Novel pathogenic mechanisms implicated in defects of neuromuscular transmission

Lead Research Organisation: Newcastle University
Department Name: Institute of Human Genetics

Abstract

Congenital myasthenic syndromes (CMS) are a group of inherited diseases that cause muscle weakness in children and adults. CMS affect the neuromuscular junction (NMJ), a link structure that is responsible for transmitting signals from nerves to muscles, causing muscles to contract. When this link breaks down, or functions less efficiently, the patient gets tired very quickly and can be severely disabled. The condition may be life-threatening, when breathing muscles are affected. We have recently discovered mutations in a new gene called GFAT1 which cause a previously unrecognised form of CMS. At the moment it is not yet understood how mutations in GFAT1 interfere with NMJ function and structure to cause CMS. We have found out that the amount of GFAT1 protein present in the muscle tissue of patients is considerably diminished compared to healthy control muscle. Our aim is therefore to study the consequences of the loss of GFAT1 for muscle function and for communication between nerve and muscle.
Many proteins carry sugar residues on their surface which are important for their function. The GFAT1 enzyme produces a metabolite that is essential for all reactions that add sugar residues onto proteins. We plan to investigate whether proteins lose their sugar modification in GFAT1 patients, which proteins are affected most by this and what the implications are for the correct function of a muscle cell.
As sugar modification of proteins is a very general mechanism in all cell types, our results may not only improve our knowledge of CMS, but also contribute to the understanding of sugar modifications on proteins of the nerve cells in diseases such as Alzheimer?s disease and in muscle for the development of type 2 diabetes.

Technical Summary

Congenital myasthenic syndromes (CMS) are inherited neuromuscular transmission defects characterised by fluctuating muscle weakness and fatigability, leading to loss of mobility and respiratory failure. A distinct form of CMS, pyridostigmine-responsive limb-girdle CMS, has not previously been associated with any gene defect. Recently, we linked this condition to a locus on chromosome 2p12-15 by genome-wide mapping and identified mutations in the GFAT1 gene as the underlying genetic defect. Subsequently we established that GFAT1 mutations lead to a loss of GFAT1 expression in patient muscle. In contrast to all other CMS genes, GFAT1 is expressed in most tissues.
This is the first association of CMS with a non-structural, ubiquitous protein, suggesting novel pathogenic mechanisms leading to NMJ dysfunction. The proposed project aims at understanding how GFAT1 mutations cause an impairment of neuromuscular transmission.
The GFAT1 enzyme regulates the flux through the hexosamine biosynthetic pathway which yields the precursor substrates for protein and lipid glycosylation. This includes N- and O- glycosylation of transmembrane and secreted proteins as well as the posttranslational modification by one O-linked beta-N-Acetylglucosamine residue of nucleocytoplasmic proteins (O-GlcNAcylation). In this project we will investigate the consequence of GFAT1 deficiency on these two types of glycosylation for skeletal muscle and NMJ proteins. We will study glycosylation in GFAT1 deficient C2C12 muscle cells, in GFAT1 deficient zebrafish as an animal model and in patient material. Several NMJ transmembrane proteins such as the acetylcholine receptor and the muscle-specific kinase MuSK are glycosylated. We will determine whether GFAT1 deficiency results in a modification of glycosyl residues on these proteins and whether this affects acetylcholine receptor clustering at the NMJ and the NMJ structure in zebrafish. Moreover, O-GlcNAcylation of nuclear and cytoplasmic proteins has been linked to stress response in cells and is thought to have a cytoprotective function. Inability of GFAT1 deficient cells to regulate their stress response might leave them more prone to stress-related damage. We will identify which proteins are O-GlcNAcylated in C2C12 cells, which of these are most affected by GFAT1 deficiency, and how this relates to stress tolerance.
Our findings will improve our understanding of posttranslational modifications of the neuromuscular junction. We will determine to what degree disturbed protein glycosylation constitutes a new basic pathogenic mechanism resulting in a synaptic defect.

Publications

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Cossins J (2013) Congenital myasthenic syndromes due to mutations in ALG2 and ALG14. in Brain : a journal of neurology

 
Description Albert Sickmann Collaboration 
Organisation Leibniz Association
Department Leibniz Institute for Analytical Sciences
Country Germany 
Sector Charity/Non Profit 
PI Contribution Proteomic studies to unravel the molecular basis of neuromuscular diseases
Collaborator Contribution Proteomic studies
Impact Not yet
Start Year 2016
 
Description Anne Dell Collaboration 
Organisation Imperial College London
Department Department of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Generation of cell material for mass spectrometric glycomic analysis with and without GFPT1
Collaborator Contribution Investigation of the effect of GFPT1 deficiency on the glycome of cell cultures
Impact Results are being analysed
Start Year 2012
 
Description Barbara Zieger Collaboration 
Organisation Albert Ludwig University of Freiburg
Country Germany 
Sector Academic/University 
PI Contribution Investigation of cytoskeletal proteins in the cause of neuromuscular diseases
Collaborator Contribution Provision of tissues, antibodies and data interpretation
Impact Not yet
Start Year 2016
 
Description David Beeson Collaboration 
Organisation University of Oxford
Department Weatherall Institute of Molecular Medicine (WIMM)
Country United Kingdom 
Sector Academic/University 
PI Contribution Identification and provision of patient material for analysis, identification of novel mutations in specific glycosylation genes,
Collaborator Contribution Sequencing data
Impact Previous joint publications (eg. Beeson et al., 2006) Upcoming joint publication on novel glycosylation gene mutations in CMS
 
Description Markus Ruegg Collaboration 
Organisation University of Basel
Department Biozentrum Basel
Country Switzerland 
Sector Academic/University 
PI Contribution Development of Gfpt1 shRNA resources for transient knowckdown in mouse muscle
Collaborator Contribution Electroporation of mouse muscle
Impact Still under development
Start Year 2012
 
Description Neuromouse Consortium 
Organisation International Mouse Phenotyping Consortium (IMPC)
Country Global 
Sector Charity/Non Profit 
PI Contribution We have been involved in the prioritisation of genes for targeting as part of a wider consortium.
Collaborator Contribution The wider consortium is providing mouse knock-outs and phenotyping data, notably on the Gfpt1 conditional mouse knock-out
Impact Mouse strains are being prepared for dispatch from Harwell.
Start Year 2010
 
Description Neuromouse Consortium 
Organisation International Mouse Phenotyping Consortium (IMPC)
Country Global 
Sector Charity/Non Profit 
PI Contribution We have been involved in the prioritisation of genes for targeting as part of a wider consortium.
Collaborator Contribution The wider consortium is providing mouse knock-outs and phenotyping data, notably on the Gfpt1 conditional mouse knock-out
Impact Mouse strains are being prepared for dispatch from Harwell.
Start Year 2010
 
Description Neuromouse Consortium 
Organisation Medical Research Council (MRC)
Department The Mary Lyon Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been involved in the prioritisation of genes for targeting as part of a wider consortium.
Collaborator Contribution The wider consortium is providing mouse knock-outs and phenotyping data, notably on the Gfpt1 conditional mouse knock-out
Impact Mouse strains are being prepared for dispatch from Harwell.
Start Year 2010
 
Description Semra Hiz Collaboration 
Organisation Izmir University
Country Turkey 
Sector Academic/University 
PI Contribution Identification of disease causative genes (for neurological disorders) in recessive patients from Turkey.
Collaborator Contribution Provision of patients DNA and clinical data
Impact Not yet
Start Year 2016