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Selective Cleavage of SNAREs in Sensory Neurons Unveils Protein Complexes Mediating Peptide Exocytosis Triggered by Different Stimuli

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Abstract

Oligomerisation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes is required for synaptic vesicle fusion and neurotransmitter release. How these regulate the release of pain peptides elicited by different stimuli from sensory neurons has not been established. Herein, K+ depolarization was found to induce multiple sodium dodecyl sulfate (SDS)-resistant SNARE complexes in sensory neurons exposed to botulinum neurotoxins (BoNTs), with molecular weights ranging from 104–288 k (large) to 38–104 k (small). Isoform 1 of vesicle-associated membrane protein 1 (VAMP 1) assembled into stable complexes upon depolarisation and was required for the participation of intact synaptosome-associated protein of relative molecular mass 25 k (SNAP-25) or BoNT/A-truncated form (SNAP-25A) in the large functional and small inactive SDS-resistant SNARE complexes. Cleaving VAMP 1 decreased SNAP-25A in the functional complexes to a much greater extent than the remaining intact SNAP-25. Syntaxin 1 proved essential for the incorporation of intact and SNAP-25A into the large complexes. Truncation of syntaxin 1 by BoNT/C1 caused /A- and/or /C1-truncated SNAP-25 to appear in non-functional complexes and blocked the release of calcitonin gene-related peptide (CGRP) elicited by capsaicin, ionomycin, thapsigargin or K+ depolarization. Only the latter two were susceptible to /A. Inhibition of CGRP release by BoNT/A was reversed by capsaicin and/or ionomycin, an effect overcome by BoNT/C1. Unlike BoNT/B, BoNT/D cleaved VAMP 1 in addition to 2 and 3 in rat sensory neurons and blocked both CGRP and substance P release. Thus, unlike SNAP-25, syntaxin 1 and VAMP 1 are more suitable targets to abolish functional SNARE complexes and pain peptide release evoked by any stimuli.

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Abbreviations

BoNT:

Botulinum neurotoxin

CGNs:

Cerebellar granule neurons

CGRP:

Calcitonin gene-related peptide

DC:

Di-chain

DMEM:

Dulbecco’s modified Eagle medium

DRGs:

Dorsal root ganglionic neurons

EIA:

Enzyme immunoassay

HC:

Heavy chain

LC:

Light chain

LDCVs:

Large dense-core vesicles

mAb:

Monoclonal antibody

SC:

Single chain

SNAP-25:

Synaptosome-associated protein of relative molecular mass 25 k

SNARE complexes:

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes

SP:

Substance P

TGNs:

Trigeminal ganglionic neurons

TRPV1:

Transient receptor potential vanilloid type 1

VAMP 1 and 2:

Vesicle-associated membrane protein isoforms 1 and 2

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Acknowledgments

This research is funded by a Principle Investigator Award (to J.O.D.) from Science Foundation Ireland which supports Dr. Meng and, in part, by Allergan Inc.

Conflict of Interest

We declare that this research is funded in part by Allergan Incorporated, P.O. Box 19534, Irvine, CA 92623, USA. The industrial sponsor had no role in study design, data collection and analysis or preparation of the manuscript.

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Authors and Affiliations

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

    Jianghui Meng, J. Oliver Dolly & Jiafu Wang

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  1. Jianghui Meng
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  2. J. Oliver Dolly
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  3. Jiafu Wang
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Corresponding authors

Correspondence to J. Oliver Dolly or Jiafu Wang.

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Supplemental Fig. S1

BoNT/D but not /B inhibited K+-evoked CGRP release from rat cultured DRG neurons, whereas both blocked its release from mouse DRGs. The latter were dissected from postnatal day 5 rats (open bars) or mice (hatched bars), dissociated and cultured for 7 days before being exposed to 100 nM recombinant BoNT/D, /B or toxin-free medium for 24 h at 37 °C; K+-evoked Ca2+-dependent CGRP release was assayed using EIA, as before, and plotted as % of the value for the respective toxin-free control. Results are means ± SEM; n ≥ 3 (PDF 11 kb)

Supplemental Fig. S2

VAMP 1 is enriched in rat TGNs unlike central CGNs; VAMP 1 forms SNARE complexes with SNAP-25 and syntaxin 1 upon depolarisation. a Equal number of cells were lysed by SDS buffer for SDS-PAGE and Western blotting. Note that TGNs express more VAMP 1 than CGNs. b Upon stimulation of rat TGNs by HK, VAMP 1 was readily detectable in large (Mr of 104–288 k) SNARE complexes (PDF 60 kb)

Supplemental Fig. S3

The presence of VAMP 3 and a negligible amount of 2 in SDS resistant SNARE complexes. Rat TGNs were depolarized with high [K+] for 5 min at 37 °C before being lysed for 2-D electrophoresis. Antibodies against syntaxin 1, SNAP-25 and VAMP 2 were used for Western blotting. Note that VAMP 2 was hardly detected in SDS-resistant complex despite the presence of its free form (upper panel). Aliquots of the same samples were subjected to separate SDS-PAGE for probing VAMP 3 (lower panel). (PDF 91 kb)

Supplemental Fig. S4

In mouse TGNs, like rat, cleavage of syntaxin 1 leads SNAP-25C1 into small complexes. After cleaving syntaxin 1 and SNAP-25 with 100 nM/C1, cells were washed and stimulated with 60 mM K+ in the presence of 2.5 mM Ca2+ for 5 min, before lysis in SDS sample buffer for 2-D electrophoresis and immunoblot analysis. Note that the residual intact SNAP-25 resides predominantly in the large complexes whereas /C1-truncated SNAP-25 occurred in the small forms. Subsequent cleavage of VAMPs by /D reduced both complexes (PDF 178 kb)

Supplemental Fig. S5

Direct comparison of the levels of the two sizes of SNARE complexes in TGNs treated with BoNTs. After incubation with 100 nM toxins, the cells were depolarized with K+ for 5 min before being harvested in SDS buffer for 2-D electrophoresis. SNARE complexes of large (104–288 k) and small (38–104 k) sizes were analysed. a BoNT/D but not /B decreased the content of both sizes of SNARE complexes. b /C1 cleavage of syntaxin 1 resulted in the appearance of the majority of SNAP-25C1 in the small SNARE complexes; subsequent exposure to/D diminished the large and small SNARE complexes. c TGNs treated with BoNT/C1 followed by BoNT/A further reduced the intact SNAP-25 in the large complexes but did not preclude the presence of SNAP-25C1 in the smaller complexes; SNAP-25 cleaved by BoNT/A alone resided predominately in the large complexes. SNAP-25C1 occurs in the small rather than the large complexes after cleavage of syntaxin 1. d Treatment with /A followed by /D decreased both the large and small complexes (PDF 175 kb)

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Meng, J., Dolly, J.O. & Wang, J. Selective Cleavage of SNAREs in Sensory Neurons Unveils Protein Complexes Mediating Peptide Exocytosis Triggered by Different Stimuli. Mol Neurobiol 50, 574–588 (2014). https://doi.org/10.1007/s12035-014-8665-1

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