title: Molecular components required for resting and stimulated endocytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons.
abstract: Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCV-releasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.
authors: International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors: Jianghui
authors: J
authors: Meng
authors: Jiafu
authors: J
authors: Wang
authors: Gary W
authors: GW
authors: Lawrence
authors: J Oliver
authors: JO
authors: Dolly
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism; D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins / Q000235:genetics / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D002478:Cells, Cultured; D002966:Clathrin / Q000235:genetics / Q000378:metabolism; D034281:Dynamins / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009498:Neurotoxins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism
chemical_list: D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D002966:Clathrin; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D010455:Peptides; D020033:Protein Isoforms; C439124:adaptor protein complex 2, alpha 2 subunit; C096323:rimabotulinumtoxinB; C075637:amphiphysin; D018698:Glutamic Acid; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D034281:Dynamins
keywords: AP2; amphiphysin; clathrin; dynamin; large dense-core vesicles; small clear synaptic vesicles