(Received for publication, June 1, 1995; and in revised form, January 6, 1996)
From the
Clostridial neurotoxins are zinc endopeptidases that block neurotransmission and have been shown to cleave, in vitro, specific proteins involved in synaptic vesicle docking and/or fusion. We have used immunohistochemistry and immunoblotting to demonstrate alterations in toxin substrates in intact neurons under conditions of toxin-induced blockade of neurotransmitter release. Vesicle-associated membrane protein, which co-localizes with synaptophysin, is not detectable in tetanus toxin-blocked cultures. Syntaxin, also concentrated in synaptic sites, is cleaved by botulinum neurotoxin C. Similarly, the carboxyl terminus of the synaptosomal-associated protein of 25 kDa (SNAP-25) is not detectable in botulinum neurotoxin A-treated cultures. Unexpectedly, tetanus toxin exposure causes an increase in SNAP-25 immunofluorescence, reflecting increased accessibility of antibodies to antigenic sites rather than increased expression of the protein. Furthermore, botulinum neurotoxin C causes a marked loss of the carboxyl terminus of SNAP-25 when the toxin is added to living cultures, whereas it has no action on SNAP-25 in in vitro preparations. This study is the first to demonstrate in functioning neurons that the physiologic response to these toxins is correlated with the proteolysis of their respective substrates. Furthermore, the data demonstrate that botulinum neurotoxin C, in addition to cleaving syntaxin, exerts a secondary effect on SNAP-25.
Clostridial neurotoxins (CNTs) ()are synthesized as
single polypeptides of 150 kDa and subsequently are cleaved to active
disulfide-linked dichain toxins. The heavy chain (100 kDa) carries the
receptor binding and transmembrane domains of the toxin, and the light
chain (50 kDa) contains the catalytic domain that blocks
neurotransmitter release(1, 2) . Clostridial
neurotoxins are zinc
endopeptidases(3, 4, 5) , which cleave
specific proteins thought to be involved in the synaptic vesicle
docking-fusion complex. Vesicle-associated membrane protein (VAMP),
also known as synaptobrevin, is cleaved by the majority of the CNTs
including tetanus toxin (TeNT) and botulinum neurotoxins (BoNTs) B, D,
F, and
G(3, 5, 6, 7, 8, 9) .
Botulinum neurotoxins A and E cleave SNAP-25 (synaptosomal-associated
protein of 25 kDa)(6, 10, 11, 12) ,
and BoNT C cleaves syntaxin(13, 14) .
VAMP, SNAP-25, and syntaxin interact with N-ethylmaleimide-sensitive fusion protein and soluble N-ethylmaleimide-sensitive fusion protein attachment proteins (SNAPs), cytosolic elements essential for intracellular membrane fusion(15) . Since SNAPs must bind to membrane receptors prior to N-ethylmaleimide-sensitive fusion protein attachment, the SNAP receptors have been designated as SNAREs, with the vesicular protein (VAMP/synaptobrevin) as the v-SNARE and the target membrane proteins (SNAP-25 and syntaxin) as the t-SNAREs(15) . Furthermore, it has been demonstrated that VAMP, SNAP-25, and syntaxin themselves form a stable complex(16, 17, 18) .
Two isoforms of VAMP
in neuronal tissue and a nonneuronal homologue cellubrevin have been
identified in a number of animal species (19, 20, 21, 22) . VAMP is comprised
of three major domains: the NH terminus is a variable
domain, the middle or B domain is highly conserved, and the COOH
terminus contains the transmembrane spanning region with a short
projection into the synaptic vesicle lumen. Tetanus toxin cleaves the
peptide bond Gln
-Phe
within the
conserved domain of VAMP(3) . Recent in vitro studies
have shown that VAMP can bind to syntaxin(17) , to SNAP-25 (17) , and to
synaptophysin(23, 24, 25) .
Syntaxin has two isoforms that are anchored to membranes by a single COOH-terminal transmembrane domain(26, 27) . Syntaxin binds to presynaptic calcium channels and to synaptotagmin located in the synaptic vesicle membrane(26, 28, 29) . Botulinum neurotoxin C cleaves syntaxin at a site near the transmembrane domain(13, 14, 30) .
SNAP-25
is a membrane-associated cytoplasmic protein implicated in the fusion
of synaptic vesicles with the presynaptic membrane (15) and in
membrane addition leading to constitutive axonal growth(31) .
