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.

抽象的 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 破坏肌肉细胞与其周围细胞外基质 (ECM) 的粘附。肌肉-ECM 粘附力是 对于肌肉发育、体内平衡、再生和抗压能力至关重要。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 α7 (Itga7) 是跨膜 ECM 受体，突变时会导致 CMD。是否和/或如何 这些跨膜受体在肌肉发育/稳态过程中相互作用尚不清楚。此外， DG 翻译后修饰在调节 ECM 本身和肌肉 ECM 中的作用 粘附力未知。我们之前发现外源 NAD+ 增强 ECM 沉积，并且 NAD+ 可改善缺乏 DG 或 Itga7 的斑马鱼的营养不良表型。基本细胞生物学 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。继发性肌营养不良症是 CMD 的一个子集，由以下基因突变引起： DG 的糖基化是必需的，而 DG 的糖基化是肌肉-ECM 粘附所必需的。 GDP-甘露糖，合成的 根据 GMPPB，对于糖基化反应至关重要。 GMPPB 突变导致 GMPPB 相关 肌聚糖病。初步数据表明，肌肉发育、体内平衡和再生是 gmppb 突变体中被破坏。与我们之前的数据相比，NAD+ 改善了 dg- 中的 ECM 沉积 对于缺乏缺陷的斑马鱼，初步数据表明 NAD+ 不会改善 gmppb 突变体的肌肉结构。在 这笔资助，我们将比较和对比 DG 糖基化和 NAD+ 影响的潜在机制 关于肌肉发育、体内平衡和再生。我们的中心假设是 NAD+ 和 gmppb 通过改变肌膜结构和 ECM 组织来调节肌肉细胞粘附。在目标 1 中我们将测试 NAD+ 通过增加 Itga7 聚类来增加 DG 突变斑马鱼的细胞粘附的假设；和 低糖基化的 DG 会破坏肌膜结构并阻止 NAD+ 介导的 Itga7 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，我们将通过以下方式识别新的肌肉细胞粘附调节剂： 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 将通过使用网络建模和网络，采取公正的方法来识别 ECM 监管节点 共表达的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 粘附如何介导脊椎动物模型中的肌肉发育和稳态 CMD。这些基本的体内细胞生物学研究对于提供对细胞的基本了解至关重要 跨膜受体、ECM 调节和细胞粘附之间的相互作用。