Micro RNA-based selection of hiPSC-derived dopaminergic neurons for genetic screening.

帕金森氏病(PD)是一种严重的、第二常见的神经退行性疾病，至今仍很严重 了解并且目前几乎没有治疗选择。帕金森病的临床表型是由选择性 中脑腹侧黑质致密部多巴胺能神经元变性。尽管 已经描述了几种与家族性帕金森病相关的基因突变，大多数帕金森病患者是特发性的， 在那里，衰老似乎是主要的风险因素。对于家族性和特发性帕金森病，氧化应激和 线粒体功能障碍被认为是帕金森病发病的基础。线粒体功能障碍如何影响 下游的神经元靶点和信号通路仍然知之甚少。基因筛查是其中之一 用于发现帕金森病新的、潜在的治疗靶点的工具。只有极少数这样的基因筛查有 到目前为止，由于没有在HiPSC衍生的DA神经元中进行，神经元群体特别影响 在警局。对实际受疾病影响的神经元进行表型筛选的一个主要限制是，大多数 分化方案导致异质细胞群体，混淆了表型读数。在这里我们 建议结合hiPSC技术产生2个新的hiPSC有丝分裂吞噬报告系 线粒体靶向、pH敏感的荧光团(mt-kema)研究PD相关突变对细胞的影响 有丝分裂，或表达mt-kema和可诱导的、DNA酶失活的Cas9融合的hiPSC系 与KRAB抑制子结构域结合用于全基因组筛选。为了运行未来的基因和 在同质的、受疾病影响的神经元亚型群体中进行表型筛选，我们的目标是实现一种细胞 一种基于细胞类型特异性microRNA表达的类型特异性选择方法。这种基于miRNA的分子 Switch将使我们能够专门选择我们将在表型筛选中使用的多巴胺能神经元 发现与帕金森病相关的线粒体吞噬功能紊乱和线粒体功能障碍的潜在靶点。这个 这项工作的结果也将为遗传扰动或表型筛选提供强大的新资源 面向更广泛的研究社区。

Parkinson’s disease (PD) is a severe, second most common neurodegenerative disorder which is still poorly understood and has few current treatment options. The clinical phenotype of PD is caused by the selective degeneration of dopaminergic neurons in the substantia nigra pars compacta in the ventral midbrain. Though several gene mutations associated with familial PD cases have been described, most PD cases are idiopathic, where aging seems to be the major risk factor. For both familial and idiopathic PD, oxidative stress and mitochondrial dysfunction are thought to underlie PD pathogenesis. How mitochondrial dysfunction effect downstream neuronal targets and signaling pathways remains less well understood. Genetic screening is one of the tools that are used to uncover new, potentially therapeutic targets for PD. Only few such genetic screens have done to date, with none carried out in hiPSC-derived DA neurons, the neuronal population specifically affected in PD. A major limitation with phenotypic screening in actual disease-affected neurons is that most differentiation protocols result in heterogenous cell populations, confounding phenotypic readouts. Here we propose to combine hiPSC-technology to generate 2 novel hiPSC mitophagy reporter lines with either expressing a mitochondrial targeted, pH-sensitive fluophore (mt-Keima) to study effect PD-associated mutations on mitophagy, or a hiPSC line expressing a combination of mt-Keima and an inducible, DNase inactive Cas9 fused with the KRAB repressor domain for genome-wide screening purposes. In order to run future genetic and phenotypic screens in homogenous, disease affected neuronal subtype populations, we aim to implement a cell type-specific selection method based on cell type-specific microRNA expression. This miRNA-based molecular switch will enable us to specifically select for dopaminergic neurons that we will use in phenotypic screens for discovery of targets underlying disturbed mitophagy and mitochondrial dysfunction, associated with PD. The outcome of this work will also provide a powerful new resource for genetic perturbation or phenotypic screens for the broader research community.