Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration are dictated by the sites of SNAP-25 truncation

Meunier, Frédéric A.; Lisk, Godfrey; Sesardic, Dorothea; Dolly, J. Oliver

doi:10.1016/s1044-7431(02)00016-7

Cited by 133 publications

(131 citation statements)

References 25 publications

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“…The addition of BoNT/E LC to permeabilized chromaffin cells led to marked changes in the localization of the target SNAREs. Following the toxin treatment, SNAP-25 immunoreactivity translocated partially into the cytosol in agreement with previous studies (19), whereas syntaxin1 immunoreactivity remained restricted to the plasma membrane (Fig. 6D).…”

Section: Resultssupporting

confidence: 91%

High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

Rickman

Meunier

Binz

et al. 2004

Journal of Biological Chemistry

105

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The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that leads to formation of the fusogenic SNARE complex. We now show that the plasma membrane syntaxin and SNAP-25 interact with high affinity and equimolar stoichiometry to form a stable dimer on the pathway to the ternary SNARE complex. In bovine chromaffin cells, syntaxin and SNAP-25 colocalize in defined clusters that average 700 nm in diameter and cover 10% of the plasma membrane. Removal of the C terminus of SNAP-25 by botulinum neurotoxin E, a known neuroparalytic agent, dissociates the target SNARE dimer in vitro and disrupts the SNARE clustering in vivo. Together, our data uncover formation of stable syntaxin/SNAP-25 dimers as a central principle of the SNARE assembly pathway underlying regulated exocytosis.Hormonal and neurotransmitter release occurs when vesicles fuse with the plasma membrane in a calcium-dependent manner. The three SNARE 1 proteins, namely synaptobrevin2 (also known as VAMP2) on the vesicular membrane and syntaxin1 and SNAP-25 on the target plasma membrane, are essential for the last step of vesicular exocytosis, the fusion of membranes (1, 2). These three proteins form a highly stable ternary complex that likely drives the fusion of the two apposing membranes (3). Botulinum toxins, which specifically cleave SNARE proteins prior to the formation of the ternary SNARE complex, block vesicle exocytosis in both neurons and endocrine cells (2, 4). The plasma membrane protein SNAP-25 is a molecular target for botulinum toxin E (5). This toxin removes the C-terminal 26 amino acids from the SNAP-25 molecule and has been used previously to dissect stages of calcium-triggered exocytosis (6, 7). Although these studies demonstrated that the ternary SNARE complex forms at a late stage of vesicle exocytosis, the molecular events preceding formation of this complex remain largely unknown.Several contradicting theories have been put forward with regard to the molecular pathway leading to SNARE complex assembly. One study suggested that SNAREs, although active, do not pre-assemble in resting cells into any intermediate complex but would form the ternary complex upon activation of exocytosis (8). It has also been proposed that SNAP-25 engages vesicular synaptobrevin in a priming step and that binding of syntaxin1 would be the pivotal event leading to membrane fusion (9). An alternative theory states that syntaxin1 and SNAP-25 pre-assemble into a stable heterodimer on the plasma membrane, and only then does vesicular synaptobrevin engage the two target SNAREs (10, 11). Importantly, it is not clear whether any SNARE intermediates actually exist in intact secretory cells as a prelude to SNARE-mediated fusion.To address these outstanding issues, we analyzed SNARE binary re...

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Section: Resultssupporting

confidence: 91%

High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

Rickman

Meunier

Binz

et al. 2004

Journal of Biological Chemistry

105

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“…Samples were run on an SDS-PAGE and then transferred to a PVDF membrane. The membrane was probed for cleaved SNAP25 using an antibody designed against the SNAP25-cleaved product, which does not recognize full-length SNAP25 (37,38). The intensity of the bands was normalized to ␤-actin, and the integrated intensity was used to determine the amount of cleavage relative to the control (ImageJ).…”

Section: Methodsmentioning

confidence: 99%

“…We next investigated whether Dyngo-4a could prevent the cleavage of SNAP25 in hippocampal neurons. Using an antibody specifically designed to recognize BoNT/A-cleaved SNAP25 (37,38), hippocampal neurons incubated with BoNT/A alone exhibited a distinct band at ϳ25 kDa (Fig. 6A).…”

Section: Dyngo-4a Prevents Bont/a-induced Snap25 Cleavage In Culturedmentioning

confidence: 99%

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

Harper

Martin

Nguyen

et al. 2011

Journal of Biological Chemistry

147

149

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The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a TM , was sufficient to abolish BoNT/ A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/ A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.

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“…These observations suggest that only a portion of functional SNAP-25 needs to be cleaved to induce a near-complete synaptic paralysis. Truncated SNAP-25 lacks only 9 C-terminal amino acids, which does not impair the forming of heterotrimeric complex with other two SNAREs (Lawrence et al, 2002;Meunier et al, 2003). Inactive SNARE complex by itself may inhibit the neurotransmitter release (Keller and Neale, 2001).…”

Section: 2 Duration and Extent Of Bont/a-induced Synaptic Paralysismentioning

confidence: 99%

Botulinum toxin A, brain and pain

Matak

Lacković

2014

Progress in Neurobiology

149

114

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Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration are dictated by the sites of SNAP-25 truncation

Cited by 133 publications

References 25 publications

High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

Botulinum toxin A, brain and pain

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