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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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Clathrin and synaptic vesicle recycling. (a) Resting control culture. Clathrin-coated pits and vesicles are seen only occasionally, whereas empty clathrin baskets (arrowheads) are more numerous. (b) K+-depolarized control culture. Clathrin-coated pits and vesicles (arrows) are seen commonly; empty clathrin baskets are rare. Note the electron lucent matrix of mitochondria (asterisks) in cultures fixed during stimulation. (c) Resting TeNT-blocked culture. Clathrin-coated pits and vesicles are almost never seen although empty clathrin baskets are found easily. (d) K+-depolarized TeNT-blocked culture. K+ stimulation causes a change in the density of the mitochondrial matrix, but no noteworthy change in the distribution of synaptic vesicles at the active zone or frequency of clathrin baskets or clathrin-coated membranes. (e) Resting BoNT A–blocked culture. This culture appears similar to the TeNT-blocked culture in the absence of clathrin-coated membranes and the persistence of empty clathrin baskets. (f) K+-depolarized BoNT A–blocked culture. This culture appears more similar to the stimulated control than to the stimulated TeNT-blocked culture. Empty clathrin baskets have all but disappeared and clathrin-coated membranes can be found. Toxin concentrations as in Fig. 5. Bar, 0.5 μm.
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Figure 7: Clathrin and synaptic vesicle recycling. (a) Resting control culture. Clathrin-coated pits and vesicles are seen only occasionally, whereas empty clathrin baskets (arrowheads) are more numerous. (b) K+-depolarized control culture. Clathrin-coated pits and vesicles (arrows) are seen commonly; empty clathrin baskets are rare. Note the electron lucent matrix of mitochondria (asterisks) in cultures fixed during stimulation. (c) Resting TeNT-blocked culture. Clathrin-coated pits and vesicles are almost never seen although empty clathrin baskets are found easily. (d) K+-depolarized TeNT-blocked culture. K+ stimulation causes a change in the density of the mitochondrial matrix, but no noteworthy change in the distribution of synaptic vesicles at the active zone or frequency of clathrin baskets or clathrin-coated membranes. (e) Resting BoNT A–blocked culture. This culture appears similar to the TeNT-blocked culture in the absence of clathrin-coated membranes and the persistence of empty clathrin baskets. (f) K+-depolarized BoNT A–blocked culture. This culture appears more similar to the stimulated control than to the stimulated TeNT-blocked culture. Empty clathrin baskets have all but disappeared and clathrin-coated membranes can be found. Toxin concentrations as in Fig. 5. Bar, 0.5 μm.

Mentions: To strengthen the hypothesis that FM1-43 staining of BoNT A–blocked terminals is related to vesicle membrane endocytosis, we examined the fine structure of synaptic terminals. In spontaneously active control cultures (Fig. 7 a), clathrin-coated pits and vesicles are seen only occasionally, although empty clathrin baskets (arrowheads; Kanaseki and Kadota 1969) are not uncommon. When control cultures are stimulated (56 mM K+ and 2 mM Ca2+ for 5 min; Fig. 7 b), small coated pits and vesicles (arrows) are observed with increased frequency and empty clathrin baskets with decreased frequency. In cultures blocked with TeNT (Fig. 7 c) or with BoNT A (Fig. 7 e), synaptic vesicles accumulate at the presynaptic membrane, empty clathrin baskets are typical, and coated pits and vesicles are extremely rare. When TeNT-blocked cultures are stimulated with K+ (Fig. 7 d), these synaptic features remain unchanged, suggesting a lack of membrane movement. When BoNT A–blocked cultures are depolarized (Fig. 7 f), the number of docked synaptic vesicles remains elevated, although clathrin-coated pits are found with increased frequency and empty clathrin baskets have disappeared. Thus, stimulated BoNT A cultures take on those features which suggest active retrieval of synaptic vesicle membrane.


