A new procedure for amyloid beta oligomers preparation enables the unambiguous testing of their effects on cytosolic and mitochondrial Ca2+ entry and cell death in primary neurons

摘要

Oligomers of the amyloid beta peptide (A beta(o)) are becoming the most likely neurotoxin in Alzheimer's disease. Controversy remains on the mechanisms involved in neurotoxicity induced by A beta(o) and the targets involved. We have reported that A beta(o) promote Ca2+ entry, mitochondrial Ca2+ 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 A beta(o) are prepared. Here we have tested the effects of different media on A beta(o) formation and on cytosolic Ca2+ concentration ([Ca2+](cyt)) in rat cerebellar and hippocampal cell cultures. We found that A beta(o) prepared according to previous protocols but solved in alternative media including saline, MEM and DMEM do not allow oligomer formation and fail to increase [Ca2+](cyt). Changes in the oligomerization protocol and supplementation of media with selected salts reported to favor oligomer formation enable A beta(o) formation. A beta(o) prepared by the new procedure and containing small molecular weight oligomers increased [Ca2+](cyt), promoted mitochondrial Ca2+ overload and cell death in cerebellar granule cells and hippocampal neurons. These results foster a role for Ca2+ entry in neurotoxicity induced by A beta(o) and provide a reliable procedure for investigating the Ca2+ entry pathway promoted by A beta(o). (C) 2015 Elsevier Ireland Ltd. All rights reserved.