Role of protein kinase M zeta in spinal nociceptive sensitization

反复或强烈的有害刺激可导致周围神经和中枢神经系统神经元的敏感化，包括脊髓背角（SCDH）。尽管多种谷氨酸受体和下游细胞内信使参与了SCDH中伤害性致敏的启动，但对脊髓伤害性致敏的维持知之甚少。蛋白激酶Mzeta （PKMzeta）是一种持续活跃的激酶，对维持海马的长期增强（LTP）和记忆储存有独特的贡献，并可能有助于脊髓伤害性致敏。我们已经证明，在持续的有害刺激或脊髓神经元的直接激活下，PKMzeta在SCDH中上调，PKMzeta抑制剂逆转了依赖于脊髓神经可塑性的降低的伤害性阈值（即异常性痛），包括由脚底辣椒素或鞘内辣椒素引起的异常性痛。DHPG，或在大鼠大腿肌内注射（i.m）酸性生理盐水后引起的后爪异常性痛。这三种刺激也诱导脊髓宽动态范围神经元对后爪感受野的机械刺激的敏化，这种敏化被PKMzeta抑制剂逆转。虽然已知各种蛋白激酶（PKs）(如PKA， PKC， Ca2+/钙调素依赖性PK- ii （CaMK-II)，丝裂原相关PK （MAPK）和磷脂酰肌醇3-激酶（pi3激酶））启动脊髓伤害性致敏，以及其他介质，如与NIMA 1相互作用的肽酰脯氨酸异构酶蛋白（PIN-1）和磷酸肌醇依赖性PK-1 （PDK1）调节海马PKMzeta活性，目前尚不清楚这些介质是否在脊髓痛觉致敏过程中调节PKMzeta的活性。通过对PKMzeta和p-PKMzeta的选择性PK抑制剂和蛋白分析，我们建议确定这些介质中的哪一种有助于外周和脊髓伤害性刺激诱导的PKMzeta的长期上调和激活。此外，已有研究表明，PKMzeta通过增加海马神经元突触后AMPA受体的水平来维持记忆，持续的伤害感受会引发SCDH AMPA受体亚细胞分布的改变。因此，为了评估有害刺激诱导的PKMzeta在脊髓中激活的可能后果，我们将使用亚细胞分离和蛋白质分析来检查PKMzeta对SCDH神经元中AMPA受体亚细胞分布改变的贡献。最后，最近的研究质疑了zeta抑制肽（迄今为止在脊髓伤害性致敏研究中使用的唯一抑制剂）的特异性，并表明PKCzeta/PKMzeta敲除小鼠的LTP和记忆加工不受影响。因此，我们建议在大鼠或标准小鼠和条件敲除小鼠中使用显性阴性PKMzeta慢病毒载体，确定基因操作对外周或脊髓伤害性刺激诱导的背角神经元异常性痛和/或致敏的维持作用。这些研究将带来新的见解和方法，有助于理解脊髓伤害性致敏的基本神经可塑性过程。理解这一过程并开发治疗方法来逆转或消除有害刺激对神经系统（特别是脊髓神经元）的影响，将比开发消除记忆的方法更实际，也更有影响力。

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.