SNAP-25 is a hydrophilic protein that is palmitylated at one to four of
its closely spaced cysteine residues and acts as an integral membrane
protein(32, 33) . Botulinum neurotoxin A cleaves SNAP
25 at a site nine amino acids from the COOH terminus between residues
Gln and
Arg
(6, 10, 11, 12) .
Tetanus toxin, BoNT A, and BoNT C act in vivo to block neurotransmitter release. Both BoNT A and TeNT have been shown previously to block synaptic transmission in spinal cord cell cultures (34, 35) . TeNT-induced disappearance of inhibitory and excitatory postsynaptic potentials coincides in the same system with the blockade of inhibitory and excitatory neurotransmitter release(36) . In the present study, we have characterized the effect of TeNT, BoNT A, and BoNT C on the vSNARE, VAMP/synaptobrevin, and the tSNARES, SNAP-25 and syntaxin, in cells in which we have demonstrated the arrest of neurotransmitter release. This is the first study to examine VAMP, SNAP-25, and syntaxin in intact functioning neurons, where it has been possible to observe the action of BoNT C on two of the three SNARE proteins.
Spinal cord cell cultures contain a heterogeneous population
of neurons growing on a monolayer of nonneuronal cells (Fig. 1A). To confirm that TeNT, BoNT A, and BoNT C
have blocked synaptic neurotransmission, spinal cord cultures are
assayed for inhibitory and excitatory neurotransmitter release.
Cultures exposed to these toxins are radiolabeled with
[H]glycine or [
H]glutamine.
Neurotransmitter release is evoked by potassium-induced depolarization
in the presence of calcium. With potassium stimulation, control
cultures release 25-30% of the total
[
H]glycine (Fig. 1B) and
4-5% of the total [
H]glutamate (Fig. 1C) taken up by cultures. Tetanus toxin, BoNT A,
and BoNT C completely block potassium-evoked release of both
neurotransmitters (Fig. 1, B and C).
Figure 1: Neurotransmitter release in spinal cord cell cultures. A, interference contrast photomicrograph shows that spinal cord cell cultures are a heterogeneous mixture of neurons. Magnification bar, 25 µm. B and C, control and toxin-exposed cultures are radiolabeled and assayed for potassium-stimulated calcium-dependent release of glycine and glutamate. Tetanus toxin and botulinum neurotoxins A and C (0.06 nM for 20 h) completely block release of the inhibitory neurotransmitter glycine (B) and the excitatory neurotransmitter glutamate (C).
Tetanus toxin abolishes synaptic immunostaining in mouse spinal cord neurons of both VAMP-1 (Fig. 2) and VAMP-2 (not shown). Loss of VAMP from neuronal terminals after TeNT proteolysis is demonstrated clearly by double-labeling experiments using mouse anti-synaptophysin, a marker for synaptic terminals, detected with fluorescein (Fig. 2, A, C, and E) and rabbit anti-VAMP-1 detected with rhodamine (Fig. 2, B, D, and F). In control cultures, VAMP-1 immunostaining of synaptic terminals (Fig. 2B) co-localizes with synaptophysin immunoreactivity (Fig. 2A). In TeNT-exposed cultures, although synaptic terminals are stained with anti-synaptophysin (Fig. 2C), there is no synaptic labeling with anti-VAMP-1 (Fig. 2D). An additional control for the specificity of TeNT action on VAMP was obtained using BoNTs A and C, which also are zinc endopeptidases, but which cleave other components of the vesicle docking-fusion complex(15) . VAMP immunoreactivity (Fig. 2F) is unaffected by BoNT A exposure at a time when neurotransmitter release is completely blocked. Similar results were observed in BoNT C-exposed cultures (data not shown). Consistent with the immunohistochemistry, VAMP is absent from immunoblots of cultures exposed to TeNT (Fig. 3).
Figure 2: Cell cultures double-labeled with antibodies against VAMP and against synaptophysin. Control cultures show synaptophysin immunostaining (A) in synaptic terminals; VAMP immunoreactivity (B) has an almost identical distribution. In cultures synaptically blocked by TeNT (0.06 nM for 20 h), synapses are identified clearly with anti-synaptophysin (C), although VAMP immunostaining (D) is totally abolished. In contrast, in BoNT A-blocked cultures (0.06 nM for 20 h), synapses are stained with both anti-synaptophysin (E) and with anti-VAMP (F) antibodies. Magnification bar, 25 µm.
