Uptake of Botulinum Neurotoxin into Cultured Neurons

Abstract

Botulinum neurotoxins (BoNTs) act within the synaptic terminal to block neurotransmitter release. The toxin enters the neuron by binding to neuronal membrane receptor(s), being taken up into an endosome-like compartment, and penetrating the endosome membrane via a pH-dependent translocation process. Once within the synaptic cytoplasm, BoNT serotypes A and E cleave separate sites on the C-terminus of the neuronal protein SNAP-25, one of the SNARE proteins required for synaptic vesicle fusion. In this study, we measured the effect of brief toxin exposure on SNAP-25 proteolysis in neuronal cell cultures as an indicator of toxin translocation. The results indicate that (1) uptake of both BoNT-A and -E is enhanced with synaptic activity induced by K+ depolarization in the presence of Ca2+ and (2) translocation of BoNT-A from the acidic endosomal compartment is slow relative to that of BoNT-E. Polyclonal antisera against each toxin protect cells when applied with the toxin during stimulation but has no effect when added immediately after toxin exposure, indicating that toxin endocytosis occurs with synaptic activity. Both serotypes cleave SNAP-25 at concentrations between 50 pM and 4 nM. IC50 values for SNAP-25 cleavage are approximately 0.5 nM for both serotypes. Inhibition of the pH-dependent translocation process by pretreating cultures with concanamycin A (Con A) prevents cleavage of SNAP-25 with IC50 values of approximately 25 nM. Addition of Con A at times up to 15 min after toxin exposure abrogated BoNT-A action; however, addition of Con A after 40 min was no longer protective. In contrast, Con A inhibited, but did not prevent, translocation of BoNT-E even when added immediately after toxin exposure, indicating that pH-dependent translocation of BoNT-E is rapid relative to that of BoNT-A. This study demonstrates that uptake of both BoNT-A and -E is enhanced with synaptic activity and that translocation of the toxin catalytic moiety into the cytosol occurs at different rates for these two serotypes.

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

“…Functional studies have clearly indicated that multiple SNARE complexes cooperate to bring about an individual vesicular fusion event (Hua and Scheller, 2001;Keller and Neale, 2001;Han et al, 2004;Keller et al, 2004;Montecucco et al, 2005), with the number of participating complexes possibly affecting the speed of opening, or the diameter of the fusion pore (Han et al, 2004). Although oligomerization may be an inherent feature of SNARE complexes, a detailed description of how such interactions take place and their relevance for membrane fusion has been lacking.…”

“…The exact number of complexes required is currently unknown, and estimates vary from three (Hua and Scheller, 2001) to five to eight (Han et al, 2004) to 10 -15 (Keller and Neale, 2001;Keller et al, 2004;Montecucco et al, 2005). Such differences may reflect the distinct experimental paradigms used and/or the types of secretory organelles studied.…”

Novel Chimeras of Botulinum Neurotoxins A and E Unveil Contributions from the Binding, Translocation, and Protease Domains to Their Functional Characteristics

“…Neurons were prepared from mouse spinal cords removed at gestation day 13, dissociated with trypsin, and seeded at a density of 10 6 cells/well on rat tail collagen-coated 24-well plates in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated horse serum and other factors (15). At 21 days in vitro, cultures were exposed for 20 min to each toxin in stimulation buffer (15), washed twice with toxin-free medium, and incubated for the periods specified in the figure legends before harvesting for analysis. In some experiments, an endosomal acidification inhibitor, ConA, was added (250 nM final concentration) at various times after toxin removal as described (15); the cells were harvested after an additional 20 h of culturing.…”

Activation of TRPV1 Mediates Calcitonin Gene-Related Peptide Release, Which Excites Trigeminal Sensory Neurons and Is Attenuated by a Retargeted Botulinum Toxin with Anti-Nociceptive Potential

“…3A) and no reduction in either 1 M capsaicin-or 60 mM K ϩ -triggered CGRP release (Fig. 3B), despite its well established rapid internalization and greater potency in blocking exocytosis from other neuron types (Foran et al, 2003;Keller et al, 2004). Because glycosylated SV2A and B, but not C, have been identified as BoNT/E acceptors (Dong et al, 2008), their relative contents in TGNs compared with CGNs were assessed by Western blotting using isoform-specific antibodies.…”