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 先天性肌营养不良症（CMD）是一种无法治愈的进行性衰弱性疾病。许多CMD 破坏肌细胞与其周围细胞外基质（ECM）的粘附。肌肉-ECM粘附是 对肌肉发育、体内平衡、再生和压力恢复至关重要。的基因突变 调节肌肉-ECM粘附经常导致CMD。例如，肌营养不良聚糖（DG）和整合素 α 7（Itga 7）是跨膜ECM受体，其在突变时导致CMD。是否和/或如何 这些跨膜受体在肌肉发育/体内平衡期间相互作用是未知的。此外该 DG的翻译后修饰在调节细胞外基质和肌肉细胞外基质中的作用 粘附力未知。我们先前发现外源性NAD+增强ECM沉积， NAD+改善缺乏DG或Itga 7的斑马鱼的营养不良表型。基本的细胞生物学 NAD+介导的肌肉-ECM粘附改善的机制尚不清楚。 我们的长期目标是了解肌肉细胞及其ECM之间的信号传导如何介导肌肉 健康继发性肌营养不良是CMD的一个子集，由以下基因突变引起： DG的糖基化是必需的，这是肌肉-ECM粘附所必需的。GDP-甘露糖，合成 由GMPPB，是必不可少的糖基化反应。GMPPB中的突变导致GMPPB相关的 营养不良性聚糖病。初步数据显示，肌肉发育，稳态和再生是 在GMPPB突变体中破坏。与我们先前的数据相比，NAD+改善了dg-1中的ECM沉积。 在缺乏NAD+的斑马鱼中，初步数据显示NAD+不会改善gmppb突变体的肌肉结构。在 在这项研究中，我们将比较和对比DG糖基化和NAD+的作用机制。 对肌肉发育、体内平衡和再生的影响。我们的中心假设是NAD+和gmppb 通过改变肌膜结构和ECM组织来调节肌细胞粘附。在目标1中，我们将测试 假设NAD+通过增加Itga 7聚集来增加DG突变斑马鱼中的细胞粘附;以及 低糖基化DG破坏肌膜结构并阻止NAD+介导的Itga 7聚集， 增加细胞粘附。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，我们将通过以下方法鉴定新的肌细胞粘附调节剂： 三种肌营养不良症斑马鱼模型中肌肉发育失调的比较研究。我们 将采取无偏的方法，通过网络建模和网络识别ECM调节节点 共表达的编码和非编码基因的弹性分析。完成这项赠款将提供新的 深入了解细胞-ECM粘附如何在脊椎动物模型中介导肌肉发育和稳态 CMD。这些基本的体内细胞生物学研究对于提供对细胞生物学的基础性理解是至关重要的。 跨膜受体、ECM调节和细胞粘附之间的相互作用。