All TSPs, i.e. TSPs 1–4 and COMP, were previously identified as synaptogenic proteins which, together with other astrocyte-derived factors, help to promote the formation of functional excitatory synapses in the CNS6,7. The α 2 δ-1 protein was demonstrated to be functionally involved in TSP-induced synaptogenesis by means of synaptic assays in retinal ganglion cells (RGCs)7, DRG/spinal cord primary neuron co-culture8,9, purified cortical neurons10 as well as in dorsal spinal cord of mice34. Biochemically, α 2 δ-1 was reported to interact in co-immunoprecipitation experiments with TSP-1, TSP-2 and TSP-4 from rat cerebral cortex7 as well as with TSP-4 from rodent spinal cord34. Similarly, a TSP-2 fragment containing all three EGF-like repeats, the calcium-binding repeats, and the C-terminal globular domain was co-purified with full-length α 2 δ-1 or its protein-binding VWA domain after co-expression in HEK293 cells7. Recently, Park et al.9,34 demonstrated for the first time a direct molecular interaction between α 2 δ-1 and recombinant full-length TSP-4 or its fragments containing EGF-like or coiled coil domains. In the present study, we investigated the biochemical properties of the direct TSP-4/α 2 δ-1 interaction. Furthermore, it was of importance to know whether other members of the TSP protein family are also able to directly bind to α 2 δ-1 in an analogous manner to that of TSP-4. Our data demonstrated that only full-length TSP-4, but not TSP-2 or COMP, is able to directly interact with immobilised soluble α 2 δ-1 variant (α 2 δ-1 S NTST) in an ELISA-style ligand binding assay (Fig. 3A), indicating the specificity of this protein-protein interaction. This observation is in direct contrast to that of Eroglu et al.7 (see above). Nevertheless, TSP-4 is remarkably the only isoform of TSP proteins reported so far to be implicated in neuropathic and joint-mediated chronic pain in rodents along with neuronal α 2 δ-18,9,31,34,35. During the processes resulting in such pain, both TSP-4 and α 2 δ-1 are up-regulated on the protein level and temporally correlate with the development of behavioural hypersensitivity in the respective animal models (for review see ref.50). In contrast, TSP-1/-2, though previously shown to be up-regulated after ischemic brain injury in rodents4,51 and promoting the subsequent synaptic recovery51, are not dysregulated on the protein level in dorsal spinal cord after spinal nerve ligation in mice, even when behavioural hypersensitivity was evident in these animals. This observation led Kim et al.31 to rule out the possibility of the involvement of these two astrocyte-secreted proteins in mediating TSP/α 2 δ-1-induced neuropathic pain. With regards to COMP, it has been reported to be expressed in several tissues including skeletal muscle, tendon, and cartilage. In the latter tissue, COMP is known to be mainly involved in chondrocyte differentiation, attachment, and cartilage extracellular matrix assembly52,53,54,55, with mutations in COMP being associated with pseudoachondroplasia56,57. COMP in skeletal muscle, tendons, and perichondrium can be theoretically in contact with nerve terminals containing α 2 δ-1. In addition, COMP was shown to have synaptogenic potential in RGCs (as discussed) and shares a high degree of both overall sequence identity (~70%) and structural similarity (Fig. 1A) with TSP-4. That is why we investigated COMP as a potential interaction partner of α 2 δ-1 in our binding studies. Nevertheless, COMP is, in contrast to TSP-4, neither abundant in neurons and astrocytes nor it is known to be dysregulated in neuropathic pain states. The fact that, beside TSP-4, all other TSPs were previously found to induce synaptogenesis through a mechanism involving neuronal α 2 δ-17 may refer to other cellular factors or scaffold proteins required to mediate their interaction with α 2 δ-1 indirectly. Furthermore, it is tempting to speculate that TSP-induced synaptogenesis might be of little relevance to neuropathic pain development since, as previously mentioned, TSP-4 is the only member of TSP family found to be up-regulated in dorsal spinal cord following nerve injury. Indeed, a recent study shows that an enhanced presynaptic NMDA receptor activity, rather than synaptogenesis, is responsible for maintaining the increased synaptic excitatory transmission in dorsal spinal cord leading to chronic pain states following nerve injury in mice58.

