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, 35 (50), 16545-60

Fbxo3-Dependent Fbxl2 Ubiquitination Mediates Neuropathic Allodynia Through the TRAF2/TNIK/GluR1 Cascade

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Fbxo3-Dependent Fbxl2 Ubiquitination Mediates Neuropathic Allodynia Through the TRAF2/TNIK/GluR1 Cascade

Tzer-Bin Lin et al. J Neurosci.

Abstract

Emerging evidence has indicated that the pathogenesis of neuropathic pain is mediated by spinal neural plasticity in the dorsal horn, which provides insight for analgesic therapy. Here, we report that the abundance of tumor necrosis factor receptor-associated factor 2 and NcK-interacting kinase (TNIK), a kinase that is presumed to regulate neural plasticity, was specifically enhanced in ipsilateral dorsal horn neurons after spinal nerve ligation (SNL; left L5 and L6). Spinal TNIK-associated allodynia is mediated by downstream TNIK-GluR1 coupling and the subsequent phosphorylation-dependent trafficking of GluR1 toward the plasma membrane in dorsal horn neurons. Tumor necrosis factor receptor-associated factor 2 (TRAF2), which is regulated by spinal F-box protein 3 (Fbxo3)-dependent F-box and leucine-rich repeat protein 2 (Fbxl2) ubiquitination, contributes to SNL-induced allodynia by modifying TNIK/GluR1 phosphorylation-associated GluR1 trafficking. Although exhibiting no effect on Fbxo3/Fbxl2/TRAF2 signaling, focal knockdown of spinal TNIK expression prevented SNL-induced allodynia by attenuating TNIK/GluR1 phosphorylation-dependent subcellular GluR1 redistribution. In contrast, intrathecal administration of BC-1215 (N1,N2-Bis[[4-(2-pyridinyl)phenyl]methyl]-1,2-ethanediamine) (a novel Fbxo3 inhibitor) prevented SNL-induced Fbxl2 ubiquitination and subsequent TFAF2 de-ubiquitination to ameliorate behavioral allodynia via antagonizing TRAF2/TNIK/GluR1 signaling. By targeting spinal Fbxo3-dependent Fbxl2 ubiquitination and the subsequent TRAF2/TNIK/GluR1 cascade, spinal application of a TNF-α-neutralizing antibody ameliorated SNL-induced allodynia, and, conversely, intrathecal TNF-α injection into naive rats induced allodynia via a spinal Fbxo3/Fbxl2-dependent modification of the TRAF2/TNIK/GluR1 cascade. Together, our results suggest that spinal TNF-α contributes to the development of neuropathic pain by upregulating TRAF2/TNIK/GluR1 signaling via Fbxo3-dependent Fbxl2 ubiquitination and degradation. Thus, we propose a potential medical treatment strategy for neuropathic pain by targeting the F-box protein or TNIK.

Significance statement: TNF-α participates in neuropathic pain development by facilitating the spinal TRAF2-dependent TNIK-GluR1 association, which drives GluR1-containing AMPA receptor trafficking toward the plasma membrane. In addition, F-box protein 3 modifies this pathway by inhibiting F-box and leucine-rich repeat protein 2-mediated TRAF2 ubiquitination, suggesting that protein ubiquitination contributes crucially to the development of neuropathic pain. These results provide a novel therapeutic strategy for pain relief.

Keywords: Fbxl2; Fbxo3; GluR1; TNIK; neuropathic; ubiquitination.

