Identification of the underlying mechanism of the c.192G>C mutation in the DJ-1 gene and functional characterisation in patient-based cellular models of Parkinson’s disease ex vivo

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disease and characterised by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta and in other brain regions. Homozygous loss-of-function mutations in the DJ-1 gene (PARK7) are a rare cause of familial early-onset PD.

The protein encoded by PARK7 is involved in a variety of biological processes including transcriptional regulation, chaperone-like functions, oxidative stress response and mitochondrial protection.

In the present study, we deciphered novel molecular mechanisms underlying the pathogenicity of the c.192G>C DJ-1 mutation predicted to lead to a p.E64D amino acid exchange in the DJ-1 protein.

To analyse the c.192G>C DJ-1 mutation, we generated and characterised different ex vivo patient-based cellular models including patient-derived primary fibroblasts, immortalised fibroblasts, induced pluripotent stem cells (iPSCs), iPSC-derived small molecule neuronal precursor cells (smNPCs) as well as iPSC-derived midbrain-specific dopaminergic (mDA) neurons.

After deciphering the pathogenic mechanism, we developed a rescue strategy. In addition, we extended our strategy by first candidate approaches aiming at a pharmacological rescue that may offer novel causative treatment options in patients carrying the c.192G>C DJ-1 mutation.

Beyond the molecular genetic characterisation, we developed different patient-based cellular models and addressed the functional effects in different patient-derived cells carrying the c.192G>C DJ-1 mutation (human fibroblasts, iPSC-derived mDA neurons).

VII These analyses revealed mitochondrial impairments in fibroblasts, including fragmentation and reduced branching of mitochondria as well as a re- duced mitochondrial membrane potential compared to healthy controls. The results support the idea of a conserved role of DJ-1 in maintaining mitochondrial function. Moreover, mDA neurons of the index patient carrying the homozygous c.192G>C DJ-1 mutation showed increased lesion rates of mtDNA and no increase in mtDNA copy numbers, suggesting a lack of compensatory capacity.

Our data substantially contribute to the understanding of mechanisms and functions of DJ-1 mutations in PD pathogenesis, in particular focusing on mitochondrial phenotypes in different human ex vivo models. This underlines the role of DJ-1 as an important key player in the response to oxidative stress and the maintenance of proper mitochondrial function and homeostasis.

Overall, we show that the fibroblasts with an inherited c.192G>C DJ-1 mutation, mDA neurons differentiated from iPSCs of these human fibroblasts and the DJ-1 knockout mice constitute excellent knockout model systems to further dissect the role of DJ-1 in neurodegeneration in PD. This also offers human DJ-1 knockout models for future isogenic control experiments with a restituted endogenous DJ-1 background. Sequentially, it is possible to test whether disease related phenotypes might be rescued by reintroducing DJ-1. Finally, the discovery of the underlying mechanism of the c.192G>C DJ-1 mutation opens up novel opportunities - for maybe even pharmacological causative treatment for PD.