CARTp/GPR160 mediates behavioral hypersensitivities in mice through NOD2

Neuropathic pain is a debilitating chronic condition that remains difficult to treat. More efficacious and safer therapeutics are needed. A potential target for therapeutic intervention recently identified by our group is the G-protein coupled receptor 160 (GPR160) and the cocaine- and amphetamine-regulated transcript peptide (CARTp) as a ligand for GPR160. Intrathecal administration of CARTp in rodents causes GPR160-dependent behavioral hypersensitivities. However, the molecular and biochemical mechanisms underpinning GPR160/CARTp-induced behavioral hypersensitivities in the spinal cord remain poorly understood. Therefore, we performed an unbiased RNA transcriptomics screen of dorsal horn spinal cord (DH-SC) tissues harvested at the time of peak CARTp-induced hypersensitivities and identified nucleotide-binding oligomerization domain-containing protein 2 ( Nod2 ) as a gene that is significantly upregulated. Nucleotide-binding oligomerization domain-containing protein 2 is a cytosolic pattern-recognition receptor involved in activating the immune system in response to bacterial pathogens. While NOD2 is well studied under pathogenic conditions, the role of NOD2-mediated responses in nonpathogenic settings is still not well characterized. Genetic and pharmacological approaches reveal that CARTp-induced behavioral hypersensitivities are driven by NOD2, with co-immunoprecipitation studies indicating an interaction between GPR160 and NOD2. Cocaine- and amphetamine-regulated transcript peptide-induced behavioral hypersensitivities are independent of receptor-interacting protein kinase 2 (RIPK2), a common adaptor protein to NOD2. Immunofluorescence studies found NOD2 co-expressed with endothelial cells rather than glial cells, implicating potential roles for CARTp/NOD2 signaling in these cells. While these findings are based only on studies with male mice, our results identify a novel pathway by which CARTp causes behavioral hypersensitivities in the DH-SC through NOD2 and highlights the importance of NOD2-mediated responses in nonpathogenic settings.