Mechanisms of cytokine-mediated viral clearance from neurons

描述（由申请人提供）：麻疹病毒（MV）感染中枢神经系统（CMS）与神经病理疾病有关，如亚急性硬化性全脑炎（SSPE），其中免疫系统无法控制MV在大脑中的复制。本实验室利用一种麻疹病毒受体（CD46）的靶向神经元表达的cns限制性MV感染转基因小鼠模型来研究MV感染神经元与免疫反应之间的相互作用。在该模型中，成年小鼠通过免疫应答（需要干扰素γ (IFN)）和向中枢神经系统募集t细胞)成功地清除了脑神经细胞中的MV，而感染MV的新生儿向大脑募集了类似水平的t细胞，但无法控制神经元中的MV复制，发展为广泛的神经病理学，并迅速死于MV感染。虽然干扰素吗?在体内对成人大脑神经元的MV清除是必需的，IFN？与更广泛研究的成纤维细胞和细胞系相比，在原代培养的神经元中触发独特的信号级联。从这些初步观察中出现了一个悖论：IFN如何？当典型信号成分（通过转录激活因子STAT1的磷酸化/激活来测量）受到限制时，如何介导神经元的病毒清除？我们假设IFN对STAT1激活的限制在神经元中有助于病毒清除，但其他信号通路有助于神经元中的抗病毒活性。为了解决这一假设，第1章剖析了IFN？介导的体外海马原代神经元MV清除。通过western blot、共聚焦成像和定量RT-PCR，原代神经元介导病毒清除的内在能力将独立于混合中枢神经系统培养中发现的神经胶质细胞的复杂贡献。Aim 2将通过确定IFN的作用来扩展原代培养神经元的观察结果。脑内复杂微环境中的信号转导。这些研究将借助转基因小鼠(如IFN？基因敲除，STAT1基因敲除)，将使我们能够解决神经发病机制的年龄依赖性。通过确定IFN的作用？信号在神经元中mv清除中的作用，这些研究将阐明IFN？在一种独特的、不可再生的细胞类型中触发抗病毒防御机制，但也旨在为细胞特异性的IFN异质性反应提供机制。此外，这些研究还将探讨神经元内在发育特性如何在决定病毒清除是否发生在中枢神经系统神经元中发挥作用。

DESCRIPTION (provided by applicant): Measles virus (MV) infection in the central nervous system (CMS) is associated with neuropathological disorders, such as subacute sclerosing panencephalitis (SSPE), in which the immune system fails to control MV replication in the brain. Our laboratory uses a transgenic mouse model of CNS-restricted MV infection via targeted neuronal expression of one of the measles virus receptors (CD46) to study interactions between MV-infected neurons and the immune response. In this model, adult mice successfully clear MV from neurons in the brain by mounting an immune response that requires interferon-gamma (IFN?) and involves T-cell recruitment to the CNS, whereas MV-infected neonates recruit similar levels of T-cells to the brain, but are unable to control MV replication in neurons, develop extensive neuropathology, and rapidly succumb to MV infection. While IFN? is necessary for MV clearance from neurons in adult brains in vivo, IFN? triggers a distinct signaling cascade in primary cultured neurons in comparison to the more extensively studied fibroblasts and cell lines. A paradox emerges from these preliminary observations: how does IFN? mediate viral clearance from neurons, when the canonical signaling components (as measured by phosphorylation/activation of the transcriptional activator STAT1) are restricted? We hypothesize that limited STAT1 activation by IFN? in neurons contributes to viral clearance, but that other signaling pathways contribute to anti-viral activity in neurons. To address this hypothesis, Aim 1 dissects the mechanisms IFN?-mediated MV clearance in explanted primary hippocampal neurons in vitro. Through western blot, confocal imaging, and quantitative RT-PCR, the intrinsic ability of primary neurons to mediate viral clearance will be addressed independently of the complicating contribution of glial cells found in mixed CNS cultures. Aim 2 will extend the observations made in primary cultured neurons by establishing the role of IFN? signal transduction in the complicated microenvironment of the brain in vivo. These studies will be aided by genetically modified mice (e.g. IFN? knockouts, STAT1 knockouts) and will allow us to address the age-dependence of neuropathogenesis. By determining the role of IFN? signaling in MV clearance in neurons, these studies will clarify how IFN? triggers anti-viral defense mechanisms in a unique, non-renewable cell type, but also aim to provide a mechanism for the cell-specific heterogeneity of responses to IFN?. In addition, these studies will also address how intrinsic developmental neuronal properties play a role in dictating whether or not viral clearance occurs in CNS neurons.