Abstract
Amyloid β peptide oligomers (AβOs), toxic aggregates with pivotal roles in Alzheimer’s disease, trigger persistent and low magnitude Ca2+ signals in neurons. We reported previously that these Ca2+ signals, which arise from Ca2+ entry and subsequent amplification by Ca2+ 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 Ca2+ release, stimulate mitochondrial Ca2+-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 Ca2+ release, mitochondrial Ca2+ uptake and fragmentation. We show here that inhibition of NADPH oxidase type-2 prevented the emergence of RyR-mediated cytoplasmic Ca2+ signals induced by AβOs in primary hippocampal neurons. Treatment with AβOs promoted mitochondrial Ca2+ 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 Ca2+-mediated noxious effects of AβOs on mitochondrial function. Our results also indicate that Ca2+ 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% Ca2+ release induced by the RyR agonist 4-chloro-m-cresol, and significantly reduced the cytoplasmic and mitochondrial Ca2+ signals and the mitochondrial fragmentation induced by AβOs. Based on our results, we propose that AβOs-induced Ca2+ entry and ROS generation jointly stimulate RyR2 activity, causing mitochondrial Ca2+ overload and fragmentation in a feed forward injurious cycle. The present novel findings highlight the specific participation of RyR2-mediated Ca2+ release on AβOs-induced mitochondrial malfunction.
Highlights
Alzheimer’s disease (AD) is currently considered a Ca2+-driven pathology (Berridge, 2013; Area-Gomez and Schon, 2017; Frazier et al, 2017; Popugaeva et al, 2017)
We suggest that amyloid β peptide oligomers (AβOs) stimulate NADPH oxidase type-2 (NOX2) activity, presumably via NMDA receptor stimulation (Brennan et al, 2009), and that the increased Ca2+ and reactive oxygen species (ROS) levels induced by AβOs jointly stimulate ryanodine receptor (RyR)-mediated Ca2+ release
We describe here novel findings highlighting the key role of the RyR2 isoform in the mitochondrial dysfunctions induced by acute AβOs treatment
Summary
Alzheimer’s disease (AD) is currently considered a Ca2+-driven pathology (Berridge, 2013; Area-Gomez and Schon, 2017; Frazier et al, 2017; Popugaeva et al, 2017). Primary hippocampal neurons from mice carrying a mutation in the amyloid precursor protein (APP) display increased intracellular Ca2+ levels (Koizumi et al, 1998). We reported previously that amyloid β peptide oligomers (AβOs) induce anomalous Ca2+ signals in primary hippocampal neurons; these signals arise initially from Ca2+ entry through N-Methyl-D-aspartate (NMDA) receptors and are subsequently amplified via RyR channels co-stimulated by Ca2+ entry signals and the increased ROS levels produced by AβOs (Paula-Lima et al, 2011; SanMartín et al, 2012a). The redox-sensitive abnormal Ca2+ signals elicited by AβOs significantly decrease RyR2 expression levels in hippocampal neurons (Paula-Lima et al, 2011; Lobos et al, 2016). It becomes important to investigate whether the RyR2 isoform is involved in the alterations in intracellular Ca2+ signaling and homeostasis induced by AβOs in hippocampal neurons
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