Deficits in Enterocyte Apical Transporters Associated with Loss of Myosin Vb

项目摘要 微绒毛包涵体病（MVID）是一种先天性腹泻，由以下基因失活突变引起： 肌球蛋白Vb（MYO 5 B）。所有MVID新生儿均接受全肠外营养（TPN）。TPN携带高 感染风险，20-30%接受TPN的患者报告败血症。1年生存率 新生儿MVID低于25%。目前MVID的唯一治疗选择是小肠 移植鉴于患有MVID的新生儿的预期寿命大大缩短， 治疗选择，了解MVID的病理学是一个重要的科学问题， 处理。这项研究建议的中心假设是MYO 5 B的缺失导致异常的 表达对营养物和水的吸收至关重要的关键顶端膜转运蛋白。我们 我们的假设基于已发表的报告，即MVID新生儿的活检组织存在心尖钠丢失， 葡萄糖转运蛋白（SGLT 1），囊性纤维化跨膜传导调节因子的亚顶端分布 （CFTR）和钠氢交换器（NHE 3）。此外，我们最近的小鼠研究 实验室已经报道了NHE 3弥漫性根尖下定位，伴有MYO 5 B的丢失。小鼠的发育 MYO 5 B的缺失为评价肠上皮细胞组成和功能的缺陷提供了新的模型。我 提出使用表达种系、组成型肠靶向和诱导型的小鼠模型 肠道靶向缺失MYO 5 B以及人肠样组织，以解决顶离子转运缺陷，其中MYO 5 B与人肠样组织一起沿着缺失 十二指肠来申请奖学金在具体目标1中，我将定义肠道的异常生理学， MVID小鼠模型中的顶端转运蛋白。将检查肠上皮细胞顶端离子转运蛋白的表达 在所有MVID小鼠模型中。将进行免疫荧光共聚焦显微镜检查，以确定 顶端离子转运蛋白是否被适当地运输到肠刷状缘。尤斯室 将用于阐明十二指肠上皮离子转运蛋白的功能。我们认为顶端离子 在MVID小鼠模型的肠上皮细胞中，转运蛋白的表达和功能均降低。在 特异性目的2 I将使用人和小鼠来源的肠样物质在体外确定MYO 5 B缺失的影响。 这种体外方法将检查两种小鼠中顶端膜蛋白的表达和定位， 和人肠上皮细胞。此外，还进行了一项功能测定来确定Na+穿过顶端的转运 肠上皮细胞膜也将进行。新生儿和成人产生的肠虫 VilCreERT 2; MYO 5 Bflx/flx小鼠将用4-羟基-他莫昔芬处理以在体外诱导MYO 5 B的缺失。的 在这些肠样组织和来源于人的肠样组织中存在和形成微绒毛内含物， 然后使用SiR-肌动蛋白（硅-罗丹明）荧光探针通过活细胞成像评估MVID。在 这些研究的完成，我们希望已经阐明MYO 5 B在肠道调节中的作用， 上皮顶端离子转运

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.