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尚不清楚。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人类神经精神疾病，如帕金森病（PD）、精神分裂症和成瘾性疾病。我们之前的研究表明，典型的WNT/b-catenin信号通路的精确调节对于小鼠中脑前体群体向致密黑质（SNc） DA神经元的正确分化是必要和充分的。此外，在遗传小鼠PD模型中，WNT/b-catenin信号通路促进了这些神经元的存活。因此，WNT/b-连环蛋白信号的靶基因的鉴定在这种情况下特别有趣。激光显微解剖的小鼠胚胎腹侧中脑wnt反应细胞主要表达编码钙（Ca2+）离子通道亚基、Ca2+传感器蛋白和其他Ca2+相关离子通道的基因。Ca2+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Ca2+介导的活性和Ca2+相关离子通道和传感器蛋白在中脑DA神经元的产生和维持过程中的功能，特别是这些神经元的特定亚群。为此，我们希望通过Ca2+成像来监测人和小鼠多能干细胞在体外分化为中脑DA神经元过程中Ca2+介导的活性，并将这些活性与相应的细胞命运联系起来。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技术配备亚群体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/阻滞剂或通过CRISPR/ cas9介导的人iPS细胞中相应基因的失活来确定特定Ca2+相关离子通道和传感器蛋白在这一过程中的功能。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，以初步了解PD来源细胞中潜在的异常Ca2+参数。通过选择性应用这些离子通道的激动剂/拮抗剂来纠正这种异常的Ca2+活性可能用于开发新的PD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.