Toxicant-induced synaptic dysfunction and neurotoxicity in Parkinson disease

描述（由申请人提供）：我们的长期目标是研究环境毒物、基因突变和潜在的基因-环境相互作用诱导的神经退行性变机制，以深入了解帕金森病（PD）的发病机制。PD遗传学的进展突出了线粒体动力学（裂变/融合/运动）在神经元功能和存活中的关键作用。然而，由于单基因家族性帕金森病只占帕金森病病例的一小部分，因此确定线粒体动力学紊乱是否也在环境神经毒物引起的黑质纹状体损伤中起作用是至关重要的。这些外源性有毒分子大多直接通过阻断线粒体呼吸或间接通过氧化应激引起线粒体功能障碍。基于我们的初步数据，本提案将利用两种互补的基于毒物的黑质纹状体神经变性动物模型：A)除草剂百草枯（PQ）主要通过氧化应激诱导细胞死亡。B)农药/杀虫剂鱼藤酮直接抑制线粒体功能。我们假设，无论是线粒体过度分裂和功能障碍是由阻断线粒体呼吸（鱼藤酮）直接引起的，还是由氧化应激（PQ）间接引起的，促进线粒体融合将减轻在这些动物模型中看到的突触前功能障碍和神经毒性。在Aim 1中，我们将研究促进线粒体融合对PQ小鼠和鱼藤酮大鼠模型的影响。由于PQ在正常小鼠中不会诱导纹状体损伤，我们将使用缺失有机阳离子转运体3 （Oct3-/-）的新型突变小鼠来创建黑质和纹状体均受损的PQ动物模型，并增强与人类基因-环境相互作用的相关性，因为Oct3变异与PD有关。小分子和基于基因的方法将用于操纵线粒体裂变/融合机制。这些策略的神经恢复和神经保护作用将在已有病变和活动性神经退行性疾病的动物中确定。纹状体线粒体功能、自由运动动物纹状体多巴胺释放、电生理学突触功能、运动功能和黑质纹状体通路的完整性将被分析。在目标2中，我们将研究PQ和鱼藤酮诱导线粒体裂变的机制，以及为什么阻断这一过程具有保护作用。在相关情况下，将同时使用动物模型和细胞培养。广泛的最先进的设备/技术，如三维电子显微镜，激光捕获显微解剖，海马细胞外通量分析仪和电流计将用于量化蛋白质/基因水平的变化，特别是在黑质多巴胺能神经元，线粒体运输/形态/功能和突触密度/功能。这些目标的实现将提供关于毒性损伤如何通过扰乱线粒体动力学影响黑质纹状体通路的关键信息，并为PD的潜在新治疗靶点提供见解。

DESCRIPTION (provided by applicant): Our long term goal is to study the mechanisms of neurodegeneration induced by environmental toxicants, genetic mutations and potential gene-environment interactions to gain insights into the pathogenesis of Parkinson's disease (PD). Advances in the genetics of PD have highlighted the critical role of mitochondrial dynamics (fission / fusion / movement) in neuronal function and survival. However, because monogenic familial PD represents only a small fraction of PD cases, it is critical to determine whether perturbed mitochondrial dynamics also plays a role in the nigrostriatal damage induced by environmental neurotoxicants. Most of these exogenous toxic molecules cause mitochondrial dysfunction either directly by blocking mitochondrial respiration, or indirectly through oxidative stress. Based on our preliminary data, this proposal will utilize two complementary toxicant-based animal models of nigrostriatal neurodegeneration: A) The herbicide paraquat (PQ) induces cell death primarily through oxidative stress. B) The pesticide/insecticide rotenone directly inhibits mitochondrial function. We hypothesize that whether excessive mitochondrial fission and dysfunction is induced directly by blocking mitochondrial respiration (rotenone) or indirectly by oxidative stress (PQ), promoting mitochondrial fusion will attenuate pre-synaptic dysfunction and neurotoxicity seen in these animal models. In Aim 1, we will investigate the impact of promoting mitochondrial fusion in the PQ mouse and rotenone rat models. Because PQ does not induce striatal damage in regular mice, we will use our novel mutant mice with deletion of the organic cation transporter 3 (Oct3-/-) to create a PQ animal model with damage in both nigra and striatum, as well as to enhance relevance to human gene-environment interactions because OCT3 variants have been associated with PD. Small molecule and gene-based approaches will be used for manipulation of mitochondrial fission/fusion machinery. Both neurorestorative and neuroprotective effects of these strategies will be determined in animals with pre-existing lesions and with active neurodegeneration. Striatal mitochondrial function, evoked striatal dopamine release in freely moving animals, synaptic function using electrophysiology, motor function and the integrity of the nigrostriatal pathway will be analyzed. In Aim 2, we will investigate the mechanisms by which PQ and rotenone induce mitochondrial fission and why blocking this process is protective. Where relevant, both animal models and cell cultures will be used. A wide range of state-of-the art equipment/techniques such as 3-dimensional electron microscopy, laser capture microdissection, the Seahorse extracellular flux analyzer and amperometry will be used to quantify alterations in levels of proteins/genes of interest specifically in nigral dopaminergic neurons, mitochondrial trafficking/morphology/function and synaptic density/function. Accomplishment of these aims will provide critical information regarding how toxic insults impact nigrostriatal pathway through perturbed mitochondrial dynamics and offer insights into a potential novel therapeutic target for PD.