Figure 3: Immunoblot analysis for VAMP and syntaxin in toxin-treated cultures. Spinal cord cell cultures were incubated with BoNT A, BoNT C, or TeNT (0.06 nM) for 16 h. Homogenates were prepared and analyzed for VAMP and syntaxin as described under ``Experimental Procedures.'' VAMP is lost completely from cultures exposed to TeNT. Syntaxin is cleaved to a lower molecular weight by BoNT C.
In control cultures, syntaxin is localized at the neuronal surface, particularly along axonal membranes, but also appears concentrated at synaptic membrane sites marked by synaptophysin immunoreactivity (Fig. 4, A and B). However, syntaxin staining persists in cultures known to be intoxicated by BoNT C (Fig. 4, C and D). This is consistent with the persistence of syntaxin on immunoblots (Fig. 3). Botulinum neurotoxin C cleaves syntaxin near the transmembrane domain producing a soluble fragment of syntaxin that is not degraded further(13) . Immunoblots of homogenates prepared from BoNT C-exposed spinal cord cell cultures show syntaxin cleaved to a lower molecular weight band. None of the other toxins have any effect on syntaxin (Fig. 3).
Figure 4: Cell cultures double-labeled with antibodies against syntaxin and against synaptophysin. A, synaptophysin immunostaining defines synaptic terminals in control cultures. B, syntaxin is associated with the neuronal membrane including that of axonal branches, although intense immunofluorescence tends to coincide with synaptic sites. In BoNT C-blocked cultures, syntaxin immunoreactivity (D) persists, both at synaptophysin-positive sites (C) and along axonal membranes. Magnification bar, 25 µm.
The localization of SNAP-25 and the effects of BoNT A, TeNT, or BoNT C on its distribution were analyzed by double-label immunohistochemistry using antibodies against synaptophysin (Fig. 5, A, C, E, and G) and against the COOH terminus of SNAP-25 (Fig. 5, B, D, F, and H). The pattern of immunostaining for SNAP-25 in control cultures is similar to that of syntaxin (Fig. 5, A and B); i.e. presence along axonal and synaptic membranes. Botulinum neurotoxin A cleaves the last nine amino acids from the COOH terminus of SNAP-25(6, 10, 11, 12) . Synaptic terminals identified by synaptophysin immunostaining in BoNT A-exposed cultures (Fig. 5C) do not stain with antibodies against the COOH terminus of SNAP-25. (Fig. 5D). SNAP-25 is lost not only from the synaptic membranes but also from the other neuronal surface membranes including those of axons and cell bodies. Unexpectedly, alterations in SNAP-25 are seen also when cultures are exposed to TeNT or BoNT C. In TeNT-exposed cultures, SNAP-25 immunofluorescence clearly is more intense than in control cultures (Fig. 5F). In contrast, the immunoreactivity of the SNAP-25 COOH terminus is markedly reduced in BoNT C-exposed neurons (Fig. 5H).
Figure 5: Cell cultures double-labeled with antibodies against the carboxyl terminus of SNAP-25 and against synaptophysin. In control cultures, SNAP-25 immunostaining (B) shows a distribution pattern similar to that for syntaxin (compare with Fig. 4B); the most intense fluorescence co-localizes with synaptic sites marked by synaptophysin immunoreactivity (A). Synapses identified by synaptophysin immunoreactivity in BoNT A-blocked cultures (C) show no staining for the COOH terminus of SNAP-25 (D). Additionally, SNAP-25 immunostaining is lost from all neuronal surface membranes in BoNT A-treated cultures (D). In TeNT-blocked cultures, SNAP-25 immunoreactivity (F) over axonal membranes is more intense than in control cultures (compare with panel B; reacted, photographed, and printed under the same conditions), whereas synaptophysin staining (E) is similar to controls. In BoNT C-blocked cultures (0.06 nM for 20 h), staining for the COOH terminus of SNAP-25 (H) is almost totally eliminated from structures that stain with anti-synaptophysin antibodies (G). Magnification bar, 25 µm.
SNAP-25 in control and toxin-treated cell
cultures was analyzed further by immunoblotting (Fig. 6).
