Mechanisms and consequences of presynaptic protein SUMOylation in the regulation of neurotransmitter release

The human brain is widely considered to be the most complex structure in the known universe. Despite this massive complexity, remarkable progress has been achieved towards understanding individual cells of the brain, how they communicate and how this communication is modified by development, experience, aging and disease. The brain is composed of neurons, which pass chemical signals to each other via specialised structures called synapses. Synapses are highly plastic and constantly undergo changes in the efficiency of information transfer. It is these changes in plasticity that underlie learning, memory and cognition. On the other hand, detrimental changes in synapses are responsible for many brain diseases including age-associated cognitive decline and dementia.

Synapses are composed of three basic components: the presynaptic terminal, which is activated by an electrical signal and converts this to a chemical signal by releasing a chemical (neurotransmitter) from the specialised structures called vesicles; the synaptic cleft, across which the neurotransmitter diffuses; and the postsynaptic membrane, which contains receptor proteins that detect the neurotransmitter and convert the chemical signal back to an electrical signal.

This project seeks to better understand the processes of neurotransmitter release at the presynaptic terminal. More specifically we are interested in a process called SUMOylation, in which a small protein, SUMO, is coupled to another 'target' protein to alter its function. We have already shown that protein SUMOylation at the presynaptic terminal affects the amount of neurotransmitter release. In this study we want to explore exactly which SUMOylated proteins cause these changes in release and how these changes are coordinated to tune synaptic function. We intend to focus on three proteins that are already well established as fundamental components of the neurotransmitter release process. Importantly, we have validated that they are modified by SUMO but the effects are entirely unknown.

We believe that our work is new, exciting and important because it directly addresses questions about how synapses operate. Increased understanding of the processes that control neurotransmitter release in normal healthy cells will also provide valuable information for what can go wrong, and potentially how to fix it, in aging and diseased synapses.

SUMOylation is the post-translational attachment Small Ubiquitin-like Modifier, a 98 amino acid peptide to target proteins. It is well established that SUMOylation plays a key role in nuclear processes such as transcriptional regulation and chromosome segregation. More recently we, and others, have demonstrated that protein SUMOylation is also a key determinant of synaptic function. For example, we showed that SUMOylation of the GluK2 subunit mediates one type of kainate receptor endocytosis to modulate synaptic excitability. We also reported that SUMOylation of presynaptic proteins can modulate neurotransmitter release but, at that time, we did not identify the proteins responsible. In subsequent work, which forms the basis of this application, we have identified syntaxin 1A, synapsin1a and RIM1a as SUMO targets that are critical to neurotransmitter release.

This purpose of this project is to determine the consequences of SUMOylation of each of these proteins for neurotransmitter release and synaptic function. We have already defined the SUMOylation sites on each of these proteins and made non-SUMOylatable mutants. Further we have successfully generated and validated shRNA constructs that effectively knock down endogenous syntaxin 1A, synapsin1a or RIM1a. In this project we will perform knockdown - rescue experiments using shRNA insensitive non-SUMOylatable mutants to investigate how SUMOylation of these substrates affects synaptic vesicle exocytosis and dynamics. Techniques will include molecular biology, protein biochemistry, immunocytochemistry, live wide-field and confocal live cell microscopy with lipophillic FM dyes and synaptophysin-pHluorin constructs, and electrophysiology.

We believe that this is a timely and important project that will increase understanding of the mechanisms of presynaptic vesicular release and neurotransmission.

Enhancement and transfer of academic knowledge: A key objective of our work is the effective and timely dissemination of results. We have a good track record of making freely available knowledge, reagents and resources. Phil Rubin, our grant funded lab technician promptly distributes requested tools and reagents, and we are acknowledged in many papers for these efforts. We will continue to publish in highly regarded journals as soon as the data permit. We shall also continue our regular participation in and organization of national and international conferences. For example, the PI is on the organizing committee of the ISN meeting in 2013. The applicants routinely present, communicate, and discuss their findings and establish new collaborations. The PI accepts regular invitations to speak at international conferences and the research co-I was an invited speaker at an international meeting in Holland in 2012. Progress summaries and links to original publications will be posted on the webpages of the School of Biochemistry.

Academic collaboration: We enjoy an extensive and active network of collaborators in Bristol and world-wide. Our findings impact on the work other labs at Bristol working on synaptic processes (e.g. ZI Bashir, J. Mellor, G. Collingridge, M. Ashby) and protein trafficking (e.g. J. Hanley, P. Cullen, G. Banting). Internationally, we have close links with S. Martin in Nice and C Mulle in Bordeaux for collaborations and access to transgenic animals, high-resolution light microscopy and specialised electrophysiology.

Information exchange with industry: We have long-standing collaborations with GSK and currently have a joint EU-funded student soon to spend 6 months at GSK in Singapore. In addition, we have had recent collaborations with UCB (Belgium) specifically on presynaptic proteins, Neurosearch and now Lundbeck on protein trafficking, and we are presently talking to Shionogi (Japan) regarding our work on SUMOylation and neuroprotection. We intend to expand such collaborations through regular networking, including with former members of the lab working in Pharma (e.g. Medimune, Novartis, Eli Lilly, GSK, Lundbeck).

Potential commercial exploitation: We do not currently foresee likely discoveries appropriate for commercialization but, if appropriate, we will consult with the Research and Enterprise Development office.

Research skills: This project will benefit researchers in our group and associated with our lab by collaborations and/or shared facilities and resources. There will be opportunities to develop their molecular, biochemistry, imaging and (through collaboration) electrophysiology skills. All eligible lab members are encouraged to attend BBSRC, University and faculty training courses to develop their general and specific skill sets resulting in enhanced career prospects.

Teaching and training: There is a skills training programme within our school and the applicants participate in formal and informal training of PhD students in molecular, biochemical and imaging techniques. Both applicants are involved in UG teaching and tutorials. The success of our teaching is assessed by student feedback and internal peer review in which we are highly ranked. This project provides excellent opportunities for research projects for final year undergraduate BSc students as well as Wellcome Trust and RCUK PhD students doing lab rotations. We regularly host overseas students, e.g. in the last two years we have had ERASMUS MSc students for 6 month placements from Germany and Holland.

Public engagement: The PI has routinely engaged in public lectures and outreach activities via
Bristol Neuroscience and the University's Centre for Public Engagement. Lab members are active in Brain Awareness Week and University open days. Press releases of our findings are, and will continue to be posted on university websites. The PIs are registered on the University's Directory of Experts.