Modulation of CNS Proinflammatory Cytokine Production

描述(申请人提供)：丝氨酸/苏氨酸蛋白激酶p381MAPK是一些外周炎症性疾病的既定治疗靶点，在这些疾病中，促炎细胞因子的产生增加有助于病理。相比之下，人们对p381MAPK在中枢神经系统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。具体地说，关于体内小胶质细胞p381MAPK信号对导致疾病相关神经病理的促炎分子上调的定量贡献，p381MAPK在激活的小胶质细胞有益的修复和重塑反应中的重要性，或者神经元p381MAPK在中枢神经系统功能障碍反应中的作用，几乎没有信息可用。我们假设p381(而不是密切相关的p382MAPK亚型)在体内是小胶质细胞炎症激活级联反应的关键贡献者，最终导致有害的促炎细胞因子过度生产和随后的神经元/突触损伤，并且抑制小胶质细胞和/或神经元中的p38MAPK信号可以导致选择性的、有益的结果。由于缺乏中枢神经系统渗透性的、选择性的小分子p381 MAPK抑制剂，该领域对这些问题的研究能力一直受到限制。我们开发了一种新型的口服生物利用型、脑穿透、选择性的小分子p381 MAPK抑制剂(化合物069a)，它可以减少海马区促炎症细胞因子的过度产生，并改善小鼠中枢神经系统损伤模型的神经学结果，现在提供了解决这些关键问题的机会，这些关键问题是p38MAPK与中枢神经系统功能障碍的体内、体外或原位研究。我们将使用这种独特的化学生物学工具和新型的基因敲除(KO)和耐药基因敲入(KI)小鼠来进行几个重要的机制研究。首先，我们将通过使用小胶质细胞p381条件性KO和p382全局性KO小鼠模型来测试p381MAPK和p382MAPK在体内的重要性。将确定小胶质细胞激活和突触功能障碍反应的时间起点和轮廓。其次，我们将通过使用细胞培养模型来补充这些体内研究，以便更详细地探索p38MAPK亚型在小胶质细胞和神经元中的相对贡献。我们将利用小胶质细胞-神经元共培养结合药理学和遗传学方法来确定小胶质细胞和神经元p38MAPK亚型在应激诱导反应中的重要性。该项目的成功完成将从机制上深入了解关键调控蛋白p381MAPK在小胶质细胞激活和诱导中枢神经系统病理生理学的应激源引起的神经元损伤中的作用，以及小胶质细胞与神经元p381MAPK在中枢神经系统功能障碍反应中的作用。此外，这些研究还将阐明高度相关的p38MAPK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改疗法类别和更全面地解释疾病进展调查提供更坚实的基础。 公共卫生相关性：该项目的成功完成将提供对关键调控蛋白p381MAPK如何参与疾病相关应激源引起的脑促炎反应和中枢神经系统功能障碍的机械性洞察。此外，研究结果还将揭示小胶质细胞与神经细胞的p381MAPK和p382MAPK在疾病相关病理反应中的相对重要性。从长远来看，拟议研究产生的洞察力和知识将为未来开发新的疾病修改疗法类别和更全面地解释疾病进展调查提供更坚实的基础。

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.