Scott F. Leiser

Skin-derived fibroblasts from long-lived species are resistant to some, but not all, lethal stresses and to the mitochondrial inhibitor rotenone

Fibroblast cell lines were developed from skin biopsies of eight species of wild‐trapped rodents, one species of bat, and a group of genetically heterogeneous laboratory mice. Each cell line was tested in vitro for their resistance to six varieties of lethal stress, as well as for resistance to the nonlethal metabolic effects of the mitochondrial inhibitor rotenone and of culture at very low glucose levels. Standard linear regression of species‐specific lifespan against each species mean stress resistance showed that longevity was associated with resistance to death induced by cadmium and hydrogen peroxide, as well as with resistance to rotenone inhibition. A multilevel regression method supported these associations, and suggested a similar association for resistance to heat stress. Regressions for resistance to cadmium, peroxide, heat, and rotenone remained significant after various statistical adjustments for body weight. In contrast, cells from longer‐lived species did not show significantly greater resistance to ultraviolet light, paraquat, or the DNA alkylating agent methylmethanesulfonate. There was a strong correlation between species longevity and resistance to the metabolic effects of low‐glucose medium among the rodent cell lines, but this test did not distinguish mice and rats from the much longer‐lived little brown bat. These results are consistent with the idea that evolution of long‐lived species may require development of cellular resistance to several forms of lethal injury, and provide justification for evaluation of similar properties in a much wider range of mammals and bird species.

Nrf2 Signaling, a Mechanism for Cellular Stress Resistance in Long-Lived Mice

ABSTRACT Transcriptional regulation of the antioxidant response element (ARE) by Nrf2 is important for the cellular adaptive response to toxic insults. New data show that primary skin-derived fibroblasts from the long-lived Snell dwarf mutant mouse, previously shown to be resistant to many toxic stresses, have elevated levels of Nrf2 and of multiple Nrf2-sensitive ARE genes. Dwarf-derived fibroblasts exhibit many of the traits associated with enhanced activity of Nrf2/ARE, including higher levels of glutathione and resistance to plasma membrane lipid peroxidation. Treatment of control cells with arsenite, an inducer of Nrf2 activity, increases their resistance to paraquat, hydrogen peroxide, cadmium, and UV light, rendering these cells as stress resistant as untreated cells from dwarf mice. Furthermore, mRNA levels for some Nrf2-sensitive genes are elevated in at least some tissues of Snell dwarf mice, suggesting that the phenotypes observed in culture may be mirrored in vivo. Augmented activity of Nrf2 and ARE-responsive genes may coordinate many of the stress resistance traits seen in cells from these long-lived mutant mice.

HIF-1 modulates longevity and healthspan in a temperature-dependent manner

The hypoxia‐inducible factor HIF‐1 has recently been identified as an important modifier of longevity in the roundworm Caenorhabditis elegans. Studies have reported that HIF‐1 can function as both a positive and negative regulator of life span, and several disparate models have been proposed for the role of HIF in aging. Here, we resolve many of the apparent discrepancies between these studies. We find that stabilization of HIF‐1 increases life span robustly under all conditions tested; however, deletion of hif‐1 increases life span in a temperature‐dependent manner. Animals lacking HIF‐1 are long lived at 25°C but not at 15°C. We further report that deletion or RNAi knockdown of hif‐1 impairs healthspan at lower temperatures because of an age‐dependent loss of vulval integrity. Deletion of hif‐1 extends life span modestly at 20°C when animals displaying the vulval integrity defect are censored from the experimental data, but fails to extend life span if these animals are included. Knockdown of hif‐1 results in nuclear relocalization of the FOXO transcription factor DAF‐16, and DAF‐16 is required for life span extension from deletion of hif‐1 at all temperatures regardless of censoring.

Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span

Aging: All in the head—and the gut The effects of hypoxia and caloric restriction, both of which extend life span in Caenorhabditis elegans, converge on the activation of an enzyme in cells of the intestine. Leiser et al. show that the life-extending effects of hypoxia begin in neurons with transcriptional activation by hypoxia-inducible factor–1 and increased serotonergic signaling. These effects lead to increased production of flavin-containing monooxygenase-2 (FMO-2) in the intestine, which increased longevity. Finding the relevant targets of FMO-2, which also accumulates in mammals under conditions that promote longevity, may elucidate further mechanisms that promote healthy aging. Science, this issue p. 1375 Two life-span–extending pathways in the worm converge to increase production of an enzyme in the intestine. Stabilization of the hypoxia-inducible factor 1 (HIF-1) increases life span and health span in nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in Caenorhabditis elegans through a cell nonautonomous signal to the intestine, which results in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This prolongevity signal requires the serotonin biosynthetic enzyme TPH-1 in neurons and the serotonin receptor SER-7 in the intestine. Intestinal FMO-2 is also activated by dietary restriction (DR) and is necessary for DR-mediated life-span extension, which suggests that this enzyme represents a point of convergence for two distinct longevity pathways. FMOs are conserved in eukaryotes and induced by multiple life span–extending interventions in mice, which suggests that these enzymes may play a critical role in promoting health and longevity across phyla.

