Mitochondrial ion channels in hypoxic neurons

描述(由申请人提供)：中风是美国发病率和死亡率的主要原因，对疾病预防策略的制定提出了挑战。剥夺脑细胞的营养和氧气供应会导致受影响最严重的细胞立即死亡，并推迟一些在首次缺血时处于危险中的细胞的死亡。后一种形式的神经元死亡是由bcl2家族蛋白调控的，bcl2家族蛋白也调节神经系统发育过程中的细胞死亡。BCL-2家族蛋白存在于线粒体膜上，在包括低浓度细胞氧在内的多种死亡刺激下被激活，形成大电导离子通道活性。通道活性与细胞死亡之间的关系目前尚不清楚。我们发展了膜片钳技术来记录活神经元内线粒体的离子通道活性，并研究了从脑分离的线粒体中bcl2家族蛋白的离子通道活性。在比较重组bcl2家族蛋白的通道活性和内源性活性时，我们发现重组的N-截短型bclxl(？n bclxl)是在死亡刺激下对全长bclxl进行急性蛋白分解而产生的，它与哺乳动物脑内短暂性全脑缺血时出现的内源性线粒体通道具有生物物理和药理学上的相似之处。？n bclxl通道活动的启动可能引发一系列分子事件，导致脆弱的海马区CA1神经元选择性、延迟性死亡。我们推测，用bclxl的特异性抑制剂ABT-737抑制bclxl通道的活性，可以阻止这些敏感神经元在缺血后的细胞死亡。此外，如果全长bclxl的蛋白水解性处理是细胞死亡所必需的，那么缺乏可切割形式bclxl的小鼠的海马神经元可能不能形成bclxl，也不能在缺血损伤后死亡。这些研究将检验这一假设，即bclxl是海马神经元细胞死亡的关键调节因子。此外，这些研究将试图阐明缺血性脑损伤后神经元细胞死亡的分子机制，以期开发抗击中风的策略。公共卫生相关性：中风是美国发病率和死亡率的主要原因。我们发现，促死亡和抗死亡bcl2蛋白家族的线粒体离子通道活性参与了神经元的生理和病理功能。我们假设，在脑缺血的背景下，这种通道活动导致脑细胞死亡，这种死亡可以通过bcl2诱导的线粒体离子通道的药物抑制剂来预防。

DESCRIPTION (provided by applicant): Stroke is a major cause of morbidity and mortality in the United States and presents challenges in the development of disease prevention strategies. Deprivation of nutrients and oxygen supply to brain cells produces immediate death in the most severely affected cells and delayed cell death in some cells that are placed at risk at the first onset of ischemia. The latter form of neuronal death is orchestrated by BCL-2 family proteins, which also regulate cell death during nervous system development. BCL-2 family proteins are present in mitochondrial membranes and become activated to form large conductance ion channel activity in response to many death stimuli, including a low concentration of cellular oxygen. The relationship of the channel activity to the onset of cell death is as yet poorly understood. We have developed patch clamp techniques to record mitochondrial ion channel activity within living neurons and to study the ion channel activity of BCL-2 family proteins in mitochondria isolated from brain. In comparing the channel activity of recombinant BCL-2 family proteins to endogenous activity, we find that the recombinant N-truncated form of BCL-xL, (?N BCL-xL) that is produced by acute proteolytic processing of full length BCL-xL in response to death stimuli, has biophysical and pharmacological similarities to an endogenous mitochondrial channel that appears during transient global ischemia in mammalian brain. The onset of channel activity of ?N BCL-xL may initiate a series of molecular events that leads to selective, delayed cell death in vulnerable CA1 neurons of the hippocampus. We hypothesize that inhibition of the ?N BCL-xL channel activity with the specific inhibitor of BCL-xL, ABT-737, will block cell death in these sensitive neurons after ischemia. Furthermore, if proteolytic processing of full length BCL-xL is necessary for cell death, then hippocampal neurons of a mouse that lacks a cleavable form of BCL-xL may fail to form ?N BCL-xL and fail to die after ischemic insult. These studies will test the hypothesis that ?N BCL-xL is the key regulator of cell death in hippocampal neurons. In addition, these studies will attempt to elucidate the molecular mechanisms underlying cell death in neurons after ischemic brain injury with an eye to developing strategies to combat stroke. PUBLIC HEALTH RELEVANCE: Stroke is a major cause of morbidity and mortality in the United States. We have found that mitochondrial ion channel activity of the pro- and anti-death BCL-2 protein family contributes to the physiological and pathological function of neurons. We hypothesize that in the setting of brain ischemia, such channel activity leads to cell death in the brain and that this death can be prevented by a pharmacological inhibitor of BCL-2-induced mitochondrial ion channels.