Treatment of cultures with BoNT A for 24 h results in the loss of the
COOH terminus of SNAP-25 (Fig. 6A). Botulinum
neurotoxin C exposure causes a similar loss of the COOH terminus of
SNAP-25, consonant with the immunohistochemistry of intact neurons. In
contrast, TeNT has no effect on SNAP-25 when analyzed by immunoblot (Fig. 6A). Immunoblots prepared from another set of
BoNT A or BoNT C-blocked cultures show two bands detected with a
monoclonal antibody against the NH terminus of SNAP-25 (Fig. 6B); the predominant band is the cleaved lower
molecular weight form of SNAP-25 and the other band corresponds to the
remaining uncleaved SNAP-25.
Figure 6:
Immunoblot analysis of SNAP-25 in
toxin-treated spinal cord cultures. Cultures are incubated with BoNT A,
BoNT C, or TeNT (0.06 nM) for 24 h (A) or 20 h (B). Homogenates are prepared and analyzed for SNAP-25
immunoreactivity using antibodies against the COOH or NH termini. In BoNT A- or BoNT C-treated cultures, SNAP-25 (COOH
terminus) immunoreactivity is lost completely (A). SNAP-25
immunoreactivity in TeNT-blocked cultures is similar to controls (A). In another set of BoNT A- or BoNT C-treated cultures,
staining of the NH
terminus confirms SNAP-25 proteolysis (B).
Cleavage of SNAP-25 was examined after
4, 8, and 16 h of toxin exposure to compare BoNT A and BoNT C effects (Fig. 7). Proteolysis of SNAP-25 by BoNT A is more rapid and
more complete than by BoNT C as evidenced with both SNAP-25 antibodies.
Some COOH terminus immunoreactivity persists after 16 h in BoNT C,
whereas there is none left with BoNT A treatment. Similarly, antibodies
against the NH terminus indicate that some uncleaved
SNAP-25 remains in BoNT C-treated cultures, although the BoNT A-exposed
cultures show a clear progression with time to the total cleavage of
SNAP-25. The time course of syntaxin cleavage demonstrates that
virtually all of syntaxin is cleaved by BoNT C in 16 h, whereas more
SNAP-25 remains intact. Thus, BoNT C action on SNAP-25 appears to
follow its proteolysis of syntaxin.
Figure 7: Time course analysis of SNAP-25 cleavage in BoNT A and BoNT C-treated cultures. Spinal cord cultures were incubated in BoNT A or BoNT C (0.3 nM) for 4, 8 and 16 h. Homogenates were prepared and analyzed for SNAP-25 and syntaxin. BoNT A cleaves SNAP-25 more rapidly than BoNT C. Whereas there is total cleavage of SNAP-25 in BoNT A-treated cultures, some uncleaved SNAP-25 remains after 16 h with BoNT C. The proteolysis of SNAP-25 in BoNT C-treated cultures appears to follow the more complete cleavage of syntaxin.
To determine if cleavage of SNAP-25 in BoNT C-exposed cultures were due to contamination with BoNT A, toxins used in these studies were immunoblotted with antibodies against BoNT A. Preparations of BoNT C were not recognized by antibodies against BoNT A, providing evidence against the possibility of contamination by BoNT A (data not shown). Additionally, immunohistochemistry and immunoblots of BoNT A and BoNT C-treated cultures were repeated using a mixture of toxin with an excess of antibodies against BoNT A. When cultures are exposed to the BoNT A preparation premixed with antibodies against BoNT A, immunoreactivity for SNAP-25 persists, and no cleavage of SNAP-25 is detected by immunoblot, i.e. BoNT A is rendered ineffective. However, BoNT C premixed with anti-BoNT A is equally as effective as BoNT C alone in altering the staining patterns of both SNAP-25 and syntaxin (data not shown). These data demonstrate that the effect of BoNT C on SNAP-25 cannot be explained by the presence of contaminating amounts of BoNT A.
BoNT C action on SNAP-25 has not been described before, although the
previous studies were carried out using subcellular preparations. We
investigated the action of BoNT C in vitro on postnuclear
supernatants prepared from spinal cord cell cultures. For in vitro studies, BoNT A and BoNT C (150 nM final concentration)
are activated (3, 4, 5, 11) prior to
addition to the postnuclear supernatants for 90 min at 37 °C. Under
these conditions, immunoblots using antibodies against either the
NH or COOH terminus demonstrate proteolysis of SNAP-25 by
BoNT A but not by BoNT C (data not shown). Thus, the action of BoNT C
on SNAP-25 is observed only when the toxin is added to intact neurons
and gains access to synaptic proteins under physiologic conditions.
