Clonally Expanded mtDNA Deletions in Human Skeletal Muscle Originate as a Proliferative Perinuclear Niche

Vincent, A.E.1, Rosa, S.H.1, Pabis, K1, Rygiel, K.A.1, Grunewald, A.1-3, Rocha, M.C.1, Reeve, A.K.1, Chen, C.1, Falkous, G,1. White, K.4, Davey, T.4, Petrof, B.5, Sayer, A.A.6, Cooper, C7., Taylor, R.W.1, Turnbull, D.M.1, Picard, M8,9.

1Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle, UK
2Institute of Neurogenetics, University of Lubeck, Lubeck, Germany
3Molecular and functional Neurobiology Group, University of Luxembourg, Luxembourgh, Luxembourg
4Electron Microscopy Research Services, Newcastle University, Newcastle, UK
5Meakins-Christie Laborotories, McGill University Health Centre, Montreal, Canada
6Newcastle NIHR Biomedical Research Centre, Newcastle University, UK
7MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton, UK
8Division of Behavioral Medicine, Columbia University Medical Center, New York, USA
9Department of Neurology and Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, USA.

Clonal expansion of mitochondrial DNA (mtDNA) deletions is of fundamental importance to the pathogenesis of certain mitochondrial diseases, neuromuscular diseases and ageing. The accumulating mutation impairs cellular bioenergetics contributes to the progression of mitochondrial disorders and age-related diseases. However, the origin and nature of this process remain incompletely resolved. Here, we investigate this question at sub-cellular resolution in patients with autosomally-driven multiple mtDNA deletions, single mtDNA deletions, inclusion body myositis and sarcopenia. Skeletal muscle biopsies were studied with histochemistry, immunofluorescence, electron microscopy, laser-capture microdissection and real-time PCR.

mtDNA deletions originate as a subsarcolemmal foci with concomitant histochemicalmcytochrome c oxidase (COX) deficiency adjacent to nuclei. Foci containing mtDNA deletions are associated with local elevations of both mitochondrial protein contentand mtDNA copy number, indicative of mitochondrial biogenesis. Given the requirement for nuclear gene products in biogenesis, perinuclear proximity may therefore account for the proliferate advantage enabling expansion of mutant mtDNA niches. In single cells with normal and mutant mtDNA, we evaluate the involvement of mito-nuclear signalling pathways using quantitative immunofluorescence against markers of mitochondrial biogenesis, mitophagy and the mitochondrial unfolded protein response. The subsequent spread of molecular and biochemical deficiency through the muscle fibres follows the three-dimensional topology of the mitochondrial network defined by electron microscopy, which is more continuous across the transverse plane of the muscle fibres. Together these data provide novel insights in to the origin and mechanism of clonal expansion of mtDNA deletion in human skeletal muscle.