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How botulinum and tetanus neurotoxins


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Biochimie 82 (2000) 427−446
© 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
S0300908400002169/FLA

How botulinum and tetanus neurotoxins block neurotransmitter release*

Yann Humeaua, Frédéric Doussaua**, Nancy J. Grantb, Bernard Poulaina***


a
Laboratoire de Neurobiologie Cellulaire, UPR 9009 du CNRS, Centre de Neurochimie,
5, rue Blaise-Pascal, 67084 Strasbourg cedex, France
b
Biologie de la Communication Cellulaire, INSERM U-338, Centre de Neurochimie,
5, rue Blaise-Pascal, 67084 Strasbourg cedex, France
(Received 17 January 2000; accepted 17 March 2000)

Abstract — Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light
chain (Mr ≈50) disulfide linked to a heavy chain (Mr ≈100). By inhibiting neurotransmitter release at distinct synapses, these toxins
cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost
been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals.
Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one
of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin),
syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific
sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations
can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins. © 2000 Société
française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS
botulinum neurotoxins / tetanus toxin / Clostridium / quantal release / neurotransmission / membrane fusion / synaptic vesicle
/ SNARE complex / VAMP / synaptobrevin / SNAP-25 / syntaxin

1. Introduction: key milestones over 2000 years toxin action was quickly deciphered in Montecucco’s
laboratory in 1992 [11, 12]. Over the next 3 years, other
Tetanus (from the greek tetanos = to contract) was first clostridial neurotoxins were also demonstrated to be
referred to more than 20 centuries ago when Greek zinc-endopeptidases that selectively cleave the SNAREs
physicians recognized this condition as a neurological (soluble NSF associated protein receptors) proteins
disease. Botulism, or sausage poisoning (from the latin (VAMP (vesicle associated membrane protein), SNAP-25
botulus = sausage), has been well documented since the (synaptosome associated protein of 25 kDa) and syntaxin)
18th century. The infectious nature of tetanus and the implicated in neurotransmitter exocytosis (reviewed in
bacterial etiology and toxicopathological origin of botu- [13, 14]). Finally, the tridimensional structure of botuli-
lism were recognized at the end of the 19th century, num neurotoxin type A has now been resolved [15].
notably by Carle and Rattone [1] and Van Ermengen [2] This article attempts to summarize what we know about
(for historical considerations see [3–6]). Major progress in the molecular actions of clostridial neurotoxins and how
understanding this disease was the discovery by Burgen differences between toxins account for their distinct ef-
and collaborators in 1949 that botulinum toxin blocks fects on neurotransmission. To simplify the text, data has
neurotransmitter release [7]. However, it was not until 30 been included in a series of tables in which the reader will
years later that Simpson proposed a cellular mechanism of find relevant references. However, only a fraction of the
action involving three sequential steps, namely binding, vast literature on this subject has been cited and we
internalization and intraneuronal action [8]. Nearly one apologize for any essential omissions.
century after their initial discovery, the amino acid se-
quences of tetanus toxin (1986) and one of the botulinum
toxins (1990) were determined in Niemann’s laboratory 2. Clostridial neurotoxins
[9, 10]. Thereafter, the intracellular mechanism of tetanus
Seven distinct serotypes of botulinum toxins (BoNTs,
* This paper is dedicated to the memory of Heiner Niemann. A-G) have been identified through the examination of
** Present address: Departement of Neurobiology, Duke Univer- botulism outbreaks in man (BoNT/A, /B, /E and /F), birds
sity Medical Center, 27710 Durham, NC, USA (BoNT/C), cattle (BoNT/D), or isolated from soils
*** Correspondence and reprints: (BoNT/G). The various BoNTs are produced by distinct
poulain@neurochem.u-strasbg.fr strains of Clostridium botulinum that display heteroge
428 Humeau et al.

