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

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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.