Modulation of CNS Proinflammatory Cytokine Production

描述（由申请人提供）：丝氨酸/苏氨酸蛋白激酶p381 MAPK是许多外周炎性疾病的既定治疗靶点，其中促炎细胞因子产生增加导致病理学。相反，对p381 MAPK在CNS功能障碍中的体内作用及其作为治疗靶点的潜力知之甚少。具体而言，关于体内小胶质细胞p381 MAPK信号传导对导致疾病相关神经病理学的促炎分子的上调的定量贡献、p381 MAPK在活化小胶质细胞的有益修复和重塑反应中的重要性或神经元p381 MAPK在CNS功能障碍反应中的作用的信息很少。我们假设p381（而不是密切相关的p382 MAPK亚型）是体内小胶质细胞炎症激活级联反应的关键贡献者，其最终导致有害的促炎细胞因子过度产生和随后的神经元/突触损伤，并且抑制小胶质细胞和/或神经元中的p38 MAPK信号传导可以导致选择性的有益结果。由于缺乏CNS渗透性的、选择性的小分子p381 MAPK抑制剂，该领域在追求这些问题的能力方面受到限制。我们开发了一种新型的口服生物可利用的、脑渗透的、选择性的小分子p381 MAPK抑制剂（化合物069 A），其在小鼠CNS损伤模型中减弱海马促炎细胞因子过度产生并导致改善的神经学结果，现在提供了在体内以及体外或原位解决关于p38 MAPK和CNS功能障碍的这些关键问题的机会。我们将使用这种独特的化学生物学工具和新的基因敲除（KO）和耐药基因敲入（KI）小鼠来进行几项重要的机制研究。首先，我们将测试的重要性，p381 MAPK和p382 MAPK在体内通过使用小胶质细胞p381条件KO和p382全球KO小鼠模型进行应激刺激。将确定小胶质细胞活化和突触功能障碍反应的时间发作和概况。其次，我们将补充这些在体内研究，通过使用细胞培养模型，以更详细地探讨在小胶质细胞和神经元中的p38 MAPK亚型的相对贡献。我们将利用小胶质细胞-神经元共培养结合药理学和遗传敲低方法来确定小胶质细胞和神经元p38 MAPK亚型对应激诱导反应的重要性。该项目的成功完成将提供对关键调节蛋白p381 MAPK在诱导CNS病理生理学的应激源引起的小胶质细胞活化和神经元损伤中的作用的机制性见解，以及小胶质细胞与神经元p381 MAPK对CNS功能障碍反应的贡献。此外，这些研究将阐明高度相关的p38 MAPK亚型p382在小胶质细胞和神经元反应中的潜在作用。从长远来看，拟议研究产生的知识将为未来开发新的疾病修饰疗法和更全面地解释疾病进展研究提供更坚实的基础。 公共卫生相关性：该项目的成功完成将为关键调节蛋白p381 MAPK如何参与疾病相关压力源引起的大脑促炎症反应和中枢神经系统功能障碍提供机制见解。此外，结果将描绘小胶质细胞与神经元p381 MAPK和p382 MAPK的疾病相关的病理反应的相对重要性。从长远来看，拟议研究产生的见解和知识将为未来开发新的疾病修饰疗法和更全面地解释疾病进展研究提供更坚实的基础。

DESCRIPTION (provided by applicant): The serine/threonine protein kinase p381 MAPK is an established therapeutic target for a number of peripheral inflammatory diseases where increased proinflammatory cytokine production contributes to pathology. In contrast, much less is known about the in vivo role of p381 MAPK in CNS dysfunction and its potential as a therapeutic target. Specifically, little information is available on the quantitative contribution of microglial p381 MAPK signaling in vivo to up-regulation of proinflammatory molecules that lead to disease- relevant neuropathology, the importance of p381 MAPK in the beneficial reparative and remodeling responses of activated microglia, or the role of neuronal p381 MAPK in CNS dysfunction responses. We hypothesize that p381 (and not the closely related p382 MAPK isoform) is a key in vivo contributor to microglial inflammatory activation cascades that culminate in detrimental proinflammatory cytokine overproduction and subsequent neuronal/synaptic damage, and that suppression of p38 MAPK signaling in the microglia and/or neuron can lead to selective, beneficial outcomes. The field has been limited in its ability to pursue these questions because of the lack of CNS-penetrant, selective, small molecule p381 MAPK inhibitors. Our development of a novel, orally bioavailable, brain-penetrant, selective, small molecule p381 MAPK inhibitor (compound 069A) that attenuates hippocampal proinflammatory cytokine overproduction and leads to improved neurologic outcomes in a mouse CNS injury model now provides the opportunity to address these critical questions about p38 MAPK and CNS dysfunction in vivo as well as in vitro or in situ. We will use this unique chemical biology tool and novel knock-out (KO) and drug-resistant knock-in (KI) mice to pursue several important mechanistic investigations. First, we will test the importance of p381 MAPK and p382 MAPK in vivo through the use of microglial p381 conditional KO and p382 global KO mouse models subjected to stressor stimuli. The temporal onset and profile of microglial activation and synaptic dysfunction responses will be determined. Second, we will complement these in vivo studies by using cell culture models in order to explore in more detail the relative contributions of p38 MAPK isoforms in microglia and neurons. We will utilize microglial-neuronal co-cultures combined with pharmacological and genetic knock-down approaches to determine the importance of microglial and neuronal p38 MAPK isoforms to stressor-induced responses. Successful completion of this project will provide mechanistic insight into the role of the key regulatory protein, p381 MAPK, in microglial activation and neuronal damage caused by stressors that induce CNS pathophysiology, and the contribution of microglial vs. neuronal p381 MAPK to the CNS dysfunction responses. In addition, the studies will elucidate the potential role of the highly related p38 MAPK isoform, p382, in the microglial and neuronal responses. Longer term, the knowledge generated by the proposed studies will provide a firmer foundation for future development of new classes of disease-modifying therapeutics and fuller interpretation of disease progression investigations. PUBLIC HEALTH RELEVANCE: Successful completion of this project will provide mechanistic insight into how the key regulatory protein, p381 MAPK, is involved in brain pro-inflammatory responses and CNS dysfunction caused by disease- relevant stressors. In addition, the results will delineate the relative importance of microglial versus neuronal p381 MAPK and p382 MAPK to the disease-relevant pathological responses. Longer term, the insights and knowledge generated by the proposed studies will provide a firmer foundation for future development of new classes of disease-modifying therapeutics and fuller interpretation of disease progression investigations.