Spinal cord protein interacting with C kinase 1 is required for the maintenance of complete Freund's adjuvant-induced inflammatory pain but not for incision-induced post-operative pain

Abstract

Protein interacting with C kinase 1 (PICK1) is a PDZ-containing protein that binds to AMPA receptor (AMPAR) GluR2 subunit and protein kinase Cα (PKCα) in the central neurons. It functions as a targeting and transport protein, presents the activated form of PKCα to synaptic GluR2, and participates in synaptic AMPAR trafficking in the nervous system. Thus, PICK1 might be involved in many physiological and pathological processes triggered via the activation of AMPARs. We report herein that PICK1 knockout mice display impaired mechanical and thermal pain hypersensitivities during complete Freund's adjuvant (CFA)-induced inflammatory pain maintenance. Acute transient knockdown of spinal cord PICK1 through intrathecal injection of PICK1 antisense oligodeoxynucleotide had a similar effect. In contrast, knockout and knockdown of spinal cord PICK1 did not affect incision-induced guarding pain behaviors or mechanical or thermal pain hypersensitivities. We also found that PICK1 is highly expressed in dorsal horn, where it interacts with GluR2 and PKCα. Injection of CFA into a hind paw, but not a hind paw incision, increased PKCα-mediated GluR2 phosphorylation at Ser880 and GluR2 internalization in dorsal horn. These increases were absent when spinal cord PICK1 was deficient. Given that dorsal horn PKCα-mediated GluR2 phosphorylation at Ser880 and GluR2 internalization contribute to the maintenance of CFA-induced inflammatory pain, our findings suggest that spinal PICK1 may participate in the maintenance of persistent inflammatory pain, but not in incision-induced post-operative pain, through promoting PKCα-mediated GluR2 phosphorylation and internalization in dorsal horn neurons.

Fig. 1

Effect of targeted disruption of PICK1 gene on CFA-induced inflammatory pain. CFA injection into a hind paw produced significant increases in paw withdrawal frequencies in response to 0.24 mN (low intensity) (A) and 4.43 mN (moderate intensity) (B) mechanical stimuli and marked decreases in paw withdrawal latencies in response to thermal stimuli (C) on the ipsilateral (ipsi) side in wild-type (WT) mice. The PICK1 knockout (KO) mice exhibited intact mechanical and thermal pain hypersensitivities at 2 h after CFA injection and significantly reduced mechanical and thermal pain hypersensitivities during the remainder of the observation period (A, B, and C). No significant changes in paw withdrawal frequencies or latencies were seen on the contralateral (con) side after CFA injection in either WT or KO mice. * P < 0.05, ** P < 0.01 vs corresponding time points in PICK1 KO mice.

Fig. 2

Effect of acute transient knockdown of spinal PICK1 on CFA-induced inflammatory pain. (A) Intrathecal injection of 10 μg PICK1 AS ODN, but not saline or 10 μg MS ODN, significantly reduced expression of PICK1, but not GluR2, PKCα, or GRIP1, in the lumbar enlargement segments compared to that in naïve rats. Top: Representative Western blots. Bottom: Statistical summary of the densitometric analysis expressed relative to naïve rats after normalization to corresponding β-actin. (B and C) Intrathecal injection of 10 μg PICK1 AS ODN, but not saline or 10 μg MS ODN, significantly attenuated the CFA-induced increase in paw withdrawal frequency in response to mechanical stimulation (B) and the CFA-induced decrease in paw withdrawal latency in response to thermal simulation (C) on the ipsilateral side at 1 d, but not 2 h, 3 d, or 5 d after CFA injection. * P < 0.05 vs the corresponding time point in the saline-treated group. Sal, saline; AS, antisense; MS, missense

Fig. 3

The targeted disruption of PICK1 gene does not affect incision-induced post-operative pain. (A) Guarding pain scores at different time points after incision. (B) Paw withdrawal latencies in response to thermal stimulation at different time points after incision. (C and D) Paw withdrawal frequencies in response to 0.24 mN (C) and 4.43 mN (D) mechanical stimuli at different time points after incision. Ipsi, ipsilateral; con, contralateral.

Fig. 4

Spinal PICK1 knockdown does not affect incision-induced post-operative pain. (A) Guarding pain scores at different time points after incision. (B) Paw withdrawal latencies in response to thermal stimulation at different time points after incision. (C) Paw withdrawal frequencies in response to mechanical stimulation at different time points after incision. Sal, saline; AS, antisense; MS, missense.

Fig. 5

Expression of PICK1 and PICK1 binding to GluR2 and PKCα in dorsal horn. (A) Western blotting showed higher levels of PICK1, GluR2, and PKCα in the dorsal horn (DH) and lower levels in the ventral horn (VH) and dorsal root ganglion (DRG). β-actin was used as a loading control. (B) Left panel: GluR2 antibody immunoprecipitated GluR2, PICK1, and PKCα in spinal cord from WT mice and only GluR2 itself in spinal cord from PICK1 KO mice. Right panel: PICK1 antibody immunoprecipitated GluR2, PICK1, and PKCα in spinal cord from WT mice, but not from PICK1 mice. IB: immunoblotting. IP: immunoprecipitation.

Fig. 6

Effect of spinal PICK1 deficiency on CFA-induced GluR2 phosphorylation at Ser880 and GluR2 internalization in dorsal horn. (A) CFA (but not saline) injection increased GluR2 phosphorylation at Ser880 (GluR2-p), but not total GluR2 or PICK1, in the ipsilateral dorsal horn of WT mice after 1 day. This increase was not seen in the ipsilateral dorsal horn of PICK1 KO mice. Top: Representative Western blots. Bottom: Statistical summary of the densitometric analysis expressed relative to naïve WT mice after normalization to corresponding β-actin. * P < 0.05 vs naive WT mice. (B) CFA (but not saline) injection increased the level of GluR2 in the 150-k-g fraction from the ipsilateral dorsal horn of WT mice after 1 day. This increase was not detected in the ipsilateral dorsal horn of PICK1 KO mice. Top: Representative Western blots. Bottom: Statistical summary of the densitometric analysis expressed relative to naïve WT mice after normalization to corresponding α-adaptin. * P < 0.05 vs naive WT mice.

Fig. 7

Paw incision does not induce GluR2 phosphorylation at Ser880 or GluR2 internalization in dorsal horn. (A) The levels of GluR2 phosphorylation at Ser880 (GluR2-p) and total GluR2 in the ipsilateral dorsal horns of WT (left panel) and PICK1 KO (right panel) mice at the different time points after a hind paw incision. Top: Representative Western blots. Bottom: Statistical summary of the densitometric analysis expressed relative to naïve (N) mice after normalization to corresponding β-actin. (B) The levels of GluR2 in the 150-k-g fraction from the ipsilateral dorsal horn of WT (left panel) and PICK1 KO (right panel) mice at the different time points after hind paw incision. Top: Representative Western blots. Bottom: Statistical summary of the densitometric analysis expressed relative to naïve (N) mice after normalization to corresponding α-adaptin.