Distinct Treatment Outcomes between MVID Patient-Modeled Mice Suggest Epithelial Cell Maturation Is Influenced by MYO5B Functional State

Abstract

Background and aims: Microvillus inclusion disease (MVID) is a devastating congenital diarrhea and malnutrition disorder caused by different point mutations in Myosin Vb (MYO5B). Specific genotype-dependent epithelial phenotypes or treatment options have not been fully investigated. Using tamoxifen-inducible epithelial-specific MYO5B-deficient (Myo5bΔIEC) mice, we have reported that MYO5B loss disrupts progenitor cell differentiation and that Lyso-Phosphatidic Acid (LPA) signaling and Notch inhibition can ameliorate the differentiation defects. We recently generated another MVID patient-modeled mouse strain, Vil1-CreERT2;Myo5bflox/G519R (Myo5b(G519R)). This study compared the epithelial pathophysiology and the effects of drug treatment between Myo5b(G519R) and Myo5bΔIEC mice. Methods: The MVID model mice and littermate controls received a single dose of tamoxifen, and their intestinal tissues were harvested on day 4 post-tamoxifen. For treatment: the small chemical LPAR5 agonist, Compound-1, or vehicle were given intraperitoneally once a day. Another set of mice received the Notch2-targeted inhibitor, Nirogacestat, on days 1 and 3. Digital image analysis scripts were developed for segmentation of immunostained tissues. Staining intensities and localization (membrane vs. cytoplasm) of markers were assessed in >300,000 epithelial cells per section. Results: Epithelial transcriptional signatures were analyzed by RNA-sequencing of enteroids generated from jejunal crypts of Myo5b(G519R) and Myo5bΔIEC mice. Enteroids from both mice showed significant upregulation of Notch signaling mediators and the downregulation of tuft cell markers and mitochondrial metabolic genes compared to control enteroids. Immunostaining of tissue sections consistently demonstrated that functional Myo5b loss decreases tuft cell numbers and mitochondrial densities in crypt and villus epithelial cells. In the vehicle treated MVID mouse tissues, sodium transporters of enterocytes, such as Na+/H+ exchanger (NHE)3 and Na+-dependent glucose transporter (SGLT)1, were mislocalized from the apical membrane and colocalized with lysosomal markers similar to what is observed in MVID patient tissues. Compound-1 treatment increased the apical localization of NHE3 and SGLT1 both in Myo5b(G519R) and Myo5bΔIEC mouse small intestines, whereas Nirogacestat improved localizations only in Myo5bΔIEC but not Myo5b(G519R) mouse tissues. Conclusions: Functional MYO5B loss, caused by the point mutation at G519R or total knockout directly disrupts Notch signaling and energy metabolic pathways in the epithelial cells, resulting in differentiation defects and malnutrition. The distinct treatment effcacies between the two MVID mouse models suggest that the total loss and impaired function of MYO5B differentially affect epithelial cell functions. NIH R01DK128190, RC2DK118640. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.