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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
Tetanus (from the greek tetanos = to contract) was first
referred to more than 20 centuries ago when Greek
physicians recognized this condition as a neurological
disease. Botulism, or sausage poisoning (from the latin
botulus = sausage), has been well documented since the
18th century. The infectious nature of tetanus and the
bacterial etiology and toxicopathological origin of botu-
lism were recognized at the end of the 19th century,
notably by Carle and Rattone [1] and Van Ermengen [2]
(for historical considerations see [3–6]). Major progress in
understanding this disease was the discovery by Burgen
and collaborators in 1949 that botulinum toxin blocks
neurotransmitter release [7]. However, it was not until 30
years later that Simpson proposed a cellular mechanism of
action involving three sequential steps, namely binding,
internalization and intraneuronal action [8]. Nearly one
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
[9, 10]. Thereafter, the intracellular mechanism of tetanus
* This paper is dedicated to the memory of Heiner Niemann.
** Present address: Departement of Neurobiology, Duke Univer-
sity Medical Center, 27710 Durham, NC, USA
*** Correspondence and reprints:
poulain@neurochem.u-strasbg.fr
toxin action was quickly deciphered in Montecucco’s
laboratory in 1992 [11, 12]. Over the next 3 years, other
clostridial neurotoxins were also demonstrated to be
zinc-endopeptidases that selectively cleave the SNAREs
(soluble NSF associated protein receptors) proteins
(VAMP (vesicle associated membrane protein), SNAP-25
(synaptosome associated protein of 25 kDa) and syntaxin)
implicated in neurotransmitter exocytosis (reviewed in
[13, 14]). Finally, the tridimensional structure of botuli-
num neurotoxin type A has now been resolved [15].
This article attempts to summarize what we know about
the molecular actions of clostridial neurotoxins and how
differences between toxins account for their distinct ef-
fects on neurotransmission. To simplify the text, data has
been included in a series of tables in which the reader will
find relevant references. However, only a fraction of the
vast literature on this subject has been cited and we
apologize for any essential omissions.
2. Clostridial neurotoxins
Seven distinct serotypes of botulinum toxins (BoNTs,
A-G) have been identified through the examination of
botulism outbreaks in man (BoNT/A, /B, /E and /F), birds
(BoNT/C), cattle (BoNT/D), or isolated from soils
(BoNT/G). The various BoNTs are produced by distinct
strains of Clostridium botulinum that display heteroge
428
neous bacteriological characteristics. Moreover, several
serotypes can be secreted by other Clostridia. In contrast,
tetanus neurotoxin (TeNT) is produced by a uniform group
of C. tetani (for reviews see [5, 16–18]). Bacteria produce
clostridial neurotoxins together with several other protein
toxins, such as the ADP-ribosyltransferase C2 binary toxin
that affects G-actin and the C3 exoenzyme which ADP-
ribosylates Rho (reviewed in [19, 20]).
BoNTs and TeNT are translated as single chain pro-
toxins and are subsequently cleaved within a disulphide
loop located about one-third of the way from the
N-terminus. This generates the fully active neurotoxin that
is both composed of a light chain of ≈50 kDa and a heavy
chain of ≈100 kDa linked by both a disulphide bridge and
non-covalent interactions [21]. During intoxication pro-
cess, the interchain bridge is reduced, and this is a
necessary prerequisite for the intracellular action of the
toxins [11, 22, 23]. Examination of the three-dimensional
structure of BoNT/A shows that when the interchain
disulphide bridge that links heavy and light chains is
intact, the N-terminal portion of the heavy chain masks the
catalytic cleft in the light chain, thereby preventing
substrate access [15].
The complete amino acid sequences of TeNT [9]and
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
sequences show an overall low homology (≈50%) with
short domains of higher homology. Only the tridimen-
sional structure of BoNT/A has been resolved [15]. Unlike
TeNT, BoNTs together with several proteins (some of
which exhibit haemagglutinin activity) are secreted as
very stable, high molecular mass complexes (up to
≈900 kDa in the case of BoNT/A) (reviewed in [5, 14,
17]). These complexes correspond to the so-called botu-
linum toxin or BoTx. The non-toxin proteins do not seem
to contribute to the cellular or molecular action of BoNTs
(discussed in [25]). Since the toxin complex is very stable
at low pH and dissociates at neutral pH [26], these
accessory proteins are probably essential for protecting
the neurotoxin moiety from proteolytic attack during
exposure to the gastrointestinal environment.
