trans-mitochondrial-cybrid-generation-from-mtdna-patient-platelets-an

Trans-Mitochondrial Cybrid Generation from mtDNA Patient Platelets: An Efficient Protocol Optimizing Colony Selection and Functional Validation

Trans-mitochondrial cybrid cell line generation represents the gold-standard method for determining pathogenicity by enabling biochemical analyses of a ...

The Children's Hospital of Philadelphia

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.

Donna M Iadarola

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