IMAGING GLUTAMATE IN THE BRAIN USING NOVEL FAST FLUORESCENT PROBES

Lead Research Organisation: St George's University of London
Department Name: Inst of Cardiovascular & Cell Science

Abstract

Glutamate is recognised as a major excitatory neurotransmitter in the central nervous system. Dysregulation of glutamate is involved in neuropsychiatric disorders like anxiety and depression, as well as acute and chronic neurodegenerative disorders such as Huntington's disease.
The development of a genetically-encoded glutamate sensor, termed iGluSnFR, which is based on the bacterial glutamate/aspartate binding protein (BP) and circular permuted enhanced green fluorescent protein (cpEGFP) represented a major breakthrough in monitoring glutamate. However, the kinetics of iGluSnFR are too slow to track high-frequency action potential (AP) firing. Our new variant iGluu with 5-fold faster kinetics was fast enough to show glutamate clearance from the synaptic cleft in between AP. In this proposal we will make use of this probe to not only visualize synaptic glutamate release but also uptake into astrocytes and compare the dynamics in health and Huntington disease using a mouse model (in collaboration with Rosemarie Grantyn, Berlin, Germany). While iGluu is a useful tool, it is still too slow to visualize the fast binding of glutamate of the AMPA receptor which is followed by the channel opening on the microsecond time scale. We generated a novel fluorescently labelled glutamate sensor, Fl-GluBP, with sub-millisecon glutamate binding kinetics. We plan to target this sensor to the extracellular site of the membrane to monitor real-time glutamate release in parallel to AMPAR response (collaboration with Thomas Oertner, Hamburg, Germany). Furthermore we will develop similar probes with altered properties (dynamic range, Kd) to match different glutamate levels (astrocytes, synaptic cleft, neurons).
The fast clearance of glutamate from the synaptic cleft is mainly done by transporters located in the plasma membrane of surrounding astrocytes. There the glutamate gets converted into glutamine and is subquentially release, taken up by the presynaptic terminal, converted into glutamate and packed into vesicles (glutamate-glutamine cycle). On the basis of the bacterial glutamine binding protein (GlNBP) we will use a similar approach as for Fl-GluBP to generate glutamine sensors (Fl-GlNBP), which will allow us to investigate the different stages of the glutamate-glutamine cycle by simultaneous imaging of astrocytes and neurons in brain cell culture. Additionally, Rosemarie Grantyn (Berlin, Germany) will use our probes to image glutamate and glutamine in astrocytes in the striatum, cerebellar cortex and subthalamic nucleus in HD and normal mice to determine their role in HD.

Technical Summary

A panel of fluorescent glutamate sensors labelled with synthetic fluorophores will be generated to have a range of affinities with fast kinetics that can be used in different cells and compartments to match the prevalent glutamate concentration. The approach developed for Fl-GluBP will be used to generate fluorescent glutamine sensors (Fl-GlNBP) by side-directed mutagenesis combined with a high through-put system. Both probes will be tested in cell cultures of HEK293T and imaged in primary neurons and astrocytes to reveal glutamate/glutamine dynamics at the cellular level. Furthermore Fl-GluBP, as well as our previously generated fast, genetically-encoding glutamate sensor iGluu, will be utilized to monitor glutamate in the synaptic cleft of hippocampal slices and corticostriatal slices derived from normal and HD model animals. These experiments will enable us to show in real-time the ultrafast release and binding of glutamate by AMPAR, while measurements in the striatum will shine light on the role of glutamate in HD.

Planned Impact

The proposed research will make both immediate and long-term academic and societal impact in a number of ways.
Academic community:
The research performed on this grant will have an impact on the academic community as it will increase our understanding of glutamate homeostasis in health and disease. The proposed research will make a contribution to the molecular mechanisms underlying signal transmission across the synaptic cleft and communication between neurons and nearby astrocytes. Furthermore, the research will provide data on the kinetics of signal transduction and insights into the AMPAR activation in situ. Additionally novel fast probes will be available for electrophysiologist to visualize glutamate as well as glutamine.
UK as international research location:
The generation of novel glutamate sensors is a highly competitive field with us as one of the few groups involved in Europe. In the past we successfully competed with our main competitor in the USA (HHMI, Janelia Research Campus) by generating faster calcium probes with altered affinities which we published in high ranking peer-reviewed journals. We will continue to strengthen UK and Europe as a research location in this field. Furthermore, the international collaboration between us and the groups in Germany will ensure that the scientific network is maintained even after Brexit. The collaborations will be enriched by visits and exchange of knowledge and technical skills among the researchers.
Students at St. George's:
Supporting research at St. George's, University of London, as one of the UK leading medical schools will allow undergraduate Bachelor's and post-graduate Master's degree students to have access to research by performing projects within the grant. This gives the students the opportunity to obtain insights into how research is conducted and make their own impact within the field, helping them to make a well informed decision on their future career. Furthermore, trained research staff can share their knowledge and techniques with the next generation of researchers.
Career of Researchers involved:
Dr Silke Kerruth is at an early stage in her career. Her first post-doctoral position was very successful with three manuscripts in preparation. Being further employed on the new BBSRC grant will give her the opportunity to further increase her skills in cell culture and imaging techniques and allow her to use her strengths by setting up a high-throughput screening system for glutamate and glutamine sensors. She will use the project to further strengthen her publication record to set her up for a career in science and to apply for a junior group leader position afterwards.
Catherine Coates will be promoted from a Research Technician to a Research Assistant giving her the opportunity to develop more independence and personal responsibly within the project. She will further expand her research skill set with dissection of brains to make primary cell cultures, patch-clamp technique and live-cell imaging. She will continue to be involved in producing data for publication and contributing to writing manuscripts for publication.
Public:
In general the function of the brain and the communication between neurons is highly interesting for the non-scientists. Our project will result in tools that make it possible to visualise the communication between nerve cells, providing and easy method to show and teach people about glutamates function as a neurotransmitter besides its commonly known use as a flavour enhancer.

