The Mode of Action of Clostridial Neurotoxins

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The structural organization of CNTs is functionally related to the fact that they intoxicate neurons via a four-step mechanism consisting of (1) binding, (2) internalization, (3) membrane translocation, and (4) enzymatic target modification (Figure 4) (Montecucco et al. 1994; Montecucco and Schiavo 1995; Rossetto et al. 2006). The LC is responsible for the intracellular catalytic activity, the amino-terminal 50kDa domain of the H chain (HN) is implicated in membrane translocation, and the carboxy-terminal part (HC) is mainly responsible for the neurospecific binding.

Gangliosides Peripheral Nerves

Fig. 4 Binding and entry of BoNTs and TeNT at peripheral nerve terminal. (1) The BoNT binding domain associates with the presynaptic membrane of a-motoneurons through interaction with ganglioside and with the exposed luminal domain of a synaptic vesicle protein upon SV membrane fusion. (2) BoNTs are endocytosed within synaptic vesicles via their retrieval to be refilled with neurotransmitter. TeNT exploits a pathway requiring lipid rafts and the clathrin machinery by binding to a lipid-protein receptor complex containing the ganglioside GD1b. Once internalized in clathrin-coated vesicles (CCV), TeNT is sorted into vesicle carriers of the axonal retrograde transport pathway. (3) At the low pH generated by the v-ATPase in the vesicle lumen, BoNTs change conformation, insert into the lipid bilayer of the vesicle membrane and translocate the L chain into the cytosol. (4) Inside the cytosol the L chain catalyses the proteolysis of one of the three SNARE proteins VAMP, SNAP-25 and syntaxin, depicted in the lower panel. The same four-step pathway of entry of BoNTs into peripheral nerve terminals is believed to be followed by TeNT in the inhibitory interneurons of the spinal cord, which are reached after retroaxonal transport and release from the peripheral motoneurons.

Fig. 4 Binding and entry of BoNTs and TeNT at peripheral nerve terminal. (1) The BoNT binding domain associates with the presynaptic membrane of a-motoneurons through interaction with ganglioside and with the exposed luminal domain of a synaptic vesicle protein upon SV membrane fusion. (2) BoNTs are endocytosed within synaptic vesicles via their retrieval to be refilled with neurotransmitter. TeNT exploits a pathway requiring lipid rafts and the clathrin machinery by binding to a lipid-protein receptor complex containing the ganglioside GD1b. Once internalized in clathrin-coated vesicles (CCV), TeNT is sorted into vesicle carriers of the axonal retrograde transport pathway. (3) At the low pH generated by the v-ATPase in the vesicle lumen, BoNTs change conformation, insert into the lipid bilayer of the vesicle membrane and translocate the L chain into the cytosol. (4) Inside the cytosol the L chain catalyses the proteolysis of one of the three SNARE proteins VAMP, SNAP-25 and syntaxin, depicted in the lower panel. The same four-step pathway of entry of BoNTs into peripheral nerve terminals is believed to be followed by TeNT in the inhibitory interneurons of the spinal cord, which are reached after retroaxonal transport and release from the peripheral motoneurons.

6.1 Binding

From the site of adsorption, BoNTs diffuse in the body fluids and eventually bind very specifically to the presynaptic membrane of cholinergic terminals. At variance, the pathway of TeNT is more complex, as it involves an additional retroax-onal transport to the spinal cord and a specific binding to inhibitory interneurons

(Lalli et al. 2003). The HC domain plays the major role in the neurospecific binding (Chai et al. 2006; Jin et al. 2006). Polysialogangliosides are involved in CNT binding (reviewed in Montecucco et al. 2004; Rossetto et al. 2006; Schiavo et al. 2000; Verderio et al. 2006; Yowler and Schengrund 2004). In fact, the HCC subdomain of BoNT bind polysialogangliosides, particularly GD1a, GT1b, and GQ1b, via the conserved peptide motif H...SXWY. ..G (Rummel et al. 2004b). Pre-incubation of BoNTs with polysialogangliosides partly prevents cell intoxication and the pre-treatment of cultured cells with polysialogangliosides increases their sensitivity to BoNT/A; conversely, depletion of gangliosides in neuroblastoma or PC12 cells impairs BoNT/A and BoNT/B internalization. Neuraminidase removes sialic-acid residues and decreases BoNT cell binding. Knockout mice defective in the production of polysialogangliosides show reduced sensitivity to BoNT/A and BoNT/B (Bullens et al. 2002; Chai et al. 2006; Kitamura et al. 2005; Schiavo et al. 2000; Yowler et al. 2002). More recently, polysialogangliosides GD1b and GT1b and phosphatidylethanolamine were reported to be the functional receptors of BoNT/C and BoNT/D, respectively (Tsukamoto et al. 2005). At variance, two sugar-binding sites have been identified within the TeNT HCC fragment and this double binding, possibly to a polysialoganglioside molecule and to a glycoprotein, is essential for the toxicity of TeNT (Rummel et al. 2003; Herreros et al. 2000).

