Peripheral tissue incision and inflammation produce persistent pain hypersensitivities in rodent animal models that mimic clinical post-operative pain and chronic inflammatory pain, respectively. Uncovering the mechanisms that lead to the hypersensitivity may lead to novel therapeutic strategies for its prevention and/or treatment. Although evidence documented over the last three decades has implicated AMPAR involvement in persistent post-operative pain and inflammatory pain, how AMPARs participate in these two disorders is not completely understood. Here, we report that spinal PICK1, an AMPAR GluR2 subunit-binding protein, might be required for the maintenance of CFA-induced inflammatory pain, but not for incision-induced post-operative pain. PICK1 may be a new target for the treatment of persistent inflammatory pain.
Our results showed that spinal PICK1 deficiency caused by the congenital knockout of PICK1 gene or acute transient knockdown of PICK1 significantly reduces mechanical and thermal pain hypersensitivities during the maintenance of CFA-induced inflammatory pain. This anti-hyperalgesic effect might be related to the functional role of spinal PICK1 in CFA-induced AMPAR GluR2 subunit internalization during inflammatory pain maintenance. PICK1 binds to both PKCα and GluR2 and can present the activated form of PKCα to synaptic GluR2 in dorsal horn [21
]. It has been shown previously that during the maintenance period of CFA-evoked pain hypersensitivities, spinal NMDAR-triggered PKCα activation leads to phosphorylation of GluR2 at Ser880, disruption of GluR2 binding to its synaptic anchoring protein ABP/GRIP, and promotion of GluR2 internalization in dorsal horn neurons [16
]. GluR2 internalization results in an increase in AMPAR Ca2+
permeability. The increase in intracellular Ca2+
in dorsal horn neurons might initiate or potentiate a variety of Ca2+
-dependent intracellular cascades that are associated with the maintenance of inflammatory pain [28
]. The present study demonstrated that PICK1 is required for CFA-induced GluR2 phosphorylation and GluR2 internalization in dorsal horn, as they were abolished by PICK1 knockout. Given that the presence or absence of synaptic GluR2 in the AMPAR complex greatly influences AMPAR Ca2+
permeability, it is very likely that the anti-hyperalgesic effect caused by PICK1 deficiency might result from the failure of activated PKCα to be recruited to GluR2. Without the resulting association, no changes would occur in GluR2 phosphorylation or trafficking in the dorsal horn.
It is noteworthy that mice and rats deficient in PICK1 still displayed some level of pain hypersensitivity during the maintenance phase of CFA-induced inflammatory pain. Preventing CFA-induced spinal GluR2 internalization through targeted mutation of the GluR2 PKC phosphorylation site also did not completely abolish CFA-induced pain hypersensitivity during the maintenance period [21
]. These findings suggest that PICK1-mediated GluR2 internalization is just one of the key factors involved in CFA-induced inflammatory pain maintenance. Moreover, PICK1 interacts with several proteins in addition to GluR2, including ASIC1, ASIC2, ephrin-B ligands, Eph receptor tyrosine kinase, metabotropic glutamate receptor subunit 7, dopamine transporter, and class I ADP-ribosylation factors [9
]. These PICK1-mediated interactions might also be involved in inflammatory pain maintenance. In addition, PICK1 is expressed in other pain-related regions of the nervous system, such as the dorsal root ganglion. Thus, the detailed mechanisms by which the deficiency of PICK1 affects CFA-induced inflammatory pain maintenance remain to be explored.
Spinal AMPARs also participate in dorsal horn sensitization that underlies incision-induced post-operative pain. Intrathecal or epidural administration of AMPA/kainate receptor antagonists reduces pain behaviors caused by incision [15
]. Intrathecal application of these antagonists also attenuates incision-induced increases in background activity of dorsal horn neurons and in the responses of dorsal horn wide dynamic range neurons to mechanical stimuli [38
]. We expected that a hind paw incision, like CFA-induced peripheral inflammation, would lead to PICK1-dependent dorsal horn GluR2 internalization and that blocking this internalization through targeted disruption of the PICK1 gene would contribute to dorsal horn sensitization after incision. However, our results showed no significant change in either PKCα-mediated GluR2 phosphorylation at Ser880 or GluR2 internalization in dorsal horn from WT and PICK1 KO mice after a hind paw incision. Neither knockdown nor knockout of spinal PICK1 affected incision-induced pain hypersensitivities during the observation period. The mechanisms that underlie these unexpected results are unclear, but they might be related to NMDAR-independent dorsal horn sensitization under incisional pain conditions. It has been demonstrated that intrathecal application of NMDAR antagonists does not affect pain behaviors or the enhanced responsiveness of dorsal horn neurons that occurs after incision [4
]. These findings indicate that spinal cord NMDARs and their downstream signal cascades (such as PKCα) might be inactivated after incision. Given that spinal PKCα activation is triggered by NMDARs in dorsal horn [21
], this might be one reason that incision does not alter the level of spinal cord GluR2 phosphorylation or internalization.
Our findings indicate that different peripheral nociceptive insults may produce distinct changes in spinal cord AMPAR subunit trafficking. This view is further supported by recent studies on different pain models. Capsaicin-induced acute inflammation increased the amount of membrane GluR1 protein and correspondingly decreased the level of cytosolic GluR1, without affecting GluR2 trafficking in dorsal horn neurons [10
]. The injection of carrageenan or formalin into a hind paw led to an increase only in the level of GluR1 in the plasma membrane of dorsal horn neurons [3
]. In addition, CFA-induced persistent inflammation was shown to produce GluR1 membrane insertion and GluR2 internalization in dorsal horn [16
]. We also found that a hind paw incision does not alter the levels of membrane or cytosolic GluR1 proteins in the dorsal horn (data not shown). It will be very interesting to further characterize spinal cord AMPAR subunit trafficking under other persistent pain conditions (such as nerve injury-induced neuropathic pain).
In conclusion, we demonstrated that knockout and knockdown of spinal cord PICK1 significantly attenuates pain hypersensitivities during CFA-induced inflammatory pain maintenance, without affecting incision-induced post-operative pain behaviors. Furthermore, we showed that spinal PICK1 deficiency abolishes CFA-induced GluR2 phosphorylation at Ser880 and GluR2 internalization in dorsal horn. Given that the latter two events contribute to CFA-induced inflammatory pain maintenance [21
], spinal cord PICK1 might participate in the maintenance of persistent inflammatory pain through promoting GluR2 phosphorylation at Ser880 and GluR2 internalization in dorsal horn neurons. It should be noted that genetic strategies, including knockout and knockdown approaches, might be impractical in the clinical setting. A recent study identified a small-molecule inhibitor (FSC231) that specifically bound to the PICK1 PDZ domain, inhibited PICK1 interaction with GluR2, and accelerated recycling of GluR2 after internalization in response to NMDAR activation [32
]. Thus, FSC231 may represent a promising novel strategy for treating persistent inflammatory pain.