Abstract
Botulinum neurotoxins (BoNTs) are the causative agent of the severe and long-lasting disease botulism. At least seven different serotypes of BoNTs (denoted A-G) have been described. All BoNTs enter human or animal neuronal cells via receptor mediated endocytosis and cleave cytosolic SNARE proteins, resulting in a block of synaptic vesicle exocytosis, leading to the flaccid paralysis characteristic of botulism. Previous data have indicated that once a neuronal cell has been intoxicated by a BoNT, further entry of the same or other BoNTs is prevented due to disruption of synaptic vesicle recycling. However, it has also been shown that cultured neurons exposed to BoNT/A are still capable of taking up BoNT/E. In this report we show that in general BoNTs can enter cultured human or mouse neuronal cells that have previously been intoxicated with another BoNT serotype. Quantitative analysis of cell entry by assessing SNARE cleavage revealed none or only a minor difference in the efficiency of uptake of BoNTs into previously intoxicated neurons. Examination of the endocytic entry pathway by specific endocytosis inhibitors indicated that BoNTs are taken up by clathrin coated pits in both non pre-exposed and pre-exposed neurons. LDH release assays indicated that hiPSC derived neurons exposed consecutively to two different BoNT serotypes remained viable and healthy except in the case of BoNT/E or combinations of BoNT/E with BoNT/B, /D, or /F. Overall, our data indicate that previous intoxication of neuronal cells with BoNT does not inhibit further uptake of BoNTs.
Highlights
Botulinum Neurotoxins (BoNTs) are produced by the gram positive anaerobic bacteria Clostridium botulinum and are the causative agent of human and animal botulism
A slight decrease in the EC50 value of BoNT/E induced SNAP-25 cleavage was observed in cells that had been pre-exposed to BoNT/A (Fig 1B)
It has been shown that BoNTs bind to gangliosides and in most cases to the luminal domain of synaptic vesicle protein receptors to associate with the neuronal cell membrane [8]
Summary
Botulinum Neurotoxins (BoNTs) are produced by the gram positive anaerobic bacteria Clostridium botulinum and are the causative agent of human and animal botulism. The LC enters the cell’s cytosol, where it cleaves a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, thereby preventing formation of a functional SNARE complex and fusion of the synaptic vesicles with the pre-synaptic cell membrane [2]. This results in an inability of the cell to release neurotransmitter, thereby causing the flaccid paralysis characteristic of botulism. The enzymatically active LC remains inside the cytosol for a prolonged period of time and continues to cleave newly synthesized SNARE proteins [3]
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