title: Brefeldin A sensitive mechanisms contribute to endocytotic membrane retrieval and vesicle recycling in cerebellar granule cells.
abstract: The recycling of synaptic vesicle (SV) proteins and transmitter release occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the adaptor protein 1 and 3 complex (AP-1/AP-3). As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7 days in vitro. We used Brefeldin A (BFA) to inhibit AP-1/AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5 min after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone (AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. Cover Image for this issue: doi. 10.1111/jnc.13801.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.|Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Rampérez
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.|Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Sánchez-Prieto
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.|Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain.
authors.forename: Magdalena
authors.identifier: 0000-0002-3893-8569
authors.initials: M
authors.lastname: Torres
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D020126:Brefeldin A / Q000494:pharmacology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D011992:Endosomes / Q000187:drug effects / Q000648:ultrastructure; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D009474:Neurons / Q000187:drug effects / Q000648:ultrastructure*; D011189:Potassium Chloride / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011726:Pyridinium Compounds / Q000378:metabolism / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000378:metabolism / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar; D013572:Synaptic Vesicles / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D013997:Time Factors; D050598:Vesicular Glutamate Transport Protein 1 / Q000235:genetics / Q000378:metabolism
chemical_list: C073804:FM1 43; C485187:PHluorin; D011500:Protein Synthesis Inhibitors; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D050598:Vesicular Glutamate Transport Protein 1; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D011189:Potassium Chloride
keywords: AP3 complex; Brefeldin A; FM1-43; cerebellar granule cells; endocytosis; synaptic vesicular cycle