Fibroblast growth factor 13 stabilizes microtubules to promote Na+ channel function in nociceptive DRG neurons and modulates inflammatory pain

Abstract

Introduction: Fibroblast growth factor homologous factors (FHFs), among other fibroblast growth factors, are increasingly found to be important regulators of ion channel functions. Although FHFs have been link to several neuronal diseases and arrhythmia, its role in inflammatory pain still remains unclear.

Objectives: This study aimed to investigate the role and mechanism of FGF13 in inflammatory pain.

Methods: Fgf13 conditional knockout mice were generated and CFA-induced chronic inflammatory pain model was established to measure the pain threshold. Immunostaining, western blot and quantitative real-time reverse transcription PCR (qRT-PCR) were performed to detect the expression of FGF13 in CFA-induced inflammatory pain. Whole-cell patch clamp recording was used to record the action potential firing properties and sodium currents of DRG neurons.

Results: Conditional knockout of Fgf13 in dorsal root ganglion (DRG) neurons (Fgf13^-/Y) led to attenuated pain responses induced by complete Freund's adjuvant (CFA). FGF13 was expressed predominantly in small-diameter DRG neurons. CFA treatment resulted in an increased expression of FGF13 proteins as well as an increased excitability in nociceptive DRG neurons which was inhibited when FGF13 was absent. The role of FGF13 in neuronal excitability of DRG was linked to its modulation of voltage-gated Na⁺ channels mediated by microtubules. Overexpression of FGF13, but not FGF13 mutant which lacks the ability to bind and stabilize microtubules, rescued the decreased neuronal excitability and Na⁺ current density in DRG neurons of Fgf13^-/Y mice.

Conclusion: This study revealed that FGF13 could stabilize microtubules to modulate sodium channel function in DRG neurons and modulate inflammatory pain. This study provides a novel mechanism for FGF13 modulation of sodium channel function and suggests that FGF13 might be a novel target for inflammatory pain treatment.

Keywords: DRG; Fibroblast growth factor 13; Inflammatory pain; Microtubules; Na+ channel.

Graphical abstract

Fig. 1

Fgf13 conditional knockout mice showed reduced response to inflammatory pain. (A) Representative confocal images of immunostaining of FGF13 in the DRG. FGF13 staining was abolished in Fgf13 conditional knockout mice. Scale bar: 50 μm. (B) Representative images of FGF13 protein expression in the DRG of Loxp and Fgf13 KO mice detected by western blot. (C) The mRNA expression levels of FGF13 detected by qPCR. Gapdh was used as reference gene. (D) In the hot plate test, the latency responses were increased in Fgf13^-/Y mice compared with controls at 49 °C, 52 °C and 55 °C. (E) In the tail immersion test, the latency responses were increased in Fgf13^-/Y mice compared with control mice at 50 °C and 52 °C. (F) Response latencies in Hargreaves test. (G) Thermal withdrawal latencies of Loxp and Fgf13 knockout mice after CFA injection. n = 8 mice/group. *P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 2

CFA injection increased the expression of FGF13 in DRG neurons. (A, B, C) Representative immunofluorescence images depicted co-localization of FGF13 with different categories of neuronal markers in the DRG of wild type mice. NF200, a marker of large sized neurons; CGRP, a marker of small diameter peptidergic neurons; IB4, a marker of small diameter non-peptidergic neurons. (D) Quantification of overlap between FGF13 and neuronal markers. Scale bar: 50 μm. n = 3 mice/group. (E) Thermal pain hypersensitivity after intraplantar CFA injection in Loxp mice. n = 8 mice/group. (F) Representative images of FGF13 protein expression in L3-L5 DRGs on day 0, 1, 3, 7 and 14 after CFA or normal saline treatment detected by Western blot. (G) Summary of panel. (H) Representative immunofluorescence images of FGF13 labeled neurons in L4/L5 DRGs on day 0, 1, 3, 7 and 14 after CFA injection. Scale bar: 50 μm. (I) Summary of the relative fluorescence intensity of FGF13 staining. n = 3 mice/group. One-way ANOVA, followed by Dunnett’s t post hoc test. (J) The relative expression levels of FGF11-14 in the ipsilateral L3-L5 DRGs on day 7 after CFA injection. All data were corrected with GAPDH and normalized to FGF13 of control group. *P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 3

Fgf13 conditional knockout decreased the excitability of nociceptive DRG neurons. (A-D) Representative responses of DRG neurons from Loxp and KO mice to 1 s, 300pA depolarizing current injection. (E) The summary of the number of action potentials elicited by depolarizing current steps of neurons from KO mice and Loxp mice under either physiological or CFA-induced chronic inflammatory pain conditions. (F) The action potential threshold in small, current-clamped DRG neurons. n = 15–20 cells/group. One-way ANOVA, followed by Bonferroni post hoc test. *P < 0.05, ^**P < 0.01.

