The role of NMDA receptor dysfunction in epileptic disorders
Lead Research Organisation:
University College London
Department Name: School of Pharmacy
Abstract
The central nervous system is an intricate network of nerve cells that transmit and receive messages. This communication occurs mostly at specialised sites of contact known as synapses found all over the surface of neurons, but typically on fine neuronal processes known as dendrites. At these sites, an arriving nerve impulse causes the release of a chemical neurotransmitter from the 'presynaptic' cell, which then interacts with protein receptor molecules in the cell membrane of a neighbouring 'postsynaptic' nerve cell. We aim to study receptors activated by two neurotransmitters, glutamate and glycine, which are classified as excitatory NMDA receptors, based on selectivity for an artificial toxin N-methyl-D-aspartate. NMDA enhances nerve cell excitability by enabling a flow of ions via an integral ion channel. The opening of these receptors alters the electrical state of the cell, either transmitting or subtly altering incoming nerve impulses. NMDA receptors are important for normal function of synapses and processes involved in learning and memory. Dysfunction of NMDA receptors has previously been implicated in neurodegeneration, pain, stroke and schizophrenia. Drugs targeting NMDA receptors have also shown clinical promise in indications such as Alzheimer's disease, Parkinson's disease, pain and depression. Recent studies involving the applicants and other researchers world-wide have shown that genetic defects in genes encoding NMDA receptor subunits cause different types of childhood epilepsy, intellectual disability, autism and schizophrenia. Initial results suggest that several of the genetic changes identified in these children appear to cause over-excitation via NMDA receptors. Our project aims to understand how such changes in NMDA receptor genes can cause such a wide range of neurological disorders. We aim to rectify this gap in knowledge, and explore new potential treatments based on targeting NMDA receptors with drugs that reduce excitation. The aims of this research project are: (i) To use biochemical, structural biology and physiological methods to classify NMDA receptor mutations as loss- or gain-of-function and to uncover how different mutations affect the basic functions of the NMDA receptor; (ii) To explore novel therapeutic routes by testing drugs that block NMDA receptor activity for their effectiveness in restoring normal function in defective NMDA receptors; iii) To create new models of epilepsy by introducing NMDA receptor mutations into the mouse genome. It is our hope that a detailed understanding of the mechanisms underlying NMDA receptor dysfunction, signalling pathways and pharmacology will enable insights into the roles of NMDA receptors in health and disease.
Technical Summary
NMDA receptors are key mediators of brain plasticity, converting neuronal activity into long-term changes in synaptic structure and function. Mutations affecting the GluN2A subunit have recently been identified in focal childhood epilepsies ranging from mild, self-limiting rolandic epilepsy to severe epileptic encephalopathies. By contrast, defects in GluN2B subunit have been linked to West syndrome, intellectual disability, autism and schizophrenia. This project aims to understand how different mutations in GluN2A and GluN2B cause such a wide spectrum of neurological disorders, defining pathogenic mechanisms, exploring new therapeutic avenues and generating new acute mouse models of NMDA receptor dysfunction. Our main objectives are to: (i) Use molecular biology to generate a series of GluN2A and GluN2B missense mutants for analysis in cellular trafficking assays. We will also use molecular modelling to predict potential pathogenic impacts of missense changes. These approaches will enable us to prioritise GluN2A and GluN2B mutations for ii) detailed electrophysiological analysis, which will examine changes in agonist binding, modulation by Zn2+, voltage-sensitive Mg2+ blockade, and changes to kinetics of NMDA currents. Preliminary data suggests that many GluN2A and GluN2B missense mutations involve gain-of-function, rather than loss-of-function; (iii) To explore the possibility of developing new treatments for epilepsy, by testing the efficacy of clinically-approved NMDA receptor antagonists such as memantine on mutant NMDARs; iv) To generate and characterise novel gain-of-function knock-in mouse models for GluN2A and GluN2B dysfunction that are defective in voltage-dependent Mg2+ blockade. This programme of research will use an integrated range of biochemical, electrophysiological, molecular biology and genetics techniques and will provide functional insights into the roles of NMDA receptors in health and disease, as well as new potential therapeutic strategies.