neous bacteriological characteristics. Moreover, several However, BoNTs cannot be considered exclusively as
serotypes can be secreted by other Clostridia. In contrast, ‘cholinergic’ toxins. Indeed, symptoms of autonomic dys-
tetanus neurotoxin (TeNT) is produced by a uniform group function (parasympathetic, i.e., cholinergic, and sympa-
of C. tetani (for reviews see [5, 16–18]). Bacteria produce thetic, i.e., adrenergic and noradrenergic) coexist with
clostridial neurotoxins together with several other protein neuro-motor alterations during botulism (reviewed in [28,
toxins, such as the ADP-ribosyltransferase C2 binary toxin 29]). Moreover, central nervous system disorders may
that affects G-actin and the C3 exoenzyme which ADP- occur during botulism or following the peripheral admin-
ribosylates Rho (reviewed in [19, 20]). istration of toxins [30–33].
BoNTs and TeNT are translated as single chain pro- Tetanus is a spastic paralysis due to motor neuron
toxins and are subsequently cleaved within a disulphide desinhibition [34]. The only etiological cause of the
loop located about one-third of the way from the disease is TeNT that is produced in a wound colonized by
N-terminus. This generates the fully active neurotoxin that C. tetani. The spastic paralysis that characterizes tetanus
is both composed of a light chain of ≈50 kDa and a heavy in mammals is due to preferential blockage of inhibitory
chain of ≈100 kDa linked by both a disulphide bridge and (GABAergic and glycinergic) synapses afferent to moto-
non-covalent interactions [21]. During intoxication pro- neurons. However, TeNT can produce flaccid paralysis in
cess, the interchain bridge is reduced, and this is a pathophysiological conditions (cranial tetanus) or after
necessary prerequisite for the intracellular action of the injection of high doses of the toxin. This effect results
toxins [11, 22, 23]. Examination of the three-dimensional from blockage of ACh release at motor nerve endings [35,
structure of BoNT/A shows that when the interchain 36]. This is consistent with the current idea that motor
disulphide bridge that links heavy and light chains is nerve terminals are the ‘gates’ giving TeNT access to the
intact, the N-terminal portion of the heavy chain masks the central nervous system. Indeed, during tetanus, TeNT is
catalytic cleft in the light chain, thereby preventing first captured by motor nerve endings followed by its
substrate access [15]. retrograde axonal transport and release into extracellular
The complete amino acid sequences of TeNT [9]and fluids in the central nervous system.
BoNTs have been determined (BoNT/A: [10], for other
types see review in [24]). Clostridial neurotoxins are
composed of ≈1300 amino acids and alignment of their 4. Quantal characteristics of the inhibitory action of
sequences show an overall low homology (≈50%) with BoNTs and TeNT on evoked and spontaneous
short domains of higher homology. Only the tridimen- synaptic transmission
sional structure of BoNT/A has been resolved [15]. Unlike
TeNT, BoNTs together with several proteins (some of BoNTs and TeNT mainly cause a dramatic reduction of
which exhibit haemagglutinin activity) are secreted as amplitude, or even elimination of the postsynaptic re-
very stable, high molecular mass complexes (up to sponses evoked by action potentials, and a large reduction
≈900 kDa in the case of BoNT/A) (reviewed in [5, 14, in the frequency of spontaneous quantal release at both
17]). These complexes correspond to the so-called botu- vertebrate and invertebrate synapses (tables I, II). After
linum toxin or BoTx. The non-toxin proteins do not seem the toxin acts, quantal events detected at neuromuscular
to contribute to the cellular or molecular action of BoNTs junctions are often of smaller amplitude and have slower
(discussed in [25]). Since the toxin complex is very stable kinetics [37–40]. However, this is mostly due to postsyn-
at low pH and dissociates at neutral pH [26], these aptic changes in ACh-receptor densities and conductance
accessory proteins are probably essential for protecting [41, 42], and not to changes in the neurotransmitter
the neurotoxin moiety from proteolytic attack during content of synaptic vesicles (for discussion see [43]).
exposure to the gastrointestinal environment. Hence, the block of neurotransmission induced by TeNT
and BoNTs is mostly due to a reduction in the number of
quanta released by the nerve impulse. Treatments known
3. Botulism and tetanus to strongly increase evoked and spontaneous release at
untreated synapses (reviewed in [44, 45]) are able to
Botulism is a severe disease affecting vertebrates (ta- induce substantial recovery of evoked release and enhance
ble I). It is characterized by flaccid paralysis originating in spontaneous exocytosis at BoNT/A-poisoned nerve termi-
the peripheral nervous system. It is induced by ingestion nals, but not at synapses inhibited by the other serotypes
of spoiled food (food-borne botulism), colonization of the (table III). Hence, in contrast to the other neurotoxins,
intestinal tract (infant botulism) or a wound (wound BoNT/A inhibits by altering the Ca2+-dependency of
botulism) by certain strains of C. botulinum. Earlier quantal release. However, neither BoNT/A nor other
studies on the pathophysiology of botulism revealed that toxins inhibit Ca2+-influx (table III). Moreover, there are
neuromuscular paralysis is due to selective inhibition of no obvious morphological modifications in the docking of
evoked acetylcholine (ACh) release at the skeletal neuro- synaptic vesicles at the active zone or the number of
muscular junction [7] (for reviews, see [8, 16, 27]). release sites (see (table III) for references). In fact, the
How botulinum and tetanus neurotoxins block neurotransmitter release 429

