Extravesicular intraneuronal migration of internalized botulinum neurotoxins without detectable inhibition of distal neurotransmission

Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. BoNTA (botulinum neurotoxin type A) is a protease that binds to peripheral nerve terminals, becomes endocytosed and causes prolonged blockade of transmitter release by cleaving SNAP-25 (synaptosome-associated protein of 25 kDa). Retrograde transport of the toxin has been suggested, but not of the transient muscle relaxant, BoNTE (botulinum neurotoxin type E). In the present study, dispersal of these proteases in compartmented cultures of rat sympathetic neurons was examined after focal application of BoNTA or BoNTE to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. BoNTE yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. Neurite transection prevented movement of BoNTA. The BoNTA protease could be detected only in the supernatants of neurites or cell body lysates, hence these proteases must move along neuronal processes in the axoplasm or are reversibly associated with membranes. Substitution into BoNTE of the BoNTA acceptor-binding domain did not alter its potency or mobility. Spontaneous or evoked transmission to cell bodies were not inhibited by retrogradely migrated BoNTA except with high doses, concurring with the lack of evidence for a direct central action when used clinically.