3. Botulism and tetanus
Botulism is a severe disease affecting vertebrates (ta-
ble I). It is characterized by flaccid paralysis originating in
the peripheral nervous system. It is induced by ingestion
of spoiled food (food-borne botulism), colonization of the
intestinal tract (infant botulism) or a wound (wound
botulism) by certain strains of C. botulinum. Earlier
studies on the pathophysiology of botulism revealed that
neuromuscular paralysis is due to selective inhibition of
evoked acetylcholine (ACh) release at the skeletal neuro-
muscular junction [7] (for reviews, see [8, 16, 27]).
Humeau et al.
However, BoNTs cannot be considered exclusively as
‘cholinergic’ toxins. Indeed, symptoms of autonomic dys-
function (parasympathetic, i.e., cholinergic, and sympa-
thetic, i.e., adrenergic and noradrenergic) coexist with
neuro-motor alterations during botulism (reviewed in [28,
29]). Moreover, central nervous system disorders may
occur during botulism or following the peripheral admin-
istration of toxins [30–33].
Tetanus is a spastic paralysis due to motor neuron
desinhibition [34]. The only etiological cause of the
disease is TeNT that is produced in a wound colonized by
C. tetani. The spastic paralysis that characterizes tetanus
in mammals is due to preferential blockage of inhibitory
(GABAergic and glycinergic) synapses afferent to moto-
neurons. However, TeNT can produce flaccid paralysis in
pathophysiological conditions (cranial tetanus) or after
injection of high doses of the toxin. This effect results
from blockage of ACh release at motor nerve endings [35,
36]. This is consistent with the current idea that motor
nerve terminals are the ‘gates’ giving TeNT access to the
central nervous system. Indeed, during tetanus, TeNT is
first captured by motor nerve endings followed by its
retrograde axonal transport and release into extracellular
fluids in the central nervous system.
4. Quantal characteristics of the inhibitory action of
BoNTs and TeNT on evoked and spontaneous
synaptic transmission
BoNTs and TeNT mainly cause a dramatic reduction of
amplitude, or even elimination of the postsynaptic re-
sponses evoked by action potentials, and a large reduction
in the frequency of spontaneous quantal release at both
vertebrate and invertebrate synapses (tables I, II). After
the toxin acts, quantal events detected at neuromuscular
junctions are often of smaller amplitude and have slower
kinetics [37–40]. However, this is mostly due to postsyn-
aptic changes in ACh-receptor densities and conductance
[41, 42], and not to changes in the neurotransmitter
content of synaptic vesicles (for discussion see [43]).
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
to strongly increase evoked and spontaneous release at
untreated synapses (reviewed in [44, 45]) are able to
induce substantial recovery of evoked release and enhance
spontaneous exocytosis at BoNT/A-poisoned nerve termi-
nals, but not at synapses inhibited by the other serotypes
(table III). Hence, in contrast to the other neurotoxins,
BoNT/A inhibits by altering the Ca2+-dependency of
quantal release. However, neither BoNT/A nor other
toxins inhibit Ca2+-influx (table III). Moreover, there are
no obvious morphological modifications in the docking of
synaptic vesicles at the active zone or the number of
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
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
block exocytotic events in a variety of non-neuronal cells
(table IV).
5. The light chains of clostridial neurotoxins are
metalloproteases
The action of extracellularly applied clostridial neuro-
toxins may be divided into three main steps, namely,
binding, internalization and intracellular action. We will
focus only on the latter step (for recent discussion of other
steps, see [18, 47, 48]). The light chains of BoNTs and
TeNT are implicated in the inhibition of transmitter
release [22, 23, 49–51]. However, the inhibitory action of
BoNTs in Aplysia neurons requires, for some unknown
reason, the presence of the heavy chain or its C-terminal
portion in the cytosol [52, 53].
The amino acid sequences of BoNT- or TeNT light
chains display low overall homology, except in the middle
portion which contains the His-Glu-x-x-His motif charac-
teristic of the catalytic pocket of all Zn-endopeptidases
[11, 12, 53, 54]. The light chains of BoNT/A, /B and /F
and TeNT bind one Zn atom [12, 55, 56], but that of
Humeau et al.
BoNT/C binds two Zn atoms, one of which plays a
structural role [57]. Zn is required for the intracellular
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].
