O-GlcNAc Signaling Orchestrates the Regenerative Response to Neuronal Injury in Caenorhabditis elegans  Daniel G. Taub, Mehraj R. Awal, Christopher V. Gabel  Cell Reports  Volume 24, Issue 8, Pages 1931-1938.e3 (August 2018) DOI: 10.1016/j.celrep.2018.07.078 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 24, 1931-1938.e3DOI: (10.1016/j.celrep.2018.07.078) Copyright © 2018 The Authors Terms and Conditions

Figure 1 O-GlcNAc Modifications Regulate Axon Regeneration (A) Representative images of axon regeneration in the ALM neuron 24 hr after laser axotomy in wild-type and O-GlcNAc mutants. Scale bar, 20 μm. (B) Average regenerative outgrowth of ALM neuron 24 hr after injury for wild-type and O-GlcNAc mutants with and without treatment of a glucose transport inhibitor (phloretin), D-glucose, or D-mannitol. (C) Representative western blot of O-GlcNAc levels in wild-type and O-GlcNAc mutant whole worm lysates. Anti-RL2 O-GlcNAc antibody primarily detects two nucleoporins. (D) Quantification of O-GlcNAc levels via western blotting. Mean density of the p62 nucleoporin band was quantified in wild-type, ogt-1, oga-1 mutants, and wild-types treated with increasing levels of glucose. n = 3–5 independent treatments per condition. (E) Average regenerative outgrowth of wild-type or ogt-1 mutants with and without the OGT inhibitor (ST045849). (F) Average regenerative outgrowth of wild-type or oga-1 with and without the OGA inhibitor (Thiamet G). (G) Average regenerative outgrowth with RNAi knockdown of ogt-1 and oga-1 in the mechanosensory neurons. Data are expressed as mean ± SEM. One-way ANOVA: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Stars compare groups with wild-type control except for brackets, which indicate specific comparisons. Cell Reports 2018 24, 1931-1938.e3DOI: (10.1016/j.celrep.2018.07.078) Copyright © 2018 The Authors Terms and Conditions

Figure 2 O-GlcNAcylation Affects a Late Stage of Axon Regeneration (A) Time lapse of axon regeneration 1, 3, 5, 12, and 24 hr after injury. (B) Percentage of neurons displaying filopodia at 1 and 3 hr post-injury. (C) Number of filopodia over time between wild-type, ogt-1, and oga-1 mutants. (D) Extent of branching in wild-type, ogt-1, and oga-1 mutants. (E) Percentage of neurons exhibiting axon regeneration from the posterior side of the neuron wild-type, ogt-1, and oga-1 mutants. (F) Genetic epistasis analysis of regeneration between dlk-1 mutants and O-GlcNAc mutants. Data are expressed as mean ± SEM. One-way ANOVA: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Stars compare groups with wild-type control except for brackets, which indicate specific comparisons. Cell Reports 2018 24, 1931-1938.e3DOI: (10.1016/j.celrep.2018.07.078) Copyright © 2018 The Authors Terms and Conditions

Figure 3 OGT Mutants Improve Regeneration by Enhancing Glycolysis Genetic epistasis analysis of ALM regeneration 24 hr after injury. (A) Between mutants of the insulin-signaling pathway and ogt-1 mutants. (B) Between ark-1, ogt-1, and akt-1. akt-1(++) indicates gain-of-function mutation. (C) Mechanosensory neuron-specific RNAi against the glycolytic enzyme phosphoglycerate kinase 1 (pgk-1) in the ogt-1 background. Data drawn from Figure S3B. (D) Mutation of phosphofructokinase-1.1 (pfk-1.1) in wild-type and ogt-1 backgrounds. (E) Treatment of wild-type and ogt-1 mutants with the non-hydrolyzable glucose analog 2-deoxy-D-glucose. (F) Mechanosensory neuron-specific RNAi against the NADH dehydrogenase iron-sulfur protein 2 (nduf-2.2) in the ogt-1 background. Data are expressed as mean ± SEM. One-way ANOVA: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Stars compare groups with wild-type control except for brackets, which indicate specific comparisons. See Table S1 for sample sizes. At least two biological replicates were performed. Cell Reports 2018 24, 1931-1938.e3DOI: (10.1016/j.celrep.2018.07.078) Copyright © 2018 The Authors Terms and Conditions

Figure 4 OGA Mutants Enhance Regeneration through FOXO/DAF-16 Activity and Alter Mitochondrial Dynamics Genetic epistasis analysis of ALM regeneration 24 hr after injury. (A) Between mutants in the insulin-signaling pathway in an oga-1 mutant background. (B) Between sgk-1 and oga-1 mutants. (C) Representative kymographs of mitochondrial movement in wild-type, ogt-1, and oga-1 mutants 24 hr after injury. (D) Quantitative analysis of mitochondrial dynamics. Shown are the percentage time in motion, the mean speed, and the total length traveled. (E) Mechanosensory neuron-specific RNAi against the NADH dehydrogenase iron-sulfur protein 2 (nduf-2.2) in the oga-1 background compared with vector control. (F) Representative ratiometric images of mitochondria expressing the mitoROGFP oxidative stress sensor within severed neurons of wild-type and oga-1 mutants. Pseudocolored yellow to represent reduced and red to represent oxidative mitochondrial environments. Blue arrowheads indicate axon cut site. White arrowheads indicate cell body. (G) Diagram summarizing our model for the action of O-GlcNAc on axon regeneration. Data are expressed as mean ± SEM. One-way ANOVA: ∗p < 0.05 and ∗∗p < 0.01. Stars compare groups with wild-type control except for brackets, which indicate specific comparisons. See Table S1 for sample sizes. At least two biological replicates were performed. Cell Reports 2018 24, 1931-1938.e3DOI: (10.1016/j.celrep.2018.07.078) Copyright © 2018 The Authors Terms and Conditions