Alphavirus-manganese interactions and dopaminergic neurodegeneration

描述（由申请方提供）：病毒感染可能是神经退行性疾病（包括帕金森病（PD））的风险因素。嗜神经性甲病毒感染可产生许多与PD中所见相同的长期退行性效应，包括蛋白质聚集、氧化应激水平升高、自噬/线粒体自噬缺陷、神经炎症和神经元死亡。因此，嗜神经性病毒可能代表了一种更好的动物模型，用于研究PD中的基因x环境相互作用，与基于药物的病变模型或遗传模型相比，该模型概括了人类疾病的更多特征，而这些模型通常在黑质中缺乏明显的表型。有证据表明，病毒感染可能与其他公认的风险因素协同作用，如衰老，遗传因素和/或先前暴露于环境神经毒素，以促进神经退行性变。在可能与嗜神经性病毒具有明显基因x环境相互作用的神经毒素中，锰（Mn）是令人感兴趣的，因为生命早期的过度暴露可能对神经功能产生持久影响，并且还可以增强甲病毒的神经毒力。在这里，我们提出了一种新的方法来测试基因x环境相互作用的帕金森病使用嗜神经甲病毒表达系统（AES）。在方便的鼻内接种后，100%的动物被感染，并且感染的进展可以使用表达内切酶的病毒和全身生物发光成像进行原位非侵入性监测。神经侵袭通过嗅觉感觉神经元发生，并且感染以模仿PD的Braak分期系统的模式沿着神经元轴沿着扩散。病毒感染的严重程度和持续性可以得到严格控制，我们使用无偏体视学的初步数据表明，AES感染导致黑质部多巴胺能神经元（SNPC）的显着损失。该提议的中心假设是，表达荧光素酶的甲病毒的鼻内感染将导致通过嗅觉和皮质下结构的神经侵袭，导致黑质中多巴胺能神经元的进行性损失，这与神经胶质细胞的炎性活化相关。此外，我们推测，在青少年发育过程中预先暴露于锰将增强对病毒介导的多巴胺能神经元损失的易感性，部分是通过增加神经炎症反应。在2年项目期间，将在以下两个特定目的中检验该假设：特定目的1 -确定最佳病毒滴度， 表达锰酶的西方马脑炎病毒（WEEV）鼻内接种后诱导基底神经节持续感染和神经变性的表型;具体目标2 -表征病毒感染前和病毒感染后暴露于Mn导致的疾病表型。我们期望这些研究将证明，所提出的基于AES的模型是诱导小鼠帕金森综合征以及将转基因递送到CNS中的有力方法，其可用于鉴定与PD相关的新基因x环境相互作用。此外，我们预计在幼年发育期间暴露于Mn将加剧成年WEEV感染后的神经变性。这些研究的结果将增加我们对环境与神经退行性疾病的联系的理解，并将为研究CNS中病毒/毒素相互作用提供一个强大的新动物模型。

DESCRIPTION (provided by applicant): Viral infection is implicated as a possible risk-factor for neurodegenerative diseases, including Parkinson's disease (PD). Infection with neurotropic alphaviruses can produce many of the same long-term degenerative effects seen in PD, including protein aggregation, increased levels of oxidative stress, autophagy/mitophagy defects, neuroinflammation, and neuronal death. Neurotropic viruses may therefore represent a better animal model for studying gene x environment interactions in PD that recapitulate more features of the human disease than drug-based lesioning models or genetic models that often lack a pronounced phenotype in the substantial nigra. Evidence suggests that viral infection may act in synergy with other recognized risk-factors, such as aging, genetic factors, and/or previous exposures to environmental neurotoxins to promote neurodegeneration. Among neurotoxins that could have a pronounced gene x environment interaction with neurotropic viruses, Manganese (Mn) is of interest because excessive exposure early in life can have lasting effects on neurological function and can also enhance the neurovirulence of alphaviruses. Here, we propose a novel method for testing gene x environment interactions in Parkinsonism using a neurotropic alphavirus expression system (AES). Following convenient intranasal inoculation, 100% of animals become infected and the progression of infection can be non-invasively monitored in situ using luciferase-expressing viruses and whole body bioluminescence imaging. Neuroinvasion occurs through olfactory sensory neurons and the infection spreads along the neuronal axis in a pattern that mimics the Braak-staging system of PD. The severity and persistence of viral infection can be tightly controlled and our preliminary data using unbiased stereology indicate that AES infection results in significant loss of dopaminergic neurons in the substantial nigra pars compacta (SNpc). It is the Central Hypothesis of this proposal that intranasal infection with luciferase- expressing alphavirus will result in neuroinvasion through olfactory and subcortical structures resulting in progressive loss of dopaminergic neurons in the substantia nigra that is associated with inflammatory activation of glial cells. Moreover, we postulate that pre-exposure to Mn during juvenile development will enhance susceptibility to viral-mediated loss of dopaminergic neurons, in part through an increased neuroinflammatory response. This hypothesis will be tested in two Specific Aims during the 2-year project period as follows: Specific Aim 1 - Determine the optimal viral titer and phenotype for inducing persistent infection and neurodegeneration in the basal ganglia following intranasal installation of luciferase-expressing Western Equine Encephalitis Virus (WEEV); Specific Aim 2 - Characterize the disease phenotype resulting from exposure to Mn pre- and post-viral infection. We expect that these studies will demonstrate that the proposed AES-based model is a powerful method for inducing parkinsonism in mice, as well as for transgene delivery into the CNS, that can be used to identify novel gene x environment interactions relevant to PD. Additionally, we expect that exposure to Mn during juvenile development will exacerbate neurodegeneration following adult infection with WEEV. The results of these studies will increase our understanding of the environmental links to neurodegenerative disease and will provide a powerful new animal model for studying virus/toxin interactions in the CNS.