Comparative functional analysis of mice after local injection with botulinum neurotoxin A1, A2, A6, and B1 by catwalk analysis.

Botulinum neurotoxins (BoNTs) are potent neurotoxins and are the causative agent of botulism, as well as valuable pharmaceuticals. BoNTs are divided into seven serotypes that comprise over 40 reported subtypes. BoNT/A1 and BoNT/B1 are currently the only subtypes approved for pharmaceutical use in the USA. While several other BoNT subtypes including BoNT/A2 and/A6 have been proposed as promising pharmaceuticals, detailed characterization using in vivo assays are essential to determine their pharmaceutical characteristics compared to the currently used BoNT/A1 and/B1. Several methods for studying BoNTs in mice are being used, but no objective and quantitative assay for assessment of functional outcomes after injection has been described. Here we describe the use of CatWalk XT as a new analytical tool for the objective and quantitative analysis of the paralytic effect after local intramuscular injection of BoNT subtypes A1, A2, A6, and B1. Catwalk is a sophisticated gait and locomotion analysis system that quantitatively analyzes a rodent's paw print dimensions and footfall patterns while traversing a glass plate during unforced walk. Significant changes were observed in several gait parameters in mice after local intramuscular injection of all tested BoNT subtypes, however, no changes were observed in mice injected intraperitoneally with the same BoNTs. While a clear difference in time to peak paralysis was observed between BoNT/A1 and/B1, injection of all four toxins resulted in a deficit in the injected limb with the other limbs functionally compensating and with no qualitative differences between the four BoNT subtypes. The presented data demonstrate the utility of CatWalk as a tool for functional outcomes after local BoNT injection through its ability to collect large amounts of quantitative data and objectively analyze sensitive changes in static and dynamic gait parameters.

Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6.

Botulinum neurotoxins (BoNTs), the most potent toxins known to humans and the causative agent of botulism, exert their effect by entering motor neurons and cleaving and inactivating SNARE proteins, which are essential for neurotransmitter release. BoNTs are proven, valuable pharmaceuticals used to treat more than 200 neuronal disorders. BoNTs comprise 7 serotypes and more than 40 isoforms (subtypes). BoNT/A1 is the only A-subtype used clinically due to its high potency and long duration of action. While other BoNT/A subtypes have been purified and described, only BoNT/A2 is being investigated as an alternative to BoNT/A1. Here we describe subtype BoNT/A6 with improved pharmacological properties compared to BoNT/A1. It was isolated from Clostridium botulinum CDC41370, which produces both BoNT/B2 and BoNT/A6. The gene encoding BoNT/B2 was genetically inactivated, and A6 was isolated to greater than 95% purity. A6 was highly potent in cultured primary rodent neuronal cultures and in human induced pluripotent stem cell-derived neurons, requiring 20-fold less toxin to cause 50% SNAP-25 cleavage than A1. Second, A6 entered hiPSCs faster and more efficiently than A1 and yet had a long duration of action similar to BoNT/A1. Third, BoNT/A6 had similar LD50 as BoNT/A1 after intraperitoneal injection in mice; however, local intramuscular injection resulted in less systemic toxicity than BoNT/A1 and a higher (i.m.) LD50, indicating its potential as a safer pharmaceutical. These data suggest novel characteristics of BoNT/A6 and its potential as an improved pharmaceutical due to more efficient neuronal cell entry, greater ability to remain localized at the injection site, and a long duration.IMPORTANCE Botulinum neurotoxins (BoNTs) have proved to be an effective treatment for a large number of neuropathic conditions. BoNTs comprise a large family of zinc metalloproteases, but BoNT/A1 is used nearly exclusively for pharmaceutical purposes. The genetic inactivation of a second BoNT gene in the native strain enabled expression and isolation of a single BoNT/A6 from cultures. Its characterization indicated that BoNT/A subtype A6 has a long duration of action comparable to A1, while it enters neurons faster and more efficiently and remains more localized after intramuscular injection. These characteristics of BoNT/A6 are of interest for potential use of BoNT/A6 as a novel BoNT-based therapeutic that is effective and has a fast onset, an improved safety profile, and a long duration of action. Use of BoNT/A6 as a pharmaceutical also has the potential to reveal novel treatment motifs compared to currently used treatments.