Distal effects of BoNT/A.

<p>Long-range effects of BoNT/A were assessed by immunofluorescence (<b>A, B</b>) and western blot, either <i>in vitro</i> (<b>A</b>–<b>C</b>) or <i>in vivo</i> (<b>D</b>), using an antibody that specifically recognises the fragment of SNAP25 generated by BoNT/A. Motor neurons grown in MFC were incubated with 10 nM BoNT/A added to the axonal compartment only of MFC. Samples were processed as described in Experimental Procedures. (<b>A</b>) Tile scan of a representative MFC treated with BoNT/A (left) and the untreated control (right). Arrowheads indicate the microgrooves whilst the hatched lines indicate the boundaries between the cell body and the axonal side of the MFC. Scale bar, 50 µm. (<b>B</b>) High magnification images of MFCs treated as in <b>A</b>. Controls were run in parallel and the microscope settings used for image acquisition were kept constant. Scale bar, 10 µm. Similar results were obtained using two independent primary motor neuron cultures, and replicated twice per condition. (<b>C</b>) Western blot revealed the presence of BoNT/A-cleaved SNAP25 (24 kDa) in the somatic compartment of MFC treated only on the axonal side with full length BoNT/A. No signal was present in control MFCs. This result demonstrates that BoNT/A undergoes axonal retrograde transport in a fully active form and was confirmed in two independent experiments. A representative western blot is shown in (<b>C</b>). β-tubulin (50 kDa) was used as loading control. (<b>D</b>) Representative western blot for BoNT/A-cleaved SNAP25 on extracts from lumbar spinal cord segments of a naive (control) animal and a rat injected with BoNT/A into the hind limb muscles 10 d earlier. These results were confirmed in two independent experiments, replicated three times. A representative western blot is shown in (<b>D</b>). α-tubulin was used as loading control (51 kDa).</p

Full length BoNT/A and BoNT/E undergo retrograde transport in motor neurons.

<p>Motor neurons were incubated with 30 nM full length AlexaFluor488-BoNT/A (<b>A</b>) or 30 nM full length AlexaFluor555-BoNT/E (<b>B</b>) for 30 min at 37°C in resting conditions, then washed and imaged. (<b>A</b>, <i>top</i>) Individual frames from a confocal time series are shown. Cell bodies are located out of view on the right. Arrowheads mark a BoNT/A-positive carrier moving towards the soma. Asterisks indicate an example of stationary carrier. (<b>A</b>, <i>bottom</i>) Kymograph corresponding to the time series described above. Scale bar, 5 µm. (<b>B</b>, <i>top</i>) Individual frames from a confocal time series are shown. Cell bodies are located out of view on the right. Arrowheads mark a BoNT/E-positive carrier moving towards the soma. Asterisks indicate an example of a stationary carrier. (<b>B</b>, <i>bottom</i>) Kymograph corresponding to the time series described above. Scale bar, 5 µm. (<b>C</b>) Speed distribution profile of full length BoNT/A (in green) or BoNT/E (in red) carriers. Note the similar transport kinetics of the two neurotoxins. (<b>D</b>) The incidence of reversals (number of changes of direction per organelle) for BoNT/E carriers is threefold higher than for BoNT/A-positive organelles. Data were obtained by analysing at least ten movies for each neurotoxin. The results were confirmed in two independent primary motor neuron cultures, and replicated twice per condition.</p

The binding fragments of BoNT/A and BoNT/E bind to the motor neuron surface.

<p>(<b>A</b>) Motor neurons were incubated with H_C fragments for 15 min at 4°C and then fixed. (<i>top)</i> Cells were incubated with 15 nM AlexaFluor488-GST-BoNT/A H_C (H_CA) and 15 nM AlexaFluor555-GST (GST) as control. Scale bar, 10 µm. <i>(bottom)</i> Motor neurons were incubated with 7.5 nM AlexaFluor488-GST-BoNT/E H_C (H_CE) and 7.5 nM AlexaFluor555-GST (GST) as control. Scale bar, 20 µm. No fluorescence signal was detectable for GST, whilst a punctate signal was present for H_CA and H_CE, indicating the ability of these binding fragments to bind to motor neurons. The signal is rather low, as expected in these experimental conditions (4°C). (<b>B</b>) Motor neurons were incubated with 15 nM H_CA <i>(top)</i> or 15 nM H_CE <i>(bottom)</i> for 15 min at 4°C, fixed, and stained for SV2C. Only limited colocalisation of H_CA and H_CE with SV2C was found in these conditions. Inset: high magnification of the indicated areas. This analysis was performed using two independent primary motor neuron cultures, and replicated at least twice for each motor neuron preparation. Shown are representative images for each condition. Scale bars, 10 µm <i>(top)</i>; 20 µm <i>(bottom)</i>.</p

H_CA and H_CE are internalised in motor neurons.