Table I. Symptoms and blocking actions of neurotransmission at vertebrate motor synapses caused by clostridial neurotoxins (a, b, c,
1
)*.

a
TeNT blocks spontaneous quantal events in the central nervous system of the rat[104]. bJ. Molgo, personal communication. cThe
electric organ of Torpedo is ontogenetically homologous to the neuromuscular junction. 1Relative neurotoxin actions have been graded
subjectively from very potent (+++) to less effective (++, +). +/-, variable effects. In some cases, toxin action was not determined (ND).
*References mentioned in the table: [29, 36, 37, 39, 40, 75, 91, 96, 98–101, 132, 153–167].

motor nerve terminal continues to release quanta all along nerve terminals is to prevent fusion of docked synaptic
its length, but the probability of release is reduced at each vesicles with the plasma membrane of nerve endings. In
site [46]. Hence, the main action of TeNT and BoNTs in agreement, when clostridial neurotoxins are applied intra
430 Humeau et al.

Table II. Blocking actions of clostridial neurotoxins at invertebrate synapses (a, +++, +, ND)*.

a
Poulain et al., unpublished observations. +++, +, ND: see table I. *References mentioned in the table: [49, 52, 80, 89, 92, 94,
105–107, 112, 113, 168–171].

cellularly to bypass the limiting membrane steps, they BoNT/C binds two Zn atoms, one of which plays a
block exocytotic events in a variety of non-neuronal cells structural role [57]. Zn is required for the intracellular
(table IV). activity of neurotoxins [12, 58–61]. However, Zn chela-
tion which mediates detoxification may also account for
an effect on the secondary structure of the light chain [62].
5. The light chains of clostridial neurotoxins are Inhibitors of metallo-endopeptidases, such as phosphora-
metalloproteases midon [12] captopril or thiorphan ([60], Deloye and
Poulain, unpublished observation), antagonize the inhibi-
The action of extracellularly applied clostridial neuro- tory action of intracellularly applied TeNT or BoNT/A, /B.
toxins may be divided into three main steps, namely, However, the effects of these pharmacological agents
binding, internalization and intracellular action. We will appear less clear in some preparations, and may depend on
focus only on the latter step (for recent discussion of other the type of cells used (PC12 cells: [59, 63], vertebrate
steps, see [18, 47, 48]). The light chains of BoNTs and neuromuscular junction: [64]). Recently, potent inhibitors
TeNT are implicated in the inhibition of transmitter of TeNT and BoNT/B have been designed, but have yet to
release [22, 23, 49–51]. However, the inhibitory action of be tested in vivo [65, 66]. Characterization of the relation-
BoNTs in Aplysia neurons requires, for some unknown ship between the structure and the function of the catalytic
reason, the presence of the heavy chain or its C-terminal pocket of TeNT light chain has revealed that His233 and
portion in the cytosol [52, 53]. His236 residues are the first two ligands of the Zn atom.
The amino acid sequences of BoNT- or TeNT light A third one is a water molecule polarized by the carboxyl
chains display low overall homology, except in the middle group of the Glu234 residue in the His-Glu-x-x-His motif.
portion which contains the His-Glu-x-x-His motif charac- A fourth ligand consists of another Glu residue outside of
teristic of the catalytic pocket of all Zn-endopeptidases the His-Glu-x-x-His motif. Tyr365 has been proposed to
[11, 12, 53, 54]. The light chains of BoNT/A, /B and /F be a fifth ligand [67]. Genetic substitution of the His and
and TeNT bind one Zn atom [12, 55, 56], but that of Glu residues in the catalytic pocket by other amino acids
How botulinum and tetanus neurotoxins block neurotransmitter release 431

Table III. Further characterization of the synaptic action of clostridial neurotoxins (a, +++, ++, +, ND)*.

a
J. Molgo, personal communication. +++, ++, +, ND: see table I. *References mentioned in the table: [36–40, 89, 92, 98–101, 104,
112, 113, 132, 157, 159, 160, 163, 165, 170, 172–190].

