The structural biology of synaptic connectivity: understanding the extracellular organizers of neurotransmission
Lead Research Organisation:
University of Oxford
Department Name: Wellcome Trust Centre for Human Genetics
Abstract
Neuronal circuits are essentially the biological substrate for all aspects of brain function. And synapses, the connecting points for neurons, hold the key to understanding these circuits. They are continuously remodelled in response to novel experiences, and this is likely the anatomical substrate for learning and formation of long-lasting memories.
Synapses have probably been studied more than any other cellular structure over the past century, with all the tools afforded by neuroscience in its broadest possible sense (from anatomy to genetics, physiology to biochemistry, cell to molecular biology). Structural biology employs a combination of methods, including the use of X-rays and electron beams, to define shapes and mechanisms of action of biological molecules. I aim to apply such techniques to study the special type of protein assemblies that span the so-called "synaptic cleft", the space that separates the outer membranes of two connected neurons. Traditionally, these proteins were studied in isolation, following purification and crystallization. This approach provided a wealth of information regarding the detailed atomic organization of receptors for small molecule neurotransmitters, for example. But if we are to understand how synapses work we must attempt to reach a higher level of complexity, that of multi-molecular assemblies, for the simple reason that in real life proteins never work alone. This is a key goal of my research.
Trans-synaptic protein assemblies are important in (at least) two ways: they provide structural support, physically tying together the pre- and post- synaptic neurons, and they provide avenues for communication between these cells. This latter aspect in particular is poorly characterised, and one of my main hypotheses is that such assemblies are highly dynamic, changing size and possibly shape in response to neuronal activity. From a basic science point of view, understanding such a mechanism should provide a completely novel view into how synaptic signalling actually works.
My work will also have an important impact in medicine, by providing snapshots into the molecular mechanisms that control synaptic stability. In humans, normal healthy aging is marked by variable degrees of neural deterioration and cognitive impairment. These are accompanied by a reduction in synapse numbers in regions of the brain involved in learning, memory and executive functions. Moreover, a malfunction of synaptic signalling and changes in synaptic morphology and number are linked to the majority of psychiatric and neurological disorders, from mental retardation and autism to Alzheimer's disease and addiction. Astonishingly, a recent report from the European Brain Council and the European College of Neuropsychopharmacology states that more than 160 million Europeans (~38% of the population in the 27 EU countries plus Switzerland, Norway and Iceland) suffer from mental disorders. For example, in the UK alone, according to Alzheimer's society, there are currently about 750,000 people suffering form dementia (one in 14 people over 65 years of age, and one in six over 80), costing the society in excess of £17 billion a year. These numbers are likely to go up as life expectancy increases. Surprisingly, recent reports revealed that a number of central nervous system disorders (including certain forms of encephalitis and ataxia) can be treated by immunotherapy. This is because they are triggered by autoantibodies against synaptic proteins. Working together with clinical immunologists and a local company specialized in high-throughput screening, my laboratory will help develop new tools for diagnosis, aiming to identify more conditions that can be tackled in this innovative and relatively straightforward way.
Synapses have probably been studied more than any other cellular structure over the past century, with all the tools afforded by neuroscience in its broadest possible sense (from anatomy to genetics, physiology to biochemistry, cell to molecular biology). Structural biology employs a combination of methods, including the use of X-rays and electron beams, to define shapes and mechanisms of action of biological molecules. I aim to apply such techniques to study the special type of protein assemblies that span the so-called "synaptic cleft", the space that separates the outer membranes of two connected neurons. Traditionally, these proteins were studied in isolation, following purification and crystallization. This approach provided a wealth of information regarding the detailed atomic organization of receptors for small molecule neurotransmitters, for example. But if we are to understand how synapses work we must attempt to reach a higher level of complexity, that of multi-molecular assemblies, for the simple reason that in real life proteins never work alone. This is a key goal of my research.
Trans-synaptic protein assemblies are important in (at least) two ways: they provide structural support, physically tying together the pre- and post- synaptic neurons, and they provide avenues for communication between these cells. This latter aspect in particular is poorly characterised, and one of my main hypotheses is that such assemblies are highly dynamic, changing size and possibly shape in response to neuronal activity. From a basic science point of view, understanding such a mechanism should provide a completely novel view into how synaptic signalling actually works.
