4E-BP1-dependent translation in microglia controls mechanical hypersensitivity in male and female mice

Abstract

Spinal microglia play a pivotal role in the development of neuropathic pain. Peripheral nerve injury induces changes in the transcriptional profile of microglia, including increased expression of components of the translational machinery. Whether microglial protein synthesis is stimulated following nerve injury and has a functional role in mediating pain hypersensitivity is unknown. Here, we show that nascent protein synthesis is upregulated in spinal microglia following peripheral nerve injury in both male and female mice. Stimulating mRNA translation in microglia by selectively ablating the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) promoted the transition of microglia to a reactive state and induced mechanical hypersensitivity in both sexes, whereas spontaneous pain was increased only in males. Conversely, inhibiting microglial translation by expressing a mutant form of 4E-BP1 in microglia attenuated their activation following peripheral nerve injury and alleviated neuropathic pain in both sexes. Thus, stimulating 4E-BP1-dependent translation promotes microglial reactivity and mechanical hypersensitivity, whereas inhibiting it alleviates neuropathic pain.

Keywords: Cell biology; Neuroscience; Pain.

Figure 1. Increase in microglial protein synthesis after peripheral nerve injury.

Protein synthesis in spinal microglia was assessed using FUNCAT. (A) Schematic illustration of FUNCAT. DIBO; Met RS, methionyl-tRNA synthetase; Met tRNA, methionine transfer RNA. (B) Mice were fed a methionine-deficient diet for 3 days before and 4 days after SNI. On day 4 after SNI, AHA was injected, and spinal cords were collected 3 hours later. (C and D) Immunostaining against Iba1 was used to identify microglia. AHA signal in Iba1⁺ microglia was assessed on the ipsilateral and contralateral sides of injury in males (representative images and quantification normalized to contralateral side in C; n = 6 mice per group) and females (representative images and quantification normalized to contralateral side in D; n = 6 mice per group). Unpaired 2-tailed t test was used for statistical analyses. Data are plotted as mean ± SEM. *P < 0.05. Scale bar: 30 μm for low-magnification images and 10 μm for high-magnification images.

Figure 2. Increase in microglial translation induces mechanical pain hypersensitivity.

(A) Schematic illustration depicting regulation of mRNA translation via the mTORC1/4E-BP1 axis. (B) Generation of mice with conditional ablation of 4E-BP1 in microglia under the TMEM119^CreERT2 promoter. (C) IHC against 4E-BP1 confirmed reduced levels of 4E-BP1 in 4E-BP1–cKO mice (n = 3 mice/group). Scale bar: 20 μm. Yellow arrows mark the location of microglia. Control (TMEM119^CreERT2) and 4E-BP1–cKO mice were tested for mechanical and thermal sensitivity. Mechanical sensitivity was tested using von Frey (males in D and females in F) and tail-clip (males in E and females in G) assays. Thermal sensitivity was tested using radiant heat paw-withdrawal (males in H and females in J) and hot-plate (males in I and females in K) assays. Spontaneous pain was assessed using the mouse grimace scale (MGS; males in L and females in M). In behavioral experiments, n = 7–10 mice per group. For analyses, unpaired 2-tailed t test was used. Two-way ANOVA was used to assess sex differences in the MGS test (L and M), revealing a significant main effect of sex [F_(1,24) = 6.548, P = 0.0172]. Tukey’s multiple-comparison test showed a difference between control and 4E-BP1–cKO males (P = 0.0041), but not between females (P = 0.8575). Data are plotted as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 3. Phenotypic changes in microglia with enhanced translation.

(A) The number of Iba1⁺ microglia in the dorsal horn is not different in 4E-BP1–cKO mice compared with control (TMEM119^CreERT2) animals (representative images in A and quantification in B, n = 4 mice per group). Scale bar: 100 μm. 3D analysis of Iba1⁺ microglia revealed that 4E-BP1–cKO microglia exhibit changes in Sholl analysis results (C: representative images, skeleton and volumetric reconstructions; D: quantification; n = 4 mice/group), branch number (E: n = 4 mice /group), process length (F: n = 4 mice /group), and volumetric analysis of CD68 (C: representative 3D volume renders reconstruction images; G: quantification, as a percentage of the total cell volume; n = 4 mice/group). Scale bar: 10 μm. Unpaired 2-tailed t test was used for B, E, F, and G. Two-way ANOVA followed by Tukey’s post hoc comparison was used for D. Data are plotted as mean ± SEM. **P < 0.01, ***P < 0.001.

Figure 4. TRAP RNA-Seq reveals differentially expressed genes in 4E-BP1–cKO microglia.

(A) Schematic illustration of the TRAP approach. (B) Imaging of spinal cord section from L10a-eGFP:TMEM119^CreERT2 mice confirmed the presence of L10a-eGFP in Iba1⁺ microglia. Scale bars: 100 μm (left) and 10 μm (right). (C) Microglial markers are enriched and nonmicroglial markers are depleted in IP fractions. (D) Heatmap of the correlation coefficients between different samples (IN, input; IP, immunoprecipitated). (E) Dual-flashlight plot shows 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 upregulated or downregulated in 4E-BP1–cKO are: increased, SSMD ≥ 0.9, BC ≥ 0.5, FC ≥ 1.33; decreased, SSMD ≤ 0.9, Bhattacharyya coefficient ≤ 0.5, FC ≤ 1.33. (F) Top 15 upregulated and downregulated genes in IP fractions in 4E-BP1–cKO versus control mice. (G) Gene Ontology (GO) analysis of the top 100 upregulated genes (Enrichr, GO Molecular Function).

Figure 5. Downregulating translation in microglia attenuates microglial reactivity after peripheral nerve injury.

(A) Generation of 4E-BP1 cMT (Tg-4EBP1mt:TMEM119^CreERT2) mice. (B and C) Peripheral nerve injury (SNI) induced a smaller increase in the number of Iba1⁺ microglia in the lumbar dorsal horn in 4E-BP1 cMT mice than in control (Tg-4EBP1mt) mice. Scale bar: 100 μm. (D–I) Analysis of microglial morphology and CD68 on the ipsilateral (ipsi) and contralateral (contra) side of injury (SNI, day 4) in 4E-BP1 cMT and control (Tg-4EBP1mt) mice. (D) Representative images of Iba1 and CD68 staining and their volumetric reconstruction. Scale bar: 20 μm. Sholl analysis (E), AUC for Sholl analysis (F), branch number (G), process length (H), and volumetric analysis of CD68 (I). n = 4 mice per condition. Two-way ANOVA followed by Tukey’s post hoc comparison. Data are plotted as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6. Downregulating translation in microglia alleviates neuropathic pain.

Paw-withdrawal thresholds (von Frey assay) were tested in 4E-BP1 cMT and control (Tg-4EBP1mt) male (A) and female (B) mice after SNI (n = 8 mice per group). BL, baseline. MGS was used to assess spontaneous pain on day 4 after SNI in male (C) and female (D) 4E-BP1 cMT and control mice (n = 7–8 mice per group). Mutant 4E-BP1 expression was induced in microglia after pain hypersensitivity was established by peripheral nerve injury via injection of tamoxifen to 4E-BP1 cMT (and control, Tg-4EBP1mt) male (E) and female (F) mice from day 4 to 9 after SNI. Two-way ANOVA followed by Tukey’s post hoc comparison was used for A, B, E, and F. One-way ANOVA followed by Tukey’s post hoc comparison was used for C and D. Data are plotted as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.