Role of atypical PKCs in spinal nociceptive sensitization

强烈的伤害性刺激导致周围神经和脊髓背角神经元的敏感化。尽管多种谷氨酸受体(R)及其下游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机制知之甚少。蛋白激酶C(PKcheeta)、PKC-lambda和PKM-Zeta是非典型的PKC(APKC)，它们有助于维持海马区的长时程增强(LTP)和记忆存储。我们已经证明，持续的伤害性刺激或脊髓神经元的直接激活会导致脊髓背角神经元中aPKCs的上调，这些激酶的抑制剂可以逆转依赖于脊髓神经可塑性的伤害阈值降低。虽然不同的激酶(PKA、PKC、CaMK-II、MAPK、PI3-Kinase和PIN-1、PIP3、p38-Kinase和Scr)在LTP过程中调节aPKCs的激活，但目前尚不清楚这些介质中是否有任何一种调节脊髓伤害性敏化过程中aPKC的活性。使用选择性的PK抑制剂和非磷酸化和磷酸化的APKCs的蛋白分析，我们建议确定这些介质中的哪一种与伤害性刺激诱导的APKCs的长时间上调和激活有关。此外，APKCs通过刺激结合伙伴如p62、PICK1、NSF与GluR1和GluR2的相互作用而增加突触后AMPAR的水平来维持LTP和记忆，并且持续的伤害性也触发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中aPKC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测aPKCs、p62、PICK1和NSF在伤害性刺激反应中SCDH神经元GluR1和GluR2亚细胞分布变化中的作用。最近的研究质疑Zeta抑制肽的特异性，并表明在PKM-Zeta基因敲除小鼠中，LTP和记忆处理不受影响。因此，我们建议确定非选择性和选择性aPKC抑制剂(包括药物、反义寡核苷酸、显性负性突变蛋白和诱导性基因消融)在维持外周或脊髓伤害性刺激诱导的机械超敏反应中的作用。最后，我们将了解细胞穿透性多肽破坏aPKC、aPKC结合伙伴和AMPA受体之间的相互作用对持续性伤害性超敏反应的影响。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，将比开发擦除记忆的方法更实用，影响也更大。

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.**