Viral Protein R (Vpr) in HIV-associated Brain Neuroinflammation and Neurotoxicity

这个拟议中的项目与退伍军人医疗保健直接相关，因为它解决了退伍军人事务部目前的研究 优先考虑HIV-1感染如何导致影响退伍军人心理健康的脑损伤，包括HIV-1， 相关的神经认知障碍（HAND）和自杀。目前约有3700万人生活在 全球艾滋病毒/艾滋病。联合抗逆转录病毒疗法（cART）的成功治疗可以消除 活跃的复制病毒，并延长患者的生命，使其接近正常的寿命。然而，新的挑战 超过一半的HIV感染者和老年患者面临的是慢性CNS神经炎症， 导致各种手。虽然由于cART，严重和进行性HAND显著减少，但慢性HAND 手通常持续存在，导致谵妄，痴呆和抑郁症的高发生率，可能导致自杀。 事实上，“艾滋病毒感染者自杀死亡的风险比未感染艾滋病毒者高3-5倍 对应方”。然而，HAND的神经发病机制尚不清楚。 HAND的典型特征是HIV介导的神经胶质炎症和神经毒性。有趣的是 一些HAND的严重程度并不总是与HIV的水平直接相关，而是与神经胶质细胞的活化相关， 这表明其他HIV相关因素，而不是整个病毒本身，导致了这些HAND。HIV-1病毒蛋白 R（Vpr）可能是病毒因子之一，因为Vpr可引起神经炎症， 凋亡此外，在cART下不存在活跃的病毒复制的情况下，可以在CNS相关性肿瘤中发现Vpr。 因为1）它可以直接从病毒颗粒中释放; 2）它可以穿过血脑屏障， 被神经胶质细胞和神经元摄取; 3）它通过与LTR结合触发潜伏感染细胞的病毒转录 启动子尽管这些强有力的证据表明Vpr在HAND中起着重要作用， 对HAND的贡献仍然难以捉摸。本提案的目的是研究Vpr在以下方面的具体作用： 激活宿主神经炎症、神经毒性和病毒再激活，以及其对HAND的贡献。 通过我们的初步研究，我们发现了HIV表达和细胞活化之间的相关性。 HIV感染的星形胶质细胞中的促炎标志物（TLR 4、TNFα、NF-κB和Sur 1-Trpm 4通道） 死后的人类和转基因小鼠脑组织。此外，Vpr单独激活相同的一组 神经胶质细胞中的标记物。Vpr和Sur 1-Trpm 4通道之间的联系可能是重要的 因为这个通道是参与各种神经认知大脑的关键神经调节器 条件事实上，通过一种改变用途并经FDA批准的药物格列本脲抑制该通道， vpr诱导细胞凋亡和改善其他神经炎症性脑疾病。因此，我们的试点研究可能 揭示了一种新的机制，涉及Vpr诱导的激活Sur 1-Trpm 4通道的手。因此，我们认为， 我们假设Vpr通过TLR 4/MyD 88和/或TNFα介导的NF-κB活化而导致HAND， 继而上调Sur 1-Trpm 4通道，导致神经炎症和神经毒性。或者，Vpr- 诱导的HAND由NF-κB和Sur 1-Trpm 4介导的神经病理学作用共同贡献。我们 进一步假设对关键调节因子如Sur 1-Trpm 4通道的靶特异性抑制减轻了 Vpr诱导的HAND。我们将用三个具体目标（SA）来检验这些假设。SA 1：描述分子 原代星形胶质细胞中Vpr诱导的神经炎症、神经毒性和病毒再活化的机制; SA 2： 测试Vpr与Sur 1-Trpm 4通道的功能联系，其与NF-kB的相互作用及其对Vpr的贡献。 诱导的HAND;和SA 3：评估Sur 1-Trpm 4的遗传和药理学抑制作用 通过使用基因敲除小鼠和靶向特异性治疗药物， 格列本脲该项目的成功完成将为1）分子机制提供新的见解 HIV-1 Vpr对HAND的贡献; 2）Vpr与Sur 1-Trpm 4通道之间的功能联系及其与HAND的关系。 对HAND的贡献，以及3）用治疗药物格列本脲治疗Vpr相关HAND的可行性。

This proposed project has direct relevance to Veteran healthcare because it addresses current VA’s research priority on how HIV-1 infection causes brain damages that affect Veteran’s mental health including HIV- associated neurocognitive disorders (HAND) and suicide. There are about 37 million people currently living with HIV/AIDS worldwide. Successful treatment with combinational antiretroviral therapies (cART) can eliminate active replicating viruses and prolong patients’ lives to nearly normal lifespans. However, the new challenge faced by more than half of those HIV-infected and aging patients is the chronic CNS neuroinflammation, which leads to various HAND. While severe and progressive HAND has decreased significantly due to cART, chronic HANDS often persists, resulting in high rates of delirium, dementia and depression that could lead to suicide. Indeed, “the risk of suicide mortality in HIV-infected persons is 3-5 times higher than in HIV-uninfected counterparts”. Nevertheless, the mechanism of neuropathogenesis underlying HAND is not well understood. HAND is typically characterized by HIV-mediated glial neuroinflammation and neurotoxicity. Interestingly, the severity of some HAND does not always directly correlate with the levels of HIV, but rather with glial activation, suggesting other HIV-associated factors, not the whole virus per