Inhibitors of metallo-endopeptidases, such as phosphora-
midon [12] captopril or thiorphan ([60], Deloye and
Poulain, unpublished observation), antagonize the inhibi-
tory action of intracellularly applied TeNT or BoNT/A, /B.
However, the effects of these pharmacological agents
appear less clear in some preparations, and may depend on
the type of cells used (PC12 cells: [59, 63], vertebrate
neuromuscular junction: [64]). Recently, potent inhibitors
of TeNT and BoNT/B have been designed, but have yet to
be tested in vivo [65, 66]. Characterization of the relation-
ship between the structure and the function of the catalytic
pocket of TeNT light chain has revealed that His233 and
His236 residues are the first two ligands of the Zn atom.
A third one is a water molecule polarized by the carboxyl
group of the Glu234 residue in the His-Glu-x-x-His motif.
A fourth ligand consists of another Glu residue outside of
the His-Glu-x-x-His motif. Tyr365 has been proposed to
be a fifth ligand [67]. Genetic substitution of the His and
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
VAMP [68–70].
6. Clostridial neurotoxins specifically recognize and
cleave VAMP, SNAP-25 or syntaxin
Only three synaptic proteins (figure 1) have been iden-
tified as targets of the eight clostridial neurotoxin sero-
types (reviewed in [14, 71]). In mammals, VAMP-1 and -2
are cleaved by TeNT, BoNT/B, /D, /F and /G (table V),
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-
ent degrees. For example, murine SNAP-23 (also termed
Syndet) can be cleaved by BoNT/E and to a reduced
extent by BoNT/A, but human SNAP-23 is resistant to
both BoNT/A and /E due to mutations of Lys185Asp and
Pro182Arg [72, 73]. Syntaxins 2 and 3, but not syntaxin 4
are cleaved by BoNT/C [74].
432
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
targets makes them resistant to proteolysis and can ac-
count for almost all cases of species insensitivity to BoNT
or TeNT (compare (tables I, II) with (tables V, VII)).
Occasionally, toxin-resistance is caused by the absence of
specific membrane ectoacceptors (i.e., man is not affected
by BoNT/D [75]). Most toxin-insensitive VAMPs are
generated by mutations in the cleavage site (table V),
while toxin-resistant SNAP-25 results from amino acid
substitutions in and around this site (table VI). This may
arise because VAMP cleavage sites are located in a
domain essential for its function (i.e., binding with other
SNAREs), whereas SNAP-25 cleavage sites, in particular
those for BoNT/A and /C are located in a less crucial
domain.
All toxins cleave their targets at distinct sites, except
for BoNT/B and TeNT which attack the same peptide
Humeau et al.
bond in VAMP. This has raised the question of what
mechanisms determine how each toxin specifically at-
tacks: i) a unique peptide bond in their target proteins; and
ii) only VAMP, SNAP-25 or syntaxin, and not other
synaptic proteins. This exquisite selectivity is due to the
presence of a highly conserved common motif in the
amino acid sequence of the target. Depending on the
protein, the motif is termed V1 and V2 in VAMP, S1 to S4
in SNAP-25 and X1 and X2 in syntaxin (figure 1).
Although BoNT/B and TeNT attack the same Gln-Phe
peptide bond, these toxins display distinct affinities for V1
and V2: TeNT, like BoNT/D requires the V1 motif,
whereas BoNT/B (as BoNT/G) binds to V2. BoNT/F
recognizes V1 and/or V2 [76–78]. Furthermore, TeNT
exploits a second exosite (figure 1), rich in positive
charges and located adjacent to the cleavage site [79].
