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Specific involvement in neuropathic pain of AMPA receptors and adapter proteins for the GluR2 subunit

https://doi.org/10.1016/S1044-7431(03)00134-9Get rights and content

Abstract

Chronic pain states arise from peripheral nerve injury and are inadequately treated with current analgesics. Using intrathecal drug administration in a rat model of neuropathic pain, we demonstrate that AMPA receptors play a role in the central sensitisation that is thought to underpin chronic pain. The GluR2 subunit of the AMPA receptor binds to a number of intracellular adapter proteins including GRIP, PICK1 and NSF, which may link the receptor to proteins with signalling, scaffolding and other roles. We implicate for the first time a possible role for GRIP, PICK1 and NSF in neuropathic sensitisation from experiments with cell-permeable blocking peptides mimicking their GluR2 interaction motifs and also demonstrate differential changes in expression of these proteins following peripheral nerve injury. These studies suggest a critical involvement of protein:protein complexes associated with the AMPA receptor in neuropathic pain, and the possibility that they may have potential as novel therapeutic targets.

Introduction

Neuropathic pain is a hypersensitive chronic pain state, which is poorly treated with current analgesics. Glutamate is important in neurotransmission from primary afferent inputs and several studies suggest a role for the AMPA subtype of glutamate receptor in sensitisation. AMPA receptor agonists depolarise nociceptive spinal neurons and increase responses to noxious and innocuous stimulation as well as enhancing acute nociceptive behaviours Aanonsen et al 1990, Dougherty et al 1992, Budai and Larson 1994, Cumberbatch et al 1994, Aanonsen and Wilcox 1987, while AMPA receptor antagonists disrupt acute nociceptive behavioural responses and chronic pain, including neuropathic pain Mao et al 1992, Chaplan et al 1997, Sorkin et al 2001, Nishiyama et al 1998. The molecular mechanisms underlying these actions of AMPA receptors are however unknown.

Of the four AMPA receptor subunits (GluR1-4; Hollmann and Heinemann, 1994), the GluR2/3 subunits have received particular attention since a number of proteins interact with a PDZ domain target motif at their C-terminus. GluR2, but not GluR3, is abundant in the dorsal spinal cord (Jakowec et al., 1995). The PDZ domain-containing proteins that can dock to GluR2 include the GRIP (Glutamate Receptor Interacting Protein) family Dong et al 1999b, Dong et al 1997, Srivastava et al 1998, Brückner et al 1999 and PICK1 (Protein Interacting with C Kinase 1) Xia et al 1999, Dev et al 1999. GRIP is enriched in the postsynaptic density (PSD) but is also cytosolic (Wyszynski et al., 1998). PICK1 (originally identified by its interaction with PKCα; Xia et al 1999, Dev et al 1999 may act to bring the kinase close to plasma membrane substrates, including the AMPA receptor Staudinger et al 1997, Perez et al 2001. Disruption of the GluR2 C-terminal SVKI motif interactions suggests that GRIP and PICK1 are important in the synaptic clustering of AMPA receptors Dong et al 1997, Osten et al 2000 for unmasking silent glutamatergic synapses in somatosensory inputs (Li et al., 1999) and for the expression of long term changes in the excitability of hippocampal and cerebellar neurons Xia et al 2000, Kim et al 2001. Both GRIP and PICK1 proteins have been implicated in the cell surface clustering of GluR2-containing AMPA receptors Xia et al 1999, Osten et al 2000, while PICK1 appears to be involved in AMPA receptor endocytosis (Perez et al., 2001). NSF (N-ethylmaleimide-Sensitive Fusion protein), a non-PDZ domain protein involved in membrane fusion, is enriched in the PSD and has been shown to dock to the GluR2 subunit at a different site on its C-terminus (VAKNAQ, Osten et al 1998, Song et al 1998. This interaction may contribute to stabilising excitatory currents in hippocampal neurons Nishimune et al 1998, Song et al 1998, to the movement of AMPA receptors to the cell surface Noel et al 1999, Lüthi et al 1999, Luscher et al 1999 and to Long Term Potentiation (LTD) (Lledo et al., 1998).

In the present study we asked whether AMPA receptors and GluR2 subunit adapter proteins might play a specific role in the sensitised responsiveness of spinal cord neurons that underlies neuropathic pain.

Section snippets

AMPA receptor antagonists attenuate neuropathic reflex sensitisation following CCI

To examine the question of a role for the AMPA receptor in neuropathic pain conditions, we assessed the contribution of spinal AMPA receptors to CCI-induced changes in somatosensory behavioural reflexes by carrying out intrathecal administration of the AMPA receptor antagonists NBQX, NS-257 and SYM 2206 (Fig. 1a and b, Table 1). Intrathecal injection of the AMPA/kainate receptor antagonist NBQX and the highly selective AMPA receptor antagonists NS-257 and SYM 2206 in rats at the peak of

Discussion

Damage to peripheral nerves results in long-lasting physiological and phenotypic alterations in the peripheral and central nervous system and brings about a state of hypersensitivity within the dorsal horn of the spinal cord. This hypersensitivity contributes to the development of the hyperalgesia and allodynia that characterise the neuropathic pain condition. We provide evidence that proteins interacting with the C-terminal domain of the GluR2 subunit of AMPA receptor play an important role in

Experimental methods

All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act, 1986. Adult male Wistar rats (200–300 g, Charles River, Kent, UK) were anaesthetised under aseptic conditions. The Bennett and Xie (Bennett and Xie, 1988) chronic constriction injury model was performed as described previously (Moss et al., 2002).

Acknowledgements

This work was supported by The Wellcome Trust (S.F.-W.), the MRC (R.M.) and by the University of Edinburgh for the award of a studentship to E.G. and the M.R.C. for the award of Studentships to A.M. and A.D., respectively. We thank staff at the Wellcome Animal Research Unit (WARU) and the Medical Faculty Animal Area (MFAA) facilities for animal husbandry as well as R. Clegg and NeuroSearch A/S for gifts of the myr-GRRNAIDHE peptide and NS 257 respectively.

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    Current address: Department of Anatomy and Developmental Biology, Medawar Building, University College London, Gower St., London, WC1E 6BT, UK.

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