Role of atypical PKCs in spinal nociceptive sensitization

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Intense noxious stimulation leads to a sensitization of peripheral nerves and spinal cord dorsal horn (SCDH) neurons. Although various glutamate receptors (R) and downstream messengers contribute to the initiation of nociceptive sensitization in SCDH, little is known about the maintenance of spinal nociceptive sensitization. Protein kinase C (PKCzeta), PKC-lambda and PKM-zeta are atypical PKCs (aPKC) that contribute to the maintenance of long-term potentiation (LTP) and memory storage in the hippocampus. We have shown that aPKCs are upregulated in SCDH in response to persistent noxious stimulation or direct activation of spinal neurons, and that inhibitors of these kinases reverse lowered nociceptive thresholds that depend on spinal neuroplasticity. Although various kinases (PKA, PKC, CaMK-II, MAPK, PI3-kinase and PIN-1, PIP3, p38-kinase and scr) regulate the activation of aPKCs during LTP, it is unknown whether any of these mediators regulate aPKC activity during spinal nociceptive sensitization. Using selective PK inhibitors and protein assays of unphosphorylated and phosphorylated aPKCs, we propose to determine which of these mediators contributes to the prolonged upregulation and activation of aPKCs induced by nociceptive stimulation. Also, aPKCs maintain LTP and memory by increasing the levels of post-synaptic AMPAR in hippocampal neurons by stimulating interactions of binding partners such as p62, PICK1, NSF with GluR1 and GluR2, and persistent nociception also triggers alterations in the subcellular distribution of SCDH AMPA receptors. Thus, to assess possible consequences of noxious stimulus-induced aPKC activation in spinal cord, we will use subcellular fractionation and protein assays to examine the contribution of aPKCs, p62, PICK1 and NSF to alterations in the subcellular distribution of GluR1 and GluR2 in SCDH neurons in response to nociceptive stimuli. Recent studies have questioned the specificity of zeta-inhibitory peptide, as well as showing that LTP and memory processing are unaffected in PKM-zeta knockout mice. Therefore, we propose to determine the effects of both non-selective and selective aPKC inhibiton (including drugs, antisense oligonucleotides, dominant negative mutated proteins, and inducible gene ablation) on the maintenance of mechanical hypersensitivity induced by peripheral or spinal nociceptive stimulation. Lastly, we will access the effects of cell penetrating peptides that disrupt interactions between aPKC, aPKC binding partners and AMPA receptors on persistent nociceptive hypersensitivity. These studies will bring new insights and approaches that should aid in understanding the fundamental neuroplastic processes that underlie spinal nociceptive sensitization. Understanding the processes and developing treatments to reverse or erase the effects of noxious stimuli on the nervous system (particularly spinal neurons) will be more practical, and have considerably more impact, than developing ways to erase memories.