Research ArticleNeuroscience

The nociceptin receptor inhibits axonal regeneration and recovery from spinal cord injury

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Science Signaling  03 Apr 2018:
Vol. 11, Issue 524, eaao4180
DOI: 10.1126/scisignal.aao4180
  • Fig. 1 NgR1 interacts with ORL1.

    (A) The levels of the ORL1 mRNA normalized to those of a Gapdh internal control in the brain stem. Tissues were collected from Ngr1+/+ or Ngr1−/− mice 4 days after sham operation or spinal cord injury (SCI) (n = 3 mice for each group). Data are means ± SE. *P < 0.05, **P < 0.001, one-way analysis of variance (ANOVA) followed by Tukey’s test. (B) Human embryonic kidney–293T (HEK293T) cells were transfected with FLAG-NgR1 alone or with hemagglutinin (HA)–ORL1, HA-p75, or HA-neuropilin. HA immunoprecipitates were immunoblotted for HA and FLAG. Means ± SE, n = 3 independent experiments. TCL, total cell lysates. (C and D) ORL1 immunoprecipitates from mouse forebrain (E) or hippocampal neurons at 21 DIV (days in vitro) (G) were immunoblotted for NgR1 and ORL1. n = 3 independent experiments. Ctrl, control. (E) Cos7 cells transfected with FLAG-NgR1 and HA-ORL1 were stained for FLAG and HA without permeabilization. Means ± SE, n = 3 independent experiments. Scale bar, 20 μm.

  • Fig. 2 ORL1 enhances O-linked glycosylation of NgR1 at Golgi body.

    (A) HEK293T cells were transfected with Myc-NgR1 or Myc-NgR2, with or without HA-ORL1. HA immunoprecipitates were immunoblotted for HA and Myc. Means ± SE, n = 3 independent experiments. (B) HEK293T cells were transfected with FLAG-NgR1 and increasing amounts of HA-ORL1 and treated without or with N-glycosidase or O-glycosidase for 1 hour. Cell lysates were immunoblotted for FLAG or actin. Means ± SE, n = 3 independent experiments. glyco, glycosidase. (C) HEK293T cells were transfected with FLAG-NgR1, with or without HA-ORL1 and treated with dimethyl sulfoxide or increasing amounts of benzyl-N-acetyl-α-d-galactosaminide (b-GalNAc) as indicated. Cell lysates were immunoblotted for FLAG, HA, and actin. Means ± SE, n = 4 independent experiments. (D) Cos7 cells cotransfected with FLAG-NgR1 and HA-ORL1 were stained with NgR1, HA, and GM130 antibodies. Means ± SE, n = 3 independent experiments. Scale bar, 20 μm.

  • Fig. 3 ORL1 increases the cell surface expression of NgR1.

    (A) HEK293T cells were transfected with FLAG-NgR1, with or without HA-ORL1, and treated with phosphatidylinositol-specific phospholipase C (PI-PLC) at the indicated amounts for 1 hour. Cell lysates were immunoblotted for FLAG, HA, and actin. Means ± SE, n = 3 independent experiments. (B and C) Cos7 cells stably expressing FLAG-NgR1 were transfected with control vector or HA-ORL1 and stained for FLAG. Cells were analyzed by flow cytometry (B). The graph shows the mean fluorescence intensity from three independent experiments with ±SE (C). ***P < 0.005, Student’s two-tailed t test. (D) HEK293T cells were transfected with FLAG-NgR1 with or without HA-ORL1 and treated with cycloheximide (CHX; 10 μg/ml) for the indicated periods. Cell lysates were immunoblotted for FLAG, HA, and actin. (E) Quantification of NgR1 upper or lower bands normalized to actin of control or ORL1-transfected cells. Means ± SE, n = 4 independent experiments.

  • Fig. 4 Reduction of ORL1 decreases NgR1 expression in Neuro2A and neurons.

