HIV Neural Apoptosis: Mechanisms, Pathways & Protection

描述(申请人提供)：HIV感染导致HIV相关性痴呆(HAD)的脑神经损伤被认为部分是通过HIV感染脑巨噬细胞引发的氧化应激相关的N-甲基-D-天冬氨酸受体(NMDAR)兴奋而发生的。富含NMDAR表达NR2A和NR2B亚单位的大脑区域，如海马体，特别容易受到艾滋病毒的影响。HIV蛋白以及激活的胶质细胞释放的谷氨酸、喹啉酸(Quin)、血小板激活因子(PAF)和活性氧(ROS)可直接或间接触发HIV诱导的NMDAR介导的兴奋性毒性。在我们最初的资助期，我们使用了我们的NT2.N神经毒性模型来表明HIV-1感染的单核细胞来源的巨噬细胞(HIV/MDM)触发了NMDAR激活的内在(线粒体)凋亡途径。此外，我们最近使用我们的原代啮齿动物海马区培养模型进行的研究表明，HIV/MDM释放兴奋毒素，通过NMDAR介导的钙蛋白激活杀死神经元，而海马区的脆弱性由NR2A和NR2B亚单位的表达决定。 我们的假设是，HIV/MDM诱导的NMDAR激活、NR亚单位磷酸化和定位的改变与巨噬细胞的应激反应有关，并决定了海马神经元对HIV诱导的损伤的易感性。 我们对HIV/MDM的神经毒性以及NMDAR和巨噬细胞综合应激反应(ISR)的作用的研究表明：i)海马神经元的脆弱性具有年龄相关性，并由NR2A和NR2B的表达模式决定；ii)HIV/MDM迅速诱导神经元钙蛋白和CDK5激活，改变NR2B的磷酸化和裂解，以及Calain和CDK5依赖的死亡；iii)艾滋病毒在MDM中的复制诱导巨噬细胞综合应激反应(ISR)；以及iv)在感染免疫缺陷病毒的猕猴脑和人脑中，ISR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变级联反应，并寻找新的神经保护靶点，我们将：1)确定HIV/MDM诱导的Calain诱导的NMDAR介导的神经毒性的通路和NR2亚单位的功能角色；2)确定NMDAR的特异性磷酸化和突触定位是否调节HIV/MDM的神经毒性以及是否在HAD中表达类似的NR磷酸化模式；以及3)确定巨噬细胞综合应激反应(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吸引人的靶点，可能被用于鉴定现有药物和开发能够有效对抗HIV诱导的神经退化的新的神经保护策略。项目简介：本项目采用体外感染模型，并对感染HIV的人脑和感染SIV的猕猴/猴脑组织进行病理分析，研究HIV-1感染巨噬细胞导致神经变性的机制。我们的假设是，基于我们的初步研究，这种感染改变了巨噬细胞综合应激反应(ISR)，导致兴奋性毒素产生和神经元NMDA受体激活，其机制决定了为什么选择性神经元群体在HIV-1感染中受到损害。我们正试图在人脑组织和SIV猕猴模型中验证我们的体外观察，以便最终能够合理地设计神经保护研究以在猕猴模型中进行测试。

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.