Department of Psychology and Neuroscience, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.
Department of Psychology and Neuroscience, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.
Exp Neurol. 2021 Jul;341:113699. doi: 10.1016/j.expneurol.2021.113699. Epub 2021 Mar 15.
Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7-11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845's effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CBR & CBR). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition.
人类免疫缺陷病毒 1 型（HIV-1）已知会引发小胶质细胞免疫反应，这可能在慢性神经炎症状态和与 HIV-1 相关的神经认知障碍（HAND）相关的神经元损伤的发展中起重要作用。特别是，HIV-1 Tat 蛋白是一种促炎神经毒素，使神经元容易受到突触树突损伤。靶向内源性大麻素降解酶的药物在减少炎症方面显示出了前景，并且在啮齿动物模型中副作用最小。考虑到神经炎症标志物可以预测 HAND 患者神经元损伤的程度，我们评估了暴露于 HIV-1 Tat 后的小胶质细胞的神经毒性作用，方法是阻断负责降解内源性大麻素（如花生四烯酸酰胺水解酶（FAAH））的代谢酶。在本研究中，培养的鼠小胶质细胞与 Tat 和/或 FAAH 抑制剂（PF3845）孵育。24 小时后，对细胞进行形态分析，并收集小胶质细胞条件培养基（MCM）。然后将培养的大脑皮层神经元（DIV7-11）暴露于 MCM，并通过活细胞钙成像和肌动蛋白阳性树突结构染色评估神经毒性。结果表明，PF3845 预处理强烈抑制了 Tat 对小胶质细胞的反应，这表现在：1）使用分形分析将小胶质细胞的形态改变为炎症表型减弱，2）使用 ELISA/多重分析测定法减少神经毒性细胞因子/趋化因子（MCP-1/CCL2）和基质金属蛋白酶（MMP；MMP-9）的释放，以及 3）使用 LC/MS/MS 增强内源性大麻素（AEA）的产生。此外，PF3845 对 Tat 诱导的小胶质细胞介导的神经毒性的作用降低了神经元细胞内钙的失调，并防止了大脑皮层神经元培养物中肌动蛋白阳性染色和点状结构的丢失。有趣的是，这些观察到的神经保护作用似乎与大麻素受体活性（CBR 和 CBR）无关。我们发现，一种假定的 GPR18 拮抗剂 CID-85469571 阻断了 PF3845 在所有实验中的神经保护作用。总的来说，这些实验增加了对 FAAH 抑制和 Tat 在 HAND 情况下介导小胶质细胞神经毒性作用的理解。
