IN VIVO CNS EFFECTS OF HIV-1 COAT PROTEINS AND NEF

HIV-1感染通常与中枢神经损伤有关 系统(CNS)。导致这一现象的确切的分子和细胞过程 损失仍是未知数。越来越多的证据表明，HIV-1衍生 蛋白质，特别是包膜蛋白和Nef，可能起重要作用。 在艾滋病痴呆症发展中的作用。研究它们对人类的影响 在体内，我们建议表达神经侵袭性HIV-1的蛋白 分离株Yu-2(包括具有和不具有切割位点突变的gp160， Gp120、标记gp120、gp41和Nef)在星形胶质细胞或巨噬细胞/小胶质细胞中的表达 转基因小鼠。这将在融合基因的帮助下完成 允许长时间、可重复地传递所选的 蛋白质(无任何污染)到特定区域/细胞 完整的中枢神经系统。神经胶质纤维酸性蛋白的调控序列 基因将用于表达上述HIV-1蛋白在星形胶质细胞和 从编码高亲和力Fc受体或溶菌酶的基因到 在巨噬细胞中表达gp120。融合基因将被显微注射 单独移植到受精的小鼠卵母细胞。由此产生的转基因小鼠 将被繁殖并在其后代中表达转基因 特色化的。来自转基因表达品系的小鼠有望表现出 中枢神经系统的结构和分子变化，这将被量化 有一系列完善的测试，包括激光共聚焦 原位杂交/免疫标记脑切片显微镜，电子 显微镜、核糖核酸酶保护试验、免疫印迹和生物测定。至 评估这些模型与人类疾病的相关性，以及 转基因小鼠的中枢神经系统将与感染HIV-1的人的中枢神经系统进行比较 脑组织，死后获取的。HIV-1诱导中枢神经系统损伤的机制 也将通过暴露非转基因小鼠的神经元进行体外分析 表达HIV-1蛋白的转基因星形胶质细胞/小胶质细胞及其检测 哪些药理试剂可以阻断由此产生的神经毒性作用。井 特征化的转基因模型将被用来在体内评估 针对有害的艾滋病毒-中枢神经系统相互作用的药物的有效性。通过 研究不同HIV-1蛋白对中枢神经系统的影响 将进一步加深我们对HIV-1相关神经疾病的了解 帮助确定治疗干预的重要目标。通过 这项研究将为药物的临床前评估提供模型 促进评估和制定治疗策略 艾滋病痴呆的治疗和预防。

HIV-1 infection is often associated with damage to the central nervous system (CNS). The precise molecular and cellular processes that cause this damage are still unknown. Increasing evidence suggests that HIV-1-derived proteins, particularly envelope proteins and Nef, may play important causal roles in the development of AIDS dementia. To study their effects on the CNS in vivo, we propose to express proteins of the neuroinvasive HIV-1 isolate YU-2 (including gp160 with and without cleavage site mutation, gp120, Flag'ed gp120, gp41 and Nef) in astrocytes or macrophages/microglia of transgenic mice. This will be accomplished with the help of fusion gene constructs which allow the prolonged, reproducible delivery of selected proteins (devoid of any contaminations) to specific areas/cells of the intact CNS. Regulatory sequences from the glial fibrillary acidic protein gene will be used to express the above HIV-1 proteins in astrocytes and sequences from genes encoding the high-affinity Fc receptor or lysozyme to express gp120 in macrophages. Fusion genes will be microinjected individually into fertilized mouse oocytes. The resulting transgenic mice will be bred and the expresssion of transgenes in their offspring characterized. Mice from transgenic expressor lines are expected to show structural and molecular alterations of the CNS, which will be quantitated with a well established battery of tests, including laser confocal microscopy of in situ hybridized/immunolabeled brain sections, electron microscopy, RNAse protection assays, Western blots, and bioassays. To assess the relevance of these models to human disease, alterations in the CNS of transgenic mice will be compared with those in HIV-1 infected human brain tissue, obtained postmortem. Mechanisms of HIV-1-induced CNS damage will also be analyzed in vitro by exposing neurons from nontransgenic mice to HIV-1 protein-expressing transgenic astrocytes/microglia and by testing which pharmacologic reagents block the resulting neurotoxic effects. Well characterized transgenic models will then be used to assess in vivo the effectiveness of drugs aimed at detrimental HIV-CNS interactions. By characterizing the CNS effects of different HIV-1 proteins, this project will further our understanding of HIV-1 associated neurologic disease and help identify important targets for therapeutic interventions. By providing models for the preclinical assessment of drugs, this study will facilitate the evaluation and development of therapeutic strategies for the treatment and prevention of AIDS dementia.