The EGF-LIKE domain of thrombospondin-4 is a key determinant in the development of pain states due to increased excitatory synaptogenesis

Abstract

Up-regulation of thrombospondin-4 (TSP4) or voltage-gated calcium channel subunit α₂δ₁ (Ca_vα₂δ₁) proteins in the spinal cord contributes to neuropathic pain development through an unidentified mechanism. We have previously shown that TSP4 interacts with Ca_vα₂δ₁ to promote excitatory synaptogenesis and the development of chronic pain states. However, the TSP4 determinants responsible for these changes are not known. Here, we tested the hypothesis that the Ca_vα₂δ₁-binding domains of TSP4 are synaptogenic and pronociceptive. We mapped the major Ca_vα₂δ₁-binding domains of TSP4 within the coiled-coil and epidermal growth factor (EGF)-like domains <i>in vitro</i> Intrathecal injection of TSP4 fragment proteins containing the EGF-like domain (EGF-LIKE) into naïve rodents was sufficient for inducing behavioral hypersensitivity similar to that produced by an equal molar dose of full-length TSP4. Gabapentin, a drug that binds to Ca_vα₂δ₁, blocked EGF-LIKE-induced behavioral hypersensitivity in a dose-dependent manner, supporting the notion that EGF-LIKE interacts with Ca_vα₂δ₁ and thereby mediates behavioral hypersensitivity. This notion was further supported by our findings that a peptide within EGF-LIKE (EGFD355-369) could block TSP4- or Ca_vα₂δ₁-induced behavioral hypersensitivity after intrathecal injections. Furthermore, only TSP4 proteins that contained EGF-LIKE could promote excitatory synaptogenesis between sensory and spinal cord neurons, which could be blocked by peptide EGFD355-369. Together, these findings indicate that EGF-LIKE is the molecular determinant that mediates aberrant excitatory synaptogenesis and chronic pain development. Blocking interactions between EGF-LIKE and Ca_vα₂δ₁ could be an alternative approach in designing target-specific pain medications.