Article Figures & Data
Figures
- Figure 1.
PARylation is induced after ON injury and suppressed by veliparib treatment in retina tissue. A, B, Immunoblots show PARylation in retinal tissue from naive state or 5 d after ON crush and treated with veliparib (10 mg/kg/d) or vehicle. RIPA soluble (A) and insoluble (B) samples were analyzed with an antibody directed against PAR protein. Molecular weight markers in kDa are at left. C, D, Quantification of PARylation in the lysate (C) or pellet (D). ON crush injury significantly increased PARylation in retina, and veliparib treatment significantly suppressed it. Data are mean ± SE from n = 3 in each condition. For one-way ANOVA followed by post hoc pairwise Tukey’s tests: *, P <0.05; **, P <0.01; ***, P <0.005.
- Figure 2.
Pharmacologic PARP inhibition does not increase ON axon regeneration or improve functional recovery from SCI. A, Mice underwent ON crush injury and were treated with veliparib or vehicle on d 3–17 postinjury. Representative images of ON from vehicle-treated and veliparib-treated mice. Multiple CTB-labeled axons proximal to the crush site are observed. Images are projections of confocal z-stacks through the entire ON. The eye is to the left, and the crush site is indicated by the blue arrow. Few axons extend centrally. B, High-magnification view of the lesion area (box) from A. C, Total number of regenerating ON fibers per mouse is presented as a function of distance central to the crush site. There is no statistically significant difference in the number of regenerating axons between the saline-treated group and the veliparib-treated group. Data are mean ± SE of n = 8 mice per group. D, Mice underwent midthoracic dorsal hemisection and then were treated with veliparib or vehicle on d 3–31 postinjury. The locomotor BMS score is plotted as a function of time after SCI. There is no statistically significant difference in the number of regenerating axons between the saline-treated group and the veliparib-treated group by one-way repeated-measure ANOVA. Data are mean ± SE for n = 8 mice per group.
- Figure 3.
Selective suppression of Parp1 improves cortical neuron axonal regeneration. A, Representative images of regenerated cortical neurons. Neurons were transduced with shRNA lentiviral particles on culture d 3 and scraped on d 8. Scraped neurons were fixed and stained at d 11. The microphotographs illustrate βIII tubulin in axons (green), phalloidin of F-actin in growth cones (phalloidin, red), and cell nuclei (DAPI, blue). Note that no neuronal cell bodies (DAPI) migrate into the scrape zone. Selectively after Parp1 knockdown, a greater number of regenerating axons and growth cones are visible in the scrape zone. Scale bars, 200 µm. B, Quantification of axon regeneration. Data are mean ± SE. shNC, n = 109; shParp1, n = 49 (each of 5 lentiviral species has 10 independent wells, with 1 cell culture lost); shParp2, n = 10 (each of 5 lentiviral species has 2 independent wells); shParp3, n = 10 (each of 5 lentiviral species has 2 independent wells); shParp9, n = 10 (each of 5 lentiviral species has 2 independent wells); shParp12, n = 10 (each of 5 lentiviral species has 2 independent wells); and shParp16; n = 16 (each of 4 lentiviral species has 4 independent wells). Each condition was compared to NC control by one-way ANOVA followed by Dunnett test. **, P <0.01; ***, P <0.005; ns, no significant difference. C, Quantification of Parp1 and Parp9 mRNA. The levels of the Parp1 and Parp9 mRNA determined by quantitative RT-PCR were normalized to those of a GAPDH internal control for cerebral cortex (n = 3 mice), spinal cord (n = 3), and retina (n = 3). Data are mean ± SE. Within each tissue, Parp1 was significantly greater than Parp9 for each tissue after one-way ANOVA with post hoc pairwise Tukey test. **, P <0.0001.
- Figure 4.
Genetic deletion of Parp1 does not increase ON axon regeneration. A, Immunoblots are shown for PARylation levels from uninjured wild-type (WT) control mice or Parp1–/– mutant mice in RIPA-insoluble fraction of retinal tissue. Molecular weight markers in kDa are at left. B, Quantification of PARylation in retinal tissue from WT mice and Parp1-null mutant mice from blots as in A. Data are mean ± SE for n = 2 mice per genotype. *, P = 0.046, Student’s two-tailed t test. C, Representative images of ON from WT control mouse and Parp1 mutant mouse. The CTB-labeled RGC axons are white. The eye is the left and the brain to the right, with crush indicated by blue arrow. Images are projections of confocal z-stacks through the entire ON. Scale bar, 500 µm. D, The total number of regenerating optic nerve fibers per mouse is presented as a function of distance central from the crush site and of genotype. Data are mean ± SE for n = 8 WT and n = 8 Parp1-null mice. No statistically significant difference was observed between the Parp1–/– and WT mice by one-way repeated-measure ANOVA.
- Figure 5.
Genetic deletion of Parp1 does not improve recovery from dorsal hemisection of thoracic spinal cord in mice. A, Wild-type (WT) and Parp1–/– mice underwent midthoracic dorsal hemisection injury. Representative images of a spinal cord sagittal section from one WT mouse and two Parp1-null mutant mice collected 6 weeks after injury. CST axons are visualized by BDA tracing (green) and astrocytic reaction by anti-GFAP staining (red). The entire depth of the spinal cord is shown. Rostral is to the left, and dorsal is up. No BDA-labeled axons are seen caudal to the lesion site in either group. Scale bar, 500 µm. B, Quantification of BDA-labeled CST axons rostral and caudal to the lesion site. For the x-axis, a positive value is rostral to the center of the lesion, and a negative value is caudal to the center of the lesion. Data are mean total number of CST axons per mouse at each location ± SE for n = 8 WT mice and n = 12 Parp1-null mice. No statistically significant difference was observed between the Parp1–/– and WT mice by one-way repeated-measure ANOVA. C, Open-field locomotion performance measured by BMS of WT and Parp1–/– mutant mice. Data are mean ± SE for n = 9 WT mice and n = 13 Parp1-null mice. No statistically significant difference was observed between the Parp1–/– and WT mice by one-way repeated-measure ANOVA.
Tables
Line Data structure Type of test P a Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0004 b Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0032 c Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.04 d Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0001 e Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0014 f Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0001 g Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0001 h Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0001 i Normal distribution One-way ANOVA, with Dunnett post hoc pairwise tests 0.0001 j Normal distribution One-way ANOVA, with Dunnett post hoc pairwise tests 0.0017 k Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0001 l Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0001 m Normal distribution One-way ANOVA, with Tukey post hoc pairwise tests 0.0001 n Normal distribution Student’s two tailed t test 0.046
In this issue
