Impacting the pathophysiology of malabsorption induced by Myosin Vb inactivating mutations

摘要 肌球蛋白VB(Myosin VB，MYO5B)是一种运动蛋白，对细胞极化和蛋白质向 上皮细胞的顶膜。失活的MYO5B突变会导致先天性腹泻， 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 基因敲除小鼠以及MVID患者肠道中介导营养和水分吸收的顶端蛋白 错误地定位于远离肠上皮细胞刷状边缘的位置。我们最近发现一种生物活性物质 磷脂，溶血磷脂酸(LPA)，可促进微绒毛成熟，并使定位正常化。 钠依赖的葡萄糖共转运体1和钠/质子交换器(NHE)3，重要的顶端钠 促进水分吸收的转运蛋白，在MYO5B基因敲除组织和有机物中都是如此。然而，LPA 注射没有显著改善条件性MYO5B基因敲除引起的小鼠体重下降。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传递 改善体内的肠道缺陷，而有效的LPA受体(LPAR)激动剂更有效。我们 合成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显著改善了MYO5B基因敲除小鼠的绒毛/隐窝比率和顶端NHE3的定位。 我们预计LPAR5的激活可以刺激肠细胞分化和顶膜转运 绕过MYO5B功能丧失导致的阻塞，导致微绒毛和绒毛的改善 结构、营养转运体定位和营养吸收。首先，我们将评估治疗 LPAR5激动剂治疗对失活MYO5B突变小鼠上皮细胞功能的影响。在……里面 除了MYO5B缺失模型外，我们还将评估化合物-1对G519R点小鼠的影响 MYO5B基因突变(在一例重症MVID患者中发现)。除了营养物质的非法运输 我们发现功能性MYO5B缺失会导致细胞谱系分化缺陷。MYO5B 基因敲除的小鼠表现出潘氏细胞的数量增加和过度增殖，而感觉簇状细胞 减少了80%。LPA处理逆转了MYO5B基因敲除小鼠的簇状细胞减少，这表明 LPA信号促进适当的细胞分化。其次，为了确定MYO5B在祖细胞中的功能， Myo5bFLOX/FLOX小鼠将与Lrig1-CreERT2小鼠杂交，并观察其对上皮细胞增殖和 无论有没有化合物-1治疗，分化的特征都将表现出来。第三，要理解 过度增殖和分化缺陷的基础机制，我们将确定 LPAR5激活前后MYO5B缺陷小鼠肠道细胞代谢途径的研究我们会 利用成像质谱学技术现场提供空间和定量代谢组学数据。 使用有效和可溶的LPAR5激动剂作为吸收不良的治疗可能提供一种更安全的替代方案 对于患有MVID和一般腹泻症状的儿童，可以进行移植或终身TPN。

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.