SanMartín Carol D, Veloso Pablo, Adasme Tatiana, Lobos Pedro, Bruna Barbara, Galaz Jose, García Alejandra, Hartel Steffen, Hidalgo Cecilia, Paula-Lima Andrea C
Department of de Neurology and Neurosurgery, Clinical Hospital Universidad de ChileSantiago, Chile.
Biomedical Neuroscience Institute, Faculty of Medicine, Universidad de ChileSantiago, Chile.
Front Mol Neurosci. 2017 Apr 25;10:115. doi: 10.3389/fnmol.2017.00115. eCollection 2017.
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.
淀粉样β肽寡聚体（AβOs）是在阿尔茨海默病中起关键作用的有毒聚集体，可在神经元中引发持续且微弱的钙信号。我们之前报道过，这些钙信号源于钙内流以及随后通过兰尼碱受体（RyR）通道的钙释放所导致的钙信号放大，会促使线粒体网络碎片化并降低RyR2的表达。在此，我们研究了AβOs是否通过诱导对氧化还原敏感的RyR介导的钙释放，刺激线粒体钙摄取、活性氧生成和线粒体碎片化，还研究了抗氧化剂N - 乙酰半胱氨酸（NAC）和线粒体抗氧化剂EUK - 134对AβOs诱导的线粒体功能障碍的影响。此外，我们研究了RyR2亚型对AβOs诱导的钙释放、线粒体钙摄取和碎片化的作用。我们在此表明，抑制2型NADPH氧化酶可防止原代海马神经元中由AβOs诱导的RyR介导的细胞质钙信号的出现。用AβOs处理可促进线粒体钙摄取并增加线粒体超氧化物和过氧化氢水平；抑制RyR活性的浓度的兰尼碱可阻止这些反应。抗氧化剂NAC和EUK - 134可抑制AβOs诱导的线粒体活性氧增加。此外，正如之前报道的NAC和兰尼碱一样，EUK - 134可防止AβOs诱导的线粒体碎片化。这些发现表明，抗氧化剂NAC和EUK - 134均可防止AβOs对线粒体功能的钙介导的有害影响。我们的结果还表明，由RyR2亚型介导的钙释放会导致AβOs对线粒体功能产生有害影响。用反义寡核苷酸敲低RyR2可使原代海马神经元中RyR2的mRNA和蛋白质水平降低约50%，使RyR激动剂4 - 氯间甲酚诱导的钙释放减少40%，并显著降低AβOs诱导的细胞质和线粒体钙信号以及线粒体碎片化。基于我们的结果，我们提出AβOs诱导的钙内流和活性氧生成共同刺激RyR2活性，在一个前馈性损伤循环中导致线粒体钙超载和碎片化。目前这些新发现突出了RyR2介导的钙释放在AβOs诱导的线粒体功能障碍中的特定作用。