se, contribute to those HAND. HIV-1 viral protein R (Vpr) might be one of those viral factors, because Vpr induces neuroinflammation and causes neuronal apoptosis. Moreover, in the absence of active viral replication under cART, Vpr can be found in CNS-associated cells because 1) it can be released directly from viral particles; 2) it crosses the blood-brain barrier that can be taken up by glia and neurons; and 3) it triggers viral transcription of latently-infected cells by binding to LTR promoter. Despite these strong evidences indicating a prominent role of Vpr in HAND, how exactly Vpr contributes to HAND remains elusive. The objective of this proposal is to study the specific role(s) of Vpr in activation of host neuroinflammation, neurotoxicity and viral reactivation, as well as its contribution to HAND. Through our pilot studies, we discovered correlations between HIV expression and activation of proinflammatory markers (TLR4, TNFα, NF-κB and the Sur1-Trpm4 channel) in astrocytes of HIV-infected postmortem human and transgenic mouse brain tissues. Furthermore, Vpr alone activate the same set of markers in glial cells. The connection between Vpr and the Sur1-Trpm4 channel could potentially be significant for understanding HAND because this channel is a key neuro-regulator involved in various neurocognitive brain conditions. Indeed, inhibition of the channel by a repurposed and FDA-approved drug glibenclamide reduces Vpr-induced apoptosis and improves other neuroinflammatory brain conditions. Thus, our pilot studies may have revealed a novel mechanism of HAND involving Vpr-induced activation of the Sur1-Trpm4 channel. Therefore, we hypothesize that Vpr contributes to HAND by TLR4/MyD88- and/or TNFα-mediated NF-κB activation, which in turn upregulate the Sur1-Trpm4 channel leading to neuroinflammation and neurotoxicity. Alternatively, Vpr- induced HAND is contributed collectively by NF-κB and Sur1-Trpm4-mediated neuropathologic effects. We further hypothesize that target-specific inhibition of key regulators such as the Sur1-Trpm4 channel mitigates Vpr-induced HAND. We will test these hypotheses with three specific aims (SA). SA1: delineate molecular mechanism of Vpr-induced neuroinflammation, neurotoxicity and viral reactivation in primary astrocytes; SA2: test the functional link of Vpr with the Sur1-Trpm4 channel, its interaction with NF-kB and its contribution to Vpr- induced HAND; and SA3: evaluate the effects of genetic and pharmacologic inhibitions of the Sur1-Trpm4 channel on Vpr-induced HAND by using knock-out mice and by target-specific therapeutic drugs such as glibenclamide. Successful completion of this project will provide novel insights into 1) the molecular mechanism and contribution of HIV-1 Vpr to HAND, 2) the functional link between Vpr and the Sur1-Trpm4 channel and its contribution to HAND, and 3) the feasibility of treating Vpr-related HAND with the therapeutic drug glibenclamide.