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)

Clathrin and synaptic vesicle recycling. (a) Resting control culture. Clathrin-coated pits and vesicles are seen only occasionally, whereas empty clathrin baskets (arrowheads) are more numerous. (b) K+-depolarized control culture. Clathrin-coated pits and vesicles (arrows) are seen commonly; empty clathrin baskets are rare. Note the electron lucent matrix of mitochondria (asterisks) in cultures fixed during stimulation. (c) Resting TeNT-blocked culture. Clathrin-coated pits and vesicles are almost never seen although empty clathrin baskets are found easily. (d) K+-depolarized TeNT-blocked culture. K+ stimulation causes a change in the density of the mitochondrial matrix, but no noteworthy change in the distribution of synaptic vesicles at the active zone or frequency of clathrin baskets or clathrin-coated membranes. (e) Resting BoNT A–blocked culture. This culture appears similar to the TeNT-blocked culture in the absence of clathrin-coated membranes and the persistence of empty clathrin baskets. (f) K+-depolarized BoNT A–blocked culture. This culture appears more similar to the stimulated control than to the stimulated TeNT-blocked culture. Empty clathrin baskets have all but disappeared and clathrin-coated membranes can be found. Toxin concentrations as in Fig. 5. Bar, 0.5 μm.
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Related In: Results  -  Collection

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Figure 7: Clathrin and synaptic vesicle recycling. (a) Resting control culture. Clathrin-coated pits and vesicles are seen only occasionally, whereas empty clathrin baskets (arrowheads) are more numerous. (b) K+-depolarized control culture. Clathrin-coated pits and vesicles (arrows) are seen commonly; empty clathrin baskets are rare. Note the electron lucent matrix of mitochondria (asterisks) in cultures fixed during stimulation. (c) Resting TeNT-blocked culture. Clathrin-coated pits and vesicles are almost never seen although empty clathrin baskets are found easily. (d) K+-depolarized TeNT-blocked culture. K+ stimulation causes a change in the density of the mitochondrial matrix, but no noteworthy change in the distribution of synaptic vesicles at the active zone or frequency of clathrin baskets or clathrin-coated membranes. (e) Resting BoNT A–blocked culture. This culture appears similar to the TeNT-blocked culture in the absence of clathrin-coated membranes and the persistence of empty clathrin baskets. (f) K+-depolarized BoNT A–blocked culture. This culture appears more similar to the stimulated control than to the stimulated TeNT-blocked culture. Empty clathrin baskets have all but disappeared and clathrin-coated membranes can be found. Toxin concentrations as in Fig. 5. Bar, 0.5 μm.
Mentions: To strengthen the hypothesis that FM1-43 staining of BoNT A–blocked terminals is related to vesicle membrane endocytosis, we examined the fine structure of synaptic terminals. In spontaneously active control cultures (Fig. 7 a), clathrin-coated pits and vesicles are seen only occasionally, although empty clathrin baskets (arrowheads; Kanaseki and Kadota 1969) are not uncommon. When control cultures are stimulated (56 mM K+ and 2 mM Ca2+ for 5 min; Fig. 7 b), small coated pits and vesicles (arrows) are observed with increased frequency and empty clathrin baskets with decreased frequency. In cultures blocked with TeNT (Fig. 7 c) or with BoNT A (Fig. 7 e), synaptic vesicles accumulate at the presynaptic membrane, empty clathrin baskets are typical, and coated pits and vesicles are extremely rare. When TeNT-blocked cultures are stimulated with K+ (Fig. 7 d), these synaptic features remain unchanged, suggesting a lack of membrane movement. When BoNT A–blocked cultures are depolarized (Fig. 7 f), the number of docked synaptic vesicles remains elevated, although clathrin-coated pits are found with increased frequency and empty clathrin baskets have disappeared. Thus, stimulated BoNT A cultures take on those features which suggest active retrieval of synaptic vesicle membrane.

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