Modulation of ceramide-induced cell death and superoxide production by mitochondrial DNA-encoded respiratory chain defects in Rattus xenocybrid mouse cells

Abstract

Mitochondria play an integral role in cell death signaling, yet how mitochondrial defects disrupt this important function is not well understood. We have used a mouse L-cell fibroblast model harboring Rattus norvegicus mtDNA (Rn xenocybrids) to examine the effects of multiple oxidative phosphorylation (OXPHOS) defects on reactive oxygen species (ROS) generation and cell death signaling. Blue native-PAGE analyses of Rn xenocybrids revealed defects in OXPHOS complex biogenesis with reduced steady-state levels of complexes I, III and IV. Isolated Rn xenocybrid mitochondria exhibited deficiencies in complex II+III and III activities, with CIII-stimulated ROS generation 66% higher than in control mitochondria. Rn xenocybrid cells were resistant to staurosporine-induced cell death, but exhibited a four-fold increase in sensitivity to ceramide-induced cell death that was caspase-3 independent and did not induce chromosomal DNA degradation. Furthermore, ceramide directly inhibited Rn xenocybrid complex II+III activity by 97%, although this inhibition could be completely abolished by exogenous decylubiquinone. Ceramide also induced a further increase in ROS output from Rn xenocybrid complex III by 42%. These results suggest that the interaction of ceramide with OXPHOS complex III is significantly enhanced by the presence of the xenotypic Rattus cytochrome b in complex III, likely due to the increased affinity for ceramide at the ubiquinone binding site. We propose a novel mechanism of altered mitochondrial cell death signaling due to mtDNA mutations whereby ceramide directly induces OXPHOS complex ROS generation to initiate cell death pathways.

Increased Diabetes Complications in a Mouse Model of Oxidative Stress Due to ‘Mismatched’ Mitochondrial DNA

“…Mitochondrial ROS production was measured in renal mitochondria using the DCFDA assay for hydroperoxide, as previously described [39].…”

The importance of sphingolipids and reactive oxygen species in cardiovascular development

“…Furthermore, ceramide analogues, which can act on isolated mitochondria, promote an increase in ROS production, inhibiting complex III and stimulating the production of H 2 O 2 at high ceramide concentrations, while NO is stimulated at low ceramide concentrations (Figure ; Nikolova‐Karakashian and Reid, ; Trounce et al., ). However, the mechanisms by which sphingolipids act on electron transporters are not fully understood.…”

“…Review concentrations (Figure 3; Nikolova-Karakashian and Reid, 2011; Trounce et al, 2013). However, the mechanisms by which sphingolipids act on electron transporters are not fully understood.…”