Mechanisms of inhibitory GABA-A and glycine receptor clustering in health and disease
Lead Research Organisation:
University College London
Department Name: School of Pharmacy
Abstract
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.
Technical Summary
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.
Planned Impact
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.
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.
Publications
Lemke JR
(2014)
GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy.
in Annals of neurology
Avila A
(2013)
Glycine receptor a2 subunit activation promotes cortical interneuron migration.
in Cell reports
Morelli G
(2017)
Cerebral Cortical Circuitry Formation Requires Functional Glycine Receptors.
in Cerebral cortex (New York, N.Y. : 1991)
Golovko T
(2015)
Control of Inhibition by the Direct Action of Cannabinoids on GABAA Receptors.
in Cerebral cortex (New York, N.Y. : 1991)
Zhang Y
(2017)
Structure-Function Analysis of the GlyR a2 Subunit Autism Mutation p.R323L Reveals a Gain-of-Function.
in Frontiers in molecular neuroscience
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
Long P
(2015)
Missense Mutation R338W in ARHGEF9 in a Family with X-linked Intellectual Disability with Variable Macrocephaly and Macro-Orchidism.
in Frontiers in molecular neuroscience
Chiou TT
(2019)
Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability.
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
Leacock S
(2018)
Structure/Function Studies of the a4 Subunit Reveal Evolutionary Loss of a GlyR Subtype Involved in Startle and Escape Responses.
in Frontiers in molecular neuroscience
Description | CW Maplethorpe Postdoctoral Fellowship in Pharmacy |
Amount | £48,113 (GBP) |
Organisation | Maplethorpe Trust Fund |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2014 |
End | 09/2015 |
Description | GW Pharma PhD studentship |
Amount | £105,000 (GBP) |
Organisation | GW Pharmaceuticals |
Sector | Private |
Country | United Kingdom |
Start | 12/2014 |
End | 11/2017 |
Description | Joint funded PhD studentship/UCL/CeGaT GmbH |
Amount | £32,500 (GBP) |
Organisation | Center for Genomics and Transcriptomics (CEGAT) |
Sector | Private |
Country | Germany |
Start | 09/2012 |
End | 09/2015 |
Description | UCL Impact studentship |
Amount | £65,000 (GBP) |
Organisation | University College London |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2013 |
End | 09/2016 |
Description | Autoimmune defects in glycine receptors and clustering proteins gephyrin/collybistin |
Organisation | University of Pennsylvania |
Department | Department of Neurology |
Country | United States |
Sector | Academic/University |
PI Contribution | Provision of cDNA clones encoding human glycine receptors and clustering proteins gephyrin and collybistin for expression in heterologous cell lines for testing human auto-antibodies found in various autoimmune disorders. |
Collaborator Contribution | Frequency of GlyR a1-IgG positivity among stiff-man syndrome phenotype cases and control subjects. |
Impact | Joint publication describing seropositive cases (12% of total cases) including 9 with stiff-man syndrome (66% were also glutamic acid decarboxylase 65 IgG positive) and 1 with progressive encephalomyelitis with rigidity and myoclonus. Immunotherapy responses were noted more frequently in GlyR a1-IgG positive cases (6 of 7 improved) than in seronegative cases (7 of 25 improved). PubMed ID: 23090334. A second paper was submitted to JAMA Neurology in Sept 2014 showing that patients with severe optic neuritis develop antibodies to the GlyR alpha1 subunit. Collaboration is multidisciplinary involving neuroscience and immunology. |
Start Year | 2012 |
Description | Genetic defects in the RhoGEF collybistin |
Organisation | Max Planck Society |
Department | Max Planck Institute for Brain Research |
Country | Germany |
Sector | Academic/University |
PI Contribution | For these three studies we: i) Generated a targeting construct for the mouse collybistin gene (arhgef9); ii) characterised mis-spliced collybistin mRNAs in a patient cell line that resulted in disruption of gephyrin and GABA-A receptor clustering in cellular and neuronal models; iii) provided data showing that the human ARHGEF9 mutation G55A disrupts neuroligin 2 induction of collybistin-mediated gephyrin clustering. |
