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

• 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
• 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

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向细胞的运输至关重要。 上皮细胞的顶膜。 MYO5B 突变失活会导致先天性腹泻病， 微绒毛包涵体病（MVID），导致危及生命的腹泻和吸收不良。在MYO5B中 敲除小鼠以及 MVID 患者肠道中介导营养和水分吸收的顶端蛋白 错误定位远离肠上皮细胞的刷状缘。我们最近发现一种具有生物活性的 磷脂、溶血磷脂酸 (LPA)，可以促进微绒毛成熟并使微绒毛定位正常化 钠依赖性葡萄糖协同转运蛋白 1 和钠/质子交换器 (NHE)3，重要的顶端钠 在 MYO5B 敲除组织和类器官中促进水吸收的转运蛋白。然而，LPA 注射并没有显着改善条件性 MYO5B 敲除引起的小鼠体重减轻。我们 假设天然 LPA 的低溶解度和快速降解限制了足够剂量的递送 改善体内肠道缺陷，并且强效 LPA 受体 (LPAR) 激动剂更有效。我们 合成了 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 MYO5B 敲除小鼠的绒毛/隐窝比率和顶端 NHE3 定位。 我们预计 LPAR5 激活可以刺激肠上皮细胞分化和顶膜运输 绕过 MYO5B 功能丧失引起的封锁，从而改善微绒毛和绒毛 结构、营养转运蛋白定位和营养吸收。首先，我们将评估治疗效果 LPAR5 激动剂治疗对具有失活 MYO5B 突变的小鼠上皮细胞功能的潜力。在 除了MYO5B缺失模型外，我们还将评估Compound-1对具有G519R点的小鼠的影响 MYO5B 突变（在严重 MVID 患者中发现）。除了营养物质的误贩运之外 转运蛋白，我们发现功能性 MYO5B 缺失会导致细胞谱系分化缺陷。 MYO5B 基因敲除小鼠表现出潘氏细胞数量增加以及过度增殖，而感觉簇细胞 减少了 80%。 LPA 治疗逆转了 MYO5B 敲除小鼠中簇细胞的减少，表明 LPA 信号传导增强适当的细胞分化。其次，为了指定祖细胞中的 MYO5B 功能， Myo5bflox/flox 小鼠与 Lrig1-CreERT2 小鼠杂交，观察对上皮增殖和 将在有或没有化合物-1治疗的情况下表征分化。三、要了解 过度增殖和分化缺陷背后的机制，我们将确定 LPAR5 激活前后 MYO5B 缺陷小鼠肠道的细胞代谢途径。我们将 利用成像质谱技术提供原位空间和定量代谢组学数据。 使用强效可溶性 LPAR5 激动剂治疗吸收不良可能提供更安全的替代方案 对于患有 MVID 以及一般腹泻症状的儿童进行移植或终身 TPN。