Neuropathogenesis of Retroviral Infections

逆转录病毒感染的神经发病机制

• 批准号: 8746854
• 负责人: Avindra Nath
• 金额: $ 180.34万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746854
• 关键词: Activated Lymphocyte; Anti-Retroviral Agents; Area; Astrocytes; Attention; Brain; CXCR4 gene; Cells; Cessation of life; Chromosomes; Clinical; Complex; Dendrites; Encephalitis; Endogenous Retroviruses; Event; Goals; Granzyme; HIV; Host Defense Mechanism; Human; Immune; Impaired cognition; In Vitro; Infection; Inflammation; Injury; Investigation; JC Virus; Laboratories; Lead; Life; Lymphocyte; Lymphocyte Activation; Lymphoid; Manuscripts; Mediating; Microglia; NF-kappa B; Neurologic; Neuronal Dysfunction; Neuronal Injury; Neurons; Neuropathogenesis; Organ; Paper; Pathway interactions; Patients; Pharmaceutical Preparations; Production; Proteins; Publishing; Receptor Cell; Retroviridae; Role; Site; Synapses; Syndrome; T-Cell Receptor; T-Lymphocyte; Tight Junctions; Transgenic Organisms; Viral; Viral Proteins; Virus; Virus Diseases; Virus Latency; cell type; env Gene Products; expression vector; gene cloning; granzyme B; immune activation; macrophage; mouse model; nerve stem cell; neurotoxic; neurotoxicity; particle; prevent; receptor; reconstitution; research study; tat Protein; trafficking

The over arching hypothesis is that the brain is an important reservoir for retroviruses because of their ability to infect long lived terminally differentiated cells and viral products released from these cells can cause immune activation and neuronal injury Aim 1: To understand the mechanism of viral persistence in brain If there is any hope to eradicate HIV, close attention to the viral reservoirs in the brain is necessary. They brain is a unique site of viral latency since it infects resident macrophages/microglia and astrocytes. These cells have very low turnover rate, and the mechanism of viral entry and persistence is very different than that of lymphocytes which are the major cell type infected by the virus in the lymphoid organs. Our laboratory has focused on studying the mechanism of viral entry in astrocytes. We have found that while free viral particles can enter these cells, cell to cell contact with lymphocytes is the most efficient way to infect astrocytes. The mechanisms by which T cells facilitate viral entry is being explored. We have discovered that the virus enters by using CXCR4 and is aided by formation of tight junctions between the cells. We have also found that upon viral entry, the virus can enter the endolysosomal pathway which acts as a host defense mechanism. Hence strategies than modulate these pathways could have a significant effect on the establishment of a reservoir in the brain. Two manuscripts are currently being prepared that detail these findings.Hoever the turnover rate of the cells in the brain (microglia and astrocytes) is also critical to the eradication of the reservoir, hence we are also undertaking a study in a mouse model of inflammation to determine if the turnover rate of these cells may be altered during the state of inflammation. These studies have recently been initiated. Aim 2: To investigate the mechanism of neuronal injury by HIV and endogenous retroviruses Despite the use of antiretroviral agents and excellent control of the virus in the periphery, HIV infected patients continue to develop cognitive impairment. Currently available antiretroviral agents have no effect on the production of early viral proteins once the virus has integrated into the chromosome. One of these proteins, Tat, has been shown to be neurotoxic. Our laboratory was one of the first to demonstrate its toxic potential and we are now investigating the mechanisms by which it causes neurotoxicity. We have found that the protein can cause synaptic injury at very low concentrations without causing neuronal death. We have characterized the proteins and the morphological changes at the level of the dendrites in human neurons and are further investigating the underlying mechanisms. Using a similar approach we are investigating the mechanisms by which the envelop protein of an endogenous retrovirus-K causes neurotoxicity. We have cloned the gene into an expression vector, created a transgenic line that expresses the protein and are characterizing the pathological findings. Several in invitro experiments are also underway to determine the mechanism by which neuronal injury takes place. Aim 3: To investigate the mechanism of HIV-induced lymphocyte activation and subsequent neuronal injury Patients treated with antiretroviral drugs may occasionally develop a T cell mediated encephalitis that can be fatal. Our group has characterized this syndrome as the CNS-immune reconstitutions syndrome and we were one of the first to characterize the clinical and pathological features of this entitity. We have also found these similar clinical syndromes can occur in patients with JC virus infection and we have published several papers characterizing the clinical entity. We have reasoned that because of the inability to control the viral replication fully in the CNS reservoirs, the lymphocytes traffic to the brain along an antigenic gradient. We have shown that the Tat protein can activate lymphocytes in vitro and these activated T cells can cause significant neurotoxicity by the release of granzymes. We have found that the Tat protein enters T cells and then activates these cells in a NF-kB dependent manner. These findings have been recently published (Johnson et al., PNAS 2013) We have also shown that the activated T cells can be detrimental to neural progenitor cells and prevent their differentiation. Similarly they are toxic to neurons which is mediated by effects of granzyme on PAR-1 on the neurons however we found that activated T cells can cause proliferation of olgioprecursor cells. These paradoxical effects are likely mediated by different receptors on these cells and suggest that the role of the T cells is very complex. This remains an active area of investigation in our lab. In summary, we have shown that astrocytes in the brain are an important reservoir for HIV and that cell to cell contact with lymphocytes is necessary for viral entry and the lysosomal pathway in these cells regulates the intracellular trafficking of the virus and its ultimate ability to successfully infect these cells. Further, we have shown that the HIV protein Tat and the env protein of endogenous retrovirus-K are neurotoxic and we are now the underlying mechanisms involved in these effects. Finally, we have also discovered that the Tat protein of HIV can stimulate T cells in a T cell receptor independent manner using a unique mechanism. These activated T cells cause neuronal injury via the release of granzyme B that acts on cell receptors causing a cascade of events leading to neuronal dysfunction.