Planned Impact
Who will benefit from this research?
Our proposed project is likely to have broad-ranging impact on several groups. These include: academics (Academic Impact) and in the longer-term, healthcare professionals associated with neuroscience and mental health, the general/lay public who are directly or indirectly affected by epileptic disorders (particularly those caused by dysfunctional NMDA receptor signalling) and pharmaceutical companies concerned with drug development for epileptic disorders (Economic/Societal Impact).
How will they benefit from this research?
Academics will gain valuable insight into the biological roles and functional operation of NMDA receptors as well as achieving a better understanding of their roles in neurological disease. In addition, we will transfer technical and managerial skills to a new generation of research scientists, either directly (to postdoctoral RAs) or indirectly (to undergraduate or Ph.D. students involved in our research). This will also help to develop an international skill base through the subsequent mobility of these new scientists. Former Ph.D. students and postdoctoral scientists have benefitted in terms of their career trajectories by training in our labs and are now in posts at many other Universities (e.g. Montreal, Oxford, UCL), industry/healthcare (e.g. Pfizer, National Health Service) or funding agencies (e.g. Wellcome Trust). Our previous studies of inhibitory synaptic transmission have provided direct evidence of translation for those suffering with inherited neurological disorders by the provision of molecular diagnostics, monitoring equipment and therapeutics (Rees et al 2006, Nat Genet 38:801-806; Carta et al 2012, J Biol Chem 287:28975-28985; James et al 2013, Neurobiol Dis 52:137-149). This leads to improved quality of life for patients, family members, carers and medical staff and can provide clinicians with insights into treatments for neurological disorders.
What will be done to ensure that they have the opportunity to benefit from this research?
The research we propose will increase our basic knowledge of the biology of synaptic transmission and translate across scientific disciplines. In the short term (1-3 years), our research will benefit research scientists and medical practitioners via the presentation of new research findings at invited lectures in the UK and internationally to scientific and lay audiences, by publishing in internationally-recognised scientific journals, and by engaging with national/international press, television and radio. The past impact of our work on inhibitory synaptic transmission is evidenced by: i) Invited talks and lectures given in this area - 30 for RJH; 13 for KH; 35 for TGS (2008-2013), including plenary lectures, awarded prizes and symposia; ii) Recent high-profile publications in this research area (e.g. Lemke et al 2014, Ann Neurology 75:147-154; Lemke et al 2013, Nat Genet 45:1067-1072; Avila et al 2013, Cell Rep 4:738-750; Manzke et al 2010, J Clin Invest 120:4118-4128; Smart 2010, Nat Neurosci 13:1043-1044; Shoubridge et al 2010, Nat Genet 42:486-488; Poulopoulos et al 2009, Neuron 63: 628-642; Miller et al 2008, Nat Struct Mol Biol 15:1084-10930). We are also working with Argenta to translate research findings into novel drug development initiatives for treating inflammatory pain and rhythmic breathing disorders, based on our previous MRC-funded research (G0500833; Harvey et al 2004, Science 304:884-887; Manzke et al 2010, J Clin Invest 120:4118-4128). At UCL, we are ideally placed to develop our findings and realise impact via: i) UCL Advances - a Centre for Entrepreneurship and Business Interactions; ii) UCL Business PLC - a dedicated technology transfer company; iii) UCL Consultants Ltd that facilitates consultancy contracts; iv) UCL Partners - one of five accredited academic health science systems in the UK translating cutting-edge research and innovation into measurable health gains for patients.
Our proposed project is likely to have broad-ranging impact on several groups. These include: academics (Academic Impact) and in the longer-term, healthcare professionals associated with neuroscience and mental health, the general/lay public who are directly or indirectly affected by epileptic disorders (particularly those caused by dysfunctional NMDA receptor signalling) and pharmaceutical companies concerned with drug development for epileptic disorders (Economic/Societal Impact).
How will they benefit from this research?
