Suppression of reactive oxygen species in cells with multiple mitochondrial DNA deletions by exogenous protein-coding RNAs
Introduction
Mutations and rearrangements of mitochondrial DNA (mtDNA) are associated with a number of human diseases as well as with the normal process of aging. Deletion derivatives of mtDNA (mtDNA-Δ) were originally observed in patients with maternally inherited or sporadic multi-system disorders such Kearns Sayre Syndrome (KSS), Pearson's Syndrome and Progressive External Opthalmoplegia (PEO) (Holt et al., 1988, Schon et al., 1989, Shoffner et al., 1989). Characteristically, single mtDNA-Δ's with different lengths and map positions are observed in different patients with a given disorder, although some, such as mtDNA4977, a 4.977 kb deletion are more common than others. Mapping of the deletion in a particular patient would enable tailor-made therapy by administration of the missing multi-gene fragment. We have recently described the phenotypic complementation of a single KSS-derived mtDNA-Δ by a polycistronic RNA directly delivered to mitochondria (Mahato et al, 2011).
Later studies of nerve, skeletal muscle, heart and other tissues from normally aging individuals revealed age-related accumulation of mtDNA-Δ, but here the situation is more complex than in the case of the overtly diseased patients described above. Two groups independently reported relatively high levels of mtDNA4977 in specific regions of aging human brain (caudate, putamen and substantia nigra) that have high dopamine metabolism (Soong et al., 1992, Corral-Debrinski et al., 1992). Subsequently, using long PCR techniques and the use of single cells or muscle fibers, it became apparent that there is considerable heterogeneity in the number and type of mtDNA-Δ. Liu et al., 1998, Kopsidas et al., 1998 reported the presence of multiple mtDNA-Δ's in whole skeletal muscle or in individual Cytochrome c Oxidase (COX)-deficient myofibers from the same or different individuals. A similar heterogeneity was observed in single cardiomyocytes from aged donors: each affected cell (up to 1 in 7) contained a single, different mtDNA-Δ, suggesting a clonal origin (Khrapko et al., 1999). Laser-assisted microdissection of single myofibers further showed the localized abundance of mtDNA-Δ in respiration-deficient regions (Cao et al., 2001). Clonality of mtDNA-Δ was also observed in single substantia nigra neurons of aged individuals (Kraytsberg et al., 2006). Taken together, these studies reveal that random genomic rearrangements generate multiple mtDNA-Δ's in different somatic tissues and that these molecules spread clonally within individual cells or intracellular domains. Heterogeneity complicates gene therapy by providing variable targets; furthermore, clonal spread of mtDNA-Δ's at the expense of wild type genomes makes it improbable that inefficient gene replacement strategies will succeed in producing a completely wild-type mitochondrial genotype.
The electron transport system of mitochondria is the predominant cellular site of generation of Reactive Oxygen Species (ROS) such as superoxide radical (O2−) and hydrogen peroxide (H2O2) which have mutagenic and other deleterious effects and are believed to contribute to aging (Balaban et al., 2005). Inhibition of respiratory complexes I or III, or reverse electron flow, results in enhanced ROS generation (Li et al., 2003, Han et al., 2008).A question of relevance in the context of mtDNA-Δ's that accumulate during aging is whether such deletions are responsible for generating ROS.
In spite of the accumulating evidence for clonality and heterogeneity of mtDNA-Δ in aging cells and tissues, there is currently no available cellular model with these features to test potential therapeutic protocols. Treatment of cultured cells with ethidium bromide (EtBr), a DNA intercalating agent that preferentially induces mtDNA-Δ, typically leads to the terminal rho-zero state, i.e. one with a total lack of mtDNA. Ideally, the cells should stably harbor multiple mtDNA-Δs, with resultant phenotypic effects. Such a cell line would enable the effects of mitochondrion-targeted therapies to be assessed.
The problem of accumulating mtDNA-Δ could be addressed by delivery of the appropriate gene or RNA to mitochondria. The delivery method is based on our previous observation that a multi-subunit protein complex from a kinetoplastid protozoon that normally binds to and translocates tRNAs across the mitochondrial membrane (the RNA Import Complex or RIC), is taken up by mammalian cells and targeted to mitochondria (Mahata et al., 2006). We have recently shown that large polycistronic RNAs (pcRNA) are transferred by a derivative of RIC (R8) to mitochondria of cells harboring a single patient-derived mtDNA-Δ (Mahato et al, 2011). In this study, we have generated a cell line with multiple mtDNA-Δs, and studied the effects of such pcRNAs on the respiratory capacity and ROS generation.
Section snippets
Isolation of EBΔ1
Cultured HepG2 (hepatocarcinoma) cells were treated with various concentrations of ethidium bromide (EtBr, 5–500 μg/ml) for 25 d in DMEM containing high uridine and pyruvate (50 and 110 μg/ml respectively; or 50/110), washed free of mutagen and cultured in the same medium for 45 d. The growth of the various cultures was then tested in DMEM lacking uridine and pyruvate (0/0). Cultures treated with 100 μg/ml or more of ethidium did not survive in this medium. However, with intermediate levels of the
Accumulation of mtDNA-Δ's in cells treated with a low dose of ethidium bromide
To develop a cellular model of mtDNA-Δ, the hepatocarcinoma cell line HepG2 was treated with low doses of EtBr. The dose was kept low in order to avoid complete deletion of mtDNA (thereby creating a rho-zero phenotype). After EtBr treatment, cells were serially diluted and each dilution grown out for 15 d before the mtDNA region between the ND1 and ND4L genes was amplified by PCR. In all the treated samples, two sub-genomic bands representing mtDNA-Δ (designated as Δ1.2 and Δ1.3 respectively)
Discussion
We show here that multiple mtDNA deletions are selected for in cultured cells treated with low doses of ethidium bromide. In these cells, there is a general down-regulation of mitochondrial transcripts resulting in loss of respiratory capacity, a switch to a glycolytic growth mode, and significantly higher superoxide levels. These features resemble the accumulation of mtDNA-Δ in cells and tissues of aging individuals, although the breakpoints of the ethidium-generated deletions (Fig. 1D) were
Acknowledgements
We thank Santu Bandyopadhyay for the FACS analysis, Siddhartha Jana and Anupam Banerjee for confocal microscopy, Ashok Giri for anti-COXIV antibody, Susanta Roychowdhury for DNA sequencing, Uday Bandyopadhyay for PBN and Tapas Chowdhury for technical assistance. Supported by Suprainstitutional Project number SIP0007 from the Council of Scientific and Industrial Research (CSIR). S.J. was supported by a Senior Research Fellowship from CSIR.
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