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，其中促炎细胞因子的产生增加有助于病理。相比之下，对于p381 MAPK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。具体来说，关于小胶质细胞p381 MAPK信号在体内对促炎分子上调导致疾病相关神经病理的定量贡献，p381 MAPK在活化小胶质细胞有益修复和重塑反应中的重要性，或神经元p381 MAPK在中枢神经系统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因素，这种级联反应最终导致有害的促炎细胞因子过量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、选择性的小分子p381 MAPK抑制剂，该领域的研究能力受到限制。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069A），它可以减少海马促炎细胞因子的过量产生，并改善小鼠中枢神经系统损伤模型的神经系统结果，现在提供了解决p38 MAPK和中枢神经系统功能障碍的关键问题的机会，无论是在体内还是在体外或原位。我们将使用这种独特的化学生物学工具和新型敲除（KO）和耐药敲入（KI）小鼠进行几项重要的机制研究。首先，我们将通过使用应激刺激下的p381条件KO和p382全局KO小鼠模型来测试p381 MAPK和p382 MAPK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特征。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p38 MAPK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-神经元共培养结合药理学和基因敲除方法来确定小胶质和神经元p38 MAPK亚型对应激源诱导反应的重要性。该项目的成功完成将提供关键调节蛋白p381 MAPK在应激源诱导的中枢神经系统病理生理引起的小胶质细胞激活和神经元损伤中的作用机制，以及小胶质细胞与神经元p381 MAPK在中枢神经系统功能障碍反应中的作用。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型疾病修饰疗法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.