methodology-to-metabolically-inactivate-bacteria-for-caenorhabditis

Methodology to Metabolically Inactivate Bacteria for <em>Caenorhabditis elegans</em> Research

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a ...

University of Michigan, Ann Arbor

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.

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

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.

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

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.

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

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.

Nrf2 signaling, a mechanism for cellular stress resistance in long-lived mice.

The hypoxic response is a well-studied and highly conserved biological response to low oxygen availability. First described more than 20 y ago, the traditional model for this response is that declining oxygen levels lead to stabilization of hypoxia-inducible transcription factors (HIFs), which then bind to hypoxia responsive elements (HREs) in target genes to mediate the transcriptional changes collectively known as the hypoxic response.(1,2) Recent work in C. elegans has forced a re-evaluation of this model by indicating that the worm HIF (HIF-1) can mediate effects in a cell non-autonomous fashion and, in at least one case, increase expression of an intestinal hypoxic response target gene in cells lacking HIF-1. 

New insights into cell non-autonomous mechanisms of the C. elegans hypoxic response.

Flavin-containing monooxygenases (FMOs) are primarily studied as xenobiotic metabolizing enzymes with a prominent role in drug metabolism. In contrast, endogenous functions and substrates of FMOs are less well understood. A growing body of recent evidence, however, implicates FMOs in aging, several diseases, and metabolic pathways. The evidence suggests an important role for these well-conserved proteins in multiple processes and raises questions about the endogenous substrate(s) and regulation of FMOs. Here, we present an overview of evidence for FMOs' involvement in aging and disease, discussing the biological context and arguing for increased investigation into the function of these enzymes.

Flavin-containing monooxygenases in aging and disease: Emerging roles for ancient enzymes.

As in other poikilotherms, longevity in C. elegans varies inversely with temperature; worms are longer-lived at lower temperatures. While this observation may seem intuitive based on thermodynamics, the molecular and genetic basis for this phenomenon is not well understood. Several recent reports have argued that lifespan changes across temperatures are genetically controlled by temperature-specific gene regulation. Here, we provide data that both corroborate those studies and suggest that temperature-specific longevity is more the rule than the exception. By measuring the lifespans of worms with single modifications reported to be important for longevity at 15, 20, or 25 °C, we find that the effect of each modification on lifespan is highly dependent on temperature. Our results suggest that genetics play a major role in temperature-associated longevity and are consistent with the hypothesis that while aging in C. elegans is slowed by decreasing temperature, the major cause(s) of death may also be modified, leading to different genes and pathways becoming more or less important at different temperatures. These differential mechanisms of age-related death are not unlike what is observed in humans, where environmental conditions lead to development of different diseases of aging.

Genetic interaction with temperature is an important determinant of nematode longevity.

HIF-1-mediated adaptation to changes in oxygen availability is a critical aspect of healthy physiology. HIF is regulated by a conserved mechanism whereby EGLN/PHD family members hydroxylate HIF in an oxygen-dependent manner, targeting it for ubiquitination by Von-Hippel-Lindau (VHL) family members, leading to its proteasomal degradation. The activity of the only C. elegans PHD family member, EGL-9, is also regulated by a hydrogen sulfide sensing cysteine-synthetase-like protein, CYSL-1, which is, in turn, regulated by RHY-1/acyltransferase. Over the last decade, multiple seminal studies have established a role for the hypoxic response in regulating longevity, with mutations in vhl-1 substantially extending C. elegans lifespan through a HIF-1-dependent mechanism. However, studies on other components of the hypoxic signaling pathway that similarly stabilize HIF-1 have shown more mixed results, suggesting that mutations in egl-9 and rhy-1 frequently fail to extend lifespan. Here, we show that egl-9 and rhy-1 mutants suppress the long-lived phenotype of vhl-1 mutants. We also show that RNAi of rhy-1 extends lifespan of wild-type worms while decreasing lifespan of vhl-1 mutant worms. We further identify VHL-1-independent gene expression changes mediated by EGL-9 and RHY-1 and find that a subset of these genes contributes to longevity regulation. The resulting data suggest that changes in HIF-1 activity derived by interactions with EGL-9 likely contribute greatly to its role in regulation of longevity.

Hypoxic response regulators RHY-1 and EGL-9/PHD promote longevity through a VHL-1-independent transcriptional response.

The rapid progress of the past three decades has led the geroscience field near a point where human interventions in aging are plausible. Advances across scientific areas, such as high throughput "-omics" approaches, have led to an exponentially increasing quantity of data available for biogerontologists. To best translate the lifespan and healthspan extending interventions discovered by basic scientists into preventative medicine, it is imperative that the current data are comprehensively utilized to generate testable hypotheses about translational interventions. Building a translational pipeline for geroscience will require both systematic efforts to identify interventions that extend healthspan across taxa and diagnostics that can identify patients who may benefit from interventions prior to the onset of an age-related morbidity. Databases and computational tools that organize and analyze both the wealth of information available on basic biogerontology research and clinical data on aging populations will be critical in developing such a pipeline. Here, we review the current landscape of databases and computational resources available for translational aging research. We discuss key platforms and tools available for aging research, with a focus on how each tool can be used in concert with hypothesis driven experiments to move closer to human interventions in aging.

