Award details

The role of transcriptional regulation in axonal and synaptic neuroprotection conferred by the Wlds gene

Reference	BB/D001722/1
Principal Investigator / Supervisor	Professor Thomas Gillingwater
Co-Investigators / Co-Supervisors
Institution	University of Edinburgh
Department	Biomedical Sciences
Funding type	Research
Value (£)	228,150
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	30/09/2008
Duration	36 months

Abstract

Axons and synapses comprise primary pathological targets in many diseases of the nervous system (including prion diseases, Alzheimer's disease and many variants of motor neuron disease). Yet, remarkably little is known about degeneration within these compartments, especially when compared to the wealth of knowledge on degenerative processes present in neuronal cell bodies (e.g. apoptosis and necrosis). However, the serendipitous discovery of the Wlds mutant mouse, whose only known phenotype is a ~10-fold delay in axon and synapse degeneration, offers the possibility of significantly enhancing our understanding of axonal and synaptic degeneration. Here we will test the hypothesis that the Wlds gene confers neuroprotection by regulating expression patterns of a specific gene, or genes that are critical regulators of axonal and synaptic degeneration. We have already undertaken gene expression experiments and demonstrated that Wlds regulates expression patterns by more than 2-fold for at least 11 other genes (out of the 23,000 examined) both in vivo and in vitro. However, the extent to which any or all of these 11 genes constitute common targets for Wlds remains unclear. In the first part of this project, we will refine our candidate list to only include those genes whose expression patterns are altered throughout the nervous system in vivo, whenever Wlds is present. In addition, we will examine the temporal characteristics of gene expression in vitro after transfection with a Wlds construct, and test for changes at the level of proteins using western blotting and immunocytochemistry. We will also identify the role that different components of the Wlds chimeric gene (comprising the N-terminal 70 amino acids of the ubiquitination factor Ube4b and the entire coding sequence for the NAD-synthesising enzyme Nmnat), and downstream pathways affected by Wlds (i.e. NAD levels) play in regulating gene expression, again using in vitro transfection systems to introduce different components of the Wlds gene to rodent cerebellar granule cells as well as HEK293 and NSC34 cell lines. The role of candidate genes, that demonstrate a conserved Wlds response in the assays above, in conferring or potentiating neuroprotection will then be addressed directly using cultured cerebellar granule cells as an in vitro assay system that we have recently validated. We will use our established rodent cerebellar granule cell cultures, in conjunction with removal of serum and potassium from culture media, to quantify any resistance to neurodegeneration. By using preparations derived from both mouse and rat tissue, we will be able to determine any inter-species dependence of the genes being tested. We will use transfection constructs to over-express, and siRNA technology to down-regulate, any given gene, developed in collaboration with specialists in these fields. Using either or both of these techniques, we will examine whether manipulations in the expression of an individual gene, or groups of genes, can confer, potentiate or suppress a neuroprotective phenotype.

Summary

The human nervous system is composed of around 100 billion nerve cells (neurons) and their supporting cells. The loss of some of these neurons - by degenerative processes - in either the central (brain and spinal cord) or peripheral (everything outside the brain and spinal cord) nervous system results in some of the most debilitating diseases known to man. For example, prion diseases, Alzheimer's disease and motor neuron disease all occur due to degeneration and subsequent loss of neurons. However, recent research findings suggest that degeneration is tightly regulated within neurons, and that each individual neuron can be subdivided into several distinct degenerative compartments (the cell body, the axon and the synapse). Each of these compartments can degenerate independently of the others. We are working on a genetic mutation, the Wlds gene, which specifically prevents degeneration occurring in the axon and the synapse. We have discovered that this mutant gene is capable of regulating other genes within the neuron, and now we want to examine this genetic regulation further. We want to know which of the altered genes we have identified should be classified as common targets of the Wlds gene, which parts of our mutant gene are required to regulate gene expression, and also, which of the genes identified are required in order to protect axons and synapses. First, we are going to undertake experiments that allow us to refine our understanding of the gene expression modifications induced by Wlds. We will quantify changes in gene expression in different regions of the nervous system to those previously studied, and also in cells from different species (including rodent and human cells). This will allow us to identify those genes that are always altered when Wlds is present, thereby identifying them as common targets. Next we will test whether these changes in gene expression are controlled by either the whole Wlds gene, or different components of the mutant gene. These experiments will be undertaken in a controlled in vitro environment (i.e. in purified and isolated cells, not in living animals), utilising neuronal and non-neuronal cells from both rodent and human origin. In vitro experimental protocols also allow us to artificially manipulate gene expression using a technique known as transfection, through which we can introduce and express foreign genes in cells, including neurons. When we have found out exactly which of our genes are common targets of Wlds, we will artificially mimic these changes in neuronal cells in vitro for each of our candidate genes using transfection technology. We will then 'kill' the neurons by removing critical components of their surrounding environment (including potassium), and see whether modifying expression of one or more of our genes replicates the protection of axons and synapses which we see when the mutant Wlds gene is present. This approach will identify those common targets of Wlds that are important for protecting axons and synapses from degeneration. It may even turn out that one or more of the genes identified by our experiments could be utilised to develop treatments for those diseases in animals and humans where axons and synapses are the first to degenerate.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Ageing, Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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