Mechanisms of cytokine-mediated viral clearance from neurons

描述(申请人提供)：麻疹病毒(MV)在中枢神经系统(CMS)的感染与神经病理疾病有关，例如亚急性硬化性泛脑炎(SSPE)，在这种疾病中，免疫系统无法控制MV在大脑中的复制。本实验室通过靶向表达麻疹病毒受体之一(CD46)的神经元，建立了中枢神经系统限制性MV感染的转基因小鼠模型，以研究MV感染神经元与免疫应答之间的相互作用。在这个模型中，成年小鼠通过建立需要干扰素-γ(干扰素？)的免疫反应，成功地清除了脑神经元中的MV。而且涉及T细胞向中枢神经系统的招募，而MV感染的新生儿向大脑招募类似水平的T细胞，但无法控制MV在神经元中的复制，发展广泛的神经病理，并迅速屈服于MV感染。而干扰素呢？在活体成人脑神经元清除MV是必需的吗，干扰素？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了明显的信号级联反应。从这些初步观察中出现了一个悖论：干扰素是如何发挥作用的？当规范的信号成分(通过转录激活物STAT1的磷酸化/激活来衡量)受到限制时，中介病毒从神经元清除吗？我们假设干扰素限制了STAT1的激活？神经元中的信号通路有助于清除病毒，但其他信号通路也有助于神经元中的抗病毒活性。为了解决这一假说，Aim 1剖析了体外移植的原代海马神经元中干扰素-1介导MV清除的机制。通过免疫印迹、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在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而得到解决。目的2通过建立干扰素的作用，扩展在原代培养神经元中的观察结果。体内复杂脑微环境中的信号转导。这些研究将得到转基因小鼠的帮助(例如，干扰素？基因敲除、STAT1基因敲除)，并将使我们能够解决神经发病机制的年龄相关性问题。通过确定干扰素的作用？信号在神经元中清除MV，这些研究将阐明干扰素是如何？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，但也旨在为对干扰素？的细胞特异性异质性反应提供一种机制。此外，这些研究还将探讨神经元的内在发育特性如何在决定中枢神经系统神经元中是否发生病毒清除方面发挥作用。

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.