Biological and therapeutic roles of glycine receptors containing the alpha2 or alpha3 subunits

Lead Research Organisation: School of Pharmacy
Department Name: Unlisted

Abstract

The central nervous system (CNS) 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) which then interacts with receptor molecules embedded in the cell membrane of a neighbouring neurone. Some types of these receptors (e.g. glycine 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. The molecular mechanisms that regulate synaptic transmission and nerve impulse activity are important in understanding normal and diseased states of the brain. Indeed, many major drugs act primarily via receptor/ion channels. The therapeutic nature of these agents provides a compelling reason for further understanding the molecular details of the structure and function of this receptor class.

This proposal will benefit research in this area by enhancing our knowledge concerning glycine receptors, inhibitory ion channels found mainly in the spinal cord and brainstem. Animal models and human studies of glycine receptors have shown that one receptor subtype (called alpha1) is involved in a rare neurological disorder (startle disease). By creating a knockout mouse where the gene for another subtype (alpha3) was disrupted, we found that this was important in inflammatory pain. New therapeutic targets for inflammatory and chronic pain are a current clinical need given that the long-term use of classical analgesics is hampered by severe side effects such as gastric ulcerations leading to significant mortality in the elderly or, for the COX2 inhibitors, a predisposition to heart attacks and stroke. Our major aims are: i) to characterise molecular variants and locations of the alpha2 and alpha3 GlyRs in the CNS; ii) to assess the contribution of the glycine receptor alpha3 subunit to inflammatory and chronic pain; iii) to discover drugs might positively modulate glycine receptors containing the alpha1 versus alpha3 subunits, the first step in developing new treatments for pain; iv) to discover the role of the alpha2 subtype by creating a knockout mouse which will lack these receptors. Taken together, our studies will inform the development of new therapeutic treatments for inflammatory and/or chronic pain and may uncover human disorders involving defects in the alpha2 subunit gene.

Technical Summary

We have recently revealed a new important role for inhibitory glycine receptors (GlyRs) containing the alpha3 subunit in prostaglandin-mediated inflammatory pain sensitisation (see Harvey et al., 2004, Science 304:884-887). This programme aims to: i) assess RNA editing and sites of expression of alpha2 and alpha3 subunit GlyRs; ii) examine the role of GlyR alpha3 in models of neuropathic pain; iii) study allosteric modulation of GlyRs by existing and novel compounds with potential therapeutic implications; and iv) generate a knockout model for the alpha2 GlyR subtype, which has been implicated in the formation of glycinergic synapses, cortical differentiation and rod photoreceptor development. This study will bring together molecular, behavioural, electrophysiological and gene knockout expertise to define the emerging biological and potential therapeutic roles of glycine receptors.

Publications

10 25 50
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Ganser LR (2013) Distinct phenotypes in zebrafish models of human startle disease. in Neurobiology of disease

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Heinze L (2007) Diversity of glycine receptors in the mouse retina: localization of the alpha4 subunit. in The Journal of comparative neurology

 
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 Glycine receptor alpha2 subunit knockout mice 
Description We successfully generated a glycine receptor alpha2 subunit mouse knockout line by homologous recombination in PC3 ES cells, removing Glra2 exon 7 by Cre-mediated recombination. After preliminary phenotyping at the University of Leeds, mice have been provided to the German Mouse Clinic (GMC) for further detailed phenotyping. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact Phenotype(s) found in Glra2 knockout mice have been characterised in detail by the GMC. We have two ongoing collaborations to examine the biological role of glra2 in more detail: i) with Catalina Betancur at the Universit Pierre et Marie Curie, we are exploring the hypothesis that glra2 defects are a rare cause of autism; ii) with Jean-Michel Rigo at Hasselt University, we are examining the role of glra2 in cortical interneuron tangential migration. The latter has recently given rise to publications showing that GlyR alpha2 subunit activation promotes cortical interneuron migration. PubMed IDs: 23954789; 24926615, 26891984. 
URL http://www.sciencedirect.com/science/article/pii/S2211124713003781
 
