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

帕金森病（PD）是一种严重的、第二常见的神经退行性疾病， 了解，目前的治疗选择很少。PD的临床表型是由选择性 中脑腹侧黑质背侧部多巴胺能神经元变性。虽然 已经描述了与家族性PD病例相关的几种基因突变，大多数PD病例是特发性的， 衰老似乎是主要的风险因素。对于家族性和特发性PD， 线粒体功能障碍被认为是PD发病机制的基础。线粒体功能障碍如何影响 下游神经元靶点和信号传导途径仍然不太清楚。基因筛查是 这些工具用于发现PD的新的潜在治疗靶点。只有少数这样的基因筛查 到目前为止，没有在hiPSC衍生的DA神经元中进行，神经元群体特异性地影响 在警局在实际的受疾病影响的神经元中进行表型筛选的一个主要限制是， 分化方案导致异源细胞群，混淆表型读数。这里我们 提出联合收割机hiPSC技术以产生2种新的hiPSC线粒体自噬报告细胞系， 一种线粒体靶向的pH敏感荧光团（mt-Keima），用于研究PD相关突变对 线粒体自噬，或表达mt-Keima和诱导型DNA酶失活Cas9融合蛋白的组合的hiPSC系 用于全基因组筛选目的。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，我们的目标是实现细胞 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 开关将使我们能够特异性地选择多巴胺能神经元，我们将在表型筛选中使用这些神经元， 发现与PD相关的线粒体自噬和线粒体功能障碍障碍的潜在靶点。的 这项工作的结果也将为遗传干扰或表型筛选提供一个强有力的新资源 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.