It is well established that both glycolipid and protein receptors are crucial for high-affinity binding to nerve cell membranes (Rummel et al. 2007). As BoNTs and TeNT are toxic at concentrations estimated to be < picomolar (Montecucco et al. 2004), binding has to be interpreted as the simultaneous occupation of the ganglio-side binding pocket(s) and of the protein binding site. Indeed, TeNT was shown to interact with a GPI-anchored glycoprotein and to bind to lipid rafts (Herreros and Schiavo 2002; Herreros et al. 2001; Munro et al. 2001). Synaptic vesicle (SV) proteins participate with their luminal domains to the neurospecific binding of BoNT/A, /B, and /G, and this is probably true for all the other BoNTs (for a recent review, see Verderio et al. 2006). BoNT/B and BoNT/G interact with the luminal domain of the SV proteins synaptotagmin I (Syt-I) and Syt-II. BoNT/B has a higher affinity for Syt-II (Dong et al. 2003; Nishiki et al. 1996), whereas BoNT/G interacts preferentially with Syt-I (Rummel et al. 2004a). The crystal structures of BoNT/B in complex with the luminal domain of Syt-II (Chai et al. 2006; Jin et al. 2006) shows that HCC accommodates the a-helical segment 45-59 of Syt-II in a cleft adjacent to the polysialogangliosides-binding site of the toxin (Figure 2). Mutations in the synaptotagmin-binding cleft and in the polysialoganglioside-binding pocket greatly reduce the toxicity of BoNT/B and /G, and double mutants at the two binding sites abolish neurotoxicity (Rummel et al. 2007).

At variance, BoNT/A binds specifically to the luminal domain of SV2 (Dong et al. 2006; Mahrhold et al. 2006). SV2 is a highly glycosylated transmembrane protein of SV implicated in vesicle recruitment to the plasma membrane in endocrine cells (Iezzi et al. 2005). Vertebrates express three distinct, but homologous, SV2 proteins termed SV2A, SV2B, and SV2C (Bajjalieh et al. 1992; Bajjalieh et al. 1994). Interestingly, motor neuron terminals express the receptor isoforms that bind BoNTs more strongly, such as Syt-II (Juzans et al. 1996; Li et al. 1994; Marqueze et al.

1995) and SV2C (Janz and Sudhof 1999). Conversely, high-affinity isoforms are poorly expressed in CNS neurons and terminals, in which Syt-I, SV2A, and SV2B are the prevalent isoforms (reviewed in Verderio et al. 2006). Recently Baldwin and Barbieri (2007) demonstrated that BoNT/A and /B binding domains associate with synaptic vesicle membrane proteins and suggested that BoNT protein receptor may be a component of a larger protein complex.

Both TeNT and BoNTs bind the presynaptic membrane of a-motoneurons, but then TeNT follows a different intracellular trafficking route and this must be determined by yet unidentified specific receptor(s).

6.2 Internalization

Available evidence indicates that CNTs do not enter the cell directly from the plasma membrane, but are endocytosed inside the lumen of vesicular structures in a temperature- and energy-dependent process (Black and Dolly 1986; Critchley et al. 1985; Dolly et al. 1984; reviewed in Schiavo et al. 2000). The finding of SV proteins as receptors of BoNTs support the proposal (Montecucco and Schiavo 1995) that, after binding, BoNTs are endocytosed within synaptic vesicles (SV) via their retrieval to be refilled with neurotransmitter, an hypothesis originally advanced to account for the increased rate of poisoning with NMJ activity (Figure 4) (Hughes and Whaler 1962).

The protein receptor of TeNT would be responsible for its inclusion in an en-docytic vesicle that moves in a retrograde direction all along and inside the axon (Figure4). TeNT-HC internalization at the motor nerve terminal occurs via a specialized clathrin-dependent pathway, which is distinct from SV endocytosis and is preceded by a lateral sorting from its lipid raft-associated ligand GD1b. (Deinhardt et al. 2006; Roux et al. 2005). The TeNT-carrying vesicles reach the cell body of the motoneurons which reside within the spinal cord and then move to dendritic terminals to release the toxin in the intersynaptic space. TeNT then binds and enters the inhibitory interneurons of the spinal cord via SV endocytosis (Matteoli et al. 1996). Consequently, it blocks the release of inhibitory neurotransmitters and impairs the system which controls the balanced movement of the skeleton (Kandel et al. 2000), giving rise to a spastic paralysis. It is not known if, and if so, to what an extent, BoNTs migrate retroaxonally. Direct measurements of retroaxonal transport indicated that 125I-BoNT/A does not reach the central nervous system (Habermann and Dreyer 1986).

