Human Dopamine Grafts in Alpha-Synuclein Models of Parkinson Disease

摘要 帕金森病（Parkinson's disease，PD）是最常见的神经退行性疾病之一。其特点是 黑质（SN）旁核中多巴胺神经元的进行性丢失，以及它们在 纹状体神经元和α-突触核蛋白聚集体的积累往往进入路易体。pd发病机制 尚未完全阐明，但有大量证据支持神经炎症级联反应的复杂循环， 线粒体功能障碍、神经元变性、持续的小胶质细胞活化和其他病理生理学变化。 这些机制共同放大了黑质及其他部位神经元丧失的无情进程。α- 突触核蛋白（α-S）聚集体直接参与作为模板， 经神经元和播种进一步聚集，和/或放大神经炎性循环，导致 这两种情况都导致神经元功能障碍和死亡。到目前为止，还没有治疗选择，导致 再生失去的神经元或恢复电路。我们的团队开创了 从人类胚胎干细胞（hES）中提取功能性多巴胺神经元，我们刚刚完成了第一阶段的研究。 这些细胞的双侧纹状体内移植的临床试验。有很多关于恢复的兴奋 干细胞衍生的神经元在PD中的潜力，但仍然存在多重挑战。在这里，我们建议研究 在2种不同小鼠模型的背景下，大脑中微环境改变的影响：3K小鼠 该模型表达基于人E46K突变的α-突触核蛋白的三重突变形式，并表现出 PD的组织学标志以及进行性运动和其他行为异常;第二种模型 包括纹状体内注射预先形成的α-突触核蛋白纤维（PFF）， 通过大脑形成致病的α-突触核蛋白包涵体，导致DA神经元和行为的丧失， 恶化这些模型基于两种不同的假设，将作为研究的重要工具。 炎症及其对行为的影响。此外，我们将用相同的hES细胞移植小鼠， 临床试验中使用的多巴胺神经元，以分析微环境对神经元功能的影响。 存活和表型，以及它们拯救行为的能力。我们将捕获移植的细胞以及宿主 在移植物的体内寿命期间的关键时间点， 细胞谱系、成熟、小胶质细胞和星形胶质细胞表型以及潜在的神经毒性信号激活。 最后一个目的是通过基因工程的方法，将编码α-synuclein的SNCA基因缺失， 在增加移植物对神经毒性的抵抗力和潜在的致病性α- 共核蛋白本研究所获得的数据将有助于进一步加深我们对神经炎症的理解 在不同的PD微环境中，并可能导致增强的细胞移植策略，包括使用 基因编辑的细胞可以抵抗神经炎症。

ABSTRACT Parkinson's disease (PD) is one of the most common neurodegenerative disorders. It is characterized by the progressive loss of dopamine neurons in the substantia nigra (SN) pars compacta, and their projections onto striatal neurons and the accumulation of α-Synuclein aggregates often into Lewy bodies. The pathogenesis of PD is not fully elucidated but there is vast evidence supporting complex loops of neuroinflammatory cascades, mitochondrial dysfunction, degenerating neurons, sustained microglial activation and other pathophysiological mechanisms that together amplify a relentless progression towards neuronal loss in the nigra and beyond. α- Synuclein (α-S) aggregates are implicated either directly in serving as a template that is transmitted transneuronally and seeding further aggregation, and/or in amplifying the neuroinflammatory loop, leading in both cases to neuronal dysfunction and death. To date, there are no therapeutic options that lead to the regeneration of lost neurons or to the restoration of circuitry. Our group has pioneered the derivation of functional dopamine neurons from human embryonic stem cells (hES) and we have just completed a Phase 1 clinical trial for the bilateral intrastriatal grafting of these cells. There is much excitement about the restorative potential of stem cell derived neurons in PD, but there remain multiple challenges. Here we propose to study the impact of microenvironmental alterations in the brain in the context of 2 different mouse models: the 3K mouse model which expresses a triple mutant form of α-synuclein based on the human E46K mutation, and exhibits histological hallmarks of PD as well as progressive motor and other behavioral abnormalities; the second model consists of the intrastriatal injection of preformed α-synuclein fibrils (PFF) which spread transneuronally through the brain to form pathogenic α-synuclein inclusions, leading to loss of DA neurons and behavioral deterioration. These models are predicated on two different hypotheses and will serve as great tools to study inflammation and its impact on behavior. In addition, we will graft the mice with the same hES cell derived dopamine neurons used in the clinical trial to analyze the impact of the microenvironment on the neurons' survival and phenotype, as well as on their ability to rescue behavior. We will capture grafted cells as well as host microglia and astrocytes at key timepoints during the in vivo lifespan of the grafts to establish dynamic maps of cell lineages, maturation, microglial and astrocytic phenotypes and potentially activation of neurotoxic signals. In the last aim, we will engineer the human ES cells to delete the SNCA gene encoding α-synuclein in an attempt at increasing the resistance of the grafts to neurotoxicity and potentially the transmission of pathogenic α- synuclein. Data obtained in this proposal will serve to further enhance our understanding of neuro-inflammation in different PD microenvironments and could result in enhanced strategies for cell grafting including the use of gene edited cells that are resistant to neuroinflammation.