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

一号工程。HIV-1对Rho GTP酶介导的神经细胞发育的影响 现在普遍认为HIV-1调节蛋白达特具有神经毒性作用， 达特对人类和啮齿类动物神经元的作用通过尚不完全清楚的机制诱导细胞凋亡。 细胞蛋白Pur-alpha是达特指导HIV-1转录调节的关键靶点， 翻译和病毒RNA转运。事实上，在相同的细胞中已经检测到Pur-alpha和达特 感染的病灶内。Pur-alpha基因敲除小鼠模型的结果显示， 在发育过程中，特别是在神经元的协调发育和分化中， 细胞遍布大脑。在过去的几年里，已经清楚地表明RhoGTP酶和 相关分子在神经元细胞发育，包括神经突生长， 分化、轴突寻路、树突棘形成以及神经元细胞维持。结果 我们的研究表明，达特有能力通过刺激RhoA激活来干扰这一通路， 伴随RhoA <$GDP通过与鸟嘌呤核苷酸相互作用转化为RhoA <$GTP的事件 交换因子（GEF）家族成员，包括PDZ-RhoGEF。事实上，我们的初步结果表明， 达特可能通过与PDZ-RhoGEF相互作用介导RhoA活性。在其激活时，RhoA通常是 定位在质膜上，并通过一系列涉及Rock，MAPK和 JNK 1、NFkB诱导神经突收缩。此外，RhoA通过异聚G蛋白的激活，包括 Ga 12和Gx 13可以刺激GSK 3 β，其也已知参与HIV-1神经毒性。上 另一方面，达特可以阻断另一个关键的RhoGT 3的激活，即Rac 1及其伴侣p21激活的 激酶1（PAK 1），其似乎调节肌动蛋白细胞骨架动力学，维持神经元细胞的完整性， 促进神经突生长。此外，达特最近已被证明可以抑制这一途径， 通过PAK 1和JNK 1。最后，发现Rac 1和RhoA显示异常表达和亚细胞表达。 在Pur-alpha基因敲除小鼠模型中的定位，表明Pur-alpha是其适当的表达所必需的。 功能基于这些初步的观察，我们假设Pur-alpha在细胞凋亡中起着核心作用。 RhoA和Rac的活性平衡良好，其基因消融或其功能改变， 与达特结合可以破坏RhoA和Rac信号通路中的几个关键参数， p115/PDZ和PAK，并导致在神经元中常见的病理特征， 中枢神经系统疾病的艾滋病患者的细胞。在这个研究项目中，我们将进行一系列的细胞生物学实验， 和分子研究，以解开达特与Rho GTdR的串扰的分子基础及其对 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.