Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the preferential loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Several clues key to understanding disease pathogenesis come from mutations in genes which have been linked to inherited forms of PD. Among these, mutations in the acid β-glucocerebrosidase (GBA1) gene, responsible for the lysosomal storage disorder Gaucher’s disease (GD), are the strongest genetic risk factor for PD known to date. To elucidate the mechanisms underlying neurodegeneration in these patients, we generated induced pluripotent stem cells (iPSCs) from subjects with GD and PD harboring GBA1 mutations as well as neurologically healthy individuals. To control for the influence of the patient genetic background and perform genotype-phenotype correlations, we generated isogenic GBA1 corrected lines by using zinc finger nuclease (ZFN)-induced homologous recombination. All iPSC lines were successfully differentiated to midbrain DA neurons and enriched by fluorescence-activated cell sorting. Importantly, such purified human iPSC-derived neurons mirrored the sphingolipid content of the adult human brain. Diseased neurons show a reduction of glucocerebrosidase activity and protein levels, increased glucosylceramide and α-synuclein levels and defects in the lysosomal/autophagic machinery. A large-scale quantitative proteomic profile on purified neurons and imaging studies revealed dysregulation of calcium homeostasis and increased vulnerability to cellular stress responses involving elevation of cytosolic calcium in mutant neurons. Importantly such phenotypes were reverted upon gene correction, thus supporting a direct link between GBA1 mutations and clinical relevant phenotypes. In summary, our findings provide evidence for a link between GBA1 mutations and complex changes in the lysosomal function and intracellular calcium homeostasis, which may underlie vulnerability to neurodegeneration.