TLR Gene Expression in HIV Neurocognitive Disorder

描述（由申请人提供）：尽管引入了高效抗逆转录病毒治疗（HAART），但HIV相关神经认知障碍（HAND）仍然是发病的重要原因。先前的工作已经证明，HAND与HIV相关的神经退行性变的严重程度有关。在初步的微阵列工作中，我们注意到与先天免疫系统toll样受体（TLR）信号通路相关的许多蛋白质的基因表达与HIV相关的神经变性强烈相关，最显著的是TLR 3和-4。我们已经进一步证明，用gp 120（Bal）处理星形胶质细胞导致TLR 4表达的增加。在本申请中，我们希望检查TLR 3和4基因表达与暴露于HIV之间的潜在关系。首先在暴露于取自HIV感染的（Bal和SF 162）单核细胞衍生的巨噬细胞（MDM）的上清液的原代人神经胶质细胞培养物中评估TLR 3和-4表达。将通过定量真实的时间聚合酶链反应（qRT-PCR）测量mRNA表达，通过免疫印迹和免疫组织化学（IHC）测量蛋白质表达。在本提案的第二部分中，我们将研究阻断TLR 3和TLR 4表达对HIV相关神经毒性的影响。与具体目标1相似，并使用该目标产生的数据，我们将用HIV感染（Bal和SF 162）MDM的上清液处理原代人神经元培养物，浓度和时间点显示会引起毒性。这将是在不存在和存在针对TLR 3和-4的siRNA的情况下。然后，我们将使用qRT-PCR验证这种敲除，并评估其对HIV神经毒性作用的影响。作为TLR诱导的神经变性的潜在机制，我们将在最后一组实验中使用ELISA评估GSK 3b活性以及特异性GSK 3b抑制剂A014418和B6 B30预防或减弱HIV相关神经毒性的能力。从该提案中产生的数据将为阐明TLR基因产物导致HIV相关神经变性的机制迈出第一步，并确定改善这种毒性过程的潜在新靶点。减少或预防与HIV相关的神经元损失的能力有可能预防或减弱HAND的影响。该提案的重点是进一步表征TLR 3和4失调在HIV相关神经变性中的作用以及GSK 3b活性在调节该机制中的贡献。抑制TLR 3和4活性可能是预防HIV相关神经变性的一种可能方法，因此可以减轻HAND的症状。

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.