Regulation of Astrocyte TIMP-1 in HIV-Associated Dementia

描述(由申请人提供)：本提案的目标是表征人类免疫缺陷病毒(HIV)相关性痴呆(HAD)期间人类星形胶质细胞金属蛋白酶组织抑制物(TIMP)表达的调节。HIV相关神经认知障碍是一组疾病，其中HAD是最严重的。在美国，HAD影响了10%-27%的艾滋病毒感染者。这种疾病的特点是激活和感染的巨噬细胞/小胶质细胞在中枢神经系统积聚，这些细胞分泌细胞因子、感染性病毒粒子和病毒蛋白，激活周围的星形胶质细胞，导致星形胶质细胞增生和基因表达改变。神经功能障碍被认为是导致痴呆症的关键因素。星形胶质细胞在中枢神经系统损伤和修复过程中维持神经动态平衡是必不可少的，这部分是通过产生神经保护性TIMP来实现的。基质金属蛋白酶和TIMPs之间的失衡被认为是导致几种神经退行性疾病的原因，包括HAD。TIMP-1是一个由4个TIMP组成的家族的诱导形式，是一种多功能蛋白，通过维持体内平衡和可能通过抑制细胞凋亡来表现出神经保护特性。与HIV-1血清阴性对照组相比，HAD患者TIMP-1水平降低，HAD相关刺激刺激原代人类星形胶质细胞TIMP-1表达增加，但暴露3天后TIMP-1水平下降。此外，删除TIMP-1启动子中-310处的CAAT位点会增加转录。因此，我们认为HAD过程中星形胶质细胞TIMP-1的下调是通过TIMP-1启动子上的CAAT位点的差异转录调控来实现的，从而导致星形胶质细胞TIMP-1表达降低，最终加剧神经退行性变。针对HAD过程中星形胶质细胞TIMP-1表达的调控，人们设计了两个特异的靶点：目的1检测CAAT增强子结合蛋白2在原代人星形胶质细胞中的表达及其对TIMP-1转录的影响；目的2确定导致原代人脑星形胶质细胞TIMP-1下调的信号转导途径。为完成目标1，将使用原代人星形胶质细胞的基本细胞培养和HAD相关刺激，如细胞因子和病毒蛋白，以及免疫印迹、实时聚合酶链式反应、免疫细胞化学和酶联免疫吸附试验等分析工具。目的2通过将途径特异性抑制物与一组荧光素酶表达质粒、CAAT结合因子过表达质粒和带有和不带有HAD相关刺激的通路指示质粒相结合来完成。这些研究的完成将有助于揭示人类星形胶质细胞TIMP-1在HAD中的表达调控。 公共卫生相关性：HIV-1相关性痴呆(HAD)是HIV-1相关性神经认知障碍的最严重表现，是HIV-1感染的重要神经系统并发症，以认知、行为和运动功能障碍为特征。据估计，尽管有高效的抗逆转录病毒疗法，但在美国等发达国家，估计有10%-27%的艾滋病毒血清阳性患者有进展。我们的研究将更好地理解激活的星形胶质细胞在HIV-1神经致病和神经炎症中的具体作用机制。

DESCRIPTION (provided by applicant): The goal of this proposal is to characterize regulation of primary human astrocyte tissue inhibitors of metalloproteases (TIMP) expression during Human Immunodeficiency Virus (HIV)-associated dementia (HAD). HIV-associated neurocognitive disorders are a group of disorders of which HAD is the most severe. HAD affects 10-27% of HIV-infected individuals in the United States. The disease is characterized by an accumulation of activated and infected macrophages/microglia in the central nervous system, which secrete cytokines, infectious virions and viral proteins that activate surrounding astrocytes leading to astrogliosis and altered gene expression. Neural dysfunction is believed to be the key component causing dementia. Astrocytes are essential in maintaining neural homeostasis in the CNS during injury and repair, in part through the production of neuroprotective TIMPs. An imbalance between matrix metalloproteases and TIMPs is thought to contribute to several neurodegenerative disorders, including HAD. TIMP-1, the inducible form of a family of 4 TIMPs, is a multifunctional protein that displays neuroprotective properties via homeostatic maintenance and possibly by inhibiting apoptosis. TIMP-1 levels are decreased in HAD patients compared to HIV-1 seronegative controls and primary human astrocytes acutely stimulated with HAD-relevant stimuli increase TIMP-1 expression, but levels fall after 3 days of exposure. Additionally, deleting the CAAT site at -310 in the TIMP-1 promoter increases transcription. Thus, we propose that down regulation of astrocyte TIMP-1 during HAD is mediated through differential transcriptional regulation via a CAAT site in the TIMP-1 promoter leading to reduced astrocyte TIMP-1 expression and ultimately exacerbating neurodegeneration. Two specific aims have been designed to address regulation of astrocyte TIMP-1 expression during HAD: Aim 1 determine the expression profile of CAAT enhancer binding protein 2 in activated primary human astrocytes and their influence on TIMP-1 transcription and Aim 2 identify signal transduction pathways leading to TIMP-1 down regulation in primary human astrocytes. To complete Aim 1 basic cell culture of primary human astrocytes and HAD-relevant stimuli like cytokines and viral proteins will be used along with analysis tools such as western blot, real-time polymerase chain reaction, immunocytochemistry and enzyme linked immunosorbent assay. Aim 2 will be completed by combining pathway-specific inhibitors with a panel of luciferase expression plasmids, CAAT-binding factor overexpression plasmids and pathway-indicating plasmids with and without HAD-relevant stimuli. Completion of these studies will shed light on the regulation of human astrocyte TIMP-1 expression in HAD. PUBLIC HEALTH RELEVANCE: HIV-1-associated dementia (HAD), the most severe manifestation of HIV-1-associated neurocognitive disorders, is an important neurological complication of HIV-1 infection and is characterized by cognitive, behavioral and motor dysfunction. An estimated 10-27% of HIV-seropositive patients progress to develop HAD in developed worlds such as the United States, despite the availability of highly active antiretroviral therapy. Our studies will provide a better understanding of the specific mechanistic contributions of activated astrocytes to HIV-1-neuropathogensis and neuroinflammation.