A Role of NOD2 in CARTp-mediated Behavioral Hypersensitivities

Abstract

<b>Abstract ID 24468</b> <b>Poster Board 402</b> Neuropathic pain is a debilitating chronic condition that is severe and remains difficult to treat. While opioids are widely used to treat chronic pain, long-term use can cause severe side-effects and the risk of overdose. Therefore, there is a high priority to identify novel non-opioid-based therapeutic targets. Our lab discovered G-protein coupled receptor 160 (GPR160) as a potential novel therapeutic target for neuropathic pain and deorphanized it as a receptor for cocaine- and amphetamine-regulated transcript peptide (CARTp). The molecular and biochemical mechanisms underpinning CARTp-induced behavioral hypersensitivities remain poorly understood. Intrathecal (i.th.) injections of CARTp in mice lead to GPR160-dependent and dose- and time-dependent behavioral hypersensitivities similar to those seen in neuropathic pain states (i.e. mechano-and cold allodynia). Unbiased RNA transcriptomics analysis of dorsal horn spinal cord (DH-SC) tissues harvested at time of peak hypersensitivities identified nucleotide-binding oligomerization domain-containing protein 2 (<i>Nod2)</i> as a gene that was significantly upregulated in response to CARTp. This led to our hypothesis that CARTp causes behavioral hypersensitivities through NOD2. NOD2 is a cytosolic pattern recognition receptor that is involved in activating the immune system in response to bacterial pathogens by directly binding to a bacterial peptidoglycan component, muramyl dipeptide (MDP). Once NOD2 is bound to MDP, RIPK2, a kinase shown to be critical for MDP-induced NOD2 signaling, binds to NOD2, activating multiple proinflammatory pathways. When compared to wild type mice (WT, C57BL/6), the development of mechano- and cold-allodynia following i.th. injections of CARTp was significantly attenuated in NOD2^-/- mice, implicating NOD2 in the pharmacological actions of CARTp. To further validate our findings, we used a genetic approach to inhibit NOD2 in the DH-SC of mice. Knocking down <i>Nod2</i> using targeted-specific siRNA delayed CARTp-induced behavioral hypersensitivities, further confirming our knockout results. To explore further the CARTp/NOD2 signaling pathway, we tested whether knocking down RIPK2 attenuates the CARTp behavioral phenotype. Unexpectedly, genetic (i.e. <i>Ripk2</i>-targeted siRNA) or pharmacological (i.e. using a selective RIPK2 antagonist, WEHI-345) failed to block CARTp-induced behavioral hypersensitivities suggesting that RIPK2 is not required in CARTp/NOD2 signaling. In contrast and as expected, blocking RIPK2 blocked MDP-induced NOD2 signaling. These results indicate that CARTp causes sensitization through NOD2 but independent of RIPK2. Our results identify a novel pathway by which CARTp causes behavioral hypersensitivities and a novel mechanism by which NOD2 is activated in non-pathogenic settings. Support/Funding Information: T32 GM 8306-29 and NIH/NINDS grant R01NS1132257 (DS).