Figures

Figure 1.
Figure 1.
SNL upregulates TNIK expression in dorsal horn neurons. A, Representative WB and statistical analyses (normalized to GAPDH) revealing SNL-increased TNIK expression in the ipsilateral (I and IPSI), but not contralateral (C and CONTRA), dorsal horn. Two-way ANOVA with repeated measures over time: treatment, F(3,20) = 45.8, p = 0.002; time, F(4,80) = 4.672, p < 0.001; and treatment × time, F(12,80) = 5.322, p < 0.001. **p < 0.01 versus Sham IPSI; ##p < 0.01 versus SNL day 1. n = 6. IB, Immunoblotting. B, Time course of SNL-reduced withdrawal threshold of the hindpaw (von Frey test). Two-way ANOVA with repeated measures over time: treatment, F(3,24) = 56.39, p < 0.001; time, F(5,120) = 2.912, p = 0.016; and treatment × time, F(15,120) = 4.282, p < 0.001. **p < 0.01 versus Sham IPSI; ##p < 0.01 versus day 1. n = 7. C, Images and statistical analysis at day 7 after surgery revealing SNL-increased TNIK immunofluorescence (red) in the ipsilateral dorsal horn (SNL 7D), which is colocalized with neuronal (NeuN, green), but not microglial (OX-42, green) or astrocytic (GFAP, green), markers. One-way ANOVA, post hoc Tukey's test: F(3,24) = 24.68, p < 0.001. **p < 0.01 versus Sham IPSI; ##p < 0.01 versus SNL CONTRA. n = 7. Scale bar, 50 μm.
Figure 2.
Figure 2.
Knockdown of spinal TNIK expression relieves SNL-induced allodynia. A, Representative WB and statistical analyses (normalized to GAPDH) demonstrating intrathecal administration with TNIK mRNA-targeting siRNA (TNIK RNAi; 1, 3, and 5 μg, 10 μl), but not missense siRNA (TNIK MS; 5 μg, 10 μl) or polyethylenimine (PEI; 10 μl), dose dependently decreased spinal TNIK expression. One-way ANOVA, post hoc Tukey's test: F(6,35) = 12.90, p < 0.001. **p < 0.01 versus Naive. n = 6. IB, Immunoblotting; it, implantation of an intrathecal catheter. B, Intrathecal TNIK mRNA-targeting siRNA (TNIK RNAi; 5 μg, 10 μl) resulted in no motor deficits in the animals (rotarod test). Two-way ANOVA with repeated measures over time: treatment, F(3,24) = 0.482, p = 0.698; time, F(4,96) = 0.379, p = 0.823; treatment × time, F(12,96) = 0.565, p = 0.865. n = 7. C, D, Although it exhibited no effect on that of sham-operated animals, intrathecal TNIK mRNA-targeting siRNA (5 μg, 10 μl) increased the withdrawal threshold of SNL animals at days 5 and 7 after surgery (von Frey test). In sham-operated animals, two-way ANOVA with repeated measures over time: treatment, F(3,24) = 0.059, p = 0.981; time, F(4,96) = 0.572, p = 0.683; treatment × time, F(12,96) = 0.091, p > 0.999. n = 7; In SNL animals, two-way ANOVA with repeated measures over time: treatment, F(3,24) = 21.26, p < 0.001; time, F(4,96) = 598.8, p < 0.001; treatment × time, F(12,96) = 11.19, p < 0.001. **p < 0.01 versus SNL. n = 7.
Figure 3.
Figure 3.
