Neuronal survival, HIV-1 and Astrocyte TIMP-1

描述（由申请方提供）：金属蛋白酶（MMP）及其抑制剂（金属蛋白酶组织抑制剂（TIMP））之间的失衡加剧了神经退行性疾病（包括人类免疫缺陷病毒（HIV）-1相关痴呆（HAD-现称为HAND：HIV-1相关神经认知障碍））的发病机制。由于TIMP表现出包括抗凋亡作用在内的多种非经典功能，因此在神经炎性病症中TIMP-1的诱导可能除了调节MMP活性之外还发挥多种作用。我们在上一个资助周期的工作表明，急性与慢性星形胶质细胞和HAD脑组织激活中TIMP-1的表达差异。我们发现，在急性激活IL-12上调TIMP-1通过多种机制，而慢性激活导致TIMP-1下调主要是通过转录控制。我们发现了一种新的CCAAT置换蛋白（CDP）结合阻遏元件参与星形胶质细胞TIMP-1的调节。此外，使用星形孢菌素、嗜巨噬细胞的HIV-1病毒和谷氨酸盐进行神经毒性测定，以诱导培养的人神经元中的细胞死亡。在这些测定中，TIMP-1具有与脑源性神经营养因子（BDNF）相当的神经保护作用。这些神经保护作用不依赖于MMP结合，如TIMP-1 T2 G突变体所示。通过Bcl-2/Bcl-xL的神经营养信号传导是TIMP-1神经保护的可能途径，并且最近四跨膜蛋白已被鉴定为潜在的TIMP-1结合伴侣。因此，该提议的总体假设是星形胶质细胞-TIMP-1在疾病中通过CDP抑制被差异调节，并且有助于通过神经元上的四跨膜蛋白受体提供的TIMP-1介导的神经保护的丧失。此外，将使用新型纳米医学方法探索TIMP-1中枢神经系统递送策略。将使用组合的分子、细胞、体外、体内和翻译方法。在这个竞争性的更新中，我们继续深入研究通过CDP调节星形胶质细胞-TIMP-1的机制，TIMP-1的神经保护作用，并根据我们在上一轮资金中的进展探索潜在的治疗性纳米医学策略。我们将首先研究CDP在HAD（Aim 1）中调节星形胶质细胞TIMP-1的作用。接下来，我们将研究TIMP-1通过四跨膜蛋白信号传导介导的神经保护机制（目的2）。具体而言，我们将描述TIMP-1神经元四跨膜蛋白受体和相关的信号转导机制，导致神经保护。最后，我们将探索使用CNS靶向纳米颗粒的GFAP驱动的TIMP-1表达的治疗应用（目的3）。将Tat缀合的PLGA纳米颗粒中的GFAP-TIMP- 1/荧光素酶构建体静脉内施用给小鼠，并评价CNS特异性TIMP-1和荧光素酶表达。因此，本次更新中提出的研究将揭示HAD慢性神经炎症期间星形胶质细胞反应的新机制，在涉及MMP/TIMP失衡的其他炎症性疾病中具有更广泛的意义，并为未来的治疗策略提供关键的概念验证数据。公共卫生相关性：HIV-1相关性痴呆（HAD），现称为HIV-1相关性神经认知障碍，是HIV-1感染的重要神经系统并发症，其特征在于认知、行为和运动功能障碍。据估计，在发达国家如美国，10-15%的HIV血清阳性（HIV+）患者进展为HAD，尽管可获得高效抗逆转录病毒治疗。反应性星形胶质细胞增生，即损伤部位星形胶质细胞的募集和增殖，是所有神经炎性疾病的病理标志，并在HAD的炎症区域中观察到。星形胶质细胞的神经保护反应如何在神经炎症中改变并导致疾病是有趣的，是我们和许多其他最近研究的焦点。我们的研究将提供一个更好的理解的具体机制的贡献活化星形胶质细胞的HIV-1-神经发病和神经炎症。

DESCRIPTION (provided by applicant): The pathogenesis of neurodegenerative disorders, including Human Immunodeficiency Virus (HIV)-1 associated dementia (HAD-now called HAND: HIV-1-associated neurocognitive disorder), is exacerbated by an imbalance between metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). As the TIMPs exhibit diverse non-classical functions including anti-apoptotic effects, the induction of TIMP-1 in neuroinflammatory conditions likely serves multiple roles in addition to modulating MMP activity. Our work in the previous funding cycle demonstrated that differential TIMP-1 expression in acute versus chronic activation of astrocytes and HAD brain tissues. We showed that in acute activation IL-12 upregulates TIMP-1 through multiple mechanisms, while chronic activation leads to TIMP-1 downregulation primarily through transcriptional control. We identified a novel CCAAT displacement protein (CDP) binding repressor element involved in astrocyte TIMP-1 regulation. Further, neurotoxicity assays were performed using staurosporine, macrophage-tropic HIV-1 virus and glutamate to induce cell death in cultured human neurons. In these assays, TIMP-1 had equivalent neuroprotective effects as brain derived neurotrophic factor (BDNF). These neuroprotective effects were independent of MMP binding as shown with the TIMP-1 T2G mutant. Neurotrophic signaling