Mechanisms of NAD+ action during muscle development and homeostasis in a zebrafish dystroglycanopathy model

斑马鱼肌聚糖病模型肌肉发育和稳态过程中 NAD 的作用机制

基本信息

批准号：
10450828
负责人：
Clarissa A Henry
金额：
$ 31.8万
依托单位：
UNIVERSITY OF MAINE ORONO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-02 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10450828
关键词：
Adhesions Architecture Biological Biology CRISPR/Cas technology Cell Adhesion Cell membrane Cells Chemosensitization Code Comparative Study Data Deposition Development Disease Dominant-Negative Mutation Dystroglycan ECM receptor Elastases Elastin Extracellular Matrix Extracellular Matrix Proteins Foundations Genes Goals Grant Guanosine Diphosphate Mannose Health Homeostasis ITGA7 gene Lead Light Mediating Microscopy Mitochondria Modeling Muscle Muscle Cells Muscle Development Muscular Dystrophies Mutate Mutation Myopathy Natural regeneration Nuclear Phenotype Play Post-Translational Protein Processing Reaction Regulation Resolution Role Sarcolemma Signal Transduction Stress Testing Untranslated RNA Up-Regulation Zebrafish congenital muscular dystrophy dystroglycanopathy experimental study glycosylation improved in vivo insight muscle degeneration muscular structure mutant network models prevent receptor resilience

项目摘要

ABSTRACT Congenital muscular dystrophies (CMDs) are progressive debilitating diseases without cures. Many CMDs disrupt the adhesion of muscle cells to their surrounding extracellular matrix (ECM). Muscle-ECM adhesion is critical for muscle development, homeostasis, regeneration, and resilience to stress. Mutations in genes that modulate muscle-ECM adhesion frequently lead to CMDs. For example, Dystroglycan (DG) and Integrin alpha7 (Itga7) are transmembrane ECM receptors that, when mutated, result in CMDs. Whether and/or how these transmembrane receptors interact during muscle development/homeostasis is not known. In addition, the roles that post-translational modification of DG plays in modulating both the ECM proper and muscle-ECM adhesion are not known. We previously found that exogenous NAD+ potentiates ECM deposition and that NAD+ improves dystrophic phenotypes in zebrafish lacking either DG or Itga7. The basic cell biological mechanisms that underlie NAD+-mediated improvement in muscle-ECM adhesion are not well understood. Our long-term goal is to understand how signaling between muscle cells and their ECM mediates muscle health. Secondary Dystroglycanopathies are a subset of CMDs that result from mutations in genes that are necessary for glycosylation of DG, which is necessary for muscle-ECM adhesion. GDP-mannose, synthesized by GMPPB, is essential for glycosylation reactions. Mutations in GMPPB result in GMPPB-associated Dystroglycanopathy. Preliminary data show that muscle development, homeostasis, and regeneration are disrupted in gmppb mutants. In contrast to our previous data showing NAD+ improves ECM deposition in dg- deficient zebrafish, preliminary data show that NAD+ does not improve muscle structure in gmppb mutants. In this grant, we will compare and contrast the mechanisms underlying the effects of DG glycosylation and NAD+ on muscle development, homeostasis, and regeneration. Our central hypothesis is that both NAD+ and gmppb regulate muscle cell adhesion by altering sarcolemma architecture and ECM organization. In Aim 1 we will test the hypothesis that NAD+ increases cell adhesion in DG mutant zebrafish by increasing Itga7 clustering; and that hypoglycosylated DG disrupts sarcolemma architecture and prevents NAD+-mediated Itga7 clustering and increased cell adhesion. We will do this with a combination of longitudinal light sheet microscopy studies and super-resolution microscopy. In Aim 2 we will identify new muscle cell adhesion regulators through comparative studies of dysregulated muscle development in three zebrafish models of muscular dystrophy. We will take an unbiased approach to identify ECM regulatory nodes by using network modeling and network resilience analysis of co-expressed coding and non-coding genes. Completion of this grant will provide new insight into how cell-ECM adhesion mediates muscle development and homeostasis in vertebrate models of CMDs. These basic in vivo cell biological studies are crucial to provide a foundational understanding of the interplay between transmembrane receptors, ECM regulation, and cell adhesion.

抽象的先天性肌肉营养不良（CMDS）是无治疗方法的渐进衰弱疾病。许多CMD 破坏肌肉细胞对周围细胞外基质（ECM）的粘附。肌肉-ECM粘附是对于肌肉发育，体内平衡，再生和对压力的韧性至关重要。基因突变调节肌肉ECM粘附经常导致CMD。例如，dystroglycan（DG）和整联蛋白 α7（itga7）是跨膜ECM受体，在突变后会导致CMD。是否和/或如何这些跨膜受体在肌肉发育/稳态期间相互作用。另外， DG的翻译后修饰的角色在调节ECM和肌肉ECM中粘附尚不清楚。我们以前发现外源性NAD+增强ECM沉积，并且 NAD+改善了缺乏DG或ITGA7的斑马鱼中营养不良的表型。基本细胞生物学 NAD+介导的肌肉ECM粘附的改善的机制尚不清楚。我们的长期目标是了解肌肉细胞与其ECM之间的信号如何介导肌肉健康。次生肿瘤肿瘤病是由基因突变引起的CMD的子集 DG糖基所必需的，这对于肌肉ECM粘附是必需的。 GDP甘露糖，合成通过GMPPB，对于糖基化反应至关重要。 GMPPB突变导致GMPPB相关 dystroglycanopathy。初步数据表明，肌肉发育，稳态和再生是在GMPPB突变体中破坏。与我们以前的数据相反，NAD+改善了DG-的ECM沉积缺乏斑马鱼的初步数据表明，NAD+不能改善GMPPB突变体的肌肉结构。在这笔赠款，我们将比较和对比DG糖基化和NAD+的作用的基础机制关于肌肉发育，稳态和再生。我们的中心假设是NAD+和GMPPB 通过改变肌体系结构和ECM组织来调节肌肉细胞的粘附。在AIM 1中，我们将测试 NAD+通过增加ITGA7聚类来增加DG突变体斑马鱼中细胞粘附的假设；和该降解糖基化的DG破坏了肌膜结构，并防止NAD+介导的ITGA7聚类和细胞粘附增加。我们将通过纵向轻度显微镜研究和超分辨率显微镜。在AIM 2中，我们将通过在肌肉营养不良的三种斑马鱼模型中，肌肉发育失调的比较研究。我们将采用一种公正的方法来通过使用网络建模和网络来识别ECM调节节点共表达编码和非编码基因的弹性分析。这笔赠款的完成将提供新的深入了解细胞-ECM粘附如何介导肌肉发育和稳态的脊椎动物模型 CMD。这些基本的体内细胞生物学研究对于提供对跨膜受体，ECM调节和细胞粘附之间的相互作用。