Neuronal and glial AMPARs: auxiliary subunits as targets for ion channel regulation

Lead Research Organisation: University College London
Department Name: Neuroscience Physiology and Pharmacology

Abstract

The functions of our brain rely on the chemical communication that takes place between its myriad cells. One of the most important of these chemicals is glutamate, a neurotransmitter responsible for a majority of fast excitatory signaling. Glutamate acts by binding to receptor proteins at points of contact between nerve cells, and most key features of this excitatory signaling are shaped by the basic properties of one specific class of glutamate receptor - the AMPA receptors (AMPARs). AMPARs are ubiquitous; their activity is integral to all fundamental brain functions, including the processes of sensation, thought, emotion and memory. Because of this, dysfunction of AMPARs causes serious neurological disorders. In particular, one subclass of AMPARs that allow the entry of calcium ions into cells is implicated in the aetiology of several conditions, including multiple sclerosis, motor neuron disease, epilepsy, brain damage following stroke or during birth, and brain tumor growth. As AMPARs play a key role in normal cognition, they are also targets for drug treatments aimed at enhancing cognitive function, notably in neurodegenerative conditions such as Parkinson's and Alzheimer's disease.

Understanding AMPARs is essential if we are to dissect their role in disease pathogenesis. Specifically, there is an urgent need to determine the principles that govern AMPAR function at a cellular and molecular level. Recently, our understanding of these processes advanced significantly, with the identification of proteins that directly associate with AMPARs and dictate their properties and behaviour. Two families of proteins have received particular attention - the transmembrane AMPAR regulatory proteins (TARPs) and the cornichons (CNIHs). Our research is aimed at understanding how these 'auxiliary subunits' control the function of AMPA receptors, with the goal of identifying potential avenues for therapeutic intervention. We will use a combination of proven electrophysiology, imaging and molecular approaches to address three specific goals: (1) to reveal how different auxiliary proteins affect the way in which endogenous molecules and exogenous drugs block or potentiate different classes of AMPARs; (2) to identify the specific auxiliary molecules important for the regulation of calcium-permeable AMPARs, and (3) to elucidate the role played by auxiliary proteins in AMPAR changes thought to underlie pathological conditions involving nerve cells and glia (inflammatory pain and white matter damage).

Potential applications and benefits of this research stem from the fact that it will provide fundamental information about the regulation of an important class of glutamate receptors in nerve cells and glia. It will consolidate our understanding of normal brain function and highlight strategies for treatment of debilitating conditions. By establishing how different auxiliary proteins affect the pharmacology of specific AMPAR subtypes it has the potential to inform new efforts in drug discovery.

Technical Summary

We will examine how pharmacological, biophysical and molecular properties of neuronal and glial AMPARs are regulated by TARP and cornichon (CNIH) proteins. The identity and role of these auxiliary subunits will be dissected by comparing recombinant and native AMPARs, by examining mutant and transgenic mice, and by targeted molecular intervention to manipulate protein expression or structure. We will use: (1) recombinant AMPARs expressed with specific TARPs and/or CNIHs in tsA201 cells; (2) identified neurons and glia in dissociated cultures, prepared from wild-type (WT) and mutant or transgenic mice; (3) neurons and glia in acute brain slices.

We will use patch-clamp methods to record pharmacologically isolated AMPAR-mediated macroscopic and single-channel currents, and synaptic currents. We will determine defining kinetic properties, voltage-dependence, calcium-permeability and conductance of recombinant, synaptic and extrasynaptic receptors, as well as the efficacy of blockers and allosteric potentiators. We will use a variety of molecular approaches, and antibody-linked quantum dot single particle tracking to explore the influence of specific auxiliary subunits on AMPAR targeting/trafficking. In collaboration with the groups of Dickenson (UCL) and Diamond (NIH) we will examine the contribution of auxiliary subunits to the properties and regulation of calcium-permeable AMPARs in retinal amacrine cells, and in the processing of nociceptive signals in dorsal horn of the spinal cord. Roger Nicoll (UCSF) will collaborate in provision of TARP knockout mice, and related experiments. Experiments using lentiviral constructs will be carried out in collaboration with Stephanie Schorge (UCL). Our research will provide fundamental information about the regulation of AMPARs in neurons and glia. It will contribute to our understanding of normal brain function, highlight potential strategies for treatment of debilitating conditions and inform new efforts in drug discovery.

