Involvement of HIV-1 Vpr in neuronal degeneration.

描述(由申请人提供)： 即使在病毒载量低于检测水平的HAART时代，由于潜伏的病毒重新激活和药物无法有效穿越血脑屏障(BBB)等多种原因，HIV-1相关神经认知障碍(HAND)的患病率仍然很高。因此，了解在HAART时代导致HIV-1感染患者神经元去调节的机制是重要的。 细胞，或潜伏活性病毒的储存库细胞，导致了这种神经元的去调节。病毒蛋白R(Virus Protein R，VPR)是HIV-1编码的蛋白质之一，已被证明可以改变感染和未感染细胞中各种重要细胞因子和炎性蛋白的表达。VPR导致神经元损伤的机制和细胞因子仍不清楚。利用人神经细胞系SH-SY5Y，我们发现VPR可以被神经元摄取(免疫组织化学和Western印迹分析)，这为我们测量神经元中VPR的量(几乎100pg)提供了理论基础。我们还证实了VPR可破坏钙稳态，促进内质网钙释放和应激，激活氧化应激途径，线粒体功能障碍和轴突运输改变。这些效应在全长VPR中被特别注意到，但在VPR突变体(R73A)中没有。为了寻找相关的细胞因子，我们使用从原代培养的人神经元和/或经重组VPR蛋白处理的SH-SY5Y收集的RNA进行了microRNA和基因阵列分析。有趣的是，VPR解除了几个microRNA(例如miR-34a)及其靶基因(例如CREB)的水平调控，这可能导致神经元功能障碍。这些因子在HIV感染者的人脑组织和VPR转基因小鼠的脑组织中被解除调控。有趣的是，在用TAT、gp120或从感染细胞收集的上清液处理的神经细胞中，这些因子也被解除了调控。因此，我们建议利用分子、病毒学和细胞方法进行一项综合研究，以揭示VPR的机制，并确定VPR使用的细胞因素以及它与microRNAs的相互作用，以导致神经元功能障碍。这些研究将在动物模型(VPR转基因小鼠)中得到验证。这些研究的结果将促进对HIV-1发病机制的理解，并将破译VPR使用的导致神经元退化的机制，即使在HAART时代也是如此。

DESCRIPTION (provided by applicant): Even in the HAART era where the viral load is below detection levels, the prevalence of HIV-1 associated neurocognitive disorders (HAND) remains high due to many reasons such as latent virus reactivation and drugs inability to efficiently cross the blood brain barrier (BBB). Therefor, it is important to understand the mechanisms leading to neuronal deregulation in HIV-1-infected patients in the HAART era. The lack of productive infection of neurons by HIV-1 suggests that viral and cellular proteins with neurotoxic activities that are released from HIV-1 infected target cells, or reservoirs cells for latent active virus, cause this neuronal deregulation. The viral protein R (Vpr) is one of the proteins encoded by HIV-1 and has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells. The mechanisms and the cellular factors used by Vpr to cause neuronal damage remain unclear. Using human neuronal cell line, SH-SY5Y, we found that Vpr can be taken up by neurons (immunohistochemistry and Western blot analysis), which gave us the rationale to measure the amount of Vpr in neurons (HPCE) (almost 100 pg). We also demonstrated that Vpr deregulates calcium homeostasis, promotes endoplasmic reticulum (ER)-calcium release and stress, activation of the oxidative stress pathway, mitochondrial dysfunction and axonal transport alteration. These effects were specifically noted with the full length Vpr but not with Vpr mutant (R73A). In search for the cellular factors involved, we performed microRNA and gene array assays using RNA collected from primary human cultures of neurons and/or SH-SY5Y-treated with recombinant Vpr protein. Interestingly, Vpr deregulates the levels of several microRNA (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. These factors were deregulated in human brain tissues of HIV-infected patients and in the brain tissues of Vpr-transgenic mice. Interestingly, these factors were also deregulated in neuronal cells treated with Tat, gp120 or supernatant collected from infected cells. Therefore, we propose a comprehensive study utilizing molecular, virological, and cellular approaches to unravel the mechanisms and identify the cellular factors used by Vpr as well as its interplay with microRNAs to cause neuronal dysfunction. These studies will be validated in an animal model (Vpr-transgenic mice). The outcome of these studies will advance the understanding of HIV-1 pathogenesis and will decipher the mechanisms used by Vpr that lead to neuronal degeneration even in the HAART era.