Byron C. Knowles

Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease

Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea.

Rab11a regulates syntaxin 3 localization and microvillus assembly in enterocytes.

Rab11a is a key component of the apical recycling endosome that aids in the trafficking of proteins to the luminal surface in polarized epithelial cells. Utilizing conditional Rab11a‐knockout specific to intestinal epithelial cells, and human colonic epithelial CaCo2‐BBE cells with stable Rab11a knockdown, we examined the molecular and pathological impact of Rab11a deficiency on the establishment of apical cell polarity and microvillus morphogenesis. We demonstrate that loss of Rab11a induced alterations in enterocyte polarity, shortened microvillar length and affected the formation of microvilli along the lateral membranes. Rab11a deficiency in enterocytes altered the apical localization of syntaxin 3. These data affirm the role of Rab11a in apical membrane trafficking and the maintenance of apical microvilli in enterocytes.

TLR sorting by Rab11 endosomes maintains intestinal epithelial‐microbial homeostasis

Compartmentalization of Toll‐like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. Here we provide genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a, a gene adjacent to a Crohns disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell‐intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild‐type controls, germ‐free Rab11a‐deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host‐microbial homeostasis at least partially via sorting TLRs.

Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells.

Ser-227 phosphorylation of Rab11-FIP2 by Par1b/MARK2 regulates the establishment of polarized epithelial monolayers in three-dimensional MDCK cell cultures and has an ongoing influence on the composition of both adherens and tight junctions in polarized epithelial cells.

Rab25 regulates integrin expression in polarized colonic epithelial cells

Rab25 is a tumor suppressor in the colon, but the mechanisms underlying the influence of Rab25 on polarity are unknown. Findings on changes in polarity in Caco2-BBE cells with knockdown and rescue of Rab25 expression indicate that Rab25 regulates integrin gene expression mediated by ETV4.

Loss of MYO5B in Mice Recapitulates Microvillus Inclusion Disease and Reveals an Apical Trafficking Pathway Distinct to Neonatal Duodenum

Background & Aims Inactivating mutations in myosin Vb (MYO5B) cause severe neonatal diarrhea in microvillus inclusion disease. Loss of active MYO5B causes the formation of pathognomonic inclusions and aberrations in brush-border enzymes. Methods We developed 3 mouse models of germline, constitutively intestinal targeted, and inducible intestinal targeted deletion of MYO5B. The mice were evaluated for enterocyte cellular morphology. Results Germline MYO5B knockout mice showed early diarrhea and failure to thrive with evident microvillus inclusions and loss of apical transporters in the duodenum. IgG was present within inclusions. Apical transporters were lost and inclusions were present in the duodenum, but were nearly absent in the ileum. VillinCre;MYO5BF/F mice showed similar pathology and morphologic changes in duodenal enterocytes. In contrast, when MYO5B KO was induced with tamoxifen treatment at 8 weeks of age, VillinCreERT2;MYO5BF/F mice developed severe diarrhea with loss of duodenal brush-border enzymes, but few inclusions were observed in enterocytes. However, if tamoxifen was administered to 2-day-old VillinCreERT2;MYO5BF/F mice, prominent microvillus inclusions were observed. Conclusions The microvillus inclusions that develop after MYO5B loss show the presence of an unrecognized apical membrane trafficking pathway in neonatal duodenal enterocytes. However, the diarrheal pathology after MYO5B loss is caused by deficits in transporter presentation at the apical membrane in duodenal enterocytes.

Apical vesicle trafficking takes center stage in neonatal enteropathies.

