Key Laboratory of Human Disease Comparative Medicine, National Health Commission of China (NHC), Institute of Laboratory Animal Science, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Institute of Laboratory Animal Science, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
Animal Model Exp Med. 2023 Apr;6(2):155-167. doi: 10.1002/ame2.12318.
Multiple mitochondrial dysfunction syndromes (MMDS) are rare mitochondrial diseases caused by mutation of mitochondrial iron-sulfur cluster synthesis proteins. This study established a rat model simulating MMDS5 disease in the nervous system to investigate its pathological features and neuronal death.
We generated neuron-specific Isca1 knockout rat (Isca1 -NeuN-Cre) using CRISPR-Cas9 technology. The brain structure changes of CKO rats were studied with MRI, and the behavior abnormalities were analyzed through gait analysis and open field tests, Y maze tests and food maze tests. The pathological changes of neurons were analyzed through H&E staining, Nissl staining, and Golgi staining. Mitochondrial damage was assessed by TEM, western blot and ATP assay, and the morphology of neurons was assessed by WGA immunofluorescence to detect the death of neurons.
This study established the disease model of MMDS5 in the nervous system for the first time, and found that after Isca1 loss, the rats suffered from developmental retardation, epilepsy, memory impairment, massive neuronal death, reduced number of Nissl bodies and dendritic spines, mitochondrial fragmentation, cristae fracture, reduced content of respiratory chain complex protein, and reduced production of ATP. Isca1 knockout caused neuronal oncosis.
This rat model can be used to study the pathogenesis of MMDS. In addition, compared with human MMDS5, the rat model can survive up to 8 weeks of age, effectively extending the window of clinical treatment research, and can be used for the treatment of neurological symptoms in other mitochondrial diseases.
多种线粒体功能障碍综合征（MMDS）是由线粒体铁硫簇合成蛋白突变引起的罕见线粒体疾病。本研究建立了一种模拟神经系统 MMDS5 疾病的大鼠模型，以研究其病理特征和神经元死亡。
我们使用 CRISPR-Cas9 技术生成神经元特异性 Isca1 敲除大鼠（Isca1-NeuN-Cre）。使用 MRI 研究 CKO 大鼠的脑结构变化，通过步态分析和旷场试验、Y 迷宫试验和食物迷宫试验分析行为异常。通过 H&E 染色、Nissl 染色和高尔基染色分析神经元的病理变化。通过 TEM、western blot 和 ATP 测定评估线粒体损伤，通过 WGA 免疫荧光评估神经元形态，以检测神经元死亡。
本研究首次建立了神经系统 MMDS5 疾病的模型，发现 Isca1 缺失后，大鼠发育迟缓、癫痫、记忆障碍、大量神经元死亡、Nissl 体和树突棘减少、线粒体碎片化、嵴断裂、呼吸链复合物蛋白含量减少、ATP 产生减少。Isca1 敲除导致神经元胀亡。
该大鼠模型可用于研究 MMDS 的发病机制。此外，与人类 MMDS5 相比，该大鼠模型可存活至 8 周龄，有效延长了临床治疗研究的窗口期，可用于治疗其他线粒体疾病的神经症状。
