Persistence of botulinum neurotoxin a subtypes 1-5 in primary rat spinal cord cells

Abstract

Botulinum neurotoxins (BoNTs) are the most poisonous substances known and cause the severe disease botulism. BoNTs have also been remarkably effective as therapeutics in treating many neuronal and neuromuscular disorders. One of the hallmarks of BoNTs, particularly serotype A, is its long persistence of 2-6 months in patients at concentrations as low as fM or pM. The mechanisms for this persistence are currently unclear. In this study we determined the persistence of the BoNT/A subtypes 1 through 5 in primary rat spinal neurons. Remarkably, the duration of intracellular enzymatic activity of BoNT/A1, /A2, /A4 and /A5 was shown to be at least 10 months. Conversely, the effects of BoNT/A3 were observed for up to ∼5 months. An intermittent dosing with BoNT/E showed intracellular activity of the shorter acting BoNT/E for 2-3 weeks, followed by reoccurrence and persistence of BoNT/A-induced SNAP-25 cleavage products.

Figure 1. Recovery of SNAP-25 after exposure to BoNT/A subtypes.

Primary rat spinal cord (RSC) cells were exposed individually to sufficient concentrations of the BoNT/A subtypes 1 through 5 to generate ∼100% cleavage of SNAP-25 after 48 h. Excess toxin was aspirated and cells were washed to remove any remaining extracellular toxin. Cells lysates were collected at the time points specified and were analyzed for SNAP-25 cleavage by Western blot, and a representative blot is shown (A). Data from 4 Western blots were quantified by densitometry, and data plots generated using PRISM software (B).

Figure 2. Secondary intoxication with BoNT/E to monitor persistence of BoNT/A light chain activity.

Primary rat spinal cord (RSC) cells that had been exposed to BoNT/A1, /A2, /A4, and /A5 and maintained for 9 months were exposed to 10 U/50 µL of BoNT/E, a sufficient concentration of toxin to cleave ∼100% of both uncleaved SNAP-25 and SNAP-25^A after 48 h. Excess toxin was aspirated and cells were washed to remove any remaining extracellular toxin. Cells lysates were collected at the time points specified and analyzed for SNAP-25 cleavage by Western blot. A representative Western blot is shown (A). Data from 4 Western blots were quantified by densitometry, and a graph showing the percentage of SNAP-25^A before and 30 days after BoNT/E exposure (when no SNAP-25^E remained) was generated using PRISM software (B).

Figure 3. Sensitivity of RSC cells cultured for 10 months to BoNT/A1 after 48 h exposure.

Cells were exposed to the indicated concentrations of BoNT/A1 and cell lysates were analyzed for SNAP-25 cleavage by Western blotting. Data from three Western blots were quantified by densitometry, and data plot and an EC₅₀ were generated.