Collaborator Contribution | Our collaborators generated and characterised of a knockout mouse for the RhoGEF collybistin. Our collaborators characterised a balanced chromosomal translocation affecting the human collybistin gene. Our collaborators discovered that neuroligin 2 binds to gephyrin and functions as a specific activator of the RhoGEF collybistin. |
Impact | These three studies have revealed a pivotal role of collybistin in clustering of gephyrin at selected GABAergic synapses. Mutations in the collybistin gene (ARHGEF9) are now unlikely to represent a significant risk factor for hyperekplexia, but rather produce complex phenotypes in humans, encompassing epilepsy, anxiety, aggression and mental retardation. PubMed IDs: 17690689, 18615734, 19755106. |
Start Year | 2007 |
Description | Genetic defects in the RhoGEF collybistin |
Organisation | Max Planck Society |
Department | Max Planck Institute for Experimental Medicine |
Country | Germany |
Sector | Academic/University |
PI Contribution | For these three studies we: i) Generated a targeting construct for the mouse collybistin gene (arhgef9); ii) characterised mis-spliced collybistin mRNAs in a patient cell line that resulted in disruption of gephyrin and GABA-A receptor clustering in cellular and neuronal models; iii) provided data showing that the human ARHGEF9 mutation G55A disrupts neuroligin 2 induction of collybistin-mediated gephyrin clustering. |
Collaborator Contribution | Our collaborators generated and characterised of a knockout mouse for the RhoGEF collybistin. Our collaborators characterised a balanced chromosomal translocation affecting the human collybistin gene. Our collaborators discovered that neuroligin 2 binds to gephyrin and functions as a specific activator of the RhoGEF collybistin. |
Impact | These three studies have revealed a pivotal role of collybistin in clustering of gephyrin at selected GABAergic synapses. Mutations in the collybistin gene (ARHGEF9) are now unlikely to represent a significant risk factor for hyperekplexia, but rather produce complex phenotypes in humans, encompassing epilepsy, anxiety, aggression and mental retardation. PubMed IDs: 17690689, 18615734, 19755106. |
Start Year | 2007 |
Description | Genetic defects in the RhoGEF collybistin |
Organisation | Max Planck Society |
Department | Max Planck Institute for Molecular Cell Biology and Genetics |
Country | Germany |
Sector | Academic/University |
PI Contribution | For these three studies we: i) Generated a targeting construct for the mouse collybistin gene (arhgef9); ii) characterised mis-spliced collybistin mRNAs in a patient cell line that resulted in disruption of gephyrin and GABA-A receptor clustering in cellular and neuronal models; iii) provided data showing that the human ARHGEF9 mutation G55A disrupts neuroligin 2 induction of collybistin-mediated gephyrin clustering. |
Collaborator Contribution | Our collaborators generated and characterised of a knockout mouse for the RhoGEF collybistin. Our collaborators characterised a balanced chromosomal translocation affecting the human collybistin gene. Our collaborators discovered that neuroligin 2 binds to gephyrin and functions as a specific activator of the RhoGEF collybistin. |
Impact | These three studies have revealed a pivotal role of collybistin in clustering of gephyrin at selected GABAergic synapses. Mutations in the collybistin gene (ARHGEF9) are now unlikely to represent a significant risk factor for hyperekplexia, but rather produce complex phenotypes in humans, encompassing epilepsy, anxiety, aggression and mental retardation. PubMed IDs: 17690689, 18615734, 19755106. |
Start Year | 2007 |
Description | Localisation of IQSEC2/IQSEC3 |
Organisation | King's College London |
Department | Department of Anatomy |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Defined synaptic markers at excitatory and inhibitory synapses and antibodies recognising these targets. |
Collaborator Contribution | Exploring the synaptic localization patterns of guanine nucleotide exchange factors IQSEC1 and IQSEC3 in the mouse retina. |
