Abstract
A set of herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the light chains (LC) of botulinum neurotoxins (BoNT) A, B, C, D, E and F was constructed. Their properties have been assessed in primary cultures of rat embryonic dorsal root ganglia (DRG) neurons, and in organotypic cultures of explanted DRG from adult rats. Following infection of primary cultures of rat embryonic DRG neurons, the different BoNT LC induced efficient cleavage of their corresponding target Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) protein (VAMP, SNAP25, syntaxin). A similar effect was observed following infection by BoNT-A LC of organotypic cultures of adult rat DRG. To quantify and compare the functional activities of the different BoNT LC, the inhibition of calcitonin gene-related protein (CGRP) secretion was assessed in DRG neurons following infection by the different vectors. All BoNT-LC were able to inhibit CGRP secretion although to different levels. Vectors expressing BoNT-F LC displayed the highest inhibitory activity, while those expressing BoNT-D and -E LC induced a significantly lower CGRP release inhibition. Cleavage of SNARE proteins and inhibition of CGRP release could be detected in neuron cultures infected at less than one transducing unit (TU) per neuron, showing the extreme efficacy of these vectors. To our knowledge this is the first study investigating the impact of vector-expressed transgenic BoNT LC in sensory neurons.
Highlights
The clostridial neurotoxin family includes tetanus toxin (TeNT), produced by Clostridium tetani, and at least seven antigenically distinct botulinum neurotoxins (BoNTs) produced by different strains ofC. botulinum (BoNT-A to -G)
Our results indicate that the different BoNT light chains (LC) display differential efficacies in the inhibition of calcitonin gene-related protein (CGRP) neurosecretion
The first expresses the reporter gene green fluorescent protein (GFP) driven by the herpes simplex virus type 1 (HSV-1) immediate-early (IE) 4/5 promoter, which is useful for measuring vector titers, to identify infected cells, and as an internal reference control for transgene expression levels
Summary
These proteins, which are amongst the most potent biological neurotoxins, are responsible for the conditions of tetanus and botulism, respectively. These diseases are a direct result of inhibition of calcium-dependent neurotransmitter release, a mechanism of action common to all these toxins [1]. Clostridial neurotoxins are disulphide bridge-linked proteins composed of a light chain (50 kDa), responsible for the cleavage of substrates through a zinc-dependent endoprotease activity, and a heavy chain (100 kDa) that is involved in neurospecific binding and delivery of the neurotoxin to the neuronal cytosol [2,3]. BoNT-A, BoNT-C and BoNT-E cleave synaptosomal-associated protein of
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