FABP5 deletion in nociceptors augments endocannabinoid signaling and suppresses TRPV1 sensitization and inflammatory pain

Abstract

The endocannabinoid anandamide (AEA) produces antinociceptive effects by activating cannabinoid receptor 1 (CB1). However, AEA also serves as an agonist at transient receptor potential vanilloid receptor 1 (TRPV1) in nociceptive sensory neurons, which may exacerbate pain. This potential functional duality is highlighted by the failure of an inhibitor of the AEA catabolic enzyme fatty acid amide hydrolase (FAAH) to afford pain relief in a clinical trial. Consequently, it remains to be determined whether elevating AEA levels in nociceptors leads to antinociceptive or pro-nociceptive effects. Fatty acid binding protein 5 (FABP5) is an intracellular carrier that mediates AEA transport to FAAH for inactivation. Leveraging the abundant expression of FABP5 in TRPV1⁺ nociceptors, we employed a conditional knockout strategy to demonstrate that FABP5 deletion in nociceptors augments AEA levels, resulting in the emergence of antinociceptive effects mediated by CB1. Mechanistically, FABP5 deletion suppresses inflammation- and nerve growth factor-mediated TRPV1 sensitization via CB1, an effect mediated by calcineurin. Unexpectedly, inhibition of FAAH failed to blunt TRPV1 sensitization, uncovering functionally distinct outputs resulting from FABP5 and FAAH inhibition. Collectively, our results demonstrate that FABP5 serves a key role in governing endocannabinoid signaling in nociceptors to disrupt TRPV1 sensitization and pain, and position FABP5 as a therapeutic target for the development of analgesics.

Figure 1

Characterization of FABP5 cKO mice. (A) Schematic showing the generation of FABP5^Flox mice and placement of LoxP sites. (B) PCR analysis of WT, heterozygous, and homozygous FABP5^Flox mice. (C) qPCR analysis of relative FABP5 expression in DRGs of FABP5^Flox and FABP5 cKO mice. **p < 0.01 calculated using unpaired t test (n = 3). (D) FABP5 distribution in DRGs of FABP5^Flox and FABP5 cKO mice.

Figure 2

Deletion of FABP5 in nociceptors suppresses inflammatory hyperalgesia. (A) Thermal withdrawal latencies in male and female WT, FABP5 KO, FABP5^Flox, and FABP5 cKO mice at baseline and 4 h after intraplantar carrageenan injection. ***p < 0.001 calculated using one-way ANOVA followed by Tukey post-hoc test (n = 6). (B) Behavioral spectrometer analysis of still behavior at baseline and after carrageenan injection in WT male and female mice administered naproxen (30 mg/kg, i.p.) or vehicle. **p < 0.01 calculated using paired t test (n = 6). (C) Still behavior in male and female WT, FABP5 KO, FABP5^Flox, and FABP5 cKO mice at baseline and 4 h after carrageenan injection. **p < 0.01; ***p < 0.001 calculated using paired t test (n = 6).

Figure 3

CB1 and PPARα mediate the antinociceptive effects in FABP5 cKO mice. (A) Levels of AEA, PEA, OEA, and 2-AG in DRGs of male and female FABP5^Flox and FABP5 cKO mice at baseline and 4 h after intraplantar carrageenan injection. **p < 0.01; ***p < 0.001 vs WT and FABP5^Flox calculated using two-way ANOVA followed by Bonferroni post-hoc test (n = 5). (B) Percent expression of CB1, PPARα, FAAH, NAPE-PLD, and ABHD4 in DRGs of male and female FABP5 cKO mice relative to FABP5^Flox controls. (C) FAAH and NAPE-PLD activity in DRGs. Left: DRGs were incubated with the selective FAAH substrate AMC-AEA (100 µM). Right: DRGs were incubated with the phospholipase A1 substrate PED-A1 (10 µM) in the presence or absence of the selective NAPE-PLD inhibitor LEI-401 (30 µM) (n = 3). (D) Thermal withdrawal latencies in FABP5 cKO mice receiving vehicle, AM251 (3 mg/kg, i.p.), or GW6471 (4 mg/kg, i.p.). ***p < 0.001 calculated using one-way ANOVA followed by Dunnett’s post-hoc test (n = 6).

Figure 4

FABP5 deletion attenuates carrageenan-induced and TRPV1-evoked spinal CGRP release. (A) WT, FABP5 KO, and FABP5 cKO mice received intraplantar injections of vehicle or carrageenan and lumbar spinal cords were obtained 4 h later. The sections were incubated for 30 min and CGRP release was subsequently quantified. *p < 0.05 calculated using unpaired t test (n = 5). (B) WT, FABP5 KO, and FABP5 cKO mice were administered vehicle, 3 mg/kg AM251, or 4 mg/kg GW6471 prior to intraplantar injections of carrageenan. Lumbar spinal cord sections were collected 4 h later and incubated with vehicle followed by 1 µM capsaicin in the presence or absence of 3 µM AM251 or 10 µM GW6471 for 30 min. CGRP release was quantified prior to and after the addition of capsaicin. **p < 0.01; ***p < 0.001 calculated using one-way ANOVA followed by Dunnett’s post-hoc test (n = 5).

Figure 5

Co-expression of FABP5 with (A) TrkA and (B) CB1 in DRG neurons. Representative images of FABP5, CB1, and TrkA distribution in L4-L5 DRGs. DAPI is shown in blue.

Figure 6

FABP5 deletion blunts NGF-mediated TRPV1 sensitization via CB1 and PPARα. (A) Design of calcium imaging experiment and representative calcium traces in WT and FABP5 KO DRGs incubated with NGF and capsaicin. DRG neurons received two 30 s pulses of 100 nM capsaicin (Cap, gray bars) interspersed with a 5 min incubation of vehicle (black trace) or 100 ng/ml NGF (blue trace). KCl was employed to confirm neuronal identity. (B) Capsaicin response ratio (2nd/1st peak) in WT (white bars) and FABP5 KO (gray bars) DRGs incubated with vehicle or NGF in the presence or absence of 3 µM AM251, 10 µM GW6471, or 200 nM cyclosporin A. The effect of FAAH inhibition upon capsaicin responses was evaluated by incubating WT DRGs with 1 µM PF3845 for 2 h prior to capsaicin application. ***p < 0.001 calculated using one-way ANOVA followed by Dunnett’s post-hoc test. (C) qPCR analysis of relative expression levels of TRPV1, TrkA, CB1, and PPARα in male and female FABP5 KO DRGs relative to WT controls (n = 3). (D) Levels of AEA in WT DRGs incubated for 2 h with vehicle or 1 µM PF3845. *p < 0.05 calculated using unpaired t test (n = 3). (E) Capsaicin-evoked CGRP release in WT mice administered vehicle or URB937 (1 mg/kg, i.p.). Mice received URB937 1 h prior to intraplantar carrageenan administration and spinal CGRP release elicited by 1 µM capsaicin was examined 4 h later (n = 5). (F) AEA levels in DRGs and lumbar spinal cords (LSC) of mice injected with vehicle or URB937 (1 mg/kg, i.p.). *p < 0.05 calculated using unpaired t test (n = 3).

Figure 7

Capsaicin-induced nocifensive behavior in WT and FABP5 cKO mice. Mice received an intraplantar injection of vehicle or 200 ng NGF followed by vehicle or 1 µg capsaicin. The duration of nocifensive behavior defined as paw licking or biting was subsequently scored for 5 min. *p < 0.05 calculated using unpaired t test (n = 6).