Mechanisms and Interventions for Methamphetamine and HIV-1 Induced CNS Injury

摘要： 甲基苯丙胺是一种使人上瘾的兴奋剂，对中枢神经系统有长期的毒性作用。 (CNS)。临床研究表明，冰毒依赖对神经心理缺陷有相加作用。 与HIV-1感染有关。氧化应激、兴奋性毒性、血脑屏障损伤和胶质细胞激活 已经独立地与冰毒和艾滋病毒-1相关的神经毒性机制有关。这 提案将调查导致艾滋病毒-1中枢神经系统感染和冰毒滥用的具体机制 氧化应激和核因子-kB信号的全面增加导致星形胶质细胞和 内皮细胞功能。我们认为冰毒介导的星形胶质细胞氧化应激导致星形胶质细胞 调控星形胶质细胞谷氨酸的主要清除剂EAAT-2，而在 对内皮细胞来说，这种氧化应激的增加会导致血脑屏障完整性的丧失。事实上，我们初步的 数据表明，冰毒暴露导致星形胶质细胞和人原代BMVEC产生ROS， 星形胶质细胞中EAAT-2表达的降低降低了体外经抗氧化剂恢复的血脑屏障完整性 治疗，导致紧密连接蛋白表达/重新分布减少，粘附性增强， BMVEC中单核细胞在内皮单层和激活的小GTP酶之间的迁移 此前曾与HIV-1脑炎(HIVE)期间的血脑屏障损伤有关。新的初步数据显示 体内暴露于冰毒的动物体内血脑屏障通透性增加，抗氧化剂可以预防 治疗。这项提案将调查一种新的机械性共性概念，即冰毒中介 氧化应激在星形胶质细胞和内皮细胞中发挥细胞特异性作用加重损伤 由HIV-1中枢神经系统感染引起的，并描述这些靶点的体内治疗选择 学习。 使用体外试验和冰毒/蜂房动物模型相结合，我们将解决以下问题 问题：增强的活性氧、ROS的产生和核因子-kB信号在其中的作用是什么(S) 星形胶质细胞中EAAT-2的表达/功能减弱？(目标1)血脑屏障的基本机制是什么 脑内皮细胞功能障碍和单核细胞黏附/迁移增强(通过氧化应激， 对核因子-kB和GTPase信号的干扰)？(目标2)；以及治疗药物能否降低氧化应激 使EAAT-2功能正常化可改善麻疹动物模型的血脑屏障功能障碍和神经毒性 还有滥用冰毒？(目标3)我们将利用原代人类星形胶质细胞和大脑来解决这些问题 微血管细胞，体外血脑屏障模型及评价冰毒和HIV-1相关刺激物的联合作用 EAAT-2和BBB功能。抗氧化剂和特定的信号抑制剂将被用来描述通路。 与这些效应有关。我们的蜂箱动物模型将被用来研究 这些在认知功能、血脑屏障损伤和神经毒性方面的细胞特异性机制。我们相信， 拟议的工作具有非常重要的意义，因为它们将揭示参与综合效应的新机制 并在这些研究的基础上提出治疗方法。

ABSTRACT: Methamphetamine (METH), an addictive stimulant has long lasting toxic effects on the central nervous system (CNS). Clinical studies indicated that METH dependence has an additive effect on neuropsychological deficits associated with HIV-1 infection. Oxidative stress, excitotoxicity, BBB impairment and glial cell activation, all have been independently implicated in the mechanisms of METH- and HIV-1-associated neurotoxicity. This proposal will investigate specific mechanisms operative in HIV-1 CNS infection and METH abuse that lead to an overall increase in oxidative stress and NF-kB signaling resulting in impairment of astrocytes and endothelial cell function. We propose that METH-mediated oxidative stress in astrocytes leads to a down regulation exitotoxic amino acid transporter (EAAT) -2, the primary astrocyte glutamate scavenger, while in endothelial cells, such an increase in oxidative stress results in loss of BBB integrity. Indeed, our preliminary data suggest that METH exposure caused ROS generation in astrocytes and human primary BMVEC, diminished EAAT-2 expression, in astrocytes, decreased BBB integrity in vitro that was restored by antioxidant treatment, caused decreased expression/re-distribution of tight junction proteins, enhanced adhesion and migration of monocytes across endothelial monolayers and activated small GTPases in BMVEC that were previously implicated in the BBB injury during HIV-1 encephalitis (HIVE). New preliminary data demonstrated increased permeability of BBB in vivo in animals exposed to METH that was prevented by antioxidant treatment. This proposal will investigate a novel concept of mechanistic commonality, i.e. METH-mediated oxidative stress exerts its cell-specific effects in astrocytes and endothelial cells aggravating injury caused by HIV-1 CNS infection and delineate therapeutic options for these targets using in vivo studies. Using a combination of in vitro assays and METH/HIVE animal model, we will address the following questions: What is the role(s) of enhanced reactive oxygen species, ROS production and NF-kB signaling in diminished expression/function of EAAT-2 in astrocytes? (Aim 1) What are underlying mechanisms of BBB dysfunction and enhanced monocyte adhesion/migration across brain endothelium (via oxidative stress, interference with NF-kB and GTPase signaling)? (Aim 2); and Can therapeutics decreasing oxidative stress and normalizing EAAT-2 function ameliorate BBB dysfunction and neurotoxicity in an animal model for HIVE and METH abuse? (Aim 3) We will address these questions utilizing primary human astrocytes and brain microvascular cells, in vitro BBB models and evaluate combined effects of METH and HIV-1 relevant stimuli on EAAT-2 and BBB function. Antioxidants and specific signaling inhibitors will be utilized to delineate pathways involved in these effects. Our HIVE animal model will be employed to investigate the biological outcomes of these cell-specific mechanisms in cognitive function, BBB damage and neurotoxicity. We believe that the proposed works are highly significant, as they will uncover novel mechanisms involved in the combined effects of HIV-1 and METH in the CNS and propose therapeutic approaches based on these investigations.