Mitochondrial Dysfunction Causes Telomere Dysfunction and Thus Heterogeneity in Replicative Senescence

Are mitochondria at all causally involved in the process of replicative senescence? There is increasing evidence that they do play an important role. Using fluorescent dyes which measure cellular peroxides, some studies showed that senescent cells

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Fig. 3.2 Confocal image of live young fibroblasts staining with JC-1. The dye emits green fluorescence at low concentrations and red fluorescence at high density. It is transported into mitochondria in a membrane potential (MMP)-dependent fashion, so that green indicates low MMP, while mitochondria stained red have high MMP. Note the cell-to-cell heterogeneity in JC-1 ratios, as well as intracellular heterogeneity (See Color Plate)

Fig. 3.2 Confocal image of live young fibroblasts staining with JC-1. The dye emits green fluorescence at low concentrations and red fluorescence at high density. It is transported into mitochondria in a membrane potential (MMP)-dependent fashion, so that green indicates low MMP, while mitochondria stained red have high MMP. Note the cell-to-cell heterogeneity in JC-1 ratios, as well as intracellular heterogeneity (See Color Plate)

were associated with high levels of endogenous reactive oxygen species (Hutter et al. 2002, Allen et al. 1999). Accumulation of oxidation products, such as protein carbonyls and lipofuscin, occurs in senescent fibroblasts grown in vitro (Sitte et al. 2000, Sitte et al. 2001). More recently, we have shown that senescent cells display mitochondrial dysfunction, characterized by lower mitochondrial membrane potential, mtDNA damage, and increased superoxide production (Passos et al. 2007). This was in accordance with earlier reports showing that senescent fibroblasts have impaired metabolism, with strong reduction of ATP and other nucleotide triphosphates (Zwerschke et al. 2003).

Beyond mere correlation, there is evidence supporting a causal role for mitochondrial dysfunction in telomere-dependent senescence. In one study, mitochondria from fibroblasts of 21 individuals between 20 weeks and 103 years of age were introduced into human mtDNA-less cells. The authors found both a slight age-dependent decrease in growth rate and a decline in respiratory rate in the transformants, suggesting a causal role of mitochondria in the senescence process (Laderman et al. 1996). Selective targeting of antioxidants directly to the mitochondria counteracted telomere shortening and increased life span in fibroblasts under mild oxidative stress (Saretzki et al. 2003). Continuous treatment with nicotinamide, which led to lower ROS generation and changes in mitochondrial function, has been reported to extend life span (a remarkable 1.6-fold increase) and decelerate telomere shortening (Kang et al. 2006). Also, pharmacological mild chronic uncoupling of mitochondria that reduced the production of superoxide anion improved telomere maintenance and extended telomere-dependent life span (Passos et al. 2007).

Accordingly, mitochondrial dysfunction generated by severe mitochondrial depolarization using an uncoupling agent (FCCP) led to an increased production of ROS, telomere attrition, telomere loss, and chromosome fusion in mouse embryos (Liu et al. 2002). Moreover, Oexle and Zwirner (1997) showed that patients with mitochondrial diseases MEL AS and LHON had, on average, 1.5 kb shorter telomeres than those of age-matched controls.

Thus, there is good evidence for a causal role of mitochondria in telomere-dependent senescence. Based on these data, it is reasonable to assume that cell-to-cell variation in mitochondrial function would impact telomere length and might explain heterogeneity in the replicative potential of cells. A stochastic network model, combining telomere attrition with other mechanisms such as oxidative stress and mitochondrial and nuclear mutations, was able to show good agreement with published data on heterogeneity in division potential (Sozou and Kirkwood 2001).

In a series of FACS sorting experiments, we have recently proven the interconnection between mitochondrial dysfunction, ROS generation, and accelerated telomere shortening as cause for cell-to-cell heterogeneity of division potential of human fibroblasts (Martin-Ruiz et al. 2004, Passos et al. 2007, von Zglinicki et al. 2003).

Senescent cells present in a culture of early passage fibroblasts have been shown to have higher levels of reactive oxygen species, together with decreased mitochondrial membrane potential, which is indicative of metabolic inefficiency. These sorted cells showed high frequency of telomeric y-H2A.X foci and shorter telomeres than the remainder of the population, indicating that mitochondrial ROS generation could be targeting telomeres specifically. Additionally, sorting of cells at early passage according to mitochondrial superoxide production (fibroblasts were stained with MitoSOX) showed that the cells which produced higher levels of superoxide were also the ones which contained the highest frequency of telom-eric Y-H2A.X foci (Passos et al. 2007).

These results support a model in which telomeres are sensors of mitochondrial function/dysfunction (Passos and von Zglinicki 2005) and replicative life span of individual cells is determined by a network of processes involving mitochondrial dysfunction, oxidative stress, and telomere shortening (Sozou and Kirkwood 2001). It is clear that stress levels (external and/or internal) will determine the relative importance of the different components of the network for induction of senescence. Thus, at very high levels of stress (for instance, caused by ionizing radiation), sufficient unrepairable DNA damage will occur to arrest cells in telomere-independent senescence. Under relatively mild stress conditions (typically cell culture under 20%-40% oxygen partial pressure) accelerated telomere shortening due to mitochondrial^ derived ROS appears to be the major route toward senescence and the main cause for its heterogeneity. Under constant low oxidative stress conditions or improved mitochondrial function, the "end replication problem" becomes more important as a cause of telomere shortening and there is less heterogeneity in the life span of cells (Passos et al. 2007).

However, the proximal cause for mitochondrial dysfunction in senescence and its heterogeneity remains still unclear. Is it to do with random accumulation of mtDNA mutations, mtDNA damage, or other changes (like differential gene expression) that can affect mitochondrial function and assembly? This is an important area of research which has yet to be further explored.

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