title: Quantitative quadruple-label immunofluorescence of mitochondrial and cytoplasmic proteins in single neurons from human midbrain tissue.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK; Institute of Neurogenetics, University of Lübeck, Lübeck, Germany. Electronic address: anne.gruenewald@newcastle.ac.uk.
authors.forename: Anne
authors.initials: A
authors.lastname: Grünewald
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: nichola.lax@newcastle.ac.uk.
authors.forename: Nichola Z
authors.initials: NZ
authors.lastname: Lax
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: m.rocha@newcastle.ac.uk.
authors.forename: Mariana C
authors.initials: MC
authors.lastname: Rocha
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: amy.reeve@newcastle.ac.uk.
authors.forename: Amy K
authors.initials: AK
authors.lastname: Reeve
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: philippa.hepplewhite@newcastle.ac.uk.
authors.forename: Philippa D
authors.initials: PD
authors.lastname: Hepplewhite
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: karolina.rygiel@newcastle.ac.uk.
authors.forename: Karolina A
authors.initials: KA
authors.lastname: Rygiel
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: Robert.taylor@newcastle.ac.uk.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Taylor
authors.affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: doug.turnbull@newcastle.ac.uk.
authors.forename: Doug M
authors.initials: DM
authors.lastname: Turnbull
mesh_terms: D000328:Adult; D000368:Aged; D042967:Electron Transport Complex I / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008875:Middle Aged; D028361:Mitochondrial Diseases / Q000473:pathology*; D009474:Neurons / Q000378:metabolism*; D018272:Porins / Q000378:metabolism; D013378:Substantia Nigra / Q000378:metabolism / Q000473:pathology*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
chemical_list: D018272:Porins; D014446:Tyrosine 3-Monooxygenase; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I
keywords: Complex I; Complex IV; Immunofluorescent labelling; Midbrain neurons; Mitochondria; Single cell analysis