胞分化中的功能； (iii) 通过产生条件性失活 Pur(α) 功能的转基因小鼠，研究 Pur(α) 在神经元细胞分化和大脑发育各个阶段的皮质神经元分层发育中的重要性。这些研究的结果应该可以深入了解神经元分化中涉及的分子事件，并进一步帮助我们了解在几种使人衰弱的中枢神经系统疾病和神经艾滋病中看到的神经元损伤和神经病理学。