<p>Motor neurons were incubated with 15 nM H_CA or 7.5 nM H_CE for 30 min at 37°C, either under resting conditions (<b>A</b>) or after stimulation of synaptic vesicle exo/endocytosis by adding 60 mM KCl to the medium, just before the addition of H_Cs (<b>B</b>). Motor neurons were placed on ice, acid washed, fixed, and stained for SV2C. Inset: high magnification of the indicated areas. This analysis was performed using three independent primary motor neuron cultures. Shown are representative images for each condition. Scale bars, 20 µm (A, <i>top</i>; B, <i>top</i>); 15 µm (A, <i>bottom</i>); 40 µm (B, <i>bottom</i>). Quantification of the uptake of H_CA and H_CE is shown in (<b>C</b>) and (<b>D</b>), respectively. Bars represent the mean ± standard deviation (SD) of the fluorescence intensity determined from a representative experiment. Ten to thirty fields were analysed for each condition. Although the internalisation of H_CA (Mann-Whitney test; ***, p<0.001), and H_CE (Mann-Whitney test; ***, p<0.001), is significantly increased under stimulation, it is extensive also in resting conditions.</p

H_CA- and H_CE-positive carriers do not colocalise with acidic organelles.

<p>Motor neurons were incubated with 15 nM H_CA (<b>A</b>) or 7.5 nM H_CE (<b>B</b>) and 50 nM Lysotracker Red for 30 min at 37°C, under stimulatory conditions. Cells were then washed and imaged. (<b>A</b>) Individual frames from a confocal time series are shown. Cell bodies are located out of view on the right. Arrowheads mark H_CA-positive carriers, whilst arrows point out Lysotracker-positive organelles. Asterisks indicate a small structure containing both H_CA and Lysotracker. Scale bar, 10 µm. (<b>B</b>) Individual frames from a confocal time series are shown. Cell bodies are located out of view on the right. Arrows mark H_CE-positive carriers, whilst arrowheads point out Lysotracker-positive organelles. Scale bars, 10 µm. (<b>C</b>) Kymographs correspondent to the time series described above. The graphs resulted from the merge between H_CA (in green; left) or H_CE (in green; right) and Lysotracker (in red). Note the virtual absence of double-positive organelles. (<b>D</b>) Quantification of H_C-Lysotracker carriers. The percentage of double-positive structures is very low, indicating that H_CA and H_CE were transported in non-acidic organelles. Bars represent the mean ± standard deviation (SD) and were obtained by analysing at least five movies per condition. (<b>E</b>) Negligible colocalisation between H_CA or H_CE and Lysotracker is also observed in the soma. Scale bars, 10 µm. The results reported in this study were obtained using two independent primary motor neuron cultures, and replicated twice per condition.</p

H_CA, H_CE and H_CT share axonal retrograde carriers.

<p>Motor neurons were incubated with 15 nM H_CA and 40 nM AlexaFluor555-TeNT H_C (H_CT) for 30 min at 37°C, either under resting (5 mM KCl) or stimulating conditions (60 mM KCl; data not shown), and then imaged. (<b>A</b>, <i>top</i>) Individual frames from a confocal time series are shown. Cell bodies are located out of view on the right. Arrows point to double-positive carriers for H_CA and H_CT moving towards the soma. Scale bar, 10 µm. (<b>A</b>, <i>bottom</i>) Kymographs of motor neuron axons treated as described above. The soma is out of view on the right. H_CT-positive carriers are frequent and fast, whilst several H_CA-positive organelles are stationary or oscillate. Carriers containing only H_CA or H_CT are also present. (<b>B</b>) Speed profile of H_CA (in green), H_CE- (in red) and H_CT (in black) carriers. Note the overlap between the three curves, which display a speed peak around 0.8–1 µm/s. (<b>C</b>) Motor neurons were incubated with 15 nM H_CA and 7.5 nM H_CE for 30 min at 37°C under stimulating conditions (60 mM KCl), and then imaged. (<b>C</b>, <i>top</i>) Individual frames from a confocal time series are shown. Cell bodies are located out of view on the right. Arrowheads point to a carrier moving towards the soma containing both H_CA and H_CE. Asterisks indicate an example of stationary double-positive carrier. Scale bar, 10 µm. (<b>C</b>, <i>bottom</i>) Kymographs of motor neuron axons correspond to the stills above. Note the high number of double-positive carriers, either moving towards the soma or oscillatory. This analysis was performed using three (H_CA, H_CT) or two (H_CE) independent primary motor neuron cultures, and replicated at least twice. At least ten (H_CA, H_CT) or five (H_CE) movies per conditions were used to assemble the speed distribution curves shown in (<b>B</b>).</p

H_CA undergoes axonal retrograde transport with the neurotrophin receptor p75^NTR in microfluidic chambers.

