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Link Between a Ubiquitous Motor Protein and Intestinal Health

By: Carol A. Rouzer, VICB Communications
Published:  September 17, 2018


New studies reveal how abnormal distribution of key intestinal transport proteins leads to severe diarrhea in patients carrying a mutation of the gene for myosin 5B


Myosin 5B (MYO5B) is a ubiquitously expressed motor protein that is involved in transporting endosomal vesicles along the actin cytoskeleton. Some mutations of the gene encoding MYO5B cause microvillus inclusion disease (MVID), characterized by severe life-threatening watery diarrhea that begins shortly after birth. Patients born with MVID must receive all of their nutrition intravenously, a process referred to as total parenteral nutrition (TPN). TPN is associated with a high risk of infection and metabolic imbalances that can exacerbate the illness. The only other alternative is an intestinal transplant. The fact that TPN leads to degenerative changes in the intestinal tract has made it difficult for researchers to clearly identify the mechanism of the diarrhea in MVID. However, the availability of genetically engineered mouse models of the disease has now enabled studies of MVID pathogenesis in the absence of TPN-induced changes. The cells lining the intestines of MVID model mice exhibit many of the hallmarks of the human disease, including blunted microvilli (Figure 1), inclusions beneath the apical membrane (Figure 2), excessive staining with periodic acid Schiff reagent, and displacement of proteins normally found in the apical membrane to subapical regions and/or inclusions (Figure 3). Now, Vanderbilt Institute of Chemical Biology member James Goldenring, his laboratory, and colleagues in Houston, TX, Phoenix, AZ, New Haven, CT, Chicago, IL, Insbruck Austria, and Würzburg, Germany report the use of MYO5B knockout mouse models to determine how key intestinal ion and solute transporters contribute to the diarrhea observed in patients [A. C. Engevik, et al., (2018) Gastroenterology, published online Aug. 22, DOI: 10.1053/j.gastro.2018.08/025].



Link Between a Ubiquitous Motor Protein and Intestinal Health_image1

FIGURE 1. Electron micographs showing the normal microvilli forming the brush border in enterocytes from wild-type (WT) mice and the stunted, poorly formed microvilli in the brush border of enterocytes from MYOB5 KO mice. Note the presence of multiple tubulovesicular elements below the apical membrane in the enterocytes from KO mice. Image adapted from V. Weis, et al., (2016) Cell Mol Gastroenterol Hepatol. 2:131-157.



FIGURE 2. Electron micrograph showing inclusion-containing microvilli observed in an enterocyte from an MYO5B KO mouse. Image adapted from V. Weis, et al., (2016) Cell Mol Gastroenterol Hepatol. 2:131-157.



FIGURE 3. Micrographs of the intestinal mucosa from wild-type (Control) and MYO5B KO mice stained for phospho-ERM (P-ERM, indicative of phospho-ezrin, a protein normally found in the apical membrane) and p120 catenin (a protein found in cell-cell junctions) (upper panels) or alkaline phosphatase (an apical membrane protein) (lower panels). All images are stained with DAPI to show nuclei).  Higher magnification insets are shown at right.  Arrows indicate the presence of microvillus inclusions in MYO5B KO enterocytes.



Watery diarrhea is most often caused by failure to absorb solutes properly and/or the secretion of solutes and water across the intestinal epithelium into the lumen. Patients with MVID exhibit abnormal sodium and glucose absorption, suggesting potential malfunctions of the sodium hydrogen exchanger isoform 3 (NHE3) and/or the sodium glucose co-transporter (SGLT1). Chloride ion absorption is dependent on the down-regulated in adenoma (DRA) transporter, whereas the cystic fibrosis transmembrane conductance regulator (CFTR) is the protein most frequently involved in excessive active secretion of chloride ions in diarrheal illnesses. In addition, aquaporins, channels that transport water and small uncharged solutes, can also contribute to diarrhea. The researchers hypothesized that the localization or function of some or all of these proteins could be altered by MYOB5 deficiency and that this might explain the mechanism of the diarrhea observed in MVID.

