Presynaptic targeting of botulinum neurotoxin type A requires a tripartitePSG‐Syt1‐SV2plasma membrane nanocluster for synaptic vesicle entry

Abstract

The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live‐cell super‐resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor‐binding‐deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG‐synaptotagmin‐1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1‐SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A‐ and BoNT/E‐induced neurointoxication as quantified by SNAP‐25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.

Saturated fatty acid-Coenzyme A supplementation restores neuronal energy levels, synaptic function, and protein homeostasis in hereditary spastic paraplegia

“…D5905) and hydrogen peroxidase (H 2 O 2 ) cytochemistry using standard protocols. Samples were contrasted with 1% osmium tetroxide and 2% uranyl acetate before dehydration and embedded in LX-112 or EPON resin using a BioWave tissue processing system (Pelco) as previously described 55 . An enzymatic reaction of HRP with hydrogen peroxidase and DAB substrates yields an insoluble reaction product that becomes electron dense during osmium tetroxide treatment.…”