Anthranilate fluorescence marks a calcium-propagated necrotic wave that promotes organismal death in C. elegans
Authors: Sally-Anne Edwards
Rosina E Pryor
Keith Nehrke
Abraham Mandel
Grahame Fischer
Parag Mahanti
Erik Allman
Nektarios Tavernarakis
Ailsa Stevens
Manolis Vlachos
Alexander Davidson
Caroline Araiz
Filipe Cabreiro
David Gems
Alexandre Benedetto
Bart P Braeckman
Zachary Pincus
Cassandra Coburn
Frank J Slack
Filip Matthijssens
Frank C Schroeder
Journal: PLoS Biology (2013)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: cell death; organismal death; necrosis pathway; fluorescence; intestinal cells
Affiliations: Institute of Healthy Ageing, and Research Department of Genetics, Evolution and Environment, University College London, London, United Kingdom.

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. 

Retracted: No

PubPeer comments:
 A more recent paper calls into question the conclusion that anthranilic acid is the cause of the blue emission of nematodes at the moment of their death (Gagarinskyi Vekshin, 2018; doi: 10.1134 S2079057018020042). Could authors possibly comment on this discrepancy? As quoted:  This paper demonstrates that the aging and death of nematodes, which is accompanied by the emission of a blue glow under a fluorescent microscope, are not directly associated with either lipofuscin (aging pigment) or with anthranilic acid (degradation product of tryptophan residues). It turns out that the parasitic light scattered on the worm cuticle and organs substantially contributes to the blue fluorescence of live and dead nematodes, as well as to blue flashes in dying worms. , 
 #1 Dear Panicum, This is an intriguing paper. Unfortunately, as I do not have free access to the content of this specific paper, I am unable to comment fully on the specifics of this study. Notably, I do not know how the authors studied the blue fluorescence experimentally, and if it is comparable to what we were studying in our PLoS Biology 2013, Cell Reports 2018 and Aging Cell 2019 papers. From their abstract the authors state that (in their setup) the  worm cuticle and organs substantially contributes to the blue fluorescence of live and dead worms . This is not a clear rejection of the conclusions of our first article (PLoS Biology 2013), but it does highlights the fact that other factors contribute to C. elegans blue autofluorescence in general, which I would not dispute. There are well-known other sources of blue (and green) autofluorescence in the worm (the fact that the cuticle autofluoresces is well-known to all C. elegans mircoscopists for decades) but the type of blue fluorescence that is specifically increased at death is what we studied, and we provided details about the optical setup (notably excitation and emission wavelength ranges) in our paper. To be clear, we can categorically exclude cuticle autofluorescence as a confounding factor in the imaging setups we have used (we did check for this before starting the study, back in 2010). Note that we have been studying a dynamic process (dying) and not merely two states of the worm (dead or alive). The blue fluorescence that appears at death can be short-lived as it is photosensitive (it photobleaches at a fast rate). It is therefore possible to miss it or underestimate its contribution if not using an appropriate experimental setup. As I cannot access the contents of the article you mention, I can not ascertain that the authors actually studied the same type of autofluorescence or if they repeated our experiments using a similar setup to demonstrate that other processes contribute significantly to the death fluorescence wave. Current work on the kynurenine pathway and organsimal death has so far further confirmed the findings of our PLoS biology paper and has not given us any reason to doubt our initial conclusions. We therefore stand by our early interpretation that the blue death fluorescence we have been studying comes primarily from modified anthranalic acid (including gucosyl esters identified in our PLoS Biology paper), and we believe the conclusions and interpretations of the original paper remain parcimonious and verifiable. Should convincing contradicting evidence emerge, it would obviously warrant revising our interpretations. However at this time, we do not believe such evidence is available yet. Best wishes, Alex ,