6.3 Membrane Translocation

The L chains of CNTs block neuroexocytosis by acting in the cytosol, and therefore at least this toxin domain must reach the cell cytosol. In order to do so, the L chain must cross the hydrophobic barrier of the vesicle membrane, and compelling evidence indicates that CNTs have to be exposed to low pH for this step to occur (Matteoli et al. 1996; Simpson et al. 1994; Williamson and Neale 1994). In fact, acidic pH causes a conformational change from a water-soluble "neutral" structure to an "acid" structure with exposure of hydrophobic patches on the surface of both the H and L chains which then enter into the hydrocarbon core of the lipid bilayer (Montecucco et al. 1989; Puhar et al. 2004; Schiavo et al. 2000; Shone et al. 1987). Following this low pH-induced membrane insertion, BoNTs and TeNT form transmembrane ion channels (reviewed in Montecucco and Schiavo 1995) and in PC12 membranes (Sheridan 1998). The cleavages of SNAP-25 by BoNT/A and BoNT/E is differentially affected by inhibitors of the vacuolar ATPase proton pump (Keller et al. 2004), but this is unlikely to be due to a difference in the toxin acid-triggered conformational change, as the low pH-driven toxin conformational change takes place in a very narrow range of pH: 4.4-4.6 for TeNT and BoNT/A, /B, /C, /E, and /F (Puhar et al. 2004). This similarity indicates that the key residues for the con-formational change are conserved among CNTs and that they behave very similarly with respect to lowering pH. Currently, membrane translocation is the least understood step of the mechanism of cell intoxication of CNTs, and further investigation is required to uncover the extent and implication of the structural changes that occur at low pH (for a recent discussion see Rossetto and Montecucco 2004). Koriazova and Montal (2003) have proposed that the H chain channel acts as a trans-membrane chaperone for the L chain, preventing its hydrophobicity-driven aggregation and maintaining its unfolded conformation during translocation. The L chain is then released into the neutral cytosol where it refolds into its native enzymatically active conformation. In this vision, additional chaperones are not implicated. However, a complex of cytosolic chaperones and thioredoxin present on the cytosolic face of endosomes was recently found to assist the translocation of the enzymatic chain of other bacterial toxins acting in the cytosol (Haug et al. 2003; Ratts et al. 2003). Four different chaperone systems have been found so far within neuron terminals and are essential for normal function of the synaptic vesicle cycle during neurotransmitter release (Evans et al. 2003; Zinsmaier and Bronk 2001). In light of the proposed role of SVs as vesicular carriers of BoNTs inside peripheral neurons, it is noteworthy that two out of four synaptic terminals chaperone complexes includes the cystein string protein of SVs. It is therefore possible that the membrane translocation and refolding of the L chain is assisted by SV chaperones in addition to the H chain, but this remains to be proven.

6.4 SNARE Proteins' Specific Metalloproteolytic Activity

The L chains of BoNTs and TeNT are highly specific proteases that recognize and cleave only three proteins, the so-called SNARE proteins, which form the core of the neuroexocytosis apparatus (Figure 4) (Schiavo et al. 2000). TeNT, BoNT/B, /D, /F and /G cleave VAMP, a protein of the SV membrane, at different single peptide bonds; BoNT/C cleaves both syntaxin and SNAP-25, two proteins of the presynaptic membrane; BoNT/A and /E cleave SNAP-25 at different sites within the COOH-terminus (reviewed in Humeau et al. 2000; Schiavo et al. 2000). BoNT-poisoned nerve terminals have a normal size and appearance with normal content and shape of synaptic vesicles and mitochondria (Duchen 1971; Thesleff 1960). Possibly the only morphological sign of BoNT/A poisoning is noticeable at the frog NMJ with the disappearance of the small outward curvatures of the membrane close to the active zones (Harlow et al. 2001; Pumplin and Reese 1977).

