Biochem. J. (2012) 441, 443–452 (Printed in Great Britain) doi:10.1042/BJ20111117 443 Extravesicular intraneuronal migration of internalized botulinum neurotoxins without detectable inhibition of distal neurotransmission Gary W. LAWRENCE*, Saak V. OVSEPIAN*, Jiafu WANG*, K. Roger AOKI† and J. Oliver DOLLY* 1 *International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland, and †Allergan Inc., Irvine, CA 9262, U.S.A. Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. BoNT A (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, BoNT E (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 BoNT A or BoNT E to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. BoNT E yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. Neurite transection prevented movement of BoNT A . The BoNT A 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 BoNT E of the BoNT A acceptor- binding domain did not alter its potency or mobility. Spontaneous or evoked transmission to cell bodies were not inhibited by retrogradely migrated BoNT A except with high doses, concurring with the lack of evidence for a direct central action when used clinically. Key words: botulinum neurotoxin, intracellular protein transport, soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE), synaptosome-associated protein of 25 kDa (SNAP-25). INTRODUCTION Elucidation of the largely unknown processes for the intracellular trafficking and localization of proteins has been aided by bacterial and plant di-chain toxins that bind susceptible cells via one of their polypeptides and deliver inside an associated enzymatic chain [1]. In this regard, intense interest has been kindled in BoNT A (botulinum neurotoxin type A) because its targeting and internalization into peripheral nerves culminates in proteolytic inactivation of a protein essential for neuroexocytosis, leading to potent and selective inhibition of acetylcholine release. Moreover, this array of unique properties has been exploited with astounding success for relaxing overactive muscles (e.g. in dystonias, spasticity and overactive bladder) and normalizing secretory disorders (e.g. hyperhydrosis and sialorrhoea) for 3–12 months after a single treatment [2]. Seven serotypes of BoNT (A–G) from Clostridium botulinum contain a 100 kDa HC (heavy chain) linked by a disulfide and non-covalent bonds to a 50 kDa LC (light chain). Their HCs mediate binding to neuronal ecto-acceptors and internalization, whereas the metalloprotease activities of the LCs inactivate SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins essential for synaptic vesicle fusion [3]. Another advantage of BoNTs as probes for investigating intracellular protein trafficking is that their intriguing uptake route involves translocation of the inhibitory LC across the limiting membrane to the cytosolic site of action via a channel created by H N (N-terminal half of the HC) [4]. Research on BoNT A is increasing due to its numerous successful clinical applications noted above, particularly its remarkable ability to cause prolonged neuroparalysis. It deletes nine C-terminal residues from SNAP-25 (synaptosome-associated protein of 25 kDa). Another serotype, E, cleaves the same SNARE target further from the C-terminus, removing 26 amino acids [3]. Despite evidence that BoNT E (botulinum neurotoxin type E) disables SNAP-25 more effectively than BoNT A in vitro [5–7], it is rarely used clinically due its neuroparalysis recovering rapidly [8]. Transfection of neuroendocrine cells with a gene fragment for LC of BoNT A fused to that of a fluorescent protein provided evidence that it associates with the plasmalemma via, in part, its unique dileucine motif [9] and its N-terminus binding to SNAP-25 [10], although susceptibility of BoNT E to ubiquitination has been reported recently to contribute to a faster degradation than BoNT A [11]. Concurrently, the dileucine motif in BoNT A was shown to underlie its extraordinarily long duration of action because substituting both of the leucine residues dramatically shortened the persistence of neuromuscular paralysis in vivo [12]. However, selective and complete retention of exogenously applied BoNTs at the site of uptake into neurons has not been established. Although the majority of 125 I-labelled BoNT A localized within a few microns of the non-myelinated synaptic terminals after intramuscular injection, a trace of radiolabel could be detected inside the myelinated nerve trunk [13] and spinal cord [14,15]; nevertheless, transport of disconjugated iodine or BoNT-degradation products were not excluded. Later, cleaved SNAP-25 was visualized distant from neuronal sites of uptake and attributed to long-distance axonal transport of BoNT A [16] inside vesicles [17]. It has been speculated that transfer of BoNT A in this way may reach the CNS (central nervous system) and cause central effects in peripherally Abbreviations used: ACSF, artificial cerebrospinal fluid; BoNT, botulinum neurotoxin; BoNT A , BoNT type A; BoNT E , BoNT type E; CV, variation coefficient; Cy3, indocarbocyanine; eEPSP, evoked excitatory postsynaptic potential; HC, heavy chain; H C , C-terminal half of the HC; H N , N-terminal half of the HC; LC, light chain; MLD 50 , median lethal dose; PPR, paired-pulse response ratio; SCGN, superior cervical ganglion neuron; sEPSC, spontaneous excitatory postsynaptic current; SNAP-25, synaptosome-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor-attachment protein receptor; SV2, synaptic vesicle protein 2. 1 To whom correspondence should be addressed (email oliver.dolly@dcu.ie). c  The Authors Journal compilation c  2012 Biochemical Society