Thrombospondin-4 (TSP-4) belongs to the extracellular matrix glycoprotein family of thrombospondins (TSPs). The multidomain, pentameric structure of TSP-4 allows its interactions with numerous extracellular matrix components, proteins and signaling molecules that enable its modulation to various physiological and pathological processes. Characterization of TSP-4 expression under development and pathogenesis of disorders has yielded important insights into mechanisms underlying the unique role of TSP-4 in mediating various processes including cell-cell, cell-extracellular matrix interactions, cell migration, proliferation, tissue remodeling, angiogenesis, and synaptogenesis. Maladaptation of these processes in response to pathological insults and stress can accelerate the development of disorders including skeletal dysplasia, osteoporosis, degenerative joint disease, cardiovascular diseases, tumor progression/metastasis and neurological disorders. Overall, the diverse functions of TSP-4 suggest that it may be a potential marker or therapeutic target for prognosis, diagnosis, and treatment of various pathological conditions upon further investigations. This review article highlights recent findings on the role of TSP-4 in both physiological and pathological conditions with a focus on what sets it apart from other TSPs.

Voltage-gated calcium channels (VGCCs) play important roles in physiological functions including the modulation of neurotransmitter release, neuronal network activities, intracellular signalling pathways and gene expression. Some pathological conditions, including nerve injuries, can cause the dysregulation of VGCCs and their subunits. This in turn can lead to a functional maladaptation of VGCCs and their subunits, which can contribute to the development of disorders such as pain sensations. This review has summarized recent findings related to maladaptive changes in the dysregulated VGCC α₂ δ₁ subunit (Ca_v α₂ δ₁ ) with a focus on exploring the mechanisms underlying the contribution of Ca_v α₂ δ₁ to pain signal transduction. At least under neuropathic pain conditions, the dysregulated Ca_v α₂ δ₁ can modulate VGCC functions as well as other plasticity changes. The latter includes abnormal excitatory synaptogenesis resulting from its interactions with injury-induced extracellular matrix glycoprotein molecule thrombospondins, which is independent of the VGCC functions. Blocking Ca_v α₂ δ₁ with gabapentinoids can reverse neuropathic pain significantly with relatively mild side effects, but only in a small population of neuropathic pain patients due to reasons yet to be explored. There are emerging data suggesting that early preventive treatment with gabapentinoids can prevent aberrant excitatory synapse formation and the development of chronic pain. If these findings are confirmed clinically, this could be an attractive approach for neuropathic pain management.

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This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Studying synapse formation, maintenance, and plasticity in adaptation to developmental and pathological changes is critical in our understanding of cellular mechanisms of biological processes and disease states. However, a major barrier in getting cell-type-specific detail information in these studies is the complexity of in vivo environment in high-density tissue or organs, such as spinal cord, that is packed with different types of cells, connecting tissues and structural components. In this chapter, we describe a co-culture system in which dorsal root ganglion sensory neurons and spinal cord neurons are cultured in separate compartments without culture medium diffusion between compartments, but allowing sensory neuron axons to outgrowth to adjacent chambers to establish synaptic connections with dendrites of spinal cord neurons. This provides an in vitro environment that mimics the in vivo synaptogenic environment between sensory neurons and spinal cord neurons and enables manipulation of specific neuronal populations and studying their detail contribution to synaptic formation, maintenance, and plastic changes.

The spinal dorsal horn is the first relay structure coding for pain transmission and modulation. Previous anatomical and electrophysiological studies have examined spinal dorsal horn circuit connections and network activity. Further work is required to understand spinal cord sensory information processing that underlies pathological neuropathic pain states. Our previous studies suggest that peripheral nerve injury enhances presynaptic excitatory input onto spinal superficial dorsal horn neurons, which in turn contributes to pathologic nociception. The potential changes in local postsynaptic circuits in the dorsal horn that lead to pathologically heightened behavioral responses to pain remain largely unexplored. We combined whole-cell electrophysiological recordings with laser-scanning photostimulation to test whether peripheral nerve injury in the spinal nerve ligation (SNL) mouse model of neuropathic pain leads to alterations in the functional connectivity of spinal cord circuits including lamina II excitatory interneurons. Here we show that SNL enhances excitation and decreases inhibition to lamina II excitatory interneurons along with their increased glutamate-evoked excitability. The enhanced excitatory postsynaptic input and connectivity evoked by SNL eventually return to normal levels concurrently with the resolution of the neuropathic pain states. The physiological pattern highly correlates with mouse pain behaviors following SNL, supporting a neurophysiological mechanism of central sensitization and neuropathic pain that is functionally localized to the spinal dorsal horn. Together, these data support that SNL induces functional changes in synaptic input and connectivity to lamina II excitatory interneurons that code for pain perception, and thus provide new insights into the mechanism and locus of pain hypersensitivity.

