Protein kinase mechanisms for chronic pain in sickle cell disease

 描述（由申请人提供）：我们的长期目标是提高对SCD慢性疼痛神经机制的认识，并开发有效的药物治疗。疼痛不仅是一个终身伴侣，也是10万美国镰状细胞病（SCD）患者死亡率的预测因素，这些患者主要是非洲人或拉丁裔。虽然现在认识到SC疼痛的特征在于慢性疼痛伴急性疼痛发作，但慢性疼痛的神经生物学研究不足，没有很好地表征，并且对目前可用的疗法是难治的。一个潜在的富有成效的调查线，以了解疼痛的SCD是丝氨酸/苏氨酸蛋白激酶介导的细胞信号传导机制的作用。蛋白激酶C（PKC）的几种亚型与某些慢性疼痛病症有关。我们最近报道，PKCδ是化疗诱导的周围神经病变中致敏伤害感受器的关键细胞调节因子。12后者可通过PKCδ抑制剂或PKCδ-siRNA治疗抑制，并且在PKCδ-null小鼠中不存在。在镰状细胞转基因小鼠模型（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，但其确切作用仍有待确定。在本研究中，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。我们的策略是使用实时qPCR、细胞运输/易位、免疫组织化学、免疫印迹和酶动力学方法系统地检测TOW小鼠中PKCδ的表达和活性，并将PKCδ表达和活性的变化与慢性疼痛的发生和发展相关联（目的1）。为了直接检验脊髓PKCδ是维持（目的2）和促进（目的3）SCD慢性疼痛表现的分子机制这一假设，我们将通过采用互补方法抑制PKCδ（亚型特异性肽抑制剂、siRNA敲除、造血干细胞移植和小鼠基因缺失）进行药理学研究。在整个研究过程中，将使用年龄/性别匹配的非镰状野生型同窝小鼠作为对照。虽然目前的建议是机械驱动的，它可能最终导致靶向PKCδ-通路的SCD疼痛的药物干预。在上一个资助周期中，我们最近将另一种激酶抑制剂从实验室转移到I期研究，我们的团队非常适合（当前）机制研究和（未来）最终使SCD患者受益的转化工作。

 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.