In further experiments, we observed a significantly increased binding of TSP-4 to α 2 NTST when compared to α 2 δ-1 S NTST (Fig. 3C), confirming previous data by Eroglu et al.7 demonstrating the TSP-4 binding site to be localised within the α 2 region of α 2 δ-1 (VWA domain). The observed enhancement in binding towards α 2 might be attributed to more exposed TSP-4 binding motif(s) in the immobilised α 2 NTST compared to the non-proteolytically processed α 2 δ-1 S NTST. This result is in agreement with recent findings from Lana et al.38 where wild-type α 2 δ-1 was very weakly co-immunoprecipitated with TSP-4, but no co-immunoprecipitation of the mutant α 2 δ-1 (MIDASAAA) with TSP-4 was detected in lysates of co-transfected tsA-201 cells. In addition, the binding to α 2 NTST seems again to be TSP-4-specific since negligible binding signals were detected when equimolar concentrations of either COMP or a truncated TSP-4 fragment were utilized in a pilot experiment (data not shown). Although our results are supported by reported data, we cannot rule out the possibility that the enhanced TSP-4 binding signal is due to improperly expressed α 2 NTST since unpaired cysteine residues, normally involved in the formation of disulphide bridges with other cysteine residues in the deleted regions of wild-type α 2 δ-159, become available. It is worth mentioning here that a very high tendency for multimerisation was observed for a recombinant VWA domain of α 2 δ-1 generated in this study (data not shown) due to the formation of intermolecular disulphide bonds. Therefore, it might be appropriate to consider the expression of α 2 and VWA fragments in which the unpaired cysteines are replaced by other isosteric residues (e.g. serine) for future binding studies.

One of the reported small molecules capable of interfering with the TSP/α 2 δ-1 interaction is GBP, an approved analgesic against neuropathic pain60,61,62 and a known ligand of α 2 δ-120. Biochemically, co-immunoprecipitation experiments showed that the interaction between a truncated TSP-2 fragment and α 2 δ-1 FLAG in a co-culture of two populations of HEK293 cells was diminished in the presence of GBP7. In addition, as previously mentioned, TSP-4 modestly but significantly reduces the binding affinity of 3H-GBP to α 2 δ-1, suggesting rather an allosteric than a pure competitive mode of inhibition38. Furthermore, in vivo data revealed the ability of GBP to block TSP-4-induced neuronal sensitisation and behavioural hypersensitivity as well as changes in Ca2+ currents and intracellular Ca2+ transients after injuries to peripheral nerves or facet-joint in rodents8,9,34,35,63. Similarly, several studies in neuropathic pain models demonstrated the ability of GBP to inhibit α 2 δ-1-induced26 or TSP-induced7,8,34,35 synaptogenesis. Most recently, GBP was also shown to inhibit TSP-2-induced synapse formation in purified culture of cortical neurons10. Despite the multidimensional evidence of GBP interference with TSP/α 2 δ-1 interaction, a direct GBP inhibition of this interaction on the molecular level has never been investigated before, to our knowledge. In the current study, we did not observe any inhibition of the direct TSP-4/α 2 δ-1 S NTST interaction in the presence of increasing concentrations (up to 1 mM) of GBP (Fig. 4B). Furthermore, the highest GBP concentration used (1 mM) did not shift the TSP-4/α 2 δ-1 S NTST binding curve (Fig. 4C). Although the utilised α 2 δ-1 S NTST was mostly expressed as uncleaved form of the protein (in agreement with the original work describing a similar porcine α 2 δ-1 mutant40), we were able to demonstrate the ability of this α 2 δ-1 S mutant to retain high affinity for GBP (Fig. 4A). For this purpose, a newly developed SPR-based binding assay suitable for detecting and quantifying the binding of small molecules to immobilized recombinant α 2 δ-1 S was used. This SPR assay has the advantage of being radiolabel-free and can easily be used to determine the binding kinetics unlike the previously used 3H-GBP binding assay38,40,64,65. Taken together, our data confirmed that the proteolytic cleavage of α 2 δ-1 is not crucial for the formation of the GBP binding pocket40. The complete lack of GBP inhibition towards the interaction of purified TSP-4 with α 2 δ-1 S NTST raises questions regarding the exact mechanism by which GBP can block the above-mentioned TSP-induced changes. It is possible that other unknown factors in the cellular environment are essential for GBP to interfere with α 2 δ-1/TSP-4 interaction and thereby mediating the known GBP inhibitory effects. Another possible explanation based on the recent findings of Chen et al.58,66 is that the α 2 δ-1/NMDA receptor complex, rather than α 2 δ-1/TSP-4 binding, represents the molecular target of gabapentinoid drugs to alleviate neuropathic pain.

