The Introverted Intestine : Pathophysiology and future treatment of microvillus inclusion disease

Abstract

The intestinal epithelium is responsible for digestion and nutrient uptake. It is highly organized with new cells being generated in the crypts, and differentiated cells occupying the villi. The most abundant differentiated cell type in the small intestine is the enterocyte. Enterocytes are polarized cells and contain an apical and a basolateral plasma membrane domain with distinct lipid and protein compositions. The apical membrane contains finger-like protrusions (microvilli). If polarization is disturbed, intestinal diseases such as microvillus inclusion disease (MVID) can occur. MVID is an orphan disease that affects newborns. Patients present in the first weeks of life with intractable diarrhoea, which is accompanied by a failure to absorb nutrients, metabolic acidosis and failure to thrive. Current treatment options are limited to total parenteral nutrition and ultimately small bowel transplantation. In about 90% of the cases, MVID is caused by mutations in the MYO5B gene. MYO5B encodes the myosin Vb (MYO5B) motor protein, which mediates transport of endosomal vesicles along actin filaments. A loss of MYO5B in enterocytes leads to disturbed apical polarity, including microvillus atrophy, intracellular microvillus inclusions, and a subapical accumulation of secretory vesicles.

This thesis aimed to provide new insights into the genetic cause and the pathophysiology of microvillus inclusion disease, and to evaluate organoids as a potential future treatment for intestinal epithelial diseases.

We analysed biopsies and intestinal organoids from two patients classified as variant MVID. We found that these patients did not have a mutation in MYO5B. Instead, we identified loss-of-function mutations in STX3 as the cause of the disease in both patients.

STX3 is a t-SNARE protein, which mediates fusion of endosomes to the apical membrane. Our identification of STX3 as an important player in variant MVID substantiates the role of the intracellular trafficking and polarity machinery in the pathophysiology of (classic and variant) MVID.

To analyse the pathophysiology of MVID in more detail, we have generated a novel mouse model, which recapitulates human MVID. The mice become severely ill as soon as day 4 after induction of MYO5B-deficiency, comparable to the severe phenotype in MVID patients. We identified that the intracellular accumulations of apical proteins and microvillus inclusions were a consequence of disturbed apical recycling. Furthermore, we show mislocalization of basolateral markers, and an intracellular accumulation of the tight junction protein claudin1 in the MYO5B-deficient mouse model.

Next, we evaluated prerequisites for small intestinal organoid transplantations as a potential future treatment for MVID. To this end, we showed that the location-specific function of the intestinal epithelium is intrinsically programmed in the stem cells, since organoids derived from a certain location, retain the matching functional properties during culture. This implies that in case of organoid transplantations, organoids from duodenum, jejunum and ileum would have to be transplanted in order to restore all intestinal functions. However, we also show that the differentiation towards a certain cell type can also be directed in vitro. As such, we could direct the stem cells towards an M-cel phenotype, by the addition of RANK ligand to the organoid cultures.