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First published online 31 July 2007
doi: 10.1242/jcs.012211

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Synaptobrevin I mediates exocytosis of CGRP from sensory neurons and inhibition by botulinum toxins reflects their anti-nociceptive potential

Jianghui Meng^*, Jiafu Wang^*, Gary Lawrence and J. Oliver Dolly

International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland

View larger version (61K): [in this window] [in a new window]   Fig. 1. Visualisation of the morphology and markers of sensory neurons in cultured TGNs. Samples were viewed in an inverted microscope by phase contrast (A,B) or in fluorescence mode (C-F), and by confocal microscopy (G-I). Bright-field views of neurons from (A) rat postnatal day 5 (P5) and (B) mouse (P5) after 7 DIV. Antibodies specific for VR1 (1:1000) stained the majority of rat TGNs (C); note that BR2 (1:500) was detected at one pole of VR1-positive cells (D); CGRP (1:500) and substance P (1:1000) appeared highly colocalized when visualised under low-magnification microscopy (E, F) but showed some distinct distribution in confocal microscopy (G-I). Fluorescently labelled secondary antibodies (goat anti-mouse Alexa Fluor-488, 1:200 or goat anti-rabbit Alexa Fluor-546, 1:200) were used. In some cases, the specimens were counterstained with DAPI. Bars, 20 µm (A-F) and 5 µm (G-I).

View larger version (62K): [in this window] [in a new window]   Fig. 2. Microscopic demonstration of the presence of SNAREs and CGRP in rat TGNs. Cells were grown on coverslips for 7 DIV, fixed and permeabilised prior to labelling with specific primary and secondary antibodies (as described for Fig. 1). Confocal images after labelling with antibodies against SNAP25 (1:500), CGRP (1:500), syntaxin I (1:500) and Sbr II (1:1000) demonstrated punctate staining of these proteins and the neuropeptide in cell bodies and/or their processes. Merged images are shown below each pair of singly stained micrographs.

View larger version (18K): [in this window] [in a new window]   Fig. 3. CGRP release evoked by K+, capsaicin or bradykinin from TGNs is Ca2+-dependent. (A-C) After 7 DIV, neurons were incubated for 30 minutes at 37°C in BR-HBS alone or with vehicle added (see Materials and Methods) for measurement of basal release of CGRP (white bars), and then incubated in BR-HBS in the presence of varying concentrations of (A) K+, (B) capsaicin or (C) bradykinin to determine stimulated release of CGRP (hatched bars). Release stimulated by K+ (60 mM), capsaicin (1 µM) or bradykinin (0.1 µM) represents 30, 20 and 15% of the total content, respectively. Likewise, Ca2+-independent release was determined, except in Ca2+-free BR-HBS containing 2 mM EGTA. Buffer in which cells were bathed was carefully removed and the concentration of CGRP determined by enzyme immuno-assay (mean ± s.e.m., n=4). Note that the basal efflux is higher when either 0.1% ethanol or dimethyl sulphoxide was included (Fig. 3B,C) than when absent (Fig. 3A). Note that each stimulus gave significant increments of evoked release over their basal level.

View larger version (30K): [in this window] [in a new window]   Fig. 4. BoNT/A differentially inhibits Ca2+-dependent CGRP release evoked from rat TGNs by three stimuli and cleaves SNAP25: receptors of BoNT/A occur on VR1-positive cells. TGNs were exposed to BoNT/A and release of CGRP was assayed over 30 minutes. Cells were then solubilised in SDS-sample buffer and equal volumes subjected to SDS-PAGE and western blotting, using an antibody that recognises intact and truncated SNAP25. The proportion of remaining substrate was calculated relative to an internal syntaxin control, using digital images of the gels. (A) Immunoblot showing the cleavage by the neurotoxin of SNAP25 but not syntaxin I. (B). Dose-response curve for BoNT/A-induced blockade of CGRP release evoked by 60 mM K+ (), which correlates with the percent of remaining SNAP25 (). Lesser extents of inhibition by BoNT/A were observed for release evoked by 0.1 µM bradykinin () and, especially, 1 µM capsaicin (). Data plotted are the mean ± s.e.m.; n=5. (C) Western blot of TGNs visualised with antibodies specific against syntaxin I or SNAP23 (*). (D) Representative micrographs demonstrating VR1 and SV2A, SV2B and SV2C in rat TGNs. Fluorescent images were obtained after labelling the cells with antibodies raised in guinea pig specific for VR 1 (1:1000) and in rabbit for SV2A or SV2B (1:1000) or in goat for SV2C (1:100). The controls were treated similarly except in the absence of primary antibodies but incubated with fluorescently labelled secondary IgGs against rabbit (as in Fig. 1) and guinea pig (goat anti-guinea-pig Alexa Fluor-488, 1:200) (1) or goat (donkey anti-goat Cy3, 1:800) and guinea pig (donkey anti-guinea-pig Cy2, 1:200) (2). Bars, 20 µm. Note that all the SV2 isoforms are present in VR1-positive neurons.

