Molecular mechanisms underlying HIV & Cocaine-mediated microglial activation: Targeting NLRP3 inflammasome

摘要： 在目前的联合抗逆转录病毒治疗（cART）时代，艾滋病毒相关性 神经认知障碍（HAND），约30-50%的HIV感染者患有不同程度的 神经认知障碍，严重影响他们的生活质量。众所周知，尽管有cART， 早期HIV蛋白的持续存在和存在，如 转录（达特），这有助于神经炎症。毒品滥用的并发症更是火上浇油 在HIV感染者中，这进一步加剧了小胶质细胞的激活，从而导致HAND的发生。 直到最近，中枢神经系统被认为是一个免疫特权网站，然而，最近的范式转变，在我们的研究中， 了解揭示了细胞溶质的作用，多蛋白复合物，属于核苷酸结合 结构域富含亮氨酸重复序列（NLR）蛋白超家族，称为“炎性小体”，在各种 神经炎性疾病这些多蛋白复合物的组装激活了促炎性半胱天冬酶 （特别是半胱天冬酶1），反过来导致IL 1 β和1 L 18的切割和释放，从而导致IL 1 β和1 L 18的释放。 强烈的炎症反应。在各种NLR中，NLRC 5和NLRP 3在小胶质细胞和神经胶质细胞中高度表达。 差异调节炎症反应。在我们努力了解艾滋病毒达特的组合效应时， 和可卡因对镁激活的影响，我们已经做了几个令人兴奋的初步观察：1）暴露于啮齿动物 HIV达特和可卡因的原代Mg均显示NLRC 5表达显著下调， 与暴露于任一试剂的细胞相比，NLRP 3的伴随上调和Mg活化加剧 2）暴露于HIV达特的啮齿动物Mg表现出microRNA（miR）-34 α的时间依赖性上调， 导致其靶点NLRC 5下调; 3）可卡因介导的Mg激活涉及诱导 活性氧（ROS），这是伴随着有缺陷的线粒体自噬和随后的 NLRP 3炎性体复合物的寡聚化，导致成熟（m）IL 1 β和IL 18的诱导表达; 4） NLRP 3抑制剂-MCC 950减轻可卡因介导的Mg激活。根据这些观察，我们 假设HIV达特和可卡因对Mg激活的协同作用涉及两个过程： 下调NLRC 5的表达，导致NFκB（信号1）的激活，进而导致转录水平的降低。 NLRP 3、pro IL 1 β和IL 18的上调，以及B）ROS介导的缺陷性线粒体自噬的诱导（信号2），其， 进而诱导NLRP 3炎性体的活化，导致mIL 1 β和IL 18的裂解和分泌增加。 该假设将在两个SA中进行测试：1）探索HIV和可卡因的分子机制- 介导的NLRP 3炎性体诱导导致Mg活化，和2）为了在体内验证 NLRC 5/NFκB和ROS/缺陷性线粒体自噬/NLRP 3轴是HIV达特和可卡因介导的Mg激活的基础。 了解HIV和可卡因诱导的小胶质细胞活化的机制将为我们建立一个平台， 用于未来开发旨在抑制神经炎症反应的新疗法。

Abstract: In the current era of combination antiretroviral therapy (cART), there is increased prevalence of HIV-associated neurocognitive disorders (HAND) with about 30-50% of HIV-infected individuals afflicted with varying degrees of neurocognitive impairments, substantially affecting their quality of life. It is well-recognized that despite cART, there is unabated persistence & presence of early HIV protein(s) such as the cytotoxic transactivator of transcription (TAT), that contributes to neuroinflammation. Adding fuel to the mix is the comorbidity of drug abuse in HIV-infected individuals, which further exacerbates microglial activation & thus symptomatology of HAND. Until recently, CNS was considered as an immune-privileged site, however, a recent paradigm shift in our understanding has revealed the role of cytosolic, multiprotein complexes, belonging to the nucleotide-binding domain leucine-rich repeat containing (NLR) protein superfamily, termed as “inflammasomes,” in various neuroinflammatory diseases. Assembly of these multiprotein complexes activates the proinflammatory caspases (specifically caspase 1), leading, in turn, to the cleavage & release of IL1β & 1L18, consequently resulting in a potent inflammatory response. Among the various NLRs, NLRC5 & NLRP3 are highly expressed in microglia & differentially regulate inflammatory responses. In our efforts to understand the combinatorial effects of HIV Tat & cocaine on Mg activation, we have made several exciting preliminary observations: 1) Exposure of rodent primary Mg to both HIV Tat & cocaine demonstrated significantly downregulated expression of NLRC5, with a concomitant upregulation of NLRP3 and, exacerbated Mg activation compared to cells exposed to either agent alone; 2) Rodent Mg exposed to HIV TAT demonstrated time-dependent upregulation of microRNA (miR)-34a, leading, in turn, to downregulation of its target - NLRC5; 3) Cocaine-mediated activation of Mg involved induction of reactive oxygen species (ROS), that was accompanied with defective mitophagy & subsequent oligomerization of NLRP3 inflammasome complex, leading to induced expression of mature (m)IL1β & IL18; 4) NLRP3 inhibitor - MCC950 mitigated cocaine-mediated activation of Mg. Based on these observations, we hypothesize that the co-operative effects of HIV TAT & cocaine on Mg activation involve two processes: a) downregulated expression of NLRC5 resulting in activation of NFκB (signal 1), leading, in turn, to transcriptional upregulation of NLRP3, pro IL1β & IL18, & b) induction of ROS-mediated defective mitophagy (signal 2), which, in turn, induces activation of NLRP3 inflammasome, resulting in increased cleavage & secretion of mIL1β & IL18. The hypothesis will be tested in two SA: 1) Explore the molecular mechanism(s) underlying HIV & cocaine- mediated induction of the NLRP3 inflammasome leading to Mg activation and 2) To validate in vivo the role of NLRC5/NFκB & ROS/defective mitophagy/NLRP3 axes underlying HIV Tat & cocaine-mediated activation of Mg. Understanding the mechanisms responsible for microglial activation induced by HIV & cocaine will set the stage for the future development of novel therapeutics aimed at dampening the neuroinflammatory responses.