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
that V1 and V2 domains are not necessarily exposed under
the same physiological circumstances. For efficient cleav-
age of SNAP-25, both BoNT/A and /E require the
presence of the C-terminal region of SNAP-25 which
includes the motif S4. However, the three N-terminal
motifs (S1, S2 and S3) can partially substitute for a
modified S4 motif [72]. Functional consequences of these
observations have been provided by the finding that the
target proteins are protected from proteolytic attack when
they are bound in ternary complex [81–83]. This is
probably due to masking of cleavage sites and/or recog-
nition motifs. This concept can be extended to several
other complexes involving SNAREs. For example, when
SNAP-25 is dimerised together with syntaxin 1, it is
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
the SNARE protein can be cleaved. For example, when
VAMP-2 interacts with syntaxin 1 via its transmembrane
domains, it can be attacked by TeNT [85]. Similarly,
SNAP-25 bound to synaptotagmin can be cleaved by
either BoNT/A or /E [86]. In general, SNAREs should be
protected from proteolysis when a closed configuration
(by folding or multimerisation) is generated. In this view,
it is likely that when VAMP binds to synaptophysin [87],
it is probably protected against proteolysis. Clostridial
neurotoxins are likely to have free access to their substrate
only during a short ‘physiological window’ (figure 2),
possibly during docking of synaptic vesicles at active
zones, when the SNAREs are dissociated from their
chaperons and prior to their assembly into the fusogenic
SNARE complex. Although the physiological window for
proteolytic attack is relatively brief, the neurotoxins are
able to progressively cleave the majority of the SNARE
population. This is consistent with the evidence that all the
steps prior to the assembly of SNAREs into tight com-
plexes are reversible [88] and tethered vesicles can be-
come undocked.
7. Cause-effect relationship between SNARE cleavage
and blockage of neurotransmitter release
7.1. SNARE proteolysis and inhibition of exocytosis
The relationship between target proteolysis and block-
age of neurotransmitter release was first established for
434
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
peptides including the VAMP cleavage site for TeNT or
BoNT/B diminishes their inhibitory action [11, 89], and
pre-injection of specific anti-VAMP/synaptobrevin anti-
bodies into Aplysia cholinergic neurones prevents the
blocking action of TeNT and BoNT/B, but not that of
BoNT/A which cleaves SNAP-25 [90]. Immunocy-
tochemical studies at the frog and crayfish neuromuscular
junctions show that the inhibition of neurotransmitter
release correlates with the disappearance of VAMP immu-
noreactivity [91, 92]. Transfection of insulin-secreting
cells with mutated VAMP that cannot be cleaved by TeNT
allows partial restoration of secretion [93]. Leech
SNAP-25 cannot be cleaved by BoNT/A, and accordingly,
cultured leech neurones are BoNT/A resistant. However,
sensitivity of neurotransmitter release to BoNT/A could be
conferred just by replacing amino acids 196–209 of leech
SNAP-25 with those of the rat protein [94]. A further
Humeau et al.
demonstration of the causal link between SNAP-25 cleav-
age and exocytosis blockage has been obtained with
insulin-secreting cells: human SNAP-23 can replace
SNAP-25 in SNARE complex, and overexpression of
SNAP-23 which is resistant to BoNT/A rescues insulin
secretion after blockage by BoNT/A [95]. Intriguingly,
although Torpedo SNAP-25 is not cleaved by BoNT/A in
vitro [72], BoNT/A abolishes ACh release at Torpedo
electric organ [96, 97].
7.2. Difference in susceptibility of spontaneous
exocytosis to toxins suggests distinct roles for VAMP-1
and VAMP-2
In general, the toxins that attack VAMP have little
effect on spontaneous exocytosis. Moreover, the degree of
inhibition seems to correlate with the different suscepti-
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
VAMP-1 and VAMP-2 can be cleaved by TeNT or
BoNT/B (table V) and there is a significant decrease in
spontaneous release frequency [40, 98, 99]. Unlike
VAMP-1, rat VAMP-2 can be cleaved by TeNT and
BoNT/B (table V), and strikingly, very little decrease in
quantal frequency is observed with TeNT [36]. BoNT/D
and /F cleave both VAMP-1 and -2 rat isoforms, and both
toxins strongly diminish quantal frequency at rat motor
nerve endings [100, 101]. VAMP-1 is preferentially ex-
pressed in the motor system, whereas VAMP-2 is pre-
dominantly present in nuclei associated with sensory and
integrative functions [102, 103]. Along this line, TeNT is
relatively potent in inhibiting both evoked and spontane-
ous exocytosis at rat hippocampal synapses [104]. Inter-
estingly, similar qualitative observations have been made
at arthropod synapses. At the crayfish motor synapses,
BoNT/D strongly reduces the frequency of spontaneous
quantal events, but BoNT/B and TeNT-L chains do not
[92]. At larval neuromuscular synapses in transgenic
Drosophila expressing TeNT-L chain, spontaneous quan-
tal release is moderately reduced [105]. This is consistent
with similar observations made at n-syb deleted transgenic
Drosophila larval neuromuscular junction [106–108].
Taken together, these results suggest that VAMP-2 is more
specialized in mediating evoked release, while VAMP-1
or a toxin-insensitive VAMP isoform is principally impli-
cated in spontaneous release.
