Untangling the interactions between telomeres, oxidative stress, and aging

In vitro human cells have a limited replicative capacity before they enter a non-dividing state, senescence. Senescence is inducible by the presence of artificially shortened telomeres. A number of immortalized cell lines contain the enzyme telomerase, which functions to add telomeric repeats to the distal tip of the telomere as they are lost. Introduction of telomerase into normal human cells in vitro extends the cells' replicative capacity indefinitely¹. Since telomere lengths are, on average, shorter in older humans than younger humans, this raises the question of whether telomere length plays a causal role in the aging process. There are, however, a number of facts that argue against this. Mice, for example, have very long telomeres that do not shorten with age, but they age rapidly compared to humans.

The reactive oxygen species (ROS) aging hypothesis states that cumulative oxidative damage causes aging. The results of experiments attempting to test this hypothesis have been mixed. Overexpression of two key antioxidative enzymes, superoxide dismutase (SOD) and catalase, in Drosophila melanogaster results in increased longevity; however, this effect only seems to exist in strains that are unusually short-lived². In Drosophila strains that naturally have a relatively long life-span, there is no increase in life-span due to increased antioxidant enzyme activity³. In Caenorhabditis elegans, antioxidant enzyme mimetics increase the life-span of the organism by an average of 44% in wild-type organisms⁴. In mice, a decrease in antioxidant enzyme activity is linked to an apparent increase in the rate of aging⁵.

When exposed to hydrogen peroxide, single-strand breaks accumulate in unusually high numbers on telomeres in cultured human cells due to a specific lack of function of normal DNA repair mechanisms on the telomeres. This translates directly into accelerated telomere attrition, which is ameliorated by the introduction of an ROS scavenger into the cells⁶. Therefore, telomere attrition in human cells is affected by oxidative stress. This creates a link between the telomere and ROS hypotheses of aging. The age-dependent telomeric length difference may be caused, at least in part, by accumulated ROS damage.

In C. elegans, it has been shown that telomere length and aging are independent of each other⁷. This then raises the question of what the general relationship is between ROS levels and telomere attrition. Does an increase in ROS level correlate with telomeric attrition rate in other organisms, and, if so, is that process coupled to aging, or is it an unrelated effect?

Aim 1: To measure telomeric DNA single-strand breaks and oxidative stress as a function of age in human fibroblasts in vitro.

Hypothesis: Telomeric single-strand breaks may show an ROS-mediated increase with age, due to the correlation previously observed between these two factors.

Experimental Approach: I propose to measure telomeric DNA single-strand breaks and ROS levels in a wide age range of human fibroblasts, the same cell type used by Von Zglinicki et al⁶. Assay of ROS levels will be done by measuring levels of superoxide anion, hydrogen peroxide, and hydroxyl radical via a chemiluminescence assay, as described by Aam and Fonnum⁸: the oxidation of luminol by ROS releases photons that can be measured with a luminometer. This assay is performed on live cell cultures and is non-toxic to the cells. A similar assay will be used to measure the levels of SOD. Oxidation of the purine base xanthine to uric acid by the enzyme xanthine oxidase generates superoxide anions. SOD functions to catalyze the breakdown of superoxide anion, so it is possible to gauge SOD activity by measuring the decrease in intensity of light from the reaction of these superoxide anions with luminol.

Telomere lengths will be measured by isolating and restricting genomic DNA from the fibroblasts and running the DNA out on an agarose gel. This will be followed by hybridization of Southern-blotted genomic DNA to a radiolabeled telomeric repeat sequence probe (5'-TTAGGG-3'). Telomeric single-strand breaks will be quantified by a nuclease protection assay, measuring the relative sensitivities to S1 nuclease, an endonuclease that cuts single-stranded nucleic acids, with a strong preference for DNA.

Outcomes: The results obtained here will provide a clear picture of how ROS levels affect telomere length with age in human fibroblasts. If there is a correlation between these measurements, this will indicate that there is a true link between the telomere and ROS aging hypotheses. This data will also present a starting point for unraveling the mechanism for the age-dependence of telomere length in human cells. If no correlation exists, this data will serve as another nail in the coffin for the already beleaguered telomere aging hypothesis.

Aim 2: Compare telomeric ROS and antioxidant enzyme activity levels to telomeric attrition in C. elegans as a function of age.

Hypothesis: Oxidative stress and telomeric attrition may not be coupled in C. elegans, as they are in in vitro human cells, so that there would not be a correlation between ROS/antioxidant enzyme measurements and levels of telomeric single-strand breaks.

Experimental Approach: The approach here will be essentially to replicate the experiment described in (6) as well as that in aim 1 for C. elegans. We will gauge telomeric DNA single-strand breaks and attrition rate for worms exposed to paraquat, an ROS generator, over the life-span of the worms. Assay of ROS levels will be done using the Amplex Red hydrogen/hydrogen peroxide assay, as described by Chavez et al⁹. Telomere length and single-strand break data will be gauged in the same way as for human cells in aim 1.

Outcomes: This data will give a clearer indication of what the interrelations between telomere attrition, oxidative stress, and aging are, and whether they are similar to these processes in human cells. Since telomere length is not age-dependent in C. elegans, if these results show a similar pattern as those for human cells, this would be a piece of evidence that the age-dependence of human telomeres is not a causal factor in aging. If the pattern is different, this indicates that aging in humans and in C. elegans follow different mechanisms in this respect, and that the decoupling of telomere length and age in C. elegans cannot necessarily be extrapolated to humans.

References:

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