title: Botulinum Neurotoxins Can Enter Cultured Neurons Independent of Synaptic Vesicle Recycling.
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.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States of America.
authors.forename: Sabine
authors.initials: S
authors.lastname: Pellett
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States of America.
authors.forename: William H
authors.initials: WH
authors.lastname: Tepp
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States of America.
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Scherf
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, United States of America.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Johnson
mesh_terms: D000818:Animals; D001681:Biological Assay; D001692:Biological Transport; D001905:Botulinum Toxins / Q000008:administration & dosage / Q000145:classification / Q000378:metabolism* / Q000633:toxicity; D001906:Botulism / Q000139:chemically induced; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D007274:Injections, Intraperitoneal; D007928:Lethal Dose 50; D051379:Mice; D008813:Mice, Inbred ICR; D055495:Neurogenesis; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D012703:Serotyping; D013572:Synaptic Vesicles / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
chemical_list: C494208:SNAP25 protein, human; D050825:Synaptosomal-Associated Protein 25; C494807:VAMP2 protein, human; D050988:Vesicle-Associated Membrane Protein 2; D001905:Botulinum Toxins