Neuronal Deficiency of Cytochrome c Oxidase Engineered by Mitochondrial DNA Editing Recapitulates Amyotrophic Lateral Sclerosis [snRNA-seq]. Neuronal Deficiency of Cytochrome c Oxidase Engineered by Mitochondrial DNA Editing Recapitulates Amyotrophic Lateral Sclerosis [snRNA-seq]

Amyotrophic lateral sclerosis (ALS) is categorized into 10% familial and 90% sporadic cases. While the space of 10% familial ALS is crowded with mutations in many genes of diverse functions, most ALS-associated mutations could not faithfully recapitulate the disease phenotype in animal models. Remarkably, nearly half of sporadic ALS patients exhibit defective mitochondrial respiratory complex Ⅳ (CⅣ). To establish the causal role of defective CⅣ in inducing ALS, we employed TALE-based mtDNA editing to mimic ALS-linked mutations in CⅣ compared to other respiratory complexes in rat neurons and demonstrated that mutations exclusively introduced to mtDNA-encoded CⅣ subunits are sufficient to cause a full spectrum of ALS-like phenotypes, including selective motor neuron loss, SOD1 overexpression and cytosolic TDP-43 aggregation. These findings reveal a broad basis for sporadic ALS, provide critical insights into the selective motor neuron vulnerability, and present a faithful animal model for advancing ALS therapy. Overall design: To investigate the effects of mitochondrial respiratory complex Ⅳ deficiency in rat neurons , the spinal cord WT and COXⅢNeuN-Cre rats, were dissected , and the RNA was isolated to perform the single-nucleus RNA sequencing study (n=3 per group).

肌萎缩侧索硬化症（Amyotrophic lateral sclerosis, ALS）可分为10%的家族性病例与90%的散发性病例。尽管10%的家族性ALS病例中，多种功能各异的基因均存在突变，但多数ALS相关突变无法在动物模型中忠实重现疾病表型。值得注意的是，近半数散发性ALS患者存在线粒体呼吸复合物Ⅳ（CⅣ，mitochondrial respiratory complex Ⅳ）功能缺陷。为明确CⅣ功能缺陷在ALS发病中的因果作用，我们采用基于转录激活因子样效应物（Transcription Activator-Like Effector, TALE）的线粒体DNA编辑技术，在大鼠神经元中模拟与ALS相关的CⅣ突变，并与其他呼吸复合物进行对照；实验证实，仅对线粒体DNA编码的CⅣ亚基引入突变，即可引发全套类ALS表型，包括选择性运动神经元丢失、超氧化物歧化酶1（SOD1）过表达以及胞浆内TDP-43聚集。这些发现为散发性ALS的发病机制提供了广泛的理论依据，为阐释选择性运动神经元的易感性提供了关键见解，同时也为推进ALS治疗构建了可靠的动物模型。整体实验设计：为探究线粒体呼吸复合物Ⅳ缺陷对大鼠神经元的影响，我们对野生型（WT，wild type）脊髓组织及COXⅢNeuN-Cre大鼠进行解剖取材，分离总RNA后开展单细胞核RNA测序研究（每组样本量n=3）。