Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

Abstract

Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

A Heterologous Reporter Defines the Role of the Tetanus Toxin Interchain Disulfide in Light-Chain Translocation

“…TrxR-1 has been postulated to act as a chaperonin to refold the LC prior to reduction; however, inhibition of ␤lac translocation upon treatment with auranofin implies reduction is prerequisite to ␤lac translocation into the cytosol (54). A requirement for reduction of the interchain disulfide prior to translocation of the LC also would explain why inhibitors of the Trx-TrxR system are not neuroprotective once the LC is in the cytosol (52). Trypsin-nicked diphtheria toxins like TeNT contain a dichain connected by an interchain disulfide bridge.…”

“…Auranofin inhibits TrxR-1 more efficiently than TrxR-2 (53). Auranofin also inhibits TeNT and BoNT/A, BoNT/B, BoNT/C, BoNT/D, and BoNT/E intoxication of cerebellar granular neurons, presumably by inhibiting reduction of the interchain disulfide (52)(53)(54). Thus, the ability of auranofin to inhibit ␤lac translocation by ␤lac-TeNT(RY) was tested.…”

“…Two common isoforms of the thioredoxin system that are present in mammalian cells, including rat cortex, are cytosolic Trx-TrxR-1 and mitochondrion-associated Trx-TrxR-2 (51). These enzymes associate with the cytosolic face of purified synaptic vesicles in cerebellar granular neurons (52). Auranofin inhibits TrxR-1 more efficiently than TrxR-2 (53).…”

Exploiting endocytic pathways to prevent bacterial toxin infection

“…BoNT/C-induced paralysis is significantly increased upon stimulation of synaptic vesicle exo-endocytosis [56,66], suggesting that BoNT/C may also be taken up into synaptic vesicles through an as-yet-unestablished mechanism. In response to acidification of the endocytosed synaptic vesicle, the translocation domain of BoNTs undergoes a conformational change, which results in the toxin creating a pore in the endosomal membrane [68][69][70][71]. This enables release of a light chain, which is responsible for the endopeptidase activity, into the cytosol [70,71], a process that was recently shown to be dependent on the thioredoxin reductase(TrxR)-thioredoxin (Trx) redox system [69].…”

“…In response to acidification of the endocytosed synaptic vesicle, the translocation domain of BoNTs undergoes a conformational change, which results in the toxin creating a pore in the endosomal membrane [68][69][70][71]. This enables release of a light chain, which is responsible for the endopeptidase activity, into the cytosol [70,71], a process that was recently shown to be dependent on the thioredoxin reductase(TrxR)-thioredoxin (Trx) redox system [69]. The light chain catalyzes the cleavage of specific amino acids from SNARE proteins, leading to a blockade of neurotransmitter release and flaccid paralysis.…”