Abstract
Respiratory chain (RC) deficiencies are found in primary mtDNA diseases. Focal RC defects are also associated with ageing and neurodegenerative disorders, e.g. in substantia nigra (SN) neurons from Parkinson's disease patients. In mitochondrial disease and ageing, mtDNA mutational loads vary considerably between neurons necessitating single cell-based assessment of RC deficiencies. Evaluating the full extent of RC deficiency within SN neurons is challenging because their size precludes investigations in serial sections. We developed an assay to measure RC abnormalities in individual SN neurons using quadruple immunofluorescence. Using antibodies against subunits of complex I (CI) and IV, porin and tyrosine hydroxylase together with IgG subtype-specific fluorescent labelled secondary antibodies, we quantified the expression of CI and CIV compared to mitochondrial mass in dopaminergic neurons. CI:porin and CIV:porin ratios were determined relative to a standard control. Quantification of expression of complex subunits in midbrain sections from patients with mtDNA disease and known RC deficiencies consistently showed reduced CI:porin and/or CIV:porin ratios. The standard histochemical method to investigate mitochondrial dysfunction, the cytochrome c oxidase/succinate dehydrogenase assay, measures CIV and CII activities. To also study CI in a patient, immunohistology in additional sections, i.e. in different neurons, is required. Our method allows correlation of the expression of CI, CIV and mitochondrial mass at a single cell level. Quantitative quadruple-label immunofluorescence is a reliable tool to measure RC deficiencies in individual neurons that will enable new insights in the molecular mechanisms underlying inherited and acquired mitochondrial dysfunction.
Highlights
The oxidative phosphorylation (OXPHOS) system comprises approximately 85 polypeptides
New method: Using antibodies against subunits of complex I (CI) and IV, porin and tyrosine hydroxylase together with IgG subtype-specific fluorescent labelled secondary antibodies, we quantified the expression of CI and complex IV (CIV) compared to mitochondrial mass in dopaminergic neurons
In primary mitochondrial DNA (mtDNA) disease, different mtDNA mutations and mutation heteroplasmy levels cause great variability in the degree of mitochondrial dysfunction in different cell populations. Under these circumstances functional analyses in single cells are preferred over investigations in tissue homogenates
Summary
The oxidative phosphorylation (OXPHOS) system comprises approximately 85 polypeptides. The passage of electrons from CI or CII via CIII and CIV catalyzes the transfer of protons from the mitochondrial matrix across the inner mitochondrial membrane to the inter-membrane space building an electrochemical gradient This gradient is the driving force for CV, ATP synthase, to generate ATP from ADP and inorganic phosphate (Hatefi, 1985; Saraste, 1999). In primary mitochondrial DNA (mtDNA) diseases, dysfunction of RC enzyme complexes is due to inherited mutations in the mitochondrial genome with the level of heteroplasmy (mutational burden) varying considerably between cells (Taylor and Turnbull, 2005). In mitochondrial disease and ageing, mtDNA mutational loads vary considerably between neurons necessitating single cell-based assessment of RC deficiencies. Results: Quantification of expression of complex subunits in midbrain sections from patients with mtDNA disease and known RC deficiencies consistently showed reduced CI:porin and/or CIV:porin ratios. Conclusion: Quantitative quadruple-label immunofluorescence is a reliable tool to measure RC deficiencies in individual neurons that will enable new insights in the molecular mechanisms underlying inherited and acquired mitochondrial dysfunction
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