The role of NMDA receptor dysfunction in epileptic disorders

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.

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.

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.