Exocytosis neurotoxins

The most ingenious exocytosis toxins, however, come from the anaerobic bacteria Clostridium botulinum and Clostridium tetani. The former produces the seven botulinum neurotoxins (BoNTs) A-G the latter produces tetanus neurotoxin (TeNT). All eight toxins consist of a heavy (H) chain and a light (L) chain that are associated by an interchain S-S bond. The L-chains enter the cytosol of axon terminals. Importantly, BoNT L-chains mainly enter peripheral cholinergic terminals, whereas the TeNT L-chain mainly enters cerebral and spinal cord GABAergic and glycinergic terminals. The L-chains are the active domains of the toxins. They are zinc-endopeptidases and specifically split the three core proteins of exocytosis, i.e. the SNAREs (Fig. 1 inset). Each ofthe eight toxins splits a... 

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. 5 The snake PLA2 neurotoxin is depicted here as a snake, which binds to an active zone, i.e., a synaptic vesicle (SV) release site, and hydrolyses the phospholipids of the external layer of the presynaptic membrane (green) with formation of the inverted-cone shaped lysophospholipid (yellow) and the cone-shaped fatty acid (dark blue). Fatty acids rapidly equilibrate by trans-bilayer movement among the two layers of the presynaptic membrane. In such a way lysophospholipids, which induce a positive curvature of the membrane, are present in trans and fatty acid, which induce a negative curvature, are present also in cis, with respect to the fusion site. This membrane conformation facilitates the transition from a hemifusion intermediate to a pore. Thus, the action of the toxin promotes exocytosis of neurotransmitter (NT) (from the left to the right panel) and, for the same membrane topological reason, it inhibits the opposite process, i.e., the fission of the synaptic vesicle.

Bleck (1989) Clinical aspects of tetanus. In Simpson LL (ed) Botulinum neurotoxin and tetanus toxin. Academic Press, San Diego, CA, pp 379-98 Bonanomi D, Pennuto M, Rigoni M, Rossetto O, Montecucco C et al. (2005) Taipoxin induces synaptic vesicle exocytosis and disrupts the interaction of synaptophysin I with VAMP2. Mol Pharmacol 67 1901-8... 

Rigoni M, Schiavo G, Weston AE, Caccin P, AUegrini F et al. (2004) Snake presynaptic neurotoxins with phospholipase a2 activity induce punctate swellings of neurites and exocytosis of synaptic vesicles. J Cell Sd 117 3561-70... 

Graham ME, Fisher RJ, Burgoyne RD (2000) Measurement of exocytosis by amperometry in adrenal chromaffin cells effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics. Biochimie 82 469-79... 

Xu T, Binz T, Niemann H et al (1998) Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nat Neurosci 1 192-200... 

These paralytic effects have been attributed to the proteolytic activity of BoNT light chain (LC) on protein substrates required for vesicular exocytosis. BoNT LC inhibits neurotransmitter exocytosis through its zinc-dependent endoproteolytic activity. The LCs of the various neurotoxin serotypes possess distinct molecular targets within the peripheral cholinergic nerve termainals (Schiavo et al, 1992, 1993a, b, 1994, 1995 Yamasaki et al, 1994). The endoproteolytic activities of the different toxin LCs produce similar flaccid paralytic effects. 

The first report in this area compared the actions of botulinum neurotoxin and botulinum binary toxin on transmission in the phrenic nerve-hemidiaphragm preparation (Simpson, 1982). There was the expected finding that neurotoxin blocked transmission by blocking acetylcholine release from nerve terminals, but the binary toxin had no effect. Apart from showing that C2 toxin did not block exocytosis, this study showed that the toxin did not act on the diaphragm to block muscle twitch. This observation is in keeping with the fact that the predominant form of actin in striated muscle is not that which is ADP-ribosylated by C2 toxin. 

Bittner MA, DasGupta BR, Holz RW (1989 a) Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells. In J. Biol. Chem. 264 10354-60... 

Sanchez-Prieto J, Shira TS, Evans D, Ashton A, Dolly JO, Nicholls DG (1987) Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes. In Eur. J. Biochem. 165 675-81 Schiavo G, Montecucco C (1995) Tetanus and botulism neurotoxins isolation and assay. Methods Enzymol. 248 643-52... 

Tetanus toxin poisoning produces tetanus, i.e. muscle contractions resulting in spastic paralysis. In contrast, Botulinum neurotoxins cause botulism, which is characterized by flaccid paralysis. This difference reflects differences in the anatomical level of action of these toxins. TeTx acts primarily on the CNS where it blocks exocytosis from inhibitory glycinergic synapses in the spinal cord. Loss of inhibitory control results in motoneuron firing. BoNTs act primarily in the periphery where they inhibit acetylcholine release at the neuromuscular junctions. 

Link E, Blasi J, Chapman ER eta/. (1994) Tetanus and botulinal neurotoxins tools to understand exocytosis in neurons. Advances in Second Messenger and Phos-phoprotein Research 29 47-58. 

Niemann H, Blasi J, John R (1994) Clostridial neurotoxins new tools for dissecting exocytosis. Trends Cell Biol. 4 179-85. 

The t-SNARE SNAP-25 is also expressed in the P-cell and has been localized mainly to the plasma membrane (Sadoul etal., 1995). SNAP-25 was cleaved by treatment of SLO-permeabilized cells with botulinum toxins (BoNT) A or E. The two neurotoxins inhibited Ca "-induced insulin exocytosis but failed to abolish the process completely (Sadoul ef al., 1995). This could be due to the failure of the toxins to cleave SNAP-25 already complexed to other fusion proteins (escaping detection by Western blotting) or to the requirement for SNAP-25 at a penultimate step in insulin exocytosis. 