    (A and B) Neuro2A cells stably expressing FLAG-NgR1 were transfected with control, ORL1 #1, or ORL1 #2 small interfering RNA (siRNA). Cell lysates were immunoblotted for FLAG and actin (A). Quantification of FLAG-NgR1 protein levels in the lysates normalized to actin from three independent experiments (B). Means ± SE. ***P < 0.005, one-way ANOVA followed by Tukey’s test. (C and D) Neuro2A cells stably expressing FLAG-NgR1 were transfected with control or ORL1 siRNA. After 36 hours, cells were fixed, stained for FLAG, and analyzed by flow cytometry (C). The graph shows the mean fluorescence intensity from three independent experiments ±SE (D). ***P < 0.005, Student’s two-tailed t test. siNC, nontargeting control small interfering RNA. (E and F) Cortical neurons were transduced with lentiviral vector for control, ORL1 short hairpin RNA (shRNA) #1, or ORL1 shRNA #2 at 3 DIV. Cell lysates were immunoblotted for NgR1 and actin (E). The graph shows the quantification of NgR1 protein levels in the lysates normalized to actin from four independent experiments (F). Means ± SE. ***P < 0.005, **P < 0.01, one-way ANOVA followed by Tukey’s test. (G and H) Cortical neurons were nucleofected with control vector or HA-ORL1. Cell lysates were immunoblotted for NgR1 and actin (G). The graph shows the quantification of NgR1 protein levels in the lysates normalized to actin from three independent experiments (H). Means ± SE. *P < 0.05, Student’s two-tailed t test. (I and J) Cortical neurons at 10 DIV were treated with the indicated concentrations of b-GalNAc for 24 hours (I) and PI-PLC for 1 hour (J). Cell lysates were immunoblotted for NgR1 and actin. n = 4 independent experiments. (K) Cortical neurons at 10 DIV were treated with indicated concentrations of b-GalNAc for 24 hours, and culture medium was replaced with PI-PLC–containing medium for 1 hour. NgR1 immunoprecipitates from the culture medium and cell lysates were immunoblotted for NgR1 and actin, respectively. n = 3 independent experiments. (L) Cortical neurons at 10 DIV were treated with CHX (10 μg/ml) for indicated times. Cell lysates were immunoblotted for NgR1 and actin. n = 4 independent experiments. (M) Cortical neurons at 10 DIV were treated with CHX (10 μg/ml) for an indicated time, and culture medium was replaced with PI-PLC–containing medium for 1 hour. NgR1 immunoprecipitates from the culture medium and cell lysates were immunoblotted for NgR1 and actin, respectively. n = 4 independent experiments.

  • Fig. 5 ORL1 inhibits axon regeneration.

    (A and B) Cortical neurons were transduced with lentiviral vector for control or ORL1 shRNA at 3 DIV. Axons were scraped at 8 DIV and allowed to regenerate for 3 days in the presence of vehicle or Nogo22 (100 nM). The microphotographs show ßIII-tubulin (in axons; green) and phalloidin (to stain F-actin; red) to illustrate the growth cones of cortical neurons in the middle of the scraped area (A). Scale bar, 100 μm. The graph shows quantification of axon regeneration (B). Means ± SE. n = 10 biological replicates, **P < 0.01, #not significant, one-way ANOVA followed by Tukey’s test. (C and D) Cortical neurons were transduced with lentiviral vector for control or ORL1 shRNA at 3 DIV. Axons were scraped at 8 DIV and allowed to regenerate for 3 days. Images are of the scraped areas from Ngr1+/+ and Ngr1−/− cultures (C). Scale bar, 100 μm. The graph shows quantification of axon regeneration (D). Means ± SE. n = 4 biological replicates, ***P < 0.005, *P < 0.05, Student’s two-tailed t test. (E and F) Cortical neurons were nucleofected with control vector or HA-ORL1. Axons were scraped at 8 DIV and allowed to regenerate for 3 days. Images are of the scraped area from Ngr1+/+ cultures (E). Scale bar, 100 μm. The graph shows quantification of axon regeneration (F). Data are means ± SE. n = 3 biological replicates, ***P < 0.005, Student’s two-tailed t test.

  • Fig. 6 Nociceptin inhibits axonal regeneration in the absence of NgR1.

    (A and B) Cortical neurons were scraped and treated with an indicated amount of nociceptin at 8 DIV for 3 days (A). Scale bar, 100 μm. The graph shows quantification of axonal regeneration (B). Means ± SE. n = 4 biological replicates, *P < 0.05, **P < 0.01, one-way ANOVA followed by Dunnett’s test (Ngr1+/+), Student’s two-tailed t test (Ngr1−/−). (C and D) Cortical neurons were scraped and treated with J113397 at 8 DIV for 3 days (C). Scale bar, 100 μm. The graph shows quantification of axon regeneration (D). Means ± SE. n = 4 biological replicates, ***P < 0.005, Student’s two-tailed t test. (E and F) Cortical neurons were scraped and treated with vehicle, Nogo22 (100 nM), nociceptin (300 nM), or Nogo22 (100 nM) + nociceptin (noci; 300 nM) at 8 DIV for 3 days (E). Scale bar, 100 μm. The graph shows quantification of axonal regeneration (F). Means ± SE. n = 5 biological replicates, ***P < 0.005, *P < 0.05, one-way ANOVA followed by Tukey’s test. (G and H) Cortical neurons were scraped and treated with vehicle, nociceptin (300 nM), or nociceptin (300 nM) + Y27632 (100 nM) at 8 DIV for 3 days (G). Scale bar, 100 μm. The graph shows quantification of axonal regeneration (H). Means ± SE. n = 5 biological replicates, *P < 0.05, ***P < 0.005, #not significant, one-way ANOVA followed by Tukey’s test.

