Human Dopamine Grafts in Alpha-Synuclein Models of Parkinson Disease

摘要 帕金森病(PD)是最常见的神经退行性疾病之一。它的特点是 黑质致密部多巴胺神经元进行性丢失及其投射到 纹状体神经元和α-突触核蛋白聚集物常常聚集成路易小体。帕金森病的发病机制 尚未完全阐明，但有大量证据支持神经炎性级联反应的复杂循环， 线粒体功能障碍、神经元变性、持续的小胶质细胞激活和其他病理生理学 这些机制共同放大了黑质及其以外的神经元不断丧失的进程。α- 突触核蛋白(α-S)聚集体要么直接作为传递的模板 跨神经和播撒进一步聚集，和/或放大神经炎性环路，导致 两例均可导致神经元功能障碍和死亡。到目前为止，还没有治疗方案可以导致 对于丢失的神经元的再生或电路的恢复。我们的团队率先推出了 人类胚胎干细胞(HES)中的功能性多巴胺神经元，我们刚刚完成了第一阶段 双侧纹状体内移植这些细胞的临床试验。关于恢复剂有很多令人兴奋的事情 干细胞来源的神经元在帕金森病中的潜力，但仍然存在多重挑战。在这里，我们建议研究 脑内微环境改变对两种不同小鼠模型的影响：3K小鼠 表达基于人E46K突变的三重突变形式的α-突触核蛋白的模型，并展示了 帕金森病的组织学特征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)，这种纤维可跨神经扩散 通过大脑形成致病的α-突触核蛋白包涵体，导致DA神经元和行为学的丧失 恶化。这些模型基于两种不同的假设，将成为研究的有力工具 炎症及其对行为的影响。此外，我们将用同样的HES细胞来源的小鼠移植 临床试验中用多巴胺神经元分析微环境对神经元的影响 生存和表型，以及他们的拯救行为的能力。我们将捕获移植的细胞以及宿主 移植物体内生命周期中关键时间点的小胶质细胞和星形胶质细胞的动态图谱 细胞谱系、成熟、小胶质细胞和星形胶质细胞表型，以及神经毒性信号的潜在激活。 在最后一个目标中，我们将工程人类ES细胞，以删除编码α-突触核蛋白的SNCA基因 在增加移植物对神经毒性的抵抗力和潜在的致病性α的传播方面- 突触核蛋白。在这项建议中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，并可能导致细胞移植的增强策略，包括使用 能抵抗神经炎症的基因编辑细胞。

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.