Academics will gain valuable insight into the biological roles and functional operation of NMDA receptors as well as achieving a better understanding of their roles in neurological disease. In addition, we will transfer technical and managerial skills to a new generation of research scientists, either directly (to postdoctoral RAs) or indirectly (to undergraduate or Ph.D. students involved in our research). This will also help to develop an international skill base through the subsequent mobility of these new scientists. Former Ph.D. students and postdoctoral scientists have benefitted in terms of their career trajectories by training in our labs and are now in posts at many other Universities (e.g. Montreal, Oxford, UCL), industry/healthcare (e.g. Pfizer, National Health Service) or funding agencies (e.g. Wellcome Trust). Our previous studies of inhibitory synaptic transmission have provided direct evidence of translation for those suffering with inherited neurological disorders by the provision of molecular diagnostics, monitoring equipment and therapeutics (Rees et al 2006, Nat Genet 38:801-806; Carta et al 2012, J Biol Chem 287:28975-28985; James et al 2013, Neurobiol Dis 52:137-149). This leads to improved quality of life for patients, family members, carers and medical staff and can provide clinicians with insights into treatments for neurological disorders.
What will be done to ensure that they have the opportunity to benefit from this research?
The research we propose will increase our basic knowledge of the biology of synaptic transmission and translate across scientific disciplines. In the short term (1-3 years), our research will benefit research scientists and medical practitioners via the presentation of new research findings at invited lectures in the UK and internationally to scientific and lay audiences, by publishing in internationally-recognised scientific journals, and by engaging with national/international press, television and radio. The past impact of our work on inhibitory synaptic transmission is evidenced by: i) Invited talks and lectures given in this area - 30 for RJH; 13 for KH; 35 for TGS (2008-2013), including plenary lectures, awarded prizes and symposia; ii) Recent high-profile publications in this research area (e.g. Lemke et al 2014, Ann Neurology 75:147-154; Lemke et al 2013, Nat Genet 45:1067-1072; Avila et al 2013, Cell Rep 4:738-750; Manzke et al 2010, J Clin Invest 120:4118-4128; Smart 2010, Nat Neurosci 13:1043-1044; Shoubridge et al 2010, Nat Genet 42:486-488; Poulopoulos et al 2009, Neuron 63: 628-642; Miller et al 2008, Nat Struct Mol Biol 15:1084-10930). We are also working with Argenta to translate research findings into novel drug development initiatives for treating inflammatory pain and rhythmic breathing disorders, based on our previous MRC-funded research (G0500833; Harvey et al 2004, Science 304:884-887; Manzke et al 2010, J Clin Invest 120:4118-4128). At UCL, we are ideally placed to develop our findings and realise impact via: i) UCL Advances - a Centre for Entrepreneurship and Business Interactions; ii) UCL Business PLC - a dedicated technology transfer company; iii) UCL Consultants Ltd that facilitates consultancy contracts; iv) UCL Partners - one of five accredited academic health science systems in the UK translating cutting-edge research and innovation into measurable health gains for patients.
Publications
Chen X
(2020)
Effects of GluN2A and GluN2B gain-of-function epilepsy mutations on synaptic currents mediated by diheteromeric and triheteromeric NMDA receptors.
in Neurobiology of disease
Comhair J
(2018)
Alpha2-Containing Glycine Receptors Promote Neonatal Spontaneous Activity of Striatal Medium Spiny Neurons and Support Maturation of Glutamatergic Inputs.
in Frontiers in molecular neuroscience
Harvey RJ
(2017)
Editorial: Molecular, Cellular and Model Organism Approaches for Understanding the Basis of Neurological Disease.
in Frontiers in molecular neuroscience
Kalscheuer VM
(2015)
Novel Missense Mutation A789V in IQSEC2 Underlies X-Linked Intellectual Disability in the MRX78 Family.
in Frontiers in molecular neuroscience
Lowrie M
(2017)
Levetiracetam in the management of feline audiogenic reflex seizures: a randomised, controlled, open-label study.
