Abstract
Botulinum Neurotoxin (BoNT) is one of the deadliest toxins known to man. Its lethal dose (LD) varies, depending on the serotype, the form of toxin (isolated vs. complex), and the route of intoxication. The extreme toxicity of BoNTs lead to the need for a sensitive detection system. Due to its high lethality and potency, BoNTs pose a higher risk as bioterrorism weapon. In cases of an outbreak, reliable and fast detection systems are needed for clinical diagnosis, identification of the source of contamination, and environmental/food testing for food safety or epidemiology studies. Type A is responsible for the highest mortality rate in humans, therefore in this study we have focused on developing a sensitive and effective model for the detection of BoNT/A. Under native and physiological conditions, BoNT/A enzymatic activity is known to exist in three different enzymatically active forms, Light chain (LC), isolated holotoxin, and Complex. The LC is the catalytic domain responsible for the enzymatic activity in all the three forms of the BoNT/A neurotoxin. In this work, we demonstrated the enzymatic activity of the three forms of BoNT/A neurotoxin using fluorescently labelled synthetic peptide (SNAPtide) by showing that the sizes of both the substrate and enzyme play crucial roles in its catalytic activity and protein membrane dynamics may be at play in the unique action mechanism. As non-cell-based assays do not employ all the steps necessary for intoxication, thereby reducing the sensitivity and relevance of the assay, we have developed a tripartite culture assay system employing human neuron progenitor cells (hNPC), differentiating into neurons and astrocytes to create a 3D relevant in vitro functional human brain model for detection and to test the functionality of the system for the neuroprotective/neurodegenerative effects triggered by BoNT/A. Compartmentalization of the device further facilitates the visualization of microglial cells establishing a tri culture model. These studies have shown that the neurons are the only cell type that are directly affected by the BoNT/A treatment, and mechanistic biochemical pathways and their response to different doses may connect the repeated treatment of BoNT/A to tau pathology and synaptic impairment.