icrovillus inclusion disease (MVID) was initially Mcharacterized in 1978 in 5 infants experiencing chronic, unremitting, secretory diarrhea. Studies of initial patient biopsy samples from the small intestine revealed profound villus atrophy, crypt hypoplasia, absence of microvilli, malabsorption of glucose, and defective sodium transport. In these MVID patients feeding was discontinued and total parenteral nutrition was started, but secretory diarrhea persisted. Later investigations revealed that the pathognomonic microvillus inclusions were observed in 10% of enterocytes, and these inclusions contained apical enterocyte proteins such as sucrase isomaltase, alkaline phosphatase, and sodium hydrogen exchanger 3. Moreover, membrane proteins that normally traffic to the apical domain were mislocalized to a subapical compartment, whereas sodium potassium ATPase (Na/K-ATPase) basolateral localization was unaffected in the biopsy samples. The causative agent of this disease remained obscure until 2007 when Sato et al demonstrated that Rab8a knockout mice displayed phenotypic similarities to MVID, suggesting that defects in trafficking to the apical domain may underpin this disease. However, no mutations in Rab8a were identified in humans. Concurrently, it was demonstrated that MYO5B regulates intracellular trafficking and endocytic recycling by localizing with specific Rab small GTPases (Rab8a, Rab10, and Rab11) in subapical vesicles. These 2 studies taken together culminated in 2008 with reports from Europe and the United States that mutations in the Myosin Vb (MYO5B) gene cause MVID. However, in the original report from Europe, there was 1 MVID patient without any MYO5B mutation. Since then, 41 published mutations in the MYO5B gene have been identified in MVID patients. In this issue of Gastroenterology, Wiegerinck et al identify patients with mutations in syntaxin-3 (STX3) that also give rise to MVID-like phenotypes. The brief report by Wiegerinck et al describes 2 cases involving toddlers who present with watery diarrhea and severe metabolic acidosis. Interestingly, both of these children developed increased tolerance for enteral feeding, but still required partial total parenteral nutrition and sodium bicarbonate supplementation. Biopsy samples from these “atypical” MVID patients revealed accumulation of periodic acid-Schiff–positive vesicles subapically, microvillus inclusions, and lateral lumens with microvilli via histology and transmission electron microscopy. Whole exome sequencing revealed that mutations in STX3, rather than MYO5B, were the underlying cause of “atypical” MVID in these cases. Stable expression of mutant STX3 in CaCo2 cells and organoid cultures derived from crypts of 1 patient recapitulated the “atypical” MVID phenotype. Most interestingly, the clinical signs and treatment used for these 2 patients differ significantly from MVID, and support the classification of these patients into a distinct but related class of enteropathy (Figure 1A). Wiegerinck et al have provided novel insights into the pathophysiology of neonatal enteropathies by supporting the likely role for aberrations in apical trafficking. Both Rab8a and Rab11a bind to MYO5B directly on recycling vesicles, and MYO5B most likely functions as a dynamic tether for both Rab8a and Rab11a and normally maintains these proteins at their appropriate subapical membrane location. MYO5B interaction with Rab8a promotes the initiation of microvilli growth, and MYO5B interaction with Rab11a is required for apical recycling of membranes internalized through apical macropinocytosis and also facilitates the maintenance of microvilli. Thus, MYO5B in concert with Rab8a and Rab11a regulates the polarity of intestinal epithelial cells, and uncoupling of these Rabs from MYO5B causes gross changes in cellular polarity and mucosal integrity in patients with MVID. These results together with previous work demonstrated that MYO5B interacts with Rab8a and Rab11a to regulate enterocyte polarity, apical trafficking, and microvilli growth. Rab11a regulates actin dynamics, apical trafficking, and recycles the apical brush border contents from macropinosomes in a MYO5B-dependent manner. Cdc42 also associates with both Rab8aand Rab11a-positive vesicles, and is activated via Rab8a through Tuba, a Cdc42 GEF, to facilitate regulation of cellular polarity and actin polymerization. Rab11a-positive vesicles utilize STX3, an apically targeted t-SNARE, to facilitate vesicle fusion of apically trafficked proteins to the plasma membrane. The process of vesicular fusion to the plasma membrane is likely the last step in Rab11a-dependent apical trafficking in enterocytes. Interestingly, patients with mutations in the STX3 binding protein STXBP2/Munc18-2, after treatment of their primary disease, have persistent chronic diarrhea, loss of microvilli, and histologic findings consistent with the “atypical” MVID phenotype described in the present report. All of these processes in concert aid in the formation and maintenance of apical surface and microvilli (Figure 1B). It is not clear from the present report how STX3 mutations affect the full range of apical trafficking events and epithelial polarity. Further studies will be required to compare the deficits present in STX3 mutants compared with MYO5B mutants and how MYO5B mutations may impact STX3 function in typical MVID patients.

