Basic NeuroscienceQuantitative quadruple-label immunofluorescence of mitochondrial and cytoplasmic proteins in single neurons from human midbrain tissue

- We developed an assay to quantify respiratory chain deficiencies in single neurons.
- Quadruple-label immunofluorescence was combined with quantitative image analysis.
- The single-cell assay was applied to tyrosine hydroxylase-positive midbrain neurons.
- The expression of complexes I and IV was determined relative to mitochondrial mass.
- The assay proved specific in patients with known respiratory chain deficiencies.

BackgroundRespiratory 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.New methodUsing 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.ResultsQuantification 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.Comparison with existing method(s)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.ConclusionQuantitative 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.