Project Summary Microvillus inclusion disease (MVID) is a form of congenital diarrhea that is caused by inactivating mutations in myosin Vb (MYO5B). All neonates with MVID are placed on total parenteral nutrition (TPN). TPN carries a high risk of infections, with sepsis being reported in 20-30% of patients receiving TPN. The 1-year survival rate of neonates with MVID is less than 25%. Currently the only treatment option for MVID is small bowel transplantation. Given the greatly reduced life expectancy of neonates with MVID and lack of alternative treatment options, understanding the pathology of MVID represents an important scientific question to be addressed. The central hypothesis of this research proposal is that loss of MYO5B results in aberrant expression of key apical membrane transporters that are critical for the absorption of nutrients and water. We base our hypothesis on published reports that biopsies from neonates with MVID have loss of apical sodium glucose transporter (SGLT1), sub-apical distribution of cystic fibrosis transmembrane conductance regulator (CFTR) and sodium hydrogen exchanger (NHE3) in enterocytes. Furthermore, recent mouse studies from our lab have reported diffuse sub-apical localization of NHE3 with loss of MYO5B. The development of mice with loss of MYO5B provides a new model to evaluate defects in enterocyte cellular composition and function. I propose the use of mouse models expressing germline, constitutively intestinal targeted and inducible intestinal targeted deletion of MYO5B along with human enteroids to address deficits in apical ion transport in the duodenum for this fellowship application. In Specific Aim 1, I will define the aberrant physiology of intestinal apical transporters in MVID mouse models. Expression of enterocyte apical ion transporters will be examined in all mouse models of MVID. Immunofluorescence confocal microscopy will be performed to determine whether apical ion transporters are being properly trafficked to the intestinal brush border. Ussing chambers will be used to elucidate the function of duodenal intestinal epithelial ion transporters. We expect that apical ion transporter expression and function are both decreased in enterocytes from mouse models of MVID. In Specific Aim 2 I will determine the effects of loss of MYO5B in vitro using human and mouse derived enteroids. This in vitro approach will examine the expression and localization of apical membrane proteins in both mouse and human enteroids. Additionally, a functional assay to determine the transport of Na+ across the apical membrane of enterocytes will also be performed. Enteroids generated from neonatal and adult VilCreERT2;MYO5Bflx/flx mice will be treated with 4-hydroxy-tamoxifen to induce deletion of MYO5B in vitro. The presence and formation of microvillus inclusions in these enteroids and in enteroids derived from a human with MVID will then be assessed with live cell imaging using the SiR-actin (silicon-rhodamine) fluorogenic probe. At the completion of these studies we expect to have elucidated the role of MYO5B in the regulation of intestinal epithelial apical ion transport.