7.3. Cleavage of syntaxin or/and SNAP-25 during
BoNT/C action
BoNT/C cleaves both syntaxin 1 and SNAP-25
[109–111]. Thus, an important issue is to determine which
of these two target proteins is cleaved during BoNT/C-
induced blockage. In vitro, cleavage of SNAP-25 by
BoNT/C occurs with a low efficiency (≈1000-fold less) as
compared to SNAP-25 cleavage by BoNT/A or /E [73,
109]. Immunocytochemical studies of the frog nerve-
motor junction have shown that carboxy-terminal
SNAP-25 immunoreactivity (this epitope is removed by
BoNT/A, /E or /C) remains nearly intact at a time when
syntaxin immunoreactivity is strongly decreased [91].
However, BoNT/C is very efficient in removing nearly all
SNAP-25 immunoreactivity in cultured hippocampal
slices or cultured spinal neurones [104, 110]. A clear
436
Table VII. Cleavage of syntaxin *#.
*Expected. #References mentioned in the table: [57, 74, 111–113].
demonstration of the causal relationship between syntaxin
cleavage and blockage of neurotransmitter exocytosis has
been made at squid synapses: syntaxin 1, but not SNAP-
25, can be cleaved by BoNT/C, and this toxin completely
inhibits neurotransmission [112, 113].
8. Alterations in SNARE binary interactions and
complex assembly
In vitro reconstitution of the SNARE complex with
SNARE proteins cleaved by clostridial neurotoxins [81,
114–116] or mutated SNAREs [117] have provided impor-
tant clues for understanding the blocking action of toxins.
The consequences of the proteolytic action of the toxins
may be divided in two groups (table VIII).
8.1. Creation of a SDS-resistant SNARE complex
After truncating SNAP-25 with BoNT/A (and by infer-
ence by BoNT/C), binary interactions between SNAREs
Humeau et al.
are almost unaffected, and a stable SDS-resistant complex
still forms. However, this complex is more efficiently
dissociated by α-SNAP/NSF than a one made with intact
SNAP-25. When VAMP is cleaved by TeNT, BoNT/B or
/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-
quences: binary interactions between syntaxin and
SNAP-25 or VAMP-2 are preserved and ternary interac-
tions allow formation of a complex which can shift, with
reduced ability, into a SDS-resistant state [81].
8.2. No assembly of SDS-resistant SNARE complex
When SNAP-25 is cleaved by BoNT/E, its interaction
with VAMP-2 is strongly affected. In the absence of the
26mer C-terminal peptides, a thermally stable SNARE
complex cannot be easily formed, and is readily dissoci-
ated when formed [81, 114]. Since the two arms of
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
65mer peptide comprising the C-terminal coil) to BoNT/E
poisoned PC12 cells allows formation of stable SNARE
complexes of VAMP-2, syntaxin 1, the N-terminal arm of
the truncated SNAP-25 and the 65mer peptide [118].
When VAMP is cleaved by BoNT/D or /F, binary inter-
actions between VAMP and SNAP-25 or syntaxin are
strongly reduced. In these cases, assembly of SDS-
resistant SNARE complexes are nearly abolished [81],
and they are poorly dissociated by α-SNAP/NSF [114].
9. The steps of exocytosis blocked by clostridial
neurotoxins
The molecular mechanisms used by clostridial neuro-
toxins to block exocytosis appear to fall into three
categories.
9.1. BoNT/A blocks a post-docking priming step
As synaptic vesicles continue to dock at active zones
after BoNT/A poisoning, the step affected must be down-
stream from docking (table VIII). The step blocked by
BoNT/A is prior to fusion per se because assembly of
SDS-resistant SNARE complexes is almost preserved
[81]. In agreement, kinetic analysis of exocytosis in
chromaffin cells suggests that BoNT/A acts on a step in
the secretory pathway that is earlier than the step affected
by BoNT/E [119, 120]. Alterations in the Ca2+-
dependency of quantal release after BoNT/A see (see
Section 4) may be accounted for by allosteric modification
of synaptotagmin triggering function (see further discus-
sion in [120]), or direct effects on the SNARE complex
[121]. Moreover, dysfunction of Snapin binding must now
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
with the view that BoNT/A acts by reducing the rate of
supply of ready-releasable granules, as suggested in chro-
maffin cells [120]. To summarize, cleavage of SNAP-25
by BoNT/A seems to perturb a Ca2+-dependent priming
step for docked vesicles that can be bypassed or forced to
evolve towards fusion by any treatment that strongly
increases intracellular Ca2+ or release probability.
