Botulinum toxin intoxication requires retrograde transport and membrane translocation at the ER

Botulinum neurotoxin A (BoNT/A) is a highly potent proteolytic toxin specific for neurons with numerous clinical and cosmetic uses. After uptake at the synapse, the protein is proposed to translocate from synaptic vesicles to cytosol through a self-formed channel. Surprisingly, we found that after intoxication proteolysis of a fluorescent reporter occurs in the neuron soma first and then centrifugally in neurites. To investigate the molecular mechanisms at play, we use a genome-wide siRNA screen in genetically engineered neurons and identify over three hundred genes. An organelle-specific split-mNG complementation indicates BoNT/A traffic from the synapse to the soma-localised Golgi in a retromer dependent fashion. The toxin then moves to the ER and appears to require the Sec61 complex for retro-translocation to the cytosol. Our study identifies genes and trafficking processes hijacked by BoNT/A, revealing a complex route for efficient intoxication that contradicts the currently accepted model of BonT intoxication.