RGS4 Maintains Chronic Pain Symptoms in Rodent Models

Abstract

Regulator of G-protein signaling 4 (RGS4) is a potent modulator of G-protein-coupled receptor signal transduction that is expressed throughout the pain matrix. Here, we use genetic mouse models to demonstrate a role of RGS4 in the maintenance of chronic pain states in male and female mice. Using paradigms of peripheral inflammation and nerve injury, we show that the prevention of RGS4 action leads to recovery from mechanical and cold allodynia and increases the motivation for wheel running. Similarly, RGS4KO eliminates the duration of nocifensive behavior in the second phase of the formalin assay. Using the Complete Freud's Adjuvant (CFA) model of hindpaw inflammation we also demonstrate that downregulation of RGS4 in the adult ventral posterolateral thalamic nuclei promotes recovery from mechanical and cold allodynia. RNA sequencing analysis of thalamus (THL) from RGS4WT and RGS4KO mice points to many signal transduction modulators and transcription factors that are uniquely regulated in CFA-treated RGS4WT cohorts. Ingenuity pathway analysis suggests that several components of glutamatergic signaling are differentially affected by CFA treatment between RGS4WT and RGS4KO groups. Notably, Western blot analysis shows increased expression of metabotropic glutamate receptor 2 in THL synaptosomes of RGS4KO mice at time points at which they recover from mechanical allodynia. Overall, our study provides information on a novel intracellular pathway that contributes to the maintenance of chronic pain states and points to RGS4 as a potential therapeutic target.SIGNIFICANCE STATEMENT There is an imminent need for safe and efficient chronic pain medications. Regulator of G-protein signaling 4 (RGS4) is a multifunctional signal transduction protein, widely expressed in the pain matrix. Here, we demonstrate that RGS4 plays a prominent role in the maintenance of chronic pain symptoms in male and female mice. Using genetically modified mice, we show a dynamic role of RGS4 in recovery from symptoms of sensory hypersensitivity deriving from hindpaw inflammation or hindlimb nerve injury. We also demonstrate an important role of RGS4 actions in gene expression patterns induced by chronic pain states in the mouse thalamus. Our findings provide novel insight into mechanisms associated with the maintenance of chronic pain states and demonstrate that interventions in RGS4 activity promote recovery from sensory hypersensitivity symptoms.

Keywords: RGS; RNA sequencing; genetic mouse models; inflammatory pain; neuropathic pain; viral gene transfer.

Figure 1.

RGS4KO mice rapidly recover from mechanical allodynia in a model of long-term peripheral inflammation. a, Male RGS4WT and RGS4KO mice show mechanical allodynia in the Von Frey assay in response to CFA injection to the left hindpaw, but RGS4KO mice recover by day 13 (two-way ANOVA followed by Bonferroni post hoc tests: F_(1,28) = 16.39, **p = 0.001, ***p < 0.001; n = 15). b, RGS4KO mice show identical responses to their RGS4WT controls in the Hargreaves assay of thermal hyperalgesia (two-way ANOVA: F_(1,20) = 0.0009, n = 11/group). c, qPCR analysis reveals that RGS4 mRNA expression in the PAG, THL, DR, NAc, and ipsilateral SC does not change at early time points (4 d) after the induction of peripheral inflammation (CFA injection to the left hindpaw). Unpaired t test: PAG, t₍₂₀₎ = 0.1893; THL, t₍₉₎ = 0.966; DR, t₍₁₀₎ = 1.153; NAc, t₍₁₀₎ = 0.8572; SC ipsilateral, t₍₁₈₎ = 0.8891; n = 5–11/group; for all comparisons, p > 0.05). d, At later time points (17 d post-CFA), there is an increase in Rgs4 transcript in the PAG and in the THL (unpaired t test: PAG: t₍₁₁₎ = 6.629, ***p < 0.001, n = 6–7/group; THL: t₍₁₀₎ = 2.64, *p = 0.02, n = 6/group; DR: t₍₁₁₎ = 1.521, n = 6–7/group, NAc: t₍₁₃₎ = 1.778, n = 7–8/group; SC: t₍₂₄₎ = 3.604 **p = 0.001, n = 13/group).