Apical Membrane Alterations in Non-intestinal Organs in Microvillus Inclusion Disease

ObjectivesMicrovillus inclusion disease (MVID) is a severe form of neonatal diarrhea, caused mainly by mutations in MYO5B. Inactivating mutations in MYO5B causes depolarization of enterocytes in the small intestine, which gives rise to chronic, unremitting secretory diarrhea. While the pathology of the small intestine in MVID patients is well described, little is known about extraintestinal effects of MYO5B mutation.MethodsWe examined stomach, liver, pancreas, colon, and kidney in Navajo MVID patients, who share a single homozygous MYO5B-P660L (1979C>T p.Pro660Leu, exon 16). Sections were stained for markers of the apical membrane to assess polarized trafficking.ResultsNavajo MVID patients showed notable changes in H/K-ATPase-containing tubulovesicle structure in the stomach parietal cells. Colonic mucosa was morphologically normal, but did show losses in apical ezrin and Syntaxin 3. Hepatocytes in the MVID patients displayed aberrant canalicular expression of the essential transporters MRP2 and BSEP. The pancreas showed small fragmented islets and a decrease in apical ezrin in pancreatic ducts. Kidney showed normal primary cilia.ConclusionsThese findings indicate that the effects of the P660L mutation in MYO5B in Navajo MVID patients are not limited to the small intestine, but that certain tissues may be able to compensate functionally for alterations in apical trafficking.

176 Loss of Functional MYO5B in CaCo2-BBe Cells and Navajo MVID Patient Enterocytes Leads to Loss of Polarity and Deficits in Maintenance of Microvilli

Myosin Vb (MYO5B) is an actin based motor that localizes specific Rab small GTPases (Rab8a, Rab10, Rab11, and Rab25) to sub-apical domains in polarized epithelial cells. Microvillus Inclusion Disease (MVID) represents a pathophysiologic window into the apical trafficking process, because it arises as a result of inactivating mutations in MYO5B that leads to the loss of microvilli in intestinal enterocytes and chronic unremitting diarrhea in the affected newborns. Understanding how mutations in MYO5B lead to aberrant enterocyte phenotype will provide novel insights into the fundamental mechanisms governing apical recycling system trafficking, microvilli formation, and MVID. We hypothesize that MYO5B plays a crucial role in apical trafficking and polarization in enterocytes, and that defective MYO5B in MVID patients mislocalizes Rab small GTPases leading to disruption in apical transport and loss of polarity in enterocytes. To test this hypothesis we have established cellular models of aberrant enterocyte apical trafficking by stably knocking down MYO5B in CaCo2-BBE cells. The cells grown on permeable filters for 15 days were analyzed using immunostaining, Scanning EM, and Transmission EM to examine markers of apical and basolateral polarity, intracellular trafficking, and the establishment of apical microvilli. MYO5B KD knockdown (KD) caused a loss of microvilli and dispersal of Rab8a and Rab11acontaining vesicles from their usual subapical distribution to diffusely cytoplasmic in the cells. MYO5B KD elicited an increase in claudin-2 and a decrease in claudin-1 expression as well as a decrease in lateral membrane staining for p120-catenin (p120). MYO5B KD also caused a loss of apical active GTP-bound cdc42 in the cells. Rescue of theMYO5B KDwith synthetic MYO5B wild type elicited recovery of normal microvilli. However, re-expression of MYO5B(P660L), the Navajo MVID mutation, failed to rescue and in addition caused the formation of microvillus inclusions. Navajo MVID MYO5B(P660L) patient samples demonstrated a loss of p120 and a pseudostratified columnar epithelium at the villus tips where ezrin-staining microvillus inclusions were most predominant. Patient biopsies also showed aberrant localization of MYO5B, Rab8a, Rab11a, Rab11-FIP2, DPPIV, and Ezrin. Rab11a and MYO5B localized around microvillus inclusions, while Rab8a staining was diffuse. Importantly, while a severe loss of brush border was observed in cells at the tips of microvilli, the cells in the mid-villus appeared to have a preserved normal brush border. Our results suggest that MYO5B regulates the polarity of intestinal epithelial cells, thereby maintaining the functional brush border. The normal brush border seen in the proximal villi in the Navajo MVID patients suggests that the pathogenesis of MVID lies in a loss of polarity in enterocytes at the tips of intestinal villi.