(Gly, Ala, etc.) abolishes the proteolytic activity against are cleaved by TeNT, BoNT/B, /D, /F and /G (table V),
VAMP [68–70]. SNAP-25 by BoNT/A, /C and /E (table VI) and syntaxin
only by BoNT/C (table VII). Several homologues of these
proteins (cellubrevin, SNAP-23) can be cleaved to differ-
6. Clostridial neurotoxins specifically recognize and ent degrees. For example, murine SNAP-23 (also termed
cleave VAMP, SNAP-25 or syntaxin Syndet) can be cleaved by BoNT/E and to a reduced
extent by BoNT/A, but human SNAP-23 is resistant to
Only three synaptic proteins (figure 1) have been iden- both BoNT/A and /E due to mutations of Lys185Asp and
tified as targets of the eight clostridial neurotoxin sero- Pro182Arg [72, 73]. Syntaxins 2 and 3, but not syntaxin 4
types (reviewed in [14, 71]). In mammals, VAMP-1 and -2 are cleaved by BoNT/C [74].
432 Humeau et al.

Table IV. Clostridial neurotoxins block other exocytic events than neurotransmitter release*.

*References mentioned in the table: [22, 50, 74, 93, 95, 119, 120, 128, 186, 191–210].

Mutations in the amino acid sequence of the toxin bond in VAMP. This has raised the question of what
targets makes them resistant to proteolysis and can ac- mechanisms determine how each toxin specifically at-
count for almost all cases of species insensitivity to BoNT tacks: i) a unique peptide bond in their target proteins; and
or TeNT (compare (tables I, II) with (tables V, VII)). ii) only VAMP, SNAP-25 or syntaxin, and not other
Occasionally, toxin-resistance is caused by the absence of synaptic proteins. This exquisite selectivity is due to the
specific membrane ectoacceptors (i.e., man is not affected presence of a highly conserved common motif in the
by BoNT/D [75]). Most toxin-insensitive VAMPs are amino acid sequence of the target. Depending on the
generated by mutations in the cleavage site (table V), protein, the motif is termed V1 and V2 in VAMP, S1 to S4
while toxin-resistant SNAP-25 results from amino acid in SNAP-25 and X1 and X2 in syntaxin (figure 1).
substitutions in and around this site (table VI). This may Although BoNT/B and TeNT attack the same Gln-Phe
arise because VAMP cleavage sites are located in a peptide bond, these toxins display distinct affinities for V1
domain essential for its function (i.e., binding with other and V2: TeNT, like BoNT/D requires the V1 motif,
SNAREs), whereas SNAP-25 cleavage sites, in particular whereas BoNT/B (as BoNT/G) binds to V2. BoNT/F
those for BoNT/A and /C are located in a less crucial recognizes V1 and/or V2 [76–78]. Furthermore, TeNT
domain. exploits a second exosite (figure 1), rich in positive
All toxins cleave their targets at distinct sites, except charges and located adjacent to the cleavage site [79].
for BoNT/B and TeNT which attack the same peptide Recent observations that VAMP cleavage by TeNT is
How botulinum and tetanus neurotoxins block neurotransmitter release 433

Figure 1. Molecular targets of clostridial neurotoxins. The three synaptic proteins cleaved by TeNT and BoNTs light chains are
shown. The cleavage sites are indicated by arrows. The residues delimited by a box denote the sites that determine the binding
specificity of the toxins light chains (also termed ‘SNARE motif’, see text (Section 6) and [72, 73, 76, 77]). An additional binding
site for the TeNT light chain on VAMP, rich in + charges, is indicated [79].

activity-dependent, but not that by BoNT/B [80], suggests (by folding or multimerisation) is generated. In this view,
that V1 and V2 domains are not necessarily exposed under it is likely that when VAMP binds to synaptophysin [87],
the same physiological circumstances. For efficient cleav- it is probably protected against proteolysis. Clostridial
age of SNAP-25, both BoNT/A and /E require the neurotoxins are likely to have free access to their substrate
presence of the C-terminal region of SNAP-25 which only during a short ‘physiological window’ (figure 2),
includes the motif S4. However, the three N-terminal possibly during docking of synaptic vesicles at active
motifs (S1, S2 and S3) can partially substitute for a zones, when the SNAREs are dissociated from their
modified S4 motif [72]. Functional consequences of these chaperons and prior to their assembly into the fusogenic
observations have been provided by the finding that the SNARE complex. Although the physiological window for
target proteins are protected from proteolytic attack when proteolytic attack is relatively brief, the neurotoxins are
they are bound in ternary complex [81–83]. This is able to progressively cleave the majority of the SNARE
probably due to masking of cleavage sites and/or recog- population. This is consistent with the evidence that all the
nition motifs. This concept can be extended to several steps prior to the assembly of SNAREs into tight com-
other complexes involving SNAREs. For example, when plexes are reversible [88] and tethered vesicles can be-
SNAP-25 is dimerised together with syntaxin 1, it is come undocked.
protected against BoNT/A [84]. However, when the pep-
tide interacting domains do not involve the SNARE motif
or cleavage site, the toxin still has access to these sites and 7. Cause-effect relationship between SNARE cleavage
the SNARE protein can be cleaved. For example, when and blockage of neurotransmitter release
VAMP-2 interacts with syntaxin 1 via its transmembrane
domains, it can be attacked by TeNT [85]. Similarly, 7.1. SNARE proteolysis and inhibition of exocytosis
SNAP-25 bound to synaptotagmin can be cleaved by
either BoNT/A or /E [86]. In general, SNAREs should be The relationship between target proteolysis and block-
protected from proteolysis when a closed configuration age of neurotransmitter release was first established for
434 Humeau et al.