SNL provokes spinal TNIK–GluR1 coupling and subsequent phosphorylation-dependent GluR1–AMPAR trafficking. A, Representative WB and statistical analyses (normalized to GAPDH or N-cadherin) demonstrating SNL-increased pGluR1 and GluR1(m), but not tGluR1 expression, in the ipsilateral (IPSI) dorsal horn. One-way ANOVA, post hoc Tukey's test: pGluR1, F(2,15) = 16.03, p < 0.001; GluR1(m), F(2,15) = 5.965, p = 0.012; tGluR1, F(2,15) = 0.024, p = 0.976. *p < 0.05, **p < 0.01 versus Sham IPSI. #p < 0.05, ##p < 0.01 versus SNL day 1. n = 6. IB, Immunoblotting. B, C, Intrathecal TNIK mRNA-targeting siRNA (SNL 7D+TNIK RNAi, 5 μg, 10 μl), but not missense small-interfering RNA (TNIK MS, 10 μl), reduced SNL-enhanced pGluR1 and GluR1(m) expression. However, both conditions exhibited no effect on tGluR1 expression. One-way ANOVA, post hoc Tukey's test; pGluR1, F(3,20) = 10.36, p < 0.001; GluR1(m), F(3,20) = 11.46, p < 0.001; tGluR1, F(3,20) = 0.285, p = 0.836. **p < 0.01 versus Sham 7D. #p < 0.05 versus SNL 7D. n = 6. D, SNL increased the levels of tGluR1–TNIK and pGluR1, which were both reduced by intrathecal administration with TNIK mRNA-targeting siRNA. E, Administration with neither CNQX (daily for 4 d from day 3 to day 6 after SNL, 10 μm, 10 μl) nor vehicle (Veh; 10 μl) affected SNL-upregulated spinal TNIK expression. One-way ANOVA, post hoc Tukey's test: F(3,20) = 5.747, p = 0.005 F, Intrathecal administration with CNQX (SNL 7D+CNQX; 1, 3, and 10 nm, 10 μl), but not the vehicle solution (SNL 7D+Veh, 10 μl), increased the withdrawal threshold of the ipsilateral hindpaw. Two-way ANOVA with repeated measures over time: treatment, F(4,30) = 93.76, p < 0.001; time, F(6,180) = 34.15, p < 0.001; treatment × time, F(24,180) = 8.539, p < 0.001. *p < 0.05, **p < 0.01 versus SNL 7D. n = 7.
Figure 4.
Figure 4.
Knockdown of spinal TNIK expression reduces SNL-enhanced TNIK, pGluR1, and TNIK/pGluR1 colocalization in the dorsal horn. A, Images and statistical analyses at day 7 after surgery, SNL increased TNIK (red), pGluR1 (green), and TNIK/pGluR1 double-labeled (yellow) immunofluorescence in the ipsilateral dorsal horn (SNL 7D), which were all reduced by intrathecal administration with TNIK mRNA-targeting siRNA (SNL 7D+TNIK RNAi, 5 μg, 10 μl). One-way ANOVA, post hoc Tukey's test: TNIK, F(2,18) = 11.60, p < 0.001; pGluR1, F(2,18) = 27.18, p < 0.001; TNIK/pGluR1, F(2,18) = 32.45, p < 0.001. **p < 0.01 versus Sham 7D. ##p < 0.01 versus SNL 7D. n = 7. Scale bar, 50 μm.
Figure 5.
Figure 5.
SNL induces spinal TRAF2/TNIK/pGluR1-dependent GluR1–AMPAR trafficking. A, Representative WB and statistical analyses (normalized to GAPDH) demonstrating SNL increased TRAF2 expression in the ipsilateral (IPSI) dorsal horn. One-way ANOVA, post hoc Tukey's test: F(2,15) = 14.97, p < 0.001. **p < 0.01 versus Sham IPSI. ##p < 0.01 versus SNL day 1. n = 6. B, Administration with neither TNIK mRNA-targeting siRNA (SNL 7D+TNIK RNAi, 5 μg, 10 μl) nor missense siRNA (SNL 7D+TNIK MS, 5 μg, 10 μl) affected SNL-enhanced spinal TRAF2 expression at day 7 after surgery (normalized to GAPDH). One-way ANOVA, post hoc Tukey's test: F(3,20) = 12.83, p < 0.001. **p < 0.01 versus Sham 7D. n = 6. C, SNL increased spinal tGluR1–TRAF2/TNIK/pGluR1 coprecipitations, which were reduced with TNIK mRNA-targeting siRNA treatment. D, Images and statistical analyses demonstrated that SNL increased TRAF2 (blue), TNIK (red), pGluR1 (green), and TRAF2/TNIK/pGluR1 triple-labeled (white) immunofluorescence in the ipsilateral dorsal horn. TNIK mRNA-targeting siRNA treatment reduced SNL-enhanced TNIK (red), pGluR1 (green), and TRAF2/TNIK/pGluR1 triple-labeled (white), but not TRAF2 (blue), immunofluorescence. One-way ANOVA, post hoc Tukey's test: TRAF2, F(2,18) = 14.08, p < 0.001; TNIK, F(2,18) = 13.87, p < 0.001; pGluR1, F(2,18) = 19.62, p < 0.001; TRAF2/TNIK/pGluR1, F(2,18) = 18.58, p < 0.001. **p < 0.01 versus Sham 7D. ##p < 0.01 versus SNL 7D. n = 7. Scale bar, 50 μm.
