Cellular and Structural Changes in the Small Intestinal Epithelium After Knockout of MAMDC4 an Endosomal Membrane Protein

Abstract

MAMDC4 is an endosomal transmembrane protein that is highly expressed during development of the intestine and continues to be expressed at lower levels in the adult. MAMDC4 is required for membrane trafficking and enterocyte morphogenesis during development. However, the function of MAMDC4 in adult intestine is unknown. We performed intestine-specific-inducible knockout of MAMDC4 in adult intestine and examined the effects on various cellular and subcellular structures related to intestinal health and disease. MAMDC4 knockout mice have a decreased weight and shorter intestine length. MAMDC4 knockout mice also have both shorter villi and crypts when compared to control mice, consistent with previous findings showing the importance of MAMDC4 in proliferation of the small intestine. Multiple epithelial cell types line the crypt-villus axis of the small intestine, including enterocytes and secretory cells. The secretory cells of the small intestine are an integral part of the maintenance and homeostasis of the intestinal epithelium and include Paneth cells, goblet cells and enteroendocrine cells. In addition, Paneth cell dysfunction has been correlated with an increased risk of Crohn’s Disease. Goblet cells secrete mucin and provide a protective gel-forming mucus layer that creates a physical barrier between the microbiome and the intestinal epithelium. While the intestinal barrier is dynamic, disruption of either of these cell types can lead to inflammation. To observe secretory granule morphology, Paneth Cells were labeled with antibodies against the granule protein lysozyme. MAMDC4 knockout results in diffuse lysozyme granule labeling, but no clear change in the number of Paneth cells. In addition, MAMDC4 knockout causes an increase in both number and individual cell area of goblet cells. Changes in Paneth cells and goblet cells are associated with changes in microbiome and changes in microbiota composition is known to contribute to inflammatory disease. We found that cecal beta diversity of the microbiome showed significant change after MAMDC4 knockout. A change in cecal beta diversity could be due to changes in AMP secretions from resident Paneth cells of the small intestine. While there are clear changes in secretory cell morphology, there are also changes in tight junction protein localization. Previous studies in developing intestine and in kidney epithelial cells in culture have shown defects in localization of tight junction proteins in the absence of MAMDC4. MAMDC4 is suggested to regulate trafficking of the polarity proteins involved in tight junctions. Importantly, there were also changes in mitochondrial morphology, which include fragmentation and rounded aspect which could be precursors to mitophagy. The absence of MAMDC4 could be affecting lysosome biogenesis and maturation in the mitophagy pathway. These results suggest that MAMDC4 plays a role in packaging of secretory granules and loss or dysfunction may result in increased inflammation and risk for development of Crohn’s disease.