Abstract
Charcot–Marie–Tooth neuropathy type 2A (CMT2A) is associated with heterozygous mutations in the mitochondrial protein mitofusin 2 (Mfn2) that is intimately involved with the outer mitochondrial membrane fusion machinery. The precise consequences of these mutations on oxidative phosphorylation are still a matter of dispute. Here, we investigate the functional effects of MFN2 mutations in skeletal muscle and cultured fibroblasts of four CMT2A patients applying high-resolution respirometry. While maximal activities of respiration of saponin-permeabilized muscle fibers and digitonin-permeabilized fibroblasts were only slightly affected by the MFN2 mutations, the sensitivity of active state oxygen consumption to azide, a cytochrome c oxidase (COX) inhibitor, was increased. The observed dysfunction of the mitochondrial respiratory chain can be explained by a twofold decrease in mitochondrial DNA (mtDNA) copy numbers. The only patient without detectable alterations of respiratory chain in skeletal muscle also had a normal mtDNA copy number. We detected higher levels of mtDNA deletions in CMT2A patients, which were more pronounced in the patient without mtDNA depletion. Detailed analysis of mtDNA deletion breakpoints showed that many deleted molecules were lacking essential parts of mtDNA required for replication. This is in line with the lack of clonal expansion for the majority of observed mtDNA deletions. In contrast to the copy number reduction, deletions are unlikely to contribute to the detected respiratory impairment because of their minor overall amounts in the patients. Taken together, our findings corroborate the hypothesis that MFN2 mutations alter mitochondrial oxidative phosphorylation by affecting mtDNA replication.
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Alexander C, Votruba M, Pesch UE, Thiselton DL, Mayer S, Moore A, Rodriguez M, Kellner U, Leo-Kottler B, Auburger G, Bhattacharya SS, Wissinger B (2000) OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28. Nat Genet 26:211–215
Amati-Bonneau P, Valentino ML, Reynier P, Gallardo ME, Bornstein B, Boissière A, Campos Y, Rivera H, de la Aleja JG, Carroccia R, Iommarini L, Labauge P, Figarella-Branger D, Marcorelles P, Furby A, Beauvais K, Letournel F, Liguori R, La Morgia C, Montagna P, Liguori M, Zanna C, Rugolo M, Cossarizza A, Wissinger B, Verny C, Schwarzenbacher R, Martín MA, Arenas J, Ayuso C, Garesse R, Lenaers G, Bonneau D, Carelli V (2008) OPA1 mutations induce mitochondrial DNA instability and optic atrophy ‘plus’ phenotypes. Brain 131:338–351
Amiott EA, Lott P, Soto J, Kang PB, McCaffery JM, DiMauro S, Abel ED, Flanigan KM, Lawson VH, Shaw JM (2008) Mitochondrial fusion and function in Charcot–Marie–Tooth 2A patient fibroblasts with mitofusin 2 mutations. Exp Neurol 211:115–127
Casasnovas C, Banchs I, Cassereau J, Gueguen N, Chevrollier A, Martínez-Matos JA, Bonneau D, Volpini V (2010) Phenotypic spectrum of MFN2 mutations in the Spanish population. J Med Genet 47:249–256
Chen H, Chomyn A, Chan DC (2005) Disruption of fusion results in mitochondrial heterogeneity and dysfunction. J Biol Chem 280:26185–26192
Chen H, McCaffery JM, Chan DC (2007) Mitochondrial fusion protects against neurodegeneration in the cerebellum. Cell 130:548–562