Figure 2.

A role of RGS4 in symptoms of peripheral inflammation in female mice. a, Female RGS4KO mice show attenuated mechanical allodynia by day 8 after CFA injection to the ipsilateral hindpaw (two-way ANOVA followed by Bonferroni post hoc tests: F_(1,27) = 14.08, *p = 0.03, ***p < 0.001, n = 14–15/group). b, Female RGS4KO mice show attenuated responses to cold (0°C) at 9 d after CFA treatment compared with the RGS4WT group (unpaired t test: t₍₂₇₎ = 3.219, **p = 0.003, n = 14–15/group). c, Prevention of RGS4 action improves voluntary wheel running. Female RGS4KO mice show higher running wheel activity compared with RGS4WT controls at 13 d after left hindpaw CFA injection, suggesting recovery from chronic pain states. Baseline responses are indistinguishable between phenotypes. Bars represent the percentage of naive baseline responses (two-way ANOVA followed by Bonferroni post-tests: F_(1,26) = 5.106, *p = 0.04, n = 7–8/group). d, In contrast, both RGS4WT and RGS4KO female mice show identical responses in the Hargreaves assay for thermal hyperalgesia (two-way ANOVA: F_(1,27) = 0.2289, n = 14–15/group). e, Female RGS4WT and RGS4KO mice show similar licking/flinching levels during the first 40 min of the formalin test, but after this time point RGS4KO mice show recovery from nocifensive states (two-way ANOVA followed by Bonferroni post hoc tests: F_(1,14) = 3.256, **p = 0.008, n = 7–9/group).

Figure 3.

A role of RGS4 in the maintenance of neuropathic states. a, Using the SNI model of neuropathic pain, we show that female RGS4KO mice recover from mechanical allodynia at 21 d after nerve injury, while their RGS4WT controls show maximal allodynia in the Von Frey test throughout the 1 month monitoring period (two-way ANOVA followed by Bonferroni post-tests: F_(1,18) = 17.28, ***p < 0.001, n = 9–11/group). b, Mechanical thresholds are indistinguishable in sham-treated (control) female groups, which do not develop mechanical allodynia (two-way ANOVA: F_(1,19) = 0.6013, n = 10–11/group). c, SNI-treated female RGS4KO mice show attenuated responses to cold when tested at 13 d post SNI (two-way ANOVA followed by Bonferroni post hoc tests: F_(3,35) = 5.817; RGS4WT SNI vs sham, **p = 0.005; RGS4KO sham vs RGS4WT SNI, *p = 0.01; RGS4KO SNI vs RGS4WT SNI, **p = 0.003; n = 9–11 female mice/group). d, Paclitaxel-treated female RGS4WT and RGS4KO mice show identical responses in the Von Frey assay at 10 d after the onset of paclitaxel (PTX) injections, but RGS4KO mice show reduced allodynia on day 33 (two-way ANOVA followed by Bonferroni post hoc tests: F_(1,13) = 2.225 for RGS4KO PTX day 10 vs day 33; *p = 0.019; n = 7–8/group).

Figure 4.

