Molecular mechanisms and synaptic functions of kainate receptor SUMOylation

Lead Research Organisation: University of Bristol
Department Name: Anatomy

Abstract

Efficient communication between neurones at synapses is required for proper brain function. This communication uses chemical transmitters and relies on the presence of receptor proteins at the synapse surface. A key feature of this system is that the number of receptors changes depending on the activity at the synapse. We have found that for one type of receptor called the kainate receptor, binding of the transmitter leads to attachment of a small protein called SUMO and this results in the receptor being removed from the surface into the cell (where it can no longer function) by a mechanism called endocytosis. Preventing SUMO attachment to kainate receptors blocks endocytosis. We go on to demonstrate that this process regulates communication between neurones. This is significant because kainate receptors regulate the general excitability of neurones and synaptic transmission and their dysfunction has been implicated in diseases such as epilepsy and Huntington's disease. In addition, we show that in addition to the receptor we have characterised, there are many other proteins present at synapses that undergo similar addition of SUMO. The aim of this proposal is to go on to work out some of the details of how this process works. Specifically we want to find out what change in the receptor allows it to bind SUMO and what molecular changes take place once the receptor is SUMOylated. We believe these experiments will have very important implications for understanding how synapses function. Furthermore, once we shed light on how kainate receptors are handled in normal cells we can then gain insight into what happens in injured or diseased cells and try to formulate strategies to compensate for or repair the problems.

Technical Summary

Kainate receptors (KARs) play crucial roles in the regulation of both excitatory and inhibitory neurotransmission. Their dysfunction has been proposed to underlie or contribute to many conditions that have a major impact on the quality and length of human life associated with profound economic and social consequences for society as a whole. Protein SUMOylation has important roles in the regulation of nuclear function. Far less well investigated are the targets and roles of SUMO modification outside the nucleus. In a paper in press at Nature, we have shown that SUMOylation of the GluR6a KAR subunit following agonist activation evokes kainate receptor endocytosis and modulation of synaptic transmission. The specific implications of our observations are that regulation of kainate receptors by SUMOylation will be involved in synaptic plasticity and neuronal excitability. In a wider context, our findings raise the prospect that protein SUMOylation, similar to other posttranslational modifications such as phosphorylation and ubiquitination, may be of fundamental importance in controlling the interactions and functions of synaptic target proteins. The aim of this proposal is to elucidate the upstream and downstream events and processes that lead to and result from GluR6 SUMOylation. We intend to explore the roles of phosphorylation as a potential trigger for SUMOylation and the effects SUMOylation has on PDZ interactions and protein complex formation. In addition, we aim to identify proteins specifically involved in the SUMOylation-dependent endocytic pathway and investigate the role of GluR6 SUMOylation in certain types of synaptic plasticity. We believe that these are novel and important issues that we are in a unique position to address.

Publications

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Wilkinson KA (2017) Commentary: Analysis of SUMO1-conjugation at synapses. in Frontiers in cellular neuroscience

 
Description In this project we have built on our previous data demonstrating the importance of ainate receptor SUMOylation and achieved significant advances towards understanding some of the key details underlying how the number of receptors at synapses change depending on the activity at that synapse.



We have characterised the role of SUMO in the regulation of one type of receptor, the kainate receptor that is critical for normal brain function. We demonstrated that for these receptors SUMOylation is downstream of phosphorylation that occurs on agonist binding, indicating the complex orchestration of protein interactions and modifications that occurs at synapses. We went on to show that this regulatory cascade is essential for kainate receptor synaptic plasticity.

In addition, we made good progress in uncovering other synaptic processes and systems that are regulated by SUMO. In particular, we show that functional expression of another critical receptor in the brain, the AMPA receptor, is indirectly regulated by SUMOylation and that overexpression of SENP1, an enzyme that deSUMOylates proteins, prevents homeostatic plasticity.



Overall, we feel this grant has been extremely successful and allowed us to discover and publish important new findings about how SUMO is involved in synaptic protein trafficking.