in Journal of feline medicine and surgery
Lowrie M
(2016)
Audiogenic reflex seizures in cats.
in Journal of feline medicine and surgery
Shoubridge C
(2019)
IQSEC2 mutation update and review of the female-specific phenotype spectrum including intellectual disability and epilepsy.
in Human mutation
Stödberg T
(2015)
Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures.
in Nature communications
Description | Horizon 2020 Research and Innovation grant |
Amount | € 6,000,000 (EUR) |
Funding ID | 666918 |
Organisation | European Commission |
Department | Horizon 2020 |
Sector | Public |
Country | European Union (EU) |
Start | 01/2016 |
End | 12/2018 |
Description | UCLB Proof of Concept award |
Amount | £25,000 (GBP) |
Funding ID | 530510 |
Organisation | UCL Business |
Sector | Private |
Country | United Kingdom |
Start | 06/2015 |
End | 07/2016 |
Title | Expression constructs for human NMDAR subtypes |
Description | We have generated a complete collection of full-length human NMDAR cDNAs for GluN1, GluN2A, GluN2B, GluN2C, GulN2D, GluN3A and GluN3B, as well as a collection of epilepsy mutants for GluN1, GluN2A and GluN2B. |
Type Of Material | Technology assay or reagent |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | We are conducting high-throughput electrophysiological screening of GluN1, GluN2A and GluN2B epilepsy mutants with Professor Joe Lynch at the University of Queensland to triage gain of function from loss of function mutations. |
Title | GluN2A and GluN2B knock-in mice |
Description | We have successfully generated knock-in mouse models for two missense mutations in different NMDA receptor subunits that removed voltage-sensitive Mg2+ blockade. Mutation N615K in GluN2A is associated with early-onset epileptic enchephalopathy, while mutation N615I in GluN2B is associated with West syndrome. Initial phenotyping of these mouse models has now taken place, and heterozygous GluN2A-N615K mice appear to be more agitated and have altered hind limb clasping upon suspension. By contrast, homozygous GluN2B N615I mice appear to show embryonic lethality - consistent with knockout models of Grin2b. |
Type Of Material | Model of mechanisms or symptoms - human |
Year Produced | 2018 |
Provided To Others? | No |
Impact | Further characterisation of these mouse lines is ongoing, they represent in vivo models for testing FDA-approved drugs (e.g. memantine) targeting gain-of-function mutations in NMDARs, with the aim of decreasing symptoms or preventing embryonic lethality. They will also be released to collaborators in due course for further phenotyping, e.g. using electrophysiology. |
Description | High-throughput screening for selective modulators of NMDA receptors containing the GluN2A and GluN2B subunits |
Organisation | University of Queensland |
Country | Australia |
Sector | Academic/University |
PI Contribution | Provision of human NMDAR GluN1, GluN2A, GluN2B subunit cDNAs (wild-type and mutant) |
Collaborator Contribution | Collaborators on this project are experts on the study of ligand-gated ions channels using patch-clamp electrophysiology and automated high-throughput screening using fluorescence-based approaches (QBI) and compound libraries derived from Australian natural products (IMB). Together, we will use high-throughput screening for selective modulators of NMDA receptors containing the GluN2A and GluN2B subunits. |
Impact | The collaboration resulted in a ULCB proof of concept award (£25,000) matched by Uniquest at the University of Queensland (https://www.uniquest.com.au/). The collaboration is mutlidisciplinary, involving genetics, electrophysiology and drug discovery. |
Start Year | 2015 |
Description | Homology modelling of ligand-gated ion channels |
Organisation | Birkbeck, University of London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provision of parts of GABA receptor structure, including information on those components necessarily deleted from the structure to improve the probability of crystallisation. |
Collaborator Contribution | Used homology modelling methods to compete the receptor structure, by including those parts of the structure previously deleted |
Impact | Fedele et al 2018 Nature Communications, in press |
Start Year | 2015 |
Description | School lecture on neurological disorders |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Debate in neurological disorders focused on epilepsy as a result of gene mutations |
Year(s) Of Engagement Activity | 2015,2018 |