The Role of Glycyl-tRNA Synthetase in Neurodegeneration

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.

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.