Age-related changes of mitochondrial structure and function in Caenorhabditis elegans

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Abstract

A number of observations have been made to examine the role that mitochrondrial energetics and superoxide anion production play in the aging of wild-type Caenorhabditis elegans. Ultrastructural analyses reveal the presence of swollen mitochondria, presumably produced by fusion events. Two key mitochondrial functions – the activity of two electron transport chain complexes and oxygen consumption – decreased as animals aged. Carbonylated proteins, one byproduct of oxidative stress, accumulated in mitochondria much more than in the cytoplasm. This is consistent with the notion that mitochondria are the primary source of endogenous reactive oxygen species. However, the level of mitochondrially generated superoxide anion did not change significantly during aging, suggesting that the accumulation of oxidative damage is not due to excessive production of superoxide anion in geriatric animals. In concert, these data support the notion that the mitochondrial function is an important aging determinant in wild-type C. elegans.

Introduction

It is known that energy metabolism figures prominently in the aging process (Balaban et al., 2005). In aerobic organisms, mitochondria are intimately responsible for ATP production via the electron transport chain [oxidative phosphorylation (OXPHOS) system]. Mitochondria also produce reactive oxygen species (ROS) such as superoxide anion (O2), hydrogen peroxide (H2O2), and hydroxyl radicals (radical dotOH) as byproducts of energy metabolism. Oxygen is converted to O2 by electron leakage from complex I and, to even a larger degree, complex III (Turrens et al., 1985, Lenaz, 1998, Finkel and Holbrook, 2000, Raha and Robinson, 2000). Such endogenously generated ROS readily attack a wide variety of cellular entities, resulting in damage that compromises cell integrity and function (Vuillaume, 1987, Collins et al., 1997). Aging and age-related degenerative diseases may be due to oxidative damage that results from an unfavorable balance between oxidative stress and antioxidant defenses, although it is difficult to distinguish causal events from the myriad of their secondary consequences (Beckman and Ames, 1998, Lenaz, 1998, Finkel and Holbrook, 2000, Raha and Robinson, 2000). For example, both carbonylated protein and lipofusucin accumulate with advancing age, but there is little straightforward evidence to suggest that they strongly act to limit life span (Gerstbrein et al., 2005, Yan et al., 1997; Yan and Sohal, 1998). However, accumulation is more rapid in short-lived mutants and slower in long-lived mutants, thus making these molecules excellent biomarkers of aging if nothing else (Hosokawa et al., 1994, Adachi et al., 1998, Yasuda et al., 1999, Gerstbrein et al., 2005).

Using oxygen consumption as a proxy of metabolism, several groups have demonstrated that metabolism in Caenorhabditis elegans decreases rapidly with aging (Braeckman et al., 2002a, Braeckman et al., 2002b, Houthoofd et al., 2002, Suda et al., 2005). We employed the novel method of Suda and co-workers for the measurement of energy metabolism, because this method is very simple and accurate. Interestingly, the decrease in respiration began soon after maturation, which is long before significant mortality occurred. In this study, we systematically examined age-related changes in mitochondrial structure and function, including energy metabolism and ROS production in aging populations of wild-type C. elegans. We document significant changes in mitochondrial structure and functionality with aging, thus supporting the notion that this organelle plays a key role in life span determination.

Section snippets

General methods

Wild-type C. elegans (N2) animals were cultured as previously described (Brenner, 1974). Embryos (eggs) were collected from nematode growth medium (NGM) agar plates using alkaline sodium hypochlorite (Emmons et al., 1979). The released eggs were allowed to hatch by overnight incubation at 20 °C in S basal buffer (100 mM NaCl, 50 mM potassium phosphate (pH 6.0)) (Sulston and Brenner, 1974). The newly hatched larvae (Ll-stage larvae) were cultured on NGM agar plates (3-fold Bacto-peptone) seeded

Ultrastructural examination of mitochondria in aging C. elegans

The structures of mitochondria in the body wall muscles of 4-, 10-, and 15-day-old animals were observed using transmission electron microscopy (Fig. 1). In the 4-day-old animals, most mitochondria were relatively small while only a few had long and thin morphologies. In the 10-day-old animals, the structure of some mitochondria was enlarged and swollen. The mitochondria in the 15-day-old animals, which still constituted 90% of the initial population, were more conspicuously enlarged and

Discussion

Mitochondria play an indispensable role in the generation of ATP in aerobic eukaryotes. Almost paradoxically, mitochondria are also the primary source of cellular oxidative stress, owing to endogenously generated ROS that result from the combination with free electrons with molecular oxygen. Once generated, ROS can readily attack a wide variety of cellular entities, resulting in cellular, tissue and organ damage that ultimately compromises organismal viability (Beckman and Ames, 1998, Demple

Acknowledgements

C. elegans wild type N2 animals were obtained from the Caenorhabditis Genetics Center. This work is supported by grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and for Aging Research from the Ministry of Health, Labor and Welfare, Japan.

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