Dysfunction of GABA and glycine transporters in human neurological disease

Lead Research Organisation: School of Pharmacy
Department Name: Pharmacology

Abstract

The central nervous system is a complex, intricate network of nerve cells (neurones) whose primary function is to 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 neurone. Some types of these receptors (e.g. glycine and GABA-A receptors) possess specific ion-permeable channels. The opening of these channels in response to neurotransmitter alters the electrical state of the cell either transmitting or subtly altering the incoming nerve impulse. Neurotransmitters are then recovered from the synapse by transporters located in neighbouring glial cells or the corresponding presynaptic cell. The mechanisms that regulate synaptic transmission and nerve impulse activity are important in understanding normal and diseased states of the brain. Indeed, many drugs in use or under development act primarily via GABA or glycine receptors and their transporters. The therapeutic nature of these agents provides a compelling reason for further understanding the molecular details of the structure and function of these proteins. This proposal will benefit research in this area by enhancing our knowledge concerning transporters for GABA and glycine. In a recent study we were able to show that genetic defects in the glycine transporter GlyT2 were responsible for causing a rare illness called hyperekplexia. This affects newborn children and is characterised by noise or touch-induced seizures which result in breath-holding episodes. In some instances hyperekplexia can lead to brain damage or sudden infant death. Our major aims are: i) to study the consequences of GlyT2 mutations to reveal how these defects disable the transporter, and to investigate whether defects in a glycine transporter found on synaptic vesicles (VIAAT) or proteins that associate with GlyT2 can also cause hyperekplexia; ii) to determine whether genetic mutations in a second glycine transporter, GlyT1, is responsible for cases of a different childhood illness, glycine encephalopathy, which can lead to severe brain damage or death; iii) since defects in GABA receptors are found in some types of epilepsy, we will investigate whether mutations in GABA transporter genes also cause epilepsy. It is our hope that a detailed understanding of the genetic defects responsible for these illnesses will enable better diagnosis and treatment of affected individuals.

Technical Summary

We have recently proven that a human neurological disorder, hyperekplexia, can be caused by mutations in the gene encoding the presynaptic glycine transporter GlyT2 (Rees et al 2006, Nature Genetics 38:801-806). Prior to this, hyperekplexia was typically associated with mutations in the genes for postsynaptic inhibitory glycine receptor subunits and associated clustering proteins. Having conclusively demonstrated that hyperekplexia can be triggered by presynaptic deficits, this raises the intriguing possibility that presynaptic causes of disease may also exist in related disorders, such as idiopathic generalised epilepsies, where mutations in postsynaptic inhibitory GABA-A receptor subunit genes have already been identified. This programme aims to examine the role of GABA and glycine transporters in hyperekplexia, glycine encephalopathy and idiopathic generalised epilepsies. For glycinergic synapses, we will examine the functional consequences of newly identified GlyT2 hyperekplexia mutations and variants of GlyT2 generated by alternative splicing. We will also perform genetic screening of genes required for presynaptic vesicular packaging of glycine (e.g. the vesicular inhibitory amino acid transporter VIAAT) and two GlyT2 interacting proteins (ULIP-6 and syntenin-1) thought to be important for localisation and turnover of GlyT2. Since knockout mice for a second glycine transporter (GlyT1) show phenotypic similarities to glycine encephalopathy, we will establish whether defects in the human GlyT1 gene underlie this severe disorder. For GABAergic synapses, we will examine genes crucial for GABA vesicular packaging and uptake (i.e. VIAAT and neuronal/glial GABA transporters) in a cohort of ~1000 patients with idiopathic generalised epilepsies. Structural and functional deficits in these proteins will be studied using appropriate cellular models and functional assays (e.g. neurotransmitter uptake assays and electrophysiology). This study will bring together genetic and molecular expertise to define the causes of these neurological disorders, while providing functional insights into inhibitory neurotransmitter transporters.

Publications

10 25 50
 
Description Action Medical Research grant
Amount £124,835 (GBP)
Funding ID 1966 
Organisation Action Medical Research 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2012 
End 02/2014
 
Description Bloomsbury Colleges PhD studentship/Bloomsbury Consortium
Amount £58,020 (GBP)
Organisation University College London 
Department School of Pharmacy
Sector Academic/University
Country United Kingdom
Start 10/2009 
End 09/2012
 
Description Epilepsy Research UK Fellowship
Amount £198,520 (GBP)
Organisation Epilepsy Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2011 
End 05/2014
 
Description Epilepsy Research UK Project grant
Amount £98,230 (GBP)
Organisation Epilepsy Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2011 
End 05/2013
 
Description Joint funded PhD studentship/UCL/Uni Research AS Norway
Amount £35,000 (GBP)
Organisation Uni Research AS 
Sector Charity/Non Profit
Country Norway
Start 10/2012 
End 09/2015
 
Description Joint funded PhD studentship/UCL/VU University Amsterdam
Amount £30,000 (GBP)
Organisation Free University of Amsterdam 
Sector Academic/University
Country Netherlands
Start 10/2010 
End 09/2013
 
