Abstract
Introduction: Mitochondrial dysfunction is a prominent pathogenic mechanism in neurodegenerative disorders. One of such entities, Parkinson’s disease (PD) is characterized by progressive loss of dopaminergic neurons and accumulation of proteinaceous Lewy body inclusions. A number of nuclear germline genetic drivers of PD, such as PRKN, are linked to mitochondrial quality control (1). Progressive mitochondrial DNA (mtDNA) damage has been observed in neuronal models of PD and blood DNA from early-stage PD patients with LRRK2 loss-of-function mutations (2). The contribution of mtDNA damage to PD progression, however, has not been clearly proven.
Aim: We sought to investigate mtDNA damage in human induced pluripotent stem cell (hiPSC)-derived neuronal models of PD (hiPSC-derived neurons) by combining a quantitative PCR (qPCR)-based method with digital droplet PCR (ddPCR) to quantify mtDNA copy numbers per cell.
Methods: iPSC lines and iPSC-derived dopaminergic neurons at early (20 days) and late (40 days) stages of differentiation, obtained from controls (n=4), asymptomatic heterozygous PRKN variant carriers (n=2), and PD patients with homozygous PRKN variants (n=2) were assessed. ND1 copy number quantification by ddPCR was used to determine the number of mtDNA copies used as input for each sample. We then performed long-range mtDNA qPCR and interpolated the obtained Ct values into a standard curve to determine the number of amplified intact mtDNA copies. Results were normalized to the input mtDNA copy number and DNA damage was compared among experimental conditions.
Results: hiPSC-derived neurons showed increased mtDNA damage per 10 kb (1.14±0.41) compared to their parental hiPSCs (0.51±0.19, p=0.003). This difference remained significant for all genotype categories. Furthermore, mtDNA damage was increased in hiPSC-derived neurons for both the early (0.99±0.28, p=0.0007) and the late (1.28±0.48, p<0.0001) stages of differentiation, compared to hiPSCs. There was a statistical trend towards increased damage in hiPSC-derived neurons at late compared to the early stage (p=0.071). Interestingly, no differences were observed pertaining the PD-related genotype of the cells.
Conclusion: Our adapted assay provides an accurate method for mtDNA damage assessment and illustrates that mtDNA damage progressively accumulates during hiPSC-derived neuronal differentiation. The assessment of non-specific DNA damage complements mtDNA sequencing and copy number analyses and will thus contribute to define a finer picture of the mtDNA alterations that underlie mitochondrial defects in PD.