Metabolic reprogrammed primary skin fibroblasts as patient specific cellular models for Parkinson's disease

Mitochondria are crucial organelles which undergo fusion and fission processes responsible for mitochondrial network rearrangements. Once mitochondria are damaged, they are selectively degraded by mitophagy. Collectively, these processes are referred to as mitochondrial dynamics, and dysregulations that hamper their function are often associated with neurodegenerative diseases like Parkinson’s disease (PD). Mutations in mitophagy-related PRKN gene, which encodes the E3 ubiquitin ligase Parkin, have been linked to autosomal recessive juvenile PD. Recently, molecular players of the endosomal-lysosomal pathway such as Rab proteins, have been linked to the mitophagic pathway, indeed, mutations in RAB32 and RAB39B have been identified to cause familial PD. In order to elucidate the interplay between Rab proteins and dysfunctional mitophagy in PD, we exploited PRKN-mutated and control human skin fibroblasts to evaluate levels and sub-cellular localization of a Rab protein subset. First of all, to make fibroblasts more sensitive to mitochondrial damages and metabolically more similar to neurons, we induced a metabolic reprogramming toward oxidative phosphorylation. The changes in CS and COX5B protein levels suggested that the protocol succeeded in the metabolic reprogramming of both control and PRKN-mutated fibroblasts. Moreover, we exploited mitochondrial toxins related to PD, Rotenone and 1-methyl-4-phenylpyridinium (MPP+), to induce mitophagy. Both treatments induced mitochondrial depolarisation and alterations in mitochondrial marker levels, but MPP+ was the selected toxin since it induced the higher mitochondrial depolarisation. Exploiting our final model, reprogrammed skin fibroblasts treated with a mitochondrial toxin, we evaluated the mitochondrial network morphology, the mitochondrial mass, and the Rab protein levels in PRKN-mutated fibroblasts. We observed increased RAB9 levels in PRKN-mutated fibroblasts especially after MPP+ treatment. RAB9 is usually associated to alternative PINK1/Parkin-mediated mitophagy, thus hinting a role of Rab proteins in mitochondrial dynamics related to PD. In conclusion, this thesis established a set-up (metabolic reprogramming and MPP+ treatment) that enables the use of skin fibroblasts as personalized cellular models to study mitochondrial dynamics related to PD. Furthermore, the preliminary study on Rab proteins involved in mitophagy revealed altered levels of these vesicular trafficking regulators in PRKN-mutated fibroblasts, particularly following mitophagy induction.