Table V. Cleavage of VAMP (a, b)*#.

a
Sequence corrected in position 93 (f > s) according to [219]. bSequence corrected in position 68 (t > s)according to [105]. *Expected.
#
References mentioned in the table: [11, 69, 79, 89, 94, 105, 121, 167, 170, 211–218].

TeNT and BoNT/B. Indeed, intraneuronal application of demonstration of the causal link between SNAP-25 cleav-
peptides including the VAMP cleavage site for TeNT or age and exocytosis blockage has been obtained with
BoNT/B diminishes their inhibitory action [11, 89], and insulin-secreting cells: human SNAP-23 can replace
pre-injection of specific anti-VAMP/synaptobrevin anti- SNAP-25 in SNARE complex, and overexpression of
bodies into Aplysia cholinergic neurones prevents the SNAP-23 which is resistant to BoNT/A rescues insulin
blocking action of TeNT and BoNT/B, but not that of secretion after blockage by BoNT/A [95]. Intriguingly,
BoNT/A which cleaves SNAP-25 [90]. Immunocy- although Torpedo SNAP-25 is not cleaved by BoNT/A in
tochemical studies at the frog and crayfish neuromuscular vitro [72], BoNT/A abolishes ACh release at Torpedo
junctions show that the inhibition of neurotransmitter electric organ [96, 97].
release correlates with the disappearance of VAMP immu-
noreactivity [91, 92]. Transfection of insulin-secreting 7.2. Difference in susceptibility of spontaneous
cells with mutated VAMP that cannot be cleaved by TeNT exocytosis to toxins suggests distinct roles for VAMP-1
allows partial restoration of secretion [93]. Leech and VAMP-2
SNAP-25 cannot be cleaved by BoNT/A, and accordingly,
cultured leech neurones are BoNT/A resistant. However, In general, the toxins that attack VAMP have little
sensitivity of neurotransmitter release to BoNT/A could be effect on spontaneous exocytosis. Moreover, the degree of
conferred just by replacing amino acids 196–209 of leech inhibition seems to correlate with the different suscepti-
SNAP-25 with those of the rat protein [94]. A further bilities of the VAMP isoforms to cleavage in various
How botulinum and tetanus neurotoxins block neurotransmitter release 435

Table VI. Cleavage of SNAP-25 and related proteins (a, b, c, d, e)*#

a
In vitro cleavage of SNAP-25 requires 1000-fold higher BoNT/C concentration than BoNT/A or /E[73]. bSubstitution of p182r, or
k185d (boxes) induces susceptibility toward BoNT/E. cResistance to BoNT/A possibly due to d189 or e189 substitution by v189[72],
see box. dNote that Torpedo is susceptible to BoNT/A[96]. eNote the presence of several non-conservative mutations around putative
cleavage sites. *Expected. #References mentioned in the table: [72, 73, 94, 109, 110, 215, 220, 221].

