title: A new procedure for amyloid β oligomers preparation enables the unambiguous testing of their effects on cytosolic and mitochondrial Ca(2+) entry and cell death in primary neurons.
abstract: Oligomers of the amyloid β peptide (Aβo) are becoming the most likely neurotoxin in Alzheimer's disease. Controversy remains on the mechanisms involved in neurotoxicity induced by Aβo and the targets involved. We have reported that Aβo promote Ca(2+) entry, mitochondrial Ca(2+) overload and apoptosis in cultured cerebellar neurons. However, recent evidence suggests that some of these effects could be induced by glutamate receptor agonists solved in F12, the media in which Aβo are prepared. Here we have tested the effects of different media on Aβo formation and on cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in rat cerebellar and hippocampal cell cultures. We found that Aβo prepared according to previous protocols but solved in alternative media including saline, MEM and DMEM do not allow oligomer formation and fail to increase [Ca(2+)]cyt. Changes in the oligomerization protocol and supplementation of media with selected salts reported to favor oligomer formation enable Aβo formation. Aβo prepared by the new procedure and containing small molecular weight oligomers increased [Ca(2+)]cyt, promoted mitochondrial Ca(2+) overload and cell death in cerebellar granule cells and hippocampal neurons. These results foster a role for Ca(2+) entry in neurotoxicity induced by Aβo and provide a reliable procedure for investigating the Ca(2+) entry pathway promoted by Aβo.
authors.affiliation: Institute of Molecular Biology and Genetics (IBGM), Spanish National Research Council (CSIC), Valladolid, Spain.
authors.forename: Erica
authors.initials: E
authors.lastname: Caballero
authors.affiliation: Institute of Molecular Biology and Genetics (IBGM), Spanish National Research Council (CSIC), Valladolid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Calvo-Rodríguez
authors.affiliation: Laboratory of Neuroanatomy, Institute of Neurosciences of Castilla y León, University of Valladolid (Soria Campus), Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Gonzalo-Ruiz
authors.affiliation: Institute of Molecular Biology and Genetics (IBGM), Spanish National Research Council (CSIC), Valladolid, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Villalobos
authors.affiliation: Institute of Molecular Biology and Genetics (IBGM), Spanish National Research Council (CSIC), Valladolid, Spain; Department of Biochemistry and Molecular Biology and Physiology, School of Medicine, University of Valladolid, Spain. Electronic address: nunezl@ibgm.uva.es.
authors.forename: Lucía
authors.initials: L
authors.lastname: Núñez
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D003470:Culture Media; D003600:Cytosol / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology; D017208:Rats, Wistar
chemical_list: D016229:Amyloid beta-Peptides; D003470:Culture Media; D010446:Peptide Fragments; C075222:amyloid beta-protein (1-42); D002118:Calcium
keywords: Alzheimer’s disease; Amyloid β peptide; Calcium; Mitochondria; Oligomers