4E-BP1-dependent translation in nociceptors controls mechanical hypersensitivity via TRIM32/type I interferon signaling

Abstract

Activation of the mechanistic target of rapamycin complex 1 (mTORC1) contributes to the development of chronic pain. However, the specific mechanisms by which mTORC1 causes hypersensitivity remain elusive. The eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is a key mTORC1 downstream effector that represses translation initiation. Here, we show that nociceptor-specific deletion of 4E-BP1, mimicking activation of mTORC1-dependent translation, is sufficient to cause mechanical hypersensitivity. Using translating ribosome affinity purification in nociceptors lacking 4E-BP1, we identified a pronounced translational up-regulation of tripartite motif-containing protein 32 (TRIM32), an E3 ubiquitin ligase that promotes interferon signaling. Down-regulation of TRIM32 in nociceptors or blocking type I interferon signaling reversed the mechanical hypersensitivity in mice lacking 4E-BP1. Furthermore, nociceptor-specific ablation of TRIM32 alleviated mechanical hypersensitivity caused by tissue inflammation. These results show that mTORC1 in nociceptors promotes hypersensitivity via 4E-BP1-dependent up-regulation of TRIM32/interferon signaling and identify TRIM32 as a therapeutic target in inflammatory pain.

Fig. 1.. Nociceptor-specific deletion of 4E-BP1 causes mechanical but not thermal hypersensitivity.

(A) Schematic of the mTORC1 pathway. (B) Generation of 4E-BP1 conditional knockout (cKO) mice. (C) Western blots and quantification showing decreased 4E-BP1 in DRG lysates of 4E-BP1 cKO mice (n = 4 to 5 mice per group, Student’s t test, two-tailed). Baseline testing showed increased mechanical hypersensitivity in the von Frey (D) and tail clip (E) tests but no difference in thermal sensitivity in the radiant heat paw withdrawal (F), tail flick assay (G), and hot plate test (H) (n = 8 to 10 mice per group, Student’s t test, two-tailed). Analysis revealed no main effects of sex or interactions for (D) to (H). (I) Immunostaining of DRG from wild-type mice revealed increased total 4E-BP1 in nonpeptidergic nociceptors compared to peptidergic nociceptors. (J) Quantification (n = 4 mice per group, Student’s t test, two-tailed; scale bar, 100 μm). All data are presented as mean ± SEM. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.. TRAP RNA-sequencing reveals differentially expressed genes and TRIM32 as translationally up-regulated mRNA.

(A) Schematic of translating ribosome affinity purification approach. (B) Immunostaining of DRG, from Scn10a Cre mice injected with AAV-eGFP-L10a, with IB4, CGRP, and NF200 to confirm tagging of nociceptors but not large-diameter neurons. (C) Quantification (n = 4 mice per group, Student’s t test, two-tailed; scale bar, 200 μm). (D) Neuronal markers are enriched and nonneuronal markers are depleted in IP fractions. (E) Heatmap of the correlation coefficients between different samples (input, IN; immunoprecipitated, IP). (F) Dual-flashlight plot depicting strictly standardized mean difference (SSMD) versus log₂ FC for genes in IP samples. Positive Log₂ FC indicates increased expression in 4E-BP1 cKO mice. Parameters for defining data as up-regulated or down-regulated in 4E-BP1 KO are indicated on the top.

Fig. 3.. Down-regulation of TRIM32 alleviates mechanical hypersensitivity in 4E-BP1 cKO.

(A) Western blots and quantification showing increased TRIM32 in DRG lysates of 4E-BP1 cKO mice (n = 4 to 5 mice per group, Student’s t test, two-tailed). (B) Schematic of AAV-DIO-TRIM32-shRNAmir intraperitoneal injections. (C) Western blots and quantification showing corrected TRIM32, IFN-α, and IFN-β in DRG lysates of 8-week-old 4E-BP1 cKO mice that received AAV-DIO-TRIM32-shRNAmir (intraperitoneally, postnatal days 2 to 4, n = 4 mice per group, one-way ANOVA followed by Bonferroni’s post hoc comparison). (D) Mechanical hypersensitivity in the von Frey test was rescued in 8-week-old 4E-BP1 cKO mice that received AAV-DIO-TRIM32-shRNAmir (n = 13 to 21 mice per group, one-way ANOVA followed by Bonferroni’s post hoc comparison). (E) Mechanical hypersensitivity in the von Frey test was corrected in 4E-BP1 cKO mice that received IFNAR1 neutralizing antibody (n = 12 mice per group, two-way ANOVA followed by Bonferroni’s post hoc comparison). Analysis revealed no main effects of sex or interactions for (D) and (E). (F) 4E-BP1 cKO and Scn10a Cre control mice were injected intraperitoneally with AAV-CAG-Flex-tdTomato at P2 to P4 and their DRGs were collected 8 weeks later and cultured for 24 hours. Cultured neurons were stained with IB4 to identify nonpeptidergic nociceptors and incubated in either immunoglobulin G (IgG) control or IFNAR1 neutralizing antibody for 60 min before recordings. Representative traces are shown for 200 pA ramp current injection. (G) 4E-BP1 cKO receiving IgG control had increased number of action potentials at 100 pA and 200 pA ramp intensities (n = 10 to 13 cells from six animals per group, one-way ANOVA followed by Bonferroni’s post hoc comparison). (H) Individual data points for 100-, 200-, and 400-pA stimulations are shown. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.. Down-regulation of TRIM32 alleviates CFA inflammatory pain.

(A) Western blots and quantification showing increased TRIM32, IFN-α, IFN-β, p-S6, and p-4E-BP1 in DRG lysates of animals 1 day after CFA intraplantar injection compared to vehicle control (n = 4 to 5 mice per group, Student’s t test, two-tailed). Animals that received AAV-DIO-TRIM32-shRNAmir showed alleviated mechanical (B) but not thermal (C) hypersensitivity compared to scrambled controls after intraplantar injection of CFA (n = 8 to 10 mice per group, two-way ANOVA followed by Bonferroni’s post hoc comparison). Animals that received IFNAR1 neutralizing antibody showed alleviated mechanical (D) but not thermal (E) hypersensitivity compared to IgG controls after intraplantar injection of CFA (n = 6 to 8 mice per group, two-way ANOVA followed by Bonferroni’s post hoc comparison). All data are presented as mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 5.. Proposed model.

Ablation of 4E-BP1, mimicking the hyperactivation of the mTORC1 pathway, in DRG nociceptors, results in increased eIF4E-dependent mRNA translation, including of TRIM32 mRNA. TRIM32 promotes type I IFN signaling by nonproteolytic ubiquitination of STING. Increased production of type I IFN and its autocrine signaling enhances nociceptor excitability, resulting in mechanical hypersensitivity.