ndufs2-/- zebrafish have impaired survival, neuromuscular activity, morphology, and one-carbon metabolism treatable with folic acid

Abstract

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 ndufs2-/- 16 bp deletion zebrafish strain. ndufs2-/- 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 ndufs2-/- larvae revealed dysregulation of the electron transport chain, TCA cycle, fatty acid beta-oxidation, and one-carbon metabolism. Similar transcriptomic profiles were observed in ndufs2-/- missense mutant C. elegans (gas-1(fc21)) and two human CI-disease fibroblast cell lines stressed in galactose media. ndufs2-/- 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 ndufs2-/-larvae. Overall, ndufs2-/- zebrafish recapitulate severe CI deficiency, complex metabolic pathophysiology, and relevant LSS neuromuscular and survival phenotypes, enabling future translational studies of therapeutic candidates.