title: Fusion of Golgi-derived vesicles mediated by SNAP-25 is essential for sympathetic neuron outgrowth but relatively insensitive to botulinum neurotoxins in vitro.
abstract: Sympathetic neurons ramify to innervate multiple cells in target tissues. In compartmentalized cultures of rat superior cervical ganglion neurons, cleavage of synaptosomal-associated protein of Mr  = 25 000 (SNAP-25) in neurites exposed to botulinum neurotoxin type A (BoNT/A) arrested their growth and collapsed interstitial branches, but this required large, nonclinical doses. A protease-inactive mutant proved ineffective, confirming involvement of SNAP-25 in neurite extension and arborization. BoNT/C1 acted like BoNT/A, but BoNT/E caused only mild inhibition, likely due to transient SNAP-25 proteolysis. Near-total lack of susceptibility to BoNT/B or BoNT/D revealed that vesicle-associated membrane protein (VAMPs) isoforms 1-3 are not essential. Neurite length was not reduced when either BoNT/A or BoNT/C1 was applied to the somata, with no detrimental effect on neuron viability being observed. Treatments that protect cells from deprivation of nerve growth factor failed to prevent the toxin-induced loss of neurites. Inactivation of SNAP-25 caused the accumulation at neurite branch sites of Golgi-derived organelles labelled with N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sphingosine conjugated to bovine serum albumin, prior to the collapse of arbors. Notably, neurite growth was ~ 1000-fold less susceptible to BoNT/A than cholinergic transmission in these neurons. Accordingly, a BoNT/A acceptor synaptic vesicle protein 2 (SV2) was found to be colocalized with VAMP 1-3, but not with VAMP 7, which is implicated in the growth of neurites. In conclusion, neurites depend on SNAP-25 for extension but this is quite resistant to BoNT/A, possibly, because of a low density of SV2 at growth sites that are distant from the highly susceptible regions of neurotransmitter release.
authors.affiliation: International Centre for Neurotherapeutics, Dublin City University, Ireland.
authors.forename: Gary W
authors.initials: GW
authors.lastname: Lawrence
authors.forename: Jiafu
authors.initials: J
authors.lastname: Wang
authors.forename: Mitchell F
authors.initials: MF
authors.lastname: Brin
authors.forename: K Roger
authors.initials: KR
authors.lastname: Aoki
authors.forename: Larry
authors.initials: L
authors.lastname: Wheeler
authors.forename: J Oliver
authors.initials: JO
authors.lastname: Dolly
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism; D019274:Botulinum Toxins, Type A; D002478:Cells, Cultured; D006056:Golgi Apparatus / Q000378:metabolism / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D016501:Neurites / Q000378:metabolism / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D051381:Rats; D017783:Superior Cervical Ganglion / Q000166:cytology; D009435:Synaptic Transmission; D050825:Synaptosomal-Associated Protein 25 / Q000502:physiology
chemical_list: D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C494210:Snap25 protein, rat; C489618:Sv2a protein, rat; D050825:Synaptosomal-Associated Protein 25; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A
keywords: SNAP-25; neurite growth; neurotransmission; soluble N-ethylmaleimide-sensitive factor-attachment protein; vesicle-associated membrane protein