Role of protein kinase M zeta in spinal nociceptive sensitization

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Repeated or intense noxious stimulation leads to a sensitization of both peripheral nerves and neurons in the central nervous system, including the spinal cord dorsal horn (SCDH). Although various glutamate receptors and downstream intracellular messengers contribute to the initiation of nociceptive sensitization in SCDH, little is known about the maintenance of spinal nociceptive sensitization. Protein kinase M zeta (PKMzeta) is a persistently active kinase that contributes uniquely to the maintenance of long-term potentiation (LTP) and memory storage in the hippocampus, and may contribute to spinal nociceptive sensitization. We have shown that PKMzeta is upregulated in SCDH in response to persistent noxious stimulation or direct activation of spinal neurons, and that a PKMzeta inhibitor reverses lowered nociceptive thresholds (i.e., allodynia) that depend on spinal neuroplasticity, including allodynia induced by intraplantar (i.pl.) capsaicin or intrathecal (i.t.) DHPG, or referred hind paw allodynia after intramuscular (i.m.) injections of acid saline in the rat thigh. These three stimuli also induced a sensitization of spinal wide dynamic range neurons to mechanical stimulation of their hind paw receptive fields, which was reversed by an inhibitor of PKMzeta. Although it is known that various protein kinases (PKs) (such as PKA, PKC, Ca2+/calmodulin-dependent PK-II (CaMK-II), mitogen-associated PK (MAPK) and phosphatidylinositol 3-kinase (PI3-kinase)) initiate spinal nociceptive sensitization, and other mediators such as peptidyl-prolyl isomerase protein interacting with NIMA 1 (PIN-1) and phosphoinositide-dependent PK-1 (PDK1) regulate PKMzeta activity in hippocampus, it is unknown whether any of these mediators regulate PKMzeta activity in during spinal nociceptive sensitization. Using selective PK inhibitors and protein assays of PKMzeta and p-PKMzeta, we propose to determine which of these mediators contributes to the prolonged upregulation and activation of PKMzeta induced by peripheral and spinal nociceptive stimulation. Also, it has been shown that PKMzeta maintains memory by increasing the levels of post-synaptic AMPA receptors in hippocampal neurons, and that persistent nociception triggers alterations in the subcellular distribution of SCDH AMPA receptors. Thus, to assess possible consequences of noxious stimulus-induced PKMzeta activation in spinal cord, we will use subcellular fractionation and protein assays to examine the contribution of PKMzeta to alterations in the subcellular distribution of AMPA receptors in SCDH neurons. Lastly, recent studies have questioned the specificity of zeta-inhibitory peptide (the only inhibitor used thus far in studies of spinal nociceptive sensitization), as well as showing that LTP and memory processing are unaffected in PKCzeta/PKMzeta knockout mice. Therefore, we propose to determine the effects of gene manipulation of PKMzeta, using a dominant-negative PKMzeta lentiviral vector in rats or standard and conditional knock-out mice, on the maintenance of allodynia and/or sensitization of dorsal horn neurons induced by peripheral or spinal nociceptive stimulation. 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.