title: A novel immunofluorescent assay to investigate oxidative phosphorylation deficiency in mitochondrial myopathy: understanding mechanisms and improving diagnosis.
abstract: Oxidative phosphorylation defects in human tissues are often challenging to quantify due to a mosaic pattern of deficiency. Biochemical assays are difficult to interpret due to the varying enzyme deficiency levels found in individual cells. Histochemical analysis allows semi-quantitative assessment of complex II and complex IV activities, but there is no validated histochemical assay to assess complex I activity which is frequently affected in mitochondrial pathology. To help improve the diagnosis of mitochondrial disease and to study the mechanisms underlying mitochondrial abnormalities in disease, we have developed a quadruple immunofluorescent technique enabling the quantification of key respiratory chain subunits of complexes I and IV, together with an indicator of mitochondrial mass and a cell membrane marker. This assay gives precise and objective quantification of protein abundance in large numbers of individual muscle fibres. By assessing muscle biopsies from subjects with a range of different mitochondrial genetic defects we have demonstrated that specific genotypes exhibit distinct biochemical signatures in muscle, providing evidence for the diagnostic use of the technique, as well as insight into the underlying molecular pathology. Stringent testing for reproducibility and sensitivity confirms the potential value of the technique for mechanistic studies of disease and in the evaluation of therapeutic approaches.
authors.affiliation: Newcastle University Centre for Ageing and Vitality, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.|Wellcome Trust Centre for Mitochondrial Research, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Mariana C
authors.initials: MC
authors.lastname: Rocha
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: John P
authors.initials: JP
authors.lastname: Grady
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Anne
authors.initials: A
authors.lastname: Grünewald
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Amy
authors.initials: A
authors.lastname: Vincent
authors.affiliation: Newcastle University Centre for Ageing and Vitality, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Philip F
authors.initials: PF
authors.lastname: Dobson
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Taylor
authors.affiliation: Newcastle University Centre for Ageing and Vitality, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.|Wellcome Trust Centre for Mitochondrial Research, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Doug M
authors.initials: DM
authors.lastname: Turnbull
authors.affiliation: Newcastle University Centre for Ageing and Vitality, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.|Wellcome Trust Centre for Mitochondrial Research, Institute for Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: Karolina A
authors.initials: KA
authors.lastname: Rygiel
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D019069:Cell Respiration / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D004272:DNA, Mitochondrial / Q000235:genetics; D004579:Electron Transport / Q000235:genetics; D045222:Electron Transport Chain Complex Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D017240:Mitochondrial Myopathies / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D015588:Observer Variation; D010085:Oxidative Phosphorylation; D010641:Phenotype; D015203:Reproducibility of Results; D013385:Succinate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D055815:Young Adult
chemical_list: D004272:DNA, Mitochondrial; D045222:Electron Transport Chain Complex Proteins; D013385:Succinate Dehydrogenase