Role of atypical PKCs in spinal nociceptive sensitization

强烈的有害刺激导致周围神经和脊髓背角（SCDH）神经元的敏感化。尽管各种谷氨酸受体(R)和下游信使参与了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C (PKCzeta)， PKC-lambda和PKM-zeta是非典型PKCs (aPKC)，有助于维持海马的长期增强（LTP）和记忆储存。我们已经证明，aPKCs在SCDH中对持续的有害刺激或脊髓神经元的直接激活作出上调反应，并且这些激酶的抑制剂逆转了依赖于脊髓神经可塑性的降低的伤害感受阈值。尽管各种激酶（PKA、PKC、CaMK-II、MAPK、pi3激酶和PIN-1、PIP3、p38激酶和scr）在LTP期间调节aPKC的激活，但尚不清楚这些介质是否在脊髓伤害性致敏过程中调节aPKC的活性。通过选择性PK抑制剂和未磷酸化和磷酸化aPKCs的蛋白测定，我们建议确定哪些介质有助于伤害性刺激诱导的aPKCs的长期上调和激活。此外，aPKCs通过刺激结合伙伴（如p62、PICK1、NSF）与GluR1和GluR2的相互作用，增加海马神经元突触后AMPAR的水平，从而维持LTP和记忆，持续的伤害感受也会引发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查aPKCs、p62、PICK1和NSF对SCDH神经元中GluR1和GluR2亚细胞分布变化的贡献，以响应伤害性刺激。最近的研究质疑了zeta抑制肽的特异性，并表明PKM-zeta敲除小鼠的LTP和记忆加工不受影响。因此，我们建议确定非选择性和选择性aPKC抑制（包括药物、反义寡核苷酸、显性负突变蛋白和诱导基因消融）对外周或脊髓伤害性刺激诱导的机械超敏反应维持的影响。最后，我们将探讨破坏aPKC、aPKC结合伙伴和AMPA受体之间相互作用的细胞穿透肽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。

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.