Fusion of Golgi-derived vesicles mediated by SNAP-25 is essential for sympathetic neuron outgrowth but relatively insensitive to botulinum neurotoxins in vitro.

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.