Control of autophagosome axonal retrograde flux by presynaptic activity unveiled using botulinum neurotoxin type a

J Neurosci. 2015 Apr 15;35(15):6179-94. doi: 10.1523/JNEUROSCI.3757-14.2015.

Abstract

Botulinum neurotoxin type A (BoNT/A) is a highly potent neurotoxin that elicits flaccid paralysis by enzymatic cleavage of the exocytic machinery component SNAP25 in motor nerve terminals. However, recent evidence suggests that the neurotoxic activity of BoNT/A is not restricted to the periphery, but also reaches the CNS after retrograde axonal transport. Because BoNT/A is internalized in recycling synaptic vesicles, it is unclear which compartment facilitates this transport. Using live-cell confocal and single-molecule imaging of rat hippocampal neurons cultured in microfluidic devices, we show that the activity-dependent uptake of the binding domain of the BoNT/A heavy chain (BoNT/A-Hc) is followed by a delayed increase in retrograde axonal transport of BoNT/A-Hc carriers. Consistent with a role of presynaptic activity in initiating transport of the active toxin, activity-dependent uptake of BoNT/A in the terminal led to a significant increase in SNAP25 cleavage detected in the soma chamber compared with nonstimulated neurons. Surprisingly, most endocytosed BoNT/A-Hc was incorporated into LC3-positive autophagosomes generated in the nerve terminals, which then underwent retrograde transport to the cell soma, where they fused with lysosomes both in vitro and in vivo. Blocking autophagosome formation or acidification with wortmannin or bafilomycin A1, respectively, inhibited the activity-dependent retrograde trafficking of BoNT/A-Hc. Our data demonstrate that both the presynaptic formation of autophagosomes and the initiation of their retrograde trafficking are tightly regulated by presynaptic activity.

Keywords: Botulinum neurotoxin; autophagosome; axon; presynaptic; retrograde transport; synaptic vesicle.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Androstadienes / pharmacology
  • Animals
  • Animals, Newborn
  • Autophagy / drug effects*
  • Autophagy / physiology
  • Axonal Transport / drug effects
  • Axonal Transport / physiology
  • Botulinum Toxins, Type A / metabolism*
  • Botulinum Toxins, Type A / pharmacology
  • Cells, Cultured
  • Enzyme Inhibitors / pharmacology
  • Female
  • Hippocampus / cytology*
  • In Vitro Techniques
  • Macrolides / pharmacology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Microtubule-Associated Proteins / metabolism
  • Neurons / cytology*
  • Neurons / drug effects
  • Neurons / ultrastructure
  • Neurotoxins / metabolism*
  • Neurotoxins / pharmacology
  • Organ Culture Techniques
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, Nerve Growth Factor / metabolism
  • Synaptosomal-Associated Protein 25 / metabolism
  • Wortmannin

Substances

  • Androstadienes
  • Enzyme Inhibitors
  • Macrolides
  • Map1lc3b protein, mouse
  • Microtubule-Associated Proteins
  • Neurotoxins
  • Receptors, Nerve Growth Factor
  • Synaptosomal-Associated Protein 25
  • TNFRSF16 protein, mouse
  • bafilomycin A1
  • Botulinum Toxins, Type A
  • Wortmannin