My work will also have an important impact in medicine, by providing snapshots into the molecular mechanisms that control synaptic stability. In humans, normal healthy aging is marked by variable degrees of neural deterioration and cognitive impairment. These are accompanied by a reduction in synapse numbers in regions of the brain involved in learning, memory and executive functions. Moreover, a malfunction of synaptic signalling and changes in synaptic morphology and number are linked to the majority of psychiatric and neurological disorders, from mental retardation and autism to Alzheimer's disease and addiction. Astonishingly, a recent report from the European Brain Council and the European College of Neuropsychopharmacology states that more than 160 million Europeans (~38% of the population in the 27 EU countries plus Switzerland, Norway and Iceland) suffer from mental disorders. For example, in the UK alone, according to Alzheimer's society, there are currently about 750,000 people suffering form dementia (one in 14 people over 65 years of age, and one in six over 80), costing the society in excess of £17 billion a year. These numbers are likely to go up as life expectancy increases. Surprisingly, recent reports revealed that a number of central nervous system disorders (including certain forms of encephalitis and ataxia) can be treated by immunotherapy. This is because they are triggered by autoantibodies against synaptic proteins. Working together with clinical immunologists and a local company specialized in high-throughput screening, my laboratory will help develop new tools for diagnosis, aiming to identify more conditions that can be tackled in this innovative and relatively straightforward way.
Technical Summary
Central nervous system neurons engaged in excitatory synapses are connected by an intricate protein network that spans the synaptic cleft, provides structural support and modulates neurotransmission. The key target of this proposal is to understand the molecular structures, principles of higher order organization and functional mechanisms of two prototypical trans-synaptic assemblies. We will focus on complexes built around type IIa receptor protein tyrosine phosphatases (in particular RPTPsigma) as well as Delta-type glutamate receptors. Our key questions are: What are the rules of assembly in these trans-synaptic complexes? How dynamic are such structures? How do they influence synapse formation, stability and synaptic transmission?
We will combine structural biology techniques (X-ray crystallography, cryo-electron tomography) and quantitative live cell fluorescence microscopy to define the architecture of these complexes in an environment as close as possible to physiological, i.e. a cell membrane context. We will create structure-guided mutant constructs and test them in synapse formation assays and other functional paradigms (electrophysiology in whole cells and brain slices, and mouse models) in collaboration with specialist laboratories.
Furthermore, by exploiting the modular architecture of soluble "synaptic organizer" proteins, we will use structural information to create molecules with novel intermolecular interaction properties through domain recombination. As an alternative, similar tools will be designed by specifically crosslinking nanobodies raised against synaptic receptors. These macromolecular probes may allow the manipulation of the number and properties of excitatory and inhibitory synapses in neuronal circuits and the implementation of novel rules of synaptic communication and homeostatic plasticity, revealing the importance of specific neuronal connections.
We will combine structural biology techniques (X-ray crystallography, cryo-electron tomography) and quantitative live cell fluorescence microscopy to define the architecture of these complexes in an environment as close as possible to physiological, i.e. a cell membrane context. We will create structure-guided mutant constructs and test them in synapse formation assays and other functional paradigms (electrophysiology in whole cells and brain slices, and mouse models) in collaboration with specialist laboratories.
Furthermore, by exploiting the modular architecture of soluble "synaptic organizer" proteins, we will use structural information to create molecules with novel intermolecular interaction properties through domain recombination. As an alternative, similar tools will be designed by specifically crosslinking nanobodies raised against synaptic receptors. These macromolecular probes may allow the manipulation of the number and properties of excitatory and inhibitory synapses in neuronal circuits and the implementation of novel rules of synaptic communication and homeostatic plasticity, revealing the importance of specific neuronal connections.
Planned Impact
Defining the mechanisms of cognitive functions (such as learning, memory, thought, speech or consciousness) in cellular and molecular terms is certainly one of the big challenges in science but also of huge interest to society. We need to satisfy fundamental introspective questions (such as "How does my brain work?") but also hope to become capable of preventing, or delaying, age-related cognitive decline as well as curing neurological and psychiatric disorders.
