Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons

PLoS Pathog. 2012 Dec;8(12):e1003087. doi: 10.1371/journal.ppat.1003087. Epub 2012 Dec 27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / metabolism
  • Animals
  • Axonal Transport / drug effects*
  • Botulinum Toxins / metabolism
  • Botulinum Toxins / pharmacology*
  • Botulinum Toxins, Type A / metabolism
  • Botulinum Toxins, Type A / pharmacology*
  • Cells, Cultured
  • Central Nervous System / drug effects
  • Central Nervous System / metabolism
  • Endocytosis / drug effects
  • Mice
  • Motor Neurons / drug effects*
  • Motor Neurons / metabolism
  • Neuromuscular Junction / metabolism
  • Neurotransmitter Agents / antagonists & inhibitors*
  • Paralysis / metabolism
  • Rats
  • Rats, Long-Evans
  • Rats, Sprague-Dawley
  • Spinal Cord / metabolism
  • Synaptic Transmission / drug effects
  • Tetanus Toxin / metabolism
  • Tetanus Toxin / pharmacology

Substances

  • Neurotransmitter Agents
  • Tetanus Toxin
  • Botulinum Toxins
  • Botulinum Toxins, Type A
  • Acetylcholine
  • botulinum toxin type E

Grants and funding

This work was supported by the Silvia Mion Prize, University of Verona (LR), Cancer Research UK (GS, KB), the Weizmann-UK Making Connections program (GS), Italian Ministry of Research (FIRB2010-RBFR10ZBYZ_003 to LR, PRIN2008 to OR and MC), Tuscany Region (Health Program 2009 to MC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.