Novel Targets and Therapeutic Interventions against Cerebral Ischemia-Reperfusion Injury

项目总结/摘要 脑缺血/再灌注（I/R）损伤是急性脑梗死患者治疗的主要挑战 通过静脉（IV）溶栓和血管内治疗缺血性卒中。目前，没有有效的 可用于治疗/预防脑I/R损伤的干预。新出现的证据表明，血栓性和 炎症反应（血栓-炎症）和异常内质网（ER）应激 脑I/R诱发的脑缺血再灌注损伤是继发性脑损伤和神经功能恶化的重要原因。在这 我们希望开发一种新的基于miRNA的治疗策略，同时靶向这些 脑I/R损伤的病理过程通过不同的分子机制。最近，减少 在患者和动物模型中，miR-30 c的表达与许多病理状况有关。 在初步研究中，我们发现miR-30 c在血小板、脑微血管和脑胶质细胞中高度表达。 皮质/海马神经元中，但其水平随年龄而下降。血液和血液中的miR-30 c水平 脑缺血性卒中后显著降低，并通过单次IV注射合成的 miR-30 c模拟物显著保护脑I/R损伤。我们进一步表明，增加的表达 miR-30 c的直接靶基因（包括血液和脑中的派-1，脑中的eIF 2 α和caspase-3） 通过IV miR-30 c模拟物处理显著降低了由脑I/R损伤诱导的脑缺血再灌注损伤。基于这些 我们从年轻成年小鼠中获得了令人兴奋的新发现，我们提出了一个创新的假设，即miR-30 c的功能是 脑I/R损伤引起的缺血性脑中血栓炎症和ER应激的关键调节剂，和 因此，靶向miR-30 c代表了对抗脑I/R损伤的新的治疗方法。以下 更新的卒中治疗学术行业圆桌会议（STAIR）临床前指南，我们将对此进行测试 老年雄性和雌性小鼠的假设。具体而言，我们将确定IV miR-30 c的有效性和安全性。 模拟为新型中风治疗剂（Aim 1）。使用互补方法，即“功能获得性”（miR-125）， 30 c模拟物）和“功能丧失”（设计为特异性竞争30 c模拟物的反义吗啉代寡核苷酸）。 miR-30 c用于每个单独靶基因的3 'UTR的结合位点，因此充当“靶保护剂”），我们将 鉴定派-1作为miR-30 c调节卒中后血栓炎症的关键分子靶点（目的2）， 并确定eIF 2 α和caspase 3是miR-30 c调节卒中后神经元ER的关键分子靶点 应激和神经元细胞死亡（目标3）。这些研究的长期目标是评估靶向miR-30 c是否 在中风高危的男性和女性老年人群中中风治疗的可行选择。

Project Summary/Abstract The cerebral ischemia/reperfusion (I/R) injury is a major challenge for the treatment of patients with acute ischemic stroke by intravenous (IV) thrombolysis and endovascular therapy. Currently, there is no effective intervention available to treat/prevent cerebral I/R injury. Emerging evidence suggests that thrombotic and inflammatory responses (thrombo-inflammation) and aberrant endoplasmic reticulum (ER) stress in the brain elicited by cerebral I/R contributes importantly to secondary brain injury and neurologic deterioration. In this proposal we wish to develop a novel miRNA-based therapeutic strategy to simultaneously target these pathological processes of cerebral I/R injury through distinct molecular mechanisms. Recently, decreased expression of miR-30c has been implicated in many pathological conditions in both patients and animal models. In preliminary studies, we show that miR-30c is highly expressed in blood platelets, cerebral microvessels, and cortical/hippocampal neurons in normal mice but its levels decline with age. miR-30c levels in both blood and brain are markedly decreased after ischemic stroke and elevating miR-30c by single IV injection of synthetic miR-30c mimic significantly protects against cerebral I/R injury. We further show that the increased expressions of the direct target genes of miR-30c (including PAI-1 in both blood and brain, elF2α and caspase-3 in the brain) induced by cerebral I/R injury were significantly decreased by IV miR-30c mimic treatment. Based on these exciting new findings from young adult mice, we propose the innovative hypothesis that miR-30c functions as a critical regulator of thrombo-inflammation and ER stress in the ischemic brain elicited by cerebral I/R injury, and thus targeting miR-30c represents a novel therapeutic approach for combating cerebral I/R injury. Following updated stroke Therapy Academic Industry Roundtable (STAIR) pre-clinical guidelines, we will test this hypothesis in aged male and female mice. Specifically, we will determine the efficacy and safety of IV miR-30c mimic as novel stroke therapeutics (Aim1). Using complementary approaches, i.e. the “gain-of-function” (miR- 30c mimic) and “loss-of-function” (anti-sense Morpholino oligos that are designed to specifically compete with miR-30c for binding sites of 3’UTR of each individual target gene and thus acts as a “target protector”), we will identify PAI-1 as a key molecular target of miR-30c in regulation of post-stroke thrombo-inflammation (Aim 2), and identify elF2α and caspase 3 as key molecular targets of miR-30c in regulation of post-stroke neuronal ER stress and neuronal cell death (Aim 3). The long-term goal of these studies is to evaluate if targeting miR-30c is a viable option for stroke therapy in both male and female older populations at high risk for stroke.