Mechanisms of inhibitory GABA-A and glycine receptor clustering in health and disease

The central nervous system is a complex, intricate network of nerve cells that transmit and receive messages. This communication occurs at specialised sites of contact known as synapses. At these sites, an arriving nerve impulse causes the release of a chemical neurotransmitter from the 'presynaptic' cell which then interacts with receptor molecules embedded in the cell membrane of a neighbouring 'postsynaptic' nerve cell. Some types of these receptors, called glycine and GABA-A receptors, are classified as inhibitory receptors because they have an integral chloride ion channel. The opening of these channels in response to the neurotransmitter alters the electrical state of the cell either transmitting or subtly altering the incoming nerve impulse. The mechanisms that regulate synaptic transmission are important in understanding normal and diseased states of the brain. Indeed, many anaesthetics, sedatives and anti-anxiety drugs in use or under development act primarily via GABA-A or glycine receptors. The therapeutic nature of these agents provides a compelling reason for understanding how these receptors are localised at synapses. We have previously shown that mutations in genes associated with synaptic clustering proteins, such as collybistin, cause anxiety, aggressive behaviour, epilepsy, sleep disturbances and intellectual disability. The reason that disruption of a single gene can cause such a range of symptoms is that loss of collybistin causes disruption and mislocalisation of several synaptic components, including a scaffolding protein known as gephyrin and certain inhibitory GABA-A receptors. However, the location of other inhibitory receptors was unaffected, suggesting that other mechanisms of synaptic clustering must exist. The aims of this research project are: i) To investigate the molecular basis of GABA-A receptor interactions with gephyrin and collybistin; ii) To establish whether motor proteins called kinesins that transport proteins to synaptic sites are crucial for this clustering process; iii) To find out if two additional synaptic proteins, IQSEC2 and IQSEC3, also contribute to the synaptic clustering of inhibitory receptors. It is our hope that a detailed understanding of the mechanisms underlying synaptic receptor clustering in health and disease will enable better diagnosis and treatment of individuals with anxiety, aggressive behaviour, epilepsy, sleep disturbances and intellectual disability. Our study will also have important implications for research into human disorders affecting the synaptic location of other neurotransmitter receptors.

Selected inhibitory GABA-A receptor and glycine receptor subtypes are clustered at inhibitory synapses via interactions with the scaffolding protein gephyrin, which in turn is targeted to inhibitory synapses by collybistin, a GDP-GTP exchange factor with neuroligin-dependent activity. We have shown that genetic defects in the human collybistin gene (ARHGEF9) give rise to a range of symptoms consistent with loss of several GABA-AR subtypes, including anxiety, seizures and intellectual disability. Consistent with this hypothesis, studies in knockout mice suggest that collybistin is dispensable for glycine receptor clustering but is required for clustering GABA-ARs containing the alpha1 and alpha2 subunits in the hippocampus and the basolateral amygdala. Loss of these GABA-AR subtypes is accompanied by increased anxiety and reduced spatial learning. Taken together, these findings strongly suggest that additional clustering factors are required for the synaptic localisation of other GABA-AR and GlyR subtypes. This project aims to examine the role of GEFs and gephyrin in inhibitory receptor clustering in health and disease. Our main objectives are to: i) Define the molecular nature of GABA-AR alpha1, alpha2 and alpha3 subunit interactions with gephyrin and collybistin; ii) Establish the role of collybistin-kinesin interactions in synaptic gephyrin clustering; iii) Characterise two novel GEFs (IQSEC2 and IQSEC3) that may be responsible for clustering GlyRs and other GABA-AR subtypes. This will be accomplished using a range of biochemical, molecular biology and confocal imaging techniques to provide key functional insights into the mechanisms underlying inhibitory GABA-AR and GlyR clustering in health and disease.

Who will benefit from this research?
The work proposed here is likely to have a wide-ranging impact on: i) research scientists and medical practitioners; ii) those in the general public who are directly or indirectly affected by pathologies involving dysregulation of GABAergic or glycinergic transmission (e.g. intellectual disability); iii) commercial entities engaged in drug development programmes (e.g. the pharmaceutical industry).

How will they benefit from this research?
The groups identified above will benefit when we present our new research findings in the public domain. In addition, we will transfer technical and managerial skills to a new generation of research scientists and practitioners either: i) directly (postdoctoral scientists, technical staff) or ii) indirectly (M. Pharm. and Ph.D. students, other postdoctoral scientists and staff) 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 in the lab have moved on to other University (e.g. UCL) and industry (e.g. IMS Health, Choice Pharma) positions. Our previous genetic studies inhibitory synaptic transmission have translated into positive impacts for individuals with inherited neurological disorders, such as improved molecular diagnostics and treatments (Rees et al 2006, Nat Genet 38:801-806; Kalscheuer et al 2009, Hum Mutat 30:61-68; Shoubridge et al 2010, Nat Genet 42:486-488) and indirect positive outcomes for the quality of life of family members, carers and medical staff.

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 large international meetings, invited lectures in the UK and abroad and publications in internationally recognised peer-reviewed journals. The past impact of our work on inhibitory synaptic transmission is evidenced by: i) the number of invited talks and lectures given on this subject area alone - thirty in the period 2004-2011 for RJH; three for KH including the 2011 Gordon Conference on 'Inhibition in the CNS'; ii) recent high-profile publications in this research area (e.g. Saiepour et al 2010, J Biol Chem 285:29623-29631; Shoubridge et al 2010, Nat Genet 42:486-488), iii) the level of citations by fellow scientists (RJH has an independently calculated H-index of 30, total citations: 3,011; including 172 citations for Harvey et al 2004, Science 304:884-887; KH has an H-index of 14, total citations 1,616; including 49 citations for Harvey et al 2004, J Neurosci 24:5816-5826). In the medium term (3-5 years), information on new disease genes and pathogenic mechanisms will translate into improved DNA-based diagnostics for individuals directly affected by pathologies involving dysregulation of GABAergic or glycinergic transmission. In the long term (5-10 years) benefits could include new therapeutics based on identified roles of inhibitory receptors and clustering proteins in health and disease. To foster these medium- and long-term goals, we will ensure that pharmaceutical companies are aware of our research and utilise the small molecule drug discovery programmes operated by MRC technology (MRCT). We have recently submitted inhibitory glycine receptors for consideration as targets for novel therapeutics in inflammatory pain and rhythmic breathing, based on our MRC-funded research (G0500833; Manzke et al 2010, J Clin Invest 120:4118-4128). Translating research outcomes into drug development programmes will assist the global economic performance and economic competitiveness of the UK.