Mitochondrial Dysfunction Accounts for the Stochastic Heterogeneity in Telomere-Dependent Senescence

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Abstract
Aging is an inherently stochastic process, and its hallmark is heterogeneity between organisms, cell types, and clonal populations, even in identical environments. The replicative lifespan of primary human cells is telomere dependent; however, its heterogeneity is not understood. We show that mitochondrial superoxide production increases with replicative age in human fibroblasts despite an adaptive UCP-2–dependent mitochondrial uncoupling. This mitochondrial dysfunction is accompanied by compromised [Ca2+]i homeostasis and other indicators of a retrograde response in senescent cells. Replicative senescence of human fibroblasts is delayed by mild mitochondrial uncoupling. Uncoupling reduces mitochondrial superoxide generation, slows down telomere shortening, and delays formation of telomeric γ-H2A.X foci. This indicates mitochondrial production of reactive oxygen species (ROS) as one of the causes of replicative senescence. By sorting early senescent (SES) cells from young proliferating fibroblast cultures, we show that SES cells have higher ROS levels, dysfunctional mitochondria, shorter telomeres, and telomeric γ-H2A.X foci. We propose that mitochondrial ROS is a major determinant of telomere-dependent senescence at the single-cell level that is responsible for cell-to-cell variation in replicative lifespan. After a limited number of cell divisions, somatic cells lose the capacity for proliferation, called cellular replicative senescence. Senescence, which is triggered by the loss of DNA sequences at the ends of chromosomes (telomeres), is often seen as an example of a regular “biological clock.” However, cell senescence is heterogeneous, with large differences in lifespan between individual cell lineages. This heterogeneity is clearly related to stress, specifically oxidative stress. It was not known, however, whether stress-induced “premature” senescence involves telomeres or is caused by telomere-independent DNA damage responses. Mitochondria are the most important source of reactive oxygen species (ROS) in cells under physiological conditions. We found that mitochondrial function deteriorated while cells approached senescence, leading to increased ROS production. Delaying mitochondrial dysfunction led to postponed replicative senescence and slowing of telomere shortening. Prematurely senescing cells sorted out of young cultures displayed mitochondrial dysfunction, increased oxidative stress, and short telomeres. We propose that replicative telomere-dependent senescence is not “clocked,” but rather is a stochastic process triggered largely by random mitochondrial dysfunction.