Histochemical Methods for the Diagnosis of Mitochondrial Diseases

Boel De Paepe1, Jan L. De Bleecker1, Rudy Van Coster2

1 Department of Neurology and Neuromuscular Reference Center, Ghent University Hospital, Ghent, Belgium, 2 Department of Pediatrics, Division of Child Neurology and Metabolism, and Neuromuscular Reference Center, Ghent University Hospital, Ghent, Belgium
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 19.2
DOI:  10.1002/0471142905.hg1902s63
Online Posting Date:  October, 2009
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Abstract

Through the process of oxidative phosphorylation (OXPHOS), mitochondria provide cells with required energy in the form of ATP. The organelle possesses its own genome (mtDNA), which encodes for part of the components needed (37 genes encoding either OXPHOS structural subunits or tRNAs and rRNAs). Nonetheless, the majority of structural OXPHOS components (as well as accessory proteins and proteins required for maintenance, replication, and expression of the mtDNA) are encoded by nuclear genes. Due to the dual genetic control and the large number of proteins involved, biogenesis and assembly of the OXPHOS system is complicated, and identifying a specific gene defect can be a difficult and time consuming task. This unit describes procedures for obtaining tissue sections and cell material suitable for histological evaluation of OXPHOS activity and integrity and immunodetection of the complexes in tissue from patients suspected of mitochondrial disease. Emphasis lies on the diagnostic potential of these techniques to differentiate mtDNA from nuclear mutations. Curr. Protoc. Hum. Genet. 63:19.2.1‐19.2.19. © 2009 by John Wiley & Sons, Inc.

Keywords: histochemistry; immunocytochemistry; immunohistochemistry; mitochondrial disorders; oxidative phosphorylation (OXPHOS); ragged‐red fibers (RRFs)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Frozen Skeletal Muscle Sections
  • Basic Protocol 2: Paraffin‐Embedded Liver Sections
  • Basic Protocol 3: Enzymatic Histology Using NADH Tetrazolium Reductase Staining
  • Basic Protocol 4: Enzymatic Histology Using Succinate Dehydrogenase Staining
  • Basic Protocol 5: Enzymatic Histology Using COX Activity Staining
  • Basic Protocol 6: Enzymatic Histology Using ATPase Staining
  • Basic Protocol 7: Immunohistochemical Staining of OXPHOS in Frozen Skeletal Muscle
  • Basic Protocol 8: Immunohistochemical Staining of OXPHOS in Paraffin‐Embedded Liver
  • Basic Protocol 9: Immunocytochemical Staining of OXPHOS in Cultured Skin Fibroblasts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Frozen Skeletal Muscle Sections

  Materials
  • Isopentane (2‐methyl‐butane)
  • Liquid nitrogen
  • Embedding medium: polyvinyl alcohol/polyethylene glycol or gelatin (e.g., CryoBlock, Klinipath)
  • Biopsied skeletal muscle tissue
  • 100‐ and 500‐ml containers (one should fit inside the other)
  • Binder clip
  • Cork disks (e.g., Klinipath)
  • Long‐handled forceps
  • Aluminum foil
  • Cryostat (microtome), −20°C
  • SuperFrost Plus glass slides (e.g., Menzel‐Glaser)

Basic Protocol 2: Paraffin‐Embedded Liver Sections

  Material
  • Biopsied liver tissue
  • Formalin: 40% (w/v) formaldehyde in PBS (see recipe for PBS)
  • 60%, 65%, 95%, and 100% ethanol in distilled water
  • Xylene
  • Paraffin wax, ∼50°C
  • Embedding cassettes (e.g., IMEB)
  • Microtome
  • SuperFrost Plus glass slides (e.g., Menzel‐Glaser)

Basic Protocol 3: Enzymatic Histology Using NADH Tetrazolium Reductase Staining

  Materials
  • β‐nicotinamide adenine dinucleotide disodium salt (NADH; e.g., Sigma)
  • Saline (see recipe)
  • Phosphate buffer (see recipe)
  • Nitro blue tertrazolium (NBT; see recipe)
  • Sections of biopsied skeletal muscle (or other tissue of interest), snap‐frozen and unfixed (see protocol 1)
  • Aqueous mounting medium (e.g., Aquatex, Merck)
  • Humidity chamber
  • Coverslips

