Altered MYO5B Function Underlies Microvillus Inclusion Disease: Opportunities for Intervention at a Cellular Level

Abstract

Microvillus inclusion disease (MVID) is a congenital diarrheal disorder resulting in life-threatening secretory diarrhea in newborns. Inactivating and nonsense mutations in myosin Vb (MYO5B) have been identified in MVID patients. Work using patient tissues, cell lines, mice, and pigs has led to critical insights into the pathology of MVID and a better understanding of both apical trafficking in intestinal enterocytes and intestinal stem cell differentiation. These studies have demonstrated that loss of MYO5B or inactivating mutations lead to loss of apical sodium and water transporters, without loss of apical CFTR, accounting for the major pathology of the disease. In addition, loss of MYO5B expression induces the formation of microvillus inclusions through apical bulk endocytosis that utilizes dynamin and PACSIN2 and recruits tight junction proteins to the sites of bulk endosome formation. Importantly, formation of microvillus inclusions is not required for the induction of diarrhea. Recent investigations have demonstrated that administration of lysophosphatidic acid (LPA) can partially reestablish apical ion transporters in enterocytes of MYO5B KO mice. In addition, further studies have shown that MYO5B loss induces an imbalance in Wnt/Notch signaling pathways that can lead to alterations in enterocyte maturation and tuft cell lineage differentiation. Inhibition of Notch signaling leads to improvements in those cell differentiation deficits. These studies demonstrate that directed strategies through LPA receptor activation and Notch inhibition can bypass the inhibitory effects of MYO5B loss. Thus, effective strategies may be successful in MVID patients and other congenital diarrhea syndromes to reestablish proper apical membrane absorption of sodium and water in enterocytes and ameliorate life-threatening congenital diarrhea.

Keywords: Congenital diarrhea; MVID; MYO5B; Microvillus inclusion disease; Myosin VB; animal models; enteroids.

Figure 1

Mutations in MYO5B cause MVID. (A) MYO5B mutations and conformations. MYO5B consists of a motor domain, a neck domain that can bind 6 calmodulins to increase rigidity, and a tail domain that can bind Rab proteins. The open active conformation of MYO5B allows for ATP driven transport along actin. In the proposed closed inactive MYO5B conformation, the head and tail domains directly interact, and the motor cannot translocate along actin. (B) Model of MYO5B trafficking at the apical surface of normal and MVID affected enterocytes. (Left) In normal enterocytes, MYO5B works to apically traffic transporters, enzymes, and brush boarder components. (Right) In MVID, transporters, enzymes, and brush boarder components are not apically localized, but CFTR is retained at the apical surface, suggesting that it is delivered independently of MYO5B. Figure created using Biorender. (C) Abnormal PAS staining and inclusion formation in MYO5B knockout mice. (i) PAS staining in wild-type mice showing normal PAS-positive brush border and gross morphology. (ii) PAS staining in MYO5B knockout mouse showing blunted microvilli and accumulation of PAS stain below the apical surface. (iii) Fracture transmission electron microscopy showing microvillus inclusions formed on the interior of knockout mouse enterocytes.

Figure 2

Loss of MYO5B induces apical bulk endocytosis and deficits in apical transporter trafficking: a target for therapeutic intervention. (A) Molecular mechanism of microvillus inclusion formation through apical bulk endocytosis induced by MYO5B loss. (Upper panels) F-actin (phalloidin, green) staining shows uniform brush borders in healthy control mouse intestine. Microvillus inclusions form from invaginations of the apical membranes of enterocytes through a process of apical bulk endocytosis in the MYO5B KO mouse (white arrows). Microvillus inclusions that are attached to the apical membrane demonstrate the process of apical bulk endocytosis. Lower diagram shows the topologies of intracellular proteins that are involved in apical bulk endocytosis and microvillus inclusion formation (adapted from Engevik et al⁷⁷). (B) LPA treatment ameliorates brush border structure and SGLT1 localization in vivo and in mouse enteroids. Immunostaining for SGLT1 (magenta), ACTG1 (green), and LAMP1 (yellow) indicate localization of the Na-glucose co-transporter, cell membrane and brush borders, and lysosomes, respectively. Mature brush borders containing SGLT1 are shown in white in control tissues. Tamoxifen-induced MYO5B KO mice with vehicle treatment show expanded lysosomes and disrupted brush border. Both mouse tissues and enteroids treated with LPA partly recover brush border structure and SGLT1 localization on the apical membrane (adapted from Kaji et al⁸⁴). It is notable that LPA treatment does not alter inclusion formation, suggesting that apical bulk endocytosis and trafficking defects are separable.

Figure 3

Summary of cell differentiation and maturation deficits in MYO5B-deficient intestine and treatment strategy. Functional MYO5B loss disrupts stem cell differentiation and enterocyte maturation. LPA receptor (LPAR) activation and/or rebalancing of Wnt/Notch signaling may rescue proper differentiation and maturation and consequently improve nutrient absorption.