animal species. Indeed, at mouse motor terminals, both Drosophila larval neuromuscular junction [106–108].
VAMP-1 and VAMP-2 can be cleaved by TeNT or Taken together, these results suggest that VAMP-2 is more
BoNT/B (table V) and there is a significant decrease in specialized in mediating evoked release, while VAMP-1
spontaneous release frequency [40, 98, 99]. Unlike or a toxin-insensitive VAMP isoform is principally impli-
VAMP-1, rat VAMP-2 can be cleaved by TeNT and cated in spontaneous release.
BoNT/B (table V), and strikingly, very little decrease in
quantal frequency is observed with TeNT [36]. BoNT/D 7.3. Cleavage of syntaxin or/and SNAP-25 during
and /F cleave both VAMP-1 and -2 rat isoforms, and both BoNT/C action
toxins strongly diminish quantal frequency at rat motor
nerve endings [100, 101]. VAMP-1 is preferentially ex- BoNT/C cleaves both syntaxin 1 and SNAP-25
pressed in the motor system, whereas VAMP-2 is pre- [109–111]. Thus, an important issue is to determine which
dominantly present in nuclei associated with sensory and of these two target proteins is cleaved during BoNT/C-
integrative functions [102, 103]. Along this line, TeNT is induced blockage. In vitro, cleavage of SNAP-25 by
relatively potent in inhibiting both evoked and spontane- BoNT/C occurs with a low efficiency (≈1000-fold less) as
ous exocytosis at rat hippocampal synapses [104]. Inter- compared to SNAP-25 cleavage by BoNT/A or /E [73,
estingly, similar qualitative observations have been made 109]. Immunocytochemical studies of the frog nerve-
at arthropod synapses. At the crayfish motor synapses, motor junction have shown that carboxy-terminal
BoNT/D strongly reduces the frequency of spontaneous SNAP-25 immunoreactivity (this epitope is removed by
quantal events, but BoNT/B and TeNT-L chains do not BoNT/A, /E or /C) remains nearly intact at a time when
[92]. At larval neuromuscular synapses in transgenic syntaxin immunoreactivity is strongly decreased [91].
Drosophila expressing TeNT-L chain, spontaneous quan- However, BoNT/C is very efficient in removing nearly all
tal release is moderately reduced [105]. This is consistent SNAP-25 immunoreactivity in cultured hippocampal
with similar observations made at n-syb deleted transgenic slices or cultured spinal neurones [104, 110]. A clear
436 Humeau et al.

Table VII. Cleavage of syntaxin *#.

*Expected. #References mentioned in the table: [57, 74, 111–113].

demonstration of the causal relationship between syntaxin are almost unaffected, and a stable SDS-resistant complex
cleavage and blockage of neurotransmitter exocytosis has still forms. However, this complex is more efficiently
been made at squid synapses: syntaxin 1, but not SNAP- dissociated by α-SNAP/NSF than a one made with intact
25, can be cleaved by BoNT/C, and this toxin completely SNAP-25. When VAMP is cleaved by TeNT, BoNT/B or
inhibits neurotransmission [112, 113]. /G, binary interactions between VAMP and SNAP-25 or
syntaxin are reduced, and the cleavage products form less
complexes. Cleavage of syntaxin has no dramatic conse-
8. Alterations in SNARE binary interactions and quences: binary interactions between syntaxin and
complex assembly SNAP-25 or VAMP-2 are preserved and ternary interac-
In vitro reconstitution of the SNARE complex with tions allow formation of a complex which can shift, with
SNARE proteins cleaved by clostridial neurotoxins [81, reduced ability, into a SDS-resistant state [81].
114–116] or mutated SNAREs [117] have provided impor-
tant clues for understanding the blocking action of toxins. 8.2. No assembly of SDS-resistant SNARE complex
The consequences of the proteolytic action of the toxins
may be divided in two groups (table VIII). When SNAP-25 is cleaved by BoNT/E, its interaction
with VAMP-2 is strongly affected. In the absence of the
8.1. Creation of a SDS-resistant SNARE complex 26mer C-terminal peptides, a thermally stable SNARE
complex cannot be easily formed, and is readily dissoci-
After truncating SNAP-25 with BoNT/A (and by infer- ated when formed [81, 114]. Since the two arms of
ence by BoNT/C), binary interactions between SNAREs SNAP-25 are functionally independent, the addition of an
How botulinum and tetanus neurotoxins block neurotransmitter release 437

Figure 2. Clostridial neurotoxins exert their proteolytic action during a short physiological window. Exocytosis follows a sequence
of steps during which free vesicles (A) undergo docking at active zones (B and C), and fuse with the plasma membrane after a Ca2+
influx (D1). During this sequence of events, the clostridial neurotoxin targets (the three SNAREs VAMP, SNAP-25 and syntaxin)
assemble in complexes organised in a fusogenic ring (D2). D2 represents a cross section of D1 at the junction of the vesicle and plasma
membranes. An open configuration is adopted by VAMP, SNAP-25 or syntaxin only during step B. This would determine the
‘physiological window’ during which the clostridial neurotoxins can cleave their targets. Cleaved SNAREs can form complexes (here
denoted by faded colors) and incorporation of these into the fusogenic ring (D1, D2) is sufficient to prevent fusion process.