Description MRC DTG four-year PhD studentship
Amount £81,750 (GBP)
Funding ID G0800111 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2008 
End 09/2012
 
Description MRC Project Grant
Amount £1,015,020 (GBP)
Funding ID G0800498 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2009 
End 12/2011
 
Title Hyperekplexia/Epilepsy DNA samples 
Description During this study we have recruited a significant number of additional DNA samples from patients with hyperekplexia from clinical referrals. We have also taken receipt of the collaborative idiopathic generalised epilepsy cohorts from Australia, Scotland and London. These are currently being analysed for mutations in GABA transporters. 
Type Of Material Biological samples 
Provided To Others? No  
Impact These samples are proving valuable in genetic and structure/function studies and have resulted in additional outputs in 2012 and 2013. See PubMed IDs: 22753417, 22700964, 23184146 and 24108130. 
URL http://europepmc.org/abstract/MED/22753417
 
Description GABA-A receptor gamma2 subunit gene mutations in Dravet syndrome and epilepsy 
Organisation Vanderbilt University
Department Department of Neurology
Country United States 
Sector Academic/University 
PI Contribution While pre-screening genomic DNA samples from Dravet syndrome and GEFS+ epilepsy for analysis in presynaptic GABA transporter genes, we have uncovered likely disease-causing variants in the GABA-A receptor gamma2 subunit gene (GABRG2).
Collaborator Contribution Our collaborators will work on in-depth electrophysiology of GABRG2 mutations found in Dravet syndrome and GEFS+ epilepsy
Impact New pathogenic variants in causative genes, leading to feedback to health professionals, patients and carers.
Start Year 2010
 
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 Glycine transporter defects in cattle 
Organisation University of Liege
Country Belgium 
Sector Academic/University 
PI Contribution Congenital muscular dystonia 2 (CMD2) is a recessive disorder in Belgian Blue cattle that has a severe phenotype, similar to hereditary hyperekplexia in humans, resulting in postnatal death due to respiratory failure. Consistent with the effect of previously characterised missense mutations in human GlyT2, our team found that the L270P substitution did not affect GlyT2 membrane trafficking, but abolished [3H]glycine uptake.
Collaborator Contribution Our collaborators used whole-genome screening approaches to reveal that CMD2 is caused by a missense mutation (T809C) in exon 4 of the glycine transporter GlyT2 gene, resulting in a L270P substitution in the third membrane-spanning domain of GlyT2.
Impact Identification of this mutation had an immediate translation into breeding practice, allowing marker-assisted selection against CMD2 by avoiding 'at risk' matings. We have also recently identified CMD2 cases in the UK in collaboration with the Animal Health and Veterinary Laboratories Agency (AHVLA). PubMed IDs: 18344998, 18707791.
Start Year 2008
 
Description Identification of startle disease genes in dogs 
Organisation Clemson University
Department Department of Genetics and Biochemistry
Country United States 
Sector Academic/University 
PI Contribution One aspect of our project was the identification of new genetic causes of startle disease/hyperekplexia. However, sequencing of candidate genes related to presynaptic glycine uptake (VGAT, syntenin-1 and ULIP6) was unproductive. In collaboration with researchers at UCSD and the University of Clemson, examined two unresolved canine disorders with similarity to startle disease. Our contributions to these studies included candidate gene analysis, mapping of microdeletions, breakpoint cloning, Southern blots and MLPA analysis.
Collaborator Contribution Provision of genomic DNA samples for canine startle disease and Episodic Falling Syndrome. Use of SNP arrays to map the physical location of the canine Episodic Falling Syndrome locus and provision of control genomic DNA samples.
Impact We demonstrated that a 4.2 kb microdeletion in the glycine transporter GlyT2 gene (SLC6A5) caused startle disease in Irish Wolfhounds (PubMed ID: 21420493) and that a 15.7 kb deletion in BCAN, encoding the brain-specific extracellular matrix proteoglycan brevican, is associated with Episodic Falling Syndrome (PubMed ID: 21821125). These studies have allowed the introduction of diagnostic tests for these disorders via Laboklin (http://www.laboklin.co.uk/), improving animal welfare. The Episodic Falling Syndrome study also represents the first report of a genetic disorder involving a neuronal-specific extracellular matrix proteoglycan. For this reason, BCAN is now a candidate gene for genetic analysis in human disorders with similar clinical presentations, such as startle disease, paroxysmal exercise-induced dyskinesia and episodic ataxia. This collaboration was multidisciplinary, involving genetics and veterinary phenotyping.
Start Year 2010
 
