Clostridial neurotoxins are proteins that are produced by the anaerobic bacteria Clostridium tetani (tetanus toxin) and Clostridium botulinum (botulinum neurotoxins). Whereas tetanus toxin (TeTx) comprises a single molecular species, different strains of Clostridium botulinum produce seven different types of botulinum neurotoxin (desig-
noted BoNT/A , B, CI, D, E, F, and G) that were originally differentiated using immunological methods, and that represent different proteins. TeTx and BoNTs differ in their clinical manifestations, clinical symptoms 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 ¡unctions.
structure Despite the differences in their clinical manifestations, the clos tridial neurotoxins comprise a group of homologous proteins with similar mechanisms of action. They are synthesized as single-chain precursors of 150 kDa which are biologically inactive. Active toxin is generated by proteolytic cleavage at a position approximately one third downstream from the N-terminus. A di-chain molecule is generated that consists of the smaller N-terminal portion (approx. 50 kDa, designated light, or L chain) and the larger C-terminal fragment (approx. 100 kDa, designated heavy, or H chain). The chains are held together by a single disulfide bond and by non-covalent forces. During poisoning of neurons, each of the two chains has a different function. The H chain is responsible for recognition and uptake of the toxin by the peripheral nerve terminal, whereas the L chain is responsible for the final toxic effect (Mochida et a/., 1989; Ahnert-Hilger et a/., 1990; Poulain eta/., 1990; Niemann, 1991). poisoning pathway Numerous studies using a variety of experimental model systems have contributed to the elucidation of the poisoning pathway of the toxins (see Niemann et a/., 1994; Ahnert-Hilger and Bigalke, 1995; and this volume, Chapter 14, for more details). Briefly, the following steps can be summarized:
1. Recognition and binding of the toxin to the plasma membrane of the nerve terminal: Binding occurs spontaneously and with high affinity to specific receptors which include both ganglioside and protein components (Montecucco, 1986; Middlebrook, 1989; Nie-mannn, 1991; Nishiki eta/., 1994).
2. Entry of the toxin into the nerve terminal: Both TeTx and BoNTs enter the nervous system preferentially at the neuromuscular ¡unction. Bound toxins are internalized by the nerve terminal, by means of endocytosis. This process requires energy and probably delivers the toxin to an endosomal compartment within the terminal. H chains mediate specific binding, internalization and intra-neuronal sorting (Niemann, 1991).
3. Sorting to the final destination: TeTx is translocated predominantly by retrograde axonal transport to the axodendritic area of the motoneurons in the spinal cord. Here, the toxin is released, probably by a transcytotic mechanism, crosses the synaptic cleft, and poisons the nerve endings of inhibitory interneurons. BoNTs mostly remain in the peripheral nerve terminals.
4. Reduction of the toxin and translocation of the L chain into the cytoplasm: To gain access to the cytoplasm, the L chain needs to cross the membrane of the endocytic compartment. For this translocation, the H chain is required, probably by forming a pro-teinaceous translocation complex in the membrane that exposes (and possibly releases) the L chain to the cytoplasm. In the reductive intracellular environment, the disulfide bond linking the H and L chains is reduced (Kistner and Habermann, 1992).
5. Inhibition of exocytosis by cleaving essential proteins of the exo-cytotic fusion complex: The reduced L chains are Zn2+-dependent metalloendoproteases with exquisite substrate specificities.
The molecular targets for these toxins are synaptobrevin (TeTx, molecular targets BoNT/B, D, F and G; Schiavo et a/., 1992, Link et a/., 1992; Schiavo et for toxins a/., 1993c; Yamasaki et a/., 1994a, b), SNAP-25 (BoNT/A, E, and CI; Blasi eta/., 1993a; Binz eta/., 1994, Schiavo eta/., 1993a, b; Foran et a/., 1996; Williamson eta/., 1996), and syntaxin (BoNT/Cl; Blasi eta/., 1993b; Schiavo et a/., 1995). Cleavage results in an essentially irreversible block of membrane fusion, whereas vesicle docking and ion currents remain unaffected (for a comprehensive overview see Simpson (1989), DasGupta (1993)).