Publications

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Title Ultrafast glutamate sensors 
Description We have made vectors for the expression of our ultrafast variants of iGluSnFR in cells and tissues. They are suitable for incorporation into viral vectors for in vivo imaging, for introducing into transgenic animals. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses. N. Helassa, C.D. Dürst, C. Coates, S. Kerruth, U. Arif, C. Schulze, J.S. Wiegert, M. Geeves, T. Oertner and K Török. (citations 9) Proc. Natl. Acad. Sci. U S A (2018) 115 (21) 5594-5599. doi: 10.1073/pnas.1720648115. High-speed imaging of glutamate release with genetically encoded sensors. C.D. Dürst, J.S. Wiegert, C. Schulze, N. Helassa, S. Kerruth, C. Coates, M. Geeves, K Török and T.G. Oertner. Nature Protocols (2019) In press 
 
Description Ultrafast glutamate sensors for synaptic release monitoring 
Organisation University Medical Center Hamburg-Eppendorf
Department Center For Molecular Neurobiology Hamburg
Country Germany 
Sector Academic/University 
PI Contribution Torok's group has generated fast variant glutamate sensors iGlu(fast) and iGlu(ultrafast) by rational design and site directed mutagenesis. The fast sensors were charactersied with respect to their biophysical properties: fluorescence dynamic ranges, equilibrium constants, response kinetics, temperature dependencies of the response rates were determined. The kinetic mechanisms was resolved. Glutamate binding was titrated on cell surfaces and the in situ affinities were determined.
Collaborator Contribution Thomas Oertner's group (Hamburg) has applied our fast glutamate sensors iGlu(fast) and iGlu(ultrafast) to hippocampal slices and monitored glutamate release at individual synapses of Shaffer collaterals. In response to high frequency stimulation protocols, the probes were found to resolve high frequency (100 Hz) glutamate release events revealing new information about the dynamics of synaptic glutamate. iGlu(ultrafast) allowed demonstration of complete clearing of synaptic glutamate between each release. Moreover, postsynaptic AMPA receptor response depression was found to be correlated with depression of glutamate release during high frequency action potential firing. Greater recovery of AMPAr response than that of glutamate release further indicated sensitisation of receptor response on recovery.
Impact Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses Nordine Helassa, Celine Durst, Catherine Coates, Silke Kerruth, Urwa Arif, Christian Schulze, J. Simon Wiegert, Michael Geeves, Thomas Oertner, Katalin Torok Proc. Natl. Acad. Sci. U S A (2018) 115 (21) 5594-5599. doi: 10.1073/pnas.1720648115. Epub 2018 May 7. PMID: 29735711 High-speed imaging of glutamate release with genetically encoded sensors. C.D. Dürst, J.S. Wiegert, C. Schulze, N. Helassa, S. Kerruth, C. Coates, M. Geeves, K Török and T.G. Oertner. Nature Protocols (2019) In press
Start Year 2015
 
Description Ultrafast glutamate sensors for synaptic release monitoring in Huntington's Disease 
Organisation Charité - University of Medicine Berlin
Country Germany 
Sector Academic/University 
PI Contribution We generated a virus construct of our ultrafast glutamate sensor.
Collaborator Contribution Prof Rosemarie Grantyn's group compared glutamate retrieval at single synapses in the striatum of normal and HD mice by fast imaging.
Impact Single synapse indicators of impaired glutamate clearance derived from fast iGluu imaging of cortical afferents in the striatum of normal and 2 Huntington (Q175) mice. A. Dvorzhak, N. Helassa, K. Török, D. Schmitz and R. Grantyn. J. Neuroscience (2019) Feb 28. pii: 2865-18. doi: 10.1523/JNEUROSCI.2865-18.2019. [Epub ahead of print] PMID: 30819797
Start Year 2018