Planned Impact

Academic impact: this work has the potential to provide new knowledge and scientific advancement. The beneficiaries of this research will be medical researchers (including those in university departments worldwide) and members of the pharmaceutical industry. By providing a clearer understanding of the impact of auxiliary proteins on the mechanisms of block of calcium-permeable AMPARs and identifying auxiliary AMPAR subunits involved in the regulation and targeting of CP-AMPARs, our work may held identify specific research avenues that will lead to strategies to limit calcium entry and cell damage in diseases where this is a central issue. Similarly, understanding the influence of auxiliary proteins on the efficacy and selectivity of AMPAR potentiators may aid the identification of strategies or drugs to enhance cognition in disease states. In addition, academic benefit may come from our development of any new methodologies and techniques to achieve our goals. In the broader sense, our work will also be of benefit to neuroscience researchers wishing to understand normal brain function. Immediate benefits (through exchange of scientific knowledge) are likely to accrue in the short to medium term, and certainly within the lifetime of the project. The programme of research will also have capacity-building impact through the training of highly skilled researchers, who will acquire specific competencies and broader transferable skills.

Economic and societal impact: Given an ageing population, neurological conditions will assume an ever-growing economic and social burden. In the long term, by informing drug development or otherwise helping to facilitate rational approaches to therapy, our studies may ultimately lead to enhanced health and well-being.

Publications

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Coombs ID (2019) Homomeric GluA2(R) AMPA receptors can conduct when desensitized. in Nature communications

 
Description CASE award (PhD Student Sarah Pearce)
Amount £157,000 (GBP)
Funding ID Not known (please note amount stated above is approx) 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 10/2017
 
Description Programme Grant
Amount £1,200,000 (GBP)
Funding ID 086185/Z/08/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2009 
End 10/2015
 
Description Hannah Mitchison: Identification of the locus of the neurological defect in the cerebellum of a model of human juvenile Batten disease 
Organisation University College London
Department Institute of Child Health
Country United Kingdom 
Sector Academic/University 
PI Contribution Functional (patch clamp) and structural experiments on the cerebellum
Collaborator Contribution Provided us with a transgenic mouse model of human Batten disease - the most common progressive neurodegenerative disorder of childhood. Provided expert advice and discussion.
Impact Currently still working on this. A paper is in the pipeline.
Start Year 2012
 
Description Ingo Greger - collaboration 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Mapping the interaction sites between AMPA receptors and TARPs which has revealed a role for the receptor N-terminal domain in channel gating
Collaborator Contribution Structural studies at the LMB in Cambridge
Impact PMID: 25373908 PMID: 28521126
Start Year 2010
 
Description John Isaac - collaboration 
Organisation Wellcome Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Calcium permeable AMPA receptors and auxiliary subunits: functional studies
Collaborator Contribution Intellectual and experimental contribution during weekly lab visits
Impact None yet.
Start Year 2016
 
Description Kenji Sakimura, University of Niigata Medical School, Japan (g-7 knockout) 
Organisation Niigata University
Department School of Medicine
Country Japan 
Sector Academic/University 
PI Contribution Functional electrophysiological analysis and molecular biology
Collaborator Contribution Generation of specific types of transgenic mice for our functional studies on AMPA receptor auxiliary subunits.
Impact None
Start Year 2016
 
Description Michael Maher, Janssen R&D, Johnson & Johnson, San Diego, CA 
Organisation Janssen Research & Development
Country Global 
Sector Private 
PI Contribution Functional patch clamp experiments
Collaborator Contribution Provided novel molecules that have selective effects on particular subtypes of AMPA receptors in a part of the brain that plays a role in memory formation, and in epilepsy.
Impact none
Start Year 2016
 
Description 5th European Synapse meeting (Bristol 2015) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Furthering scientific understanding and research internationally.
Year(s) Of Engagement Activity 2016
 
Description Dr Ian Coombs (University College London) "A conducting desensitized AMPA receptor" 6th Annual iGluR Meeting, University Pittsburgh 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Present data to receive feedback and disseminate information, listen to the latest cutting edge research in the field, and exchange ideas and important new techniques, develop research collaborations and contacts worldwide.
Year(s) Of Engagement Activity 2018
URL https://www.medicine.mcgill.ca/pharma/dbowielab/pdf_files/Schedule_iGluReatreat2018.pdf
 
Description Gordon Conference 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Gordon Conference Invited speaker. Cerebellum. NH, USA (Aug 11-16, 2013) (Chair: Indira Raman & Michael Mauk)

Highly regarded international venue. Valuable discussions with many other workers in the field.
Year(s) Of Engagement Activity 2013
 
Description Yale University Speaker at iGluR Meeting in honor of the scientific contributions of Prof James Howe 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Speaker at Yale University Med School iGluR meeting
Year(s) Of Engagement Activity 2017