Vesicle Synaptobrevin

Tetanus is a disease caused by the release of neurotoxins from the anaerobic, spore-forming rod Clostridium tetani. The clostridial protein, tetanus toxin, possesses a protease activity which selectively degrades the pre-synaptic vesicle protein synaptobrevin, resulting in a block of glycine and y-aminobutyric acid (GABA) release from presynaptic terminals. Consistent with the loss of neurogenic motor inhibition, symptoms of tetanus include muscular rigidity and hyperreflexia. The clinical course is characterized by increased muscle tone and spasms, which first affect the masseter muscle and the muscles of the throat, neck and shoulders. Death occurs by respiratory failure or heart failure. 

All botulin neurotoxins act in a similar way. They only differ in the amino-acid sequence of some protein parts (Prabakaran et al., 2001). Botulism symptoms are provoked both by oral ingestion and parenteral injection. Botulin toxin is not inactivated by enzymes present in the gastrointestinal tracts. Foodborne BoNT penetrates the intestinal barrier, presumably due to transcytosis. It is then transported to neuromuscular junctions within the bloodstream and blocks the secretion of the neurotransmitter acetylcholine. This results in muscle limpness and palsy caused by selective hydrolysis of soluble A-ethylmalemide-sensitive factor activating (SNARE) proteins which participate in fusion of synaptic vesicles with presynaptic plasma membrane. SNARE proteins include vesicle-associated membrane protein (VAMP), synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP-25). Their degradation is responsible for neuromuscular palsy due to blocks in acetylcholine transmission from synaptic terminals. In humans, palsy caused by BoNT/A lasts four to six months. 

The sequence of events that result in neurotransmission of information from one nerve cell to another across the s)mapses begins with a wave of depolarization which passes down the axon and results in the opening of the voltage-sensitive calcium charmels in the axonal terminal. These charmels are frequently concentrated in areas which correspond to the active sites of neurotransmitter release. A large (up to 100 M) but brief rise in the calcium concentration within the nerve terminal triggers the movement of the synaptic vesicles, which contain the neurotransmitter, towards the synaptic membrane. By means of specific membrane-bound proteins (such as synaptobrevin from the neuronal membrane and synaptotagrin from the vesicular membrane) the vesicles fuse with the neuronal membrane and release their contents into the synaptic gap by a process of exocytosis. Once released of their contents, the vesicle membrane is reformed and recycled within the neuronal terminal. This process is completed once the vesicles have accumulated more neurotransmitter by means of an energy-dependent transporter on the vesicle membrane (Table 2.3). 

The decisive element in exocytosis is the interaction between proteins known as SNAREs that are located on the vesicular membrane (v-SNAREs) and on the plasma membrane (t-SNAREs). In the resting state (1), the v-SNARE synaptobrevin is blocked by the vesicular protein synaptotagmin. When an action potential reaches the presynaptic membrane, voltage-gated Ca "" channels open (see p. 348). Ca "" flows in and triggers the machinery by conformational changes in proteins. Contact takes place between synaptobrevin and the t-SNARE synaptotaxin (2). Additional proteins known as SNAPs bind to the SNARE complex and allow fusion between the vesicle and the plasma membrane (3). The process is supported by the hydrolysis of GTP by the auxiliary protein Rab. 

Synaptic exocytosis involves three SNARE proteins the R-SNARE synaptobrevin/VAMP (isoforms 1 and 2) on the vesicle, and the Q-SNAREs syntaxin (isoforms 1 and 2) and SNAP-25 on the plasma membrane (Figure 4). Since SNAP-25 has two SNARE-motifs, synaptobrevin, syntaxin, and SNAP-25 together have four SNARE-motifs. Synaptobrevins and SNAP-25 are relatively simple SNARE proteins that are composed of little else besides SNARE motifs and membrane-attachment sequences (a transmembrane region for synaptobrevin, and a cysteine-rich palmitoylated sequence for SNAP-25). Syntaxins, in contrast, are complex proteins. The N-terminal two-thirds of syntaxins include a separate, autonomously folded domain (the so-called Habc-domain), while the C-terminal third is composed of a SNARE motif and transmembrane region just like synaptobrevin. 

The experiments using these optical tools consistently supported the premise that synaptic vesicle exocytosis and endocytosis are tightly coupled processes. This largely kinetic coupling is also backed up by recent molecular evidence that proteins critical for exocytosis such as synaptotagmin and synaptobrevin, are also essential for triggering endocytosis (Poskanzer et al., 2003 Deak et al., 2004 Nicholson-Tomishima and Ryan, 2004). 

The SNAREs involved in the fusion of synaptic vesicles and of secretory granules in neuroendocrine cells, referred to as neuronal SNAREs, have been intensely studied and serve as a paradigm for all SNAREs. They include syntaxin 1A and SNAP-25 at the presynaptic membrane and synaptobrevin 2 (also referred to as VAMP 2) at the vesicle membrane. Their importance for synaptic neurotransmission is documented by the fact that the block in neurotransmitter release caused by botulinum and tetanus neurotoxins is due to proteolysis of the neuronal SNAREs (Schiavo et al. 2000). Genetic deletion of these SNAREs confirmed their essential role in the last steps of neurotransmitter release. Intriguingly, analysis of chromaffin cells from KO mice lacking synaptobrevin or SNAP-25 showed that these proteins can be at least partially substituted by SNAP-23 and cellubrevin, respectively (Sorensen et al. 2003 Borisovska et al. 2005), i.e., the corresponding SNAREs involved in constitutive exocytosis. 