intact C-terminal arm of SNAP-25 (aa 142-206, i.e., the [81]. In agreement, kinetic analysis of exocytosis in
65mer peptide comprising the C-terminal coil) to BoNT/E chromaffin cells suggests that BoNT/A acts on a step in
poisoned PC12 cells allows formation of stable SNARE the secretory pathway that is earlier than the step affected
complexes of VAMP-2, syntaxin 1, the N-terminal arm of by BoNT/E [119, 120]. Alterations in the Ca2+-
the truncated SNAP-25 and the 65mer peptide [118]. dependency of quantal release after BoNT/A see (see
When VAMP is cleaved by BoNT/D or /F, binary inter- Section 4) may be accounted for by allosteric modification
actions between VAMP and SNAP-25 or syntaxin are of synaptotagmin triggering function (see further discus-
strongly reduced. In these cases, assembly of SDS- sion in [120]), or direct effects on the SNARE complex
resistant SNARE complexes are nearly abolished [81], [121]. Moreover, dysfunction of Snapin binding must now
and they are poorly dissociated by α-SNAP/NSF [114]. be considered as it binds to SNAP-25 and thereby,
regulates the binding of the Ca2+-sensor synaptotagmin to
the SNARE complex [122]. All these results are consistent
9. The steps of exocytosis blocked by clostridial with the view that BoNT/A acts by reducing the rate of
neurotoxins supply of ready-releasable granules, as suggested in chro-
maffin cells [120]. To summarize, cleavage of SNAP-25
The molecular mechanisms used by clostridial neuro- by BoNT/A seems to perturb a Ca2+-dependent priming
toxins to block exocytosis appear to fall into three step for docked vesicles that can be bypassed or forced to
categories. evolve towards fusion by any treatment that strongly
increases intracellular Ca2+ or release probability.
9.1. BoNT/A blocks a post-docking priming step
9.2. BoNT/D, /E, and /F prevent formation of fusion
As synaptic vesicles continue to dock at active zones complex
after BoNT/A poisoning, the step affected must be down-
stream from docking (table VIII). The step blocked by From in vitro data, it can be inferred that truncation of
BoNT/A is prior to fusion per se because assembly of SNAP-25 by BoNT/E prevents or destabilizes the four-
SDS-resistant SNARE complexes is almost preserved helical bundle of the synaptic fusion complex in such a
438 Humeau et al.

Table VIII. Exocytotic steps blocked by clostridial neurotoxins (a, b, c).

a
According to [81, 86, 114–116]. bFor details see table III. cNote that binding via transmembrane domain is unaffected after TeNT
cleavage of VAMP [85].

way that it cannot be tight enough to force the synaptic 9.3. BoNT/B, /C, /G, and TeNT uncouple fusion particle
vesicle membrane and plasma membrane to fuse. The from vesicle or plasma membrane
blocking action of BoNT/D and /F is rather similar.
Indeed, although the synaptic vesicle is coupled to Although the SDS-resistant SNARE complex can form
SNARE complexes by remaining VAMP-fragments, this after VAMP cleavage by BoNT/B or TeNT, the vesicle
complex cannot assemble into a SDS-resistant, fusogenic membrane and fusion particle are no longer physically
state. This explains why several of the treatments used to linked. In the case of BoNT/C action, the cleavage site in
enhance release probability, including black widow spider syntaxin is very close to the transmembrane domain.
venom or α-latrotoxin are unable to promote exocytosis at Hence, the vesicle and the fusion particle are tethered to
motor nerve terminals poisoned with BoNT/E, /D or /F. the plasma membrane only by SNAP-25, and this link is
How botulinum and tetanus neurotoxins block neurotransmitter release 439

