Atoxic derivative of botulinum neurotoxin A as a prototype molecular vehicle for targeted delivery to the neuronal cytoplasm.
We have previously described genetic constructs and expression systems that enable facile production of recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. In this report we describe the properties of one such derivative, BoNT/A ad, which was rendered atoxic by introducing two amino acid mutations to the light chain (LC) of wt BoNT/A, and which is being developed as a molecular vehicle for delivering drugs to the neuronal cytoplasm. The neuronal binding, internalization, and intracellular trafficking of BoNT/A ad in primary hippocampal cultures was evaluated using three complimentary techniques: flow cytometry, immunohistochemistry, and Western blotting. Neuronal binding of BoNT ad was significantly increased when neurons were incubated in depolarizing medium. Flow cytometry demonstrated that BoNT/A ad internalized into neurons but not glia. After 24 hours, the majority of the neuron-bound BoNT/A ad became internalized, as determined by its resistance to pronase E-induced proteolytic degradation of proteins associated with the plasma membrane of intact cells. Significant amounts of the atoxic LC accumulated in a Triton X-100-extractable fraction of the neurons, and persisted as such for at least 11 days with no evidence of degradation. Immunocytochemical analysis demonstrated that the LC of BoNT/A ad was translocated to the neuronal cytoplasm after uptake and was specifically targeted to SNARE proteins. The atoxic LC consistently co-localized with synaptic markers SNAP-25 and VAMP-2, but was rarely co-localized with markers for early or late endosomes. These data demonstrate that BoNT/A ad mimics the trafficking properties of wt BoNT/A, confirming that our platform for designing and expressing BoNT derivatives provides an accessible system for elucidating the molecular details of BoNT trafficking, and can potentially be used to address multiple medical and biodefense needs.
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, United States of America.
Edwin J
EJ
Vazquez-Cintron
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, United States of America.
Maksim
M
Vakulenko
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, United States of America ; Department of Orthopaedic Surgery, New York University Hospital for Joint Diseases, New York, New York, United States of America.
Philip A
PA
Band
USDA, Agriculture Research Service, Albany, California, United States of America.
Larry H
LH
Stanker
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Eric A
EA
Johnson
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, United States of America.
Konstantin
K
Ichtchenko
D000818:Animals; D015153:Blotting, Western; D019274:Botulinum Toxins, Type A / Q000008:administration & dosage* / Q000235:genetics / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D005260:Female; D005434:Flow Cytometry; D006624:Hippocampus / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D009474:Neurons / Q000378:metabolism*; D009498:Neurotoxins / Q000008:administration & dosage / Q000235:genetics / Q000493:pharmacokinetics; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
D009498:Neurotoxins; D050988:Vesicle-Associated Membrane Protein 2; D019274:Botulinum Toxins, Type A