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

通过使用现代血管重建疗法，缺血性中风的治疗已经有了显著的进步。然而，大多数患者不适合接受这些治疗，或者尽管进行了血运重建，但预后不佳。然而，对缺血性损伤病理生理学的日益增长的认识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认为是有希望的治疗靶点，与经典的神经保护策略相比，它可以有效地调节缺血后的急性期。在此背景下，在调节炎症过程中起重要作用的核受体及其配体成为治疗干预的候选对象。我们的初步数据首次显示，核受体NR4A1参与了急性卒中的病理生理过程。在实验性中风后，NR4A1-/-小鼠有更大的梗塞和更差的神经结局，反之亦然，接受NR4A1激动剂Cytosporone B治疗的野生型小鼠有更好的结果，这表明NR4A1具有强大的保护模式作用。我们发现，在NR4A1-/-小鼠中，中性粒细胞在缺血脑中的渗透增加，以及T细胞的增殖和细胞因子的产生增加是潜在的潜在机制。这些发现令人惊讶，因为NR4A1-/-在中性粒细胞或T细胞功能中的主要作用尚未被证明。NR4A1首先是中性粒细胞功能的调节器，还是以T细胞依赖的方式增加中性粒细胞向大脑的募集，目前尚不清楚，将在拟议的项目中进行研究。这项建议的主要目的是阐明NR4A1在缺血性卒中的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基因敲除小鼠来鉴定NR4A1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，将对这些已识别的NR4A1-/-细胞介体进行全面的分析，包括功能分析和细胞代谢研究。此外，SPECT成像将用于详细描述脑中性粒细胞的渗透情况。为了评估治疗的适用性，NR4A1激动剂Cytosporone B将在小鼠缺血后的不同时间点开始治疗。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NR4A1的表达进行全面的研究，并评估Cytosporone B对患者血液免疫细胞的影响。总而言之，拟议的项目允许在一般情况下和在脑缺血的背景下确定NR4A1的新功能，并将确定其作为治疗靶点的适宜性。

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.