The use of permeabilized cells in the study of exocytosis is not restricted to the investigation of agents which do not cross normally the plasma membrane, such as the clostridial neurotoxins, peptides or recombinant proteins. This approach can be combined with the transient overexpression of proteins of interest, e.g., components of the fusion machinery. The method described below applies to insulin-secreting HIT-T15 cells (Lang efal., 1995). 

The protocol described above for TeTx has been applied successfully in our laboratory to study the effect of other clostridial neurotoxins on the exocytosis of insulin. Thus we have shown that pretreatment of insulin-secreting cells with BoNT/B, which cleaves VAMP-2 and cellu-brevin between the same amino acid residues as TeTx (Niemann et al., 1994), blocks Ca -induced secretion (Regazzi et al., 1995). The same protocol was also used to investigate the effect on insulin secretion of BoNT/A and BoNT/E, which cleave the t-SNARE SNAP-25 (Sadoul etai, 1995), and of BoNT/Cl which cuts syntaxin (Lang, Niemann and Wollheim, unpublished). 

For each BoNT serotype, the dichain form constimtes the active configuration of the neurotoxin the isolated LC and HC are devoid of systemic toxicity. The absence of toxicity is consistent with findings that the LC cannot gain access to the cytosol unless it is coupled to the HC and that the HC lacks the ability to inhibit neurotransmitter release (Stecher et al., 1989 Goodnough et al., 2002). The isolated LC does, however, remain enzymatically active as evidenced by its ability to inhibit exocytosis from permeabilized chromaffin cells (Stecher et al., 1989), by its ability to cleave SNARE proteins in cell-free assays (Adler et al., 1998), and by its capacity to inhibit ACh release in skeletal muscle when delivered by liposomes (de Paiva and Dolly, 1990). It is not clear whether any portion of the HC is translocated along with the LC, and if so, whether it exerts a role in enhancing the catalytic activity or stability of the LC. 

Anne, C., Turcaud, S., Blommaert, A.G., Darchen, F., Johnson, E.A., and Roques, B.P. 2005. Partial protection against Bomhnum B neurotoxin-induced blocking of exocytosis by a potent inhibitor of its metallopep-tidase activity. Chembiochem 6 1375-1380. 

Spider venom peptides, toxic peptides from spiders that mainly modulate neurotransmission. Spider venoms are rich in neurotoxins that influence ion channels, interfere with neurotransmitter exocytosis, or affect neurotransmitter binding. The most important families are the atracotoxins (36-68 amino acids) and the latrotoxins. Many spider venom peptides are translated as prepropeptides and post-translationally modified, e.g., by disulfide bridge formation and C- or N-terminal modification. Because of the high diversity of its constituents, the spider venom is sometimes regarded as a biogenic structurally constrained combinatorial peptide library where nearly all amino acids of the mature sequence may be mutated, with the exception of a few strictly conserved cysteine residues responsible for the three-dimensional fold of the toxin [G. Estrada etal, Nat. Prod. Rep. 2007, 24,145]. 

Type Cl and D botulinum neurotoxins as ADP-ribosyl transferases. As shown by Knight et al. (3), type D botulinum neurotoxin was able to inhibit exocytosis in cultured chromaffin cells. Fig. 1 represents the results of our experiments showing the time course of this inhibition. When cultured bovine adrenal chromaffin cells were incubated with type D botulinum neurotoxin, inhibition of acetylcholine-evoked catecholamine release appeared. This inhibition, however, did not occur instantaneously but appeared and increased with days of incubation, suggesting involvement... 

Amperometry at single PC 12 cells has also been used in conjunction with a genetic cell transfection protocol to examine the effects of toxin expression on basal and evoked exocytosis. PC 12 cells have been transfected with the specific endoprotease Botulinum neurotoxin Cl light chain (BoNT/Cl), which cleaves the proteins syntaxin and SNAP-25 [5], The molecular dissection of the mechanisms underlying exocytosis has been motivated by the SNARE hypothesis, which postulates that exocytosis requires the assembly of the plasma membrane proteins syntaxin 1, SNAP-25, and the vesicle associated membrane protein (VAMP) into a complex [5], This SNARE complex then acts as a receptor for cytosolic components of the proposed fusion machinery. Direct evidence for the role of the SNARE proteins in neurotransmission comes from molecular genetic studies in which syntaxin and VAMP have been shown to be required for neurotransmission in Drosophila [47 9] and Caenorhabditis elegans [50,51]. To assess the effects of the disruption of SNARE proteins on exocytosis in PC 12 cells, amperometry has been used in conjunction with a genetic cell transfection assay to establish a... 

An effective Botulinum neurotoxin-based drug delivery vehicle can be used to directly deliver toxin inhibitors into the intoxicated nerve terminal cytosol. The concept may possibly be utilized for drug delivery for other neuronal and neuromuscular disorders. Besides a BoNT therapeutic approach, this report also provides new fundamental knowledge of endocytosis and exocytosis as well as of BoNT trafficking in neurons. 

Neurotoxin A Blocks Synaptic Vesicle Exocytosis but Not Endocytosis at the Nerve Terminal. 1999, 147 1249-1260. 

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