Impact of HIV-1 on Rho GTPase mediated neuronal cell development

项目1：HIV-1对Rho GTP酶介导的神经细胞发育的影响。 现在人们普遍认为HIV-1调节蛋白Tat具有神经毒性作用，这种治疗方法 在人类和啮齿动物神经元中，TAT诱导细胞凋亡的机制尚不完全清楚。 细胞蛋白PUR-α是TAT指导HIV-1转录调控的关键靶点， 翻译，以及病毒RNA的运输。事实上，在同一个细胞中检测到了pur-α和Tat 在蜂房感染的病变内。在Pur-α基因敲除小鼠模型中的结果显示了一个关键的作用 在发育过程中，特别是在神经元的协调发展和分化过程中 遍布整个大脑的细胞。在过去的几年里，很明显，RhoGTP酶和 相关分子在神经细胞发育中发挥重要作用，包括轴突生长， 分化、轴突寻路、树突棘的形成以及神经细胞的维持。结果 我们的研究表明，TAT具有通过刺激RhoA激活来干扰这一途径的能力，并且 伴随着通过与鸟嘌呤核苷酸相互作用将RhoA？GDP转化为RhoA？GTP的事件 交换因子(全环基金)家族成员，包括PDZ-罗伊夫全环基金。事实上，我们的初步结果表明 TAT可能通过与PDZ-Rhogenf的相互作用来调节RhoA的活性。一旦被激活，RhoA通常是 定位于质膜，并通过一系列涉及Rock、MAPK和 JNK1、NFkB诱导轴突回缩。此外，RhoA通过异构体G蛋白激活，包括 GA12和G&lt；x13可以刺激GSK3(3)，这也是已知参与HIV-1神经毒性的因素。论 另一方面，TAT可以阻止另一个关键的RhoGTP酶的激活，即rac1及其伙伴p21激活 激酶1(PAK1)，它似乎调节肌动蛋白细胞骨架的动力学，维持神经细胞的完整性，以及 促进轴突生长。此外，TAT最近被证明通过作用抑制这一途径。 通过PAK1和JNK1。最后发现，rac1和RhoA表现出异常的表达和亚细胞 在Pur-α基因敲除小鼠模型中的定位，表明Pur-Alpha是其正常发育所必需的 功能。基于这些初步观察，我们假设pur-α在 RhoA和Rac活性的平衡，以及它的基因消融或功能改变 关联TAT可以破坏RhoA和RAC信号通路中的几个关键参数，包括 P115/PDZ和PAK，并导致在神经元中常见的病理特征 伴有中枢神经系统紊乱的艾滋病患者的细胞。在这个研究项目中，我们将进行一系列细胞生物学 以及分子研究以揭示TAT与Rho GTP酶相互作用的分子基础及其对 神经细胞的完整性。

Project #1. Impact of HIV-1 on the Rho GTPase-mediated neuronal cell development. It is now well accepted that the HIV-1 regulatory protein, Tat, has a neurotoxic effect and that treatment of both human and rodent neurons with Tat induces apoptosis by mechanisms that are not fully understood. The cellular protein, Pur-alpha, is a key target of Tat for directing the regulation of HIV-1 transcription, translation, and viral RNA transport. Indeed, both Pur-alpha and Tat have been detected in the same cells within HIVE infected lesions. Results in the Pur-alpha knockout mouse model have revealed a critical role for Pur-alpha during development, particularly in the coordinated development and differentiation of neuronal cells throughout the brain. Over the past several years, it has become clear that the RhoGTPases and related molecules play an important role in neuronal cell development, including neurite outgrowth, differentiation, axon pathfinding, dendritic spine formation, as well as neuronal cell maintenance. Results from our studies indicate that Tat has the ability to disturb this pathway by stimulating RhoA activation, an event that is accompanied by conversion of RhoA¿GDP to RhoA¿GTP by interacting with guanine nucleotide exchange factor (GEF) family members including PDZ-RhoGEF. Indeed, our preliminary results suggest that Tat may mediate RhoA activity via interaction with PDZ-RhoGEF. Upon its activation, RhoA is usually positioned in the plasma membrane, and via a series of signaling pathways that involve Rock, MAPK, and JNK1, NFkB induces neurite retraction. Moreover, activation of RhoA via heteromeric G-proteins including Ga12 and G<x13 can stimulate GSK3(3 which is also known to be involved in HIV-1 neurotoxicity. On the other hand, Tat can block activation of another key RhoGTPase, i.e. Rac1 and its partner p21-activated kinase 1 (PAK1), which appears to regulate actin cytoskeletal dynamics, maintain neuronal cell integrity, and promote neurite outgrowth. In addition, Tat has recently been shown to inhibit this pathway by acting through PAK1 and JNK1. Finally, Rac1 and RhoA were found to display aberrant expression and subcellular localization in the Pur-alpha knockout mouse model, suggesting that Pur-alpha is required for their proper function. Based on these preliminary observations, we hypothesize that Pur-alpha has a central role in the well-balanced activities of RhoA and Rac, and that its genetic ablation or its functional alteration by associating with Tat can derail several key parameters in RhoA and Rac signaling pathways including p115/PDZ and PAK, respectively, and results in pathological features that are commonly seen in neuronal cells of AIDS patients with CNS disorders. In this research project, we will perform a series of cell biological and molecular studies to unravel the molecular basis of Tat cross-talk with Rho GTPase and its impact on neuronal cell integrity.