9.2. BoNT/D, /E, and /F prevent formation of fusion
complex
From in vitro data, it can be inferred that truncation of
SNAP-25 by BoNT/E prevents or destabilizes the four-
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
vesicle membrane and plasma membrane to fuse. The
blocking action of BoNT/D and /F is rather similar.
Indeed, although the synaptic vesicle is coupled to
SNARE complexes by remaining VAMP-fragments, this
complex cannot assemble into a SDS-resistant, fusogenic
state. This explains why several of the treatments used to
enhance release probability, including black widow spider
venom or α-latrotoxin are unable to promote exocytosis at
motor nerve terminals poisoned with BoNT/E, /D or /F.
9.3. BoNT/B, /C, /G, and TeNT uncouple fusion particle
from vesicle or plasma membrane
Although the SDS-resistant SNARE complex can form
after VAMP cleavage by BoNT/B or TeNT, the vesicle
membrane and fusion particle are no longer physically
linked. In the case of BoNT/C action, the cleavage site in
syntaxin is very close to the transmembrane domain.
Hence, the vesicle and the fusion particle are tethered to
the plasma membrane only by SNAP-25, and this link is
How botulinum and tetanus neurotoxins block neurotransmitter release
probably too loose to make fusogenic the SNARE alpha-
helical bundle. This explains why any increase in the
release probability is almost always unsuccessful with
these toxins.
9.4. Alteration of synchronisation of SNARE complexes
during fusion
At vertebrate neuromuscular junctions, the blockade of
quantal ACh release induced by BoNTs or TeNT exhibits
several differences that are characteristic for a given set of
serotypes: after near completion of the blocking action of
the toxins, the few quanta that can still be evoked remain
synchronized after BoNT/A and /E cleavage of SNAP-25
(table III). In contrast, evoked quanta are strongly dis-
persed, seemingly desynchronized, after TeNT, BoNT/B,
/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
phenotype. One possibility relates to an alteration in the
synchronisation of SNARE complexes during the fusion
process. Indeed, fusion of vesicles with plasma membrane
is likely to involve simultaneous bundling of several
SNARE complexes creating a ring between the vesicle
membrane and the plasmalemma [123]. Hence, the pres-
ence of a few altered SNARE complexes in the ring (i.e.,
made with truncated VAMP) would be sufficient to
prevent fusion in normal conditions (figure 2). However,
when the release probability is strongly enhanced, this
blockade can sometimes be overcome (table III). Possibly
due to heterogeneous responses of altered/intact SNARE
complexes in the ring to Ca2+ rises, the fusion reaction is
slowed thus accounting for the dispersed quanta that can
be observed after TeNT, BoNT/B, /D or /F. It is not clear
why quantal dispersion is not observed after BoNT/E
whereas, as discussed in the last paragraph, the fusion
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
may represent evoked release from a small vesicle popu-
lation whose VAMP is resistant to toxins. Toxin resistant
exocytosis has been reported in mammalian cells possibly
due to the presence of other VAMP isoforms [124, 125].
10. SNARE cleavage may alter steps of the vesicle
cycle other than fusion
After VAMP cleavage by TeNT, the SNARE complex
can be dissociated upon ATP hydrolysis by NSF, whereas
the complex formed with VAMP fragments after BoNT/F
is much more resistant [114, 116, 117]. Thus, each step of
the synaptic vesicle cycle involving SNARE complex
dissociation may be affected by the toxin action. After
cleavage of VAMP-2 on the vesicle membrane, the inter-
action between VAMP-2 transmembrane domains with
439
syntaxin is not abolished [84]. Hence, truncated
C-terminal fragment of VAMP can sequester a certain
amount of syntaxin on vesicles. VAMP cleavage abolishes
the interaction of VAMP with the adaptator protein AP3
[126] and may affect synaptic vesicle recycling via early
endosomes. The observation that vesicle-associated syn-
taxin is cleaved by BoNT/C [111] raises the question of
the step altered by this toxin. In addition to the direct
consequences of altering SNARE integrity, clostridial
toxins act by releasing into the cytosol soluble target
fragments that can compete with intact molecules. Intra-
neuronal injection of VAMP fragments encompassing the
VAMP domain released by BoNT/B or TeNT blocks
exocytosis [89, 127]. Intracellular application of the 9mer
and 26mer peptides generated by BoNT/A and /E block
exocytosis [128, 129].