Impact | Publication describing distribution patterns of IQSEC1 and IQSEC3 in retina. Confirmation that IQSEC3 colocalises with gephyrin and a subpopulation of inhibitory synapses expressing glycine receptors or GABA-ARs in retina. PMID: 22886754. |
Start Year | 2012 |
Description | Novel collybistin mutations in XLID |
Organisation | Greenwood Genetic Center (GGC) |
Country | United States |
Sector | Charity/Non Profit |
PI Contribution | Our group is functionally characterising a missense mutation (c.1012C-T; p.R338W) in in the collybistin gene (ARHGEF9) identified by a collaborating group. |
Collaborator Contribution | Identification of the ARHGEF9 c.1012C-T; p.R338W missense mutation. |
Impact | The ARHGEF9 p.R236W mutation is present in affected members of a published XLID family (Johnson et al 1998, J Med Genet 35:1026-1030) and has provided a resolution of the likely cause of disease for the family concerned, which has been studied since the early 1990s. This collaboration resulted in a joint publication, PubMed ID: 26834553. Collaboration is multidisciplinary, involving genetics and structure/function studies. |
Start Year | 2013 |
Description | BBC Health article |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Coverage of startle disease research outcomes and new research funding in awareness article on BBC Health website. Increased awareness of startle disease impacts on affected children and families; Enquiries from scientists, clinicians and members of the public. |
Year(s) Of Engagement Activity | 2013 |
URL | http://www.bbc.co.uk/news/health-18911272 |
Description | France Culture radio broadcast |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Radio broadcast 'Grande Traversée Agatha Christie' speaking about the 'Poisons of Agatha Christie', aired on France Culture 19th-23rd August 2013. Not applicable. |
Year(s) Of Engagement Activity | 2013 |
URL | http://www.franceculture.fr/emission-grande-traversee-agatha-christie-doc-appelez-un-doctueur-2013-0... |
Description | Highlighting MRC-funded research on UCL School of Pharmacy web portal |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Type Of Presentation | Paper Presentation |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | We have publicised new research findings via the UCL School of Pharmacy web portals, see e.g. http://www.ucl.ac.uk/pharmacy/news/startle Parents of individuals with hyperekplexia or referring clinicians contacting PIs by E-mail or letter regarding genetic screening. |
Year(s) Of Engagement Activity | 2006,2007,2008,2009,2010,2011,2012 |
Description | Outreach Event: 'Brain, Drugs and Rock 'N' Roll' |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Outreach Event: 'Brain, Drugs and Rock 'N' Roll' (12th March 2014): 'A level' students at selected London schools learned about careers in neuroscience, careers in pharmacy and neuroscience research at the UCL School of Pharmacy. Higher than expected interest in science degrees for 'A level' students from these local schools. |
Year(s) Of Engagement Activity | 2014 |
URL | http://www.ucl.ac.uk/pharmacy/research/neuroscience-in-health-and-disease/neuroscience-health-diseas... |
Description | Talk at Café Scientifique Event, UCL |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Type Of Presentation | Keynote/Invited Speaker |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Talk on genetics and wellbeing at a Café Scientifique Event, Roberts Building, UCL (19th April 2013) organised through the UCL Public Engagement Unit. The audience consisted of 30 members of the University of The Third Age from North London. Further invitation to talk at the UCL Science Society (http://www.ucl.ac.uk/science-society/), whose members are drawn from all branches of the Sciences |
Year(s) Of Engagement Activity | 2013 |
Description | Talk at the UCL Science Society |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Lecture at the UCL Science Society - Defective inhibitory neurotransmission in startle disease: some surprising findings (27th Feb 2014) audience made up of UCL students, staff, alumni and guests. After talk members of the UCL Science Society made further requests for information about my work and asked to visit my laboratory. |
Year(s) Of Engagement Activity | 2014 |
URL | http://www.ucl.ac.uk/pharmacy/research/disease-models-and-clinical-pharmacology/disease-models-news/... |