Neurobiology of Aging 24 (2003) 829–838 High mutational burden in the mtDNA control region from aged muscles: a single-fiber study Roberto Del Bo a,∗ , Marco Crimi a , Monica Sciacco a , Giulia Malferrari b , Andreina Bordoni a , Laura Napoli a , Alessandro Prelle a , Ida Biunno b , Maurizio Moggio a , Nereo Bresolin a,c , Guglielmo Scarlato a,† , Giacomo Pietro Comi a a Centro Dino Ferrari, Centro di Eccellenza per le Malattie Neurodegenerative, Dipartimento di Scienze Neurologiche, Università degli Studi di Milano, I.R.C.C.S. Ospedale Maggiore Policlinico, Via F. Sforza, 35, 20122 Milan, Italy b Istituto Tecnologie Biomediche Avanzate (ITBA)-CNR, Centro Interdisciplinare Studi bio-molecolari e applicazioni Industriali (CISI), Milan, Italy c I.R.C.C.S. E. Medea de La Nostra Famiglia, Bosisio Parini, Italy Received 19 June 2002; received in revised form 17 October 2002; accepted 20 November 2002 Abstract The ageing process is associated with the accumulation of somatic mutations of mitochondrial DNA (mtDNA). The aged human skeletal muscle tissue presents a mosaic of fibers when stained histochemically for cytochrome c oxidase (COX) activity with a proportion of COX negative fibers. Given the potential relevance of any alteration in the mtDNA control region for replication, we analysed the correlation between the presence of mutations and their degree of heteroplasmy and the COX phenotype in individual muscle fibers of aged healthy donors. A region of the mtDNA D-loop was cloned from single fiber-derived DNA and multiple clones were analysed. This strategy showed that a high level of mutational burden is present in all fibers and that several types of mtDNA rearrangements are detectable: recurrent (A189G, T408A and T414G) and rare point mutations, length variations affecting the homopolymeric tract and the (CA) n repeat and macrodeletions. The aggregate mutational load in the D-loop region correlated with the single fiber COX phenotype, suggesting that the cumulative burden of multiple, individually rare, mtDNA alterations might functionally impair the mitochondrial genetic machinery. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Mitochondrial DNA; Somatic point mutations; Ageing; Muscle; Single fiber 1. Introduction Human mitochondrial DNA (mtDNA) is a circular double-stranded DNA of 16,569 bp in size. The genome contains genes coding for 13 polypeptides involved in res- piration and oxidative phosphorylation, 2 rRNAs and a set of 22 tRNAs that are essential for protein synthesis of the mitochondria. In contrast to the nuclear DNA, mtDNA is a naked compact DNA molecule without introns and is replicated at a much higher rate without an efficient DNA repair mechanism. Therefore, mtDNA is more vulnerable to attacks by reactive oxygen species and free radicals that are generated by electron leak of the respiratory chain of mitochondria [38]. In the past few years a number of point † G. Scarlato recently died. ∗ Corresponding author. Tel.: +39-02-55033817; fax: +39-02-50320430. E-mail address: gpcomi@mailserver.unimi.it (R. Del Bo). mutations, deletions and insertions have been found in mtDNA from patients with specific neuromuscular diseases [4]. Several mtDNA deletions have also been identified in various tissues of old humans [12–14]. Since mutant mtDNA, or mitochondria containing high levels of mu- tant mtDNA, may, in certain situations, have a replicative advantage within the cell, they might, with time, cause a specific biochemical effect [44]. Somatic point mutations and deletions in mtDNA have been established to increase in an age-dependent manner in humans: the accumulation of deleterious alterations in the mitochondrial genome has been proposed to be the major cause of the respiratory function decline during human ageing process [15,41]. Several reports show that skeletal muscle develops a his- tochemical mosaic during the process of normal ageing; in particular, the frequency of cytochrome c oxidase (COX) deficient muscle fibers is highly dependent on the age of the subject [6,31]. It has been hypothesised that multiple mtDNA rearrangements occurring in muscle tissue from 0197-4580/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0197-4580(02)00233-6