Microvillus inclusion disease-associated MYO5B deficiency impairs endosome-to-mitochondrion iron transfer

Abstract Background MYO5B deficiency causes microvillus inclusion disease (MVID), characterized by the inability to absorb dietary nutrients and secretory diarrhea. MVID intestinal tissue shows metabolic abnormalities, but the causality with MYO5B and the underlying mechanism are unknown. The aim of this study was to determine the effects of MYO5B deficiency on mitochondria as key regulators of cellular metabolism and the underlying mechanism. Methods Intestinal tissue from MVID patients and inducible intestine-specific myo5b-knockout (KO) mouse were examined by using light and large-scale scanning transmission electron microscopy. CRISPR-Cas9 was used to generate MYO5B KO intestinal Caco2 cells. Site-directed mutagenesis was performed to generate MYO5B mutants. Fluorescence-based indicators of mitochondrial membrane potential and iron levels, analyses of carbonylated protein residues from isolated mitochondria, and high-resolution respirometry were used to assess mitochondrial homeostasis and function. Results MYO5B-deficient Caco2 cells showed fragmented and swollen mitochondria, reduced intra-mitochondrial cristae, defective aerobic respiration, reduced mitochondrial membrane potential, and increased mitochondrial oxidative stress. Introduction of a myc-tagged full-length MYO5B in MYO5B KO cells restored membrane potential, whereas the MVID-causing MYO5B-p.(Pro660Leu) variant and the MYO5B-p.(Lys1534Ala) and -p.(Leu1597Pro) mutants did not, demonstrating causality. Quantitative 3D fluorescence microscopy revealed close associations between mitochondria and MYO5B-positive endosomes carrying the iron-transporting transferrin. Associations between transferrin-loaded endosomes and mitochondria were diminished in MYO5B-depleted Caco2 cells. MYO5B-deficient Caco2 cells showed reduced mitochondrial iron content and an accumulation of iron in the endosomal system. Conclusion MYO5B deficiency impairs endosome-to-mitochondrial iron transfer, leading to mitochondrial dysfunction. These results offer a novel therapeutic avenue aimed at restoring mitochondrial function in MVID.