GPR160 de-orphanization reveals critical roles in neuropathic pain in rodents

Abstract

Treating neuropathic pain is challenging and novel non-opioid-based medicines are needed. Using unbiased receptomics, transcriptomic analyses, immunofluorescence, and in situ hybridization, we found that the expression of the orphan GPCR Gpr160 and GPR160 increased in the rodent dorsal horn of the spinal cord following traumatic nerve injury. Genetic and immunopharmacological approaches demonstrated that GPR160 inhibition in the spinal cord prevented and reversed neuropathic pain in male and female rodents without altering normal pain response. GPR160 inhibition in the spinal cord attenuated sensory processing in the thalamus, a key relay in the sensory discriminative pathways of pain. We also identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a GPR160 ligand. Inhibiting endogenous CARTp signaling in spinal cord attenuated neuropathic pain, whereas exogenous intrathecal CARTp evoked painful hypersensitivity through GPR160-dependent ERK and cAMP response element-binding protein (CREB). Our findings de-orphanize GPR160, identify it as a determinant of neuropathic pain and potential therapeutic target, and provide insights into its signaling pathways. CARTp is involved in many diseases including depression and reward and addiction; de-orphanization of GPR160 is a major step forward understanding the role of CARTp signaling in health and disease.

Keywords: G-protein coupled receptors; Neuroscience; Pain.

Figure 1. Gpr160 and GPR160 upregulation in the spinal cord following CCI.

(A) Quantitative real-time PCR analysis of oGPCR mRNA expression in the dorsal and ventral horns of the spinal cord from rats with CCI on day 7 (n = 5). (B and C) RNA-Seq analyses of rat DH-SC ipsilateral to CCI on day 9. (B) Differential expression of 60 GPCRs between CCI and sham (n = 3/group). (C) Gpr160 in CCI and SHAM. TPMs, total reads per million. (D) Immunolabeled GPR160 (red) in lamina I/II spinal cord of rats with CCI. Ipsilateral (Ipsi), contralateral (Contra), GFAP (green), and NeuN (blue). (E–G) RNAScope analyses of the rat DH-SC on day 10 after CCI. (E) Quantitation of total Gpr160. (F and G) Association (white arrows; F) of Gpr160 (magenta) and Aif1 (microglia; yellow) increased ipsilateral to CCI (G). Nuclei were stained with DAPI (cyan). Scale bars: 100 μm (D) or 10 μm (F). Data are expressed as (A) median, interquartile range, and minimum/maximum values or (E and G) mean ± SD. (A–C, E, and G) Data analyzed by 2-tailed Student’s t test; (B and C) adjusted by Benjamini-Hochberg false discovery rate. *P < 0.05 versus Contra and ^#P < 0.05 and q < 0.05 versus sham.

Figure 2. GPR160 inhibition attenuated and reversed neuropathic pain.

(A) CCI-induced and (B) spared nerve injury–induced (SNI-induced) mechano-allodynia in male rats were prevented by daily i.th. siGpr160 (A, n = 6; B, n = 4), but not sieGfp control (A, n = 7; B, n = 4). (C) CCI-induced mechano-allodynia in male rats was reversed by i.th. siGpr160, but not sieGfp (n = 3/group). CCI-induced (D) mechano-allodynia (n = 6/group) and (E) cold-allodynia (n = 3/group) in male rats was reversed with i.th. GPR160 Ab, but not with nonspecific IgG. (F) Intrathecal GPR160 Ab (n = 4), but not IgG (n = 5), reversed CCI-induced mechano-allodynia in female rats. (G) Intrathecal GPR160 Ab or IgG (n = 7/group) in normal male rats had no effect on tail-flick nociceptive responses. (H and I) Intrathecal CARTp Ab (H, n = 11; I, n = 3), but not IgG (H, n = 8; I, n = 4), reversed CCI-induced mechano-allodynia in male mice (H) and rats (I). When compared with baseline, i.th. GPR160 Ab (n = 5) attenuated neuronal responses to punctate mechanical stimuli (J), but not ongoing neuronal activity (K) in spinal nerve ligation (SNL), but not sham, rats. No effects on neuronal responses to punctate mechanical stimuli (L) or ongoing neuronal activity (M) were observed with IgG (n = 4) in naive rats. Data are expressed as mean ± SD (A–I) or mean ± SEM (J–M) and analyzed by 2-tailed, 2-way repeated-measures ANOVA with Bonferroni’s multiple-comparisons test (A–F and H–L) or 2-tailed t test (G and M). *P < 0.05 versus day 0 (D0), ^#P < 0.05 versus D7, and ^†P < 0.05 versus baseline (BL). PWT, paw withdrawal threshold.

Figure 3. CARTp is a GPR160 ligand.

(A and B) CARTp-stimulated (A) cFOS in human KATO III cells and (B) ERK phosphorylation in PC-12 cells; events attenuated with siGpr160, but not control siRNA (A, sieGfp or B, siCON [noncoding scrambled siRNA]; n = 3 experiments/group with 3 replicates/experiment). tERK, total ERK. (C) In KATO III cell lysates, exogenous CARTp coimmunoprecipitated with GPR160 (n = 3 experiments/group with 3 replicates/experiment). (D) FAM-labeled CARTp (green) colocalized (yellow; white arrows) with GPR160 (red) in KATO III cells (n = 3 experiments with 1 replicate/experiment). (E and F) Proximity ligation assay revealed the close proximity (red) of CARTp and GPR160 (n = 3 experiments with 2 replicates/experiment) in CARTp-treated cells (F), but not in untreated cells (E). Blue = nuclear staining. Data are expressed as mean ± SD and were analyzed by 2-tailed 1-way ANOVA with Dunnett’s multiple-comparisons test. *P < 0.05 versus Veh and ^†P < 0.05 versus CARTp + sieGfp/siCON.

Figure 4. CARTp induced mechano-hypersensitivity in mice through GPR160-dependent ERK-CREB activation in the spinal cord.

(A) Time-dependent development of mechano-allodynia in mice (n = 4) after i.th. CARTp 55–102 or CARTp 62–102. (B and C) Mechano-allodynia measured 1 hour after i.th. CARTp 55–102 (B, n = 6; C, n = 4) or CARTp 62–102 (B, n = 7; C, n = 5) was reduced with i.th. GPR160 Ab (B, n = 10 and n = 8, respectively) or i.th. CARTp Ab (C, n = 6 and n = 5, respectively). (D and E) When compared with vehicle (D, n = 8; E, n = 4), i.th. CARTp 55–102 induced phosphorylation of ERK (D; p-ERK, n = 9) and CREB (E; p-CREB, n = 6) in the DH-SC, which was attenuated with i.th. coinjections of MEK inhibitor U0126 (D, n = 9; E, n = 5), CREB inhibitor 666-15 (E, n = 5), or GPR160 Ab (D, n = 6). (F) CARTp 55–102–induced mechano-allodynia (n = 20) was attenuated with coinjection of U0126 (n = 20) or 666-15 (n = 6). Vehicles for CARTp, U0126, and 666-15 (n = 17) had no effect on behavior. (G) Proposed model of CARTp/GPR160–induced signaling. Data are expressed as mean ± SD and were analyzed by (A) 2-tailed, 2-way ANOVA with Bonferroni’s multiple-comparisons test or (B–F) 2-tailed, 1-way ANOVA with Dunnett’s multiple-comparisons test. *P < 0.05 versus 0 hours; ^#P < 0.05 versus Veh; and ^†P < 0.05 versus respective CARTp plus Veh. PWT, paw withdrawal threshold.