Computational tools for geroscience.

As the demographics of the modern world skew older, understanding and mitigating the effects of aging is increasingly important within biomedical research. Recent studies in model organisms demonstrate that the aging process is frequently modified by an organism's ability to perceive and respond to changes in its environment. Many well-studied pathways that influence aging involve sensory cells, frequently neurons, that signal to peripheral tissues and promote survival during the presence of stress. Importantly, this activation of stress response pathways is often sufficient to improve health and longevity even in the absence of stress. Here, we review the current landscape of research highlighting the importance of cell non-autonomous signaling in modulating aging from  to mammals. We also discuss emerging concepts including retrograde signaling, approaches to mapping these networks, and development of potential therapeutics.

Cell non-autonomous regulation of health and longevity.

Caenorhabditis elegans is an instrumental research model used to advance our knowledge in areas including development, metabolism, and aging. However, research on metabolism and/or other measures of health/aging are confounded by the nematode's food source in the lab, live E. coli bacteria. Commonly used treatments, including ultraviolet irradiation and antibiotics, are successful in preventing bacterial replication, but the bacteria can remain metabolically active. The purpose of this study is to develop a metabolically inactive food source for the worms that will allow us to minimize the confounding effects of bacterial metabolism on worm metabolism and aging. Our strategy is to use a paraformaldehyde (PFA) treated E. coli food source and to determine its effects on worm health, metabolism and longevity. We initially determine the lowest possible concentrations of PFA necessary to rapidly and reproducibly kill bacteria. We then measure various aspects of worm behavior, healthspan and longevity, including growth rate, food attraction, brood size, lifespan and metabolic assessments, such as oxygen consumption and metabolomics. Our resulting data show that worms eat and grow well on these bacteria and support the use of 0.5% PFA-killed bacteria as a nematode food source for metabolic, drug, and longevity experiments.

An alternative food source for metabolism and longevity studies in Caenorhabditis elegans.

Flavin-Containing Monooxygenases are conserved xenobiotic-detoxifying enzymes. Recent studies have revealed endogenous functions of FMOs in regulating longevity in  and in regulating aspects of metabolism in mice. To explore the cellular mechanisms of FMO's endogenous function, here we demonstrate that all five functional mammalian FMOs may play similar endogenous roles to improve resistance to a wide range of toxic stresses in both kidney and liver cells. We further find that stress-activated c-Jun N-terminal kinase activity is enhanced in FMO-overexpressing cells, which may lead to increased survival under stress. Furthermore, FMO expression modulates cellular metabolic activity as measured by mitochondrial respiration, glycolysis, and metabolomics analyses. FMO expression augments mitochondrial respiration and significantly changes central carbon metabolism, including amino acid and energy metabolism pathways. Together, our findings demonstrate an important endogenous role for the FMO family in regulation of cellular stress resistance and major cellular metabolic activities including central carbon metabolism.

Article	Year
Correlated resistance to glucose deprivation and cytotoxic agents in fibroblast cell lines from long-lived pituitary dwarf mice. Mechanisms of ageing and development\| PubMed ID: 16979221
Skin-derived fibroblasts from long-lived species are resistant to some, but not all, lethal stresses and to the mitochondrial inhibitor rotenone. Aging cell\| PubMed ID: 17156084
Mechanisms of stress resistance in Snell dwarf mouse fibroblasts: enhanced antioxidant and DNA base excision repair capacity, but no differences in mitochondrial metabolism. Free radical biology & medicine\| PubMed ID: 19439226
Nrf2 signaling, a mechanism for cellular stress resistance in long-lived mice. Molecular and cellular biology\| PubMed ID: 19933842
New insights into cell non-autonomous mechanisms of the C. elegans hypoxic response. Worm\| PubMed ID: 27383456	2016
Flavin-containing monooxygenases in aging and disease: Emerging roles for ancient enzymes. The Journal of biological chemistry\| PubMed ID: 28515321	2017
Genetic interaction with temperature is an important determinant of nematode longevity. Aging cell\| PubMed ID: 28940623	2017
Hypoxic response regulators RHY-1 and EGL-9/PHD promote longevity through a VHL-1-independent transcriptional response. GeroScience\| PubMed ID: 32399915
Computational tools for geroscience. Translational medicine of aging\| PubMed ID: 33241167
Cell non-autonomous regulation of health and longevity. eLife\| PubMed ID: 33300870
An alternative food source for metabolism and longevity studies in Caenorhabditis elegans. Communications biology\| PubMed ID: 33637830
Flavin-Containing Monooxygenases Are Conserved Regulators of Stress Resistance and Metabolism. Frontiers in cell and developmental biology\| PubMed ID: 33644069

Scott F. Leiser

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