PLATELET ACTIVATING FACTOR AND HIV-1 DEMENTIA

血小板激活因子与 HIV-1 痴呆

• 批准号: 2890906
• 负责人: HARRIS A GELBARD
• 金额: $ 26.79万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2890906
• 关键词: AIDS dementia complex; G protein; HIV envelope protein gp120; NMDA receptors; apoptosis; astrocytes; autoradiography; cytokine receptors; embryo /fetus tissue /cell culture; human fetus tissue; laboratory rat; malonates; membrane potentials; mitochondrial membrane; neurotoxicology; oxidative stress; platelet activating factor; radioimmunoassay; tissue /cell culture

DESCRIPTION (adapted from the Abstract): In this application the Principal Investigator focuses on the activity of platelet activating factor (PAF) in mediating neuronal damage and death in HIV-associated dementia. His rationale is based on studies that have correlated levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in brain tissue and PAF in cerebrospinal fluid (CSF) with neurologic disease in patients infected with HIV. Studies have demonstrated that activated HIV-infected monocytes secrete increased amounts of TNF-alpha and PAF. Exogenous TNF-alpha and PAF induce neuronal apoptosis. TNF-induced neuronal apoptosis can be blocked by PAF receptor antagonism, and neurotoxicity of conditioned media from HIV-infected monocytes can be ameliorated by enzymatic hydrolysis of PAF. PAF-induced neurotoxicity, in turn, can be blocked by NMDA receptor antagonists. In the proposed studies the researchers will test the hypotheses that (1) the HIV glycoprotein gpl20 induces PAF production by monocytes, and (2) subsequent PAF receptor activation increases intracellular oxidative stress and NMDA receptor over-activation ("weak" excitotoxicity), resulting in neuronal apoptosis. In in vitro studies the researchers will test whether treatment with different species of the HIV glycoprotein gpl20 affect PAF production from uninfected monocytes. They will apply conditioned media from PAF-treated microglia or astrocytes to neuronal cultures depleted of glia to determine whether PAF receptor activation in glia is necessary for neurotoxicity. In studies with PAF-treated neuronal cultures they will test the hypothesis that mitochondrial dysfunction and increased oxidative stress are necessary prerequisites for lethal NMDA receptor activation in vulnerable neurons by measuring mitochondrial membrane potential and reactive oxygen species with confocal and fluorescence microscopy, as well as measuring NMDA receptor channels with cellular autoradiography. They will measure neuronal apoptosis in cultures treated with PAF and antioxidants. To determine which G protein and intracellular effector system mediates PAF receptor activation in neural cells, the researchers will measure intracellular calcium and neuronal apoptosis in PAF- or vehicle-treated neuronal cultures in the presence and absence of specific agents that stimulate or inhibit G protein subtypes/second messenger enzymes. To determine whether PAF induces "weak" excitotoxicity in vivo, they will inject stereotactically varying doses of PAF plus mitochondrial toxin (malonate) into subcortical brain regions of rats, and will examine brain tissue for changes in glutamate receptors and neuronal apoptosis. These studies will help provide insights into the mechanisms of PAF-induced neuronal apoptosis.

描述（改编自摘要）：在本申请中，校长 研究人员重点关注血小板激活因子（PAF）的活性 介导艾滋病毒相关痴呆的神经元损伤和死亡。 他的 基本原理基于具有相关水平的研究 脑中促炎细胞因子肿瘤坏死因子α（TNF-α） 患有神经系统疾病的脑脊液 (CSF) 中的组织和 PAF 感染艾滋病毒的患者。 研究表明，激活 感染 HIV 的单核细胞会分泌更多的 TNF-α 和 PAF。 外源性 TNF-α 和 PAF 诱导神经元凋亡。 TNF诱导的神经元 PAF受体拮抗作用可阻断细胞凋亡，并且其神经毒性 来自 HIV 感染的单核细胞的条件培养基可以通过 PAF 的酶水解。 PAF 诱导的神经毒性反过来可以是 被 NMDA 受体拮抗剂阻断。 在拟议的研究中 研究人员将测试以下假设：(1) HIV 糖蛋白 gpl20 诱导单核细胞产生 PAF，以及 (2) 随后的 PAF 受体 激活增加细胞内氧化应激和 NMDA 受体 过度激活（“弱”兴奋毒性），导致神经元凋亡。 在体外研究中，研究人员将测试是否使用 不同种类的 HIV 糖蛋白 gpl20 影响 PAF 的产生 未感染的单核细胞。 他们将使用经过 PAF 处理的条件培养基 将小胶质细胞或星形胶质细胞加入到耗尽胶质细胞的神经元培养物中以确定 神经胶质细胞中PAF受体的激活是否是神经毒性所必需的。 在 他们将用 PAF 处理的神经元培养物进行研究来检验这一假设 线粒体功能障碍和氧化应激增加是必要的 脆弱神经元中致命的 NMDA 受体激活的先决条件 测量线粒体膜电位和活性氧 共聚焦和荧光显微镜，以及测量 NMDA 受体 细胞放射自显影通道。 他们将测量神经元 用 PAF 和抗氧化剂处理的培养物中的细胞凋亡。 确定哪个 G蛋白和细胞内效应系统介导PAF受体激活 在神经细胞中，研究人员将测量细胞内的钙和 PAF 或媒介物处理的神经元培养物中的神经元凋亡 是否存在刺激或抑制 G 蛋白的特定药物 亚型/第二信使酶。 确定PAF是否诱发“弱” 体内兴奋毒性，他们会立体定向注射不同剂量的 PAF 加上线粒体毒素（丙二酸）进入皮层下大脑区域 老鼠，并将检查脑组织中谷氨酸受体的变化 神经元凋亡。 这些研究将有助于深入了解 PAF诱导神经细胞凋亡的机制。