Description Identification of startle disease genes in dogs 
Organisation University of California, San Diego (UCSD)
Department Department of Pathology
Country United States 
Sector Academic/University 
PI Contribution One aspect of our project was the identification of new genetic causes of startle disease/hyperekplexia. However, sequencing of candidate genes related to presynaptic glycine uptake (VGAT, syntenin-1 and ULIP6) was unproductive. In collaboration with researchers at UCSD and the University of Clemson, examined two unresolved canine disorders with similarity to startle disease. Our contributions to these studies included candidate gene analysis, mapping of microdeletions, breakpoint cloning, Southern blots and MLPA analysis.
Collaborator Contribution Provision of genomic DNA samples for canine startle disease and Episodic Falling Syndrome. Use of SNP arrays to map the physical location of the canine Episodic Falling Syndrome locus and provision of control genomic DNA samples.
Impact We demonstrated that a 4.2 kb microdeletion in the glycine transporter GlyT2 gene (SLC6A5) caused startle disease in Irish Wolfhounds (PubMed ID: 21420493) and that a 15.7 kb deletion in BCAN, encoding the brain-specific extracellular matrix proteoglycan brevican, is associated with Episodic Falling Syndrome (PubMed ID: 21821125). These studies have allowed the introduction of diagnostic tests for these disorders via Laboklin (http://www.laboklin.co.uk/), improving animal welfare. The Episodic Falling Syndrome study also represents the first report of a genetic disorder involving a neuronal-specific extracellular matrix proteoglycan. For this reason, BCAN is now a candidate gene for genetic analysis in human disorders with similar clinical presentations, such as startle disease, paroxysmal exercise-induced dyskinesia and episodic ataxia. This collaboration was multidisciplinary, involving genetics and veterinary phenotyping.
Start Year 2010
 
Description Mapping glycine and GABA-A receptor expression in human brain 
Organisation University of Auckland
Country New Zealand 
Sector Academic/University 
PI Contribution Our team provided antibodies against components of inhibitory synapses (e.g. glycine receptors) to enable accurate mapping of inhibitory receptor distributions in human brain.
Collaborator Contribution Our collaborators provided postmortem human brain tissue from the New Zealand Neurological Foundation Human Brain Bank and expertise and training in immunohistochemistry on human brain sections.
Impact Collaboration with the New Zealand Neurological Foundation Human Brain Bank, known internationally for the very high quality of formalin-fixed and fresh frozen tissues. These studies have also provided valuable insights into the inhibitory receptor subtypes implicated in hyperekplexia and epilepsy. PubMed IDs: 17444490, 18085588, 19915682, 19950251.
Start Year 2007
 
Title Genetic tests for canine neurological disorders 
Description Genetic tests for canine neurological disorders including startle disease in Irish Wolfhounds and Episodic Falling Syndrome in Cavalier King Charles spaniels. See PubMed IDs: 21420493 and 21821125. These tests have been made available to the general public via commercialisation (UCL Business) with Laboklin Laboratory for Clinical Diagnostics. The Episodic Falling test has been protected under patent EP2522744 (http://www.google.com/patents/EP2522744A1). 
IP Reference EP2522744 
Protection Patent application published
Year Protection Granted 2012
Licensed Yes
Impact These tests have seen good take-up and have impacted on breeding practices and animal welfare, with the Irish Wolfhound Foundation recently commenting in a letter to the School: 'this is the first clinically useful test for a genetic disease in our breed'. See also awareness articles at: http://www.iwfoundation.org/articles_detail.html?item_id=38&year=2009
 
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 Brain Awareness Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact The School of Pharmacy currently hosts the London Chapter of the Society for Neuroscience (http://www.sfnlondonchapter.org/) which organises activities such as annual meetings, seminars and young scientist workshops during Brain Awareness Week, with the aim of engaging and educating a new generation of neuroscientists and raising public awareness and interest in neuroscience research.

Staff and PhD students becoming members of the London Chapter of the Society for Neuroscience.
Year(s) Of Engagement Activity 2010
 
Description Highlighting MRC-funded research on ILS Swansea website 
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 MRC-funded research resulting in notable publications or achievements is routinely highlighted on the home page of the ILS Swansea website (http://www.swan.ac.uk/ils/), see e.g. http://www.swan.ac.uk/ils/research/biomed/neuroscienceandmolecularpsychiatry/molecularneuroscience/

Raising awareness of MRC-funded research with the general public, staff, other stakeholders and M.Pharm. students. Parents of individuals with hyperekplexia or referring clinicians contacting PIs by E-mail or letter regarding genetic screening.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
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
 
Description School of Pharmacy Annual Review 
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 In an article entitled 'Focus on Neuroscience: Genetic research brings new insights into CNS disorders', we reviewed our work on glycine receptors and hyperekplexia in lay language.

Increased awareness of MRC-funded research into glycine receptors and hyperekplexia at the School of Pharmacy and other report recipients.
Year(s) Of Engagement Activity 2008
 
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/...
 
Description Wales Epilepsy Research Network (WERN) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact Additional public engagement activity was undertaken via the Patient Research Development Group (RDG) within the Wales Epilepsy Research Network (WERN). This project was discussed at quarterly meetings involving service users, patient carers, parents of Sudden Unexpected Death in Epilepsy (SUDEP) cases and health professionals involved in epilepsy care. Approximately 30 people are involved in each meeting to discuss how to develop health and social outcomes in biomedical studies, e.g. the impact of genetic diagnosis/explanations.

Increased awareness of potential presynaptic and postsynaptic causes of startle disease/idiopathic epilepsies.
Year(s) Of Engagement Activity 2009,2010,2011