through Bcl-2/Bcl-xL is a possible pathway for TIMP-1 neuroprotection and recently a tetraspanin has been identified as a potential TIMP-1 binding partner. Thus, the overarching hypothesis for this proposal is that astrocyte-TIMP-1 is differentially regulated in disease through CDP repression and contributes to loss of TIMP-1-mediated neuroprotection rendered via tetraspanin receptors on neurons. Furthermore, TIMP-1 CNS delivery strategies will be explored using novel nanomedicine approaches. Combined molecular, cellular, in vitro, in vivo and translational approaches will be used. In this competing renewal, we continue an in depth investigation into the mechanisms of astrocyte-TIMP-1 regulation via CDP, neuroprotective effects of TIMP-1 and explore a potential therapeutic nanomedicine strategy, based on our progress in the previous round of funding. We will first investigate the role of CDP in the regulation of astrocyte-TIMP-1 in HAD (Aim 1). Next, we will study the mechanisms of TIMP-1-mediated neuroprotection via tetraspanin signaling (Aim 2). Specifically, we will delineate the TIMP-1 neuronal tetraspanin receptors and related signal transduction mechanism leading to neuroprotection. Finally, we will explore the therapeutic application of GFAP-driven TIMP-1 expression using CNS-targeted nanoparticles (Aim 3). GFAP-TIMP- 1/luciferase constructs in Tat-conjugated PLGA nanoparticles will be intravenously administered to mice and CNS-specific TIMP-1 and luciferase expression will be evaluated. Thus, studies proposed in this renewal will unravel novel mechanisms underlying astrocyte responses during chronic neuroinflammation in HAD, have broader implications in other inflammatory diseases that involve MMP/TIMP imbalance and provide critical proof-of-concept data for future therapeutic strategies. PUBLIC HEALTH RELEVANCE: HIV-1-associated dementia (HAD), now referred to as HIV-1-associated neurocognitive disorder, is an important neurological complication of HIV-1 infection and is characterized by cognitive, behavioral and motor dysfunction. An estimated 10-15% of HIV-seropositive (HIV+) patients progress to develop HAD in developed worlds such as the United States, despite the availability of highly active antiretroviral therapy. Reactive astrogliosis, recruitment to and proliferation of astrocytes at the site of injury, is the pathological hallmark of all neuroinflammatory conditions and is observed in areas of inflammation in HAD. How neuroprotective responses of astrocytes are altered in neuroinflammation and contribute to disease is intriguing and is the focus of our and many other recent investigations. Our studies will provide a better understanding of the specific mechanistic contributions of activated astrocytes to HIV-1-neuropathogensis and neuroinflammation.