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Botulinum toxin’s axonal transport from periphery to the spinal cord

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Abstract

Axonal transport of enzymatically active botulinum toxin A (BTX-A) from periphery to the CNS has been described in facial and trigeminal nerve, leading to cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal transport of peripherally applied BTX-A to spinal cord via sciatic nerve.

We employed BTX-A-cleaved SNAP-25 immunohistochemistry of lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal intrasciatic injection of colchicine (5 mM, 2 μl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was localized within cholinergic neurons.

These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs commonly following peripheral application.

Graphical abstract

Occurrence of cleaved SNAP-25 in ipsilateral lumbal ventral horn following botulinum toxin A (30 U/kg) subcutaneous injection into the rat hind paw.

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Highlights

► Peripheral BTX-A cleaves SNAP-25 in dorsal and ventral horn of the spinal cord. ► Axonal transport of BTX-A occurs at low intramuscular dose. ► BTX-A retrograde transport occurs via peripheral nerves and is microtubule-dependent. ► Truncated SNAP-25 is localized within cholinergic neurons of the ventral horn.

Introduction

It is a textbook knowledge that botulinum toxin type A (BTX-A) in botulism, as well as in therapeutic applications, exerts the neuromuscular paralysis by enzymatic cleavage of peripheral synaptosomal-associated protein 25 (SNAP-25), involved in neuroexocytosis. Unlike related tetanus toxin, which is known to be retrogradely transported and transcytosed to second-order synapses (Schwab et al., 1979), it was generally accepted that BTX-A acts directly only on peripheral nerve endings. Nevertheless, already in the 70-ties some authors reported axonal transport of radioactively labeled botulinum toxin A (BTX-A) within peripheral nerves to spinal cord (Habermann, 1974, Wiegand et al., 1976). Those observations remained forgotten and questioned in later studies (Tang Liu et al., 2003). Main objections to these early studies were that it was not known if radioactively labeled BTX-A retained the enzymatic activity by the time it reached spinal cord. However, axonal transport of functional BTX-A molecules was recently found in hippocampus, visual system and in facial motoneurons (Antonucci et al., 2008). BTX-A axonal transport followed by enzymatic cleavage in CNS has been demonstrated in trigeminal sensory neurons (Matak et al., 2011). Recent in vitro study suggests spread of BTX-A within cell bodies and distal processes of cultured sympathetic neurons (Lawrence et al., 2012).

Retrograde axonal transport of low dose BTX-A in spinal sensory neurons has been suggested by behavioral experiments in models of bilateral muscular hyperalgesia (Bach-Rojecky and Lacković, 2009) and diabetic neuropathy (Bach-Rojecky et al., 2010).

In present study we found the enzymatic activity of BTX-A in rat motor and sensory regions of the spinal cord after intramuscular, subcutaneous, or intraneural toxin application.

Section snippets

Animals

Eighteen male Wistar rats (University of Zagreb School of Medicine, Croatia), weighing 300–400 g, kept on 12 h/12 h light and dark cycle with unlimited access to food and water, 3 months old, were used. Experiments were conducted according to the European Communities Council Directive (86/609/EEC). Animal procedures were approved by the Ethical Committee of University of Zagreb, School of Medicine (Permit No. 07-76/2005-43). All efforts were made to reduce the suffering of animals and the number of

Results

Slight ipsilateral flaccidity of rat distal hind limb was visible only following the i.m. treatment (5 and 30 U/kg of BTX-A), but not following the i.n. or s.c. injections.

BTX-A-cleaved SNAP-25 immunoreactivity appeared in ipsilateral lumbal ventral horns of s.c., i.m. and i.n.- BTX-A treated animals (Fig. 1, Fig. 2). Cleaved SNAP-25 immunoreactivity was visible around motoneuronal nuclei of lamina 9 in the form of dense, small fibers, and long neuronal processes. Following i.m. and i.n.

Occurrence of cleaved SNAP -25 in the spinal cord after BTX-A peripheral injections

Cleavage of central SNAP-25 in ipsilateral spinal cord segments after single peripheral application of BTX-A suggests the long-distance axonal traffic of enzymatically active BTX-A fragments from periphery to the spinal cord. These results extend recent findings on retrograde axonal transport of functionally active BTX-A in cranial nerves (Antonucci et al., 2008, Matak et al., 2011). Importantly, apart from cranial nerves in facial region with relatively short axons which project to the rat

Conclusion

Appearance of truncated SNAP-25 in spinal cord following low dose i.m. BTX-A (5 U/kg) administration suggests that the axonal transport of toxin to CNS commonly occurs following peripheral administration.

Acknowledgements

Experiments were performed at the Department of Pharmacology, University of Zagreb School of Medicine, and Clinics and Polyclinics of Psychiatry, University of Würzburg. This work was supported by Croatian Ministry of Science, Education and Sport, (Project No. 108-1080003-0001 to Z. Lackovic) and Deutscher Academischer Austauch Dienst (DAAD, to J. Deckert and Z. Lackovic). Antibody to BTX-A–cleaved SNAP-25 was a kind gift from Assist. Prof. Ornella Rossetto (University of Padua, Italy). We also

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