CONICET | Buscador de Institutos y Recursos Humanos

Objectives: Establish a correlation between autosomal PARK gene mutations and neuronal dysfunction in Parkinsons disease (PD). Methods: Neuronal stem cell (NSC) clones were generated from induced pluripotent stem cells iPSCs derived from fibroblasts from patients with a (rare) triplication of the SNCA (α-synuclein, aSyn) gene, the prevalent G2019S mutation of LRRK2, and age-matched controls. The susceptibility to oxidative stress was investigated by assessing mitochondrial function with the Seahorse XF24, and ROS production by Mitotracker sensors. The concentration-dependent interactions and distribution of aSyn in isolated mitochondrial preparations were characterized by superresolution microscopy and functional assessment of the respiratory chain. The neuronal differentiation of patient-derived and control NSCs in defined media and in astrocyte co-cultures were compared. Differentiated cells were analyzed for the expression of neuronal markers by immunofluorescence and biochemical techniques. Results: Cells with the SNCA triplication express 2-fold more aSyn compared to the control lines. Elevated expression of aSyn correlates with a reduced mitochondrial spare respiratory capacity and the LRRK2 mutant lines show an increased production of ROS in the undifferentiated state. In vitro experiments suggest that aSyn binds to and is imported into isolated mitochondria and has an inhibitory effect on the function of Complex IV. The SNCA triplication clones show a reduced capacity to differentiate compared to age-matched controls. However, co-culture with astrocytes improved the differentiation phenotype in the all the cell lines. Conclusions: NSCs obtained from PD patient-derived iPSCs exhibit several disease-related phenotypic features such as overexpression of aSyn, higher vulnerability to 6OHDA and increased production of ROS, and then serve to define the biochemical dysfunctions underlying neuronal cytotoxicity