RyR2-Mediated Ca Release and Mitochondrial ROS Generation Partake in the Synaptic Dysfunction Caused by Amyloid β Peptide Oligomers.

Amyloid β peptide oligomers (AβOs), toxic aggregates with pivotal roles in Alzheimer's disease, trigger persistent and low magnitude Ca signals in neurons. We reported previously that these Ca signals, which arise from Ca entry and subsequent amplification by Ca release through ryanodine receptor (RyR) channels, promote mitochondrial network fragmentation and reduce RyR2 expression. Here, we examined if AβOs, by inducing redox sensitive RyR-mediated Ca release, stimulate mitochondrial Ca-uptake, ROS generation and mitochondrial fragmentation, and also investigated the effects of the antioxidant -acetyl cysteine (NAC) and the mitochondrial antioxidant EUK-134 on AβOs-induced mitochondrial dysfunction. In addition, we studied the contribution of the RyR2 isoform to AβOs-induced Ca release, mitochondrial Ca uptake and fragmentation. We show here that inhibition of NADPH oxidase type-2 prevented the emergence of RyR-mediated cytoplasmic Ca signals induced by AβOs in primary hippocampal neurons. Treatment with AβOs promoted mitochondrial Ca uptake and increased mitochondrial superoxide and hydrogen peroxide levels; ryanodine, at concentrations that suppress RyR activity, prevented these responses. The antioxidants NAC and EUK-134 impeded the mitochondrial ROS increase induced by AβOs. Additionally, EUK-134 prevented the mitochondrial fragmentation induced by AβOs, as previously reported for NAC and ryanodine. These findings show that both antioxidants, NAC and EUK-134, prevented the Ca-mediated noxious effects of AβOs on mitochondrial function. Our results also indicate that Ca release mediated by the RyR2 isoform causes the deleterious effects of AβOs on mitochondrial function. Knockdown of RyR2 with antisense oligonucleotides reduced by about 50% RyR2 mRNA and protein levels in primary hippocampal neurons, decreased by 40% Ca release induced by the RyR agonist 4-chloro-m-cresol, and significantly reduced the cytoplasmic and mitochondrial Ca signals and the mitochondrial fragmentation induced by AβOs. Based on our results, we propose that AβOs-induced Ca entry and ROS generation jointly stimulate RyR2 activity, causing mitochondrial Ca overload and fragmentation in a feed forward injurious cycle. The present novel findings highlight the specific participation of RyR2-mediated Ca release on AβOs-induced mitochondrial malfunction.