Role of protein kinase M zeta in spinal nociceptive sensitization

反复或强烈的伤害性刺激会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。尽管多种谷氨酸受体和下游的细胞内信使参与了脊髓伤害性敏感化的启动，但对脊髓伤害性敏感化的维持知之甚少。蛋白激酶M Zeta(PKMzeta)是一种持久的活性蛋白，在维持长时程增强(LTP)和记忆存储方面具有独特的作用，并可能参与脊髓伤害性敏感化。我们已经证明，在持续的伤害性刺激或脊髓神经元的直接激活下，PKMzeta在SCDH中上调，并且PKMzeta抑制剂逆转依赖于脊髓神经可塑性的降低的伤害性阈值(即，超敏)，包括由足底(i.pl)诱导的超敏。辣椒素或鞘内(I.T.)DHPG，或肌肉注射后牵涉性后爪痛觉过敏(I.M.)大鼠大腿注射酸性生理盐水。这三种刺激还诱导了脊髓大动态范围神经元对机械刺激其后爪感受野的敏化，这种敏化可被PKMzeta的抑制剂逆转。 虽然已知多种蛋白激酶(PKs)(如PKA、PKC、钙/钙调素依赖的PK-II(CaMK-II)、丝裂原相关PK(MAPK)和磷脂酰肌醇3-激酶(PI3-Kinase))可启动脊髓伤害性敏感化，但目前尚不清楚这些介导物中是否有任何一种在脊髓伤害性敏化过程中调节PKMzeta的活性。利用选择性的PK抑制剂和PKMzeta和p-PKMzeta的蛋白分析，我们建议确定这些介质中的哪一种与外周和脊髓伤害性刺激引起的PKMzeta的长时间上调和激活有关。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，并且持续的伤害性刺激触发了SCDHAMPA受体亚细胞分布的改变。因此，为了评估伤害性刺激诱导的脊髓内PKMzeta激活的可能后果，我们将使用亚细胞分级和蛋白质分析来研究PKMzeta在SCDH神经元中AMPA受体亚细胞分布变化中的作用。最后，最近的研究质疑Zeta抑制肽(迄今为止在脊髓伤害性敏化研究中使用的唯一抑制剂)的特异性，并表明LTP和记忆加工在PKcheeta/PKMzeta基因敲除小鼠中没有受到影响。因此，我们建议在大鼠或标准和条件性基因敲除小鼠中使用显性负性PKMzeta慢病毒载体来确定PKMzeta基因操作对外周或脊髓伤害性刺激诱导的背角神经元的痛觉异常和/或敏化的维持作用。 这些研究将带来新的见解和方法，有助于理解脊髓伤害性敏感化的基本神经可塑性过程。了解这一过程并开发出逆转或消除有害刺激对神经系统(特别是脊髓神经元)影响的治疗方法，将比开发擦除记忆的方法更实用，影响也更大。

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.