RyR2-Mediated Ca(2+) 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(2+) signals in neurons. We reported previously that these Ca(2+) signals, which arise from Ca(2+) entry and subsequent amplification by Ca(2+) 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(2+) release, stimulate mitochondrial Ca(2+)-uptake, ROS generation and mitochondrial fragmentation, and also investigated the effects of the antioxidant N-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(2+) release, mitochondrial Ca(2+) uptake and fragmentation. We show here that inhibition of NADPH oxidase type-2 prevented the emergence of RyR-mediated cytoplasmic Ca(2+) signals induced by AβOs in primary hippocampal neurons. Treatment with AβOs promoted mitochondrial Ca(2+) 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(2+)-mediated noxious effects of AβOs on mitochondrial function. Our results also indicate that Ca(2+) 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(2+) release induced by the RyR agonist 4-chloro-m-cresol, and significantly reduced the cytoplasmic and mitochondrial Ca(2+) signals and the mitochondrial fragmentation induced by AβOs. Based on our results, we propose that AβOs-induced Ca(2+) entry and ROS generation jointly stimulate RyR2 activity, causing mitochondrial Ca(2+) overload and fragmentation in a feed forward injurious cycle. The present novel findings highlight the specific participation of RyR2-mediated Ca(2+) release on AβOs-induced mitochondrial malfunction.