Modelling Parkinson´s disease with patient induced pluripotent stem cells
Résumé
Mutations in PRKN are the most common genetic cause for familial Parkinson’s disease (PD). The discovery of Induced pluripotent stem cells (iPSCs) makes it possible to generate patient-specific dopaminergic (DA) neurons to study how PRKN mutation causes PD. iPSCs can be differentiated into DA neurons, and be used as a human disease model for PD. However, one of the limitations is that culture models can typically last only several weeks not long enough to reproduce disease-specific phenotypes. One of the most important issues is the lack of in vivo cues necessary for the development and maturation of DA neurons. Some of these problems can be resolved by transplanting the cells derived-patient iPSCs to an animal brain. In this study, in a first step, we have investigated the impact of PRKN mutations in vitro on cell survival, differentiation, maturation and mitochondrial function of DA neurons derived patient with PRKN mutation and control subjects. After differentiation, more than 70% of the generated cells expressed DA precursor markers such as FOXA2, OTX2 & LMX1A in both subjects. However, there is an important reduction in the number of cells expressing mature DA markers in patient. The mitochondrial health has been analyzed by using Cytochrome-C antibody and a membrane potential marker: MitoTracker. At DA precursor stage, around 80% of mitochondria were metabolically actives in both subjects. However, at maturation stage, we observed a 15-25% decrease in mitochondrial activity in patient cells. Apoptosis detection using TUNEL or annexin-V assay showed 20% higher apoptosis in patient cells. Moreover, the mitophagy was 2-fold higher in patient compared to control. In a second step, we transplanted DA precursors derived patients and control into the substantia nigra in a mouse model of PD. One month after grafting, we found a 5-fold reduction in the number of DA mature neurons in grafts from patient cells compared to control. Interestingly, there was no difference between cell proliferation and neuronal maturation between both subjects. Experiments are underway to study the impact of PARK2 mutation on survival, axon elongation, pathogenic protein accumulation, cell-type-specific vulnerability and mitochondrial dysfunction of grafted DA neurons.