Vincent O'Connor

Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.

A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP‐25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP‐25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS‐resistant SNARE core was compromised. This SDS‐resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP‐dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmins proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin‐containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.

On the structure of the ‘synaptosecretosome’ Evidence for a neurexin/synaptotagmin/syntaxin/Ca2+ channel complex

Recent experiments have identified interactions between presynaptic and synaptic vesicle membrane proteins, that might be important in organizing the components of the fast neurotransmitter release mechanism to ensure that the process follows a rapid time course. Here we extend previous investigations to show that in addition to the α‐latrotoxin receptor (neurexin) and synaptotagmin another presynaptic protein, syntaxin, co‐purifies on a α‐latrotoxin affinity column. This implies that syntaxin is associated with these two molecules in a complex; a conclusion supported by the immunoprecipitation of [125I]latrotoxin binding by syntaxin antibodies. In addition, antibodies against syntaxin and the α‐latrotoxin receptor immunoprecipitate [125I]ω‐conotoxin binding sites, indicating that calcium channels are associated with this complex. Thus, neurexin, synaptotagmin, syntaxin, and calcium channels can be found in a structure we propose to call the ‘synaptosecretosome’. The components of the synaptosecretosome, in association with additional proteins, are postulated to organize the process of neurotransmitter release.

SNARE protein recycling by αSNAP and βSNAP supports synaptic vesicle priming.

Neurotransmitter release proceeds by Ca(2+)-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca(2+) uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca(2+) sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.

The N-ethylmaleimide-sensitive fusion protein (NSF) is preferentially expressed in the nervous system

NSF and SNAPs (soluble NSF attachment proteins), originally identified as cytosolic components of intracellular vesicular transport mechanisms, have recently been implicated in Ca2+‐triggered neurotransmitter release from synaptic terminals. Here, we have investigated the temporal and spatial expression pattern of the rodent NSF and SNAP genes. A single transcript of 4.5 kb is highly expressed in rat brain, whereas only minor amounts of NSF mRNA are found in liver, kidney, heart, lung and skeletal muscle. In situ hybridisation revealed NSF transcripts as early as embryonic day 10 preferentially in the nervous system of mouse embryos. In the adult brain NSF is widely expressed with particularly high levels in the hippocampus. An identical expression profile was observed for α/β‐SNAP. Our data are consistent with a central function of NSF and SNAPs in neurotransmission.

Fusion complex formation protects synaptobrevin against proteolysis by tetanus toxin light chain

The clostridial neurotoxin, tetanus toxin, is a Zn2+‐dependent protease which inhibits neurotransmitter exocytosis by selective cleavage of the synaptic vesicle protein, synaptobrevin. Synaptobrevin is thought to serve as a receptor for two neuronal plasma membrane proteins, syntaxin and SNAP‐25, which in the presence of non‐hydrolyzable ATP analogs form a 20 S fusion complex with the soluble fusion proteins NSF and α‐SNAP. Here we show that synaptobrevin, when in this 20 S complex, or its 7 S precursor, is protected against proteolysis by the enzymatically active tetanus toxin light chain. Our data define distinct pools of synaptobrevin, which provide markers of different steps of vesicle/plasma membrane interaction.

Molecular Approaches to Neurotransmitter Release

The nervous system relies on the integration of electrical and chemical messages to signal information. The basic unit for this integration is the synapse. Here, an action potential conducted along the axon invades the presynaptic terminal, causing neurotransmitter release. The neurotransmitter released into the synaptic cleft activates receptor molecules that transduce this chemical signal into electrical events. The most extreme form of this process is found at fast chemical synapses where the delay between presynaptic transmitter release and postsynaptic response is less than 1 millisecond. This highly ordered process is fundamental to efficient point-to-point communication in the nervous system. In contrast to the events underlying action potential conduction and chemical signal transduction, our understanding of the molecular process responsible for neurotransmitter release is less clearly defined.’.* Despite large gaps in our understanding, electrophysiological analysis of the neuromuscular junction has provided a clear context into which components of the release process must fit. Studies of Katz and associates have indicated that synaptic release is characterized by a rapid Ca2+-dependent, quantal ~ecretion.~ This quantal nature implies that neurotransmitter is released in packets of defined size. As presynaptic terminals are rich in small synaptic vesicles (ssv),~ quantal release was postulated to result from a Ca2+-triggered exocytosis of neurotransmitter from ssv. Rapid freezing of stimulated neurons allowed ultrastructural definition of synaptic vesicles captured at various stages of fusion with the plasma membrane and went some way to confirming the “vesicle hypothesis” of transmitter release? In addition, a population of ssv are clustered at specialized regions of the presynaptic plasma membrane. These “active zones” are thought to contain a pool of readily releasable vesicles that are “docked” close to the channels that mediate the Ca2+ influx that triggers From the above description, it is clear that the process of neurotransmitter release requires (1) molecular components that target and dock ssv to release sites to ensure rapid release, (2) proteins that sense rising CaZ+ concentrations to trigger exocytosis in response to CaZ+ influx, and (3) fusion proteins that enable ssvs to fuse specifically with the presynaptic plasmalemma, the vesicle acceptor membrane in neurotransmit-

PROTEIN INTERACTIONS IMPLICATED IN NEUROTRANSMITTER RELEASE

Biochemical evidence indicates that the exocytotic release of neurotransmitters involves both evolutionary conserved membrane proteins, the SNAREs, as well as ubiquitous cytosolic fusion proteins, NSF and SNAPs. We have analyzed the biochemical properties and the physiological effects of these proteins. Our data suggest models how NSF, SNAPs and SNAREs may function in neurotransmitter exocytosis.