Basic Protocol 4: Enzymatic Histology Using Succinate Dehydrogenase Staining

  Materials
  • NBT (see recipe)
  • Sodium succinate (see recipe)
  • Phosphate buffer (see recipe)
  • K‐EGTA (see recipe)
  • Sections of biopsied skeletal muscle (or other tissue of interest), snap‐frozen and unfixed (see protocol 1)
  • Aqueous mounting medium (e.g., Aquatex, Merck)
  • Humidity chamber
  • 37°C incubator
  • Coverslips

Basic Protocol 5: Enzymatic Histology Using COX Activity Staining

  Materials
  • 3,3′‐Diaminobenzidine tetrahydrochloride (DAB; e.g., Sigma), store at −20°C
  • Phosphate buffer (see recipe)
  • Catalase: 2 mg catalase from bovine liver (e.g., Sigma) in 10 ml distilled water, store at −20°C
  • Cytochrome c type III from horse heart (e.g., Sigma), store at −20°C
  • 1 N NaOH
  • Sections of biopsied skeletal muscle (or other tissue of interest), snap‐frozen and unfixed (see protocol 1)
  • 70%, 95%, and 100% ethanol solutions in distilled water
  • Xylene
  • Non‐aqueous mounting medium (e.g., Merckoglas, Merck)
  • pH meter
  • Humidity chamber
  • 37°C incubator
  • Coverslips

Basic Protocol 6: Enzymatic Histology Using ATPase Staining

  Materials
  • Biopsied skeletal muscle (or other tissue of interest), snap‐frozen and unfixed (see protocol 1)
  • pH 4.2 or pH 4.6 buffer (see reciperecipes)
  • Barbiturate buffer (see recipe)
  • 1% CaCl 2
  • 1 N NaOH
  • ATP disodium salt hydrate (e.g., Sigma)
  • 2% CoCl 2
  • 20% (NH 4) 2S stock in distilled water
  • 70%, 95%, and 100% ethanol in distilled water
  • Xylene
  • Non‐aqueous mounting medium (e.g., Merckoglas, Merck)
  • pH meter
  • Humidity chamber
  • Coverslips

Basic Protocol 7: Immunohistochemical Staining of OXPHOS in Frozen Skeletal Muscle

  Materials
  • Frozen muscle sections (see protocol 1)
  • Acetone, 4°C
  • Phosphate buffered saline (PBS; see recipe)
  • 2.5% BSA (see recipe)
  • Mouse monoclonal anti‐OXPHOS antibodies (e.g., Invitrogen or MitoSciences; see Table 19.2.1)
  • Biotinylated anti‐mouse antibody (e.g., LSAB2 kit, Dako)
  • HRP‐coupled streptavidin (e.g., LSAB2 kit, Dako)
  • 3,3′‐Diaminobenzidine (DAB; e.g., Dako)
  • Hematoxylin (e.g., Gill no. 2, Sigma, cat. no. GHS216)
  • Aqueous mounting medium (e.g., Aquatex, Merck)
  • Humidity chamber: plastic slide storage box with lid and a few drops of water under slides
  • Coverslips

Basic Protocol 8: Immunohistochemical Staining of OXPHOS in Paraffin‐Embedded Liver

  Materials
  • Paraffin sections (see protocol 2)
  • Xylene
  • 80%, 95%, and 100% ethanol solutions in distilled water
  • 2.5% BSA (see recipe)
  • Mouse monoclonal anti‐OXPHOS antibodies (e.g., Invitrogen or MitoSciences; see Table 19.2.1)
  • Phosphate buffered saline (PBS; see recipe)
  • AP‐labeled polymer (e.g., EnVision, Dako)
  • Fast red (e.g., Dako)
  • Hematoxylin (e.g., Gill no. 2, Sigma)
  • Aqueous mounting medium (e.g., Aquatex, Merck)
  • Humidity chamber
  • Coverslips

