Parkin mutation decreases neurite complexity and maturation in neurons derived from human fibroblasts

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, and mainly characterized by the progressive degeneration of dopaminergic (DA) neurons in the midbrain substantia nigra and non-DA neurons in many other parts of the brain. Previous studies have shown that several genes associated with the causes of PD can influence neurite outgrowth. Mutations of PRKN (encoding parkin, an E3 ubiquitin ligase) are the most frequent cause of recessively inherited PD. The lack of a PD phenotype in Prkn-knockout mice may imply a unique vulnerability of neurons to parkin mutations.

CRISPR/Cas9 technology was used to target random mutations into exon3 of PRKN in human fibroblasts cell line MRC-5. The induced DA neurons were achieved from direct conversion of fibroblasts (with or without PRKN mutations) via a cocktail of transcriptional factors (Ascl1, Nurr1, Lmx1a, miRNA124, p53 shRNA) and chemicals (CHIR99021, Purmorphamine, TGFβ3, BDNF, GDNF, NGF and Y27632).

Herein, we successfully established human neuronal cell models with parkin mutations from fibroblast-reprogrammed neurons. In these neurons, not only were the induced ratio and number of mature neurons markedly decreased, but also the complexity of the neuronal processes, measured by total neurite length and number of terminals, was greatly reduced, in TH⁺ and TH^-neurons with PRKN mutations.

The results suggest that parkin not only maintains the morphological complexity of human neurons, but also influences maturation and differentiation in the fibroblast reprogramming process.