Fig 1.
BoNT/C triple mutants retain a residual activity against SNAP-25.
(A) Proteolytic activity of BoNT/C variants in cultured cerebellar granule neurons (CGNs). Full-length BoNT/C-wt or BoNT/C α-51 or BoNT/C α-3W were added to cultured CGNs at indicated concentrations for 12 hours. The cleavage of syntaxin-1A/1B and SNAP-25 was assayed in western blot by using two antibodies recognizing both the intact and the cleaved forms of the proteins. (B) Recombinant syntaxin 1A fusion protein (1 μM, upper panel) or SNAP-25 (10 μM, lower panel) spiked with the corresponding radiolabeled protein generated by in vitro transcription/translation in the presence of [35S]-Met were incubated with various concentrations of LC/C-wt (cyan) or its mutants (α-51, green; α-3W, red) in toxin assay buffer. After 1 h of incubation at 37°C, samples were analyzed by SDS-PAGE. Percentage of cleavage was quantified by means of the radiolabeled substrate by phosphorimaging. Data are mean values of three to six independent experiments. Statistical significance was determined by a Student's t-test comparing the mean values between groups (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n.s. not significant).
Fig 2.
BoNT/C mutants display a different neurodegenerative effect on cultured neurons.
CGNs were treated with the indicated concentrations of BoNT/C-wt or mutants for 12 hours. Neurons were then fixed and stained with an antibody against cleaved SNAP-25 (SNAP-25c, in red) and neurofilament-200 (NF200, in green). Neurodegeneration was evaluated following the appearance of varicosities along neurites and the loss of NF200 staining. Images are representative of at least three independent experiments. Scale bar, 10 μm.
Fig 3.
BoNT/C mutants display noticeable lower potency than wild type BoNT/C.
(A) Activity of BoNT/C variants at the MPN hemidiaphragm assay. The black trace represents a dose-response calibration curve reporting the T50 value obtained at indicated bath concentration of a reference wild type BoNT/C [45]. Recombinant BoNT/C-wt (black diamond), tested at 100 pM displays a T50 comparable to the previous BoNT/C-wt used at the same concentration. BoNT/C α-3W (white square) and BoNT/C α-51 (black square) need much higher concentrations to achieve a T50 within the calibration curve. Error bars represent SD of n = 3–4 technical replicates. (B) Calculation of potency of BoNT/C mutants employing a power function fitted to the dose-response calibration curve in A. (C) Immunofluorescent analysis of hemidiaphragms derived from MPN assays. Hemidiaphragms treated with the indicated toxin and concentration were fixed immediately upon completion of paralysis and stained for cleaved SNAP-25 (SNAP-25c, red). NMJs were spotted with α-Bungarotoxin (α-BTX, in green). Images shown are representative of at least three independent experiments. Scale bar, 10 μm.
Fig 4.
BoNT/C mutants are poorly lethal in vivo.
(A) Mouse bioassay for BoNT/C variants. CD1 female mice weighting 20–24 grams were injected intraperitoneally with the indicated doses of BoNT/C-wt (cyan) or BoNT/C α-51 (green) or BoNT/C α-3W (red). Survival after 96 hours is reported as the percentage of mice survived with respect to the total group treated with the same amount of toxin. (B) Animals of the mouse bioassay were monitored every 4 hours and their survival reported as a Kaplan-Meier plot. Top panel shows BoNT/C-wt, middle panel is for BoNT/C α-3W and bottom panel is for BoNT/C α-51.
Table 1.
Summary of the lethality of the different BoNT/C variants used in this study.
Fig 5.
Time course of neuroparalysis recovery upon a local injection of BoNT/C variants in the mouse hind limb.
(A) Digit Abduction Score (DAS) assay. 1 LD50 of BoNT/C-wt (cyan), or BoNT/C α-51 (green) or BoNT/C α-3W (red) were injected intramuscularly in the mice hind limb and neuroparalysis was evaluated according to [49]. The rescue from paralysis was monitored daily until complete recovery was attained. Traces are representative of three independent experiments with at least 5 mice per condition. Error bars represent SEM (B) Analysis of evoked post synaptic junction potentials (EJP) on injected soleus muscles. Mice were treated as in A and at indicated time points soleus muscles were collected and processed for recordings of EJPs, as previously reported [27]. Data are presented as a percentage of EJPs of control muscles. Each point represents an average EJP amplitude obtained from at least 45 muscle fibers from three different mice per condition. Statistical significance at each time point was determined by a Student's t-test comparing the mean values between either BoNT/C α-51 (green) or BoNT/C α-3W (red) compared to BoNT/C-wt (cyan) (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n.s. not significant). Error bars represent SEM.
Fig 6.
Imaging of cleaved SNAP-25 and syntaxin-1 in muscles treated with BoNT/C-wt.
Soleus muscles of mice treated with BoNT/C-wt and used for the analysis of EJPs of Fig 5B were fixed immediately after the electrophysiological recordings and stained for (A) cleaved SNAP-25 (SNAP-25c) or (B) Syntaxin-1A/1B (Stx-1A/1B), both shown in red. NMJs were spotted with α-Bungarotoxin (α-BTX, in green). The first row of panels represents the staining of a control muscle. Scale bar, 10 μm.
Fig 7.
Imaging of cleaved SNAP-25 and syntaxin-1 in muscles treated with BoNT/C α-3W.
Soleus muscles of mice treated with BoNT/C α-3W and used for the analysis of EJPs of Fig 5B were fixed immediately after the electrophysiological recordings and stained for (A) cleaved SNAP-25 (SNAP-25c) or (B) Syntaxin-1A/1B (Stx-1A/1B), both shown in red. NMJs were spotted with α-Bungarotoxin (α-BTX, in green). The first row of panels represents the staining of a control muscle. Scale bar, 10 μm.
Fig 8.
Imaging of cleaved SNAP-25 and syntaxin-1 in muscles treated with BoNT/C α-51.
Soleus muscles of mice treated with BoNT/C α-51 and used for the analysis of EJPs of Fig 5B were fixed immediately after the electrophysiological recordings and stained for (A) cleaved SNAP-25 (SNAP-25c) or (B) Syntaxin-1A/1B (Stx-1A/1B), both shown in red. NMJ were spotted with α-Bungarotoxin (α-BTX, in green). The first row of panels represents the staining of a control muscle. Scale bar, 10 μm.
Fig 9.
Time course of neurotransmission recovery in soleus muscles upon a local injection of a low dose of BoNT/C α-51.
(A) The black trace shows the analysis of evoked post synaptic junction potentials (EJP) on soleus muscles injected intramuscularly with 10 ng/kg of BoNT/C α-51. At indicated time points soleus muscles were collected and processed for the electrophysiological recordings of EJPs, as previously reported [27]. Data are presented as a percentage of EJPs of control muscles. Each point represents an average EJP amplitude obtained from at least 45 muscle fibers from three different mice per condition. Error bars represent SEM. As a comparison, dotted trace shows the time course of EJP recovery obtained with 1 LD50 of BoNT/C α-51. Statistical significance at each time point was determined by a Student's t-test comparing the mean values (**** p<0.0001, n.s. not significant). Error bars represent SEM. (B and C) Soleus muscles coming from the EJP analyses were fixed and stained for (B) cleaved SNAP-25 (SNAP-25c) or (C) Syntaxin-1A/1B (Stx-1A/1B), both shown in red. NMJ were spotted with α-Bungarotoxin (α-BTX, in green). The first row of panels represents the staining of a control muscle.