View larger version (48K): [in this window] [in a new window]   Fig. 5. BoNT/C1 incompletely cleaves SNAP25 and syntaxin I, and partially inhibits Ca2+-dependent CGRP release evoked by three stimuli. TGNs were treated with BoNT/C1, and release of CGRP was assayed and western blotting performed. Results were calculated as described for Fig. 4 relative to Sbr I and Sbr II control. (A) Partial cleavage of SNAP25 by BoNT/C1, visualised with the IgG used in Fig. 4. Decrease in syntaxin I was revealed with an antibody only reactive with this intact SNARE. Syntaxin II and, especially, syntaxin III, proved difficult to quantify and, thus, cleavage by toxin was not detectable. (B) Dose-response curves for BoNT/C1-induced cleavage of SNAP25 () and syntaxin I (). (C) BoNT/C partially inhibits CGRP release evoked by 60 mM K+ (), 0.1 µM bradykinin () or 1 µM capsaicin (). Data plotted are the mean ± s.e.m.; n3.

View larger version (22K): [in this window] [in a new window]   Fig. 6. BoNT/D blocks evoked Ca2+-dependent CGRP release and cleaves three Sbr isoforms. The amount of Ca2+-dependent basal and evoked release of CGRP for each stimulus was measured in cells treated with BoNT/D, as described for Fig. 4. (A) Three Sbr isoforms were detected using antibodies against Sbr I or Sbr II, or Sbr I, Srb II and Srb III together (HV-62); Srb I and Srb II co-migrate. (B) Dose-response curves for the remaining intact Sbr II () and Sbr I (), and for inhibition of release evoked by 60 mM K+ (); inset shows the amount of CGRP in TGNs after overnight incubation with or without BoNT/D. (C) Dose-response curves for BoNT/D-induced inhibition of CGRP release evoked by 1 µM capsaicin () or 0.1 µM bradykinin (). Inset shows the reduction of basal release by BoNT/D. The values are the mean ± s.e.m.; n=8.

View larger version (63K): [in this window] [in a new window]   Fig. 7. BoNT/B proteolyses Sbr II and SbrIII in rat TGNs but does not reduce K+-evoked CGRP release despite the presence of its receptor; in mouse TGNs, Sbr I is also cleaved and exocytosis is blocked. TGNs were cultured from rat (left panels) and mouse (right panels) for 7 DIV before exposure to BoNT/B; enzyme immuno-assay of CGRP release and western blotting were carried out as described for Fig. 4. Values are the mean ± s.e.m., n=8. (A,C) Immuno-blots showing the disappearance of Sbr isoforms [two (Srb II and Srb III) for rat and three (Srb I, Srb II and Srb III) for mouse] relative to the internal standard (SNAP25) that remained unchanged. (B,D) Dose-response curves for inhibition of CGRP release evoked by 60 mM K+ (), capsaicin () and remaining Sbr II (), Sbr I and Srb II (), and Sbr I (). Inset in D illustrates the inhibition by BoNT/D of K+-evoked CGRP release from mouse TGNs, for comparison. (E,F) Representative fluorescence micrographs showing that the putative protein receptors of BoNT/B, synaptotagmin I and II, are present in CGRP-positive neurons; as well as CGRP and Sbr I are highly colocalized in cell bodies and their fine fibres in rat TGNs. Specimens were stained using (E) rabbit anti-CGRP (1:500) (and donkey anti-rabbit IgG Cy2, 1:200) and goat anti-synaptotagmin I/II (1:100) (and donkey anti-goat IgG Cy3, 1:800) or (F) rabbit anti-Sbr I (1:1000) (and donkey anti-rabbit IgG Cy2, 1:200) and mouse anti-CGRP (1:500) (and donkey anti-mouse IgG Cy3, 1:800). Notice the striking punctate co-staining in the two right hand panels in F. The control was treated in the absence of primary antibodies but incubated with secondary fluorescently labelled IgGs against goat and rabbit. Bars, 20 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Immuno-absorption of vesicles from TGNs by antibodies against Sbr I and Sbr II. Equal amounts of the total membrane fraction from lysed TGNs were incubated overnight at 4°C with protein A beads coupled to IgGs specific for Sbr I or SbrII, or rabbit non-immune IgG (control). After extensive washing, equivalent aliquots of beads were sedimented and pellets dissolved in SDS sample buffer for SDS-PAGE and western blotting. Alternatively, 2 M acetic acid with 0.1% TFA was added to replicate samples for CGRP determination. (A) CGRP content (± s.e.m.) measured in two separate preparations show the large enrichment in vesicles obtained using beads containing Sbr I or Sbr II relative to the control. (B) Western blots of the two vesicle preparations and the control, using antibodies specific for Sbr I or Sbr II.

View larger version (69K): [in this window] [in a new window]   Fig. 9. Sbr I complexes with SNAP25 and syntaxin 1 in TGNs. The pelleted cells were extracted in 1 ml of buffer containing 1% (v/v) Triton X-100 (see Materials and Methods) for 1 hour at 4°C, followed by centrifugation. The supernatant was incubated at 4°C overnight with anti-Sbr I IgG coupled to protein A agarose. After sedimentation and extensive washing with the extraction buffer, beads were suspended in SDS sample buffer for SDS-PAGE (with and without boiling the samples for 10 minutes) under non-reducing conditions, followed by western blotting using antibodies specific for each SNARE. Only the lower halves of the gels blotted for Sbr I or Sbr II are shown because of excessive staining of the rabbit IgG that overlapped the SNARE complex. Note that boiling raised the proportions of dissociated SNAP25, syntaxin I and Sbr I; this corresponds to the decrease in the complex.