N-Linked Glycan Modification in Synaptic Development and Function

描述(申请人提供)：细胞间的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多糖成分。糖链修饰大多数细胞外基质和细胞表面蛋白，而糖基化通常在蛋白质的加工、分类、运输和功能中发挥关键作用&GT；20人类N-连锁糖基化途径组分的不同基因突变会导致先天性糖基化紊乱(CDGs)家族的庞大且不断增长，并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mGAT1突变条件为研究N-糖链的神经作用提供了一个特别强大的切入点，因为它阻止了所有成熟的、复杂的、分支的N-糖基化的产生。在这里，拟议的研究将使用mGat1突变体和相关的转基因工具在特征良好的果蝇NMJ上研究突触机制和人类疾病状态模型中N-糖基化的要求。嵌套的假说如下：1)依赖于MGAT1的N-糖链成熟丧失，损害了NMJ的结构和功能发育，导致协调运动受损。2)突触基质中依赖于镁的N-糖基化的缺失损害了跨突触信号，从而限制了控制NMJ突触发生的细胞内突触前和突触后支架的募集。3)在mGat1缺失的NMJ中，ALK的丢失是由于mGat1依赖的N-糖基化缺失引起的，并且是mGat1突变型NMJ突触发生缺陷的原因。测试这些假说的功能方面将涉及各种电生理学技术，包括双电极电压钳、自发的微小兴奋结电流记录和巨型光纤马达电路记录。为了识别突触结构和蛋白质定位缺陷，免疫细胞化学将同时使用共聚焦显微镜和电子显微镜。将产生新的突变系来鉴定特定蛋白质上N-连接糖基化缺失的结果。利用这一强大的 将在神经系统中确定N-糖基化的一系列技术、新要求和作用。这项工作的具体重点是进一步了解N-糖基化在人类先天性疾病中引起的突触发生和突触功能障碍中的作用。

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.