The project aims for development and implementation of a phenotypic approach exploiting a physiologically highly relevant disease model and the identification of small molecules lead to the phenotypic rescue of the pathogenic mutations of the SNCA gene, targeting mitochondrial functionality and the autophagy/mitophagy pathways using human patient-derived midbrain dopaminergic neurons (mDANs) using our private/public partner Ksilink.We aim to employ 5 pathogenic patient-derived SNCA mutations, namely the point mutations: p.A30P, p.E46K and p.A53T, and the duplication and triplication of the SNCA gene. The cellular disease model will be complemented via isogenic controls where we will use gene editing to create isogenic cell lines, correcting the pathogenic point mutations, and inserting these point mutations into age- and gender-matched controls. The disease model will be based on a multi-parametric imaging algorithm to recognize validated profiles of mDANs expressing pathogenic and wild-type SNCA, respectively. The primary screen will aim for the identification of compounds according to their ability to rescue a WT-like profile in iPSC-derived midbrain dopaminergic neurons expressing the pathologic SNCA variants. These candidates will then enter a screening tree to determine if these compounds rescue neuronal cytotoxicity following noxious insult. CrispR-Cas9 technology will be used to engineer the iPSC lines harboring the SNCA variants to express autophagy and mitophagy pH-sensors. Within the FNR Bridges program we will differentiate these engineered lines to midbrain dopaminergic neurons to identify screenable phenotypes. Additionally we will adapt the differentiation and phenotyping protocols to our high throughput screening (HTS) platform to provide targeted pathway analysis of the hit compounds identified in the initial screenings by Ksilink. Mitochondrial and synaptic functionality will be assessed additionally using end-point assays. Furthermore, we will use our own libraries consisting mainly of FDA-approved compounds to screen on these lines for drugs suitable for repurposing. Compounds that can be validated in this project as pre-clinical candidates (PCCs) will enter in-vivo modelling using transgenic SNCA rats overexpressing the human SNCA gene that exhibit pathological disease phenotypes. The successful PCC that rescue disease phenotypes will advance to clinical trials for the treatment of PD.