HIV Neural Apoptosis: Mechanisms, Pathways & Protection

描述（由申请方提供）：HIV感染脑中的神经元损伤导致HIV相关痴呆（HAD），认为部分通过脑巨噬细胞HIV感染引发的氧化应激相关N-甲基-D-天冬氨酸受体（NMDAR）兴奋发生。富含表达NR 2A和NR 2B亚基的NMDAR的脑区域，如海马，特别容易受到HIV的攻击。HIV蛋白以及谷氨酸、喹啉酸（QUIN）、血小板活化因子（PAF）和从活化的胶质细胞释放的活性氧（ROS）可以作为HIV诱导的NMDAR介导的兴奋性毒性的直接或间接触发剂。在我们最初的资助期间，我们使用我们的NT2.N神经毒性模型来证明HIV-1感染的单核细胞衍生的巨噬细胞（HIV/MDM）触发NMDAR激活的内在（线粒体）凋亡途径。此外，我们最近的研究使用我们的主要啮齿动物海马培养模型表明，HIV/MDM释放兴奋性毒素，杀死神经元的NMDAR介导的钙蛋白酶激活，海马的脆弱性是由NR 2A和NR 2B亚基表达。 我们的假设是，HIV/MDM诱导的NMDAR激活，改变NR亚基磷酸化和定位与巨噬细胞应激反应相关，并决定海马神经元对HIV诱导的损伤的脆弱性。 我们对HIV/MDM的神经毒性以及NMDAR和巨噬细胞整合应激反应（ISR）的作用的研究表明：i）海马神经元的脆弱性是年龄依赖性的，并由NR 2A和NR 2B表达模式决定：ii）HIV/MDM迅速诱导神经元钙蛋白酶和cdk 5激活，改变NR 2B磷酸化和切割，以及钙蛋白酶和cdk 5依赖性死亡; iii）MDM中的HIV复制诱导巨噬细胞整合应激反应（ISR）;和iv）ISR在感染免疫缺陷病毒的个体的猕猴脑和人脑中被激活。为了更好地确定HIV感染巨噬细胞如何触发神经退行性级联反应，并确定神经保护的新靶点，我们将：1）确定HIV/MDM诱导的钙蛋白酶诱导的NMDAR介导的神经毒性的途径和NR 2亚基的功能作用; 2）确定NMDAR的特异性磷酸化和突触定位是否调节HIV/MDM神经毒性，以及HAD中是否表达类似的NR磷酸化模式;（3）确定巨噬细胞整合应激反应（ISR）在HIV/MDM诱导的神经毒性中的作用。这些研究在HIV感染引发的神经变性级联反应的多个步骤中提供了有吸引力的靶点，这些靶点可能被用于鉴定目前可用的药物和开发新的神经保护策略，这些策略可以有效对抗HIV诱导的神经变性。项目叙述：该项目研究了巨噬细胞的HIV-1感染诱导神经变性的机制，使用体外感染模型和HIV感染的人脑和猴免疫缺陷病毒（SIV）感染的猕猴/猴脑组织的病理分析。基于我们的初步研究，我们的假设是，这种感染改变了巨噬细胞整合应激反应（ISR），导致兴奋性毒素的产生和神经元NMDA受体激活的机制，确定为什么选择性神经元群体在HIV-1感染中受损。我们正试图验证我们在人脑组织和SIV猕猴模型中的体外观察结果，以便最终可以合理设计神经保护研究，用于猕猴模型中的测试。

DESCRIPTION (provided by applicant): Neuronal damage in HIV-infected brain resulting in HIV-associated dementia (HAD) is thought to occur in part through oxidative stress-associated N-methyl-D-aspartate receptor (NMDAR) excitation initiated by HIV infection of brain macrophages. Brain regions enriched in NMDAR expressing the NR2A and NR2B subunits, such as the hippocampus, are particularly vulnerable to HIV. HIV proteins as well as glutamate, quinolinic acid (QUIN), platelet activating factor (PAF) and reactive oxygen species (ROS) released from activated glia can act as direct or indirect triggers for HIV-induced NMDAR-mediated excitotoxicity. In our initial funding period, we used our NT2.N neurotoxicity model to show that HIV-1-infected monocyte-derived macrophages (HIV/MDM) trigger the NMDAR-activated intrinsic (mitochondrial) apoptosis pathway. Furthermore, our recent study using our primary rodent hippocampal culture model demonstrates that HIV/MDM release excitotoxins that kill neurons by NMDAR-mediated calpain activation, and that hippocampal vulnerability is determined by NR2A and NR2B subunit expression. Our hypothesis is that HIV/MDM-induced NMDAR activation, altered NR subunit phosphorylation & localization are associated with macrophage stress responses and determine hippocampal neuronal vulnerability to HIV induced damage. Our studies of HIV/MDM neurotoxicity and the roles of NMDAR and the macrophage integrated stress response (ISR) have shown that i) hippocampal neuronal vulnerability is age-dependent and is determined by NR2A and NR2B expression patterns; ii) HIV/MDM rapidly induce neuronal calpain and cdk5 activation, altered NR2B phosphorylatioh & cleavage, and calpain- and cdk5-dependent death; iii) HIV replication in MDM induces the macrophage integrated stress response (ISR); and iv) the ISR is activated in macaque brain and human brain in individuals infected with immunodeficiency virus. To better define how HIV infection of macrophages triggers neurodegeneration cascades and to identify new targets for neuroprotection we will: 1) Define the pathways of calpain-induced NMDAR-mediated neurotoxicity induced by HIV/MDM and the functional roles for NR2 subunits; 2) Determine whether specific phosphorylation and synaptic localization of NMDAR modulate HIV/MDM neurotoxicity and whether similar NR phosphorylation patterns are expressed in HAD; and 3) Determine the role of the macrophage Integrated Stress Response (ISR) in HIV/MDM-induced neurotoxicity. These studies offer appealing targets at multiple steps in the neurodegeneration cascade initiated by HIV infection that can potentially be exploited for identfication of currently available drugs and for the development of new neuroprotective strategies that can be effective against HIV-induced neurodegeneration. Project Narrative: This project investigates the mechanisms by which HIV-1 infection of macrophages induces neurodegeneration, using an in vitro infection model and pathological analyses of HIV-infected human brain and simian immunodeficiency virus (SIV)-infected macaque/monkey brain tissues. Our hypothesis, based on our preliminary studies, is that such infection alters the macrophage integrated stress response (ISR), leading to excitotoxin production and neuronal NMDA receptor activation by mechanisms that determine why selective neuronal populations are damaged in HIV-1 infection. We are attempting to validate our in vitro observations in human brain tissue and in the SIV macaque model so that neuroprotection studies can eventually be rationally designed for testing in the macaque model.