Research ArticlePain

Palmitoylation of δ-catenin promotes kinesin-mediated membrane trafficking of Nav1.6 in sensory neurons to promote neuropathic pain

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Science Signaling  27 Mar 2018:
Vol. 11, Issue 523, eaar4394
DOI: 10.1126/scisignal.aar4394
  • Fig. 1 The palmitoylation of δ-catenin contributes to the neuropathic pain.

    (A and B) δ-Catenin palmitoylation was examined in the dorsal root ganglion (DRG) (A) and spinal dorsal cord (B) isolated on days 1, 4, 7, and 10 after rats were treated with oxaliplatin (Oxal). n = 6 rats per time point. One-way analysis of variance (ANOVA) followed by Tukey post hoc test, *P < 0.05, **P < 0.01 versus vehicle. (C) Mechanical allodynia in the rats treated with oxaliplatin and intrathecal injection of various doses of palmitoylation inhibitor 2-bromopalmitate (2-BP) for 10 consecutive days before oxaliplatin. n = 12 rats per group. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test, F5,264 = 120.0, P < 0.0001. **P < 0.01 versus vehicle and ##P < 0.01 versus the corresponding oxaliplatin group. (D and E) δ-Catenin palmitoylation in the DRG (D) and spinal dorsal cord (E) from rats isolated on days 1, 5, 10, and 15 after lumbar 5 ventral root transection (L5-VRT). n = 6 rats per time point. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the sham group. (F to H) Effects of intrathecal application of 2-BP (F) or δ-catenin short hairpin RNA (shRNA) (G and H) on mechanical allodynia induced by L5-VRT (F and H) or oxaliplatin (G). n = 12 rats in per group. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test, F2,99 = 235.4 (F), F3,176 = 550.8 (G), F3,132 = 246.5 (H), P < 0.0001. **P < 0.01 versus sham/scramble and ##P < 0.01 versus the corresponding L5-VRT/oxaliplatin group. (I) Immunohistochemistry assessing the colocalization of δ-catenin with neurofilament 200 (NF200), calcitonin gene–related peptide (CGRP), and IB4 (neuronal markers) and Nav1.6 in DRG tissues. n = 3 rats. Scale bar, 50 μm.

  • Fig. 2 TNF-α mediates the δ-catenin palmitoylation in DRG.

    (A and B) Tumor necrosis factor–α (TNF-α) abundance in DRG tissues from rats after oxaliplatin treatment (A) or L5-VRT (B). n = 6 rats per time point. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus controls. (C) Effect of intraperitoneal application of thalidomide before oxaliplatin for 10 consecutive days on oxaliplatin-induced palmitoylation of δ-catenin assessed 10 days after oxaliplatin treatment. n = 6 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus vehicle, ##P < 0.01 versus the corresponding oxaliplatin group. (D) Effect of TNF-α (1 ng/ml for 4 hours) on the abundance of palmitoylated δ-catenin in cultured DRG neurons. n = 6 per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus vehicle.

  • Fig. 3 DHHC3 was involved in the δ-catenin palmitoylation in the development of neuropathic pain.