<p>(<b>A</b>) Axonal compartment of a MFC. Motor neurons differentiated from an embryonic stem (ES) cell line expressing GFP under the control of the motor neuron-specific promoter (HB9::GFP) have been seeded in the somatic compartment (on the left; not shown). Upon differentiation, their axons elongated through the microgrooves (arrow in the <i>merge</i> panel) and reached the axonal compartment (hatched lines indicate the boundary of this compartment). Cells were stained for the neuronal marker βIII tubulin (blue) and the axonal marker SMI32 (red). Cell bodies are out of view on the left. Scale bar, 50 µm. (<b>B</b>) Primary mouse motor neuron cultures in MFC were used to assess the axonal retrograde transport of H_CA. Motor neurons were incubated with fluorescent H_CA (red, on the left) and with a fluorescent antibody for p75^NTR (green, middle), for 30 min at 37°C in resting conditions, then washed and imaged. Both H_CA and the antibody have been added to the axonal side only of the MFC. Representative stills from a confocal time series are shown in the top part of the panel whilst the relative kymographs are shown at the bottom. Arrowheads indicate double-positive retrogradely transported organelles. Cell bodies are located out of view on the right. The analysis shown in (<b>B</b>) is representative of two independent primary motor neuron cultures. Scale bar, 20 µm.</p

The internalisation of H_CA and H_CE in motor neurons occurs in absence of synaptic vesicle exocytosis and recycling.

<p>Motor neurons were incubated with 15 nM H_CA (<b>A</b>) or 7.5 nM H_CE (<b>B</b>) for 30 min at 37°C, under stimulating conditions (60 mM KCl). Cells were then placed on ice, acid washed, fixed, and stained for VAMP2. Inset: high magnification of the indicated areas. (A, B, C; <i>bottom panels</i>) Motor neurons were pre-treated with 2 nM BoNT/D to cleave VAMP2, thus blocking synaptic vesicle exo/endocytosis. Treatment with BoNT/D did not prevent internalization of H_CA and H_CE. (<b>C</b>) The colocalisation of H_CA and H_CE is largely independent of BoNT/D treatment. The results reported in this figure are representative of experiments performed using two independent primary motor neuron cultures. Scale bars, 20 µm (A, <i>top and bottom</i>; B, <i>top</i>); 10 µm (B, <i>bottom</i>); 5 µm (C).</p

Monitoring peripheral neuroparalytic and central proteolytic effects of BoNT/A and TeNT <i>in vivo</i>.

<p>BoNT/A (or TeNT) was injected unilaterally into the whisker pad of adult rats. Behavioural analysis of whisking and biochemical detection of proteolytic activity in the brainstem were performed at different times following toxin injection. (<b>A,C</b>) Longitudinal assessment of time spent whisking for BoNT/A- or TeNT-treated rats and naïve (control) animals. Note that whisker movements were completely abolished in BoNT/A-treated animals as early as 1 d after injection, while the neuroparalytic effect in TeNT animals was fully apparent at day 3. Quantification reported here is from a representative experiment, which included three animals per group. Data points represent the mean ± standard deviation (SD). Significance was assessed by two-way ANOVA followed by Holm-Sidak test; ***, p<0.001. (<b>B</b>) Representative western blot for cleaved SNAP25 (24 kDa) on protein extracts from ipsilateral facial nuclei of BoNT/A-treated rats at different time points after a single toxin injection. SNAP25 cleavage within the facial nucleus (containing motor neuron somas) was detectable starting from day 3 and further increased at day 10. (<b>D</b>) Representative immunoblotting for VAMP2 on protein extracts from ipsilateral facial nuclei of TeNT-treated rats at different times after a single toxin injection. Loss of intact VAMP2 (13 kDa) within the facial nucleus was apparent starting from day 3. (<b>B</b>, <b>D</b>) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003087#s2" target="_blank">Results</a> were confirmed in two independent experiments, replicated three times. Representative western blots are shown in (<b>B</b>) and (<b>D</b>). Each lane represents one animal. Control, naive uninjected rat. Total protein loaded per lane, 50 µg (<b>B</b>), 10 µg (<b>D</b>). α-tubulin, internal standard, (51 kDa).</p

Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission

<div><p>Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absence of neuromuscular damage. Two triple mutants of BoNT/C, namely BoNT/C <i>S51T/R52N/N53P</i> (BoNT/C α-51) and BoNT/C <i>L200W/M221W/I226W</i> (BoNT/C α-3W), were recently reported to selectively cleave syntaxin and have been used here to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to the effect of BoNT/C <i>in vivo</i>. Although BoNT/C α-51 and BoNT/C α-3W toxins cleave syntaxin with similar efficiency, we unexpectedly found also cleavage of SNAP-25, although to a lesser extent than wild type BoNT/C. Interestingly, the BoNT/C mutants exhibit reduced lethality compared to wild type toxin, a result that correlated with their residual activity against SNAP-25. In spite of this, a local injection of BoNT/C α-51 persistently impairs neuromuscular junction activity. This is due to an initial phase in which SNAP-25 cleavage causes a complete blockade of neurotransmission, and to a second phase of incomplete impairment ascribable to syntaxin cleavage. Together, these results indicate that neuroparalysis of BoNT/C at the neuromuscular junction is due to SNAP-25 cleavage, while the proteolysis of syntaxin provides a substantial, but incomplete, neuromuscular impairment. In light of this evidence, we discuss a possible clinical use of BoNT/C α-51 as a botulinum neurotoxin endowed with a wide safety margin and a long lasting effect.</p></div