Cytochrome c release from brain mitochondria in response to respiratory inhibition
Apoptosis is the programmed cell death mechanism responsible for regulation of cell number during tissue development and maturity. However, dysregulation of apoptosis has pathological consequences, particularly in the brain, where 'inappropriate' apoptosis leads to neurodegeneration. Neuronal apoptosis is primarily initiated by release of the respiratory protein cytochrome c from mitochondria, the organelles responsible for aerobic respiration. Considerable interest has been focussed on the mechanism of cytochrome c release, but surprisingly little on the nature of the cytochrome c signal itself. It has been claimed that release is all-or-none, however this is inconsistent with accumulating evidence of cytosolic mechanisms for 'buffering' cytochrome c. This study has addressed two primary methodological weaknesses in the field, by modelling an underlying disease pathology, rather than artificially inducing apoptosis, and by using a truly quantitative assay for measuring cytochrome c concentration. These experimental advantages have yielded the first demonstration that cytochrome c is released in proportion to the severity of pathological insult. The model adopted was reduced activity of the mitochondrial NADH:ubiquinone oxidoreductase, a recognised feature of Parkinson's disease, induced in this case by rotenone titration. The controlled, proportional release observed invited the hypothesis that a low level of cytochrome c release is insufficient to induce apoptosis. In support of this suggestion, the mechanism of release was determined to be conducive to cell survival given hypothetical 'sub-threshold' release. It was also demonstrated that release is likely to be induced following mitochondrial generation of reactive oxygen species, of which there is believed to be a constitutive basal level. Finally it was established that in primary cortical neurons, respiratory inhibition can induce cytochrome c release which is sub-threshold for induction of apoptosis. The study has generated considerable support for therapeutic exploitation of the cytosolic 'Inhibitor of Apoptosis Proteins', for delaying progression of Parkinson's and other neurodegenerative diseases.