This study is the first to demonstrate in physiologically relevant cells, i.e. in intact functioning neurons, a direct correlation between the clostridial neurotoxin-induced block in neurotransmitter release and the cleavage of toxin-specific protein substrates, VAMP, SNAP-25, or syntaxin. The cleavage of synaptic proteins may not be the only mechanism whereby these toxins induce their prolonged neuroparalysis(41, 42, 43) . Nonetheless, our data clearly demonstrate by immunohistochemistry and immunoblot analysis that VAMP, SNAP-25, and syntaxin are cleaved by TeNT, BoNT A, or BoNT C, respectively, in the same neurons in which neurotransmitter release is shown to be blocked. These findings confirm that the principal mechanism of action of clostridial neurotoxins is proteolytic cleavage of specific synaptic proteins necessary for neurotransmitter release.
Additionally, this study provides insight
into the interaction of these proteins preceding synaptic vesicle
exocytosis in intact neurons. Treatment of cultures with TeNT results
in the cleavage of VAMP and an increase in the intensity of SNAP-25
immunoreactivity as detected by immunohistochemistry with the COOH
terminus antibody. This suggests that VAMP/synaptobrevin binds to the
COOH terminus of SNAP-25. In contrast, immunohistochemistry of the
NH terminus of SNAP-25 in intact neurons remains unchanged
(data not shown). Furthermore, immunoblot analysis of SNAP-25 (COOH
terminus) did not show any difference between control and TeNT-exposed
cultures. Thus, the increased immunoreactivity of the COOH terminus of
SNAP-25 in TeNT-treated cultures is not due to increased levels of
SNAP-25 but rather to increased accessibility to the antibody. Direct
binding of SNAP-25 to VAMP and to syntaxin was demonstrated in an in vitro system using purified fusion proteins and deletion
analysis (17, 18) . Furthermore, Chapman et al.(17) reported that deletion of nine amino acids from the
COOH terminus of SNAP-25 reduced VAMP binding by 73%. Our observations
in spinal cord neurons are consistent with these findings.
When BoNT
C is added to intact spinal cord neurons in culture, not only is
syntaxin cleaved, as expected, but there is a concomitant loss of
immunostaining for the COOH terminus of SNAP-25. The reduction in
SNAP-25 immunoreactivity in intact neurons is seen both by
immunohistochemistry and by immunoblotting. Furthermore, in support of
this result, an antibody against the NH terminus of SNAP-25
recognizes two bands due to SNAP-25 cleavage in BoNT C-treated
cultures. Possible explanations for the effect include 1) that syntaxin
proteolysis by BoNT C changes the conformation or accessibility of
SNAP-25, increasing its susceptibility to endogenous proteases or 2)
that BoNT C itself proteolytically cleaves SNAP-25 albeit at a slower
rate than it cleaves syntaxin. The action of BoNT C on SNAP-25
apparently occurs only when living neurons are exposed to this toxin.
SNAP-25 is not affected when BoNT C is added to spinal cord cell
culture homogenates, although BoNT A cleaves SNAP-25 in the same
homogenates. Furthermore, BoNT C does not cleave recombinant SNAP-25. (
)Thus, the action of BoNT C on SNAP-25 in vivo might occur only when SNAP-25 is complexed with another protein
and/or plasma membranes. Alternatively, SNAP-25 may be protected from
proteolysis by endogenous proteases when it is complexed to syntaxin,
and the cleavage of syntaxin by BoNT C increases SNAP-25's
susceptibility to proteolysis.
Recent findings demonstrate the presence of syntaxin (proposed as a t-SNARE) in purified synaptic vesicle fractions(37) . Although the majority of syntaxin appeared in a plasma membrane fraction, BoNT C preferentially cleaved vesicular syntaxin leaving the plasma membrane syntaxin largely unaffected. In the spinal cord cultures, however, much lower concentrations of BoNT C cleaved almost all syntaxin. Differences between in vivo and in vitro preparations as well as differences in the conditions of BoNT C incubation might account for this discrepancy.
In summary, the findings reported here demonstrate the actions of the clostridial neurotoxins in vivo, seen together as the blockade of neurotransmitter release with the proteolytic cleavage of the respective toxin substrates. The data also provide evidence for the first time that, in addition to cleavage of syntaxin, BoNT C has a secondary action on the COOH terminus of SNAP-25. The finding that BoNT C is the only clostridial neurotoxin that acts on two of the three SNARE proteins might be significant in terms of its efficacy for the clinical treatment of muscle spasm disorders.