11. Duration of the block of neurotransmitter release
due to cleavage of SNAREs
The paralysis induced by BoNT/E or by BoNTs that
target VAMP or syntaxin has a shorter duration than that
caused by BoNT/A. In the rat, BoNT/A-induced muscle
paralysis remains for several weeks [100, 130, 131],
whereas the inhibition caused by the other toxins is
reversed within a few days [100, 132]. In man, similar
results are obtained [133]. Immunoreactivity for SNAP-25
cleaved by BoNT/A, but not by BoNT/E, can be detected
long after the toxin application [134, 135]. Thus, it
remains unclear whether the truncated forms of SNAP-25
have a different turnover rate, and/or BoNT/A has a longer
lifetime than other toxins. Indeed, only cleavage-resistant
SNAP-25 proved efficient in rescuing catecholamine exo-
cytosis in chromaffin cells after 3 weeks of BoNT/A-
poisoning [136].
12. Do clostridial neurotoxins exert additional
actions?
12.1. Non-proteolytic blocking action
In this last part of the review, we want to briefly
mention several controversial observations that support
the idea that additional mechanisms may contribute to the
inhibitory action of toxins. Intracellular application of
TeNT mutants lacking proteolytic activity [68–70] had no
effect on vasopressin secretion at isolated permeabilised
neurohypophysial endings [60]. However, when the same
TeNT mutants or their corresponding mRNA were intro-
duced into intact neurons by injection, neurotransmitter
release was substantially blocked [68, 137]. This suggests
that TeNT-mutants have non-proteolytic effects on a yet,
to be identified, synaptic target. The latter is likely to be
soluble as it seems to be lost by dialysis during the
440
permeabilisation procedure (thus explaining the opposing
results obtained at intact and permeabilised nerve end-
ings).
12.2. Activation of transglutaminase
TeNT has been found to activate GTP-binding protein
type h, also termed tissue transglutaminase type II (abbre-
viated as Gh/TGase) [138]. Gh/TGase is found in the
soluble cytosolic fraction of nerve terminals or associated
with vesicle membranes [139]. The inhibitory activity of
non-proteolytic mutants of TeNT has been proposed to
involve Gh/TGase [137, 140].
12.3. Cytoskeletal alterations
Another matter of controversy is the action of TeNT on
the cytoskeleton. TeNT inhibits the rearrangements of
subcortical microfilaments that accompanies secretion in
chromaffin cells [141], and expression of TeNT light chain
in mouse Sertoli cells alters the actin cytoskeleton [142].
TeNT affects the depolarisation-stimulated phosphoryla-
tion and redistribution of synapsin I [143]. Furthermore,
DasGupta and Tepp [144] reported that BoNT/E light
chain cleaves actin in vitro, and all of the 11 cleavages
sites identified involved Arg or Lys residues in P1
position, exactly as it does in SNAP-25 (table VI).
12.4. Changes in PKC activity
In a possible link with the cytoskeletal alterations,
TeNT action has also been reported to be associated with
changes in synaptic phosphoinositidesty levels and/or
protein kinase C activity [145–149].
12.5. Inhibition of neurotransmitter re-uptake
Unexpectedly, TeNT but not BoNT/A, inhibits sodium
dependent 5HT uptake, and this action is mediated by the
C-terminal half of its heavy chain [150, 151].
13. Conclusion
The question was: what are the clostridial neurotoxins
doing that is so deadly? ‘Blocking neurotransmission’ is
the answer which prompted many physicians to use
botulinum toxins as therapeutic tools to treat dystonia and
related diseases following Alan Scott’s example ([152]
and reviews in [16, 153]). ‘SNARE cleavage’ is the
answer that stimulated many cell biologists to use
clostridial neurotoxins as research tools for solving fun-
damental questions on secretion [13]. What will the next
answer and application be?
Acknowledgments
Work on botulinum and tetanus neurotoxins was supported by
grants from Association Française contre les Myopathies, Pro-
Humeau et al.
gramme de Recherche Fondamentale en Microbiologie et Mala-
dies Infectieuses et Parasitaires and Direction des Systèmes des
Forces et de la Prospective (Contract no. 99-34-038) to B.P.
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