Alterations in cellular metabolic pathway and epithelial cell maturation induced by MYO5B defects are partially reversible by LPAR5 activation

Functional loss of the motor protein, Myosin Vb (MYO5B), induces various defects in intestinal epithelial function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). Utilizing the MVID model mice, Vil1-CreERT2;Myo5bflox/flox (MYO5B?IEC) and Vil1-CreERT2;Myo5bflox/G519R (MYO5B(G519R)), we previously reported that functional MYO5B loss disrupts progenitor cell differentiation and enterocyte maturation that result in villus blunting and deadly malabsorption symptoms. In this study, we determined that both absence and a point mutation of MYO5B impair lipid metabolism and alter mitochondrial structure, which may underlie the progenitor cell malfunction observed in MVID intestine. Along with a decrease in fatty acid oxidation, the lipogenesis pathway was enhanced in the MYO5B?IEC small intestine. Consistent with these observations in vivo, RNA-sequencing of enteroids generated from the two MVID mouse strains showed similar downregulation of energy metabolic enzymes, including mitochondrial oxidative phosphorylation genes. In our previous studies, lysophosphatidic acid (LPA) signaling ameliorates epithelial cell defects in MYO5B?IEC tissues and enteroids. The present study demonstrated that the highly soluble LPAR5-preferred agonist, Compound-1, improved sodium transporter localization and absorptive function, and tuft cell differentiation in patient-modeled MVID animals that carry independent mutations in MYO5B. Body weight loss in male MYO5B(G519R) mice was ameliorated by Compound-1. These observations suggest that Compound-1 treatment has a trophic effect on intestine with MYO5B functional loss through epithelial cell-autonomous pathways that can accelerate the differentiation of progenitor cells and the maturation of enterocytes. Targeting LPAR5 may represent an effective therapeutic approach for treatment of MVID symptoms induced by different point mutations in MYO5B. Overall design: To assess the epithelial cell-autonomous effects of MYO5B inactivation, bulk RNA-sequencing analysis was performed in enteroids that were generated from MYO5B?IEC and MYO5B(G519R) mouse jejunum. Enteroids were generated from jejunal crypts of adult Vil1-CreERT2; Myo5bflox/flox (for in vitro induced KO (iKO) and control) and Vil1-CreERT2; Myo5bflox/G519R (G519R) mice without tamoxifen treatment and passaged twice with mechanical breakdown by P200 pipette. One day after the second passage, 1 µM 4-OH-tamoxifen (SML1666, Sigma) or vehicle (EtOH) was added into the Mouse Organoid Growth Medium (IntestiCult; Stem Cell Technology) and incubated for 24 hours to induce Cre recombinase. Following day, the medium was replaced with the differentiation medium, which contains 5% Noggin conditioned medium and 5% R-spondin conditioned medium (generated in Vanderbilt Organoid Core) to withdraw Wnt ligands and EGF for enhancing cell differentiation. Some iKO enteroids were incubated with 100 nM Compound-1 for 2 days in the differentiation conditions.