Title New mouse models of osteoarthritis and neuropathy 
Description New experimentally-induced mouse models of osteoarthritis and neuropathy were developed in C57Bl/6 mice and characterised at the behavioural and electrophysiological levels. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact These models enabled us to characterise the role of the glycine receptor alpha3 subunit in mouse models of inflammatory and chronic pain including: i) Neuropathic models - Partial Sciatic Nerve Ligation; ii) Inflammatory models - Persistent inflammatory pain: Freund's complete adjuvant (CFA), Acute inflammatory pain: carrageenan, capsaicin; iii) Arthritis models - rheumatoid arthritis (kaolin/carageenan), osteoarthritis (monosodium iodoacetate) and iv) cancer-induced bone pain. See PubMed IDs 19379487, 19915732. 
URL http://europepmc.org/abstract/MED/19379487
 
Description Mapping of glycine receptor expression patterns in retina 
Organisation Max Planck Society
Department Max Planck Institute for Brain Research
Country Germany 
Sector Academic/University 
PI Contribution Our research team developed subunit-specific antibodies for the GlyR alpha2, alpha3 and alpha4 subunits, which were provided to Professor Waessle for this collaborative project.
Collaborator Contribution Our collaborators mapped the expression patterns of GlyR alpha2, alpha3 and alpha4 subunits in retina
Impact These studies have revealed that the GlyR alpha4 subunit potentially forms a heteromeric receptor with GlyR alpha2. This co-distribution has since been confirmed in other tissues and has implications for our Glra2 knockout study, since GlyR alpha4 may compensate for loss of GlyR alpha2. PubMed IDs: 17154252, 19924257.
 
Description Phenotyping Glra2 knockout mice 
Organisation National Institute of Health and Medical Research (INSERM)
Department Inserm U1130 Neuroscience Department Paris Seine
Country France 
Sector Public 
PI Contribution Our team generated Glra2 knockout mice, where exon 7 had been removed by homologous recombination and devised this collaboration, to examine the role of GlyR alpha2 in cortical migration during synaptogenesis.
Collaborator Contribution Our collaborators at the University of Hasselt found that GlyRs are expressed by embryonic cortical interneurons in vivo, and that genetic disruption of Glra2 leads to interneuron migration defects. In a follow-up study, we found that ablation of Glra2 disrupts dorsal cortical progenitor homeostasis, resulting in an overall reduction of projection neurons that settle in upper or deep layers of the cerebral cortex. This depletion of cortical neurons leads to moderate microcephaly in newborn Glra2 knockout mice. Collaborators at INSERM U1130 found that Glra2 knockout mice exhibited deficits in object recognition memory and impaired long-term potentiation in the prefrontal cortex.
Impact The collaboration with the University of Hasselt was pivotal in understanding the biological role of GlyRs containing the a2 subunit. We discovered that extrasynaptic activation of a2 subunit GlyRs in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx. This signalling pathway modulates actomyosin contractility to fine-tune nuclear translocation during migration. The studies also suggest that defects in the human GlyR alpha2 subunit gene may lead to microcephaly in humans. PubMed IDs: 23954789; 24926615, 26891984. Collaboration with INSERM U1130 partners revealed a link between GlyRs a2 mutations and autism spectrum disorder. PubMed ID: 26370147. These collaborations collaboration are multidisciplinary, involving mouse genetics and behaviour, and neuronal migration analysis via imaging and electrophysiology.
Start Year 2011
 
Description Phenotyping Glra2 knockout mice 
Organisation University of Hasselt
Department BIOMED Research Institute
Country Belgium 
Sector Academic/University 
PI Contribution Our team generated Glra2 knockout mice, where exon 7 had been removed by homologous recombination and devised this collaboration, to examine the role of GlyR alpha2 in cortical migration during synaptogenesis.
Collaborator Contribution Our collaborators at the University of Hasselt found that GlyRs are expressed by embryonic cortical interneurons in vivo, and that genetic disruption of Glra2 leads to interneuron migration defects. In a follow-up study, we found that ablation of Glra2 disrupts dorsal cortical progenitor homeostasis, resulting in an overall reduction of projection neurons that settle in upper or deep layers of the cerebral cortex. This depletion of cortical neurons leads to moderate microcephaly in newborn Glra2 knockout mice. Collaborators at INSERM U1130 found that Glra2 knockout mice exhibited deficits in object recognition memory and impaired long-term potentiation in the prefrontal cortex.
Impact The collaboration with the University of Hasselt was pivotal in understanding the biological role of GlyRs containing the a2 subunit. We discovered that extrasynaptic activation of a2 subunit GlyRs in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx. This signalling pathway modulates actomyosin contractility to fine-tune nuclear translocation during migration. The studies also suggest that defects in the human GlyR alpha2 subunit gene may lead to microcephaly in humans. PubMed IDs: 23954789; 24926615, 26891984. Collaboration with INSERM U1130 partners revealed a link between GlyRs a2 mutations and autism spectrum disorder. PubMed ID: 26370147. These collaborations collaboration are multidisciplinary, involving mouse genetics and behaviour, and neuronal migration analysis via imaging and electrophysiology.
Start Year 2011
 