Our efforts were as well focused on the investigation of the TSP-4/α 2 δ-1 interaction in a cellular system to get closer to the physiological/pathological situation in the CNS. We over-expressed full-length α 2 δ-1 in HEK293-EBNA cells and demonstrated both its intracellular and plasma membrane localisation in transfected cells (Fig. 5A). Treatment with increasing concentrations (up to 909 nM) of fluorescently labelled A555-TSP-4, however, showed no differences in binding of the protein to α 2 δ-1 overexpressing cells when compared to control cells (Fig. 5C, Supplementary Fig. S5). Furthermore, fluorescent signals of A555-TSP-4 did not co-localise with those of immunostained α 2 δ-1 on the cells (Supplementary Fig. S5). The observed loss of binding cannot be attributed to an impairment of the interaction of the two proteins by the fluorescent label of TSP-4 since the fluorescent protein was generated with a minimal dye-to-protein molar ratio and showing substantial α 2 δ-1 S NTST binding in the ELISA-style assay (Fig. 5B). A possible explanation could instead be arising from the weak interaction of the two proteins under the conditions of the cell-based assay, unlike the ELISA-style assay. This means that very high local concentrations of TSP-4 in the proximity of cell-surface α 2 δ-1 would be required to enable the detection of their interaction by simulating the pathological situation (e.g. dramatic up-regulation following nerve injury). This could not, however, be achieved with the range of A555-TSP-4 concentrations (up to 909 nM) used in the assay. In our experiments, we over-expressed α 2 δ-1 in HEK293-EBNA cells without co-expression of α 1 subunit, which interacts intracellularly with α 2 δ-1 before trafficking of the complex to the cell surface67. However, we assume that the lack of α 1 subunit did not hamper the putative binding of TSP-4 to α 2 δ-1 on the cell surface. This assumption is based on recent data showing the ability of wild-type α 2 δ-1, expressed in HEK293 cells without co-expression of α 1 subunits, to be efficiently transported to the cell surface and thereby become accessible to extracellular ligands like TSP10. Functionally, this α 2 δ-1 over-expression construct alone was able to rescue synapses in cortical organotypic slices from α 2 δ-1 knockout mice. This effect is found to be mediated through activation of the small Rho GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1) and is independent of α 1 subunits of the postsynaptic L-type calcium channels Ca V 1.2 and Ca V 1.310.

The α 2 δ subunits are thought to promote membrane trafficking of the pore subunits of voltage-gated calcium channels17 and α 2 δ-1-driven allodynia in mice can be reversed by blockers of voltage-gated calcium channels like ω-conotoxin GVIA68. However, other findings suggest that the maladaptive changes contributing to chronic pain in rodents following nerve injuries and resulting from the interaction of dysregulated TSP-4 with α 2 δ-1 are partially independent of the role of the latter protein in regulating voltage-gated calcium channels’ trafficking and function50.

As previously mentioned, our data align with those of Lana et al.38 who reported no interaction of secreted TSP-4 with membrane-localised α 2 δ-1 on tsA-201 cells when subjected to immunocytochemical analysis. On the other hand, the same study showed weak intracellular interaction of both proteins in co-immunoprecipitation experiments from cells over-expressing both proteins38. It has therefore been postulated that the weak TSP-4/α 2 δ-1 interaction may occur in an intracellular compartment rather than on the cell surface38,69. This postulation is in contrast to the previous data showing synaptogenic effect of secreted TSPs which is mediated by neuronal α 2 δ-1 thought to be located either pre-8 or post-7,10 synaptically. To our knowledge, there are no data so far showing the co-localisation of TSP(-4) and α 2 δ-1 in neuronal cell cultures or spinal cord tissue where TSP-induced changes (e.g. synaptogenesis) were demonstrated. To reveal the exact cellular localisation of TSP-4/α 2 δ-1 interaction it would be very helpful to simultaneously analyse co-immunostained TSP-4 and α 2 δ-1 proteins in cultures of neurons utilized in in vitro synapse assays6,7,10.

In summary, our results provide substantial in vitro biochemical evidence for a direct and specific Ca2+-insensitive TSP-4/α 2 δ-1 interaction which is rather weak. Importantly, GBP does not inhibit this interaction on a molecular level, indicating the possible involvement of other unknown factors or targets in mediating GBP inhibitory effects in neuropathic pain.