Although this is essentially a basic science proposal, without doubt the long-term beneficiary of this work is the general public. According to the World Health Organization (http://www.who.int/mental_health/policy/services/integratingmhintoprimarycare/en/index.html), disorders linked to synaptic dysfunction currently affect hundreds of millions of people worldwide. This poses a huge burden to the world's economic output. Without the concerted efforts of academia and industry, this situation is likely to exacerbate considering the predicted increase of life expectancy worldwide. For example, within the UK, research commissioned by the Alzheimer's Society projects that the 2011 number of 750,000 people suffering from dementia may increase to 1 million by 2021 and 1.7 million by 2051.
A detailed understanding of the mechanisms that drive the formation, stability and function of CNS synapses would provide the solid grounding needed to develop such therapies. The crystal structures we will solve have the potential to guide the development of novel drugs, that may trigger fewer side effects compared to those designed using the traditional "silver bullet" approach. While my own laboratory is not focused on drug design, I am actively involved in a collaboration with MRC-Technology and MRC-LMB scientists to exploit our recent crystallographic results on AMPA receptors. Being funded by an MRC grant, there is an added benefit that patents for any therapies designed in this way would be owned by the public, thus ensuring that money spent by the NHS will go directly to a government agency and support further research.
Furthermore, I am particularly excited about the potential impact onto the "synaptic repair" field that may emerge from the use of synthetic synaptic "connectors", molecules that can cross-link pre- and post-synaptic receptors and as a result promote (or indeed reduce, depending on the choice of interactions) the number and/or strength of neuronal synapses.
Although this is essentially a basic science proposal, without doubt the long-term beneficiary of this work is the general public. According to the World Health Organization (http://www.who.int/mental_health/policy/services/integratingmhintoprimarycare/en/index.html), disorders linked to synaptic dysfunction currently affect hundreds of millions of people worldwide. This poses a huge burden to the world's economic output. Without the concerted efforts of academia and industry, this situation is likely to exacerbate considering the predicted increase of life expectancy worldwide. For example, within the UK, research commissioned by the Alzheimer's Society projects that the 2011 number of 750,000 people suffering from dementia may increase to 1 million by 2021 and 1.7 million by 2051.
A detailed understanding of the mechanisms that drive the formation, stability and function of CNS synapses would provide the solid grounding needed to develop such therapies. The crystal structures we will solve have the potential to guide the development of novel drugs, that may trigger fewer side effects compared to those designed using the traditional "silver bullet" approach. While my own laboratory is not focused on drug design, I am actively involved in a collaboration with MRC-Technology and MRC-LMB scientists to exploit our recent crystallographic results on AMPA receptors. Being funded by an MRC grant, there is an added benefit that patents for any therapies designed in this way would be owned by the public, thus ensuring that money spent by the NHS will go directly to a government agency and support further research.
Furthermore, I am particularly excited about the potential impact onto the "synaptic repair" field that may emerge from the use of synthetic synaptic "connectors", molecules that can cross-link pre- and post-synaptic receptors and as a result promote (or indeed reduce, depending on the choice of interactions) the number and/or strength of neuronal synapses.
People |
ORCID iD |
Alexandru Aricescu (Principal Investigator / Fellow) |
Publications
Altemose Nicolas
(2017)
A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis
in ELIFE
Aricescu AR
(2013)
Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology.
in Current opinion in structural biology
Berridge G
(2018)
Glutamate receptor d2 serum antibodies in pediatric opsoclonus myoclonus ataxia syndrome.
in Neurology
Chang VT
(2016)
Initiation of T cell signaling by CD45 segregation at 'close contacts'.
in Nature immunology
Coles CH
(2015)
Extracellular regulation of type IIa receptor protein tyrosine phosphatases: mechanistic insights from structural analyses.
in Seminars in cell & developmental biology
Davies B
(2017)
A point mutation in the ion conduction pore of AMPA receptor GRIA3 causes dramatically perturbed sleep patterns as well as intellectual disability.
in Human molecular genetics
Doody KM
(2015)
Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy.
in Science translational medicine
Elegheert J
(2016)
Structural basis for integration of GluD receptors within synaptic organizer complexes.
in Science (New York, N.Y.)