    (A) Expression of mRNA encoding palmitoyl acyltransferases in DRG tissues after oxaliplatin (day 10) or L5-VRT (day 15) treatment. n = 5 rats per group. Two-tailed, independent Student’s t test. (B) Co–immunoprecipitation (IP) of DHHC3 (Asp-His-His-Cys 3), DHHC8, and DHHC9 with antibody to δ-catenin in lysates from DRG tissues isolated from rats treated with vehicle or oxaliplatin (day 10). n = 6 rats per group. IgG, immunoglobulin G; IB, immunoblot. (C) Significant increase in δ-catenin was immunoprecipitated with DHHC3 antibody on day 10 after oxaliplatin treatment. n = 6 rats per group. (D) Representative blots and histogram showed the up-regulation of DHHC3 induced by oxaliplatin treatment. n = 5 rats per time point. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group. (E) High-resolution image from structured illumination microscopy (SIM) assessing the colocalization between δ-catenin and DHHC3 in DRG tissues of rats treated with vehicle or oxaliplatin (day 10). n = 4 rats per group. Two-tailed, independent Student’s t test, **P < 0.01 versus the vehicle group. White box, zoomed right. Scale bar, 1 μm. (F) δ-Catenin palmitoylation in DRG tissues from rats treated with oxaliplatin (day 10) after intrathecal injection of DHHC3 shRNA. n = 5 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus vehicle and ##P < 0.01 versus the corresponding scramble + oxaliplatin group. (G) δ-Catenin palmitoylation in DRG tissues from L5-VRT rats (day 15) after intrathecal injection of DHHC3 shRNA. n = 5 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the sham group and ##P < 0.01 versus the corresponding scramble + L5-VRT group. (H and I) Mechanical allodynia induced by oxaliplatin treatment (H) or L5-VRT (I) after intrathecal injection of DHHC3 shRNA. n = 12 rats in per group. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test, F3,176 = 463.2 (H), F3,132 = 349.4 (I), P < 0.0001. **P < 0.01 versus the control group and ##P < 0.01 versus the corresponding oxaliplatin or L5-VRT group. (J) Representative blots and histogram assessing DHHC3 abundance in cultured DRG cells cultured with TNF-α (1 ng/ml for 4 hours). n = 7 per group. Two-tailed, independent Student’s t test, **P < 0.01 versus vehicle. (K) High-resolution image assessing the colocalization between DHHC3 and δ-catenin in cultured DRG neurons incubated with vehicle or TNF-α (1 ng/ml for 4 hours). White box, zoomed right. n = 3 per group. Two-tailed, independent Student’s t test, **P < 0.01 versus the vehicle group. Scale bar, 1 μm.

  • Fig. 4 Nav1.6 membrane trafficking contributed to the development of neuropathic pain.

    (A) Co-IP of Nav1.6, Nav1.7, Nav1.8, and Nav1.9 with antibody to δ-catenin in lysates from DRG tissues isolated from rats treated with vehicle, oxaliplatin (day 10), or 2-BP + oxaliplatin (day 10). n = 6 rats per group. (B) High-resolution image showing the colocalization between Nav1.6 and δ-catenin in DRG tissues from rats treated with vehicle, oxaliplatin (day 10), or 2-BP + oxaliplatin (day 10). n = 4 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding oxaliplatin group. Scale bar, 1 μm. (C) Abundance of membrane and total Nav1.6 in DRG tissues from vehicle or oxaliplatin rats and the proportion of Nav1.6 trafficking in vehicle or oxaliplatin rat’s DRG tissues. n = 6 rats per time point. One-way ANOVA followed by Tukey post hoc test, *P < 0.05, **P < 0.01 versus the vehicle group. TfR, transferrin receptor. (D) Surface expression of Nav1.6 induced by oxaliplatin (day 10) after intrathecal injection of Nav1.6 shRNA. n = 6 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group; #P < 0.05, ##P < 0.01 versus the corresponding oxaliplatin group. (E and F) Mechanical allodynia induced by oxaliplatin (E) and L5-VRT (F) after intrathecal injection of Nav1.6 shRNA. n = 12 rats per group. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test, F3,176 = 343.8 (E), F3, 132 = 392.8 (F), P < 0.0001. **P < 0.01 versus the corresponding control group and ##P < 0.01 versus the corresponding oxaliplatin or L5-VRT group. (G) Surface accumulation of Nav1.6 in DRG tissues of oxaliplatin (day 10) rats after intrathecal injection of brefeldin A (BFA) (15 nmol/10 μl). n = 6 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding oxaliplatin group.

  • Fig. 5 δ-Catenin palmitoylation-mediated Nav1.6 membrane trafficking.

