Botulinum Neurotoxins A and E Undergo Retrograde Axonal Transport in Primary Motor Neurons

Abstract

The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.

Author Summary

Botulinum neurotoxins are the most toxic molecules known to mankind, and as a result, are currently listed among the top bio-threats. However, their ability to bind specifically to neurons and their inhibitory effects on regulated secretion prompted their clinical use in pathologies characterised by increased muscular tone, such as dystonia and various forms of spasticity, or abnormal secretion, such as drooling and excessive sweating, to cite a few. As a consequence, botulinum neurotoxin A, which is the serotype most commonly used in human therapy, has become the treatment of choice for an ever-expanding number of pathological and non-pathological (e.g. cosmetic) conditions. All current indications show that the systemic effects and toxicity of botulinum neurotoxin A are minimised by the specific route of administration (local injection) and the low diffusion of this molecule in tissues. However, recent reports suggest that in contrast to this common belief, botulinum neurotoxin A is able to reach distal sites in the body and may have previously unanticipated effects in the central nervous system. In this study, we demonstrate that botulinum neurotoxin A and E enter alternative endocytic pathway(s) in addition to synaptic vesicle recycling, and undergo long-range transport in a non degradative compartment in spinal cord motor neurons. Our results show that axonal retrograde transport is a common pathway for the dissemination in the central nervous system of pathogens and virulence factors important for human and animal health.