Otmakhov Nikolai, Gorbacheva Elena V, Regmi Shaurav, Yasuda Ryohei, Hudmon Andy, Lisman John
Biology Department, Brandeis University, Waltham, Massachusetts, 02454, United States of America.
Max Planck Florida Institute, One Max Planck Way, Jupiter, Florida, 33458, United States of America.
PLoS One. 2015 Mar 20;10(3):e0120881. doi: 10.1371/journal.pone.0120881. eCollection 2015.
Over-activation of excitatory NMDA receptors and the resulting Ca2+ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1-3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca2+ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca2+-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults.
兴奋性N-甲基-D-天冬氨酸（NMDA）受体的过度激活以及由此导致的Ca2+超载是中风期间神经元毒性的主要原因。在此类事件中，钙/钙调蛋白依赖性蛋白激酶II（CaMKII）的调节会出现异常。生化研究表明，中风后CaMKII活性要么急剧丧失，要么持续自主激活，这两个过程都与细胞毒性有关。为了补充生化数据，我们使用CaMKIIα荧光共振能量转移（FRET）传感器Camui，通过高空间/时间分辨率的双光子成像，监测了脑片培养中活海马神经元内CaMKII的激活情况。通过测量Camui荧光寿命来估计CaMKII的激活状态。短暂的NMDA刺激导致Camui激活，随后其蛋白质定位发生重新分布：棘突中增加，树突干中减少，并在细胞体中聚集形成许多簇。Camui激活要么持续（>1 - 3小时），要么短暂（约20分钟），并且一般与其蛋白质重新分布相关。然而，在更长时间的NMDA刺激后，Camui的重新分布比其激活持续的时间更长，这表明这些过程存在不同的调节/阶段。突变和药理学分析表明，Camui的持续激活是由于Ca2+升高时间延长所致，T286自身磷酸化和/或C280/M281氧化产生的自主状态对此影响较小。使用可表达的线粒体标记物线粒体-红色荧光蛋白（mito-dsRed）监测细胞损伤。在Camui激活和聚集后不久，NMDA处理导致线粒体肿胀，肿胀的持续时间暂时与Camui激活的持续时间相关。结果表明，在活神经元中，兴奋性毒性损伤会产生长期的Ca2+依赖性CaMKII活性状态，该状态暂时与细胞损伤相关。然而，由于强烈的聚集，CaMKII的功能受到限制。这项研究首次描述了兴奋性毒性损伤期间活神经元中CaMKII激活动力学的特征。
