title: Clostridium botulinum neurotoxins act with a wide range of potencies on SH-SY5Y human neuroblastoma cells.
abstract: We have described, in undifferentiated SH-SY5Y human neuroblastoma cells, the relative potency of Clostridium botulinum neurotoxin (BoNT) serotypes A-F Sensitivity of stimulated [3H]-noradrenaline ([3H]-NA) release to the toxins had a rank order of potency of: C > D > A > B > F after 3 days exposure. The difference between the most potent (BoNT/C: IC50 0.54 nM) and the least (BoNT/F: IC50 > 300 nM) was approximately 1,000-fold. Though fluid phase endocytosis may have been the mechanism of entry for low potency toxins the far higher potency of BoNT/C would suggest receptor-driven entry. Potency was not a reflection of the dependence of the release mechanism on a particular SNARE since the substrate specificities were mixed throughout the potency order. This indicated that the toxins differed in their efficiency of 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 [3H]-NA release. Cleavage of the appropriate substrate proteins was observed for all serotypes. SNAP-25 cleavage by BoNT/A was shown to be a dose-dependent and correlated closely with reduction of release, supporting proteolysis as the mechanism by which toxin inhibited secretion. Comparison of the SH-SY5Y cell line sensitivity to BoNT/A with glycine releasing rat primary spinal cord neuron cultures, revealed a massive difference in potency; the primary cultures being approximately 200,000-fold more sensitive. The demonstration, using BoNTs, of the crucial role of SNAP-25, VAMP and syntaxin in SH-SY5Y cells suggests the use of this neuroblastoma as a model in the study of these proteins in neurotransmitter release.
authors.affiliation: Centre for Applied Microbiology and Research, Salisbury, Wiltshire, UK. john.purkiss@camr.org.uk
authors.forename: J R
authors.initials: JR
authors.lastname: Purkiss
authors.forename: L M
authors.initials: LM
authors.lastname: Friis
authors.forename: S
authors.initials: S
authors.lastname: Doward
authors.forename: C P
authors.initials: CP
authors.lastname: Quinn
mesh_terms: D000818:Animals; D018726:Anti-Dyskinesia Agents / Q000494:pharmacology*; D001905:Botulinum Toxins / Q000494:pharmacology*; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism*; D009638:Norepinephrine / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins; D013116:Spinal Cord / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D033921:Vesicular Transport Proteins
chemical_list: D018726:Anti-Dyskinesia Agents; D008565:Membrane Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D001905:Botulinum Toxins; D009638:Norepinephrine