Neuronal survival, HIV-1 and Astrocyte TIMP-1

描述（由申请人提供）：神经退行性疾病，包括人类免疫缺陷病毒(HIV)-1相关痴呆（had -现在称为HAND: HIV-1相关神经认知障碍）的发病机制，由于金属蛋白酶（MMPs）及其抑制剂，金属蛋白酶组织抑制剂（TIMPs）之间的不平衡而加剧。由于timp表现出多种非经典功能，包括抗凋亡作用，除了调节MMP活性外，在神经炎症条件下诱导TIMP-1可能还具有多种作用。我们在上一个资助周期的工作表明，在星形胶质细胞和HAD脑组织的急性和慢性激活中，TIMP-1的表达存在差异。我们发现，在急性激活时，IL-12通过多种机制上调TIMP-1，而慢性激活主要通过转录控制导致TIMP-1下调。我们发现了一个新的CCAAT位移蛋白（CDP）结合抑制元件参与星形胶质细胞TIMP-1的调节。此外，使用staurosporine、嗜巨噬细胞HIV-1病毒和谷氨酸进行神经毒性试验，诱导培养的人类神经元细胞死亡。在这些实验中，TIMP-1具有与脑源性神经营养因子（BDNF）相当的神经保护作用。TIMP-1 T2G突变体显示，这些神经保护作用与MMP结合无关。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，最近发现了一种潜在的TIMP-1结合伙伴。因此，该建议的总体假设是星形胶质细胞- timp -1在疾病中通过CDP抑制受到差异调节，并导致timp -1通过神经元上的四种蛋白受体介导的神经保护作用丧失。此外，TIMP-1 CNS递送策略将探索使用新的纳米医学方法。结合分子，细胞，体外，体内和翻译的方法将被使用。在这一竞争更新中，我们继续深入研究星形胶质细胞-TIMP-1通过CDP调节的机制，TIMP-1的神经保护作用，并探索潜在的治疗纳米药物策略，基于我们在上一轮融资中的进展。我们将首先研究CDP在HAD中星形胶质细胞- timp -1的调节中的作用（目的1）。接下来，我们将研究timp -1通过tetraspanin信号通路介导的神经保护机制（Aim 2）。具体来说，我们将描述TIMP-1神经元四跨蛋白受体和导致神经保护的相关信号转导机制。最后，我们将探索使用cns靶向纳米颗粒驱动gmap驱动TIMP-1表达的治疗应用（目的3）。gmap - TIMP-1 /荧光素酶构建物将通过静脉注射给小鼠，并评估cns特异性TIMP-1和荧光素酶的表达。因此，本文提出的研究将揭示HAD慢性神经炎症中星形胶质细胞反应的新机制，对其他涉及MMP/TIMP失衡的炎症性疾病具有更广泛的意义，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相关痴呆（HAD），现在被称为HIV-1相关神经认知障碍，是HIV-1感染的重要神经系统并发症，以认知、行为和运动功能障碍为特征。在美国等发达国家，尽管有高效抗逆转录病毒治疗，估计仍有10-15%的HIV血清阳性（HIV+）患者发展为HAD。反应性星形胶质细胞增生，即损伤部位星形胶质细胞的聚集和增殖，是所有神经炎症的病理标志，在HAD的炎症区域也可以观察到。星形胶质细胞的神经保护反应如何在神经炎症中发生改变并导致疾病是有趣的，也是我们和许多其他近期研究的重点。我们的研究将更好地理解激活星形胶质细胞对hiv -1神经发病和神经炎症的具体机制贡献。

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.