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Latrohilin in the synapse: localisation trafficking and role in the regulation of synaptic vesicle pools. A fine structure study

Reference	BB/D523078/1
Principal Investigator / Supervisor	Professor Yuri Ushkaryov
Co-Investigators / Co-Supervisors	Dr Otto Berninghausen, Professor Colin Hopkins
Institution	Imperial College London
Department	Biological Sciences
Funding type	Research
Value (£)	324,604
Status	Completed
Type	Research Grant
Start date	01/09/2005
End date	31/08/2008
Duration	36 months

Abstract

We are interested in the molecular mechanisms regulating exocytosis of neurotransmitters and trafficking of synaptic vesicles. As a specific tool for these studies we are using alpha latrotoxin, a potent secretagogue from the venom of the black widow spider. This toxin binds to its presynaptic membrane receptor, latrophilin, and provokes intracellular signalling. Simultaneously, the wild type toxin forms membrane pores. To avoid this effect and to enable the studies of signal transduction and vesicle trafficking induced by the receptor action, we have started to employ a mutant toxin (LTXN4C), which binds and stimulates latrophilin but fails to form pores. Using this mutant, we have shown that activation of latrophilin leads to stimulation of G proteins and phosopholipase C. This enzyme cleaves membrane phosphoinositides and produces IP3 and diacylglycerol. These second messengers trigger two distinct signalling pathways: release of Ca2+ from intracellular stores, which serves as a trigger of vesicle exocytosis, and activation of protein kinase C and/or Munc13, which sensitise the vesicle fusion machinery to Ca2+. Although latrophilin regulates secretory activity via G protein-coupled signal transduction mechanisms, it is a very unusual receptor, whose molecular architecture has been revealed only recently, in our work. Latrophilin contains a cell adhesion-like extracellular N-terminal domain and a signalling (7-transmembrane) C-terminal domain. However, soon after its synthesis, this receptor cleaved into two fragments, corresponding to the two structural domains, which behave as independent cell-surface proteins. The fragments of this receptor interacts with opposite cells and/or extracellular matrix and provides docking sites on the cell surface for the second, signalling fragment and for endogenous agonists, thereby spatially an/or temporally defining sites for C-terminal signalling. We have also shown that this two-part, on-off receptor architecture allows eachfragment and their complex to follow distinct internalisation routes. Thus, the turnover of the latrophilin components cam be used to explore signalling and endocytotic pathways in the synapse. Upon stimulation by alpha latrotoxin, the latrophilin signalling mechanisms act specifically on the readily releasable pool (RRP) of synaptic vesicles in central synapses. At neuromuscular junctions, they cause burst-like exocytosis, a process that is also likely to involve RRP vesicles only. Very few other secretagogues (e.g. hypertonic sucrose) have such an exquisite specificity for the RRP, but they act through completely different molecular mechanisms. Thus, LTXN4C represents a unique tool for dissecting the presynaptic regulatory mechanisms and vesicle behaviour. In addition to the RRP, all nerve terminals contain other populations of synaptic vesicles, including the depot and resting pools, which are normally less active but play an important role in synaptic transmission. To date, vesicle exchange between the pools and been poorly understood but it can now be successfully studied using LTXN4C in comparison with other secretagogues. The results from these studies will be compared with experiments employing synaptic terminals from animals where latrophilin has been removed by gene knockout. The main experimental approach we are planning to apply in this study is fine morphological analysis using electron microscopy in conjunction with electrophysiological and biochemical detection of transmitter secretion triggered either by the wild type or the mutant alpha latrotoxins and by other exocytotic stimuli. Electron microscopical structural analysis use by us previously has proved instrumental in the elucidation of the toxin¿s mode of action. The proposed research is expected to further improve and extend our knowledge of the intricate molecular mechanisms involved in the regulation of synaptic vesicle exocytosis and recycling.

Summary

Neurones send thin processes (axons) terminating in thousands of tiny buttons (synaptic terminals) that organise the millions of neurones in the brain into an intricate array containing billions of connections (synapses). Each synaptic terminal behaves as a tiny but complex relay station where incoming electrical impulses are quickly converted into chemical signals ¿ packets of neurotransmitter molecules that are puffed into the cleft between the cells. When neurotransmitters reach the next cell, they stimulate it, leading to the propagation and modulation of the signal. Synaptic terminals are tightly controlled and yet very flexible: synapses can grow and shrink; nerve impulses of similar size may cause larger or smaller, faster of slower, secretion of neurotransmitter, depending on the internal characteristics of a particular synapse, additional external signals, and previous history of firing. To date, neuroscientists have compiled a large body of data on how synapses are built and which molecules are important for their work. However, the precise molecular mechanisms that control and modulate the incessant activity and flexibility of synaptic terminals remain unclear. In particular, it is not known how synapses can work so efficiently when vesicles often appear to be somewhat reluctant to react to incoming signals. We also would like to know more about how synaptic vesicles actually release their content of neurotransmitter and how they are renewed. In our attempts to understand these regulatory mechanisms, we use a very specific molecular tool, a neurotoxin from the black widow spider. This toxin (alpha latrotoxin) has be designed by nature to stimulate synaptic vesicles to release their neurotransmitter payload. We have identified a molecule in the membrane of synaptic terminals that mediates the toxin¿s action. This protein, latrophilin, is normally involved in the regulation neurotransmitter release, but it is hijacked by alpha latrotoxin to overstimulate synapses. We have modified this toxin to limit and control its poisonous effect. Now we are planning to use this novel tool to dissect the tiny synapse and try to understand how its vesicles are normally controlled. In order to be able to see the vesicles and any changes in their behaviour, we will use a very powerful technology that allows one to examine the structure of the synapse at high (macromolecular) resolution in the electron microscope. As a result of this study, we will make a further step in our quest to comprehend this complex and fascinating computer that works in our heads, the brain.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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