D'Orsi Beatrice, Kilbride Seán M, Chen Gang, Perez Alvarez Sergio, Bonner Helena P, Pfeiffer Shona, Plesnila Nikolaus, Engel Tobias, Henshall David C, Düssmann Heiko, Prehn Jochen H M
Department of Physiology and Medical Physics, Centre for the Study of Neurological Disorders and 3U-COEN, Royal College of Surgeons in Ireland, Dublin 2, Ireland; and.
Department of Physiology and Medical Physics, Centre for the Study of Neurological Disorders and 3U-COEN, Royal College of Surgeons in Ireland, Dublin 2, Ireland; and Institute for Stroke and Dementia Research, DZNE, Groβhadern, D-81377 Munich, Germany.
J Neurosci. 2015 Jan 28;35(4):1706-22. doi: 10.1523/JNEUROSCI.2453-14.2015.
Excessive Ca(2+) entry during glutamate receptor overactivation ("excitotoxicity") induces acute or delayed neuronal death. We report here that deficiency in bax exerted broad neuroprotection against excitotoxic injury and oxygen/glucose deprivation in mouse neocortical neuron cultures and reduced infarct size, necrotic injury, and cerebral edema formation after middle cerebral artery occlusion in mice. Neuronal Ca(2+) and mitochondrial membrane potential (Δψm) analysis during excitotoxic injury revealed that bax-deficient neurons showed significantly reduced Ca(2+) transients during the NMDA excitation period and did not exhibit the deregulation of Δψm that was observed in their wild-type (WT) counterparts. Reintroduction of bax or a bax mutant incapable of proapoptotic oligomerization equally restored neuronal Ca(2+) dynamics during NMDA excitation, suggesting that Bax controlled Ca(2+) signaling independently of its role in apoptosis execution. Quantitative confocal imaging of intracellular ATP or mitochondrial Ca(2+) levels using FRET-based sensors indicated that the effects of bax deficiency on Ca(2+) handling were not due to enhanced cellular bioenergetics or increased Ca(2+) uptake into mitochondria. We also observed that mitochondria isolated from WT or bax-deficient cells similarly underwent Ca(2+)-induced permeability transition. However, when Ca(2+) uptake into the sarco/endoplasmic reticulum was blocked with the Ca(2+)-ATPase inhibitor thapsigargin, bax-deficient neurons showed strongly elevated cytosolic Ca(2+) levels during NMDA excitation, suggesting that the ability of Bax to support dynamic ER Ca(2+) handling is critical for cell death signaling during periods of neuronal overexcitation.
在谷氨酸受体过度激活（“兴奋毒性”）过程中，过量的Ca(2+)内流会导致急性或延迟性神经元死亡。我们在此报告，bax基因缺失对小鼠新皮质神经元培养物中的兴奋毒性损伤和氧/葡萄糖剥夺具有广泛的神经保护作用，并可减小小鼠大脑中动脉闭塞后的梗死面积、坏死性损伤和脑水肿形成。对兴奋毒性损伤期间神经元Ca(2+)和线粒体膜电位（Δψm）的分析表明，bax基因缺失的神经元在NMDA兴奋期的Ca(2+)瞬变显著减少，并且未表现出在野生型（WT）对应神经元中观察到的Δψm失调。重新引入bax或一种无法进行促凋亡寡聚化的bax突变体，同样能在NMDA兴奋期间恢复神经元Ca(2+)动力学，这表明Bax独立于其在凋亡执行中的作用来控制Ca(2+)信号传导。使用基于FRET的传感器对细胞内ATP或线粒体Ca(2+)水平进行定量共聚焦成像表明，bax基因缺失对Ca(2+)处理的影响并非由于细胞生物能量学增强或线粒体Ca(2+)摄取增加。我们还观察到，从WT或bax基因缺失细胞中分离的线粒体同样会发生Ca(2+)诱导的通透性转变。然而，当用Ca(2+)-ATP酶抑制剂毒胡萝卜素阻断Ca(2+)摄取到肌浆网/内质网时，bax基因缺失的神经元在NMDA兴奋期间表现出胞质Ca(2+)水平大幅升高，这表明Bax支持内质网Ca(2+)动态处理的能力对于神经元过度兴奋期间的细胞死亡信号传导至关重要。
