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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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HRP uptake stimulated by K+ depolarization. (a) Control. Recycled synaptic vesicles contain peroxidase reaction product. (b) TeNT (10 ng/ml for 18 h). Only occasional synaptic vesicles contain reaction product, indicating the failure of synaptic vesicles to undergo recycling. (c) BoNT A (200 ng/ml for 18 h). A large number of synaptic vesicles contain reaction product, evidence that these vesicles have been formed from surface membrane. (insets) Reaction product labeling of clathrin-coated vesicles implicates these structures in the process of vesicle recycling. Note the larger labeled structures (arrows) in a and c. That these structures are not seen in TeNT-exposed cultures suggests that they are an intermediate in the vesicle recycling pathway. Double arrowheads in b mark coated pits associated with conventional endocytosis that is unaffected by TeNT. These micrographs are the high resolution equivalents of Fig. 6, a, d, and f. Bar: (a–c) 1 μm. Insets are enlarged to 135,000×.
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Figure 8: HRP uptake stimulated by K+ depolarization. (a) Control. Recycled synaptic vesicles contain peroxidase reaction product. (b) TeNT (10 ng/ml for 18 h). Only occasional synaptic vesicles contain reaction product, indicating the failure of synaptic vesicles to undergo recycling. (c) BoNT A (200 ng/ml for 18 h). A large number of synaptic vesicles contain reaction product, evidence that these vesicles have been formed from surface membrane. (insets) Reaction product labeling of clathrin-coated vesicles implicates these structures in the process of vesicle recycling. Note the larger labeled structures (arrows) in a and c. That these structures are not seen in TeNT-exposed cultures suggests that they are an intermediate in the vesicle recycling pathway. Double arrowheads in b mark coated pits associated with conventional endocytosis that is unaffected by TeNT. These micrographs are the high resolution equivalents of Fig. 6, a, d, and f. Bar: (a–c) 1 μm. Insets are enlarged to 135,000×.

Mentions: Purified TeNT (2 × 107 mouse lethal doses/mg of protein) was a gift from Dr. William Habig (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD). Except as noted, purified BoNT A was purchased from List Biological Laboratories, Inc. and BoNT C was from the Centre for Applied Microbiology and Research (5.2 × 107 and 1.0 × 107 mouse LD50/mg protein, respectively). BoNT A (9.4 × 106 mouse LD50/mg protein) from Calbiochem-Novabiochem Corp. was used for the experiments presented in Fig. 5, a–c, and Fig. 8. BoNT A provided by Drs. Eric Johnson and Michael Goodenough (University of Wisconsin, Madison, WI) was used for the experiments in Fig. 4 and Fig. 5. Concentration of BoNT A was adjusted based on the IC50 for blockade of glycine release; the effects of each preparation on exo- and endocytosis were tested and found to be identical. BoNT B (1.26 × 106 LD50/mg protein) was purchased from Calbiochem-Novabiochem Corp. [3H]Glycine (sp act 12.2 Ci/mmol) was purchased from Amersham Corp. Affinity-purified rabbit polyclonal antisynapsin 1a was obtained from Chemicon International, Inc. Affinity-purified rabbit polyclonal antibodies against amino acids 1–32 of the variable domain of vesicle-associated membrane protein (VAMP) 1 and against the COOH-terminal 12 amino acids of synaptosomal-associated protein of 25 kD (SNAP-25) (Rossetto et al. 1996) were a gift of Dr. Cesare Montecucco (University of Padova, Padova, Italy). FM1-43 was obtained from Molecular Probes and HRP Type VI from Sigma Chemical Co.


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)

HRP uptake stimulated by K+ depolarization. (a) Control. Recycled synaptic vesicles contain peroxidase reaction product. (b) TeNT (10 ng/ml for 18 h). Only occasional synaptic vesicles contain reaction product, indicating the failure of synaptic vesicles to undergo recycling. (c) BoNT A (200 ng/ml for 18 h). A large number of synaptic vesicles contain reaction product, evidence that these vesicles have been formed from surface membrane. (insets) Reaction product labeling of clathrin-coated vesicles implicates these structures in the process of vesicle recycling. Note the larger labeled structures (arrows) in a and c. That these structures are not seen in TeNT-exposed cultures suggests that they are an intermediate in the vesicle recycling pathway. Double arrowheads in b mark coated pits associated with conventional endocytosis that is unaffected by TeNT. These micrographs are the high resolution equivalents of Fig. 6, a, d, and f. Bar: (a–c) 1 μm. Insets are enlarged to 135,000×.
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Related In: Results  -  Collection

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Figure 8: HRP uptake stimulated by K+ depolarization. (a) Control. Recycled synaptic vesicles contain peroxidase reaction product. (b) TeNT (10 ng/ml for 18 h). Only occasional synaptic vesicles contain reaction product, indicating the failure of synaptic vesicles to undergo recycling. (c) BoNT A (200 ng/ml for 18 h). A large number of synaptic vesicles contain reaction product, evidence that these vesicles have been formed from surface membrane. (insets) Reaction product labeling of clathrin-coated vesicles implicates these structures in the process of vesicle recycling. Note the larger labeled structures (arrows) in a and c. That these structures are not seen in TeNT-exposed cultures suggests that they are an intermediate in the vesicle recycling pathway. Double arrowheads in b mark coated pits associated with conventional endocytosis that is unaffected by TeNT. These micrographs are the high resolution equivalents of Fig. 6, a, d, and f. Bar: (a–c) 1 μm. Insets are enlarged to 135,000×.
Mentions: Purified TeNT (2 × 107 mouse lethal doses/mg of protein) was a gift from Dr. William Habig (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD). Except as noted, purified BoNT A was purchased from List Biological Laboratories, Inc. and BoNT C was from the Centre for Applied Microbiology and Research (5.2 × 107 and 1.0 × 107 mouse LD50/mg protein, respectively). BoNT A (9.4 × 106 mouse LD50/mg protein) from Calbiochem-Novabiochem Corp. was used for the experiments presented in Fig. 5, a–c, and Fig. 8. BoNT A provided by Drs. Eric Johnson and Michael Goodenough (University of Wisconsin, Madison, WI) was used for the experiments in Fig. 4 and Fig. 5. Concentration of BoNT A was adjusted based on the IC50 for blockade of glycine release; the effects of each preparation on exo- and endocytosis were tested and found to be identical. BoNT B (1.26 × 106 LD50/mg protein) was purchased from Calbiochem-Novabiochem Corp. [3H]Glycine (sp act 12.2 Ci/mmol) was purchased from Amersham Corp. Affinity-purified rabbit polyclonal antisynapsin 1a was obtained from Chemicon International, Inc. Affinity-purified rabbit polyclonal antibodies against amino acids 1–32 of the variable domain of vesicle-associated membrane protein (VAMP) 1 and against the COOH-terminal 12 amino acids of synaptosomal-associated protein of 25 kD (SNAP-25) (Rossetto et al. 1996) were a gift of Dr. Cesare Montecucco (University of Padova, Padova, Italy). FM1-43 was obtained from Molecular Probes and HRP Type VI from Sigma Chemical Co.

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