The researchers began to test their hypothesis using enterocytes from the intestines in mice bearing a germline deletion of the gene encoding MYOB5 (MYOB5 KO mice). As expected, these cells exhibited typical abnormalities observed in enterocytes from MVID patients, including an accumulation of subapical inclusions along with disorganization and abnormal dispersion of lysosomes. Apical proteins, such as phosphorylated ezrin (P-ERM), intestinal alkaline phosphatase, and DPPIV, were mislocalized to the subapical region and present in inclusions (Figure 3).

A specific search for transport proteins of interest revealed that both NHE3 and SGLT1, which are normally expressed in the apical membrane, exhibited reduced apical expression and mislocalization to the cytosol and/or inclusions. The aquaporin AQP7 was similarly mislocalized. In contrast, CFTR was present in the brush border of both wild-type and MYOB5 KO cells in similar quantities, though some of the protein was also visualized in inclusions in the KO enterocytes. These findings suggested that transporter mislocalization would likely result in poor absorption of sodium and glucose with no reduction in chloride secretion. Both of these factors would contribute to the generation of diarrhea.

To further understand the contribution of CFTR to diarrhea in MVID, the researchers measured chloride levels in the intestines of MYOB5 KO and wild-type mice. Levels of the ion were higher in the duodenum of KO mice than wild-type mice, but no significant differences were noted in other regions of the intestine. The researchers then created enteroid cultures from the duodenal crypts of wild-type and MYOB5 KO mice. Following differentiation of the enteroids by removal of Wnt from the culture medium, the investigators used forskolin to activate CFTR-mediated transport. In this assay, swelling of the enteroid results from transport of chloride and water across the apical membrane. The amount of swelling was similar in enteroids from wild-type and KO mice, suggesting CFTR activity was unaffected by MYOB5 deficiency.

The researchers next investigated the effects of MYOB5 deficiency in a mouse model in which selective knockout of the gene in the intestine could be induced in otherwise normal adult animals (MYOB5 adult KO). Enterocytes from these mice exhibited many, though not all, of the abnormalities observed in those from the germline MYOB5 KO mice. Critically, however, in comparison to wild-type cells, expression of NHE3, SBLT1, and AQP7 was reduced in the apical membranes of MYOB5 adult KO enterocytes, and expression of DRA was totally lost. In contrast, expression of CFTR was retained or even increased following knockout of MYOB5 expression in the adult.

The investigators used Ussing chambers to measure the membrane properties of the intestinal epithelia of MYOB5 adult KO mice. There were no barrier defects in the jejunum or ileum in the KO mice, demonstrating that diarrhea was not the result of a fundamental mechanical abnormality. Of particular interest, however, was the finding that the greatest contributor to electrogenic ion transport in wild-type mice was SGLT1, whereas in MYOB5 adult KO mice, it was CFTR. These findings were consistent with the results of microscopic evaluations demonstrating abnormal expression and localization of SGLT1 and normal or increased expression of CFTR in the KO mice.

To determine the relevance of their findings to the human disease, the researchers examined intestinal tissues from MVID patients. They observed cytoplasmic mislocalization of NHE3 and SGLT1 along with total loss of AQP7 from the apical membrane. In contrast, expression and localization of CFTR was the same in tissues from MVID patients and those from normal controls.

The researchers concluded that MYOB5 deficiency leads to failure of proper localization of key transporters in enterocytes. The result is poor absorption of sodium, glucose, and water, all of which would contribute to the diarrhea observed in MVID. Exacerbating the problem is normal, or possibly increased function of CFTR, which would result in the secretion of chloride and more water into the intestinal lumen. These insights, gained first from studies of mouse models of MVID, appear to apply also to human patients and provide an important foundation for the better understanding of MYOB5's role in intestinal physiology.




View Gastroenterology article: Loss of MYO5B Leads to Reductions in Na+ Absorption with Maintenance of CFTR-dependent Cl-Secretion in Enterocytes





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