TLR Gene Expression in HIV Neurocognitive Disorder

描述(申请人提供)：尽管引入了高效抗逆转录病毒疗法(HAART)，但艾滋病毒相关性神经认知障碍(HAND)仍然是发病率的重要原因。以前的工作已经证明，手与HIV相关的神经变性的严重程度有关。在芯片的初步工作中，我们已经注意到与先天性免疫系统Toll样受体(TLR)信号通路相关的一些蛋白质的基因表达与HIV相关的神经变性密切相关，最显著的是TLR3和-4。我们进一步证明，用gp120(Bal)处理星形胶质细胞后，TLR4的表达增加。在目前的应用中，我们希望研究TLR3和TLR4基因表达与HIV暴露之间的潜在关系。TLR3和-4的表达最初将在暴露于HIV感染的(Bal和SF162)单核细胞来源的巨噬细胞(MDM)上清液的原代人类神经胶质细胞培养中进行评估。通过实时定量聚合酶链式反应(qRT-PCR)检测mRNA表达，免疫印迹和免疫组织化学(IHC)检测蛋白表达。在这项建议的第二部分，我们将研究阻断TLR3和-4表达对HIV相关神经毒性的影响。与具体目标1类似，并使用从这一目标产生的数据，我们将用HIV感染的MDM(Bal和SF162)的上清液处理原代人类神经元培养，处理的浓度和时间点表明会导致毒性。这将是在没有和存在针对TLR3和-4的siRNA的情况下。然后，我们将使用qRT-PCR验证这一基因敲除，并评估其对HIV神经毒性效应的影响。作为TLR诱导的神经变性的一个潜在机制，我们将在最后一组实验中使用ELISA法评估GSK3b的活性，以及特定的GSK3b抑制剂A014418和B6B30预防或减轻HIV相关神经毒性的能力。这项提议产生的数据将为阐明TLR基因产品导致HIV相关神经退化的机制提供第一步，并确定改善这一有毒过程的潜在新靶点。公共卫生相关性减少或防止与艾滋病毒相关的神经元丢失的能力有可能预防或减弱手的影响。这项建议的重点是进一步表征TLR3和TLR4在HIV相关神经变性中的作用以及GSK3b活性在调节这一机制中的作用。抑制TLR3和TLR4活性可能是预防HIV相关神经变性从而缓解手部症状的一种可能方法。

DESCRIPTION (provided by applicant): Despite the introduction of highly active antiretroviral therapy (HAART), HIV associated neurocognitive disorder (HAND) remains a significant cause of morbidity. Previous work has demonstrated that HAND is related to the severity of HIV associated neurodegeneration. In preliminary microarray work we have noted that the gene expression of a number of proteins related to the innate immune system toll-like receptor (TLR) signaling pathway correlates strongly with HIV associated neurodegeneration, most significantly TLR3 and -4. We have further demonstrated that treatment of astrocytes with gp120 (Bal) resulted in an increase in expression of TLR4. In the current application we wish to examine the potential relationship between TLR3 and 4 gene expression and exposure to HIV. TLR3 and -4 expressions will be assessed initially in primary human neuroglial cultures exposed to supernatants taken from HIV infected (Bal and SF162) monocyte derived macrophages (MDMs). mRNA expression will be measured via quantitative real time polymerase chain reaction (qRT-PCR) with protein expression measured via immunoblot and immunohistochemistry (IHC). In the second part of this proposal we will investigate the effects of blocking TLR3 and -4 expressions on HIV associated neurotoxicity. Similar to specific aim 1 and using the data generated from this aim we will treat primary human neuronal cultures with supernatants from HIV infected (Bal and SF162) MDMs at concentrations and time-points shown to cause toxicity. This will be in the absence and presence of siRNA's to TLR3 and -4. We will then verify this knockdown using qRT-PCR and assess the effects of this on HIV neurotoxic effects. As a potential mechanism for TLR induced neurodegeneration we will assess GSK3b activity in a final set of experiments using ELISA and also the ability of specific GSK3b inhibitors A014418 and B6B30 to prevent or attenuate HIV associated neurotoxicity. Data generated from this proposal will provide a first step towards elucidating the mechanism by which TLR gene products result in HIV associated neurodegeneration and also identify potential novel targets for ameliorating this toxic process. PUBLIC HEALTH RELEVANCE The ability to reduce or prevent neuronal loss associated with HIV has the potential to prevent or attenuate the effects of HAND. The focus of this proposal is to further characterize the role of TLR 3 and 4 dysregulation in HIV associated neurodegeneration and contribution of GSK3b activity in regulating this mechanism. Inhibition of TLR 3 and 4 activity may present a possible way to prevent HIV associated neurodegeneration and therefore alleviate symptoms of HAND.