GFAP as a novel HIV/neuroAIDS biomarker

描述(由申请人提供)：HIV-1感染中枢神经系统(CNS)发生在大多数艾滋病患者中，并导致各种神经功能障碍和神经病理，通常称为神经艾滋病。在中枢神经系统中，小胶质细胞/巨噬细胞和星形胶质细胞是HIV-1感染的主要靶细胞，而神经元很少感染HIV-1，但在HIV/NeuroAIDS中受影响最多。因此，对HIV/NeuroAIDS的发病机制提出了几种间接机制。其中就有HIV-1Tat蛋白。我们已经证明，在没有HIV-1感染的情况下，TAT的表达足以引起神经行为异常和病理，类似于艾滋病毒/神经艾滋病中提到的大多数。此外，我们还发现TAT激活了星形胶质细胞中胶质纤维酸性蛋白(GFAP)的表达，导致星形胶质细胞功能障碍和随后的神经元死亡。此外，我们的初步研究发现，TAT激活的GFAP表达涉及一系列转录因子，并与星形胶质细胞中GFAP聚集和内质网(ER)应激有关，并损害神经元的存活。重要的是，我们还获得了将脑脊液(CSF)水平与HIV/神经艾滋病发病机制联系起来的初步证据。作为我们研究的逻辑延伸，我们建议进一步剖析GFAP在TAT神经毒性和HIV/神经艾滋病发病机制中的作用。此外，我们还将确定将脑脊液GFAP水平作为一种新的HIV/NeuroAIDS生物标志物的可行性。因此，目前提出的假设是TAT通过GFAP激活/聚集和内质网应激对星形胶质细胞功能和神经元存活产生不利影响。换句话说，GFAP不仅是一种介质，而且是TAT神经毒性和HIV/神经艾滋病发病机制的指标。为了验证这一假设，我们提出了以下相关的具体目标：(1)研究星形胶质细胞GFAP表达与ER应激的关系；(2)确定TAT激活的GFAP表达/聚集和ER应激对星形胶质细胞的影响；(3)确定GFAP/ER应激介导的神经毒性的分子机制；以及(4)探讨GFAP作为一种新的HIV/NeuroAIDS生物标志物的可能性。在我们的研究中，我们将使用分子、细胞、生化和遗传学相结合的方法，包括使用原代小鼠皮质星形胶质细胞培养和神经元培养、TAT转基因小鼠、GFAP空/TAT转基因小鼠、原代人胎脑培养、嵌入的脑组织和大量HIV-1队列的脑脊液样本。所寻求的答案对于理解这种关键的和普遍存在的蛋白GFAP及其在HIV/NeuroAIDS发病机制中的作用具有重要意义。此外，这些答案还将有助于确定艾滋病毒/神经艾滋病生物标记物和制定抗艾滋病毒/神经艾滋病治疗策略。

DESCRIPTION (provided by applicant): HIV-1 infection of the central nervous system (CNS) occurs in a majority of AIDS patients and causes a variety of neurologic dysfunction and neuropathologies generally termed neuroAIDS. Microglia/macrophages, and astrocytes to a less extent are the main target cells for HIV-1 infection in the CNS, whereas neurons are rarely infected by HIV-1 but mostly affected in HIV/neuroAIDS. Therefore, several indirect mechanisms have been proposed for HIV/neuroAIDS pathogenesis. Among them is HIV-1 Tat protein. We have shown that Tat expression in the absence of HIV-1 infection is sufficient to cause neurobehavioral abnormalities and pathologies similar to most of those noted in HIV/neuroAIDS. Moreover, we have shown that Tat activates glial fibrillary acidic protein (GFAP) expression in astrocytes and results in astrocyte dysfunction and subsequent neuron death. Furthermore, our preliminary studies have found that Tat-activated GFAP expression involves a network of transcription factors and is associated with GFAP aggregates and endoplasmic reticulum (ER) stress in astrocytes and impaired neuron survival. Importantly, we have also obtained preliminary evidence to link the cerebrospinal fluid (CSF) levels to HIV/neuroAIDS pathogenesis. As a logical extension of our studies, we propose to further dissect the GFAP function in Tat neurotoxicity and HIV/neuroAIDS pathogenesis. Besides, we will determine the feasibility of using the CSF GFAP level as a novel HIV/neuroAIDS biomarker. Thus, the underlying hypothesis for the current proposal is that Tat adversely affects astrocyte function and neuronal survival through GFAP activation/aggregation and ER stress. In other words, GFAP is not only a mediator but also an indicator of Tat neurotoxicity and HIV/neuroAIDS pathogenesis. To test this hypothesis, we propose to address the following interrelated specific aims: (1) To characterize the relationship between GFAP expression and ER stress in astrocytes; (2) To determine effects of Tat-activated GFAP expression/aggregation and ER stress on astrocytes; (3) To define the molecular mechanisms of GFAP-/ER stress-mediated neurotoxicity; and (4) To investigate the potential of using GFAP as a novel HIV/neuroAIDS biomarker. We will use a combined molecular, cellular, biochemical, and genetic approach, including use of primary mouse cortical astrocyte cultures and neuron cultures, Tat transgenic mice, GFAP-null/Tat transgenic mice, primary human fetal brain cultures, embedded brain tissues and CSF samples of a large HIV-1 cohort in our studies. The answers sought have fundamental significance for understanding of this critical and pervasive protein GFAP, and its role in HIV/neuroAIDS pathogenesis. In addition, these answers shall also aid in identification of HIV/neuroAIDS biomarkers and development of anti-HIV/neuroAIDS therapeutic strategies.