MtDNA point mutations are associated with deletion mutations in aged rat Jeong W. Pak, Fue Vang, Chad Johnson, Debbie McKenzie, Judd M. Aiken * Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1656 Linden Drive, Madison, WI 53706, USA Received 17 September 2004; received in revised form 3 December 2004; accepted 10 December 2004 Available online 18 January 2005 Abstract The age-dependent accumulation of point mutations in the control region of human mtDNA has been suggested to contribute to aging processes. We investigated whether mtDNA point mutations accumulate to detectable levels in this region of mtDNA from aged Fischer 344 X Brown Norway F 1 hybrid rats. The control region and a portion of the major arc region (nucleotides 4386–7707) of the mtDNA were PCR- amplified and directly sequenced from microdissected single cardiomyocytes and single skeletal muscle fibers of 36-month old rats. Point mutations were not observed in these regions of the full-length mtDNA. Point mutations were, however, associated with deletion mutations, especially in cardiac cells. Approximately 40% of the deletion mutations identified in heart contained a point mutation, whereas only 1.9% of deletion mutations in skeletal muscle contained a point mutation. Point mutations were located adjacent to the deletion breakpoints and each point mutation was unique. In aged rats, point mutations are clonally expanded only when associated with deletion events suggesting that there are important differences between rats and humans in the mechanisms that cause mtDNA abnormalities. q 2005 Elsevier Inc. All rights reserved. Keywords: Mitochondrial DNA; Point mutation; Deletion mutation; Laser-capture microdissection 1. Introduction The accumulation of damage to mitochondrial macro- molecules as an underlying mechanism of aging was proposed decades ago (Harman, 1983). Screening for mitochondrial DNA (mtDNA) abnormalities identified age-induced deletion mutations in numerous species, primarily in nervous tissue, muscle and heart (Cortopassi et al., 1992; Zhang et al., 1992; Wanagat et al., 2001). The impact of these genomic alterations in skeletal muscle includes concomitant phenotypic expression of mitochon- drial electron transport system (ETS) abnormalities and subsequent fiber atrophy and muscle fiber breakage (Cao et al., 2001; Wanagat et al., 2001). Single cell analysis has identified high levels of age-related somatic point mutations in some tissues (Jazin et al., 1996; Michikawa et al., 1999; Calloway et al., 2000; Murdock et al., 2000; Taylor et al., 2001; Wang et al., 2001; Nekhaeva et al., 2002). The majority of these studies have focused primarily on the control region (D-loop region, w1 kb) of the human mitochondrial genome. The total load of mtDNA point mutations in human cardiac and buccal cells, based on those identified in the control region, was postulated to be as high as one expanded point mutation per cell on average (Nekhaeva et al., 2002). Point mutations in the control region were not linked to other mtDNA mutations and accumulated to greater than 50% in individual cardiomyo- cytes. Since the control region is crucial for replication and transcription of the mitochondrial genome, the abundance and accumulation of point mutations in the control region from aged human tissues suggested that point mutations play a role in aging. Although single nucleotide mutations were shown to accumulate with age in humans, they have not been linked to abnormal phenotypes nor is it known if the age-associated clonal accumulation of point mutations occurs in other species. Transgenic mice expressing a proof-reading deficient version of the catalytic subunit of mtDNA polymerase have provided strong evidence that mtDNA rearrangements can cause aging phenotypes (Trifunovic et al., 2004). These mice exhibited increased levels of single nucleotide mtDNA mutations as well as mtDNA deletion mutations with age. 0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2004.12.005 Experimental Gerontology 40 (2005) 209–218 www.elsevier.com/locate/expgero * Corresponding author. Tel.: C1 608 262 7362; fax: C1 608 262 7420. E-mail addresses: aiken@svm.vetmed.wisc.edu (J.M. Aiken), jma@ ahabs.wisc.edu (J.M. Aiken).