Molecular basis of neuronal dysfunction in AIDS

描述（由申请人提供）：HIV-1入侵中枢神经系统，引起各种临床异常，包括痴呆，也称为HIV-1相关痴呆，HAD。从组织学上看，患有神经系统疾病的艾滋病患者的脑活检显示出广泛的异常，最明显的是神经元细胞丢失。神经细胞感染的缺失提示了病毒诱导神经细胞损伤的间接作用。大量研究结果表明，脑内感染的巨噬细胞、小胶质细胞和星形胶质细胞的细胞因子和免疫调节剂等多种细胞蛋白的表达和分泌参与了神经元细胞的损伤。除细胞因子外，病毒调节蛋白Tat因其在没有病毒感染的情况下由感染细胞分泌、进入未感染细胞并改变细胞基因表达的独特能力而受到关注。此外，Tat蛋白已被证明具有神经毒性活性，最有可能通过对神经细胞存活至关重要的信号通路失调。通过使用PC12神经元细胞模型，以及大鼠原代神经元细胞，我们证明了Tat刺激了Id基因的表达，这是一种已知的抑制神经元细胞分化的细胞蛋白。通过影响控制细胞周期的几个因子的表达和活性，促进静止细胞非预定地进入增殖阶段，并导致抑制一组与维持分化细胞有关的蛋白质。碱性螺旋-环-螺旋，bHLH， DNA结合蛋白家族，如Ngn， NeuroD和Mash1，是Id1的主要靶点，当Id1失调时可促进神经元细胞损伤和凋亡。我们的初步数据表明，Tat激活Id1基因是通过一个新的调控途径介导的，该途径包括Egr1、C/EBP和SP1等关键转录因子，这些转录因子在物理和功能上都与Tat相互作用。在这个项目的第一个目标中，我们将进行一系列全面的功能和结构研究，以揭示Tat在神经元细胞中激活Id1的分子机制。在第二部分中，我们将研究Id1在神经元细胞周期中的重要性，其表达被Tat升高，以及控制细胞进入细胞周期和检查点的因子的表达和活性，如p21、p16、pRb和各种周期蛋白。此外，还将评估tat诱导的Id1过量产生对bHLH表达和活性的作用。这些研究的结果将为HIV-1诱导的神经细胞损伤通路的研究提供新的信息，并将有助于我们制定针对HIV-1诱导的神经细胞损伤的策略。

DESCRIPTION (provided by applicant): HIV-1 invasion of the Central Nervous System, CNS, induces a variety of clinical abnormalities including dementia, also known as HIV-1 associated dementia, HAD. Histologically, brain biopsy from AIDS patients with neurological disorders exhibit a broad ranges of abnormalities, most notably neuronal cell loss. The absence of neuronal infection suggested an indirect role for the virus to induce neuronal cell damages. Results from a large number of studies indicated that expression and secretion of several cellular proteins including cytokines and immunomodulators from the infected macrophages, microglia, and astrocytes in the brain participate in the neuronal cell damages. In addition to cellular factors, the viral regulatory protein Tat has received attention due to its unique ability to be secreted by the infected cells, enter into uninfected cells and alter the expression of cellular genes in the absence of viral infection. Moreover, Tat protein has been shown to have neurotoxic activity, most likely through dysregulating signaling pathways that are critical for neuronal cell survival. By using PC12 neuronal cell model, as well as primary rat neuronal cells, we demonstrated that Tat stimulate the expression of Id gene, a known cellular protein that inhibits differentiation of neuronal cells. Id, by affecting expression and activity of several factors which control the cell cycle promotes unscheduled entering of quiescent cells into proliferating stage and that results in suppression of a group of proteins that are implicated in the maintenance of differentiated cells. The basic helix-loop-helix, bHLH, family of DNA binding proteins such as Ngn, NeuroD, and Mash1, are the primary targets for Id1 which upon dysregulation may promote neuronal cell injury and apoptosis. Our preliminary data suggest that activation of Id1 gene by Tat is mediated via a novel regulatory pathway that includes critical transcription factors such as Egr1, C/EBP and SP1, all of which physically and functionally interact with Tat. In Aim one of this project we will perform a series of comprehensive functional and structural studies to unravel the molecular mechanisms involved in activation of Id1 by Tat in neuronal cells. In Aim two, we will investigate the importance of Id1, whose expression is elevated by Tat, in neuronal cell cycle, control of expression and activity of factors controlling cell entry into cell cycle and checkpoints such as p21, p16, pRb, and various cyclins. Furthermore, the role of Tat-induced overproduction of Id1 on the expression and activity of bHLH will be assessed. The outcome of these studies will provide a new information on the pathways which are affected by HIV-1 Tat, and will assist to improve our effort for developing strategies against HIV-1 induced neuronal cell injury.