Fig. 4

Fgf13 conditional knockout attenuated CFA-induced increase in Na⁺ currents in DRG neurons. (A, B) Representative traces of total Na_v, TTX-R Na_v and TTX-S Na_v currents in FGF13⁺/Na_v1.8⁺ neurons of Loxp mice and FGF13^-/Na_v1.8⁺neurons of KO mice. (C, D) Representative images of single cell PCR results. (E-H) Averaged current density of total Na_v, TTX-R Na_v, TTX-S Na_v and Na_v1.7 channels in L3-L5 DRG neurons 7 days after intraplantar CFA injection. n = 7–13 cells/group. One-way ANOVA, followed by Bonferroni post hoc test. *P < 0.05, ^**P < 0.01.

Fig. 5

FGF13 can modulate the stability of microtubules in DRG neurons. (A) The ability of FGF13 and FGF13 mutant to bind to tubulin was confirmed by co-IP. (B) Immunoblotting of DRG tissues showed the Ace-tubulin, Tyr-tubulin, Detyr-tubulinand α-tubulin levels in Loxp and KO mice. The lower panel showed the summarized data. n = 3 mice/group. (C) Western blots showed Ace-tubulin, Tyr-tubulin, Detyr-tubulin and α-tubulin expression in DRG neurons by overexpression of FGF13 or FGF13 mutant. The lower panel showed the summarized data. n = 3. 5 mice were included in each group. Two-way ANOVA, followed by Bonferroni post hoc test. *P < 0.05.

Fig. 6

The effect of pharmacological manipulation of microtubules on subcellular distribution of Na⁺ channel proteins and Na⁺ channel current density. (A) Subcellular localization of Na⁺ channel proteins in different conditions. Scale bar: 5 μm. The lower panel showed the fluorescence intensities of Na⁺ channel proteins along the line marked by the white arrow in the picture. (B) Representative Na⁺ channel currents of DRG neurons. (C) I-V curves of Na⁺ channel currents. (D) Maximum current density of Na⁺ currents. n = 15–20 cells/group. One-way ANOVA, followed by Dunnett’s t post hoc test. *P < 0.05; ^**P < 0.01.

Fig. 7

FGF13 overexpression rescued the decrease in Na⁺ channel current density in Fgf13 conditional knockout mice. (A-D) Averaged current density of total Na_v, TTX-R Na_v, TTX-S Na_v and Na_v1.7 currents after the adenovirus-mediated delivery of FGF13 and FGF13 mutant (FGF13 M) in DRG neurons from KO mice. N = 7–16 cells/group. One-way ANOVA, followed by Bonferroni post hoc test. *P < 0.05, ^**P < 0.01.

Fig. 8

FGF13 overexpression rescued the decreased excitability of Fgf13 conditional knockout mice. (A, G) Representative response of DRG neurons from Loxp and KO mice to 1 s, 300pA depolarizing current injection. (B, H) Summary of the number of action potentials elicited by depolarizing current steps in DRG neurons. Current steps start from 50 to 500 pA, with 50 pA increment, lasting 1 s. (C, I) Summary of the current threshold for eliciting action potentials in DRG neurons. One-way ANOVA, followed by Bonferroni post hoc test. (D, J) Representative current clamp recordings of three groups in DRG neurons under ramp current stimulation from 0 to 1000 pA of 500 ms duration (see inset). (E, K) Summary of the action potential threshold in small, current-clamped DRG neurons elicited by ramp current stimulation. One-way ANOVA, followed by Bonferroni post hoc test. (F, L) Summary of the number of action potentials elicited by ramp current stimulation. n = 15–24 cells/group. One-way ANOVA, followed by Bonferroni post hoc test. *P < 0.05, ^**P < 0.01.