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J Neurosci Methods. 2014 Jul 30;232:143-9. doi: 10.1016/j.jneumeth.2014.05.026. Epub 2014 May 29.

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

Author information

1
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.
2
Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: nichola.lax@newcastle.ac.uk.
3
Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: m.rocha@newcastle.ac.uk.
4
Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: amy.reeve@newcastle.ac.uk.
5
Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: philippa.hepplewhite@newcastle.ac.uk.
6
Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: karolina.rygiel@newcastle.ac.uk.
7
Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: Robert.taylor@newcastle.ac.uk.
8
Wellcome Trust Centre for Mitochondrial Research, Institute of Ageing and Health, Newcastle University, Newcastle upon Tyne, UK. Electronic address: doug.turnbull@newcastle.ac.uk.

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.

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 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.

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.

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.

KEYWORDS:

Complex I; Complex IV; Immunofluorescent labelling; Midbrain neurons; Mitochondria; Single cell analysis

PMID:
24880043
PMCID:
PMC4076514
DOI:
10.1016/j.jneumeth.2014.05.026
[Indexed for MEDLINE]
Free PMC Article

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