Persistence of botulinum neurotoxin action in cultured spinal cord cells

Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying the differences in persistence of botulinum neurotox in A (BoNT/A) and botulinum neurotoxin/E (BoNT/E) activities. Spinal cord c ultures were exposed to BoNT/A (0.4 pM) for 2-3 days, which converted appro ximately half of the SNAP-25 to an altered form lacking the final nine C-te rminal residues. The distribution of toxin-damaged to control SNAP-25 remai ned relatively unchanged for up to 80 days thereafter. Application of a hig h concentration of BoNT/E (250 pM) either 25 or 60 days following initial i ntoxication with BoNT/A converted both normal and BoNT/ A-truncated SNAP-25 into a single population lacking the final 26 C-terminal residues. Excess BoNT/E was removed by washout, and recovery of intact SNAP-25 was monitored by Western blot analysis. The BoNT/E-truncated species gradually diminishe d during the ensuing 18 days, accompanied by the reappearance of both norma l and BoNT/A-truncated SNAP-25, Return of BoNT/A-truncated SNAP-25 was obse rved in spite of the absence of BoNT/A in the culture medium during all but the first 3 days of exposure. These results indicate that proteolytic acti vity associated with the BoNT/A light chain persists inside cells for >11 w eeks, while recovery from BoNT/E is complete in <3 weeks. This longer durat ion of enzymatic activity appears to account for the persistence of serotyp e A action. (C) 1999 Federation of European Biochemical Societies.