Molecular control of synaptobrevin retrieval and its biological function by synaptophysin

Brain cells (neurones) communicate by releasing chemical neurotransmitters. Neurotransmitters are stored in small spherical compartments within neurones called synaptic vesicles (SVs). When neurones communicate, SVs fuse with the outer surface of the neurone causing neurotransmitter release. After neurotransmitter release these SVs are reformed by a process called endocytosis. The correct formation of SVs during endocytosis is essential for the maintenance of neurotransmitter release, since SVs with the wrong composition will be faulty for subsequent fusion. A critical part of SV generation is the packaging the correct proteins in the correct amounts into SVs. This is usually done by specific molecules called adaptor proteins, however in some cases additional molecules are required.

We have recently identified an essential role for the protein synaptophysin in coordinating the packaging of sybII into SVs during endocytosis. While these proteins are known to stick to each other, no-one knows which regions of the proteins are important for this. Other molecules are also suggested to be required to control sybII and synaptophysin packaging into SVs. These are AP180 and AP-2. We have proposed a working model whereby all four proteins stick together in a coordinated manner to control packaging of sybII and synaptophysin into SVs. This model will be tested in this application. Finally various different types of syb are found on SVs, all of which have specific jobs in the control of neurotransmitter release. Therefore synaptophysin may also control their packaging into SVs and subsequently their biological role.

We propose that synaptophysin is the central organiser in the packaging of different syb molecules into SVs during endocytosis. We will test this by a number of approaches. We will examine how syb movements differ when the gene encoding synaptophysin is removed from neurones grown on glass coverslips. We will also examine the simultaneous movement of altered forms of sybs and synaptophysin which do not stick to each other to see how their interaction controls their function. We will also monitor syb and synaptophysin movement in the absence of AP180 or AP-2. Finally we will determine how interfering with the normal function of synaptophysin in syb retrieval alters neurotransmitter release by detecting it indirectly via electrical changes in neighbouring neurones.

The combination of experiments outlined in this application will systematically dissect the role of synaptophysin in both the control of the packaging of different sybs into SVs but also its downstream functions in neurotransmitter release. This is very important, since a decrease in the efficiency of syb packaging into SVs is proposed to underlie a series of neurodegenerative and neurodevelopmental disorders such as Alzheimer's Disease, Parkinson's Disease and X-linked intellectual disability. It will also provide important leads outside this field, since the mutants created in this application can be used to examine other possible neuronal functions of synaptophysin such as the formation of connections between neurones in brain.

We have recently identified a key role for the SV protein synaptophysin protein in the retrieval of sybII during endocytosis in central nerve terminals. Synaptophysin and sybII are known interaction partners, however the molecular basis of their interaction is still undetermined. We propose a working model where synaptophysin presents sybII in a "retrieval competent" conformation, allowing a simultaneous interaction with adaptor molecules AP180 and AP-2 respectively, which will be tested in work outlined in this application. Multiple forms of syb reside on SVs, all performing specific roles in neurotransmitter release. We propose that synaptophysin may also control their retrieval and thus their neuronal function. In support we have key pilot data showing the syb homologue Vti1a is stranded at the plasma membrane of synaptophysin knockout neurones, a phenotype reversed by expression of the wild-type protein.

The major hypothesis underlying this application is that synaptophysin is the hub of a molecular complex which co-ordinates efficient retrieval of synaptobrevin homologues to direct multiple neuronal events. This will be tested via the simultaneous monitoring of sybII and synaptophysin traffic in wild-type and synaptophysin knockout neurones. The role of the reciprocal interactions between sybII, synaptophysin, AP180 and AP-2 will be determined via expression of interaction mutants, shRNA knockdown and inhibition by competitive peptides. Finally the biological role of synaptophysin in controlling neurotransmitter release via the traffic of multiple sybs will be assessed using an integration of live imaging and paired electrophysiological recordings in culture.

The outcome of these experiments will be the determination of the molecular role of synaptophysin in the control of multiple facets of neurotransmitter release.

Communications and Engagement
The major beneficiaries outside the academic community will be the commercial private sector, primarily pharmaceutical companies. Prof. Cousin has previously explored such interactions in research funded by CHDI and UCB Pharma. Work originating from this application will provide a platform for future interactions by 1) creating new links with potential private sector partners or 2) by exploiting existing partnership links to design and develop modulators of synaptophysin-dependent sybII traffic.

At key stages in the project critical junctures will arise where a commercial partner could add great value. At these points existing contacts (via Prof. Cousin) and possible new partners will be contacted with a view to entering partnership agreements. All of these discussions will be brokered by technology transfer staff at the University of Edinburgh.

Prof. Cousin will engage the wider general public in conjunction with the University of Edinburgh Press Office, which has an excellent record of communicating research highlights to the public. Information will also be circulated to current and prospective students as well as partner organisations within the public and private sectors.

Collaboration
The project will be managed by Prof. Cousin in collaboration with Dr. Gordon. The roles and responsibilities for day to day project management are explained in the justification section.

Exploitation and Application
Intellectual property management, licensing and technology and knowledge transfer will be handled by the research office at University of Edinburgh. Edinburgh Research and Innovation (ERI) has extensive expertise in the protection of intellectual property arising from grant funded research.

Capability
Prof. Cousin will be responsible for the majority of delivery of impact activities in close collaboration with ERI. The team at ERI are expert in the drafting and production of publications for the commercial sector to publicise licensing opportunities or partnership agreements. Previous patents from Prof. Cousin's research have gone through this process. Prof. Cousin has extensive experience in the commercialisation of research and in the development of links with commercial partners, with a number of outputs from his laboratory already patented. Recently he successfully negotiated a collaboration and exploitation agreement between his laboratory, Bio-Link (a life sciences commercialisation company), the Children's Medical Research Institute (Sydney, Australia) and The University of Newcastle (Australia) in the development of novel anti-epileptic pharmaceuticals.

ERI provides instruction and training for academics to prepare academics for media engagements and both Prof. Cousin and Dr. Gordon will be trained as required.

Impact activity deliverables and milestones
Pathways to impact will be evaluated every 6 months. Key milestones to be monitored will be a determination of 1) the molecular interface between synaptophysin and sybII, 2) the constituent components of the synaptophysin-sybII endocytic complex (and their roles) and 3) the physiological consequences of synaptophysin-mediated sybII retrieval in neuronal physiology. Achievement of these milestones will trigger negotiations with commercial partners and their research offices.

Resource for the activity
Resource implications have been outlined in the application. ERI has a technology transfer budget that will cover the initial costs of protecting output from the research.