Fig 1.
Atfs-1 is both necessary and sufficient for UPRmt activation and reduced death from anoxia-reperfusion.
Representative fluorescent photomicrographs of Phsp60::GFP activation in A.) vector control and B.) UPRmt inducer spg-7(RNAi) treated worms and in C.) an atfs-1(et15) gain-of-function mutant. D.) Box-and-whisker plot showing that pre-induction of the UPRmt with spg-7(RNAi) reduces death following A-R (n = 4, p*[Student’s t test] <0.05). E.) Box-and-whisker plot showing that spg-7(RNAi) does not protect atfs-1(tm4525) loss-of-function worms from A-R toxicity (n = 5, p*[Student’s t test] <0.05). F.) Box-and-whisker plot showing that atfs-1(et15) mutants are constitutively less susceptible to A-R (n = 6, p*[Student’s t test] <0.05). Experimental replicates are shown as black dots; lines between replicates indicate that they were run on the same day. Grey diamonds are means with the error shown as standard deviations.
Fig 2.
A gain-of-function in atfs-1 attenuates axonal degeneration following anoxia-reperfusion.
A.) Schematic representation of mechanosensory neurons (red dots indicate soma) in the worm, and the PLM axonal processes that were used to measure degeneration. B.) Representative fluorescent photomicrograph of an integrated Pmec-4::mCherry reporter in wildtype worms (top image) and following A-R (bottom image). Scored puncta are denoted by stars. C.) atfs-1(et15) worms develop fewer puncta following A-R (n = 4 independent trials with ~10 worms per genotype per trial), p*[Student’s t test] <0.05). The error is the standard deviation.
Fig 3.
Electron transport chain dysfunction in neurons reduces anoxia-reperfusion toxicity.
A.) Schematic of how cco-1HP interference with Complex IV leads to mitochondrial stress in neurons, which is transduced remotely through an unidentified “mitokine” to elicit adaptive signaling measures in distal cells. B.) Box-and-whisker plot showing that neuronal cco-1HP reduces death following A-R (n = 5, p*[Student’s t test] <0.05). Experimental replicates are shown as black dots; lines between replicates indicate that they were run on the same day. Grey diamonds are means with the error shown as standard deviations.
Fig 4.
A FLP-out transgenic model to restrict UPRmt activation.
A.) Schematic showing relevant genetic constructs to restrict atfs-1 expression using transgenic FLP recombinase and an atfs-1 promoter interruption. The activity of the flp and atfs-1 gene products on their target promoters is indicated by arrows. B.) Representative transmission and fluorescent photomicrographs of Patfs-1::wCherry expressed from a MosSCI single-copy, integrated atfs-1 promoter interruption in the absence of FLP recombinase. C-E.) Photomicrographs of DIC (grayscale), a fluorescent marker of transgenic FLP (red) and UPRmt reporter Phsp-60::GFP (green) in flp(-) and F-H.) flp(+) siblings following spg-7(RNAi). The genomic background is atfs-1(tm4525), a loss-of-function allele.
Fig 5.
FLP-out atfs-1(gf) and trans-cellular UPRmt signaling.
A.) Schematic showing the relevant genetic constructs and the activity of the flp and atfs-1 gene products on their target promoters. B.) Schematic representing UPRmt activation in neurons (green) in the presence of Prab-3::FLP (red). The intestine is denoted in pink, as labeled, and activation of the UPRmt is notably absent in this tissue. C-E.) Transmission and fluorescent photomicrographs of cell-autonomous UPRmt activation in a genomic atfs-1(+) background and in a F-H.) atfs-1(4525) background. Note that distal tissues do not express the Phsp-60::GFP transgene, regardless of genetic background.
Fig 6.
Protecting neurons from local anoxia-reperfusion injury in the atfs-1(gf) FLP out worm does not reduce A-R death.
Representative fluorescent photomicrographs of a FLP out atfs-1(et15) transgenic worm with neuronal FLP (Prab-3::FLP) expressing A.) the UPRmt reporter Phsp60::GFP (green) and B.) the neuronal reporter Pmec-4::mCherry in mechanosensory neurons. Labeling of the PLM cell processes can be clearly distinguished over background Patfs-1::wCherry from the un-excised promoter interruption in other cells. C.) Merge indicating co-localization of UPRmt activation and Pmec-4::mCherry. D.) Expression of the FLP out atfs-1(et15) MosSCI transgene attenuates axonal damage following A-R (n = 3 independent trials of ~10 worms per genotype per trial), p*[Student’s t test] <0.05). The error is the standard deviation. E.) Box-and-whisker plot indicating that neuronal FLP out atfs-1(et15) MosSCI transgene does not reduce death following A-R (n = 5, p*[Student’s t test] <0.05). All data was obtained in an atfs-1(+) genomic background. Experimental replicates are shown as black dots; lines between replicates indicate that they were run on the same day. Grey diamonds are means with the error shown as standard deviations.