Clostridial Neurotoxins and Substrate Proteolysis in Intact Neurons

Abstract

Clostridial neurotoxins are zinc endopeptidases that block neurotransmission and have been shown to cleave, in vitro, specific proteins involved in synaptic vesicle docking and/or fusion. We have used immunohistochemistry and immunoblotting to demonstrate alterations in toxin substrates in intact neurons under conditions of toxin-induced blockade of neurotransmitter release. Vesicle-associated membrane protein, which colocalizes with synaptophysin, is not detectable in tetanus toxin-blocked cultures. Syntaxin, also concentrated in synaptic sites, is cleaved by botulinum neurotoxin C. Similarly, the carboxyl terminus of the synaptosomal-associated protein of 25 kDa (SNAP-25) is not detectable in botulinum neurotoxin A-treated cultures. Unexpectedly, tetanus toxin exposure causes an increase in SNAP-25 immunofluorescence, reflecting increased accessibility of antibodies to antigenic sites rather than increased expression of the protein. Furthermore, botulinum neurotoxin C causes a marked loss of the carboxyl terminus of SNAP-25 when the toxin is added to living cultures, whereas it has no action on SNAP-25 in vitro preparations. This study is the first to demonstrate in functioning neurons that the physiologic response to these toxins is correlated with the proteolysis of their respective substrates. Furthermore, the data demonstrate that botulinum neurotoxin C, in addition to cleaving syntaxin, exerts a secondary effect on SNAP-25.

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

“…Samples were analyzed by SDS-PAGE (13% gel) and immunoblotting as described in Figure 3. i.e., whether the proteolytic loss of a functional domain or the generation of a toxic breakdown product is primarily responsible for the block. No synaptobrevin fragments were detected when intact neurons or synaptosomes were poisoned (Link et al, 1992;Mundigl et al, 1995;Osen-Sand et al, 1996;Williamson et al, 1996). In these studies, antibodies binding N-terminal of the cleavage site were used for detection.…”

“…However, the susceptibility of substrate proteins toward neurotoxin proteolysis in vitro may differ from that observed in vivo. For example, botulinus toxin C cleaves syntaxin and also attacks SNAP-25 in intact neurons (Blasi et al, 1993b;Williamson et al, 1996). In contrast, recombinant SNAP-25 is not cleaved in vitro even using concentrations of 500 nM BoNT/C (Foran et al, 1996).…”

Primary cell culture for evaluation of botulinum neurotoxin antagonists

“…BoNT/B and BoNT/E were nicked with trypsin type XI at pH 6 and then treated with an excess of soybean trypsin Spinal cords were dissected from fetal NIH Swiss mice inhibitor prior to use (Ohishi and Sakaguchi, 1977). Equine at gestation day E13, dissociated with trypsin and plated on antiserum to BoNT/A was an IgG preparation (> 95% pure) collagen-coated 12-well culture plates at 105 cells/cm 2 from PerImmune, Inc. (Rockville, MD), and was raised (Fitzgerald, 1989).…”

“…results agree closely with those of Hall et al (2004Hall et al ( ). 1985Williamson et al, 1992Williamson et al, , 1996. Evoked release from manuscript and Dr Janet Ransom at PerImmune for cultured spinal cord cells can be measured quantitatively providing equine polyclonal BoNT/A antiserum using elevated K+ to release radiolabeled neurotransmitter.…”

“…Following these represent a sensitive system for testing the actions of BoNT incubations, 0.2% sodium dodecyl sulfate was added, and and of potential BoNT antagonists on synaptic transmission, the radiolabel remaining in the cells was determined. Release of radiolabeled glycine or glutamate in response to Evoked release of neurotransmitter was defined as the depolarization is inhibited by tetanus neurotoxin and several quantity of 3[H]-glycine released in the presence of 56 mM BoNT serotypes in a concentration-and time-dependent K+/2 mM Ca2+ minus the fraction released in 5 mM K+/ manner (Williamson et al, 1992(Williamson et al, , 1996Keller and Neale, 0 mM Ca 2 +. The percent of total release was calculated as 2001; Keller et al, 2004;Hall et al, 2004).…”