Substrate cleavage and duration of action of botulinum neurotoxin type FA (“H, HA”)

Abstract

Botulinum neurotoxin (BoNT) type FA is the only known naturally occurring chimeric BoNT of domains of BoNT/A and BoNT/F. BoNT/FA consists of an F5-like light chain (LC), a unique heavy chain (HC) translocation domain, and a HC receptor binding domain similar to BoNT/A1. Previous analyses of purified BoNT/FA have indicated a 5-10-fold greater potency in cultured human or rat neurons as compared to BoNT/A1 and a 400-500-fold greater potency compared to BoNT/B1. However, in vivo potency in mice was about 5-fold lower than BoNT/A1 or/B1. In this report, species specificity was examined by cell-based assays using primary neurons from mice and examining VAMP1 and 2 cleavage. The data indicated similar potency of BoNT/FA in primary mouse spinal cord neurons as previously observed in primary rat and human induced pluripotent stem cell (hiPSC) derived neuronal cell models, and equal enzymatic cleavage of mouse VAMP1 and 2 isoforms. Since the duration of action of BoNTs is due to continuous enzymatic activity of the LC in the neuronal cytosol, BoNT/FA was expected to have a short duration of action due to its F-type LC. In this report the duration of action of BoNT/FA was compared to that of BoNT/F1,/F5, and/B1 in both hiPSC derived neurons and in the in vivo mouse model. The data indicate a duration of action of BoNT/FA similar to BoNT/B1, while BoNT/F5 had a short duration of action similar to BoNT/F1.

Botulinum neurotoxins A, B, C, E, and F preferentially enter cultured human motor neurons compared to other cultured human neuronal populations

“…Botulinum neurotoxins comprise a large family of protein toxins, with seven neurotoxic serotypes and several subtypes as well as chimeric toxins . While all BoNTs cause botulism, variation in potency, clinical presentation, duration of action, species specificity, and cell entry properties vary for different BoNT serotypes . These variations could be due to differences in LC activity, specific cell association, or entry of the LC into the cell's cytosol and intracellular trafficking.…”

“…[8]. While all BoNTs cause botulism, variation in potency, clinical presentation, duration of action, species specificity, and cell entry properties vary for different BoNT serotypes[7,17,31,[56][57][58][59][60][61][62][63][64]. These variations could be due to differences in LC activity, specific cell association, or entry of the LC into the cell's cytosol and intracellular trafficking.…”

Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

“…Potency determinations of different BoNTs by NCB assay and MBA do not necessarily correlate. Several BoNT types (A2, A6, FA) have been shown to enter cultured neurons more efficiently, but BoNT/FA was significantly less potent in mice [55,57,61,126,127]. Thus, greater potency of a BoNT in NCB assays doesn't necessarily mean greater toxicity in vivo.…”

“…For these applications, an assay that can detect the biologic activity of multiple BoNT seroand sub-types is required. While cell-based assays can provide human-specific and highly sensitive BoNT detection platforms fulfilling this requirement, research in the past few years has indicated that the activity of BoNTs in cultured neurons and in vivo in mice does not equally correlate for all BoNTs [55,57,61,126,128]. Thus, the mouse bioassay remains an important detection method for BoNTs, in particular, for investigations of novel BoNTs and for detection of BoNTs in complex matrices such as stool samples and food matrices.…”

“…Intramuscular and subcutaneous injections, usually of sub-lethal doses, combined with assays assessing the resulting local paralysis, are usually used to study pathology, onset, and duration of action of sub-lethal doses of BoNTs. Significant differences between BoNT types have been demonstrated using such studies [57][58][59]62,126,133].…”