Impacting the pathophysiology of malabsorption induced by Myosin Vb inactivating mutations

影响肌球蛋白 Vb 失活突变引起的吸收不良的病理生理学

基本信息

批准号：
10365488
负责人：
Izumi Kaji
金额：
$ 38.06万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-25 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10365488
关键词：
Address Agonist Alkaline Phosphatase Apical Biological Assay Body Weight decreased Brush Border Bypass Cell Differentiation process Cell Lineage Cell physiology Cells Characteristics Child Childhood Chlorides Data Defect Diarrhea Disease Electrophysiology (science)Energy Metabolism Pathway Enterocytes Epithelial Epithelial Cells Experimental Models Family suidae Functional disorder Genetic Engineering Glucose Image In Situ Injections Intestinal Mucosa Intestines Knock-out Knockout Mice Life Lipids Liquid substance Lysophosphatidic Acid Receptors Malabsorption Syndromes Mass Spectrum Analysis Mediating Membrane Proteins Metabolic Metabolic Pathway Metabolism Microvillus inclusion disease Modeling Motor Mus Mutation Myosin ATPase Navajo Nonmuscle Myosin Type IIA Nutrient Oligo-1,6-Glucosidase Organoids Paneth Cells Pathway interactions Patients Pattern Pharmaceutical Preparations Phospholipids Point Mutation Population Proteins Protons Receptor Activation Reporting Sensory Signal Transduction Sodium Solubility Structure Sucrose Symptoms Techniques Therapeutic Tissues To specify Transgenic Mice Transplantation Villus Water absorption apical membrane cellular microvillus diarrheal disease dosage drug efficacy fatty acid oxidation gastrointestinal transplantation improved in vivo intestinal crypt intestinal epithelium ketogenesis lipid biosynthesis lysophosphatidic acid metabolomics mouse model mutant nutrient absorption nutrition polarized cell protein transport self-renewal stem stem cell function stem cells therapeutic target therapy development trafficking transcriptome sequencing

项目摘要

Abstract Myosin Vb (MYO5B) is a motor protein that is critical for cell polarization and protein trafficking towards the apical membrane in epithelial cells. Inactivating MYO5B mutations cause the congenital diarrheal disease, microvillus inclusion disease (MVID), which leads to life-threatening diarrhea and malabsorption. In MYO5B knockout mice, as well as in MVID patient intestines, apical proteins that mediate nutrient and water absorption are mis-localized away from the brush border of intestinal epithelial cells. We recently found that a bioactive phospholipid, lysophosphatidic acid (LPA), can promote microvillus maturation and normalize localization of sodium-dependent glucose cotransporter 1 and sodium/proton exchanger (NHE)3, important apical sodium transporters that promote water absorption, both in MYO5B knockout tissues and organoids. However, LPA injection did not significantly improve body weight loss induced by conditional MYO5B knockout in mice. We hypothesize that the low solubility and fast degradation of natural LPA limit delivery of a sufficient dosage to ameliorate intestinal deficits in vivo, and that potent LPA receptor (LPAR) agonists are more efficient. We synthesized selective agonists for LPAR1 and LPAR5. Our preliminary data indicate that the LPAR5 agonist, Compound-1, significantly improved villus/crypt ratios and apical NHE3 localization in MYO5B knockout mice. We anticipate that LPAR5 activation can stimulate enterocyte differentiation and apical membrane trafficking that bypass the blockades induced by loss of MYO5B function, leading to improved microvillus and villus structure, nutrient transporter localization, and nutrient absorption. First, we will evaluate the therapeutic potential of LPAR5 agonist treatment on epithelial cell function in mice with inactivating MYO5B mutations. In addition to MYO5B deletion models, we will evaluate the effects of Compound-1 on mice with a G519R point mutation in MYO5B (identified in a severe MVID patient). In addition to the mis-trafficking of nutrient transporters, we have found that functional MYO5B loss induces cell lineage differentiation deficits. MYO5B knockout mice show increased numbers of Paneth cells along with hyperproliferation, while sensory tuft cells are reduced by 80%. LPA treatment reversed the tuft cell reductions in MYO5B knockout mice, suggesting that LPA signaling enhances proper cell differentiation. Second, to specify MYO5B function in progenitor cells, Myo5bflox/flox mice will be crossed with Lrig1-CreERT2 mice and the effects on epithelial proliferation and differentiation will be characterized with or without Compound-1 treatment. Third, to understand the mechanisms that underlie the hyperproliferation and differentiation deficits, we will determine the alterations in cellular metabolic pathways in MYO5B-deficient mouse intestine before and after LPAR5 activation. We will utilize imaging mass spectrometry techniques to provide spatial and quantitative metabolomics data in situ. The use of potent and soluble LPAR5 agonist as a treatment for malabsorption may provide a safer alternative to transplantation or life-long TPN in children with MVID as well as general diarrheal symptom.

抽象的肌球蛋白VB（MyO5B）是一种运动蛋白，对于细胞极化和蛋白质运输至关重要上皮细胞中的顶膜。灭活肌5b突变会导致先天性腹泻病，微绒毛膜疾病（MVID），导致威胁生命的腹泻和吸收不良。在myo5b中敲除小鼠以及MVID患者肠，介导营养和吸水的顶端蛋白将其定位远离肠上皮细胞的刷子边界。我们最近发现一种生物活性磷脂，溶物磷脂酸（LPA）可以促进微伏氏成熟并正常化的定位依赖钠的葡萄糖共转运蛋白1和钠/质子交换器（NHE）3，重要的根尖钠在Myo5b敲除组织和类器官中促进吸水的转运蛋白。但是，LPA 注射没有显着改善小鼠有条件的Myo5b敲除引起的体重减轻。我们假设自然LPA限制足够剂量的自然溶解度和快速降解至体内改善肠道缺陷，有效的LPA受体（LPAR）激动剂更有效。我们合成的LPAR1和LPAR5的选择性激动剂。我们的初步数据表明LPAR5激动剂，化合物1，显着改善了绒毛/隐窝比和Myo5b敲除小鼠中的顶端NHE3定位。我们预计LPAR5激活可以刺激肠球细胞分化和顶膜运输绕过损失Myo5b功能引起的封锁，导致微绒毛和绒毛的改善结构，营养转运蛋白定位和营养吸收。首先，我们将评估治疗性 LPAR5激动剂治疗在失活肌5b突变的小鼠上上皮细胞功能上的潜力。在除了MyO5B删除模型外，我们将评估化合物1对G519R点小鼠的影响 MyO5b突变（在严重的MVID患者中鉴定）。除了营养不足转运蛋白，我们发现功能性myo5b损失会诱导细胞谱系分化缺陷。 myo5b 敲除小鼠显示出泛池细胞的数量增加，而感觉簇细胞则显示减少80％。 LPA治疗逆转了Myo5b敲除小鼠的簇状细胞减少，表明 LPA信号传导增强了适当的细胞分化。其次，要在祖细胞中指定Myo5b功能， Myo5bflox/Flox小鼠将与LRIG1-CREERT2小鼠交叉，并对上皮增殖和影响有或没有化合物1处理的分化将被表征。第三，了解构成过度增殖和分化缺陷的机制，我们将确定变化 LPAR5激活之前和之后，肌5b缺乏小鼠肠中的细胞代谢途径。我们将利用成像质谱技术在原位提供空间和定量代谢组学数据。使用有效和可溶性LPAR5激动剂作为吸收不良的治疗可能会提供更安全的替代方案在患有MVID以及一般腹泻症状的儿童中移植或终身TPN。