Mechanisms controlling the number and location of synaptic AMPARs

Lead Research Organisation: University of Bristol
Department Name: Anatomy

Abstract

Glutamate is a molecule in the brain that transfers messages from one neurone to the next. In the receiving neurone, glutamate binds to proteins called receptors that activate that neurone which, in turn, often stimulates it to release glutamate itself to activate the next neurone in the chain. We are particularly interested in the glutamate receptor subtype called AMPA receptors (AMPARs). Information passing through AMPARs represents the vast majority of information flow in the brain.

To function correctly, AMPARs need to be precisely located on the surface of the neurone. The focus of our work is how neurones achieve the remarkable feat of getting the AMPARs to the right place at the right time. In addition, the number and location of AMPARs can be rapidly changed. It has been discovered that there are proteins that direct and anchor AMPARs; they determine the level and location of AMPAR expression and they connect to specific signalling pathways inside the neurone.

This project aims to provide a more complete picture of how AMPARs are transported (trafficked) around the neurone and define the specific roles of interacting proteins. We shall use a combination of methods that takes full advantage of the technological advances made over the last few years to elucidate the principles that govern the turnover and surface expression of AMPARs.

To do this we will look at what happens in both resting, unstimulated neurones and also in neurones that have been stimulated with physiologically relevant stimuli. We will use new microscopes and special fluorescent protein labels that allow us to actually see AMPARs moving in the cell and how this is changed by different conditions. We will then use biochemical methods to work out what proteins are involved in orchestrating these movements and what happens when we prevent them from working properly.

The public can access neuroscience research in the University of Bristol via Bristol Neuroscience (BN). There is an extensive website that allows the public to gain access to the research going on in our labs. BN is also active in arranging events to increase public understanding of science. Via BN and the widening participation scheme the PI gives talks about ?how the brain works? to A-level students in schools.

Technical Summary

Our aim is to understand how the number and specific localization of synaptic AMPARs is determined. Our hypothesis is that AMPARs with specific subunit compositions and interacting proteins undergo targeted, activity-dependent delivery to, stabilisation at and removal from pre- and postsynaptic sites. For presentation purposes we have divided the application into four objectives but these are highly interrelated in terms of conceptual and experimental design.
Our major objectives are to:

1. Define trafficking of postsynaptic AMPARs in resting and stimulated neurones to compare subunit composition, delivery pathways and properties and sites of constitutive and evoked exocytosis

2. Distinguish the roles of AMPAR interactors in intracellular transport, endocytosis, exocytosis and lateral diffusion. In particular we shall focus on GRIP and ABP isoforms and Protein kinase Mzeta (PKM?) in the regulation of synaptic AMPARs.

3. Elucidate the molecular mechanisms underlying the rapid regulation of the GluR2 content of AMPARs and define the source of GluR2-lacking AMPARs

4. Identify the mechanisms that regulate trafficking and function of presynaptic AMPARs. We shall identify their subunit compositions, interacting proteins and the parameters of their activity-dependent regulation

This is an integrated proposal that will use a combination of established and emergent molecular, cellular and imaging techniques to address questions fundamental to the basic understanding how neurones and neuronal networks modulate information transfer and storage via synaptic plasticity. Furthermore, because AMPAR function and dysfunction underlies a wide range of clinically important neurological diseases our results will influence future research into novel drug targets and therapeutic intervention strategies.

We will use rat brain tissue and cultured neurones and slices. In all cases the tissue preparation(s) most appropriate for each specific series of experiments will be used. For example, to visualise AMPAR and AMPAR-associated protein trafficking we exploit dispersed cell culture systems to optimise the tools and techniques. Then, based on advances in the equipment, reagents and experimental methods we will progress to the use of acute slice cultures and ex vivo slices that better represent the physiological situation in vivo.

Finally, an important aspect of this application is our support our MRC Centre colleagues and the wider neuroscience community. We will continue to share our existing and new tools (e.g. viral vectors, fluorophore-tagged proteins, specific targeted mutants etc) with our electrophysiology and behavioural collaborators to facilitate the focus of multiple levels of investigation on defined problems of common interest.

Publications

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Anderson DB (2009) Protein SUMOylation in neuropathological conditions. in Drug news & perspectives

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Cimarosti H (2012) Enhanced SUMOylation and SENP-1 protein levels following oxygen and glucose deprivation in neurones. in Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

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Craig TJ (2012) Protein SUMOylation in spine structure and function. in Current opinion in neurobiology

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Craig TJ (2012) Homeostatic synaptic scaling is regulated by protein SUMOylation. in The Journal of biological chemistry

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Dennis SH (2011) Oxygen/glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors. in The Journal of neuroscience : the official journal of the Society for Neuroscience

 
Description Alzheimer's Society studentship "Can protein SUMOylation be neuroprotective in Alzheimer's disease?"
Amount £80,000 (GBP)
Funding ID 170 
Organisation Alzheimer’s Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 01/2017
 
Description BRACE Studentship "Analysis of changes in protein SUMOylation in Alzheimer's and Down's syndrome brain: implications for reducing impaired AMPAR trafficking and synaptic dysfunction"
Amount £84,306 (GBP)
Organisation BRACE (Alzheimer's disease charity) 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2015 
End 01/2018
 
Description EU 7th Framework Marie Curie Initial Training Grant
Amount £80,000 (GBP)
Funding ID Grant agreement no. 238608 
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 10/2010 
End 09/2013
 
Description European Research Council - 'Mechanisms and consequences of synaptic SUMOylation in health and disease'
Amount £1,700,000 (GBP)
Funding ID 232881 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 03/2009 
End 02/2013
 
Description Manipulating protein SUMOylation for neuroprotection in Parkinson's disease
Amount £64,711 (GBP)
Funding ID G-1605 
Organisation Parkinson's UK 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2017 
End 05/2018
 
Description Royal Society-Wolfson Merit Award
Amount £75,000 (GBP)
Funding ID WRSA09R2/HLL 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start  
 
Title AMPAR subunit pHluorin cDNAs 
Description fluorophore tagged AMPAR subunits that allow real-time live cell imaging of receptor trafficking and surface expression 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Multiple papers have been published by other groups using these reagents 
 
Title SEP-GluRs 
Description flourophore tagged proteins and mutant cDNA clones 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact improved experimental design 
 
Description Collaboration via EMBO Fellowship to Bordeaux 
Organisation University of Bordeaux
Department UMR 5297 (Institute for Interdisciplinary Neuroscience)
Country France 
Sector Academic/University 
PI Contribution We have identified novel roles of metabotropic actions of kainate receptors on AMPAR trafficking and plasticity
Collaborator Contribution Access to transgenic mice
Impact None, as yet
Start Year 2011
 
Description EU Marie Curie funded PhD 
Organisation GlaxoSmithKline (GSK)
Department Neuroscience (GSK)
Country United Kingdom 
Sector Private 
PI Contribution Joint PhD
Collaborator Contribution The student will spend 6 months at GSK in China
Impact Studnet appointed Oct 2010
Start Year 2010
 
Description U Amsterdam 
Organisation University of Amsterdam
Country Netherlands 
Sector Academic/University 
PI Contribution Live cell imaging of AMPARs
Collaborator Contribution collaboration on effects of steroid on AMPARs
Impact Paper in PloS One
Start Year 2007
 
Description Media interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? Yes
Primary Audience Media (as a channel to the public)
Results and Impact Media interest in our results that we published in Nature.

e-mails and inquiries from general public
Year(s) Of Engagement Activity 2007
 
Description Schools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact Presentations to 6th form and FE students

interaction and interest
Year(s) Of Engagement Activity 2006,2007,2008,2009