RGS4 knockdown in VPL-THL neurons induces earlier recovery from mechanical allodynia. a, Conditional RGS4 downregulation in neurons of the VPL-THL of male RGS4^fl/fl mice by stereotaxic infection of AAV2-CMV-CRE vectors led to recovery from mechanical allodynia in the Von Frey assay (two-way ANOVA followed by Bonferroni post hoc tests: F_(1,14) = 19.32, ***p < 0.001; n = 8/group). b, Western blot analysis verifying RGS4 downregulation in the VPL-THL of mice infected with AAV2-CMV-Cre-EGFP compared with AAV2-CMV-EGFP vectors (t test unpaired two-tailed: t₍₆₎ = 2.528, *p = 0.04; n = 4/group). c, Representative image showing GFP fluorescence 5 weeks after AAV2-CMV-Cre-EGFP infection of the VPL-THL. d, A similar phenotype was observed in female RGS4^fl/fl mice injected with AAV2-CMV-CRE vectors in the VPL-THL. e, Downregulation of RGS4 in the female VPL-THL leads to recovery from mechanical allodynia in the Von Frey assay (two-way ANOVA followed by Bonferroni post hoc tests: F_(1,13) = 12.33; ***p < 0.001; n = 7–8/group) and alleviates cold hypersensitivity (unpaired two-tailed t test: t₍₁₃₎ = 2.552; *p = 0.0241, n = 7–8/group). f, g, Notably, the downregulation of RGS4 in the VPL-THL does not affect wheel-running activity (unpaired two-tailed t test: t₍₁₀₎ = 0.6497; n = 6/group; f) and Hargreaves responses (unpaired two-tailed t test: t₍₁₃₎ = 0.7550; n = 7–8/group; g). h, Downregulation of RGS4 in the MD-THL of male mice does not promote recovery from mechanical allodynia in the CFA model (two-way ANOVA: F_(1,12) = 0.1690; n = 7/group). i, Representative image showing GFP fluorescence in the MD-THL of mice infected with AAV2-CMV-EGFP. j, Downregulation of RGS4 in the NAc of male mice does not affect the trajectory of mechanical allodynia in the CFA model (two-way ANOVA: F_(1,13) = 0.8012; n = 6–9/group). k, Representative image showing GFP fluorescence in the NAc of mice infected with AAV2-CMV-EGFP.

Figure 5.

THL-specific RNA-Seq profiling of RGS4KO in groups of naive and CFA-treated mice. a, Experimental time line showing that THL tissue from male groups of mice was collected 18 d after the induction of peripheral inflammation (a time point that RGS4KO mice show recovery from mechanical allodynia). b–d, Venn diagram depicting the number of genes affected in each group and their direction of regulation suggest that long-term peripheral inflammation promotes distinct gene expression adaptations between genotypes (b) and associated gene ontology (GO) categories (c, d). Bar graphs show GO of genes uniquely regulated by pain (CFA) in the THL of RGS4WT (black color) and RGS4KO (gray color) mice. Only GO terms with p < 0.05 are depicted.

Figure 6.

RGS4 in the THL modulates gene expression patterns of peripheral inflammation. a, b, Heat map analysis (a) and Venn diagrams (b) showing the effect of peripheral inflammation in RGS4WT and RGS4KO THL (cutoff p value <0.05; cutoff log2fold <−0.25 and >0.25). The overall patterns of gene expression regulation were distinct between RGS4KO and RGS4WT groups. Interestingly, the commonly regulated genes are contraregulated by peripheral inflammation between genotypes (212 contraregulated genes). c, IPA of the set of genes affected by CFA in the RGS4KO group compared with RGS4WT predicts pathways that are selectively modulated upon RGS4 gene ablation in the RGS4KO group (cutoff: p value <0.003). d, qPCR validation of a subset of genes in a separate cohort of male animals (naive RGS4WT and CFA-treated RGS4WT: Glp1r (unpaired t test: t₍₁₁₎ = 2.735, *p = 0.02); CFA-treated RGS4WT and CFA-treated RGS4KO (unpaired t test: Gng11 t₍₂₄₎ = 2.486, *p = 0.02; Ppp3r2 t₍₁₀₎ = 2.364, *p = 0.04; Atf4 t₍₉₎ = 5.236, ***p < 0.001; n = 5–13/group. e, Western blot analysis in THL synaptosomes from naive and CFA groups of RGS4WT and RGS4KO mice reveals an upregulation of mGluR2 at 18 d after the induction of peripheral inflammation in the RGS4KO group. Two-way ANOVA followed by Bonferroni post hoc tests: F_(1,59) = 3.505, *p = 0.04, n = 15–16/group.