Thrombospondin-4 (TSP4) belongs to a family of large, oligomeric extracellular matrix glycoproteins that mediate interactions between cells and interactions of cells with underlying matrix components. Recent evidence shows that TSP4 might contribute to the generation of neuropathic pain. However, there has been no systematic examination of TSP4 expression in the dorsal root ganglia (DRG) after injury. This study, therefore, investigates whether TSP4 protein level is changed in DRG after injury following spinal nerve ligation (SNL) and spared nerve injury in rats by performing Western blotting, immunohistochemistry, and immunocytochemistry. After nerve ligation, TSP4 protein level is upregulated in the axotomized somata of the fifth lumbar (L5) DRG. There is substantial additional TSP4 in the nonneuronal compartment of the L5 DRG that does not costain for markers of satellite glia, microglia, or Schwann cells and appears to be in the interstitial space. Evidence of intracellular overexpression of TSP4 persists in neurons dissociated from the L5 DRG after SNL. These findings indicate that, following peripheral nerve injury, TSP4 protein expression is elevated in the cytoplasm of axotomized sensory neurons and in the surrounding interstitial space.

Painful nerve injury disrupts Ca²⁺ signaling in primary sensory neurons by elevating plasma membrane Ca²⁺-ATPase (PMCA) function and depressing sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) function, which decreases endoplasmic reticulum (ER) Ca²⁺ stores and stimulates store-operated Ca²⁺ entry (SOCE). The extracellular matrix glycoprotein thrombospondin-4 (TSP4), which is increased after painful nerve injury, decreases Ca²⁺ current (I_Ca) through high-voltage-activated Ca²⁺ channels and increases I_Ca through low-voltage-activated Ca²⁺ channels in dorsal root ganglion neurons, which are events similar to the effect of nerve injury. We therefore examined whether TSP4 plays a critical role in injury-induced disruption of intracellular Ca²⁺ signaling. We found that TSP4 increases PMCA activity, inhibits SERCA, depletes ER Ca²⁺ stores, and enhances store-operated Ca²⁺ influx. Injury-induced changes of SERCA and PMCA function are attenuated in TSP4 knock-out mice. Effects of TSP4 on intracellular Ca²⁺ signaling are attenuated in voltage-gated Ca²⁺ channel α₂δ₁ subunit (Ca_vα₂δ₁) conditional knock-out mice and are also Protein Kinase C (PKC) signaling dependent. These findings suggest that TSP4 elevation may contribute to the pathogenesis of chronic pain following nerve injury by disrupting intracellular Ca²⁺ signaling via interacting with the Ca_vα₂δ₁ and the subsequent PKC signaling pathway. Controlling TSP4 mediated intracellular Ca²⁺ signaling in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain.

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 in vitro 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.

Facet joint injury induces persistent pain that may be maintained by structural plasticity in the spinal cord. Astrocyte-derived thrombospondins, especially thrombospondin-4 (TSP4), have been implicated in synaptogenesis and spinal sensitization in neuropathic pain, but the TSP4 response and its relationship to synaptic changes in the spinal cord have not been investigated for painful joint injury. This study investigates the role of TSP4 in the development and maintenance of persistent pain following injurious facet joint distraction in rats and tests the hypothesis that excitatory synaptogenesis contributes to such pain. Painful facet joint loading induces dorsal horn excitatory synaptogenesis along with decreased TSP4 in the DRG and increased astrocytic release of TSP4 in the spinal cord, all of which parallel the time course of sustained tactile allodynia. Blocking injury-induced spinal TSP4 expression with antisense oligonucleotides or reducing TSP4 activity at its neuronal receptor in the spinal cord with gabapentin treatment both attenuate the allodynia and dorsal horn synaptogenesis that develop after painful facet joint loading. Increased spinal TSP4 also facilitates the development of allodynia and spinal hyperexcitability, even after non-painful physiological loading of the facet joint. These results suggest that spinal TSP4 plays an important role in the development and maintenance of persistent joint-mediated pain by inducing excitatory synaptogenesis and facilitating the transduction of mechanical loading of the facet joint that leads to spinal hyperexcitability.