Calcium activity-dependent generation and maintenance of mesodiencephalic dopaminergic neurons from murine and human pluripotent stem cells in the context of Parkinson’s Disease (DACaION)

在人类和小鼠胚胎发育过程中，中脑中多巴胺释放（多巴胺能，DA）神经细胞的细胞和分子异质性的出现在很大程度上还不清楚。这些神经细胞参与不同的认知，情感/情绪和运动脑功能。它们的丧失或功能障碍导致严重的神经精神疾病，如帕金森病（PD）、精神分裂症和成瘾性疾病。我们以前的工作已经表明，一个精确的调制经典WNT/β-连环蛋白信号通路是必要的和足够的前体人口在小鼠中脑的正确分化为黑质部腹侧部（SNc）DA神经元。此外，WNT/β-连环蛋白信号通路促进这些神经元在遗传性PD小鼠模型中的存活。因此，WNT/β-连环蛋白信号传导的靶基因的鉴定在这种情况下特别令人感兴趣。激光显微切割的小鼠胚胎中脑腹侧WNT反应细胞主要表达编码钙离子通道亚基、钙离子传感器蛋白和其他钙离子相关离子通道的基因。钙离子介导的电生理活动已经在这些早期发育阶段的小鼠胚胎的腹侧中脑中得到证实，我们现在想要研究这些钙离子介导的活动和钙离子相关的离子通道和传感器蛋白在一般的中脑DA神经元和这些神经元的特定亚群的产生和维持中的功能。为此，我们希望通过钙离子成像监测人和小鼠多能干细胞体外分化为中脑DA神经元过程中钙离子介导的活动，并将这些活动与相应的细胞命运相关联。为此，我们将使用小鼠胚胎（ES）和诱导多能（iPS）报告干细胞以及使用CRISPR/Cas9技术配备亚群特异性报告基因的人类iPS细胞。此外，我们希望通过应用这些离子通道的特异性拮抗剂/阻断剂或通过CRISPR/Cas9介导的人iPS细胞中相应基因的失活来确定特定Ca 2+相关离子通道和传感器蛋白在此过程中的功能。我们还将对来自特发性PD患者和健康对照的分化iPS细胞进行这些分析，以获得对PD衍生细胞中潜在异常Ca 2+参数的初步了解。通过选择性地应用这些离子通道的激动剂/拮抗剂来校正这种异常的Ca 2+活性可能用于开发PD的新治疗方法。

The emergence of the cellular and molecular heterogeneity of dopamine-releasing (dopaminergic, DA) nerve cells in the midbrain during human and mouse embryonic development is largely not yet understood. These nerve cells participate in different cognitive, affective/emotional and motor brain functions. Their loss or dysfunction leads to severe neuropsychiatric human diseases, such as Parkinson’s Disease (PD), schizophrenias and addictive disorders. Our previous work has shown that a precise modulation of the canonical WNT/b-catenin signaling pathway is necessary and sufficient for the correct differentiation of a precursor population in the mouse midbrain into substantia nigra pars compacta (SNc) DA neurons. Moreover, the WNT/b-catenin signaling pathway promotes the survival of these neurons in a genetic mouse model of PD. The identification of the target genes of WNT/b-catenin signaling was therefore of particular interest in this context. The laser-microdissected WNT-responsive cells in the ventral midbrain of the mouse embryo express mostly genes encoding calcium (Ca2+) ion channel subunits, Ca2+ sensor proteins and other Ca2+-associated ion channels. Electrophysiological activities mediated by Ca2+ ions have already been demonstrated in the ventral midbrain of the mouse embryo at these early developmental stages.We now want to investigate the function of these Ca2+-mediated activities and Ca2+-associated ion channels and sensor proteins during the generation and maintenance of midbrain DA neurons in general and specific subsets of these neurons in particular. To this end, we want to monitor the Ca2+-mediated activities by Ca2+ imaging during the in vitro differentiation of human and mouse pluripotent stem cells into midbrain DA neurons, and to correlate these activities with the corresponding cell fate. For this purpose, we will use mouse embryonic (ES) and induced pluripotent (iPS) reporter stem cells as well as human iPS cells that have been equipped with subpopulation-specific reporter genes using CRISPR/Cas9 technology. In addition, we want to determine the function of particular Ca2+-associated ion channels and sensor proteins during this process by applying specific antagonists/blockers of these ion channels or by CRISPR/Cas9-mediated inactivation of the corresponding genes in the human iPS cells. We will also perform these analyses on differentiating iPS cells derived from idiopathic PD patients and healthy controls to obtain initial insights into potentially aberrant Ca2+ parameters in the PD-derived cells. Correction of such aberrant Ca2+ activities by the selective application of agonists/antagonists of these ion channels might be used for the development of new therapeutic approaches to PD.