Figure 6.
Figure 6.
BC-1215 relieves allodynia by preventing ubiquitination-dependent spinal Fbxl2/TRAF2/TNIK/pGluR1 signaling. A, Representative WB and statistical analyses (normalized to GAPDH) demonstrating SNL decreased Fbxl2 but increased Fbxo3 abundance in the ipsilateral (IPSI) dorsal horn. One-way ANOVA, post hoc Tukey's test: Fbxl2, F(2,15) = 20.63, p < 0.001; Fbxo3, F(2,15) = 11.21, p = 0.001. **p < 0.01 versus Sham. ##p < 0.01 versus SNL day 1. n = 6. B, C, Intrathecal administration with BC-1215 (SNL 7D+BC-1215, 10, 30, and 100 nm, 10 μl), but not the vehicle solution (SNL 7D+Veh, 10 μl), increased the withdrawal threshold of the ipsilateral hindpaw. Nevertheless, both these treatments failed to affect the contralateral hindpaw at 3 h after injection (BC-1215 100 nm; von Frey test). Ipsilateral hindpaw, two-way ANOVA with repeated measures over time: treatment, F(4,30) = 46.98, p < 0.001; time, F(6,180) = 22.40, p < 0.001; treatment × time, F(24,180) = 6.460, p < 0.001. *p < 0.05, **p < 0.01 versus SNL 7D. n = 7. Contralateral hindpaw, one-way ANOVA, post hoc Tukey's test: F(2,18) = 0.031, p = 0.970. D, At day 7 after surgery, SNL increased Fbxl2 ubiquitination in the ipsilateral dorsal horn, which was reversed markedly by spinal injection of BC-1215, which increased TRAF2 ubiquitination. E, F, Without affecting the abundances of tGluR1 and Fbxo3, BC-1215 reversed the SNL-increased TRAF2, TNIK, pGluR1, and GluR1(m) expressions and the SNL-decreased Fbxl2 expression. One-way ANOVA, post hoc Tukey's test: TRAF2, F(3,20) = 48.08, p < 0.001; TNIK, F(3,20) = 18.75, p < 0.001; Fbxl2, F(3,20) = 43.52, p < 0.001; Fbxo3, F(3,20) = 16.80, p < 0.001; pGluR1, F(3,20) = 26.66, p < 0.001; tGluR1, F(3,20) = 0.260, p = 0.854; GluR1(m), F(3,20) = 28.80, p < 0.001. **p < 0.01 versus Sham 7D. ##p < 0.01 versus SNL 7D. n = 6. G, Administration with neither TNIK mRNA-targeting siRNA (SNL 7D+TNIK RNAi, 5 μg, 10 μl) nor missense siRNA (SNL 7D+TNIK MS, 5 μg, 10 μl) affected SNL-upregulated Fbxo3 and SNL-downregulated Fbxl2 expression. One-way ANOVA, post hoc Tukey's test: Fbxl2, F(3,20) = 23.94, p < 0.001; Fbxo3, F(3,20) = 4.992, p = 0.010. *p < 0.05, **p < 0.01 versus Sham 7D. n = 6. H, SNL-enhanced tGluR1-TRAF2/TNIK/pGluR1 precipitations were reduced by spinal injection of BC-1215.
Figure 7.
Figure 7.
BC-1215 reduces SNL-enhanced TRAF2, TNIK, pGluR1, and triple-labeled immunofluorescence. A, Images and statistical analyses showing SNL enhanced TRAF2 (blue), TNIK (red), pGluR1 (green), and TRAF2/TNIK/pGluR1 triple-labeled (white) immunofluorescence in the ipsilateral dorsal horn at 7 d after surgery, which were all reduced by a spinal BC-1215 injection (SNL 7D+BC-1215, 100 nm, 10 μl). One-way ANOVA, post hoc Tukey's test: TRAF2, F(2,18) = 40.67, p < 0.001; TINK, F(2,18) = 17.53, p < 0.001; pGluR1, F(2,18) = 26.50, p < 0.001; TRAF2/TNIK/pGluR1, F(2,18) = 29.46, p < 0.001. **p < 0.01 versus Sham 7D. ##p < 0.01 versus SNL 7D. n = 7. Scale bar, 50 μm.