Our work has shed light on how kainate receptors are handled in normal cells and we have now extended this to investigate what happens in injured or diseased cells. In particular, we are using cell culture models of stroke to study how the SUMOylation systems and receptor trafficking processes in neurones respond to severe metabolic stress caused by oxygen and glucose deprivation. Although these experiments are ongoing they have already revealed important new insights into previously unsuspected processes involved in cell death and survival pathways. We anticipate that these data together with future studies may allow us to try to formulate strategies to attempt compensate for or repair SUMO dysfunction in damaged and diseased cells.

At a fundamental level the BBSRC funding enabled us to provide core, detailed information as to how synapses work under normal, activated and stressed conditions.
Exploitation Route A large number of new reagents were generated during the course of this grant and these have been published and, when requested, disseminated to scientists worldwide. More specifically, we have made a series of loss and gain of function point mutations in GluK2 subunit to investigate the relationship between phosphorylation and SUMOylation of kainate receptors.



In addition, we have constructed Sindbis virus vectors expression fluorophore tagged versions of these constructs for their efficient expression in dispersed cortical and hippocampal cell cultures as well as cite slice cultures.



As a spin off from the core aims of this project we also made tools in parallel to investigate the effects of neuronal SUMOylation on other key pre- and postsynaptic proteins. Again these comprise multiple targeted point mutants and expression constructs. We are in contact with GSK and NeuroSearch regarding likely SUMO targets and possible approaches to modulate these targets in disease. For example, with NeuroSearch we exchanged material relating to Huntingtin protein since this is a validated SUMO substrate. However, unfortunately NeuroSearch has withdrawn from all basic research to focus on clinical trials of a potential Huntington drug.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Provided a basis for possible therapeutic design targeting SUMOylation
First Year Of Impact 2014
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Alzheimer's Society studentship "Can protein SUMOylation be neuroprotective in Alzheimer's disease?"
Amount £80,000 (GBP)
Funding ID 170 
Organisation Alzheimer's Society 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2013 
End 01/2017
 
Description BBSRC - Mechanisms and consequences of presynaptic protein SUMOylation in the regulation of neurotransmitter release
Amount £398,028 (GBP)
Funding ID BB/K014358/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 10/2017
 
Description BRACE Studentship "Analysis of changes in protein SUMOylation in Alzheimer's and Down's syndrome brain: implications for reducing impaired AMPAR trafficking and synaptic dysfunction"
Amount £84,306 (GBP)
Organisation BRACE (Alzheimer's disease charity) 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2015 
End 01/2018
 
Description Can manipulating SUMOylation of PTEN correct aberrant AMPA receptor trafficking and synaptic dysfunction in Alzheimer's disease?
Amount £86,957 (GBP)
Organisation BRACE (Alzheimer's disease charity) 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2017 
End 01/2020
 
Description European Research Council - 'Mechanisms and consequences of synaptic SUMOylation in health and disease'
Amount £1,700,000 (GBP)
Funding ID 232881 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 03/2009 
End 02/2013
 
Description Manipulating protein SUMOylation for neuroprotection in Parkinson's disease
Amount £64,711 (GBP)
Funding ID G-1605 
Organisation Parkinson's UK 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2017 
End 05/2018
 
Description Protective mechanisms of protein SUMOylation in the heart
Amount £282,650 (GBP)
Funding ID PG/14/60/31014 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2015 
End 07/2018
 
Description Royal Society Newton Award "SUMOylation: novel neuroprotective approach for Alzheimer's disease"
Amount £56,500 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 05/2018
 
Description Royal Society-Wolfson Merit Award
Amount £75,000 (GBP)
Funding ID WRSA09R2/HLL 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start  
 
Title AMPAR subunit pHluorin cDNAs 
Description fluorophore tagged AMPAR subunits that allow real-time live cell imaging of receptor trafficking and surface expression 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Multiple papers have been published by other groups using these reagents 
 
Title SEP-GluRs 
Description flourophore tagged proteins and mutant cDNA clones 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact improved experimental design