Description Phenotyping Glra3 knockout mice 
Organisation University of Göttingen
Country Germany 
Sector Academic/University 
PI Contribution Our team developed a provided standard and phospho-specific GlyR alpha3 subunit antibodies for this study, as well as Glra3 knockout mice. We also reconstituted the 5HT1A-GlyR signalling pathway in HEK293 cells, showing that activation of the 5HT1A receptor was capable of enhancing glycine-mediated currents mediated by GlyRs containing alpha3.
Collaborator Contribution Our collaborators found that GlyR alpha3 is expressed in identified respiratory neurons in the pre-Boetzinger complex and that GlyR alpha3 knockout mice have defective breathing patterns.
Impact GlyRs containing alpha3 modulate the activity of the respiratory network due to modulation by 5HT1A receptor stimulation of protein kinase A. This new signaling pathway is of therapeutic significance, since serotonin receptor 1A-directed pharmacotherapy was able to reverse opioid-induced apnea in wild-type but not Glra3 knockout mice. PubMed ID: 20978350. This collaboration was multidisciplinary, involving biochemistry, molecular biology, genetics and electrophysiology.
Start Year 2007
 
Description Presynaptic compensation in Glra3 knockout mice 
Organisation Louis Pasteur University
Country France 
Sector Academic/University 
PI Contribution Our research team used quantitative RT-PCR to determine that in GlyR alpha3 knockout mice, deleting Glra3 exon 7 decreases levels of GlyR alpha3 transcripts, but surprisingly no compensatory increase in GlyR alpha1, alpha2 or alpha4 subunit gene expression was observed. This observation lead us study presynaptic compensation using electrophysiological methods.
Collaborator Contribution Our collaborators determined that glycinergic synapses in lamina II of the dorsal horn show presynaptic compensation for the loss of GlyR alpha3.
Impact This study explains why GlyR alpha3 knockout mouse display an inflammatory pain phenotype: presynaptic compensation maintains glycinergic transmission via GlyR alpha1beta receptors in the GlyR alpha3 knockout, but alpha1 does not contain the PKA phosphorylation site modified by activation of the EP2 receptor. PubMed ID: 20092571. This collaboration was multidisciplinary, involving molecular biology, genetics and electrophysiology.
Start Year 2007
 
Description Subunit-specific modulators of glycine receptors 
Organisation University of Queensland
Country Australia 
Sector Academic/University 
PI Contribution Our research team provided expression constructs for rat and human GlyR subunits and advised on possible subtype-selective modulators.
Collaborator Contribution Our collaborators examined the pharmacology of possible subtype-specific modulators of GlyRs including picrotoxin/picrotin, dihydropyridines and cannabinoids.
Impact Different GlyR subtypes show distinct profiles for potentiation or inhibition by picrotoxin/picrotin, dihydropyridines and cannabinoids. PubMed IDs: 17714449, 18657556, 18755158. This collaboration was multidisciplinary, involving molecular biology and electrophysiology.
Start Year 2006
 
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 Distinguished Gowdey Prize Lecture in Pharmacology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact Attended postgraduate posters and talked to postgraduates about research following prize lecture.

Research collaborations
Year(s) Of Engagement Activity 2009
 
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 Rhine/Mainz/Max Planck plenary: Plasticity of synaptic and extrasynaptic inhibitory transmission 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Health professionals
Results and Impact This plenary lecture was delivered at the first major symposium between these German Universities and Max Planck. The PI talked to postgraduates about research following the plenary lecture.

Postgraduates visited the PIs laboratory.
Year(s) Of Engagement Activity 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 Visit to Croydon High School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact ~150 pupils attended an informal talk on neuroscience and pharmacology.

Pupils applied and two came to gain experience in the laboratory.
Year(s) Of Engagement Activity 2010