Department of Pharmacology and Experimental Neuroscience, 985880 Nebraska Medical Center, University of Nebraska Medical Center, Omaha, Nebraska 68198.
Department of Pharmacology and Experimental Neuroscience, 985880 Nebraska Medical Center, University of Nebraska Medical Center, Omaha, Nebraska 68198
J Neurosci. 2018 Jun 6;38(23):5367-5383. doi: 10.1523/JNEUROSCI.3474-17.2018. Epub 2018 May 14.
The present study demonstrates HIV-1 Tat-mediated epigenetic downregulation of microglial miR-124 and its association with microglial activation. Exposure of mouse primary microglia isolated from newborn pups of either sex to HIV-1 Tat resulted in decreased expression of primary miR-124-1, primary miR-124-2 as well as the mature miR-124. In parallel, HIV-1 Tat exposure to mouse primary microglial cells resulted in increased expression of DNA methylation enzymes, such as DNMT1, DNMT3A, and DNMT3B, which were also accompanied by increased global DNA methylation. Bisulfite-converted genomic DNA sequencing in the HIV-1 Tat-exposed mouse primary microglial cells further confirmed increased DNA methylation of the primary miR-124-1 and primary miR-124-2 promoters. Bioinformatic analyses identified MECP2 as a novel 3'-UTR target of miR-124. This was further validated in mouse primary microglial cells wherein HIV-1 Tat-mediated downregulation of miR-124 resulted in increased expression of MECP2, leading in turn to further repression of miR-124 via the feedback loop. In addition to MECP2, miR-124 also modulated the levels of STAT3 through its binding to the 3'-UTR, leading to microglial activation. Luciferase assays and Ago2 immunoprecipitation determined the direct binding between miR-124 and 3'-UTR of both MECP2 and STAT3. Gene silencing of and and overexpression of miR-124 blocked HIV-1 Tat-mediated downregulation of miR-124 and microglial activation. findings were also confirmed in the basal ganglia of SIV-infected rhesus macaques (both sexes). In summary, our findings demonstrate a novel mechanism of HIV-1 Tat-mediated activation of microglia via downregulation of miR-124, leading ultimately to increased MECP2 and STAT3 signaling. Despite the effectiveness of combination antiretroviral therapy in controlling viremia, the CNS continues to harbor viral reservoirs. The persistence of low-level virus replication leads to the accumulation of early viral proteins, including HIV-1 Tat protein. Understanding the epigenetic/molecular mechanism(s) by which viral proteins, such as HIV-1 Tat, can activate microglia is thus of paramount importance. This study demonstrated that HIV-1 Tat-mediated DNA methylation of the miR-124 promoter leads to its downregulation with a concomitant upregulation of the MECP2-STAT3-IL6, resulting in microglial activation. These findings reveal an unexplored epigenetic/molecular mechanism(s) underlying HIV-1 Tat-mediated microglial activation, thereby providing a potential target for the development of therapeutics aimed at ameliorating microglial activation and neuroinflammation in the context of HIV-1 infection.
本研究表明，HIV-1 Tat 通过表观遗传下调小胶质细胞 miR-124 的表达，并与其激活相关。将来自新生幼仔的雄性或雌性小鼠原代小胶质细胞暴露于 HIV-1 Tat 中，导致初级 miR-124-1、初级 miR-124-2 以及成熟 miR-124 的表达降低。同时，HIV-1 Tat 暴露于小鼠原代小胶质细胞中导致 DNA 甲基转移酶（如 DNMT1、DNMT3A 和 DNMT3B）的表达增加，这也伴随着全基因组 DNA 甲基化水平的增加。在 HIV-1 Tat 暴露的小鼠原代小胶质细胞中，经亚硫酸氢盐转化的基因组 DNA 测序进一步证实了初级 miR-124-1 和初级 miR-124-2 启动子的 DNA 甲基化增加。生物信息学分析鉴定出 MECP2 是 miR-124 的一个新的 3'-UTR 靶标。这在小鼠原代小胶质细胞中得到进一步验证，其中 HIV-1 Tat 介导的 miR-124 下调导致 MECP2 的表达增加，进而通过反馈环进一步抑制 miR-124。除了 MECP2 之外，miR-124 还通过与其 3'-UTR 结合来调节 STAT3 的水平，导致小胶质细胞激活。荧光素酶测定和 Ago2 免疫沉淀确定了 miR-124 与 MECP2 和 STAT3 的 3'-UTR 之间的直接结合。和的基因沉默和 miR-124 的过表达阻断了 HIV-1 Tat 介导的 miR-124 下调和小胶质细胞激活。在 SIV 感染的恒河猴（雄性和雌性）的基底神经节中也证实了这些发现。总之，我们的研究结果表明，HIV-1 Tat 通过下调 miR-124 来激活小胶质细胞，最终导致 MECP2 和 STAT3 信号的增加，这是一种新的机制。尽管联合抗逆转录病毒疗法在控制病毒血症方面非常有效，但中枢神经系统仍然存在病毒储存库。低水平病毒复制的持续存在导致早期病毒蛋白（包括 HIV-1 Tat 蛋白）的积累。因此，了解病毒蛋白（如 HIV-1 Tat）如何通过表观遗传/分子机制激活小胶质细胞至关重要。本研究表明，HIV-1 Tat 介导的 miR-124 启动子的 DNA 甲基化导致其下调，同时 MECP2-STAT3-IL6 的表达上调，导致小胶质细胞激活。这些发现揭示了 HIV-1 Tat 介导的小胶质细胞激活的一种未被探索的表观遗传/分子机制，为开发旨在改善 HIV-1 感染中小胶质细胞激活和神经炎症的治疗方法提供了一个潜在的靶点。