最重要的假设是，大脑是逆转录病毒的重要储存库，因为它们能够感染长寿的终末分化细胞，而这些细胞释放的病毒产物会导致免疫激活和神经元损伤 目标1：了解病毒在大脑中持续存在的机制 如果有希望根除艾滋病毒，就必须密切关注大脑中的病毒库。他们的大脑是病毒潜伏的独特部位，因为它感染常驻巨噬细胞/小胶质细胞和星形胶质细胞。这些细胞的周转率非常低，病毒进入和持续存在的机制与淋巴器官中病毒感染的主要细胞类型淋巴细胞非常不同。我们实验室致力于研究病毒进入星形胶质细胞的机制。我们发现，虽然游离病毒颗粒可以进入这些细胞，但与淋巴细胞的细胞间接触是感染星形胶质细胞的最有效方式。 T 细胞促进病毒进入的机制正在探索中。我们发现病毒通过 CXCR4 进入，并通过细胞之间形成紧密连接来帮助。我们还发现，病毒进入后，病毒可以进入作为宿主防御机制的内溶酶体途径。因此，调节这些途径的策略可能对大脑中储存库的建立产生重大影响。目前正在准备两份手稿，详细说明这些发现。然而，大脑中的细胞（小胶质细胞和星形胶质细胞）的更新率对于根除储存库也至关重要，因此我们还在小鼠炎症模型中进行了一项研究确定这些细胞的周转率在炎症状态下是否会改变。这些研究最近已启动。 目标2：研究HIV和内源性逆转录病毒对神经元损伤的机制 尽管使用了抗逆转录病毒药物并且外周病毒得到了很好的控制，但艾滋病毒感染者仍继续出现认知障碍。一旦病毒整合到染色体中，目前可用的抗逆转录病毒药物对早期病毒蛋白的产生没有影响。其中一种蛋白质 Tat 已被证明具有神经毒性。我们的实验室是最早证明其潜在毒性的实验室之一，我们现在正在研究它引起神经毒性的机制。我们发现该蛋白质可以在非常低的浓度下引起突触损伤，而不会导致神经元死亡。我们已经表征了人类神经元树突水平的蛋白质和形态变化，并正在进一步研究其潜在机制。使用类似的方法，我们正在研究内源性逆转录病毒-K 的包膜蛋白引起神经毒性的机制。我们已将该基因克隆到表达载体中，创建了表达该蛋白质的转基因系，并正在表征病理结果。一些体外实验也在进行中，以确定神经元损伤发生的机制。 目标3：研究HIV诱导的淋巴细胞活化和随后的神经元损伤的机制 使用抗逆转录病毒药物治疗的患者偶尔可能会出现致命的 T 细胞介导的脑炎。我们的小组将这种综合征称为中枢神经系统免疫重建综合征，并且我们是最早描述该实体的临床和病理特征的人之一。我们还发现 JC 病毒感染患者也可能出现这些类似的临床综合征，并且我们发表了几篇描述该临床症状的论文。我们推断，由于无法完全控制中枢神经系统储存库中的病毒复制，淋巴细胞沿着抗原梯度运输到大脑。我们已经证明，Tat 蛋白可以在体外激活淋巴细胞，这些激活的 T 细胞可以通过释放颗粒酶引起显着的神经毒性。我们发现 Tat 蛋白进入 T 细胞，然后以 NF-kB 依赖性方式激活这些细胞。这些发现最近已发表（Johnson 等人，PNAS 2013）我们还表明，激活的 T 细胞可能对神经祖细胞有害并阻止其分化。类似地，它们对神经元具有毒性，这是通过颗粒酶对神经元上的 PAR-1 的作用介导的，但是我们发现活化的 T 细胞可以引起 olgioprecursor 细胞的增殖。这些矛盾的效应可能是由这些细胞上的不同受体介导的，这表明 T 细胞的作用非常复杂。这仍然是我们实验室研究的一个活跃领域。 总之，我们已经证明，大脑中的星形胶质细胞是艾滋病毒的重要储存库，细胞与淋巴细胞的接触对于病毒进入是必要的，这些细胞中的溶酶体途径调节病毒的细胞内运输及其成功成功传播的最终能力。感染这些细胞。此外，我们已经证明，HIV 蛋白 Tat 和内源性逆转录病毒 K 的 env 蛋白具有神经毒性，我们现在正在研究这些效应所涉及的潜在机制。最后，我们还发现HIV的Tat蛋白可以利用独特的机制以不依赖于T细胞受体的方式刺激T细胞。这些激活的 T 细胞通过释放颗粒酶 B 来引起神经元损伤，颗粒酶 B 作用于细胞受体，导致一系列事件导致神经元功能障碍。