title: Reduction of AP180 and CALM produces defects in synaptic vesicle size and density.
abstract: Clathrin assembly proteins AP180 and CALM regulate the assembly of clathrin-coated vesicles (CCVs), which mediate diverse intracellular trafficking processes, including synaptic vesicle (SV) recycling at the synapse. Although studies using several invertebrate model systems have indicated a role for AP180 in SV recycling, less is known about AP180's or CALM's function in the synapse of mammalian neurons. In this study, we examined synapses of rat hippocampal neurons in which the level of AP180 or CALM had been reduced by RNA interference (RNAi). Using light microscopy, we visualized synaptic puncta in these AP180- or CALM-reduced neurons by co-expressing Synaptophysin::EGFP (Syp::EGFP). We found that neurons with reduced AP180 or reduced CALM had smaller Syp::EGFP-illuminated puncta. Using electron microscopy, we further examined the ultrastructure of the AP180- or CALM-reduced presynaptic terminals. We found that SVs became variably enlarged in both the AP180-reduced and CALM-reduced presynaptic terminals. Lower AP180 and CALM also reduced the density of SVs and the size of SV clusters. Our findings demonstrate that in the presynaptic terminals of hippocampal neurons, AP180 and CALM have a similar role in regulating synaptic vesicles. This overlapping activity may be necessary for high-precision and high-efficacy SV formation during endocytosis.
authors.affiliation: Advanced Imaging Core, NIDCD/NIH, Bethesda, MD 20892, USA.
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Petralia
authors.forename: Ya-Xian
authors.initials: YX
authors.lastname: Wang
authors.forename: Fred E
authors.initials: FE
authors.lastname: Indig
authors.forename: Ittai
authors.initials: I
authors.lastname: Bushlin
authors.forename: Fangbai
authors.initials: F
authors.lastname: Wu
authors.forename: Mark P
authors.initials: MP
authors.lastname: Mattson
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Yao
mesh_terms: D000818:Animals; D002478:Cells, Cultured / Q000648:ultrastructure; D022163:Clathrin-Coated Vesicles / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006624:Hippocampus / Q000166:cytology; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D034041:Monomeric Clathrin Assembly Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000502:physiology*; D017729:Presynaptic Terminals / Q000648:ultrastructure; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure*; D016708:Synaptophysin / Q000235:genetics; D014162:Transfection
chemical_list: D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; C499980:Picalm protein, rat; D034741:RNA, Small Interfering; D016708:Synaptophysin; C569016:Syp protein, rat; C439023:clathrin assembly protein AP180; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins