Mitchell Dana V, Iadarola Donna M, Mathew Neal D, Keith Kelsey, Seiler Christoph, Yu Sanghyeon, Kim Man S, Woodard Niki, Anderson Vernon E, Nakamaru-Ogiso Eiko, Taylor Deanne M, Falk Marni J
Department of Biomedical and Health Informatics, The Children's Hospital of Philadelphia, Philadelphia, PA.
Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA.
bioRxiv. 2025 Jul 18:2025.07.16.664929. doi: 10.1101/2025.07.16.664929.
Mitochondrial complex I (CI) deficiency represents a common biochemical pathophysiology underlying Leigh syndrome spectrum (LSS), manifesting with progressive multi-system dysfunction, lactic acidemia, and early mortality. To facilitate mechanistic studies and rigorous screening of therapeutic candidates for CI deficient LSS, we used CRISPR/Cas9 to generate an 16 bp deletion zebrafish strain. larvae exhibit markedly reduced survival, severe neuromuscular dysfunction including impaired swimming capacity, multiple morphologic malformations, reduced growth, hepatomegaly, uninflated swim bladder, yolk retention, small intestines, and small eyes and pupils with abnormal retinal ganglion cell layer. Transcriptome profiling of larvae revealed dysregulation of the electron transport chain, TCA cycle, fatty acid beta-oxidation, and one-carbon metabolism. Similar transcriptomic profiles were observed in missense mutant () and two human CI-disease fibroblast cell lines stressed in galactose media. zebrafish had 80% reduced CI enzyme activity. Unbiased metabolomic profiling showed increased lactate, TCA cycle intermediates, and acyl-carnitine species. One-carbon metabolism associated pathway alterations appear to contribute to CI disease pathophysiology, as folic acid treatment rescued the growth defect and hepatomegaly in larvae. Overall, zebrafish recapitulate severe CI deficiency, complex metabolic pathophysiology, and relevant LSS neuromuscular and survival phenotypes, enabling future translational studies of therapeutic candidates.
线粒体复合体I（CI）缺乏是 Leigh 综合征谱系（LSS）潜在的常见生化病理生理学表现，伴有进行性多系统功能障碍、乳酸性酸中毒和早期死亡。为了促进对CI缺乏型LSS的机制研究和对治疗候选药物的严格筛选，我们使用CRISPR/Cas9技术构建了一个16bp缺失的斑马鱼品系。该品系幼虫的存活率显著降低，出现严重的神经肌肉功能障碍，包括游泳能力受损、多种形态畸形、生长减缓、肝肿大、鳔未充气、卵黄滞留、小肠异常以及视网膜神经节细胞层异常导致的小眼睛和小瞳孔。对该品系幼虫进行转录组分析发现电子传递链、三羧酸循环、脂肪酸β氧化和一碳代谢失调。在错义突变体（）和两种在半乳糖培养基中应激的人类CI疾病成纤维细胞系中也观察到了类似的转录组图谱。该品系斑马鱼的CI酶活性降低了80%。非靶向代谢组学分析显示乳酸、三羧酸循环中间体和酰基肉碱种类增加。一碳代谢相关途径的改变似乎导致了CI疾病的病理生理学，因为叶酸治疗挽救了该品系幼虫的生长缺陷和肝肿大。总体而言，该品系斑马鱼概括了严重的CI缺乏、复杂的代谢病理生理学以及相关的LSS神经肌肉和存活表型，为未来治疗候选药物的转化研究提供了可能。
