Heterogeneity and Mitochondria

There is a good experimental basis to consider a possible involvement of mitochondria in ageing, mainly from data obtained in postmitotic tissues. Mitochondria are the major source of oxygen-free radicals in cells, which by their very nature are highly unstable molecules with an unpaired electron in the outer orbital, which can react indiscriminately with any organic molecules present in a cell.

Moreover, one of the key observations when accessing mitochondrial function and levels of mtDNA mutations in tissues is that both vary considerably from cell to cell. Thus, changes in mitochondrial function might be the missing link explaining cell-to-cell heterogeneity in telomeres and, consequently, in cellular senescence.

The main cause for increased mitochondrial production of free radicals is still unclear. One suggestion has been accumulation of mutations in the mitochondrial (mt) DNA (Harman 1972). Mitochondria, like telomeres, are highly susceptible to oxidative damage - and for good reasons. There is close physical proximity between the sites of reactive oxygen species production and the mtDNA. Moreover, there is relative inefficiency of the repair mechanisms in mitochondria and, unlike the nuclear genome, the mitochondrial genome is not protected by histones. Accordingly, mtDNA damage was found to be more extensive and persistent than nuclear DNA damage in cultured human fibroblasts following treatment with hydrogen peroxide (Yakes and Van Houten 1997). This damage might have functional consequences, since mtDNA damage in immortalized normal human fibroblasts (NHF) after hydrogen peroxide exposure has been shown to correlate with decreased mitochondrial membrane potential (Santos et al. 2003).

MtDNA mutations are responsible for deficient activity of respiratory chain enzymes such as cytochrome C oxidase, which is a mitochondrial membrane-bound enzyme composed of subunits that are encoded in both the mitochondria (COX subunits I, II, and III) and the nucleus (all others). Muller-Hocker showed that mutant mitochondria deficient in cytochrome c oxidase (COX) are distributed in the muscle in a few fiber segments, where the surrounding fibers are normal (Muller-Hocker 1989). The frequency of COX-deficient fibers was shown to increase significantly with age, but remained always below 1%. In situ hybridization studies using mtDNA probes from various regions of the mitochondrial genome showed accumulation of mtDNA deletions in COX-negative cells (Lee et al. 1998). The availability of microdissection techniques facilitated the direct analysis of single cells in histological sections. Using this technique (Cao et al. 2001, Wanagat et al. 2001), it was shown that all electron transport chain-deficient fibers in rat skeletal muscle contained mtDNA deletion mutations. In single neurons from substantia nigra of humans showing COX deficiency, an age-dependent increase in mtDNA deletions was found in two independent studies (Bender et al. 2006, Kraytsberg et al. 2006). Interestingly, these data reveal that interneuronal variation (measured as median absolute deviation) in the percentage of mtDNA deletions increased significantly with age.

Other recent data showed that extensive, stochastic variability in the presence and level of mitochondrial DNA mutations occurs in human colon stem cells, one of the most actively proliferating cell types in the body (Taylor et al. 2003). This result demonstrates that heterogeneity in mitochondrial mutation load is not exclusive to postmitotic cells.

Nonetheless, recent work has questioned the association between mtDNA mutation load and mitochondrial dysfunction as a major cause of aging. It has been shown that homozygous knock-in mice that express a proof-reading-deficient version of the nucleus-encoded catalytic subunit of mtDNA polymerase y (PolgA) accumulated very high levels of mtDNA mutations and deletions and showed a significant decrease in life span (Trifunovic et al. 2004). However, the mechanism for this phenomenon remains unknown, since no increased markers of oxidative stress, mitochondrial dysfunction or defects in cellular proliferation were found in these animals (Kujoth et al. 2005, Trifunovic et al. 2005). Importantly, mice that are heterozygous for PolgA function show no significant reduction in life span despite a mtDNA mutation burden 30 times higher than in old wild-type animals (Vermulst et al. 2007). These studies suggest that mtDNA mutation load does not limit life span of wild-type mice and that mtDNA mutations, even at very high levels, do not necessarily lead to increased mitochondrial ROS generation. Mitochondrial dysfunction and generation of reactive oxygen species might be consequences of other still unexplored factors occurring with cell ageing.

With regard to cell senescence in culture, one can hypothesize that variation in mitochondrial function between cells could determine changes in their replicative potential. It is clear in mass populations of fibroblasts that mitochondrial function varies significantly between cells and determines their replicative life span, as we shall discuss in the next section (Passos et al. 2007 and see Fig. 3.2).

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