Chen H, Vermulst M, Wang YE, Chomyn A, Prolla TA, McCaffery JM, Chan DC (2010) Mitochondrial fusion is required for mtDNA stability in skeletal muscle and tolerance of mtDNA mutations. Cell 141:280–289
Delettre C, Lenaers G, Griffoin JM, Gigarel N, Lorenzo C, Belenguer P, Pelloquin L, Grosgeorge J, Turc-Carel C, Perret E, Astarie-Dequeker C, Lasquellec L, Arnaud B, Ducommun B, Kaplan J, Hamel CP (2000) Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat Genet 26:207–210
Dubowitz V, Sewry CA (2007) Muscle biopsy: a practical approach, 3rd edn. Saunders Elsevier, Philadelphia
Guillet V, Gueguen N, Verny C, Ferre M, Homedan C, Loiseau D, Procaccio V, Amati-Bonneau P, Bonneau D, Reynier P, Chevrollier A (2010) Adenine nucleotide translocase is involved in a mitochondrial coupling defect in MFN2-related Charcot-Marie-Tooth type 2A disease. Neurogenetics 11:127–133
Guillet V, Gueguen N, Cartoni R, Chevrollier A, Desquiret V, Angebault C, Amati-Bonneau P, Procaccio V, Bonneau D, Martinou JC, Reynier P (2011) Bioenergetic defect associated with mKATP channel opening in a mouse model carrying a mitofusin 2 mutation. FASEB J 25:1618–1627
Guo X, Popadin KY, Markuzon N, Orlov YL, Kraytsberg Y, Krishnan KJ, Zsurka G, Turnbull DM, Kunz WS, Khrapko K (2010) Repeats, longevity and the sources of mtDNA deletions: evidence from ‘deletional spectra’. Trends Genet 26:340–343
Heinrich R, Rapoport TA (1974) A linear steady-state treatment of enzymatic chains. General properties, control and effector strength. Eur J Biochem 42:89–95
Hudson G, Amati-Bonneau P, Blakely EL, Stewart JD, He L, Schaefer AM, Griffiths PG, Ahlqvist K, Suomalainen A, Reynier P, McFarland R, Turnbull DM, Chinnery PF, Taylor RW (2008) Mutation of OPA1 causes dominant optic atrophy with external ophthalmoplegia, ataxia, deafness and multiple mitochondrial DNA deletions: a novel disorder of mtDNA maintenance. Brain 131:329–337
Kacser H, Burns JA (1973) The control of flux. In: Davies DD (ed) Rate control of biological processes. Cambridge University Press, London, pp 64–109
Koshiba T, Detmer SA, Kaiser JT, Chen H, McCaffery JM, Chan DC (2004) Structural basis of mitochondrial tethering by mitofusin complexes. Science 305:858–862
Kudin AP, Kudina TA, Seyfried J, Vielhaber S, Beck H, Elger CE, Kunz WS (2002) Seizure-dependent modulation of mitochondrial oxidative phosphorylation in rat hippocampus. Eur J Neurosci 15:1105–1114
Kunz WS, Kudin A, Vielhaber S, Elger CE, Attardi G, Villani G (2000) Flux control of cytochrome c oxidase in human skeletal muscle. J Biol Chem 275:27741–27745
Kuznetsov AV, Winkler K, Kirches E, Lins H, Feistner H, Kunz WS (1997) Application of inhibitor titrations for the detection of oxidative phosphorylation defects in saponin-skinned muscle fibers of patients with mitochondrial diseases. Biochim Biophys Acta 1360:142–150
Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS (2008) Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3:965–976
Loiseau D, Chevrollier A, Verny C, Guillet V, Gueguen N, Pou de Crescenzo MA, Ferré M, Malinge MC, Guichet A, Nicolas G, Amati-Bonneau P, Malthièry Y, Bonneau D, Reynier P (2007) Mitochondrial coupling defect in Charcot–Marie–Tooth type 2A disease. Ann Neurol 61:315–323
Meeusen S, McCaffery JM, Nunnari J (2004) Mitochondrial fusion intermediates revealed in vitro. Science 305:1747–1752
