Is There Still Any Role for Oxidative Stress in Mitochondrial DNA-Dependent Aging?

Abstract

Recent deep sequencing data has provided compelling evidence that the spectrum of somatic point mutations in mitochondrial DNA (mtDNA) in aging tissues lacks G > T transversion mutations. This fact cannot, however, be used as an argument for the missing contribution of reactive oxygen species (ROS) to mitochondria-related aging because it is probably caused by the nucleotide selectivity of mitochondrial DNA polymerase γ (POLG). In contrast to point mutations, the age-dependent accumulation of mitochondrial DNA deletions is, in light of recent experimental data, still explainable by the segregation of mutant molecules generated by the direct mutagenic effects of ROS (in particular, of HO· radicals formed from H₂O₂ by a Fenton reaction). The source of ROS remains controversial, because the mitochondrial contribution to tissue ROS production is probably lower than previously thought. Importantly, in the discussion about the potential role of oxidative stress in mitochondria-dependent aging, ROS generated by inflammation-linked processes and the distribution of free iron also require careful consideration.

Keywords: aging; mitochondrial DNA; oxidative stress; reactive oxygen species.

Figure 1

Atomic force microscopy (AFM) image of mitochondrial DNA (mtDNA) isolated from human skeletal muscle (essentially as described by [36]) by phenol extraction. The DNA molecules (0.6 ng/mL) were deposited on freshly cleaved mica in 4 mM HEPES-K (pH 7.4), 2 mM MgCl₂, and 10 mM NaCl for 5 min. The surface was rinsed with ultrapure distilled water and dried by blowing nitrogen gas. AFM imaging was performed on a Solver PRO AFM system (NT-MDT, Moscow, Russia), in a semicontact (tapping) mode, using Si-gold-coated cantilevers (NT-MDT) with a resonance frequency of 80–110 kHz. Molecules 1 and 2 are supercoiled and molecule 3 is linear mtDNA, all having a contour length of 5 µm.

Figure 2

Southern blot showing the time course of native mtDNA damage caused by 1 mM H₂O₂ and followed by the DNA repair by HEK293 cells. M: molecular weight marker; 0: control DNA (from untreated cells); The cells were exposed to 1 mM H₂O₂ and harvested after 30 min, 2 h, 4 h, 6 h, 24 h, respectively. 1 µg of total DNA was loaded on a 0.6% agarose gel prepared in tris-borate-EDTA (TBE) buffer and was run in the presence of 0.5 µg/mL ethidium bromide overnight at 40 V. After alkaline treatment and re-neutralization of the gel, the DNA was blotted to a Zeta-Probe membrane (Bio-Rad, Hercules, CA, USA) and immobilized by baking at 80 °C for 30 min. The blot was hybridized with a MT-ND6 probe.