The role of the nuclear receptor NR4A1 in acute ischemic stroke: pathophysiology, therapeutic evaluation and human translation

现代血运重建疗法的应用显著改善了缺血性卒中的治疗。然而，大多数患者不适合这些治疗，或尽管进行了血运重建，但仍有不利的结局。然而，对缺血性损伤的病理生理学知识的不断增长为新的治疗选择铺平了道路。特别是，脑缺血后的局部炎症反应被确定为有希望的治疗靶点，与经典的神经保护策略相比，其可以有效地调节缺血的急性期以外。在这种情况下，核受体，这在调节炎症及其配体的重要作用是有吸引力的候选人的治疗interventions.Our初步数据首次显示，核受体NR 4A 1参与急性中风的病理生理。在实验性中风后，NR 4A 1-/-小鼠具有较大的梗塞和较差的神经学结果，反之亦然，用NR 4A 1激动剂胞孢酮B处理的野生型小鼠具有较好的结果，表明NR 4A 1的强保护模式作用。我们确定了NR 4A 1-/-小鼠缺血性脑中中性粒细胞浸润增加以及T细胞增殖和细胞因子产生增加作为潜在的潜在机制。这些发现是令人惊讶的，因为NR 4A 1-/-在中性粒细胞或T细胞功能中的主要作用尚未得到证实。NR 4A 1首先是中性粒细胞功能的调节剂，还是中性粒细胞向大脑的募集以T细胞依赖性方式增加，目前尚不清楚，将在拟议项目中进行研究。该提案的总体目标是阐明NR 4A 1在缺血性卒中病理生理学中的作用，并评估其对未来治疗干预的潜在适用性。首先，我们将使用骨髓嵌合小鼠、过继细胞转移和细胞特异性敲除小鼠鉴定NR 4A 1介导的神经保护的细胞介质（中性粒细胞或T细胞）。接下来，为了确定分子机制，将对这些鉴定的NR 4A 1-/-细胞介质进行全面分析，包括功能分析和细胞代谢研究。此外，SPECT成像将用于详细表征脑中性粒细胞浸润。为了评价治疗适用性，在小鼠缺血发作后的不同时间点开始用NR 4A 1激动剂胞孢酮B治疗。为了弥合我们的实验研究与人类中风之间的差距，我们将对患者脑组织和血液免疫细胞中的NR 4A 1表达进行全面研究，并评估Cytosporone B对患者血液免疫细胞的影响。 总之，拟议的项目允许识别NR 4A 1在一般情况下和在脑缺血的背景下的新功能，并将确定其作为治疗靶点的适用性。

The treatment of ischemic stroke has significantly improved by using modern revascularization therapies. Nevertheless, the majority of patients is not suitable for these therapies or has an unfavorable outcome despite revascularization. However, the growing knowledge on the pathophysiology of ischemic injury paved the way for new therapeutic options. In particular, the local inflammatory response after cerebral ischemia was identified as promising therapeutic target, which can be, in contrast to classic neuroprotective strategies, effectively modulated beyond the acute phase of ischemia. In this context, nuclear receptors, which play an important role in regulating inflammation and their ligands are attractive candidates for therapeutic interventions.Our preliminary data show for the first time that the nuclear receptor NR4A1 is involved in the pathophysiology of acute stroke. After experimental stroke NR4A1-/- mice have larger infarcts and a worse neurological outcome and vice versa wildtype mice treated with the NR4A1 agonist Cytosporone B have better outcomes suggesting a strong protective mode action of NR4A1. We identified increased neutrophil infiltration into the ischemic brain in NR4A1-/- mice as well as an increased proliferation and cytokine production of T cells as potential underlying mechanisms. These findings are surprising as a major role of NR4A1-/- in neutrophil or T cell functions was not yet demonstrated. Whether NR4A1 is a modulator of neutrophil function in the first place or neutrophil recruitment to the brain is increased in a T cell dependent manner is yet unknown and will be investigated in the proposed project. The overarching aim of this proposal is to elucidate the role of NR4A1 in the pathophysiology of ischemic stroke and to evaluate its potential suitability for future therapeutic interventions. First, we will identify the cellular mediators (neutrophils or T cells) of NR4A1 mediated neuroprotection using bone marrow chimeric mice, adoptive cell transfer, and cell specific knockout mice. Next, aiming at determining the molecular mechanisms, comprehensive analyses of such identified NR4A1-/- cellular mediators will be performed including functional analyses and studies on cell metabolism. In addition, SPECT imaging will be used for a detailed characterization of brain neutrophil infiltration. In order to evaluate the therapeutic applicability, a treatment with the NR4A1 agonist Cytosporone B will be initiated at different time points after ischemia onset in mice. To bridge the gap between our experimental studies and human stroke we will perform comprehensive studies on NR4A1 expression in patient brain tissue and blood immune cells as well as evaluate the effect of Cytosporone B on blood immune cells of patients. In summary, the proposed project allows for the identification of novel functions of NR4A1 in general and in the context of cerebral ischemia and will determine its suitability as therapeutic target.