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.
Mutations in 20+ human genes involved in N-linked glycosylation, the most common form of carbohydrate modification on proteins, result in Congenital Disorders of Glycosylation (CDGs) heritable diseases that impair nervous system function. This project utilizes the Drosophila neuromuscular junction (NMJ) genetic model synapse to study the effects of loss of complex and branched N-glycosylation on structural and functional development.
|Parkinson, William M; Dookwah, Michelle; Dear, Mary Lynn et al. (2016) Synaptic roles for phosphomannomutase type 2 in a new Drosophila congenital disorder of glycosylation disease model. Dis Model Mech 9:513-27|
|Parkinson, William; Dear, Mary Lynn; Rushton, Emma et al. (2013) N-glycosylation requirements in neuromuscular synaptogenesis. Development 140:4970-81|