Figure 8.
Figure 8.
TNF-α-neutralizing antibody relieves allodynia by attenuating spinal Fbxo3/Fbxl2/TRAF2/TNIK/pGluR1-dependent GluR1–AMPAR trafficking. A, B, Intrathecal administration with an TNF-α-neutralizing antibody (SNL 7D+TNF-α Ab; 10, 30, and 100 ng, 10 μl), but not nonspecific IgG (SNL 7D+IgG; 100 ng, 10 μl), dose dependently increased the withdrawal threshold of the ipsilateral hindpaw. In addition, neither treatment affected the contralateral hindpaw at 2 h after injection (TNF-α-neutralizing antibody, 100 ng; von Frey test). Ipsilateral hindpaw, two-way ANOVA with repeated measures over time: treatment, F(4,30) = 64.39, p < 0.001; time, F(6,180) = 24.44, p < 0.001; treatment × time, F(24,180) = 4.657, p < 0.001. *p < 0.05, **p < 0.01 versus SNL 7D. n = 7. Contralateral hindpaw, one-way ANOVA, post hoc Tukey's test: F(2,18) = 0.047, p = 0.954. C, D, Although it had no effect on tGluR1, administrating with TNF-α-neutralizing antibody reversed SNL-upregulated TRAF2, TNIK, Fbxo3, pGluR1, GluR1(m), and SNL-downregulated Fbxl2 (normalized to GAPDH or N-cadherin) expressions. One-way ANOVA, post hoc Tukey's test: TRAF2, F(3,20) = 75.39, p < 0.001; TNIK, F(3,20) = 29.62, p < 0.001; Fbxl2, F(3,20) = 34.78, p < 0.001; Fbxo3, F(3,20) = 22.30, p < 0.001; pGluR1, F(3,20) = 24.55, p < 0.001; tGluR1, F(3,20) = 0.086, p = 0.966; GluR1(m), F(3,20) = 10.10, p < 0.001. **p < 0.01 versus Sham 7D. ##p < 0.01 versus SNL 7D. n = 6.
Figure 9.
Figure 9.
TNF-α induces allodynia via spinal Fbxo3/Fbxl2/TRAF2/TNIK/pGluR1-dependent GluR1–AMPAR trafficking. A, Statistical analysis of von Frey test showing intrathecal application of TNF-α (1 pm, 10 μl) significantly decreased the withdrawal threshold of the ipsilateral hindpaw at 2 h after injection, which was prevented by pretreatment with TNIK mRNA-targeting siRNA (5 μg, 10 μl, once daily for 4 d before TNF-α application) and BC-1215 (100 nm, 10 μl, 2 h before TNF-α application) but not by missense siRNA (5 μg, 10 μl) or the vehicle solution (10 μl). One-way ANOVA, post hoc Tukey's test: F(6,42) = 61.05, p < 0.001. **p < 0.01 versus Naive; ##p < 0.01 versus TNF-α. n = 7. B, C, Administration with TNIK mRNA-targeting siRNA prevented TNF-α-increased TNIK, pGluR1, and GluR1(m) expression but exhibited no effect on the levels of TRAF2, Fbxl2, Fbxo3, and tGluR1. Intrathecal pretreatment with BC-1215 prevented increases in TRAF2, TNIK, Fbxo3, pGluR1, and GluR1(m) expressions and decreases in Fbxl2 expression caused by TNF-α injection. However, it had no effect on tGluR1 expression. One-way ANOVA, post hoc Tukey's test: TRAF2, F(6,35) = 21.42, p < 0.001; TNIK, F(6,35) = 23.9, p < 0.001; Fbxl2, F(6,35) = 14.28, p < 0.001; Fbxo3, F(6,35) = 51.42, p < 0.001; pGluR1, F(6,35) = 37.03, p < 0.001; tGluR1, F(6,35) = 0.422, p = 0.859; GluR1(m), F(6,35) = 29.21, p < 0.001. **p < 0.01 versus Sham 7D; ##p < 0.01 versus SNL 7D. n = 6. D, Bolus of BC-1215 prevented TNF-α-enhanced tGluR1-TRAF2/TNIK/pGluR1 coprecipitation. Daily TNIK mRNA-targeting siRNA administration exhibited similar effects with the exception that it failed to affect TNF-α-enhanced tGluR1-TRAF2 coprecipitation.

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