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: 8594073
• 负责人: William M. Parkinson
• 金额: $ 2.66万
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8594073
• 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; 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; Gene Mutation; Genes; Genetic; Genetic Models; Glass; Glutamate Receptor; Growth; Horseradish Peroxidase; Human; Human Genetics; Image; Immunoelectron Microscopy; Impairment; 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; mutant; neurotransmission; postsynaptic; presynaptic; protein distribution; protein structure; public health relevance; 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）：细胞间发育和通讯需要正确组成的细胞外基质（ECM）和细胞表面组成，以及适当的相对蛋白质和聚糖贡献。聚糖修饰大多数ECM和细胞表面蛋白质，并且糖基化通常在蛋白质加工、分选、运输和功能中起关键作用。N-连接的糖基化途径组分的20种不同的人类基因突变产生了巨大且不断增长的先天性糖基化障碍（CDG）家族，具有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Mgat 1突变条件提供了一个特别强大的切入点，研究神经功能的N-聚糖的作用，因为它阻止所有成熟的，复杂的，分支的N-糖基化的生产。在这里，拟议的研究将使用Mgat 1突变体和相关的转基因工具，在充分表征的果蝇NMJ研究突触机制和人类疾病状态模型中N-糖基化的要求。嵌套假设如下：1）Mgat 1依赖性N-聚糖成熟的丧失损害NMJ结构和功能发育，导致协调运动受损。2)突触基质的Mgat 1依赖性N-糖基化的丧失损害跨突触信号传导以限制控制NMJ突触发生的细胞内突触前和突触后支架的募集。3)Mgat 1缺失NMJ中Alk的缺失是由Mgat 1依赖性N-糖基化的缺失引起的，并且是Mgat 1突变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，自发性小兴奋性连接电流记录和巨纤维运动电路记录。为了鉴定突触结构和蛋白定位缺陷，免疫细胞化学将与共聚焦显微镜和电子显微镜一起使用。将产生新的突变株系以鉴定特定蛋白质上N-连接糖基化丧失的结果。使用这个强大 一系列的技术，新的要求和N-糖基化的作用将被确定在神经系统中。这项工作的具体重点是进一步了解N-糖基化在人类患者先天性糖基化疾病中突触发生和突触功能障碍中的作用。