The Role of Glycyl-tRNA Synthetase in Neurodegeneration

Lead Research Organisation: University of Oxford
Department Name: Physiology Anatomy and Genetics

Abstract

Degenerative disorders of the nervous system place a significant and increasing healthcare burden as the average age of the population rises. In order to develop effective treatments, an understanding of how these disorders arise is critical. Relatively rare inherited diseases such as spinal muscular atrophy are an ideal paradigm with the insights gained expected to be broadly applicable to common conditions such as Alzheimer‘s and Parkinson‘s Disease.

We are studying a neurodegenerative disorder caused by mutations in the gene for Glycyl-tRNA Synthetase (GlyRS): an enzyme required by cells to ensure proteins are synthesised correctly from their genetic templates. However, motor nerves are particularly vulnerable to mutations in this enzyme. To understand why, we will examine the cells of mice to study how GlyRS mutations might change gene expression and affect motor nerve function. We will then examine the interactions of this ubiquitous enzyme with proteins that may be specific to motor nerves. Through determination of the atomic structures, the key parts of the enzyme mediating such interactions can be identified.

This research will yield fresh insights into how neurons degenerate and why they are vulnerable. This will then form the basis of new treatments for these appalling disorders.

Technical Summary

Glycyl-tRNA synthetase (GlyRS) is an essential enzyme for ensuring the fidelity of translation of the genetic code through ligation of glycine with its cognate tRNA. Several dominant mutations have been identified in GlyRS causing lower motor neuron disorders. A mouse model with a C201R GlyRS mutation has also been identified on the basis of a distal motor neuronopathy (grip-strength) phenotype. Our preliminary structure-function studies demonstrate that mutant GlyRS forms functional enzymes but activity, which may be increased or decreased, is not consistently correlated with the disease phenotype. Furthermore a GlyRS mutation, with increased enzyme activity, reduces survival of cultured neurons.

The aim of the proposed study is to understand how GlyRS mutations lead to neurodegeneration. We hypothesize that toxicity is mediated by a neuron-specific intermediary protein and this will be explored using the C201R mouse. We have therefore designed this project with the following specific objectives:

1. Characterisation of the C201R mouse a. Histopathology of peripheral nerves and neuromuscular junction b. Motor neuron culture transfection studies c. Gene expression profiling to reveal gene networks leading to neurodegeneration

2. Identifying GlyRS binding partners to reveal protein networks leading to neurodegeneration

3. Structure-biochemistry of GlyRS-substrate complexes

We will fully characterise the nature of the neurodegenerative process in C201R mice through electron microscopy and histopathology of peripheral nerves and muscle. Primary motor neuron cultures will be transfected with tagged axonal transport and synapse proteins and visualised with confocal microscopy. Changes in gene expression will be determined using DNA microarrays and real-time PCR.

A yeast two-hybrid screen, co-immunoprecipitation and mass spectrometry will be used to identify binding partners. GlyRS will be crystallised in complex with cognate tRNA and binding partners using high-throughput sitting-drop vapour diffusion and structures determined with publicly available bioinformatics resources. This will be correlated with GlyRS enzyme activity in-vitro and in-vivo using standard radio-labelled aminoacylation assays.

This project examines the neurodegenerative process from the most fundamental level of molecular architecture to the whole organism level. It represents a unique scientific opportunity to integrate several complementary disciplines; each placing the other in context and having the potential to provide greater insights into the mechanism of disease.

If the mechanism of toxicity in GlyRS mutations can be identified, drugs could be used to disrupt this process. GlyRS inhibitors already exist as do antibiotics targeting other aminoacyl-synthetases. Modification of these agents based upon structure-function work could eventually lead to effective treatments for neurodegenerative disorders.