probably too loose to make fusogenic the SNARE alpha- syntaxin is not abolished [84]. Hence, truncated
helical bundle. This explains why any increase in the C-terminal fragment of VAMP can sequester a certain
release probability is almost always unsuccessful with amount of syntaxin on vesicles. VAMP cleavage abolishes
these toxins. the interaction of VAMP with the adaptator protein AP3
[126] and may affect synaptic vesicle recycling via early
9.4. Alteration of synchronisation of SNARE complexes endosomes. The observation that vesicle-associated syn-
during fusion taxin is cleaved by BoNT/C [111] raises the question of
the step altered by this toxin. In addition to the direct
At vertebrate neuromuscular junctions, the blockade of consequences of altering SNARE integrity, clostridial
quantal ACh release induced by BoNTs or TeNT exhibits toxins act by releasing into the cytosol soluble target
several differences that are characteristic for a given set of fragments that can compete with intact molecules. Intra-
serotypes: after near completion of the blocking action of neuronal injection of VAMP fragments encompassing the
the toxins, the few quanta that can still be evoked remain VAMP domain released by BoNT/B or TeNT blocks
synchronized after BoNT/A and /E cleavage of SNAP-25 exocytosis [89, 127]. Intracellular application of the 9mer
(table III). In contrast, evoked quanta are strongly dis- and 26mer peptides generated by BoNT/A and /E block
persed, seemingly desynchronized, after TeNT, BoNT/B, exocytosis [128, 129].
/D or /F (i.e., all the neurotoxins that cleave VAMP)
(table III) and, references, therein). This raises the ques-
tion of the molecular mechanisms accounting for this 11. Duration of the block of neurotransmitter release
phenotype. One possibility relates to an alteration in the due to cleavage of SNAREs
synchronisation of SNARE complexes during the fusion
process. Indeed, fusion of vesicles with plasma membrane The paralysis induced by BoNT/E or by BoNTs that
is likely to involve simultaneous bundling of several target VAMP or syntaxin has a shorter duration than that
SNARE complexes creating a ring between the vesicle caused by BoNT/A. In the rat, BoNT/A-induced muscle
membrane and the plasmalemma [123]. Hence, the pres- paralysis remains for several weeks [100, 130, 131],
ence of a few altered SNARE complexes in the ring (i.e., whereas the inhibition caused by the other toxins is
made with truncated VAMP) would be sufficient to reversed within a few days [100, 132]. In man, similar
prevent fusion in normal conditions (figure 2). However, results are obtained [133]. Immunoreactivity for SNAP-25
when the release probability is strongly enhanced, this cleaved by BoNT/A, but not by BoNT/E, can be detected
blockade can sometimes be overcome (table III). Possibly long after the toxin application [134, 135]. Thus, it
due to heterogeneous responses of altered/intact SNARE remains unclear whether the truncated forms of SNAP-25
complexes in the ring to Ca2+ rises, the fusion reaction is have a different turnover rate, and/or BoNT/A has a longer
slowed thus accounting for the dispersed quanta that can lifetime than other toxins. Indeed, only cleavage-resistant
be observed after TeNT, BoNT/B, /D or /F. It is not clear SNAP-25 proved efficient in rescuing catecholamine exo-
why quantal dispersion is not observed after BoNT/E cytosis in chromaffin cells after 3 weeks of BoNT/A-
whereas, as discussed in the last paragraph, the fusion poisoning [136].
block seems rather similar to that produced by BoNT/D or
/F. Note that after BoNT/F some quanta remain synchro-
nized [100]. Alternatively, desynchronized quantal events 12. Do clostridial neurotoxins exert additional
may represent evoked release from a small vesicle popu- actions?
lation whose VAMP is resistant to toxins. Toxin resistant
exocytosis has been reported in mammalian cells possibly 12.1. Non-proteolytic blocking action
due to the presence of other VAMP isoforms [124, 125].
In this last part of the review, we want to briefly
mention several controversial observations that support
10. SNARE cleavage may alter steps of the vesicle the idea that additional mechanisms may contribute to the
cycle other than fusion inhibitory action of toxins. Intracellular application of
TeNT mutants lacking proteolytic activity [68–70] had no
After VAMP cleavage by TeNT, the SNARE complex effect on vasopressin secretion at isolated permeabilised
can be dissociated upon ATP hydrolysis by NSF, whereas neurohypophysial endings [60]. However, when the same
the complex formed with VAMP fragments after BoNT/F TeNT mutants or their corresponding mRNA were intro-
is much more resistant [114, 116, 117]. Thus, each step of duced into intact neurons by injection, neurotransmitter
the synaptic vesicle cycle involving SNARE complex release was substantially blocked [68, 137]. This suggests
dissociation may be affected by the toxin action. After that TeNT-mutants have non-proteolytic effects on a yet,
cleavage of VAMP-2 on the vesicle membrane, the inter- to be identified, synaptic target. The latter is likely to be
action between VAMP-2 transmembrane domains with soluble as it seems to be lost by dialysis during the
440 Humeau et al.

permeabilisation procedure (thus explaining the opposing gramme de Recherche Fondamentale en Microbiologie et Mala-
results obtained at intact and permeabilised nerve end- dies Infectieuses et Parasitaires and Direction des Systèmes des
ings). Forces et de la Prospective (Contract no. 99-34-038) to B.P.

12.2. Activation of transglutaminase


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