Fig 1.
Sir-2.3 knock-out is protective in a model of hypoxic ischemia.
A: Photograph of the head of a wild type Caenorhabditis elegans incubated in S Basal at pH 6.5 for 5 hours and stained with the lipophilic dye DiD. DiD is taken up by 6 pairs of amphid sensory neurons (ASKs, ADLs, ASIs, ASHs, ASJs, and AWBs) exposed to the outside environment. B: Photograph of an animal treated with 100 mM azide at pH 6.5 for 5 hours and stained with DiD. Fewer stained sensory neurons are visible in this animal as a result of neuronal death. Scale bar is 20 μm. C: Number of DiD stained amphid neurons in wild type animals incubated for 8 hours in S basal pH 7.4 (control), S basal pH 6.5, S Basal pH 7.4 containing 20 mM Na-Azide, S basal at pH 6.8 and pH 6.5 plus 20 mM Na-Azide. N was of experiments was 2 with at least 12 animals for each condition. Data are expressed as mean +/- SE with P values 0.000026, 8.25E-25 and 0.003 by ANOVA with Bonferroni correction. D: Number of DiD labeled amphid neurons in wild type treated with S basal pH 6.5 and S basal pH 6.5 plus 100 mM Na-Azide for 3, 5 and 6 hours respectively. N of animals was 12 per condition. Data are expressed as mean +/- SE with P values 1.32E-52, 1.25E-80 and 0.037 by ANOVA with Bonferroni correction. E: Number DiD stained amphid sensory neurons in wild type, wild type under dietary deprivation (DD), sir-2.3 knock-out animals, and sir-2.3;SIR-2.3 rescue animals incubated for 5 hours in S Basal at pH 6.5 and in S Basal at pH 6.5 containing 100 mM Na-azide. At least 50 animals were analyzed in each of 4 separate experiments. Data are expressed as mean +/- SE with P values 0.01, 0.0015 and 0.0002 by ANOVA with Bonferroni correction.
Fig 2.
Knock-out of mitochondrial sir-2.3 protects against mec-4(d) and mec-10(d)-induced neuronal death.
A-D: Quantification of the proportion of animals with 0, 1, 2 and 3 surviving touch neurons in mec-4(d), mec-4(d);sir-2.3 knock-out and mec-4(d);sir-2.3;SIR-2.3 rescue strains. Number of experiments was 23, 23, and 4, respectively, with at least 50 animals per strain analyzed in each experiment. P was 0.0002 and 0.0001 by ANOVA with Bonferroni correction. E-H: Proportion of mec-10(d) expressing animals with 3, 4, 5 and 6 surviving touch neurons. Number of experiments was 4 each, with at least 50 animals per strain analyzed in each experiment. P was 0.01 and 0.0024 by t-Test. I: Photograph of the tail of an L1 mec-4(d) mutant Caenorhabditis elegans showing a necrotic swollen PLM touch neuron, visible as a large vacuole. Scale bar is 10 μm. J: Quantification of the number of swollen PLMs in L1 mec-4(d), mec-4(d);sir-2.3 knock-out and mec-4(d);sir-2.3;SIR-2.3 rescue strains. Number of experiments was 5, 5, and 4, respectively with at least 50 animals per strain analyzed in each experiment. Data are expressed as mean +/- SE with P value 0.014 by ANOVA with Bonferroni correction.
Fig 3.
SIR-2.1 does not mediate the protective effects of sir-2.3 knock-out.
A: Quantitative real-time PCR (qRT-PCR) from total RNA was conducted using Taqman gene expression assays in order to quantify the levels of mRNA for sirtuin genes. mRNA percentage of each target gene was calculated by comparing the cycle threshold of the target gene to that of the housekeeping gene pmp-3. The wild-type N2 (Bristol) strain was used as the calibrator. All conditions had three technical replicates. B-E: Proportion of animals with 0, 1, 2 and 3 alive touch neurons in mec-4(d), mec-4(d);sir-2.3 and mec-4(d);sir-2.1;sir-2.3 mutants. Data are expressed as mean +/- SE, N = 3. P was from left to right and from top to bottom: 0.003, 0.0029, 0.0019, 0.0005, 0.000013 and 0.00009 by ANOVA with Bonferroni correction.
Fig 4.
Block of the NAD+ salvage pathway by null mutation in pnc-1 protects neurons from hypoxic ischemia.
