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: 10673724
• 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/10673724
• 关键词: Acceleration; 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; Cytomegalovirus Infections; Data; Defect; Diarrhea; Disease; Electrophysiology (science); Energy Metabolism Pathway; Enterocytes; Epithelial Cells; Epithelium; Experimental Models; Family suidae; Functional disorder; Genetic Engineering; Glucose; Heterozygote; In Situ; Injections; Intestinal Mucosa; Intestines; Knock-out; Knockout Mice; Life; Lipids; Liquid substance; LoxP-flanked allele; Lysophosphatidic Acid Receptors; Malabsorption Syndromes; 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; Proliferating; Proteins; Protons; Receptor Activation; Reporting; Sensory; Signal Transduction; Sodium; Solubility; Specific qualifier value; Structure; Sucrose; Symptoms; Techniques; Therapeutic; Tissues; 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; mass spectrometric imaging; metabolomics; mouse model; mutant; nutrient absorption; nutrition; polarized cell; protein transport; self-renewal; stem cell function; stem cells; symporter; 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.

摘要 肌球蛋白Vb（MYO 5 B）是一种马达蛋白，其对于细胞极化和蛋白质运输朝向肌球蛋白B是关键的。 上皮细胞的顶膜。MYO 5 B突变失活导致先天性睾丸炎， 微绒毛包涵体病（MVID），其导致危及生命的腹泻和吸收不良。在MYO 5 B中 敲除小鼠以及MVID患者肠道中，介导营养和水吸收的顶端蛋白质 被错误地定位在远离肠上皮细胞刷状缘的位置。我们最近发现， 磷脂，溶血磷脂酸（LPA），可以促进微绒毛成熟和正常化的定位， 钠依赖性葡萄糖协同转运蛋白1和钠/质子交换剂（NHE）3，重要的顶端钠 在MYO 5 B敲除组织和类器官中促进水吸收的转运蛋白。然而，LPA 注射没有显著改善小鼠中由条件MYO 5 B敲除诱导的体重减轻。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，并且有效的LPA受体（LPAR）激动剂更有效。我们 合成了LPAR 1和LPAR 5的选择性激动剂。我们的初步数据表明，LPAR 5激动剂， 化合物-1显著改善MYO 5 B敲除小鼠中的绒毛/隐窝比率和顶端NHE 3定位。 我们预期，LPAR 5的激活可以刺激肠上皮细胞的分化和顶膜运输 绕过MYO 5 B功能丧失引起的阻塞，导致微绒毛和绒毛改善， 结构、营养转运蛋白定位和营养吸收。首先，我们将评估治疗 在具有失活MYO 5 B突变的小鼠中，LPAR 5激动剂治疗对上皮细胞功能的潜力。在 除了MYO 5 B缺失模型之外，我们将评估化合物-1对具有G519 R点的小鼠的作用 MYO 5 B突变（在重度MVID患者中发现）。除了营养物质的错误运输 在MYO 5 B转运蛋白中，我们发现功能性MYO 5 B缺失诱导细胞谱系分化缺陷。MYO5B 敲除小鼠显示潘氏细胞数量增加，沿着过度增殖，而感觉丛细胞 减少了80%。LPA治疗逆转了MYO 5 B基因敲除小鼠中簇状细胞的减少，这表明 LPA信号传导增强适当的细胞分化。第二，为了确定MYO 5 B在祖细胞中的功能， Myo 5 bflox/flox小鼠将与Lrig 1-CreERT 2小鼠杂交，并观察对上皮细胞增殖和 分化将在有或没有化合物-1处理的情况下表征。第三，了解 机制的基础上过度增殖和分化缺陷，我们将确定的变化， 在LPAR 5活化之前和之后MYO 5 B缺陷小鼠肠中的细胞代谢途径。我们将 利用成像质谱技术提供空间和定量代谢组学数据。 使用强效可溶性LPAR 5激动剂治疗吸收不良可能提供更安全的替代方案 对MVID伴全身消化道症状的患儿进行移植或终身TPN治疗。