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

描述(申请人提供)：甲基苯丙胺(冰毒)，一种令人上瘾的兴奋剂，对中枢神经系统(CNS)有长期毒性影响。临床研究表明，冰毒依赖对与HIV-1感染相关的神经心理缺陷具有相加作用。氧化应激、兴奋性毒性、血脑屏障损伤和胶质细胞激活都独立地参与了冰毒和HIV-1相关的神经毒性的机制。这项建议将探讨HIV-1中枢神经系统感染和冰毒滥用导致氧化应激和核因子-kB信号全面增加，从而导致星形胶质细胞和内皮细胞功能受损的具体机制。我们认为，冰毒介导的氧化应激导致星形胶质细胞主要的谷氨酸清除剂兴奋性毒性氨基酸转运体(EAAT)-2的下调，而在内皮细胞中，这种氧化应激的增加导致BBB完整性的丧失。事实上，我们的初步数据表明，冰毒暴露导致星形胶质细胞和人原代BMVEC产生ROS，减少EAAT-2在星形胶质细胞中的表达，降低体外经抗氧化处理恢复的BBB完整性，导致紧密连接蛋白的表达/重新分布减少，促进单核细胞跨内皮单分子层的黏附和迁移，并激活BMVEC中先前与HIV-1脑炎(HIVE)期间的BBB损伤有关的小GTP酶。新的初步数据表明，暴露于冰毒的动物体内血脑屏障的通透性增加，而抗氧化剂治疗可以防止这种情况。这项建议将探讨一种新的机制共性概念，即冰毒介导的氧化应激在星形胶质细胞和内皮细胞中发挥其细胞特异性作用，加重HIV-1中枢神经系统感染造成的损伤，并通过体内研究描绘这些靶点的治疗方案。结合体外实验和冰毒/蜂窝动物模型，我们将解决以下问题：在星形胶质细胞中，增强的活性氧、ROS的产生和NF-kB信号在EAAT-2的表达/功能降低中起什么作用(S)？(目的1)血脑屏障功能障碍和单核细胞跨脑内皮细胞黏附/迁移增强的潜在机制是什么(通过氧化应激、干扰核因子-kB和GTPase信号)？(目标2)：在滥用蜂巢和冰毒的动物模型中，降低氧化应激和使EAAT-2功能正常化的治疗药物能否改善血脑屏障功能障碍和神经毒性？(目的3)利用原代培养的人脑星形胶质细胞和脑微血管细胞，建立体外血脑屏障模型，评价甲硫氨酸和HIV-1相关刺激物对EAAT-2和BBB功能的影响。抗氧化剂和特定的信号抑制剂将被用来描述参与这些效应的途径。我们的蜂巢动物模型将被用来研究这些细胞特异性机制在认知功能、血脑屏障损伤和神经毒性方面的生物学结果。我们认为，拟议的工作具有非常重要的意义，因为它们将揭示HIV-1和冰毒在中枢神经系统中共同作用的新机制，并基于这些研究提出治疗方法。与公共健康相关：甲基苯丙胺是一种高度上瘾的兴奋剂，被数百万美国人滥用，对中枢神经系统有长期的毒性影响。临床研究表明，甲基苯丙胺依赖对与HIV-1感染相关的认知障碍有成瘾作用。目前的提案旨在了解HIV-1和甲基苯丙胺在大脑中联合作用的机制，并提出神经保护疗法。

DESCRIPTION (provided by applicant): 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 excitotoxic 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. PUBLIC HEALTH RELEVANCE: Methamphetamine, a highly addictive stimulant abused by millions of Americans has long lasting toxic effects on the central nervous system. Clinical studies indicated that methamphetamine dependence has an addictive effect on cognitive deficits associated with HIV-1 infection. Current proposal aims to understand mechanisms of combined effects of HIV-1 and methamphetamine in the brain and to propose neuroprotective therapies.