Mita S, Rizzuto R, Moraes CT, Shanske S, Arnaudo E, Fabrizi GM, Koga Y, DiMauro S, Schon EA (1990) Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA. Nucl Acids Res 18:561–567
Payne BA, Wilson IJ, Hateley CA, Horvath R, Santibanez-Koref M, Samuels DC, Price DA, Chinnery PF (2011) Mitochondrial aging is accelerated by anti-retroviral therapy through the clonal expansion of mtDNA mutations. Nat Genet 43:806–810
Pich S, Bach D, Briones P, Liesa M, Camps M, Testar X, Palacín M, Zorzano A (2005) The Charcot–Marie–Tooth type 2A gene product, Mfn2, up-regulates fuel oxidation through expression of OXPHOS system. Hum Mol Genet 14:1405–1415
Rocher C, Taanman JW, Pierron D, Faustin B, Benard G, Rossignol R, Malgat M, Pedespan L, Letellier T (2008) Influence of mitochondrial DNA level on cellular energy metabolism: implications for mitochondrial diseases. J Bioenerg Biomembr 40:59–67
Rouzier C, Bannwarth S, Chaussenot A, Chevrollier A, Verschueren A, Bonello-Palot N, Fragaki K, Cano A, Pouget J, Pellissier JF, Procaccio V, Chabrol B, Paquis-Flucklinger V (2012) The MFN2 gene is responsible for mitochondrial DNA instability and optic atrophy ‘plus’ phenotype. Brain 135:23–34
Samuels DC, Schon EA, Chinnery PF (2004) Two direct repeats cause most human mtDNA deletions. Trends Genet 20:393–398
Sitarz KS, Yu-Wai-Man P, Pyle A, Stewart JD, Rautenstrauss B, Seeman P, Reilly MM, Horvath R, Chinnery PF (2012) MFN2 mutations cause compensatory mitochondrial DNA proliferation. Brain 135:e219
Song Z, Ghochani M, McCaffery JM, Frey TG, Chan DC (2009) Mitofusins and OPA1 mediate sequential steps in mitochondrial membrane fusion. Mol Biol Cell 20:3525–3532
Stumpf JD, Copeland WC (2011) Mitochondrial DNA replication and disease: insights from DNA polymerase γ mutations. Cell Mol Life Sci 68:219–233
Zsurka G, Baron M, Stewart JD, Kornblum C, Bös M, Sassen R, Taylor RW, Elger CE, Chinnery PF, Kunz WS (2008) Clonally expanded mitochondrial DNA mutations in epileptic individuals with mutated DNA polymerase gamma. J Neuropathol Exp Neurol 67:857–866
Züchner S, Mersiyanova IV, Muglia M, Bissar-Tadmouri N, Rochelle J, Dadali EL, Zappia M, Nelis E, Patitucci A, Senderek J, Parman Y, Evgrafov O, Jonghe PD, Takahashi Y, Tsuji S, Pericak-Vance MA, Quattrone A, Battaloglu E, Polyakov AV, Timmerman V, Schröder JM, Vance JM (2004) Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A. Nat Genet 36:449–451
Acknowledgments
The technical assistance of Sybille Tschorn (Magdeburg), Susanne Beyer, Karin Kappes-Horn, and Manuela Stepien-Mering (Bonn) is gratefully acknowledged. We would like to thank Jacqueline Koenig for language improvements. This work was supported by the Bundesministerium für Bildung und Forschung (mitoNET 01GM0868 to WSK), the Deutsche Forschungsgemeinschaft (SFB TR3 A11 and D12 to WSK), the Stiftung für Medizinische Wissenschaft, Frankfurt am Main (to WSK and SV), and the German Center for Neurodegenerative Diseases/DZNE (to SV).
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S. Vielhaber, G. Debska-Vielhaber, and V. Peeva have contributed equally to this work.
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Vielhaber, S., Debska-Vielhaber, G., Peeva, V. et al. Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion. Acta Neuropathol 125, 245–256 (2013). https://doi.org/10.1007/s00401-012-1036-y
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DOI: https://doi.org/10.1007/s00401-012-1036-y