Abstract
Investigation of mitochondrial metabolism perturbations and successful diagnosis of patients with mitochondrial abnormalities often requires assessment of human samples like muscle or liver biopsy as well as autopsy material. Immunohistochemical and histochemical examination is an important technique to investigate mitochondrial dysfunction that combined with spectrophotometric and Blue Native electrophoresis techniques can be an important tool to provide diagnosis of mitochondrial disorders. In this chapter, we focus on technical description of the methods that are suitable to detect the activity of complex I, II, and IV of mitochondrial respiratory chain in frozen sections of brain, heart, muscle, and liver biopsies/autopsy. The protocols provided can be useful not only for general assessment of mitochondrial activity in studied material, but they are also successfully used in the diagnostic procedures in case of suspicion of mitochondrial disorders. In the age of high-performance NGS sequencing, these methods can be used to confirm whether mutations are pathogenic by proving their impact on the activity of individual respiratory chain complexes.
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References
McBride HM, Neuspiel M, Wasiak S (2006) Mitochondria: more than just a powerhouse. Curr Biol 16:R551–R560
Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787–795
Maechler P, Wollheim CB (2001) Mitochondrial function in normal and diabetic beta-cells. Nature 414:807–812
McKenzie M, Liolitsa D, Hanna MG (2004) Mitochondrial disease: mutations and mechanisms. Neurochem Res 29:589–600
Taylor RW, Turnbull DM (2005) Mitochondrial DNA mutations in human disease. Nat Rev Genet 6:389–402
Sciacco M, Bonilla E (1996) Cytochemistry and immunocytochemistry of mitochondria in tissue sections. Methods Enzymol 264:509–521
Capaldi RA, Murray J, Byrne L, Janes MS, Marusich MF (2004) Immunological approaches to the characterization and diagnosis of mitochondrial disease. Mitochondrion 4:417–426
Pronicki M, Szymańska-Dębińska T, Karkucińska-Więckowska A, Krysiewicz E, Kaczmarewicz E, Bielecka L, Piekutowska-Abramczuk D, Sykut-Cegielska J, Cukrowska B, Więckowski MR (2008) Assessment of respiratory chain function in cultured fibroblasts using cytochemistry, immunocytochemistry and SDS-PAGE. Ann Diagn Pediatr Pathol 12:53–61
Kramer KA, Oglesbee D, Hartman SJ, Huey J, Anderson B, Magera MJ, Matern D, Rinaldo P, Robinson BH, Cameron JM, Hahn SH (2005) Automated spectrophotometric analysis of mitochondrial respiratory chain complex enzyme activities in cultured skin fibroblasts. Clin Chem 51(11):2110–2116
Karkucińska-Więckowska A, Czajka K, Wasilewski M, Sykut-Cegielska J, Pronicki M, Pronicka E, Zabłocki K, Duszyński J, Więckowski MR (2006) Blue native electrophoresis: an additional useful tool to study deficiencies of mitochondrial respiratory chain complexes. Ann Diagn Pediatr Pathol 10(3–4):89–92
Lebiedzinska M, Duszynski J, Wieckowski MR (2008) Application of “blue native” electrophoresis in the studies of mitochondrial respiratory chain complexes in physiology and pathology. Postepy Biochem 54:217–223
Piekutowska-Abramczuk D, Magner M, Popowska E, Pronicki M, Karczmarewicz E, Sykut-Cegielska J, Kmiec T, Jurkiewicz E, Szymanska-Debinska T, Bielecka L, Krajewska-Walasek M, Vesela K, Zeman J, Pronicka E (2009) SURF1 missense mutations promote a mild Leigh phenotype. Clin Genet 76(2):195–204
Pronicki M, Sykut-Cegielska J, Matyja E, Musialowicz J, Karczmarewicz E, Tonska K, Piechota J, Piekutowska-Abramczuk D, Kowalski P, Bartnik E (2007) G8363A mitochondrial DNA mutation is not a rare cause of Leigh syndrome - clinical, biochemical and pathological study of an affected child. Folia Neuropathol 45(4):187–191
Pronicki M, Matyja E, Piekutowska-Abramczuk D, Szymanska-Debinska T, Karkucinska-Wieckowska A, Karczmarewicz E, Grajkowska W, Kmiec T, Popowska E, Sykut-Cegielska J (2008) Light and electron microscopy characteristics of the muscle of patients with SURF1 gene mutations associated with Leigh disease. J Clin Pathol 61(4):460–466
Seligman AM, Karnovsky MJ, Wasserkrug HJ, Honker JS (1968) Non-droplet ultrastructural demonstration of cytochrome oxidase activity whit polymerizing osmiophilic reagent, DAB. J Cell Biol 38:1
Acknowledgments
This work was supported by the Internal Projects of The Children’s Memorial Health Institute No. S125/2012 for AKW and MP and by the Polish National Science Centre grant (UMO- 2018/29/B/NZ1/00589 for MRW).
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Wieckowski, M.R., Pronicki, M., Karkucinska-Wieckowska, A. (2021). Update on the Histoenzymatic Methods for Visualization of the Activity of Individual Mitochondrial Respiratory Chain Complexes in the Human Frozen Sections. In: Palmeira, C.M., Rolo, A.P. (eds) Mitochondrial Regulation. Methods in Molecular Biology, vol 2310. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1433-4_6
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DOI: https://doi.org/10.1007/978-1-0716-1433-4_6
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