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

• 批准号: 10241349
• 负责人: Clarissa A Henry
• 金额: $ 31.16万
• 依托单位: UNIVERSITY OF MAINE ORONO
• 依托单位国家: 美国
• 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/10241349
• 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

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.

摘要 先天性肌营养不良(CMD)是一种无法治愈的进行性衰弱疾病。许多CMD 破坏肌肉细胞与其周围的细胞外基质(ECM)的黏附。肌肉-ECM粘连是 对肌肉发育、动态平衡、再生和对压力的恢复至关重要。基因的突变 调节肌肉-ECM粘连常常导致CMDs。例如，营养不良多糖(DG)和整合素 Alpha7(Itga7)是跨膜ECM受体，当突变时，会导致CMDs。是否和/或如何 这些跨膜受体在肌肉发育/内稳态过程中相互作用，目前尚不清楚。此外， DG翻译后修饰在调节细胞外基质本身和肌肉细胞外基质中的作用 粘附性是未知的。我们先前发现，外源NAD+促进细胞外基质沉积，并且 NAD+改善缺乏DG或Itga7的斑马鱼的营养不良表型。基础细胞生物学 NAD+介导的改善肌肉-ECM粘连的机制尚不清楚。 我们的长期目标是了解肌肉细胞和它们的细胞外基质之间的信号如何调节肌肉 健康。继发性糖耐量减退症是由以下基因突变引起的CMD的一个子集 DG的糖基化是必需的，而DG是肌肉-ECM粘连所必需的。GDP-甘露糖，合成 GMPPB，是糖基化反应所必需的。GMPPB基因突变导致GMPPB相关 糖营养不良症。初步数据显示，肌肉发育、动态平衡和再生 在gmppb突变体中被干扰。与我们之前的数据显示，NAD+改善了DG中ECM的沉积- 在缺乏斑马鱼的情况下，初步数据表明，NAD+不能改善gmppb突变体的肌肉结构。在……里面 在这项授权中，我们将比较和对比DG糖基化和NAD+效应的机制 肌肉发育、动态平衡和再生。我们的中心假设是NAD+和gmppb 通过改变肌膜结构和细胞外基质组织来调节肌细胞黏附。在目标1中，我们将测试 假设NAD+通过增加Itga7聚集性来增加DG突变斑马鱼的细胞黏附；以及 这种低糖基化的DG破坏了肌膜结构，阻止了NAD+介导的Itga7聚集和 细胞粘附力增强。我们将结合纵向光片显微镜研究和 超分辨率显微镜。在目标2中，我们将通过以下途径确定新的肌肉细胞黏附调节剂 三种肌营养不良斑马鱼模型肌肉发育失调的比较研究。我们 将采取不偏不倚的方法，通过使用网络建模和网络来识别ECM监管节点 共表达的编码和非编码基因的复原力分析。这笔赠款的完成将提供新的 洞察细胞-ECM黏附如何在脊椎动物模型中调节肌肉发育和动态平衡 CMDS。这些基础的活体细胞生物学研究对于提供对 跨膜受体、细胞外基质调节和细胞黏附之间的相互作用。