Study of bcl-2 function in neuronal cells

Bcl-2是一种独特的癌基因，可防止细胞凋亡。本研究的目的是阐明bcl-2在神经元细胞中的功能，我们通过显微注射的方法证明bcl-2可以保护原代培养的大鼠交感神经元免于神经生长因子耗竭引起的细胞死亡。我们在大鼠嗜铬细胞瘤细胞系PC 12中也观察到过表达bcl-2基因能有效地挽救血清耗竭诱导的细胞死亡。这些数据表明bcl-2能够在神经元细胞以及淋巴样细胞中发挥其免于死亡的活性。本研究成功地建立了bcl-2基因高表达的转基因小鼠，为bcl-2基因在神经元中的功能研究提供了第一个证据，为神经退行性疾病的研究提供了新的思路。为了阐明bcl-2在神经细胞中正常表达的作用，制备了bcl-2基因纯合缺失小鼠。尽管神经元组织表现正常，但小鼠显示出多效性异常，这表明存在补偿bcl-2缺陷的基因。尽管bcl-2功能的生化基础仍然未知，但已经提出并广泛接受了一种机制，即涉及bcl-2对活性氧（ROS）的活性，我们发现Bcl-2的表达阻止了由氧的缺乏（缺氧）诱导的细胞死亡，氧的缺乏显著地减少了氧自由基的净形成。此外，无论ROS清除剂还是ROS清除剂的抑制剂都不影响细胞死亡，无论bcl-2的表达如何。因此，我们的数据表明，bcl-2发挥抗细胞死亡功能的机制，而不是通过ROS活性的调节。

Bcl-2 is a unique oncogene that prevents apoptotic cell death. The specific aim supported by this grant was to elucidate the bcl-2 function in neuronal cells.We have shown by a microinjection procedure that bcl-2 protects rat sympathetic neurons in a primary culture from cell death induced by nerve growth factor-depletion. We have obtained a similar observation that a rat phechromocytoma cell line, PC12 was efficinetly rescued from cell death induced by serum depletion by overexpression of human bcl-2 gene. These data indicate that bcl-2 is able to exert its death-sparing activity in neuronal cells as well as lymphoid cells. We have generated the transgenic mice expressing high levels of bcl-2 in neuronal cells in which facial motor neurons did not degenerate after axotomy, providing the first evidence for in vivo function of bcl-2 in neurons and suggesting that the mice will be very useful for study of neurodegenarative disorders. To elucidate roles of bcl-2 normally expressed in neuronal cells, the mice homozygouly deleted for bcl-2 gene were generated. The mice revealed pleiotropic abnormalities although the neuronal tissues appeared normal, suggesting the presence of gene (s) compensating the bcl-2 deficiency.Although the biochemical basis of the bcl-2 fucntion remains unknown, a mechanism has been proposed and widely accepted, that involves bcl-2 activity on reactive oxygen species (ROS), we find that expression of bcl-2 prevents cell death induced by withdrawal of oxygen (anoxia), which drastically decreases the net formation of oxygen free radicals. Furthermore, neither ROS scavenger nor inhibitor of ROS scavenger affects the cell death, regardless of the expression of bcl-2. Thus, our data suggest that bcl-2 exerts an anti-cell death function by a mechanism other than through regulation of ROS activity.