Elucidation of pathogenic mechanism underlying juvenile Parkinson's disease induced by DNAJC6 mutation using isogenic hESCs

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder caused by loss of dopamine (DA) neuron in the substantia nigra. Recent studies show that a homozygous mutation in DNAJC6, which encodes the neuronal-specific clathrin endocytic pathway co-chaperone auxilin, cause recessive juvenile parkinsonism. Auxilin’s role in uncoating of clathrin from clathrin-coated vesicles is well established, but no study figures out how the deletion or reduction in expression of auxilin leads to PD phenotype. To elucidate PD pathogenic mechanism caused by impairment of DNAJC6, I conducted DNAJC6 loss-of-function study in vitro. I generated DNAJC6 knockout(KO) human embryonic stem (hES) cell lines using CRISPR/Cas9 system. The DNAJC6 KO hES cell lines have maintained pluripotent markers expressions (Oct4, Nanog, Tra1-60 and Sox2) and proliferative capacity. The hES cell differentiation was induced towards DA neurons via intermediary neural stem cell (NSC) stage. NSCs derived from the DNAJC6-KO hES cells (DNAJC6-KO hNSCs) exhibited more extensive cell death than those derived from the control hESCs (Control hNSCs), and the cell death became more severer in the differentiated dopamine neuronal stage derived from in the KO-hNSCs .In addition, DA neurons differentiated from DNAJC6-KO hNSCs (DNAJC6-KO hDA neurons) released lower level of DA neurtransmitter upon depolarization stimuli. I further observed an increased level of a-synuclein aggregation in the cultures for DNAJC6-KO hDA neurons, compared to the control counterpart. Although further studies are required to elucidate the pathogenic mechanisms, especially those as to how DNAJC6 KO causes the observed pathologic findings, this is the first report to establish a valuable human in vitro system for studying DNAJC6-meidated pathogenic mechanisms, along with crucial manifestations associated with PD.