A: schematic representation of the invertebrate NAD+ salvage pathway. In invertebrates PNC-1 converts nicotinamide into nicotinic acid. A null mutation in pnc-1 leads to reduced availability of NAD+ and accumulation of its substrate nicotinamide, which in turn inhibits the activity of sirtuins. B: Average number of DiD stained amphid sensory neurons in WT and pnc-1(ku212) mutant in control conditions (pH 6.5) and under conditions of chemical hypoxia (100 mM azide at pH 6.5). Data are expressed as mean +/- SE. Number of animals was 24 and 25 for wild type and 23 and 25 for pnc-1(ku212) in control conditions and azide respectively. P was 0.000029 by Student’s t-Test in which the 2 samples in azide were compared with each other. C-F: Proportion of animals with 0,1,2 and 3 GFP touch neurons in mec-4(d), mec-4(d);pnc-1, mec-4(d);sir-2.3, mec-4(d);sir-2.3;pnc-1 mutants. Data are expressed as mean +/- SE. Number of experiment was 7,4,3 and 3 respectively with at least 40 animals per strain analyzed in each experiment. P was from left to right and from top to bottom: 9.9E-8, 0.007, 0.001 (panel C), 3.08E-6, 9.07E-4, 0.011 (panel D), 5.04E-6, 0.0013, 2.43E-4, 0.034 (panel E), 2.76E-5, 2.76E-5, 0.013 (panel F) by ANOVA with Bonferroni correction.
Fig 5.
Protection afforded by block of glycolysis in the presence and absence of SIR-2.3.
A-D: Proportion of animals with 0, 1, 2, or 3 surviving touch neurons that were either grown on standard plates or on plates containing 5 mM 2-DG for 48 hours. Number of experiments was 4 each, with at least 30 animals in each experiment. Data are expressed as mean +/- SE. P was 0.0012, 0.018 and 0.048, by t-Test. E: Photograph of an ALM touch neuron in a mec-4(d) animals grown on 2-DG plate. The neuronal processes are still intact and show some arborization. Scale bar is 10 μm.
Fig 6.
Effect of block of glycolysis and knock-out of sir-2.3 on mec-4(d)-induced neuronal death in cultured cells.
A-F: Fluorescent images of wild type, mec-4(d), and sir-2.3;mec-4(d) touch neurons expressing Pmec-4::GFP cultured in vitro. Cells were in control media (A,C,E) or in media containing 10 mM 2-deoxy-glucose(2-DG)(B,D,F). Scale bar is 10 μm. G: Quantification of surviving touch neurons in culture for the genetic strains shown in the micrographs, in control media and in media containing 10 mM 2-DG. Data are expressed as the means +/- SE. Similar results were obtained in 5 independently performed experiments. N is 2 coverslips for each strain (wild type, mec-4(d) and sir-2.3;mec-4(d)) and for both conditions (control and 10 mM 2-DG respectively), with at least 10 fields scored per coverslip. H: Quantification of surviving touch neurons in culture for an experiment similar to the one shown in panel G for mec-4(d) and sir-2.3;mec-4(d). In this case the culturing conditions were: control, 10 mM 2-DG, 2.5 μM rotenone and 10 mM 2-DG + 2.5 μM rotenone. N is 2 coverslips each with at least 10 fields scored per coverslip. P was 0.039 and 0.015 (panel G) and 0.019, 0.007, 0.00007 and 0.00006 (panel H) by ANOVA with Bonferroni correction.
Fig 7.
Neuronal protection mediated by sir-2.3 knock-out depends on daf-16/FOXO transcription factor.
A: Number of vacuolated PLM neurons in L1 mec-4(d), daf-16;mec-4(d), mec-4(d);sir-2.3, and daf-16;mec-4(d);sir-2.3 mutants. Number of experiments is 3 each with at least 40 animals per strain analyzed in each experiment. Data are expressed as mean +/- SE. P was 1.96E-9 and 2.82E-33 by ANOVA with Bonferroni correction. B-E: Proportion of animals with 0, 1, 2 and 3 surviving touch neurons in mec-4(d), daf-16;mec-4(d);, mec-4(d);sir-2.3 and daf-16;mec-4(d);sir-2.3 mutants. Number of experiments was 3 each, with at least 45 animals per strain analyzed in each experiment. P was from left to right and from top to bottom: 0.030, 0.00045, 5.43E-9, 0.006, 2.57E-6, 0.000416, 2.84E-5, 2.01E-7, 0.027 and 0.027 by ANOVA with Bonferroni correction.
Fig 8.
ROS in sir-2.3 knock-out animals.
A: Quantification of DCF fluorescence in wild type and sir-2.3 knock-out mutants grown under standard condition, dietary deprivation (DD), or on plates containing 5 mM 2-DG for 48 hours with or without treatment with 100 mM Na-azide for 2 hours. Data are expressed as mean +/- SE, P was (from left to right) 0.0017, 0.0068, 0.042, 0.00001, and 0.00001 by ANOVA with Bonferroni correction. N was (from left to right) 22, 55, 23, 68, 25, 23, 23, 16, 38 and 35. B-E: Representative photographs of wild type and sir-2.3 mutant animals grown under standard condition (B and C) or in dietary deprivation (DD) for 48 hours (D and E) and stained with the fluorescent ROS indicator DCF. The dotted lines correspond to the animal contour. Scale bar is 50 μm.