Clostridium botulinum neurotoxins act with a wide range of potencies on SH-SY5Y human neuroblastoma cells

We have described, in undifferentiated SH-SY5Y human neuroblastoma cells, t he relative potency of Clostridium botulinum neurotoxin (BoNT) serotypes A- F Sensitivity of stimulated [H-3]-noradrenaline ([H-3]-NA) release to the t oxins had a rank order of potency of: C > D > A > B > F after 3 days exposu re. The difference between the most potent (BoNT/C: IC50 0.54 nM) and the l east (BoNT/F: IC50 > 300 nM) was approximately 1000-fold. Though fluid phas e endocytosis may have been the mechanism of entry for low potency toxins t he ftir higher potency of BoNT/C would suggest receptor-driven entry. Poten cy was not a reflection of the dependence of the release mechanism on a par ticular SNARE since the substrate specificities were mixed throughout the p otency order. This indicated that the toxins differed in their efficiency o f binding/endocytosis or enzymatic activity inside the cell. The serotypes that cleaved vesicle-associated membrane protein (VAMP) isoforms (BoNT/B, D and F) did not fully inhibit [H-3]-NA release. Cleavage of the appropriate substrate proteins was observed for all serotypes. SNAP-25 cleavage by BoN T/A was shown to be a dose-dependent and correlated closely with reduction of release, supporting proteolysis as the mechanism by which toxin inhibite d secretion. Comparison of the SH-SY5Y cell line sensitivity to BoNT/A with glycine releasing rat primary spinal cord neuron cultures, revealed a mass ive difference in potency; the primary cultures being approximately 200,000 -fold more sensitive. The demonstration, using BoNTs, of the crucial role o f SNAP-25, VAMP and syntaxin in SH-SY5Y cells suggests the use of this neur oblastoma as a model in the study of these proteins in neurotransmitter rel ease. (C), 2001 Elsevier Science Inc. All rights reserved.