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      Anthranilate Fluorescence Marks a Calcium-Propagated Necrotic Wave That Promotes Organismal Death in C. elegans

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          Abstract

          Death of the nematode Caenorhabditis elegans involves a conserved necrotic cell death cascade which generates endogenous blue anthranilate fluorescence, allowing death to be visualized.

          Abstract

          For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters—not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals—e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death.

          Author Summary

          In the nematode Caenorhabditis elegans, intestinal lysosome-related organelles (or “gut granules”) contain a bright blue fluorescent substance of unknown identity. This has similar spectral properties to lipofuscin, a product of oxidative damage known to accumulate with age in postmitotic mammalian cells. Blue fluorescence seems to increase in aging worm populations, and lipofuscin has been proposed to be the source. To analyze this further, we measure fluorescence levels after exposure to oxidative stress and during aging in individually tracked worms. Surprisingly, neither of these conditions increases fluorescence levels; instead blue fluorescence increases in a striking and rapid burst at death. Such death fluorescence (DF) also appears in young worms when killed, irrespective of age or cause of death. We chemically identify DF as anthranilic acid glucosyl esters derived from tryptophan, and not lipofuscin. In addition, we show that DF generation in the intestine is dependent upon the necrotic cell death cascade, previously characterized as a driver of neurodegeneration. We find that necrosis spreads in a rapid wave along the intestine by calcium influx via innexin ion channels, accompanied by cytosolic acidosis. Inhibition of necrosis pathway components can delay stress-induced death, supporting its role as a driver of organismal death. This necrotic cascade provides a model system to study neurodegeneration and organismal death.

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          Most cited references 72

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          Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009.

          Different types of cell death are often defined by morphological criteria, without a clear reference to precise biochemical mechanisms. The Nomenclature Committee on Cell Death (NCCD) proposes unified criteria for the definition of cell death and of its different morphologies, while formulating several caveats against the misuse of words and concepts that slow down progress in the area of cell death research. Authors, reviewers and editors of scientific periodicals are invited to abandon expressions like 'percentage apoptosis' and to replace them with more accurate descriptions of the biochemical and cellular parameters that are actually measured. Moreover, at the present stage, it should be accepted that caspase-independent mechanisms can cooperate with (or substitute for) caspases in the execution of lethal signaling pathways and that 'autophagic cell death' is a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. This study details the 2009 recommendations of the NCCD on the use of cell death-related terminology including 'entosis', 'mitotic catastrophe', 'necrosis', 'necroptosis' and 'pyroptosis'.
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            Pleiotropy, Natural Selection, and the Evolution of Senescence

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              Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans.

              The nematode Caenorhabditis elegans is an important model for studying the genetics of ageing, with over 50 life-extension mutations known so far. However, little is known about the pathobiology of ageing in this species, limiting attempts to connect genotype with senescent phenotype. Using ultrastructural analysis and visualization of specific cell types with green fluorescent protein, we examined cell integrity in different tissues as the animal ages. We report remarkable preservation of the nervous system, even in advanced old age, in contrast to a gradual, progressive deterioration of muscle, resembling human sarcopenia. The age-1(hx546) mutation, which extends lifespan by 60-100%, delayed some, but not all, cellular biomarkers of ageing. Strikingly, we found strong evidence that stochastic as well as genetic factors are significant in C. elegans ageing, with extensive variability both among same-age animals and between cells of the same type within individuals.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Biol
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                1544-9173
                1545-7885
                July 2013
                July 2013
                23 July 2013
                : 11
                : 7
                Affiliations
                [1 ]Institute of Healthy Ageing, and Research Department of Genetics, Evolution and Environment, University College London, London, United Kingdom
                [2 ]Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York, United States of America
                [3 ]Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, United States of America
                [4 ]Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America
                [5 ]Department of Biology, Ghent University, Ghent, Belgium
                [6 ]Institute of Molecular Biology and Biotechnology, University of Crete, Heraklion, Crete, Greece
                [7 ]Department of Medicine, University of Rochester Medical Center, Rochester, New York, United States of America
                St. Jude Children's Research Hospital, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: CC DG KN PM AB FC. Performed the experiments: CC AD SAE AS FS ZP PM BB FM AM GF EA KN MV NT AB CA FC REP. Analyzed the data: CC DG KN EA PM FS BB FM MV NT AB. Contributed reagents/materials/analysis tools: PM FS EA FJS KN FM BB. Wrote the paper: CC DG KN. AS worked as an undergraduate project student to identify the severity of different Glo mutant phenotypes, consequently allowing us to select glo-1(zu347) as the primary mutant of study.

                Article
                PBIOLOGY-D-12-04084
                10.1371/journal.pbio.1001613
                3720247
                23935448

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 17
                Funding
                CC, CA, FC, and DG acknowledge funding from the BBSRC, the EU (FP7-259679 IDEAL, FP6-036894 and FP6-518230), and the Wellcome Trust (Strategic Award 098565/Z/12/Z). ZP was supported by a Jane Coffin Child postdoctoral fellowship, and ZP and FJS were supported by NIH R01 AG033921. KWN acknowledges support from the NSF (IOS 0919848: KN). EA was supported in part by a Ruth L. Kirschstein National Research Service Institutional Training Grant T32 GM068411. FM is supported by a Special Research Fund fellowship from the University of Ghent. FCS and PM were supported in part by the NIH (AG033839 and GM088290). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Genetics
                Microbiology
                Model Organisms

                Life sciences

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