Basic Protocol 9: Immunocytochemical Staining of OXPHOS in Cultured Skin Fibroblasts

  Materials
  • Fibroblasts grown in culture medium in a 25‐cm2 tissue culture flask
  • Phosphate buffered saline (PBS; see recipe)
  • Acetone, 4°C
  • 2.5% BSA (see recipe)
  • Mouse monoclonal anti‐OXPHOS antibodies (e.g., Invitrogen or MitoSciences; see Table 19.2.1)
  • Biotinylated anti‐mouse secondary antibody (e.g., LSAB2, Dako)
  • HRP‐labeled streptavidin (e.g., LSAB2, Dako)
  • DAB (e.g., Dako)
  • Hematoxylin (e.g., Gill no. 2, Sigma)
  • Aqueous mounting medium (e.g., Aquatex, Merck)
  • Rubber policeman
  • Cytospin module: metal holder with SuperFrost Plus microscopic glass (e.g., Menzel‐Glaser), filters (e.g., Shandon), and funnel (e.g., Shandon)
  • Centrifuge (e.g., Cytospin, Shandon)
  • Coverslips
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Figures

Videos

Literature Cited

   Antonicka, H., Mattman, A., Carlson, C.G., Glerum, D.M., Hoffbuhr, K.C., Leary, S.C., Kennaway, N.G., and Shoubridge, E.A. 2003. Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway, causing early‐onset fatal hypertrophic cardiomyopathy. Am. J. Hum. Genet. 72:101‐114.
   Comi, G.P., Bordoni, A., Salani, S., Franceschina, L., Sciacco, M., Prelle, A., Fortunato, F., Zeviani, M., Napoli, L., Bresolin, N., Moggio, M., Ausenda, C.D., Taanman, J.W., and Scarlato, G. 1998. Cytochrome c oxidase subunit I microdeletion in a patient with motor neuron disease. Ann. Neurol. 43:110‐116.
   Copeland, W.C. 2008. Inherited mitochondrial diseases of DNA replication. Annu. Rev. Med. 59:131‐146.
   de Lonlay, P., Valnot, I., Barrientos, A., Gorbatyuk, M., Tzagoloff, A., Taanman, J.W., Benayoun, E., Chretien, D., Kadhom, N., Lombes, A., de Baulny, H.O., Niaudet, P., Munnich, A., Rustin, P., and Rotig, A. 2001. A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure. Nat. Genet. 29:57‐60.
   De Meirleir, L., Seneca, S., Lissens, W., De Clercq, I., Eyskens, F., Gerlo, E., Smet, J., Van Coster, R., Lazarou, M., Thornburn, D.R., Ryan, M.T., and McKenzie, M. 2004. Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP12. J. Med. Genet. 41:120‐124.
   De Paepe, B., Smet, J., Leroy, J.G., Seneca, S., George, E., Matthys, D., Van Malderghem, L., Scalais, E., Lissens, W., De Meirleir, L., Meulemans, A., and Van Coster, R. 2006. Diagnostic value of immunostaining in cultured skin fibroblasts from patients with oxidative phosphorylation defects. Pediatr. Res. 59:1‐5.
   De Paepe, B., Smet, J., Lammens, M., Seneca, S., Martin, J.J., De Bleecker, J.L., De Meirleir, L., Lissens, W., and Van Coster, R. 2009. Immunohistochemical analysis of the oxidative phosphorylation complexes in skeletal muscle from patients with mitochondrial DNA encoded tRNA gene defects. J. Clin. Pathol. 62:172‐176.
   DiMauro, S. and De Vivo, D.C. 1996. Genetic heterogeneity in Leigh syndrome. Ann. Neurol. 40:5‐7.
   Dunning, C.J., McKenzie, M., Sugiana, C., Lazarou, M., Silke, J., Connely, A., Fletcher, J.M., Kirby, D.M., Thornburn, D.R., and Ryan, M.T. 2007. Human CIA30 is involved in the early assembly of mitochondrial complex I and mutations in its gene cause disease. EMBO J. 26:3227‐3237.
