The mechanisms of mitochondrial damage-dependentneuroinflammation in experimental models ofParkinson’s disease. The role of Parkin dysfunction.

帕金森病（PD）涉及多巴胺能神经元的进行性死亡，并且是第二常见的神经退行性疾病。α-突触核蛋白（α-syn）的错误折叠和路易体沉积是PD病因学的标志，但其神经毒性机制尚不清楚。另一种已建立的PD相关蛋白E3泛素连接酶parkin对于线粒体质量控制至关重要，其活性的丧失导致错误折叠蛋白和受损线粒体的积累，这可以释放损伤相关分子模式（DAMP，包括mtDNA），激活先天免疫受体以促进（神经）炎症，这是PD病理学的重要驱动因素。然而，parkin在α-syn-induced，与炎症相关的炎症（mitoflammation）和多巴胺能细胞死亡中的作用尚未深入阐明。我们的主要假设是parkin功能障碍有助于α-syn-induced线粒体损伤，导致PD中的神经炎症和神经变性。为了验证这一假设，我们将研究帕金参与α-syn-evoked线粒体损伤，炎症反应，神经变性在小鼠和细胞模型的突触核蛋白病。为了诱导PD样病理学，将向12周龄C57 BL/6 J小鼠、parkin敲除小鼠（Park 2-/-）或parkin过表达（Park 2 oe）小鼠的背侧纹状体中立体定向注射人α-syn寡聚体（5μg蛋白质/5μl/侧）或溶媒。体外和离体模型将用于研究线粒体损伤、信号传导通路以及神经元、小胶质细胞和星形胶质细胞之间的相互作用的特定方面：来自PD患者的诱导多能干细胞（iPSC）分化成小胶质细胞和星形胶质细胞，条件永生化的人胎儿中脑细胞（LUHMES）分化为表现出多巴胺神经元特征以及小胶质细胞特征。耗尽的器官型小鼠中脑切片。我们假设parkin通过清除神经元中受损的线粒体来防止α-syn-induced炎症和神经变性，从而防止DAMP的释放，并减弱PD相关的神经胶质炎症反应。我们将分析α-syn神经毒性和潜在神经保护性药物治疗的对照和parkin过度表达小鼠的神经元存活率、神经炎症、多巴胺及其代谢产物、脑组织超微结构和动物行为。本项目将描述α-syn-induced parkin功能障碍调节PD临床前模型中线粒体损伤和相关免疫反应的机制。迄今为止，尚未研究α-syn传播、帕金功能障碍、有丝分裂炎症和多巴胺能细胞死亡之间的直接关联。解决parkin在α-syn传播和神经炎症的交叉路口的作用，有望更好地理解PD的病理机制和识别新的治疗靶点。

Parkinson's disease (PD) involves progressive death of dopaminergic neurons and is the second most common neurodegenerative disorder. Misfolding and deposition of α-synuclein (α-syn) as Lewy bodies is a hallmark of PD etiology, but the mechanisms of its neurotoxicity remain unclear. Another established PD-associated protein, the E3 ubiquitin ligase parkin is essential for mitochondrial quality control, and loss of its activity leads to accumulation of misfolded proteins and damaged mitochondria which can release damage-associated molecular patterns (DAMPs, including mtDNA) activating innate immune receptors to promote (neuro)inflammation, an important driver of PD pathology. However, the role of parkin in α-syn–induced, mitochondria-linked inflammation (mitoflammation) and dopaminergic cell death has not been elucidated in depth. Our main hypothesis is that parkin dysfunction contributes to α-syn–induced mitochondrial impairment that leads to neuroinflammation and neurodegeneration in PD. To test this hypothesis, we will investigate parkin’s involvement in α-syn–evoked mitochondrial damage, inflammatory response, and neurodegeneration in mouse and cellular models of synucleinopathy. To induce PD-like pathology, 12-week-old C57BL/6J mice, parkin knock-out mice (Park2-/-) or parkin overexpressing (Park2oe) mice will be stereotactically injected with human α-syn oligomers (5μg of protein/5μl/side) or vehicle, bilaterally into the dorsal striatum. In vitro and ex vivo models will be used for the investigation of specific aspects of mitochondrial damage, signaling pathways, and interactions between neurons, microglia and astrocytes: induced pluripotent stem cells (iPSC) from PD patients differentiated into microglia and astrocytes, conditionally immortalized human fetal mesencephalic cells (LUHMES) differentiated to exhibit dopamine neuron characteristics as well as microglia-depleted organotypic mouse midbrain slices. We assume that parkin prevents α-syn–induced inflammation and neurodegeneration by clearing damaged mitochondria in neurons, thereby preventing the release of DAMPs, and attenuating PD-linked glial inflammatory responses. We will analyze neuron survival, neuroinflammation, dopamine and its metabolites, brain tissue ultrastructure and animal behavior in control and parkin-overexpressing mice subjected to α-syn neurotoxicity and to potentially neuroprotective pharmacological treatments. This project will characterize the mechanisms by which α-syn–induced parkin dysfunction modulates mitochondrial damage and associated immune response in preclinical models of PD. The direct association between α-syn propagation, parkin dysfunction, mitoflammation, and dopaminergic cell death has not been investigated so far. Addressing the role of parkin at the crossroads of α-syn spreading and neuroinflammation holds the promise for better understanding of PD pathomechanism and identification of novel therapeutic targets.