Elegheert J
(2018)
Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.
in Nature protocols
Elegheert J
(2017)
Structural Mechanism for Modulation of Synaptic Neuroligin-Neurexin Signaling by MDGA Proteins.
in Neuron
Elegheert J
(2017)
Structural Mechanism for Modulation of Synaptic Neuroligin-Neurexin Signaling by MDGA Proteins.
in Neuron
Farhy-Tselnicker I
(2017)
Astrocyte-Secreted Glypican 4 Regulates Release of Neuronal Pentraxin 1 from Axons to Induce Functional Synapse Formation.
in Neuron
Gallimore AR
(2016)
A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression.
in PLoS computational biology
Laverty D
(2019)
Cryo-EM structure of the human a1ß3?2 GABAA receptor in a lipid bilayer.
in Nature
Masiulis S
(2019)
GABAA receptor signalling mechanisms revealed by structural pharmacology.
in Nature
Matsuda K
(2016)
Transsynaptic Modulation of Kainate Receptor Functions by C1q-like Proteins.
in Neuron
Miller PS
(2017)
Structural basis for GABAA receptor potentiation by neurosteroids.
in Nature structural & molecular biology
Miller PS
(2014)
Crystal structure of a human GABAA receptor.
in Nature
Nakane T
(2020)
Single-particle cryo-EM at atomic resolution.
in Nature
Nolan R
(2017)
nandb-number and brightness in R with a novel automatic detrending algorithm.
in Bioinformatics (Oxford, England)
Robinson RA
(2021)
Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling.
in Cell
Scott S
(2019)
A structural perspective on GABAA receptor pharmacology.
in Current opinion in structural biology
Seiradake E
(2015)
Production of cell surface and secreted glycoproteins in mammalian cells.
in Methods in molecular biology (Clifton, N.J.)
Sente A
(2022)
Differential assembly diversifies GABAA receptor structures and signalling.
in Nature
Taylor J
(2015)
Factors influencing success of clinical genome sequencing across a broad spectrum of disorders
in Nature Genetics
Uchanski T
(2021)
Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.
in Nature methods
Yuzaki M
(2017)
A GluD Coming-Of-Age Story.
in Trends in neurosciences
Zhang P
(2018)
Heparan Sulfate Organizes Neuronal Synapses through Neurexin Partnerships.
in Cell
Description | HFSP Research Grant |
Amount | $350,000 (USD) |
Funding ID | RGP0065/2014 |
Organisation | Human Frontier Science Program (HFSP) |
Sector | Charity/Non Profit |
Country | France |
Start | 05/2014 |
End | 11/2017 |
Description | Bridge over troubled synapses: synthetic extracellular protein scaffolds for neuronal connectivity |
Organisation | German Centre for Neurodegenerative Diseases |
Country | Germany |
Sector | Public |
PI Contribution | Design and produce extracellular synaptic organisers. |
Collaborator Contribution | Test molecules we designed in animal models (physiology and behaviour). |
Impact | Research in progress. |
Start Year | 2014 |
Description | Bridge over troubled synapses: synthetic extracellular protein scaffolds for neuronal connectivity |
Organisation | Keio University |
Department | Department of Physiology |
Country | Japan |
Sector | Academic/University |
PI Contribution | Design and produce extracellular synaptic organisers. |
Collaborator Contribution | Test molecules we designed in animal models (physiology and behaviour). |
Impact | Research in progress. |
Start Year | 2014 |
Description | Dr Radu Aricescu: A novel diagnostic method for autoimmune disorders based on cell-derived vesicle microarrays |
Organisation | University of Oxford |
Department | Nuffield Department of Clinical Medicine |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | The aim of this project was to (1) Establish an efficient, reproducible and easy to scale-up procedure to generate cell-membrane derived outside-out vesicle preparations stable for long-term storage at 4C or, if possible, even dried, (2) Demonstrate that such vesicles are suitable for detection of autoantibodies in routine diagnostics. This will build on our successful preliminary work on AQP4 (shown below) and include known autoimmune targets such as glutamate, GABAB and glycine receptors, as well as other surface proteins (LGI1, CASPR2) and (3) Develop a procedure to improve immobilization of the vesicles displaying proteins of interest, using in-vivo biotinylation and streptavidin-coated slides or other substrates (i.e. membranes for potential dip-stick development). |
