Mitochondrial ion channels in hypoxic neurons

描述（由申请人提供）：卒中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。脑细胞营养和氧气供应的减少导致最严重受影响的细胞立即死亡，并在缺血首次发作时处于危险中的一些细胞中延迟细胞死亡。后一种形式的神经元死亡是由BCL-2家族蛋白协调的，BCL-2家族蛋白也在神经系统发育期间调节细胞死亡。BCL-2家族蛋白存在于线粒体膜中，并且响应于许多死亡刺激（包括低浓度的细胞氧）而被激活以形成大电导离子通道活性。通道活性与细胞死亡开始的关系至今知之甚少。我们已经开发了膜片钳技术来记录活神经元内的线粒体离子通道活性，并研究从脑中分离的线粒体中BCL-2家族蛋白的离子通道活性。在比较重组BCL-2家族蛋白的通道活性与内源活性时，我们发现重组N-截短形式的BCL-xL，（？N BCL-xL），其与哺乳动物脑中短暂性全脑缺血期间出现的内源性线粒体通道具有生物物理学和药理学相似性。通道活动的开始？NBCL-XL可能引发一系列分子事件，导致海马脆弱的CA 1神经元选择性延迟细胞死亡。我们假设，抑制？BCL-xL通道活性与BCL-xL的特异性抑制剂ABT-737，将阻断缺血后这些敏感神经元中的细胞死亡。此外，如果全长BCL-xL的蛋白水解加工是细胞死亡所必需的，那么缺乏BCL-xL的可裂解形式的小鼠的海马神经元可能无法形成？N BCL-xL，缺血损伤后不死亡。这些研究将检验这样一个假设：NBCL-XL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，以期制定对抗中风的策略。公共卫生相关性：中风是美国发病率和死亡率的主要原因。我们已经发现，亲和抗死亡BCL-2蛋白家族的线粒体离子通道活性有助于神经元的生理和病理功能。我们假设在脑缺血的情况下，这种通道活性导致脑中的细胞死亡，并且这种死亡可以通过BCL-2诱导的线粒体离子通道的药理学抑制剂来预防。

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.