mitoNEET as a therapeutic target for mitigating ischemic brain injury following MCAO

项目摘要 越来越多的证据表明，缺血性卒中后低灌注脑组织功能丧失 由于半暗带神经元之间的电通信由于氧化性神经元的显著减少而中断， 新陈代谢.因此，很明显，线粒体功能障碍在神经细胞损伤程度中起着重要作用。 缺血性脑损伤后出现死亡;然而，线粒体被完全激活的速度很慢。 研究了通过偶然发现吡格列酮的脱靶治疗作用， 线粒体铁硫簇蛋白，mitoNEET（mNT）被鉴定为重新引起了治疗的兴趣， 靶向线粒体。MitoNEET嵌入线粒体外膜，并作为氧化还原和 pH传感器调节线粒体生物能量学，特别是响应细胞应激。使用吡格列酮 作为我们的母体化合物，我们设计了NL-1，一种对mN具有高特异性的一流配体。使用NL-1，我们 已经证明在中风后的神经病理学和功能障碍方面有明显的改善， 小鼠和大鼠的短暂大脑中动脉闭塞（MCAO）。本提案的目的是解决 关于mNT如何在脑内发挥作用以减轻急性脑缺血的知识存在根本性差距 损伤我们的中心假设是，调节mNT的作用是改善脑内脆弱的神经元。 通过减少过度铁诱导的脂质过氧化和增加神经元的恶性循环， 死亡基于大量的先前文献和试验数据，我们假设NL-1的初始活性目标 是脑微血管系统，因此，我们提出了两个具体的目标来测试我们的假设。在具体目标1中， 我们将使用血液的4细胞Transwell体外模型测试mNT选择性配体NL-1是否减轻铁凋亡， 缺氧缺糖再灌注后脑屏障损伤。而在具体目标2中，我们将测试 mNT配体NL-1可减少MCAO后的脑铁蓄积和血脑屏障功能障碍。成功 完成拟议的研究预计将提供：（1）更好地了解如何和在哪里mNT 减轻缺血性中风后的脑损伤;（2）对线粒体功能障碍的影响的新认识， 减少缺血性卒中结局的生物能量学;（3）介入治疗的强大科学基础 治疗缺血性中风的方法。通过这些获得的机制和临床前数据 这些研究将成为推进靶向治疗发展的重要里程碑， 治疗神经损伤和疾病。

PROJECT SUMMARY Accumulating evidence suggests that following ischemic stroke hypoperfused brain tissue is functionally disabled as electrical communication among penumbral neurons is disrupted due to marked reductions in oxidative metabolism. Thus, it is apparent that mitochondrial dysfunction plays a central role in the degree of neuronal cell death encountered following ischemic brain injury; however, mitochondria have been slow to be fully investigated. Through a serendipitous discovery of an off-target therapeutic effect of pioglitazone, a small mitochondrial iron-sulfur cluster protein, mitoNEET (mNT) was identified that has renewed interest in therapeutic targeting of mitochondria. MitoNEET is embedded in the outer mitochondrial membrane and acts as a redox and pH sensor to regulate mitochondrial bioenergetics, especially in response to cellular stress. Using pioglitazone as our parent compound, we designed NL-1, a first-in-class ligand with high specificity for mN. Using NL-1, we have demonstrated marked improvements in stroke neuropathology and functional impairment following transient middle cerebral artery occlusion (MCAO) in mice and rats. The objective of this proposal is to address fundamental gaps in knowledge regarding how mNT works within the brain to mitigate acute ischemic brain injury. Our central hypothesis is that modulation of mNT acts to improve vulnerable neurons within the penumbra by reducing the vicious cycle of excessive iron-induced lipid peroxidation and increased neuronal death. Based on a strong body of prior literature and pilot data, we postulate the initial target of activity for NL-1 is the cerebral microvasculature; thus, we propose two specific aims to test our hypothesis. In specific aim 1, we will test if mNT selective ligand, NL-1, mitigates ferroptosis using a 4 cell Transwell in vitro model of the blood- brain barrier following oxygen-glucose deprivation with reperfusion. Whereas, in specific aim 2, we will test if mNT ligand, NL-1, reduces brain iron accumulation and blood-brain barrier dysfunction post-MCAO. Successful completion of the proposed research is expected to provide a: (1) greater understanding of how & where mNT mitigates brain injury following ischemic stroke; (2) new insight into the impact of mitochondrial dysfunction & diminished bioenergetics on ischemic stroke outcomes; & (3) strong scientific foundation for an interventional therapeutic approach for treating ischemic stroke. The mechanistic & preclinical data obtained through these studies will serve as critical milestones for advancing the development of mitochondria-targeted therapies for treating neurological injuries & disease.