Neuronal Survival, HIV-1 and Astrocyte-TIMP-1

描述(申请人提供)：星形胶质细胞产生金属蛋白酶组织抑制物(TIMP)-1在中枢神经系统内稳态和炎症性疾病(如HIV-1相关性痴呆(HAD))中发挥重要作用。虽然TIMP-1最初被发现是一种基质金属蛋白酶(MMPs)的抑制剂，但最近作为一种促进细胞存活的多功能分子而出现。基质金属蛋白酶/组织基质金属蛋白酶失衡与多种神经炎症性疾病有关。我们之前的研究独特地证明了HAD患者大脑中TIMP-1的水平降低。星形胶质细胞-TIMP-1的表达在急性和慢性炎症条件下有不同的调节，急性炎症条件下表达升高，而慢性炎症条件下表达降低。这些观察表明，在HAD中补充TIMP-1可能具有治疗价值。我们认为，星形胶质细胞-TIMP-1通过神经营养活性和/或通过激活抗凋亡蛋白来调节HAD中神经元的存活。TIMP-1神经保护可能涉及MMPs，也可能不涉及MMPs。为了了解TIMP-1在HAD中的调控和神经保护机制，提出了以下问题： 1.TIMP-1在免疫激活的星形胶质细胞中是如何在分子水平上调节的，TIMP-1的表达与慢性胶质细胞激活之间有什么时间关系？ 2.TIMP-1介导的神经元存活机制是什么？TIMP-1是否通过与基质金属蛋白酶的相互作用介导神经保护？ 3.TIMP-1能否作为治疗药物来减轻HIV-1介导的神经变性？为了解决这些问题，HAD的细胞和动物模型将用于模拟急性和慢性炎症性疾病的过程。将使用由原代人类星形胶质细胞、人类神经元、神经毒素、病毒和病毒蛋白组成的实验室体外系统。TIMP-1启动子-荧光素酶报告基因的构建、腺病毒感染和siRNA分子的基因沉默等分子操作将被用来研究TIMP-1启动子元件和信号转导途径参与Astrocvte-TIMP-1调控和神经保护作用。将评估TIMP-1的神经保护机制，包括诱导抗凋亡通路和神经营养活性。这些现象对基质金属蛋白酶的依赖性将使用具有不同基质金属蛋白酶结合能力的TIMP-1突变体进行研究。为了验证TIMP-1是否可以减轻神经退行性变，将腺病毒表达的TIMP-1通过颅内或脑室内注射到HIV脑炎SCID小鼠模型的损伤区域。这些研究将阐明HAD中的神经胶质炎症反应，揭示神经胶质细胞-神经元相互作用的新机制，并可能揭示未来治疗神经退行性疾病的潜在策略。

DESCRIPTION (provided by applicant): Astrocyte production of tissue inhibitor of metalloproteinase (TIMP)-1 plays an important role in central nervous system homeostasis and inflammatory diseases such as HIV-1-associated dementia (HAD). While originally discovered as an inhibitor of matrix metalloproteinases (MMPs), recently, TIMP-1 has emerged as a multifunctional molecule promoting cell survival. MMP/TIMP imbalance is implicated in several neuro-inflammatory diseases. Our previous studies uniquely demonstrate that HAD patients have reduced levels of TIMP-1 in the brain. Astrocyte-TIMP-1 expression is differentially regulated in acute and chronic inflammatory conditions with elevated levels in acute and diminished levels in chronic. These observations indicate that TIMP-1 replenishment in HAD may have therapeutic value. We propose that astrocyte-TIMP-1 regulates neuronal survival in HAD, either through neurotrophic activities and/or via activation of anti-apoptotic proteins. TIMP-1 neuroprotection may or may not involve MMPs. To understand the mechanisms of TIMP-1 regulation and neuroprotection in HAD, the following questions are proposed: 1. How is TIMP-1 regulated in immune-activated astrocytes at the molecular level and what is the temporal relationship between TIMP-1 expression and chronic glial activation? 2. What is the mechanism of TIMP-1-mediated neuronal survival? Is TIMP-1 neuroprotection mediated through interactions with MMP? 3. Can TIMP-1 be utilized as a therapeutic agent to attenuate HIV-1-mediated neurodegeneration? To address these questions, cellular and animal models for HAD will be utilized to mimic acute and chronic inflammatory disease processes. Laboratory in vitro systems consisting of primary human astrocytes, human neurons, neurotoxins, virus and viral proteins will be used. Molecular manipulations such as TIMP-1 promoter-luciferase reporter constructs, adenoviral infections and gene silencing with siRNA molecules will be used to examine TIMP-1 promoter elements and signal transduction pathways involved in astrocvte-TIMP-1 regulation and neuroprotective effects. Neuroprotective mechanisms of TIMP-1 including induction of anti-apoptotic pathways and neurotrophic activities will be evaluated. MMP-dependence of these phenomena will be investigated using TIMP-1 mutants with differential MMP binding abilities. To examine if TIMP-1 can attenuate neurodegeneration, adenoviral-expressed TIMP-1 will be delivered to areas of injury in an HIV encephalitis SCID mouse model using intra-cranial or intra-ventricular injections. These studies will shed light on glial inflammatory responses in HAD, unravel novel mechanisms of glial-neuronal interactions, and may uncover potential future therapeutic strategies for neurodegenerative diseases.