Atoxic Derivative of Botulinum Neurotoxin A as a Prototype Molecular Vehicle for Targeted Delivery to the Neuronal Cytoplasm

Edwin J Vazquez-Cintron,Eric A Johnson,Maksim Vakulenko,Larry H Stanker,Konstantin Ichtchenko,Philip A Band,Michel R Popoff

doi:10.1371/journal.pone.0085517

Abstract

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.

Highlights

Botulinum neurotoxins (BoNTs) are a family of highly toxic proteins produced by Clostridium botulinum [1,2,3]
The binding of wt BoNT/A to neurons has been demonstrated to occur primarily in neurons that are actively involved in exocytosis because the BoNT/A protein receptor, Synaptic Vesicle protein 2 (SV2), is only exposed on the neuronal surface after fusion of synaptic vesicles with the plasma membrane [16]
To confirm that BoNT/A ad binding occurs via the same mechanism as wt BoNT/A, we evaluated the effect of neuronal depolarization on the binding of BoNT/A ad to primary rat hippocampal neuron cultures (Fig. 1)

Summary

Introduction

Botulinum neurotoxins (BoNTs) are a family of highly toxic proteins produced by Clostridium botulinum [1,2,3]. There are eight BoNT serotypes (A–H) and multiple sub-types, all with common structural features [4,5,6,7]. Despite their toxicity, BoNTs have become widely used as pharmaceutical agents, because small doses can be applied to paralyze local muscle groups and thereby effect targeted therapeutic paralysis. Because all currently approved pharmaceutical products are wild type (wt) toxins derived from the natural Clostridium botulinum host, a complex multi-step purification is required, and batch to batch variation with respect to overall content of active protein is difficult to attain

Methods

Results

Conclusion