VAMP, SNAP-25, and syntaxin form an heterotrimeric coiled-coil complex, termed the SNARE complex, which induces the juxtaposition of vesicle to the target membrane (Jahn et al. 2003; Sudhof 2004). Several SNARE complexes assemble into a rosette in order to bring the SV membrane close enough to the cytosolic face of the presynaptic membrane to permit their fusion with subsequent release of the vesicle neurotransmitter content into the synaptic cleft (Montecucco et al. 2005). Proteolysis of one SNARE protein prevents the formation of a functional SNARE complex and, consequently, the release of neurotransmitter. This is not true for BoNT/A and /C, which cleave SNAP-25 within few residues from the C terminus giving rise to a truncated SNAP-25, which can still form a SNARE complex but not a rosette of SNARE complexes. Given the fact that a rosette of SNARE complexes is necessary for the exocytosis of one synaptic vesicle, the presence of a single defective SNARE complex will have a dominant negative effect on neurotransmitter release. This rationale explains the experimental finding that incomplete proteolysis of SNAP-25 at nerve terminals by BoNT/A is sufficient to cause full inhibition of neurotransmitter release (Bruns et al. 1997; Foran et al. 1996; Jurasinski et al. 2001; Keller et al. 2004; Osen-Sand et al. 1996; Raciborska et al. 1998; Williamson et al. 1996). This also explains the fact that the paralysis induced by BoNT/A and /C is long lasting, as the inhibitory activity of C-terminal truncated SNAP-25 on the assembly of the rosette of SNARE complexes overlaps and functionally extends the lifetime of the metalloproteolytic activity of the L chain of these two BoNTs (for a detailed discussion see Rossetto et al. 2006). These explanations, however, may not be sufficient to account for the astonishing fact that the duration of the BoNT/A inhibition of autonomic nerve terminals is more than one year (Naumann and Jost 2004).

The molecular basis of the CNTs specificity for the three SNAREs resides on protein-protein interactions which extend well beyond the proteolysed SNARE regions, and a major role is played by a nine-residue-long motif present within the SNARE proteins. This motif is characterized by three carboxylate residues alternated with hydrophobic and hydrophilic residues (Breidenbach and Brunger 2004; Evans et al. 2005; Pellizzari et al. 1996; Rossetto et al. 1994; Vaidyanathan et al. 1999; Washbourne et al. 1997). This motif is present in two copies in VAMP and syntaxin and four copies in SNAP-25. The various CNTs differ with respect to the specific interaction with the recognition motif (Rossetto et al. 2001b). Only protein segments including at least one copy of the motif are cleaved by the toxins, and mutations within the motif inhibit the proteolysis (Fang et al. 2006; Schiavo et al. 2000). Moreover antibodies against the SNARE motif inhibit the proteolytic activity of the neurotoxins (Pellizzari et al. 1997). A recent co-crystal structure of LC/A and SNAP25-(146-204) defined additional regions of interaction external to the cleavage site and to the motif (Breidenbach and Brunger 2004; Breidenbach and Brunger 2005b). More recently, the fitting of an extended region of the substrate (residues 189-203) within the long active site cleft was defined following extensive mutagenesis of LC/A and SNAP-25 (Chen and Barbieri 2006; Chen et al. 2007).

There are no quantitative data on the number of L chains required to intoxicate a nerve terminal. In Aplyisa californica cholinergic neurons, few molecules of toxin appear to be sufficient to block neuroexocytosis within an hour at room temperature (Poulain, personal communication). It is even more likely that few copies of L chain are sufficient in warm-blooded animals. It is evident that as long as the toxin is present in an active form, the nerve signal cannot be transmitted.

The half-time of action of the different BoNTs has been estimated in mice cere-bellar granular neurons in culture by Foran et al. (2003b): BoNT/A (31 days) > BoNT/C (25) > BoNT/B (10) > BoNT/F (2) > BoNT/E (0.8). Comparable data were found in the mice NMJ in vivo (Meunier et al. 2003; Morbiato et al., 2007). These data parallel those observed in human patients injected with different BoNTs within skeletal muscles: BoNT/A (2-4 months) > BoNT/C (12-16 weeks) > BoNT/B (5-10 w) > BoNT/F (5-8 w) > BoNT/E (4-6 w). In general, humans recover more slowly than do rodents. The lifetime of LC inside the nerve terminal is clearly involved in determining the duration of paralysis. The finding that LC/A overexpressed in cultured cells binds to the cytosolic face of cell membrane, while LC/E is prevalently cytosolic (Fernandez-Salas et al. 2004), has provided a possible explanation, within the limits of the significance of protein overexpression with respect to their intracellular localization. However, it is hard to conceive that this is the sole explanation, particularly if one considers that the duration of the effect of BoNT/A in autonomic disfunction in humans is 12-15 weeks (Naumann and Jost 2004). As discussed above, experimental evidence supports the proposal that an important role is also played by the dominant negative effect on neurotransmission exerted by the BoNT/A- or /C-truncated SNAP-25 (Montecucco et al. 2005; Rossetto et al. 2006). Nevertheless, even the addition of the lifetimes of LC and of truncated SNAP-25 may not be sufficient to provide a satisfactory explanation for the very long time of recovery of intoxicated autonomic terminals of exocrine glands.

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