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