Morphological profiling in human dopaminergic neurons identifies mitochondrial uncoupling as a neuroprotective effect

Multiple pathological cell biological processes in midbrain dopaminergic (mDA) neurons contribute to Parkinson’s disease (PD). Described disease mechanisms converge upon defects in protein degradation, disruption of vesicular trafficking, endolysosomal function, mitochondrial dysfunction and oxidative stress. Current cellular PD models for in vitro drug discovery are often of non-neuronal origin and do not take complex pathological interactions into account and focus on a single readout or phenotype. Here, we used patient-derived SNCA triplication (SNCA-4x) and isogenic control (SNCA-corr) mDA neurons and applied high-content imaging-based morphological profiling with the goal to determine and rescue multiple phenotypes simultaneously. We performed compound screening using a total of 1,020 compounds with biological activity annotations relevant to PD pathobiology including some FDA-approved drugs. We scored compounds based on their ability to revert the SNCA-4x mDA neuron morphological profile towards a healthy-like isogenic control neuronal profile. Top-scoring compounds led to a morphological rescue in SNCA-4x mDA neurons including increased Tyrosine hydroxylase (TH) level and decreased total α-synuclein (αSyn) protein levels. Multiple hit compounds were also linked to mitochondrial biology and we further evaluated them by determining their effect on neuronal mitochondrial membrane potential and cytoplasmic ROS levels. Additional biochemical analysis of the protonophore and mitochondrial uncoupler Tyrphostin A9 showed decreased total ROS levels and normalized mitochondrial membrane potential, and an increase in mitochondrial respiration. We confirmed this effect in mDA neurons by using five structurally related molecules and measuring mitochondrial activity and membrane potential. Additionally, Western blotting indicated that mitochondrial uncouplers, such as Tyrphostin A9, can decrease both low and high molecular weight forms of αSyn. Based on target agnostic morphological profiling in human mDA neurons, we therefore identified a connection between the compound-induced rescue of multiple morphological features, mild mitochondrial uncoupling, and a αSyn protein level decrease.