Correlated resistance to glucose deprivation and cytotoxic agents in fibroblast cell lines from long-lived pituitary dwarf mice

Fibroblast cell lines derived from the skin of young adult mice of the long-lived Snell dwarf mutant mouse stock have been shown to be resistant to the cytotoxic effects of multiple agents, including hydrogen peroxide, cadmium, heat, ultraviolet light, and the carcinogen methyl methanesulfonate. Snell dwarf fibroblasts are here reported to differ from control cell lines in two other respects: they are relatively resistant to the metabolic inhibition induced by low glucose concentrations, and also resistant to the effects of the mitochondrial poison rotenone, a blocker of Complex I of the electron transport chain. Furthermore, analysis of cell lines derived from a group of genetically heterogeneous mice established that cell lines resistant to peroxide-induced cytotoxicity were also relatively resistant to death induced by paraquat, cadmium, and ultraviolet light. Resistance to the metabolic effects of low glucose medium was associated with resistance to peroxide and cadmium in cells from heterogeneous mice and Snell dwarf mice, though unexpectedly not associated with resistance to the lethal effects of paraquat or UV light. Further analysis of the basis for metabolic abnormalities in these cell lines may provide insights into the cause of stress resistance in dwarf-derived cultures and to the longevity and disease-resistance of these long-lived mutant mice.

Life-Span Extension From Hypoxia in Caenorhabditis elegans Requires Both HIF-1 and DAF-16 and Is Antagonized by SKN-1

Stabilization of the hypoxia-inducible factor (HIF-1) protein extends longevity in Caenorhabditis elegans. However, stabilization of mammalian HIF-1α has been implicated in tumor growth and cancer development. Consequently, for the hypoxic response to benefit mammalian health, we must determine the components of the response that contribute to longevity, and separate them from those that cause harm in mammals. Here, we subject adult worms to low oxygen environments. We find that growth in hypoxia increases longevity in wild-type worms but not in animals lacking HIF-1 or DAF-16. Conversely, hypoxia shortens life span in combination with overexpression of the antioxidant stress response protein SKN-1. When combined with mutations in other longevity pathways or dietary restriction, hypoxia extends life span but to varying extents. Collectively, our results show that hypoxia modulates longevity in a complex manner, likely involving components in addition to HIF-1.

The hypoxic response and aging.

Cells, tissues, and organisms need to adapt to varying oxygen availability in order to survive. As a consequence, most organisms have evolved a response to low-oxygen environments, dubbed the hypoxic response. In mammals and most multicellular eukaryotes, the primary transcription factors responsible for this response are the hypoxia-inducible factors. Activation of these hypoxia-inducible factors protects from the damage associated with low oxygen and leads to changes in metabolism, vasculature, and cell survival. Recent work also suggests that the hypoxic response pathway can play an important role in aging. This chapter discusses the role of the hypoxic response pathway in protecting cells and organisms from low oxygen and how this pathway can act to affect aging and disease. We will also discuss the interactions between the hypoxic response and other aging-related pathways and how these pathways fit into aging research as a whole.

A Role for SIRT1 in the Hypoxic Response

In this issue of Molecular Cell, Lim et al. (2010) show that SIRT1 deacetylates HIF-1alpha and regulates its ability to respond to hypoxia, revealing yet another important function of SIRT1 and suggesting a connection between HIF function in aging and sirtuin enzymes.

Mechanisms of stress resistance in Snell dwarf mouse fibroblasts: enhanced antioxidant and DNA base excision repair capacity, but no differences in mitochondrial metabolism.

Dermal fibroblasts from long-lived Snell dwarf mice can withstand a variety of oxidative and non-oxidative stressors compared to normal littermate controls. Here, we report differences in the levels and activities of intracellular antioxidant and DNA repair enzymes between normal and Snell dwarf mice fibroblasts cultured under a variety of conditions, including: 3% and 20% ambient O(2); the presence and absence of serum; and the addition of an exogenous oxidative stress. The only significant difference between normal and dwarf cells cultured in complete medium, at 20% O(2), was an approximately 40% elevation of glutathione peroxidase (GPx) activity in the mutant cells. Serum deprivation elicited increases in GPx in both genotypes, but these activities remained higher in dwarf mouse cells. Dwarf mouse cells deprived of serum and challenged with exposure to paraquat or hydrogen peroxide showed a generally greater upregulation of catalase and DNA base excision repair enzymes. As these toxins can interact with mitochondria to increase mitochondrial ROS production, we explored whether there were differences in mitochondrial metabolism between normal and dwarf mouse cells. However, neither mitochondrial content nor the apparent mitochondrial membrane potential differed between genotypes. Overall, the results suggest that superior hydrogen peroxide metabolism and a marginally greater DNA base excision repair capacity contribute to the stress resistance phenotype of Snell dwarf mouse fibroblasts.

Environmental Canalization of Life Span and Gene Expression in Caenorhabditis elegans

Animals, particularly poikilotherms, exhibit distinct physiologies at different environmental temperatures. Here, we hypothesized that temperature-based differences in physiology could affect the amount of variation in complex quantitative traits. Specifically, we examined, in Caenorhabditis elegans, how different temperatures (15°C, 20°C, and 25°C) affected the amount of interindividual variation in life span and also expression of three reporter genes-transcriptional reporters for vit-2, gpd-2, and hsp-16.2 (a life-span biomarker). We found the expected inverse relationship between temperature and average life span. Surprisingly, we found that at the highest temperature, there were fewer differences between individuals in life span and less interindividual variation in expression of all three reporters. We suggest that growth at 25°C might canalize (reduce interindividual differences in) life span and expression of some genes by eliciting a small constitutive heat shock response. Growth at 25°C requires wild-type hsf-1, which encodes the main heat shock response transcriptional activator. We speculate that increased chaperone activity at 25°C may reduce interindividual variation in gene expression by increasing protein folding efficiency. We hypothesize that reduced variation in gene expression may ultimately cause reduced variation in life span.