  • Fig. 7 Administration of the ORL1 antagonist J113397 improves recovery from SCI.

    (A) Open-field locomotion performance as assessed by the BMS of vehicle- and J113397-injected WT mice. Animals were scored on days 0, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 by two experienced observers who were blinded to the group. Means ± SE. n = 10 vehicle-injected mice and n = 9 J113397-injected mice. *P = 0.022, repeated-measures ANOVA for treatment effect across last five time points. (B) The extent of spared tissue at the injury site was quantified. Data are presented as means ± SE. n = 10 vehicle-injected mice and n = 9 J113397-injected mice. No significant differences between groups. Student’s two-tailed t test. (C) Open-field locomotion performance as assessed by the BMS of WT and Ngr1−/− mice after T7 dorsal overhemisection and treatment with vehicle or J113397. Animals were scored on days 0, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, and 98 by two experienced observers who were blinded to the group. Means ± SE. n = 14 mice in each group. *P = 0.010, repeated-measures ANOVA for group effect across last six time points, and *P < 0.05, **P < 0.01, significant difference between indicated pairs by repeated-measures ANOVA with Tukey’s multiple comparison test. (D) The extent of spared tissue at the injury site was quantified. Means ± SE. n = 14 mice in each group. No significant differences between groups. One-way ANOVA. (E) Gridwalk test at day 80 after injury. Means ± SE. n = 14 mice in each group. *P < 0.05, one-way ANOVA followed by Tukey’s test.

  • Fig. 8 Administration of the ORL1 antagonist J113397 increases raphespinal axon growth after SCI.

    (A and C) Raphespinal axon sprouting in WT animals. Representative image of raphespinal fibers stained for 5HT in the spinal ventral horn. Coronal sections of cervical (A) and lumbar (C) were from vehicle- and J113397-treated mice at 72 days after hemisection. Scale bar, 100 μm. (B and D) Quantification of 5HT+ fiber density in WT animals. Means ± SE. n = 7 vehicle-injected mice and n = 7 J113397-injected mice. #No significant difference (B), *P < 0.05 (D), Student’s two-tailed t test. (E and G) Representative image of raphespinal fibers stained for 5HT in the spinal ventral horn. Coronal sections of cervical (E) and lumbar (G) were from vehicle- and J113397-treated mice from each genotype at 98 days after overhemisection. Scale bar, 100 μm. (F and H) Quantification of 5HT+ fiber density. Means ± SE. n = 14 mice for each group, #no significant differences (F), **P < 0.01, ***P < 0.005 (H), one-way ANOVA followed by Tukey’s test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/524/eaao4180/DC1

    Fig. S1. NgR1 interacts with ORL1.

    Fig. S2. ORL1 interacts with NgR1 and NgR2.

    Fig. S3. Quantification of NgR1 protein in PI-PLC–treated HEK293T cells.

    Fig. S4. Identification of O-glycosylation sites in NgR1.

    Fig. S5. Reduction of ORL1 decreases NgR1 expression in Neuro2A and neurons.

    Fig. S6. Effect of J113397 on nociceptin- and Nogo-mediated inhibition of regeneration.

    Fig. S7. Motor performance after SCI and J113397 treatment.

    Fig. S8. Effect of high-dose J113397.

    Fig. S9. Model for nociception/ORL1-mediated regulation of neural repair in relation to NgR1.

    Table S1. Gene expression in brainstem after SCI in Ngr1−/− and Ngr1+/ mice.

  • Supplementary Materials for:

    The nociceptin receptor inhibits axonal regeneration and recovery from spinal cord injury

    Yuichi Sekine, Chad S. Siegel, Tomoko Sekine-Konno, William B. J. Cafferty, Stephen M. Strittmatter*

    *Corresponding author. Email: stephen.strittmatter{at}yale.edu

    This PDF file includes:

    • Fig. S1. NgR1 interacts with ORL1.
    • Fig. S2. ORL1 interacts with NgR1 and NgR2.
    • Fig. S3. Quantification of NgR1 protein in PI-PLC–treated HEK293T cells.
    • Fig. S4. Identification of O-glycosylation sites in NgR1.
    • Fig. S5. Reduction of ORL1 decreases NgR1 expression in Neuro2A and neurons.
    • Fig. S6. Effect of J113397 on nociceptin- and Nogo-mediated inhibition of regeneration.
    • Fig. S7. Motor performance after SCI and J113397 treatment.
    • Fig. S8. Effect of high-dose J113397.
    • Fig. S9. Model for nociception/ORL1-mediated regulation of neural repair in relation to NgR1.
    • Table S1. Gene expression in brainstem after SCI in Ngr1/ and Ngr1+/ mice.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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