Cyrano/S3-OPA1/Drp1：脑缺血再灌注损伤神经元线粒体动力学失衡调控新机制

神经元线粒体动力学失衡是导致脑缺血再灌注损伤的重要因素，具体调控机制需进一步研究。我们预实验提示：短暂缺血后，神经元线粒体快速分裂导致嵴结构破坏，高水平钙离子促进OPA1新剪切体S3-OPA1以lnc Cyrano依赖的方式与Drp1互作。据此我们提出假说：Cyrano/S3-OPA1/Drp1可能是脑缺血再灌注损伤神经元线粒体动力学失衡调控新机制。为证明该假说，我们利用活细胞成像和电镜检测脑缺血再灌注对神经元线粒体动力学和线粒体结构的影响；利用诱导型OPA1基因敲除小鼠和互补多酶切质谱联用证明S3-OPA1的存在；利用RIP和RNAscope探索Cyrano/S3-OPA1/Drp1互作；利用Cyrano基因干预调控S3-OPA1/Drp1互作，分析调控这一互作对脑缺血再灌注后神经元线粒体动力学失衡和脑损伤的影响。本工作旨在为开发脑缺血患者再灌注联合线粒体保护治疗新策略提供新的实验依据。

Excessive mitochondrial fission of neurons leads to cerebral ischemia-reperfusion injury, which is difficult to be explained by the classical mitochondrial dynamics regulated by OPA1/Drp1. Our preliminary experiments suggest that transient ischemic induces S3-OPA1, a newly discovered OPA1 splice, interacting with Drp1 in lnc Cyrano-dependent manner, accompanied by mitochondrial fission and cristae structural destruction. Based on previous reports and our preliminary results, we hypothesize that Cyrano/S3-OPA1/Drp1 exacerbates reperfusion neuronal injury by mediating mitochondrial fission during transient ischemia. In order to prove this hypothesis, we propose to use live cell real-time imaging and electron microscopy to verify the transient ischemia-induced mitochondrial fission and mitochondrial structural destruction in neurons. OPA1 gene knockout mice and complementary multienzyme digestion for protein C-terminal with Mass Spectrometry Sequencing are used to prove the existence of S3-OPA1. Cyrano/S3-OPA1/Drp1 interaction will be explored by RNAscope and RIP. Cyrano gene intervention is performed to regulate the interaction between S3-OPA1 and Drp1. Then, we will research the effect of Cyrano gene intervention on transient ischemia-induced mitochondrial fission and neuronal injury after reperfusion. This work is expected to provide a new experimental basis for developing a new strategy of reperfusion combined with mitochondrial protection in patients with cerebral ischemia.