Regulatory Effects Of Paxillin On Nociceptive Synaptic Transmission In Spinal Dorsal Horn

Objective:Paxillin, a signal adaptor protein comprising multiple functional domains, has been implicated in the regulation of cell adhesion process through binding to focal adhesion kinase (FAK), Src family tyrosine kinase (SFKs) and C-terminal Src kinase (CSK). Paxillin is known to localize at excitatory glutamatergic synapses in the central nervous system. However, the precise physiological roles played by Paxillin in synaptic transmission and the pathological significance remain unclear. The current study investigated the function and molecular mechanism of Paxillin in regulation of nociceptive synaptic transmission in the spinal dorsal horn.Method:To investigate the possible roles of Paxillin in nociceptive plasticity, the Complete Freund’s Adjuvant (CFA) inducing chronic inflammatory pain model, nociceptive behavioral tests, co-immunoprecipitation, immunohistochemistry, patch-clamp electrophysiological recording and C-fiber evoked field potential recording were used to explore the synaptic localization, proteins interactions and signal transmission.Results:(1) Paxillin was predominantly expressed in dorsal horn neurons, and localized at the postsynaptic density. (2) Immunohistochemistry data showed that synaptic activity regulated paxillin phosphorylation at Tyr-118. When spinal cord slices were pretreated with GABAa receptor antagonist bicuculline (100μM) to increase synaptic activity, the phosphorylation of Paxillin-Y118 was dramatically decreased, which could be blocked by N-methyl-D-aspartate (NMDA) receptor inhibitor D-APV (100μM). Similarly, peripheral tissue injury also induced dephosphorylation of Paxillin-Y118. (3) To evaluate the functional significance of altered Paxillin-Y118 phosphorylation, we generated Paxillin(Y118E) (tyrosine to glutamate mutation at residue 118) and Paxillin(Y118F) (tyrosine to phenylalanine mutation at residue 118) mutants to mimic Paxillin-Y118 phosphorylation and dephosphorylation, respectively. The recombinant adenovirus encoding these mutants was intrathecally injected to infect dorsal horn neurons. We found that overexpression of Paxillin(Y118F) induced pain hypersensitivity and enhanced the long term potentiation (LTP) magnitudes of C fiber-evoked field potentials in intact rats. Although spinal overexpression of Paxillin (Y118E) had no detectable effects on pain threshold, it decreased the LTP magnitudes of C fiber-evoked field potential. These results indicated that Paxillin-Y118 phosphorylation was associated with the spinal sensitization. (4) Paxillin might regulate the nociceptive transmission through Src and NMDA receptors signaling. We found that Paxillin(Y118F) stimulated Src and enhanced the tyrosine phosphorylation of NMDA receptor subunit GluN2B at Tyr1472. In addition, intrathecal application of SFKs inhibitor PP2 (1.5μg) effectively attenuated Paxillin(Y118F)-induced pain hypersensitivity. (5) Both in vitro and in vivo data suggested that dephosphorylation of Paxillin-Y118 disrupted Paxillin interaction with CSK, an endogenous inhibitor of Src. The resultant decrease in the synaptic expression of CSK was also observed. Therefore, the dissociation of CSK from Paxillin caused by dephosphorylation might contribute to the SFKs-depended GluN2B hyperfunction. (6) To test this hypothesis, we manipulated endogenous CSK activity by intrathecal injection of recombinant adenovirus that encoded GFP-targeted wild-type CSK [CSK(WT)] or its dominant-negative CSK(K222R) mutant. We found that overexpression of CSK(K222R) also induced pain hypersensitivity, which could be blocked by GluN2B selective inhibitor ifenprodil (1.6μg). (7) To explore the possible role of paxillin in chronic inflammatory pain, Paxillin(Y118E) was overexpressed in spinal cord of inflamed mice. We found that Paxillin(Y118E) resumed the CSK synaptic expression in inflamed mice and significantly alleviated the inflammatory mechanical allodynia.Conclusion:Peripheral tissue injury caused the dephosphorylation of spinal Paxillin at Tyr-118. The dephosphorylation of Paxillin could disrupt its interaction with CSK and decrease the CSK expression at postsynaptic density, leading to the removal of CSK-mediated inhibition and ultimate Src activation. Overexpression of Paxillin(Y118E) elevated the synaptic expression of CSK and alleviated the inflammatory mechanical allodynia.