Alphavirus-manganese interactions and dopaminergic neurodegeneration

描述(申请人提供)：病毒感染可能是神经退行性疾病的危险因素，包括帕金森氏病(PD)。感染嗜神经性甲型病毒可产生许多与帕金森病相同的长期退行性影响，包括蛋白质聚集、氧化应激水平增加、自噬/有丝分裂缺陷、神经炎症和神经元死亡。因此，嗜神经病毒可能是研究帕金森病基因x环境相互作用的更好的动物模型，与基于药物的损害模型或通常在实质黑质中缺乏明显表型的遗传模型相比，这些模型概括了人类疾病的更多特征。有证据表明，病毒感染可能与其他公认的危险因素协同作用，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化。在可能与嗜神经病毒有明显基因x环境相互作用的神经毒素中，锰(Mn)是令人感兴趣的，因为在生命早期过度暴露会对神经功能产生持久的影响，也可以增强甲型病毒的神经毒力。在这里，我们提出了一种新的方法，利用嗜神经的甲型病毒表达系统(AES)来测试帕金森病的基因x环境交互作用。在方便的鼻腔接种后，100%的动物被感染，利用表达荧光素酶的病毒和全身生物发光成像可以非侵入性地原位监测感染的进展。神经侵袭通过嗅觉感觉神经元发生，感染沿着神经轴传播，其模式类似于帕金森病的Braak分期系统。病毒感染的严重性和持久性是可以严格控制的，我们使用无偏体视学的初步数据表明，AES感染导致实质性黑质致密部(SNPC)多巴胺能神经元的显著损失。这一假设的中心假设是，鼻腔感染表达荧光素酶的甲型病毒将导致通过嗅觉和皮质下结构的神经侵袭，导致黑质多巴胺能神经元的进行性丧失，这与胶质细胞的炎症激活有关。此外，我们推测，在幼年发育期间预先暴露于锰将增加病毒介导的多巴胺能神经元丢失的易感性，部分原因是通过增加神经炎症反应。在为期两年的项目期间，这一假设将在两个具体目标中进行检验，具体目标如下：具体目标1-确定最佳病毒滴度和 在鼻腔内安装表达荧光素酶的西方马脑炎病毒(Weev)后，导致基底节持续感染和神经变性的表型；特定目的2-表征病毒感染前和病毒感染后暴露于锰引起的疾病表型。我们期望这些研究将证明所提出的基于AES的模型是一种有效的方法来诱导小鼠的帕金森病，以及转基因进入中枢神经系统，可用于识别与帕金森病相关的新的基因x环境相互作用。此外，我们预计，在青少年发育期间暴露于锰将加剧成年感染Weev后的神经退化。这些研究的结果将加深我们对环境与神经退行性疾病的联系的理解，并将为研究中枢神经系统中病毒/毒素的相互作用提供一个强有力的新的动物模型。

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.