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Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal.

Neale EA, Bowers LM, Jia M, Bateman KE, Williamson LC - J. Cell Biol. (1999)

Bottom Line: Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis.We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis.In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. eneale@codon.nih.gov

ABSTRACT
The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.

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Exocytosis of HRP stimulated by K+ depolarization. (a and c) Control; and (b and d) BoNT A (200 ng/ml for 18 h). Synaptic terminals are loaded with horseradish peroxidase (a, b). After subsequent stimulation in the absence of HRP, the percent labeled vesicles is lower in control (c) but unchanged in toxin-blocked (d) cultures. These micrographs are analogous to Fig. 6, a and i, and Fig. 6f and Fig. k, providing further demonstration of the block in exocytosis induced by BoNT A. Note coated pits and coated vesicles implicated in synaptic vesicle membrane retrieval (arrows) in b and c and conventional coated pits (double arrowheads) in a and d. Bar, 0.5 μm.
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Figure 9: Exocytosis of HRP stimulated by K+ depolarization. (a and c) Control; and (b and d) BoNT A (200 ng/ml for 18 h). Synaptic terminals are loaded with horseradish peroxidase (a, b). After subsequent stimulation in the absence of HRP, the percent labeled vesicles is lower in control (c) but unchanged in toxin-blocked (d) cultures. These micrographs are analogous to Fig. 6, a and i, and Fig. 6f and Fig. k, providing further demonstration of the block in exocytosis induced by BoNT A. Note coated pits and coated vesicles implicated in synaptic vesicle membrane retrieval (arrows) in b and c and conventional coated pits (double arrowheads) in a and d. Bar, 0.5 μm.

Mentions: To confirm that HRP-labeled vesicles in control cultures are competent for exocytosis, we loaded terminals by stimulating for 2 min in the presence of 8 mg/ml HRP. HRP-loaded terminals were subjected to a second interval of K+ depolarization and the change in percentage of labeled vesicles was determined (Fig. 9 and Table ). After HRP-loading of the control cultures, ∼10% of total synaptic vesicles contain HRP (Fig. 9 a). In cultures stimulated after loading, labeled vesicles decrease to 2.5% of total vesicles (Fig. 9 c). Without Ca2+ in the stimulation medium, the percentage of labeled vesicles remains high (10%; not shown). Thus, the loss of HRP is consistent with synaptic vesicle exocytosis.


Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal.

Neale EA, Bowers LM, Jia M, Bateman KE, Williamson LC - J. Cell Biol. (1999)

Exocytosis of HRP stimulated by K+ depolarization. (a and c) Control; and (b and d) BoNT A (200 ng/ml for 18 h). Synaptic terminals are loaded with horseradish peroxidase (a, b). After subsequent stimulation in the absence of HRP, the percent labeled vesicles is lower in control (c) but unchanged in toxin-blocked (d) cultures. These micrographs are analogous to Fig. 6, a and i, and Fig. 6f and Fig. k, providing further demonstration of the block in exocytosis induced by BoNT A. Note coated pits and coated vesicles implicated in synaptic vesicle membrane retrieval (arrows) in b and c and conventional coated pits (double arrowheads) in a and d. Bar, 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168097&req=5

Figure 9: Exocytosis of HRP stimulated by K+ depolarization. (a and c) Control; and (b and d) BoNT A (200 ng/ml for 18 h). Synaptic terminals are loaded with horseradish peroxidase (a, b). After subsequent stimulation in the absence of HRP, the percent labeled vesicles is lower in control (c) but unchanged in toxin-blocked (d) cultures. These micrographs are analogous to Fig. 6, a and i, and Fig. 6f and Fig. k, providing further demonstration of the block in exocytosis induced by BoNT A. Note coated pits and coated vesicles implicated in synaptic vesicle membrane retrieval (arrows) in b and c and conventional coated pits (double arrowheads) in a and d. Bar, 0.5 μm.
Mentions: To confirm that HRP-labeled vesicles in control cultures are competent for exocytosis, we loaded terminals by stimulating for 2 min in the presence of 8 mg/ml HRP. HRP-loaded terminals were subjected to a second interval of K+ depolarization and the change in percentage of labeled vesicles was determined (Fig. 9 and Table ). After HRP-loading of the control cultures, ∼10% of total synaptic vesicles contain HRP (Fig. 9 a). In cultures stimulated after loading, labeled vesicles decrease to 2.5% of total vesicles (Fig. 9 c). Without Ca2+ in the stimulation medium, the percentage of labeled vesicles remains high (10%; not shown). Thus, the loss of HRP is consistent with synaptic vesicle exocytosis.

Bottom Line: Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis.We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis.In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. eneale@codon.nih.gov

ABSTRACT
The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.

Show MeSH
Related in: MedlinePlus