Abstract
Background: Parkinson’s disease (PD) is a complex and progressive neurodegenerative movement disorder. Mutations in the Parkin gene (PRKN) are the most common known cause of autosomal recessive early-onset PD. Parkin is an E3 ubiquitin ligase acting in the protein quality control and the removal of damaged mitochondria in the Parkin-PINK1 pathway.
Aim: Given the complex role of Parkin in determining cell fate in response to mitochondrial damage, the aim of this study was to identify novel disease-specific phenotypes caused by PRKN mutations during the in vitro neuronal differentiation process in physiologically-relevant human-derived cellular models.
Results: Human induced pluripotent stem cell (hiPSC)-derived midbrain dopaminergic (mDA) neurons were generated for two PD patients carrying a biallelic mutation in PRKN and two control individuals using both a conventional 2D differentiation protocol and a recently developed 3D differentiation method based on the microencapsulation of hiPSCs in small alginate/fibronectin beads. Targeted expression profiling by RNA-sequencing (RNA-seq) analysis was evaluated to further elucidate the cellular impact of PRKN mutations on the molecular events linked to disease development and progression. Human iPSC-derived mDA neurons collected at four different timepoints, hiPSCs (T0), and days 10, 20, and 35 of differentiation, in both culture conditions, were used as validated cellular systems to model PD. The bioinformatic analyses of PRKN mutation-specific gene sets and functional pathways of early timepoints (D10 and D20) and of early postmitotic neurons (D35) revealed multiple dysregulated pathways involved in PRKN-pathology, including synaptic and metabolic function, inflammation, RNA metabolism, microtubule stabilization, and intracellular trafficking, reflecting a complex and layered disease development.
Discussion and conclusions: The RNA-seq analysis performed in this study took advantage of combining two alternative differentiation approaches for the generation of mDA neurons from hiPSCs that offers the potential to derive human-based models in the genetic background of the patient. Moreover, the inclusion of early timepoints (D10 and D20) allowed for the identification of pathways at early stages of disease development, while the analysis of more mature neurons (D35) revealed pathways associated with later stages of PD development and progression. Notably, many of the identified genes and pathways were consistently dysregulated at all investigated timepoints during neuronal differentiation, likely reflecting early events in the disease development. Overall, these data provide important new insights into the biochemical mechanisms linked to PRKN-PD, opening up new avenues to test markers for early diagnosis and to guide targeted interventional strategies before neurodegeneration advances.