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，HIV-1相关神经认知障碍（HAND）的患病率仍然很高，这是由于许多原因，如潜伏病毒再活化和药物无法有效穿过血脑屏障（BBB）。因此，在HAART时代，了解导致HIV-1感染患者神经元失调的机制非常重要。HIV-1缺乏对神经元的生产性感染表明，从HIV-1感染的靶细胞释放的具有神经毒性活性的病毒和细胞蛋白质， 细胞或潜伏活性病毒的储存细胞引起这种神经元失调。病毒蛋白R（Vpr）是由HIV-1编码的蛋白质之一，并且已经显示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引起神经元损伤的机制和细胞因子尚不清楚。 使用人神经元细胞系SH-SY 5 Y，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（HPCE）（几乎100 pg）提供了理论依据。我们还表明，Vpr失调钙稳态，促进内质网（ER）-钙释放和压力，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中特别明显，但在Vpr突变体（R73 A）中没有。为了寻找涉及的细胞因子，我们使用从用重组Vpr蛋白处理的神经元和/或SH-SY 5 Y的原代人类培养物收集的RNA进行microRNA和基因阵列测定。有趣的是，Vpr下调了几种microRNA（例如miR-34 a）及其靶基因（例如CREB）的水平，这可能导致神经元功能障碍。这些因子在HIV感染患者的人脑组织和Vpr转基因小鼠的脑组织中被解除调节。有趣的是，这些因子在用达特、gp 120或从感染细胞收集的上清液处理的神经元细胞中也失调。 因此，我们提出了一个全面的研究，利用分子，病毒学和细胞的方法来解开机制，并确定Vpr使用的细胞因子，以及其与microRNA的相互作用，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）中进行验证。 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使用的机制，即使在HAART时代也会导致神经元变性。

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.