    (A and B) Membrane, total Nav1.6 expression, and proportion of Nav1.6 trafficking in oxaliplatin (on day 10) rat DRG tissues after 2-BP (A) or δ-catenin shRNA (B) intrathecal application. n = 5 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding oxaliplatin group. (C and D) Abundance of Nav1.6 in membrane of L5-VRT (day 15) rat DRG tissues after intrathecal application of 2-BP (C) or δ-catenin shRNA (D). n = 5 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the sham group and ##P < 0.01 versus the corresponding L5-VRT group. (E) Abundance of Nav1.6 in membrane of DRG neurons after incubation of vehicle, TNF-α (1 ng/ml for 4 hours) or 2-BP (100 nM) 20 to 30 min before TNF-α. n = 6 per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the TNF-α group. (F) High-resolution images showing the colocalization between Nav1.6 and δ-catenin in cultured DRG neurons described in (E). n = 4 per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the TNF-α group. Scale bar, 1 μm.

  • Fig. 6 Palmitoylation of δ-catenin increased the current density of Nav1.6.

    (A) δ-Catenin palmitoylation in the human embryonic kidney (HEK) 293T cells after incubation of vehicle, TNF-α (1 ng/ml for 4 hours), or 2-BP (100 nM) 20 to 30 min before TNF-α. n = 6 per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding TNF-α group. (B) Representative current-voltage relation curve of currents recorded in the HEK293T cells described in (A). (C) Current density of Nav1.6 in cells as described in (A). n = 10 cells per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding TNF-α group. (D to F) Activation (D), inactivation (E), or recovery (F) curves of Nav1.6 in cells as described in (A). (G) Abundance of palmitoylated δ-catenin in HEK293T cells overexpressing δ-catenin. n = 6 per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the mCherry group. (H) Nav1.6 current density in HEK293T cells overexpressing δ-catenin and incubated with 2-BP (100 nM). n = 9 cells in mCherry group and n = 10 cells in Ctnnd2-mCherry and Ctnnd2-mCherry + 2-BP group, respectively. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the mCherry group and ##P < 0.01 versus the corresponding Ctnnd2-mCherry group. (I) δ-Catenin palmitoylation in cells described in (H). n = 5 experiments per group. Two-tailed, independent Student’s t test, **P < 0.01 versus the Ctnnd2-mCherry + 2-BP group.

  • Fig. 7 KIF3A was involved in palmitoylated δ-catenin–mediated membrane trafficking of Nav1.6.

    (A) DRG tissue lysates from rats treated with vehicle, oxaliplatin, or 2-BP + oxaliplatin (on day 10) were immunoprecipitated with δ-catenin–specific antibody, and the immunocomplex was further analyzed with blotting for KIF3A (kinesin 3A), KIF3B, KIF5A, KIF5B, KIF17, or KIFC2. n = 6 rats per group. (B) Blotting assessment of δ-catenin Co-IP with KIF3A antibody in DRG tissue lysates described in (A). n = 6 rats per group. (C) SIM triple-fluorescence staining of δ-catenin and KIF3A in DRG tissues from rats treated as described in (A). n = 3 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding oxaliplatin group. Scale bar, 1 μm. (D) Colocalization of δ-catenin and KIF3A in cultured DRG neurons after incubation with vehicle, TNF-α (1 ng/ml for 4 hours), or 2-BP (100 nM) 20 to 30 min before TNF-α. n = 4 per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding TNF-α group. Scale bar, 1 μm. (E and F) Co-IP analysis of KIF3A and Nav1.6 in DRG tissue lysates from oxaliplatin-treated rats (on day 10) after intrathecal application of 2-BP (E) or δ-catenin shRNA (F). n = 6 rats per group. (G) SIM triple-fluorescence staining of Nav1.6 and KIF3A in DRG tissues from rats treated as described in (E) and (F). n = 4 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding oxaliplatin group. Scale bar, 1 μm. (H) SIM triple-fluorescence staining of KIF3A and Nav1.6 in cultured neurons after incubation with vehicle, TNF-α (1 ng/ml for 4 hours), or 2-BP (100 nM) 20 to 30 min before TNF-α. n = 4 per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding TNF-α group. Scale bar, 1 μm. (I and J) Abundance of surface Nav1.6 in DRG tissues intrathecally injected with KIF3A shRNA and treated with oxaliplatin (day 10) (I) or subjected to L5-VRT (day 15) (J). n = 5 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the corresponding control group and ##P < 0.01 versus the corresponding oxaliplatin or L5-VRT group. (K and L) Mechanical allodynia in rats induced by oxaliplatin (K) or L5-VRT (L) after intrathecal injection of KIF3A shRNA. n = 12 rats per group. Two-way repeated-measures ANOVA followed by Bonferroni post hoc test, F3,176 = 346.3 (K), F3,132 = 251.6 (L), P < 0.0001. **P < 0.01 versus the corresponding control group and ##P < 0.01 versus the corresponding oxaliplatin or L5-VRT group. (M) Surface abundance of Nav1.6 in cultured DRG neurons transfected with KIF3A shRNA and incubated with TNF-α (1 ng/ml for 4 hours). n = 5 per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding TNF-α group. (N) Nav1.6 current density in HEK293T cells transfected with KIF3A shRNA and incubated with TNF-α (1 ng/ml for 4 hours). n = 10 cells per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the scramble group and ##P < 0.01 versus the corresponding TNF-α group.