Collaborator Contribution | . |
Impact | Vesicle production from Aquaporin-4 (AQ4) transfected HEK cells has been improved and relative large preparations of membrane vesicles produced, suitable for 100-200 assays. Immuno-assay with AQ4 vesicles were optimised allowing blind screening of 100 patient sera and controls with very good correlation with diagnostic cell-based assay (major project goal achieved). Myelin Oligodendrocyte Glycoprotein (MOG) vesicle is in production for use in immuno-assay with MOG vesicles. Cloning of other neuronal receptor targets for use in the vesicle immuno-assays is on-going. Construction and validation of "AviFlag" tagging for binding to streptavidin was achieved (major project goal achieved) and we are establishing use of vesicles in a Streptavidin-coated micro-array system. A patent was filed in June 2014. The CiC project funding was used to successfully demonstrate exemplification of concept that has been included in the filing. Project will be launched to coincide with publication paper. |
Start Year | 2013 |
Description | Ion Channels and Diseases of Electrically Excitable Cells |
Organisation | University of Oxford |
Department | Department of Physiology, Anatomy and Genetics |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We are actively working on the crystallographyc analysis of several synaptic ion channels; we provide protein samples to clinicians to develop novel diagnostic tools for neurological autoimmune diseases. |
Collaborator Contribution | This collaboration resulted in a successful grant application to the Wellcome Trust ""Ion Channels and Diseases of Electrically Excitable Cells" (2008-2013, £6,452,907 Principal applicant Prof FM Ashcroft, on behalf of the OXION consortium, to which my lab belongs). This grant was extended for a further 5 year period, and currently funds a DPhil student in my lab (scholarship and lab expenses). |
Impact | This is very much work in progress. It led to a key publication in the field (Miller & Aricescu, Nature, 2014) and further structural results are expected in the near future. |
Start Year | 2008 |
Title | Method of detecting the presence or absence of autoantibodies |
Description | We developed a method, based on immobilised exosomes, for diagnostic testing of autoantibodies against a broad range of cell surface proteins. |
IP Reference | US20160139119 |
Protection | Patent application published |
Year Protection Granted | |
Licensed | No |
Impact | None yet, but this is still work in progress. |
Description | Interview for Chemical and Engineering News (on behalf of the American Chemical Society) |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Interview led to online article published by Chemical and Engineering News. Further requests for information and collaboration. |
Year(s) Of Engagement Activity | 2014 |
URL | http://esciencenews.com/sources/cen/2014/06/13/gaba.receptor.structure.solved |
Description | Interview for for NeuroPod (monthly Nature podcast, neuroscience-related) |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Further requests for media engagement. |
Year(s) Of Engagement Activity | 2014 |
URL | http://www.nature.com/neurosci/neuropod/index-2014-06-26.html |
Description | LifeLab DNA bracelets activity at Science Museum |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | During LifeLab Science tair I was part of DNA bracelets activity at Cambridge Science Museum. The bracelet represents short DNA sequences from organisms including a human, chimpanzee, butterfly, carnivorous plant or flesh-eating bacteria. The activity is for kids and for parents to learn in a fun way about DNA. The key message was that by sequencing DNA of different organisms we can get better understanding of the slight differences in genetic code between organisms and the relationship between genotype and fenotype. |
Year(s) Of Engagement Activity | 2018 |
Description | Piece written for Wellcome Trust Blog. |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Work presented to public with general science interest. Coverage on social media channels. |
Year(s) Of Engagement Activity | 2014 |
URL | http://blog.wellcome.ac.uk/2014/06/20/imageof-the-week-gaba-receptor/ |