Neuronal survival, HIV-1 and Astrocyte TIMP-1

描述(申请人提供)：神经退行性疾病的发病机制，包括人类免疫缺陷病毒(HIV)-1相关性痴呆(过去称为Hand：HIV-1相关性神经认知障碍)，由于金属蛋白酶(MMPs)及其抑制物、金属蛋白酶组织抑制物(TIMPs)之间的失衡而加剧。由于TIMP具有多种非经典功能，包括抗细胞凋亡作用，TIMP-1在神经炎性条件下的诱导可能除了调节基质金属蛋白酶的活性外，还具有多种作用。我们在前一个资金周期的工作表明，TIMP-1在急性和慢性激活的星形胶质细胞和有脑组织的组织中的表达存在差异。我们发现，在急性激活中，IL-12通过多种机制上调TIMP-1，而慢性激活主要通过转录调控导致TIMP-1下调。我们发现了一个新的CCAAT置换蛋白(CDP)结合抑制元件，参与星形胶质细胞TIMP-1的调节。此外，使用星形孢子素、嗜巨噬细胞的HIV-1病毒和谷氨酸诱导培养的人神经元细胞死亡，进行了神经毒性试验。在这些检测中，TIMP-1具有与脑源性神经营养因子(BDNF)相同的神经保护作用。TIMP-1T2G突变体的神经保护作用不依赖于基质金属蛋白酶的结合。通过Bcl2/Bclxl的神经营养信号通路可能是TIMP-1神经保护的一条途径，最近发现了一个潜在的TIMP-1结合伙伴--Tetraspanin。因此，这一提议的主要假设是，星形胶质细胞-TIMP-1在疾病中通过CDP抑制而受到不同的调节，并导致TIMP-1介导的神经保护作用的丧失，这种保护作用是通过神经元上的Tetraspanin受体实现的。此外，将使用新的纳米药物方法探索TIMP-1 CNS的传递策略。将使用分子、细胞、体外、体内和翻译方法相结合的方法。在这一竞争更新中，我们继续深入研究CDP调节星形胶质细胞-TIMP-1的机制，TIMP-1的神经保护作用，并在前一轮资助的基础上探索潜在的治疗纳米药物策略。我们将首先研究CDP在HAD中星形胶质细胞-TIMP-1调节中的作用(目标1)。接下来，我们将研究TIMP-1通过Tetraspanin信号转导的神经保护机制(目标2)。具体地说，我们将描述TIMP-1神经元的TIMP-1受体和相关的导致神经保护的信号转导机制。最后，我们将探索使用CNS靶向纳米颗粒(目标3)来治疗GFAP驱动的TIMP-1表达的应用。TAT结合的PLGA纳米粒中的GFAP-TIMP-1/荧光素酶结构将被静脉注射到小鼠身上，并将评估中枢特异性TIMP-1和荧光素酶的表达。因此，本次更新中提出的研究将揭开HAD慢性神经炎症期间星形胶质细胞反应的新机制，对涉及基质金属蛋白酶/TIMP失衡的其他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相关性痴呆(HAD)，现在被称为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，以认知、行为和运动功能障碍为特征。据估计，尽管有高效的抗逆转录病毒疗法，但在美国等发达国家，估计有10%-15%的艾滋病毒血清阳性(HIV+)患者进展为HIV+。反应性星形胶质细胞增生症，即星形胶质细胞在损伤部位的募集和增殖，是所有神经炎性疾病的病理特征，在HAD的炎症区域可观察到。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，这是耐人寻味的，也是我们和许多其他最近研究的重点。我们的研究将更好地理解激活的星形胶质细胞在HIV-1神经致病和神经炎症中的具体作用机制。

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.