Programmed Cell Death In The Nervous System

我们研究了神经系统中的程序性细胞死亡和细胞凋亡的生化机制。最近，我们重点研究了在发育过程中调节神经元存活的Bcl-2蛋白家族。这个家族中的一个成员Bax是神经元正常死亡所必需的，而Bax基因的消除会导致成年动物中过多的神经元。Bax和其他Bcl-2家族成员如何调控细胞生存尚不清楚。我们研制了一系列抗Bcl2、BclxL和Bax的单抗，以探讨它们在体内外的作用机制。抗体显示Bax在细胞凋亡过程中从胞浆迁移到细胞膜。为了探索Bax、Bcl2和Bclxl在神经元中的运动，绿色荧光蛋白被用来标记和跟踪活细胞中的蛋白。我们发现Bax和Bclxl从胞浆移动到线粒体是其细胞凋亡控制机制中的一个重要步骤。Bax插入线粒体较好的点突变株毒性较大，线粒体插入较少的突变株毒性较小，表明Bax的C末端调节线粒体的结合。因此，我们确定了Bax的三维结构，并发现C-末端的尾巴适合于一个疏水口袋，该口袋被认为调节了Bcl-2家族成员之间异二聚体和同源二聚体的形成。这表明了一种新的模型，即二聚体的形成和线粒体的对接在结构上与C末端的解离有关。与Bax不同的是，Bclxl可防止因多种神经毒性损伤而导致的神经细胞死亡。与Bax一样，在细胞凋亡过程中，Bclxl也会插入线粒体。我们发现了一个控制Bclxl亚细胞定位的调控步骤。Bad是一个促凋亡的Bcl2家族成员，它与Bclxl结合并触发其与线粒体的对接。Bad与Bclxl的结合不良是由磷酸化和几种神经营养因子调节的，这些神经营养因子刺激激酶，阻止Bad与Bclxl结合，从而防止神经元死亡。我们还发现，BclxL通过增加Bax的逆转位回到胞浆，从而抑制Bax在线粒体上的积聚和细胞凋亡。此外，还研究了Bcl2家族成员与线粒体结合的后果。Bax在线粒体表面结合成大的聚集体，与线粒体分裂机制共定位，可以激活线粒体分裂，因此，管理线粒体分裂的细胞机制参与了细胞凋亡。目前的项目探索在大的Bax焦点处的线粒体膜的结构，该结构介导了分裂和细胞色素c的释放。

We have studied programmed cell death in the nervous system and the biochemical mechanism of apoptosis. Recently we have focused on the Bcl-2 family of proteins that regulates the survival of neurons during development. One member of this family, Bax, is required for the normal death of neurons and elimination of the Bax gene results in excess neurons in adult animals. How Bax and other Bcl-2 family members regulate cell survival is unknown. We developed a series of monoclonal antibodies against Bcl-2, Bcl-xl, and Bax to probe their mechanism of action in vitro and in vivo. The antibodies revealed that Bax migrates from the cytosol to cell membranes during apoptosis. To explore Bax, Bcl-2 and Bcl-xl movement in neurons the green fluorescent protein has been used to tag and follow the proteins in living cells. We discovered that Bax and Bcl-xl move from the cytosol to the mitochondria as an essential step in their mechanism of apoptosis control. Point mutants of Bax that insert better into mitochondria are more toxic and mutants with less mitochondrial insertion are less toxic indicating that the C-terminus of Bax regulates the mitochondrial association. Therefore, we determined the three dimensional structure of Bax and found that the C-terminal tail fits into a hydrophobic pocket that has been thought to regulate hetero- and homo-dimer formation among Bcl-2 family members. This suggests a new model that dimer formation and mitochondrial docking are structurally linked to disengagement of the C-terminus. In contrast to Bax, Bcl-xl prevents neuron cell death due to many neurotoxic insults. Like Bax, Bcl-xl inserts into mitochondria during apoptosis. We have found one regulatory step that controls Bcl-xl subcellular location. Bad, a pro-apoptotic Bcl-2 family member, binds to Bcl-xl and triggers its docking to mitochondria. Bad binding to Bcl-xl is in turn regulated by phosphorylation and several neurotrophic factors that stimulate kinases prevent Bad from binding to Bcl-xl and thus prevent neuron death. We also found that Bcl-xL inhibits Bax accumulation on mitochondria and apoptosis by increasing the retrotranslocation of Bax back into the cytosol. The consequences of Bcl-2 family member binding to mitochondria have been also studied. Bax coalesces into large aggregates on the mitochondrial surface that co-localize with machinery for mitochondrial division and can activate mitochondrial division.Thus, the cell machinery that governs mitochondrial division participates in apoptosis. Current projects explore the structure of the mitochondrial membranes at the large Bax foci that mediate fission and cytochrome c release.