Fig. 1 Structure of the neuronal SNAREs. Upper panel domain structure of the three neuronal SNARE proteins involved in synaptic vesicle fusion. Syntaxin 1A and SNAP-25 (contains two SNARE motifs) are associated with the presynaptic membrane, whereas synaptobrevin 2 is synaptic vesicle associated. The SNARE motifs form a stable complex (core complex) whose crystal structure has been analyzed (lower panel). In the complex, each of the SNARE motifs adopts an alpha-helical structure, and the four alpha-helices are aligned in parallel forming a twisted bundle (modified from Sutton et al. 1998). Stability of the complex is mediated by layers of interaction (—7 to +8) in which amino acids from each of the four alpha-helices participate (see text).

In addition to the proteins discussed above, neuronal SNAREs were reported to interact with numerous other proteins in a specific manner, but in most cases both the structural basis and the biological function of these interactions need to be defined. For instance, synaptophysin, a membrane protein of synaptic vesicles, forms a complex with synaptobrevin in which synaptobrevin is not available for interactions with its partner SNAREs syntaxin 1A and SNAP-25, suggesting that this complex represents a reserve pool of recruitable synaptobrevin (Becher et al. 1999) or regulates interactions between the vesicle-associated synaptobrevin and the plasmalem-mal SNAREs. Alternatively, it has been suggested that this complex is involved in synaptobrevin sorting to synaptic vesicles. 

Tomosyn is a soluble protein of 130 kDa with a C-terminal R-SNARE motif that is capable of replacing synaptobrevin in the neuronal SNARE complex. Most available data indicate that tomosyn negatively regulates exocytosis by competing with synaptobrevin in the formation of SNARE complexes (Brunger 2005), thereby leading to the inhibition of synaptic vesicle priming (McEwen et al. 2006). 

Artalejo CR, Elhamdani A, Palfrey HC (2002) Sustained stimulation shifts the mechanism of endocytosis from dynamin-1-dependent rapid endocytosis to clathrin- and dynamin-2-mediated slow endocytosis in chromaffin cells. Proc Natl Acad Sci USA 99 6358-63 Becher A, Drenckhahn A, Pahner I, Margittai M, Jahn R, Ahnert-Hilger G (1999) The synaptophysin-synaptobrevin complex a hallmark of synaptic vesicle maturation. J Neurosd 19 1922-31... 

Pellizzari R, Rossetto O, Lozzi L, Giovedi S, Johnson E et al. (1996) Structural determinants of the specificity for synaptic vesicle-associated membrane protein/synaptobrevin of tetanus and botulinum type B and G neurotoxins. J Biol Chem 271 20353-8 Pellizzari R, Mason S, Shone CC, Montecucco C (1997) The interaction of synaptic vesicle-associated membrane protein/synaptobrevin with botulinum neurotoxins D and F. FEBS Lett 409 339 12... 

Yamasaki S, Hu Y, Binz T, Kalkuhl A, Kurazono H et al. (1994) Synaptobrevin/vesicle-assodated membrane protein (VAMP) of aplysia califomica structure and proteolysis by tetanus toxin and botubnal neurotoxins type D and F. Proc Natl Acad Sci U S A 91 4688-92... 

Fig. 5 GPCR regulation of exocytosis downstream of Ca2+-entry. (a) Sequence of steps leading from recruitment to maturation of synaptic vesicles from a reserve pool (RP) to a readily-releasable pool (RRP) displaying slow (asynchronous) and fast (synchronous highly Ca2+-sensitive pool, HCSP synaptotagmin 1 (SYT 1) supported) components, (b) Protein-protein interactions of SNARES (SYX, syntaxin SYB, synaptobrevin and SNAP-2s-7S complex) and major putative regulatory proteins. Phosphoproteins are shown in shaded boxes (phosphorylation sites for PKA and PKC are indicated where known) with phosphorylation-dependent interactions depicted by arrows (increase indicated by filled arrows decrease indicated by open arrows). Circle-end connectors indicate a phosphorylation-independent or as yet unspecified interaction. Potential effects of interactions at various points of the sequence in A are discussed in the text.

The light chain, when separated from the heavy chain, behaves as an enzyme that selectively cleaves a peptide associated with synaptic vesicles. This peptide, synapt-obrevin, is required for docking and fusion of the vesicle during acetylcholine release. By enzymatically cleaving it, botulinum toxin renders vesicle docking and fusion impossible, and cholinergic neurotransmission comes to a halt. Because the light chain has enzymatic activity, just a few molecules can catalyze the destruction of synaptobrevin on thousands of vesicles. In this way, extreme potency is achieved via amplification of the process. 

Glutamate synapses are specialized for rapid vesicle fusion/neurotransmitter release. Fusion is initiated by SNARE proteins such as syntaxin-1, synaptobrevin-2, and SNAP-25 (see Chapter 2.4 this volume). Presynaptic gene expression, in general, is widely disturbed in schizophrenia (Mimics et al., 2000 Mimics et al., 2001 Hemby et al., 2002), including reduced expression of SNAP-25 RNA(Hemby et al., 2002) and... 

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