The poisoning sequence outlined above defines the conditions that are required for using the toxins as tools to block exocytosis. Accordingly, the following points should be taken into account:
Cell entry: Toxins enter into the cell that contains the respective membrane receptor. The presence of the receptor (and not the mechanism of action) is responsible for the high selectivity with which peripheral motoneurons are targeted. As discussed below in more detail, CNS synaptosomes also contain toxin receptors.
L chain or Di-chain-toxin: If the biological poisoning pathway is being used, the toxins must be in their di-chain form. Isolated L chains cannot enter the cell and are thus ineffective when added to intact cells or isolated nerve terminals. However, isolated L chains block exocytosis if they are introduced directly into the cytoplasm, e.g., by microinjection (Penner et a/., 1986; Mochida et a/., 1989; Hunt et a/., 1994), by permeabilization of the cells prior to toxin exposure (Bittner and Holz, 1988; Ahnert-Hilger eta/., 1989a,b, and this volume, Chapter 18; Dayanithi et a/., 1992), or by expression of recombinant L chains (Mochida eta/., 1990, Sweeney eta/., 1995). These techniques allow the inhibition of exocytosis in neurosecretory cells that are toxin-resistant due to a lack of receptors. Likewise, only the free L chains (reduced di-chain toxin or isolated L chain) are active towards the respective substrates in vitro (Schiavo et al., 1992; Link et a/., 1992; Schiavo eta/., 1993c; Link eta/., 1994).
Substrate specificity: When an L chain has reached the cytoplasm its potency is determined by the availability and functional relevance of cleavable substrates. As discussed above, the neuronal substrates are the synaptic vesicle protein synaptobrevin (also referred to as VAMP) and the synaptic membrane proteins syntaxin and SNAP-25. Each of these proteins is a member of a protein family with ubiquitously expressed members that function in many intracellular membrane fusion events (reviewed by Ferro-Novick and Jahn, 1994). Furthermore, the cleavage sites of all toxins are known (they are all different, with the exception of BoNT/B and TeTx), and major progress has been made recently concerning the structural and sequence requirements for toxin-mediated cleavage of the substrate proteins (Niemann et a/., 1994; Yamasaki et a/., 1994; Hayashi et a/., 1994, 1995; Blasi et a/., 1993b; Schiavo et a/., 1995; Otto et al., 1995; Pellegrini et a/., 1995). Although a full discussion of this topic is beyond the scope of this chapter, several points of importance have begun to emerge. First, synaptobrevin, SNAP-25 and syntaxin are highly conserved during evolution, with up to 80 % sequence identity between vertebrates and invertebrates. Thus, most of the toxins are active in all species tested. However, even minor sequence alterations at or near the cleavage sites are not well tolerated. For instance, synaptobrevin 1 is far less susceptible to these toxins than its isoform synaptobrevin 2, due to a single amino acid alteration at the cleavage site. In contrast, BoNT/D (which cleaves at an adjacent site that is identical in the isoforms) is equally potent. Another example is the resistance of SNAP-25 in some invertebrates towards BoNT/A and E, which is also due to substitutions around the respective cleavage sites. Second, some of the nonneuronal isoforms of the substrate proteins are susceptible to toxin cleavage including, cellubrevin (McMahon et a/., 1993) and syntaxins 2 and 3 (Schiavo et a/., 1995). This offers the exciting possibility of using toxin L chains in a wide variety of non-neuronal cells (Link et a/., 1993; Galli eta/., 1994; Gaisano etai., 1994 ; Steinhardt eta/., 1994, Ikonen et a/., 1995 ; Sadoul et a/., 1995). Third, it was recently discovered that synaptobrevin 2 is expressed in all cells, where it is apparently specific for specialized pathways whose functions are not yet fully understood (Ralston et al., 1994). Although it cannot be predicted which of the members of these growing protein families will be susceptible to toxin cleavage (e.g., none of the yeast proteins can be cleaved), one can safely predict that the toxins will become established tools for the highly selective dissection of exocytotic pathways in non-neuronal cells (Volchuk eta/., 1994; Galli etai., 1994; Ikonen et al., 1995).
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