PROJECT SUMMARY Traumatic Spinal Cord Injury (SCI) is a devastating condition currently affecting approximately 300,000 people in the United States. The prevalence of severe chronic pain in SCI is 16%-63%. Following SCI, rapid activation of microglia at the injury site likely contributes to establishment of chronic pain through the release of pro- inflammatory signaling mediators. VGF (nonacronymic) is a neuropeptide precursor that is upregulated upon neuronal injury, and VGF expression is rapidly increased after contusion SCI. Dr. Vulchanova's laboratory was the first to show that VGF-derived peptides signal to microglia, and established that the VGF-derived peptide TLQP-21 contributes to both the development and maintenance of hypersensitivity after peripheral nerve injury and inflammation. TLQP-21 activates the Complement 3a (C3aR1) receptor. Because C3aR1 is upregulated in microglia following CNS injury, I hypothesize that C3aR1-expressing microglia are the cellular target of TLQP-21. This idea is supported by my preliminary results in microglial cultures indicating that TLQP-21 evokes Ca2+ transients in microglia. Signaling via C3aR1 is known to activate the mitogen-activated protein kinase family, including ERK. Activated ERK is increased in microglia after SCI and is associated with hypersensitivity by mediating release of PGE2. My overall hypothesis is that TLQP-21 and C3aR1 increase spinal microglial ERK and PGE2 signaling, ultimately leading to SCI neuropathic pain. Specific aim 1: Characterize behavioral and spinal microglial correlates of neuropathic pain after SCI and evaluate the involvement of TLQP-21 and C3aR1 in SCI pain behavior. I will evaluate behavioral signs of hypersensitivity and spontaneous pain and will quantify Iba1, pERK, TLQP-21 and C3aR1 using immunohistochemistry and in situ hybridization. Additionally I will examine the contribution of TLQP-21 and C3aR1 to the maintenance of pain behavior following SCI. My working hypothesis is that disruption of TLQP-21 and C3aR1 signaling will attenuate the maintenance of pain-related behaviors after SCI. I will use TLQP-21 sequestration as well as a C3aR1 antagonist to examine the function of TLQP-21 and C3aR1 in the establishment of pain behaviors following SCI. Specific aim 2: Establish the function of TLQP-21 and C3aR1 on spinal microglia in our SCI model. I will use Ca2+ imaging in the adult spinal cord slice to test the working hypothesis that TLQP-21-evoked C3aR1-dependent calcium transients are potentiated in microglia after SCI. Additionally, utilizing an ELISA, I will examine PGE2 release from injured spinal cord slices to test the working hypothesis that TLQP-21 evoked PGE2 release is potentiated following SCI. These studies will have a significant impact because they represent the first examination of the function of the VGF-derived peptide TLQP-21 on spinal microglial signaling and on chronic pain following SCI. The outcomes of the proposed project have the potential to impact the identification of TLQP-21 and C3aR1 as novel therapeutic targets for SCI-induced neuropathic pain.

PROJECT NARRATIVE A large percentage of spinal cord injury patients experience chronic pain following injury. The goal of this project is to examine the contribution of a novel ligand/receptor interaction to pain following spinal cord injury. The outcomes of the proposed project have the potential to impact the identification of novel therapeutic targets for treating spinal cord injury induced neuropathic pain.