  • Fig. 8 Palmitoylation mediates the formation of a complex comprising KIF3A, Nav1.6, and δ-catenin.

    (A) SIM triple-fluorescence staining of δ-catenin, KIF3A, and Nav1.6 in DRG tissues from rats treated with vehicle, oxaliplatin, or 2-BP + oxaliplatin (at day 10). n = 4 rats per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding oxaliplatin group. Scale bar, 1 μm. (B) SIM triple-fluorescence staining of δ-catenin, KIF3A, and Nav1.6 in cultured DRG neurons incubated with vehicle, TNF-α (1 ng/ml for 4 hours), or 2-BP (100 nM) 20 to 30 min before TNF-α. n = 5 per group. One-way ANOVA followed by Tukey post hoc test, **P < 0.01 versus the vehicle group and ##P < 0.01 versus the corresponding TNF-α group. Scale bar, 1 μm.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/523/eaar4394/DC1

    Fig. S1. shRNA knockdown of δ-catenin in DRG tissues.

    Fig. S2. shRNA knockdown of DHHC3 in DRG tissues.

    Fig. S3. shRNA knockdown of Nav1.6 in DRG tissues.

    Fig. S4. Overexpression of δ-catenin in HEK293T cells.

    Fig. S5. shRNA knockdown of KIF3a in DRG tissues.

    Table S1. shRNA nucleotide sequences.

    Table S2. PCR primer sequences.

    Table S3. Antibodies.

    Movie S1. Colocalization between δ-catenin and DHHC3 in oxaliplatin-treated DRG neurons.

    Movie S2. Colocalization among δ-catenin, KIF3A and Nav1.6 in DRG neurons incubated with TNF-α.

  • Supplementary Materials for:

    Palmitoylation of δ-catenin promotes kinesin-mediated membrane trafficking of Nav1.6 in sensory neurons to promote neuropathic pain

    Xiao-Long Zhang, Huan-Huan Ding, Ting Xu, Meng Liu, Chao Ma, Shao-Ling Wu, Jia-You Wei, Cui-Cui Liu, Su-Bo Zhang, Wen-Jun Xin*

    *Corresponding author. Email: xinwj{at}mail.sysu.edu.cn

    This PDF file includes:

    • Fig. S1. shRNA knockdown of δ-catenin in DRG tissues.
    • Fig. S2. shRNA knockdown of DHHC3 in DRG tissues.
    • Fig. S3. shRNA knockdown of Nav1.6 in DRG tissues.
    • Fig. S4. Overexpression of δ-catenin in HEK293T cells.
    • Fig. S5. shRNA knockdown of KIF3a in DRG tissues.
    • Table S1. shRNA nucleotide sequences.
    • Table S2. PCR primer sequences.
    • Table S3. Antibodies.
    • Legends for movies S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Colocalization between δ-catenin and DHHC3 in oxaliplatin-treated DRG neurons.
    • Movie S2 (.mp4 format). Colocalization among δ-catenin, KIF3A and Nav1.6 in DRG neurons incubated with TNF-α.

    © 2018 American Association for the Advancement of Science

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