Publications

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Janssen BJ (2012) Neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex. in Nature structural & molecular biology

 
Description IMI
Amount € 26,000,000 (EUR)
Organisation European Commission 
Department Innovative Medicines Initiative (IMI)
Sector Public
Country Belgium
Start 09/2012 
End 09/2017
 
Description OSCI Seed Funding (James Martin Fund)
Amount £20,000 (GBP)
Organisation University of Oxford 
Department James Martin Fund
Sector Academic/University
Country United Kingdom
Start 09/2011 
End 09/2012
 
Description Oxford University John Fell Fund Award
Amount £45,000 (GBP)
Organisation University of Oxford 
Department John Fell Fund
Sector Academic/University
Country United Kingdom
Start 01/2007 
End 03/2015
 
Description Project Grants
Amount € 93,000 (EUR)
Organisation French Muscular Dystrophy Association (AFM) 
Sector Charity/Non Profit
Country France
Start 03/2013 
End 03/2015
 
Title A Fly model of Glycyl-tRNA Synthetase 
Description Flies expressing human GARS - wildtype and disease causing mutants 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact This has provide significant insights into the time course of the neuro-degenerative process and convergence points for genetic interactors 
 
Title An inducible GARS neuronal stable cell line 
Description A lentiviral transduced NSC-34 cell line expressing Tet-ON GARS. Mitochondrial and cytoplasmic as well as wild-type and mutant isoforms 
Type Of Material Cell line 
Year Produced 2012 
Provided To Others? Yes  
Impact This has allowed the characterization of the GARS transcriptome and proteome 
 
Title Stem Cell Resources 
Description Panel of cell lines from range of neurological and psychiatric disorders as well as diabetes and healthy controls. 
Type Of Material Cell line 
Year Produced 2014 
Provided To Others? Yes  
Impact This will become accessbile to the international research community 
URL http://stembancc.org/
 
Description A fly model of GlyRS Neurodegeneration 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution Leading collaborative work
Collaborator Contribution Generation of a fly model of Glycyl-tRNA synthetase to identify pathogenic mechanism and to understand interactions with other genes involved in neurodegeneration
Impact A validated fly model of neurodegeneration
Start Year 2010
 
Description StemBANCC 
Organisation European Commission
Department Innovative Medicines Initiative (IMI)
Country Belgium 
Sector Public 
PI Contribution Leading this consortium
Collaborator Contribution 25 academic institutions 10 pharmaceutical companies providing 26 million euros in-kind contribution
Impact None
Start Year 2012
 
Description StemBANCC 
Organisation F. Hoffmann-La Roche AG
Country Global 
Sector Private 
PI Contribution Leading this consortium
Collaborator Contribution 25 academic institutions 10 pharmaceutical companies providing 26 million euros in-kind contribution
Impact None
Start Year 2012
 
Description TRESK - a new gene for migraine 
Organisation University of Montreal
Country Canada 
Sector Academic/University 
PI Contribution Co-ordinating and leading collaboration.
Collaborator Contribution This collaboration involved the electrophysiological characterization of TRESK in XenopusThe sequencing of migraine probands to identify variants in ion channel genes
Impact Identification of the first gene linked to typical migraine - TRESK. Publication in Nature Medicine Significant media coverage of findings
Start Year 2008
 
Description TRESK - a new gene for migraine 
Organisation University of Oxford
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution Co-ordinating and leading collaboration.
Collaborator Contribution This collaboration involved the electrophysiological characterization of TRESK in XenopusThe sequencing of migraine probands to identify variants in ion channel genes
Impact Identification of the first gene linked to typical migraine - TRESK. Publication in Nature Medicine Significant media coverage of findings
Start Year 2008
 
Description Mechanisms of Migraine, Migraine Trust Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact 100 migraine and headache sufferers attended a talk on the mechanisms of migraine. There was a question and answer session following the talk

There was significant interest in the talk such that other patient groups have subsequently requested further talks
Year(s) Of Engagement Activity 2010
 
Description TRESK and Migraine Breaking News 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Widespread coverage of Nature Medicine publication by newspapers, radio and TV worldwide

Numerous emails and other communications with congratulations or requests of interest to participate in research
Year(s) Of Engagement Activity 2010