ROLE OF PAF IN HIV-1 NEUROPATHOGENESIS

PAF 在 HIV-1 神经发病中的作用

• 批准号: 6613501
• 负责人: HARRIS A GELBARD
• 金额: $ 27.91万
• 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 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6613501
• 关键词: AIDS dementia complex; HIV infections; NMDA receptors; apoptosis; arachidonate; cytokine receptors; enzyme activity; immunocytochemistry; laboratory rat; macrophage; microglia; neuropathology; neurotoxicology; platelet activating factor; protein kinase; receptor expression; tissue /cell culture

Platelet activating factor (PAF), a potent phospholipid mediator, is synthesized by brain-resident macrophages and microglia, and secreted at neurotoxic levels during HIV-1 infection. We have previously shown that levels of PAF in cerebrospinal fluid correlate with neurologic dysfunction and immunosuppression in patients with HIV-1 associated dementia (HIV-D). Furthermore, we have shown that PAF mediates neuronal apoptosis by activation of the NMDA subtype of glutamate receptors. These findings lead us to propose that excessive production of PAF is an initiator step in HIV-1 neuropathogenesis. In this application we will determine how PAF activates brain-resident macrophages and microglia, and vulnerable neurons to induce neuronal dysfunction and death in the following specific aims: In Aim 1, we will determine whether cultures of cortical, striatal, hippocampal, and cerebellar granule neurons have different profiles of vulnerability (i.e. cell death) based on their identity (i.e. neurotransmitter/receptor subtype) and brain region. We will then determine whether PAF receptor-mediated neuronal demise is due in part to a change is NMDA receptor subunit composition using the technique of single cell antisense RNA analysis. We will repeat these experiments in organotypic slice cultures to determine if the in vitro results remain valid with an intact extracellular matrix and supporting glial cells present in an organotypic culture slice. In Aim 2, we will determine the relative contribution of PAF receptor activation in brain- resident macrophages and microglia toward the excess production of arachidonic acid, which is known to activate NMDA receptors and thereby further potentiate any effects that PAF may have on presynaptic release of glutamate. We will correlate these findings with neuronal cell death measured in cortical, striatal, hippocampal, and cerebellar granule neurons co-cultured with carbamyl (resistant to metabolic degradation) PAF-treated microglia. In Aim 3, because PAF receptor activation mediates increased neuronal GSK-3beta activity, which phosphorylates the cytoskeletal protein beta-catenin, destabilizing it and thereby targeting it for proteasomal degradation, we will determine whether in vitro and in vivo application of carbamyl PAF results in the functional consequence of decreased neurite outgrowth in vitro and a reduction in the dendritic arbor in vivo, a neuropathologic hallmark of HIV-D. Taken together, results from these studies will advance our understanding of how excess production of PAF contributes to the neuropathogenesis of HIV-1.

血小板活化因子（PAF）是一种有效的磷脂介质，由脑内巨噬细胞和小胶质细胞合成，在HIV-1感染时以神经毒性水平分泌。 我们以前已经表明，PAF在脑脊液中的水平与神经功能障碍和免疫抑制的患者与HIV-1相关的痴呆症（HIV-D）。 此外，我们已经表明PAF通过激活谷氨酸受体的NMDA亚型介导神经元凋亡。 这些发现使我们提出，PAF的过量产生是HIV-1神经发病机制的启动步骤。 在本申请中，我们将确定PAF如何激活脑内巨噬细胞和小胶质细胞，以及脆弱的神经元，以诱导神经元功能障碍和死亡，具体目标如下：在目标1中，我们将确定皮质，纹状体，海马，和小脑颗粒神经元有不同的脆弱性基于它们的身份（即神经递质/受体亚型）和脑区域，可以确定它们的细胞死亡（即细胞死亡）。 然后，我们将确定PAF受体介导的神经元死亡是否部分是由于改变是NMDA受体亚基组成的单细胞反义RNA分析技术。 我们将在器官型切片培养物中重复这些实验，以确定在器官型培养切片中存在完整的细胞外基质和支持神经胶质细胞的情况下，体外结果是否仍然有效。 在目标2中，我们将确定脑内巨噬细胞和小胶质细胞中PAF受体活化对花生四烯酸过量产生的相对贡献，已知花生四烯酸活化NMDA受体，从而进一步增强PAF可能对谷氨酸突触前释放的任何影响。 我们将这些发现与皮质，纹状体，海马和小脑颗粒神经元与氨甲酰（耐代谢降解）PAF处理的小胶质细胞共培养的神经元细胞死亡的测量。 在目标3中，由于PAF受体活化介导神经元GSK-3 β活性增加，使细胞骨架蛋白β-连环蛋白磷酸化，使其不稳定，从而靶向其进行蛋白酶体降解，我们将确定氨甲酰PAF的体外和体内应用是否导致体外神经突生长减少和体内树突减少的功能后果，艾滋病病毒的神经病理学标志 总之，这些研究的结果将促进我们对PAF过量产生如何促进HIV-1神经发病机制的理解。