N-Linked Glycan Modification in Synaptic Development and Function

突触发育和功能中的 N 连接聚糖修饰

• 批准号: 8869061
• 负责人: William M. Parkinson
• 金额: $ 1.92万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8869061
• 关键词: 3-Dimensional; Adult; Alleles; Antibodies; Architecture; Area; Asparagine; Ataxia; Automobile Driving; Binding; Biological Assay; Boat; Carbohydrates; Cell Surface Proteins; Cell surface; Cells; Communication; Complex; Confocal Microscopy; Congenital Disorders; Coupled; DNA Sequence Alteration; Data; Databases; Defect; Development; Disease; Drosophila genus; Dyes; Dystroglycan; Electrodes; Electron Microscopy; Electrophysiology (science); Environment; Epilepsy; Epitopes; Event; Exhibits; Extracellular Matrix; Family; Fiber; Functional disorder; Genes; Genetic; Genetic Models; Glass; Glutamate Receptor; Growth; Health; Horseradish Peroxidase; Human; Human Genetics; Image; Immunoelectron Microscopy; Intellectual functioning disability; Larva; Lectin; Ligands; Link; Longevity; Maintenance; Mannosides; Mass Spectrum Analysis; Membrane; Membrane Proteins; Mind; Modeling; Modification; Molecular; Motor; Movement; Muscle; Mutation; N-Acetylglucosaminyltransferases; Nervous System Physiology; Nervous system structure; Neuroglia; Neurologic; Neuromuscular Junction; Neurons; Output; Pathway interactions; Patients; Phenocopy; Phenotype; Play; Polysaccharides; Positioning Attribute; Postsynaptic Membrane; Process; Production; Protein Glycosylation; Proteins; RNA Interference; Relative (related person); Role; Scaffolding Protein; Seizures; Shapes; Signal Pathway; Signal Transduction; Site; Sorting - Cell Movement; Symptoms; Synapses; Synaptic Cleft; System; Techniques; Testing; Transgenes; Transgenic Organisms; Vicia; WNT Signaling Pathway; Western Blotting; Work; extracellular; genetic analysis; glycosylation; human disease; immunocytochemistry; molecular assembly/self assembly; molecular imaging; motor impairment; mutant; neurotransmission; postsynaptic; presynaptic; protein distribution; protein structure; receptor; scaffold; synaptic function; synaptogenesis; tool; uptake; voltage clamp

DESCRIPTION (provided by applicant): Intercellular development and communication require correctly composed extracellular matrix (ECM) and cell surface composition, with appropriate relative protein and glycan contributions. Glycans modify most ECM and cell surface proteins, and glycosylation often plays a pivotal role in protein processing, sorting, transport and function >20 different human genetic mutations of N-linked glycosylation pathway components give rise to the large and growing family of congenital disorders of glycosylation (CDGs), with severe neurological symptoms including intellectual disability, seizures, ataxia and epilepsy. These disease states can be effectively modeled in the Drosophila genetic system. The Drosophila Mgat1 mutant condition provides a particularly powerful entry point to study the neurological roles of N-glycans, as it blocks production of all mature, complex, branched N-glycosylation. Here, the proposed study will use Mgat1 mutants and associated transgenic tools at the well-characterized Drosophila NMJ to study requirements of N-glycosylation in synaptic mechanisms and human disease state models. The nested hypotheses are as follows: 1) The loss of Mgat1-dependent N-glycan maturation compromises NMJ structural and functional development to cause impaired coordinated movement. 2) The loss of Mgat1-dependent N-glycosylation of the synaptomatrix impairs trans-synaptic signaling to limit recruitment of intracellular pre- and postsynaptic scaffolds controlling NMJ synaptogenesis. 3) Loss of Alk in Mgat1 null NMJs is caused by loss of Mgat1-dependent N-glycosylation and is causative for aspects of the Mgat1 mutant NMJ synaptogenesis defects. Testing the functional aspects of these hypotheses will involve various electrophysiology techniques including two-electrode voltage-clamp, spontaneous mini excitatory junction current recording and giant fiber motor circuit recordings. To identify synaptic structure and protein localization defects, immunocytochemistry will be used with both confocal microscopy and electron microscopy. New mutant lines will be generated to identify the result of the loss of N-linked glycosylation on specific proteins. Using this powerful array of techniques, new requirements and roles for N-glycosylation will be identified in the nervous system. The specific focus of this work is to further understanding of N-glycosylation roles in synaptogenesis and synaptic dysfunction arising in congenital diseases of glycosylation in human patients.

描述（由申请人提供）：细胞间的发育和交流需要正确组成的细胞外基质（ECM）和细胞表面成分，以及适当的相对蛋白质和聚糖的贡献。糖基化修饰大多数ECM和细胞表面蛋白，而糖基化通常在蛋白质加工、分选、运输和功能中起关键作用bbb20不同的人类n -连锁糖基化途径组分的基因突变导致大量且不断增长的先天性糖基化疾病（CDGs）家族，导致严重的神经系统症状，包括智力残疾、癫痫发作、共济失调和癫痫。这些疾病状态可以在果蝇遗传系统中有效地建模。果蝇Mgat1突变条件为研究n -聚糖的神经功能提供了一个特别有力的切入点，因为它阻断了所有成熟的、复杂的、分支的n -糖基化的产生。本研究将利用Mgat1突变体和相关的转基因工具在果蝇NMJ中研究n -糖基化在突触机制和人类疾病状态模型中的需求。嵌套的假设如下：1)mgat1依赖性n -聚糖成熟的缺失损害了NMJ的结构和功能发育，导致协调运动受损。2)突触基质中mgat1依赖性n -糖基化的缺失会损害突触间信号传导，从而限制细胞内控制NMJ突触发生的突触前和突触后支架的募集。3) Mgat1缺失的NMJs中Alk的缺失是由Mgat1依赖的n -糖基化缺失引起的，是Mgat1突变体NMJ突触发生缺陷的原因。测试这些假设的功能方面将涉及各种电生理学技术，包括双电极电压钳，自发微型兴奋结电流记录和巨型纤维马达电路记录。为了确定突触结构和蛋白质定位缺陷，免疫细胞化学将与共聚焦显微镜和电子显微镜一起使用。将产生新的突变系，以确定特定蛋白质上n -链糖基化丧失的结果。使用这个强大的