Protein kinase mechanisms for chronic pain in sickle cell disease

 描述(由申请人提供)：我们的长期目标是促进对SCD慢性疼痛神经机制的了解，并开发有效的药物治疗方法。疼痛不仅是终生的伴侣，也是10万患有镰状细胞病(SCD)的美国人死亡率的预测指标，这些人主要是非洲或拉丁裔。虽然现在认识到SC疼痛的特征是慢性疼痛并伴有急性疼痛发作，但慢性疼痛的神经生物学研究很少，特征不好，而且对现有的治疗方法难以奏效。一个潜在的富有成效的研究路线，以促进对SCD疼痛的理解，是丝氨酸/苏氨酸蛋白激酶介导的细胞信号机制的作用。蛋白激酶C(PKC)的几种异构体与某些慢性疼痛状况有关。我们最近报道，PKCδ是一种关键的细胞调节因子，在化疗引起的周围神经病变中使伤害性感受器敏化。12后者可被PKCδ抑制剂或PKCδ-siRNA抑制，在PKCδ缺失的小鼠中缺失。在小鼠镰状细胞转基因模型(TOW小鼠)中，发现PKCδ在与疼痛相关的脊髓区域被显著激活，其活性(激活或抑制)与TOW小鼠的疼痛行为相关，但其确切作用尚不明确。在这项建议中，我们假设脊髓PKCδ是一种促进和维持SCD慢性疼痛表现的神经机制。我们的策略是采用实时定量聚合酶链式反应、细胞转运/易位、免疫组织化学、免疫印迹和酶动力学等方法系统地检测两只小鼠的PKCδ的表达和活性，并将其表达和活性的变化与慢性疼痛的发生和发展联系起来(目标1)。为了直接验证这一假设，即脊髓PKCδ是维持(目标2)和促进(目标3)SCD慢性疼痛表现的分子机制，我们将通过采用互补的方法来抑制PKCδ(异构体特异性多肽抑制剂、siRNA敲除、造血干细胞移植和小鼠基因缺失)来进行药理学研究。在整个研究过程中，年龄/性别匹配的非镰刀野生型产仔小鼠将作为对照。尽管目前的提议是机械驱动的，但它最终可能导致针对蛋白激酶Cδ通路的药物干预，从而在SCD中治疗疼痛。在前一个资金周期中，我们的团队最近将另一种激酶抑制剂从BASE转移到I期研究，因此我们的团队非常适合(当前)机械性研究和(未来)最终能使SCD患者受益的翻译工作。

 DESCRIPTION (provided by applicant): Our long-term goal is to advance knowledge of the neural mechanisms of chronic pain in SCD and develop an effective pharmacologic treatment. Pain is not only a life-long companion but also a predictor of mortality rate for the 100,000 Americans living with sickle cell disease (SCD), people mainly of African or Latin descent. Though it is now appreciated that SC pain is characterized by chronic pain with episodes of acute pain, the neurobiology of chronic pain is poorly studied, not well characterized, and is refractory to currently available therapies. A potentially fruitful line of investigation to advanc understanding of pain in SCD is the role of cellular signaling mechanisms mediated by serine/threonine protein kinases. Several isoforms of the protein kinase C (PKC) have been implicated in certain chronic pain conditions. We recently reported that PKCδ is a critical cellulr regulator sensitizing nociceptors in chemotherapy-induced peripheral neuropathy.12 The latter was inhibited by treatments with PKCδ inhibitors or PKCδ-siRNA and was absent in PKCδ-null mice. In a mouse sickle cell transgenic model (TOW mice), PKCδ was found to be significantly activated in the spinal regions relevant for pain and its activity (activation or inhibition) correated with the pain behaviors in TOW mice, thought its exact role remains to be defined. In this proposal, we hypothesize that spinal PKCδ is a neuronal mechanism promoting and maintaining the manifestation of chronic pain in SCD. Our strategy is to systematically examine the expression and activity of PKCδ in TOW mice using real-time qPCR, cellular trafficking/translocation, immunohistochemistry, immunoblotting and enzymatic kinetics methods, and correlate changes in PKCδ expression and activity with the on-set and development of chronic pain (Aim 1). To directly test the hypothesis that spinal PKCδ is a molecular mechanism that maintains (Aim 2) and promote (Aim 3) the manifestation of chronic pain in SCD, we will conduct pharmacologic studies by employing complementary approaches to inhibit PKCδ (isoform-specific peptide inhibitors, siRNA knockdown, hematopoietic stem cell transplantation, and mouse gene-deletion). Throughout the study, age/sex matched non-sickle wildtype littermate mice will be used as controls. Although the current proposal is mechanistically driven, it may ultimately lead to pharmacological interventions that target the PKCδ-pathway for pain in SCD. Having recently moved another kinase inhibitor from bench to a Phase I study in the previous funding cycle, our team is uniquely suited for the (current) mechanistic study and (future) translational work that can ultimately benefit patients with SCD.