   Keightley, J.A., Kristen, C.H., Burton, M.D., Salas, V.M., Johnston, W.S.W., Penn, A.M.W., Buist, N.R.M., and Kennaway, N.G. 1996. A microdeletion in cytochrome c oxidase (COX) subunit III associated with COX deficiency and recurrent myoglobinuria. Nat. Genet. 12:410‐416.
   Meulemans, A., De Paepe, B., De Bleecker, J.L., Smet, J., Lissens, W., Van Coster, R., De Meirleir, L., and Seneca, S. 2007. Two novel mitochondrial DNA mutations in muscle tissue of a patient with limb‐gridle myopathy. Arch. Neurol. 64:1339‐1343.
   Naviaux, R.K. 2004. Developing a systemic approach to the diagnosis and classification of mitochondrial disease. Mitochondrion 4:351‐361.
   Ogilvie, I., Kennaway, N.G., and Shoubridge, E.A. 2005. A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy. J. Clin. Invest. 115:2784‐2792.
   Papadopoulou, L.C., Sue, C.M., Davidson, M.M., Tanji, K., Nichino, I., Sadlock, J.E., Krishna, S., Walker, W., Selby, J., Glerum, D.M., Van Coster, R., Lyon, G., Scalais, E., Lebel, R., Kaplan, P., Shanske, S., De Vivo, D.C., Bonilla, E., Hirano, M., DiMauro, S., and Schon, E.A. 1999. Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene. Nat. Genet. 23:333‐337.
   Valnot, I., Von Kleist‐Retzow, J.C., Barrientos, A., Rotig, A., Rustin, P., and Munnich, A. 2000a. A mutation in the human heme A:farnesyltranseferase gene (COX10) causes cytochrome c oxidase deficiency. Hum. Mol. Genet. 9:1245‐1249.
   Valnot, I., Osmons, S., Gigarel, N., Mehaye, B., Amiel, J., Cormier‐Daire, V., Munnich, A., Bonnefont, J.P., Rustin, P., and Rotig, A. 2000b. Mutations of SCO1 gene in mitochondrial cytochrome c oxidase deficiency with neonatal‐onset hepatic failure and encephalopathy. Am. J. Hum. Genet. 67:1104‐1109.
   van den Heuvel, L. and Smeitinck, J. 2001. The oxidative phosphorylation (OXPHOS) system: Nuclear genes and human genetic diseases. BioEssays 23:518‐525.
   Visapaa, I., Fellman, V., Vesa, J., Dasvarma, A., Hutton, J.L., Kumar, V., Payne, G.S., Makarow, M., Van Coster, R.V., Taylor, R.W., Turnbull, D.M., Suomalainen, A., and Peltonen, L. 2002. GRACILE syndrome, a lethal metabolic disorder with iron overload, is caused by a point mutation in BCS1L. Am. J. Hum. Genet. 71:863‐876.
   Zhu, Z., Yao, J., Johns, T., Fu, K., De Bie, I., Macmillan, C., Cuthbert, A.P., Newbold, R.F., Wang, J., Chevrette, M., Brown, G.K., Brown, R.M., and Shoubridge, E.A. 1998. SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat. Genet. 20:337‐343.
   Zhu, X., Peng, X., Guan, M.Y., and Yan, Q. 2009. Pathogenic mutations of nuclear genes associated with mitochondrial disorders. Acta Biochim. Biophys. Sin. 41:179‐187.
Key References
   Copeland, 2008. See above.
  Provides a good overview of the genetics behind mtDNA replication defects.
   De Paepe, 2006. See above.
  Describes the technique and diagnostic potential of immunostaining for OXPHOS in patient fibroblasts.
   De Paepe, 2009. See above.
  Describes OXPHOS immunostaining patterns in muscle from patients with mtDNA tRNA gene defects.
   DiMauro and De Vivo, 1996. See above.
  Describes the complex genetics of Leigh syndrome.
   Naviaux, 2004. See above.
  Develops a diagnostic strategy for mitochondrial disease, integrating clinical data with laboratory findings.
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