Mechanisms of Thrombospondin-4 in Pain Modulation
Upregulation of the thrombospondin-4 (TSP4) or calcium channel alpha-2-delta-1 subunit (Cava2d1) in the dorsal spinal cord and dorsal root ganglia plays a causal role in neuropathic pain development through an unidentified mechanism. TSP4 blockade either by antibodies or inactivation of the TSP4 gene prevents development of chronic pain states. Intrathecal injection of TSP4 proteins into naive rats can cause dorsal horn neuron hyperexcitability and allodynia, which are blocked by the Cava2d1 ligand gabapentin. These findings suggest that TSP4 and Cava2d1 may interact together in mediating pain processing. Here, I show that TSP4 binding to Cava2d1 is detectable in the same immunocomplexes from rodent spinal cords and in solid-phase binding. SPOT Peptide array analysis and in vitro binding of TSP4 recombinant truncation proteins to Cava2d1 reveal multiple binding sites within the TSP4 Epidermal Growth Factor-like domains (EGF-like) and coil-coil domain. Functionally, lumbar intrathecal injection of EGF-like domain proteins is sufficient to induce behavioral hypersensitivity. Selective ablation of Cava2d1 from Nav1.8-positive sensory neurons abolishes EGF-like domain induced thermal hyperalgesia, but not tactile allodynia. A 15-mer TSP4 peptide from the EGF-like domains can block both Cava2d1- and TSP4-induced behavioral hypersensitivity, presumably by disrupting the interaction between TSP4 and Cava2d1. My data suggest that the EGF-like domains of TSP4 interact with Cava2d1 to induce chronic pain states in a modality specific manner. Emerging data also suggest that Cava2d1 and TSP4 interact to induce synapse formation. To determine if maladaptive changes in synapses correlate with neuropathic pain states in a nerve injury model with injury-induced upregulation of TSP4 and Cava2d1, I examined synapse numbers at ultrastructure level in superficial dorsal horn of rats after trigeminal nerve injury. I found a significant increase in both excitatory and inhibitory synapses within lamina I and II of the superficial dorsal horn, respectively, and a decrease in mean synaptic length, a marker for synapse strength, of inhibitory interneurons mainly in lamina I of the injury side. Together, it is likely that injury-induced TSP4/Cava2d1 contribute to the development of chronic pain states after nerve injury through a mechanism involving dysregulation of excitatory and inhibitory synapses in the superficial dorsal horn.