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See detailIdentification 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
Obermaier, Carolin UL

Doctoral thesis (2015)

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 ... [more ▼]

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 previously 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. Analyses of DJ-1 expression in these patient-derived model systems from homozygous carriers of the c.192G>C DJ-1 mutation unexpectedly revealed that this mutation leads to the loss of DJ-1 protein in these cell types. Further experiments using qPCR and an in vitro splicing assay showed a splicing defect causing complete skipping of the mutation-carrying exon 3 in the pre-mRNA. After deciphering the pathogenic mechanism, we developed a targeted genetic rescue strategy of the pathological skipping of exon 3. This was performed by using a specific U1 snRNA that specifically binds to the mutated DJ-1 pre-mRNA and allows for the re-induction of physiological splicing. 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 as well as for other diseases caused by the same mutational mechanism. Beyond the molecular genetic characterisation, we developed different patient-based cellular models and addressed the functional effects of loss of DJ-1 protein in different patient-derived cells carrying the c.192G>C DJ-1 mutation (human fibroblasts, iPSC-derived mDA neurons). These analyses revealed mitochondrial impairments upon loss of DJ-1 protein in fibroblasts, including fragmentation and reduced branching of mitochondria as well as a reduced mitochondrial membrane potential compared to healthy controls. The results correlate with our observations in primary cells from DJ-1 knockout mice and 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 in-creased 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 upon loss of DJ-1 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 iso-genic 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 or correcting the defective splicing. Finally, the discovery of the underlying mechanism of the c.192G>C DJ-1 mutation opens up novel opportunities for a first genetic and maybe even pharmacological causative treatment for PD. [less ▲]

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