Neurobiological Basis of Neuronal Survival

在本财政年度，我们发现内质网线粒体界面对于增加细胞生物能量是重要的，通过利用抗凋亡蛋白Bcl-xL来增强内质网和线粒体之间的Ca2+信号。简介如下。Bcl-2家族出现了与细胞定位相关的非凋亡功能。特别是Bcl-xL，已经被假设通过调节内质网的Ca2+信号来刺激生物能量学。然而，Bcl-xL特异性地通过3型肌醇1,4,5-三磷酸受体（IP3R3）直接ER到线粒体Ca2+转移的功能尚未被研究过。在这里，我们发现CHO细胞中相当一部分Bcl-xL以簇状形式存在于ER-线粒体界面或MAM（线粒体相关ER膜），在那里它与IP3R3相互作用以增强线粒体Ca2+信号传导并改变生物能量学。特别是，Bcl-xL增强了线粒体Ca2+瞬态在thapsigargin （Tg）诱导的ER Ca2+耗竭，同时减少细胞质Ca2+释放。结果表明，Bcl-xL过表达细胞的TCA循环活性增强，延长Tg损伤后，TCA循环活性进一步增强。这种作用是Bcl-xL易位到MAM的结果，这是已知的首次在非凋亡应激事件中观察到Bcl-xL易位。我们的研究结果表明，MAM是一个关键的细胞信号连接，Bcl-xL可能已经进化到包括多种功能。一个作用是促进细胞代谢，促进正常细胞功能，第二个任务是通过增加细胞的生物能量能力来防止细胞凋亡的开始。

In this fiscal year we have found that the ER-mitochondrion interface is important for increasing cellular bioenergetics by utilizing an anti-apoptotic protein Bcl-xL to enhancing the Ca2+ signaling between the ER and mitochondria. The synopsis is as follows. Non-apoptotic functions related to cellular localization are emerging for the Bcl-2 family. Bcl-xL, in particular, has been hypothesized to stimulate bioenergetics by regulating Ca2+ signaling at the ER. However, the functioning of Bcl-xL specifically in direct ER to mitochondrial Ca2+ transfer, through type 3 inositol 1,4,5-trisphosphate receptors (IP3R3), has not previously been investigated. Here we found a significant fraction of Bcl-xL in CHO cells exists in clustered form at the ER-mitochondrion interface or MAM (mitochondria-associated ER membrane) where it interacts with IP3R3 to enhance mitochondrial Ca2+ signaling and alter bioenergetics. Particularly, Bcl-xL enhanced mitochondrial Ca2+ transients upon thapsigargin (Tg)-induced ER Ca2+ depletion, while concomitantly reducing cytosolic Ca2+ release. Consequently, enhanced TCA cycle activity was observed in Bcl-xL overexpressing cells, which was further increased after prolonged Tg insult. This action is the result of Bcl-xL translocation to the MAM, which is the first known observation of Bcl-xL translocating during a non-apoptotic stress event. Our results suggest the MAM is a critical cell-signaling junction whereby Bcl-xL might have evolved to include multiple functionalities. One role being to facilitate cellular metabolism to promote normal cell functioning, and the second task being to prevent the initiation of apoptosis by increasing the bioenergetics capacity of the cell.