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

HIV-1感染通常与中枢神经系统的损害有关 系统（CNS）。 造成这种情况的精确的分子和细胞过程 目前还不清楚损失情况。 越来越多的证据表明，HIV-1衍生的 蛋白质，特别是包膜蛋白和Nef，可能发挥重要的因果关系， 在艾滋病痴呆症的发展中的作用。 为了研究它们对 CNS在体内，我们建议表达神经侵入性HIV-1的蛋白质， 分离的YU-2（包括具有和不具有切割位点突变的gp 160， gp 120、Flaged艾德gp 120、gp 41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 所述构建体允许选择的药物的延长的、可再现的递送， 蛋白质（没有任何污染）的特定区域/细胞 中枢神经系统完好 胶质细胞酸性蛋白的调控序列 基因将用于在星形胶质细胞中表达上述HIV-1蛋白， 来自编码高亲和力Fc受体或溶菌酶的基因的序列， 在巨噬细胞中表达gp 120。 融合基因将被显微注射 分别植入受精的小鼠卵母细胞中。 由此产生的转基因小鼠 将被繁殖并在它们的后代中表达转基因 表征了 来自转基因表达系的小鼠预期显示出 CNS的结构和分子改变，将对其进行定量 通过一系列完善的测试，包括激光共聚焦 原位杂交/免疫标记脑切片显微镜检查，电子 显微镜、RNA酶保护测定、蛋白质印迹和生物测定。 到 评估这些模型与人类疾病的相关性， 将转基因小鼠的CNS与HIV-1感染者的CNS进行比较 死后获得的脑组织 HIV-1致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， 所述药理学试剂阻断所产生的神经毒性作用。 以及 然后将使用表征的转基因模型来体内评估 针对有害的HIV-CNS相互作用的药物的有效性。 通过 描述不同HIV-1蛋白质对中枢神经系统的影响， 将进一步加深我们对HIV-1相关神经系统疾病的理解， 帮助确定治疗干预的重要目标。 通过 为药物的临床前评估提供模型，本研究将 促进评估和制定治疗战略， 治疗和预防艾滋病痴呆症。

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.