The role of Bcl-2 family proteins in developmental neurogenesis.

哺乳动物拥有进化上最先进的神经系统，其发育受到细胞增殖、生存和死亡的严格调控。我的研究计划的长期目标是了解Bcl-2家族在哺乳动物神经系统发育中的作用。Bcl-2家族的典型作用是调节细胞凋亡，这是一种活跃的细胞死亡形式。然而，最近Bcl-2家族成员已被发现在线粒体动力学、自噬、钙调节和细胞周期等不同于凋亡的作用中发挥作用。在短期内，我建议证明Bcl-2蛋白、Mcl-1和Bcl-xL的抗凋亡作用与发育性神经发生过程中细胞周期调节的新作用有关。***神经系统起源于神经前体细胞（NPCs），这是一种由神经干细胞和祖细胞组成的多样化增殖细胞群。在神经发生过程中，npc退出细胞周期并分化为有丝分裂后的神经元。细胞周期失调导致细胞凋亡死亡。抗凋亡蛋白Mcl-1的表达在细胞周期退出或再进入之前的m期达到峰值，而抗凋亡蛋白Bcl-xL的表达在有丝分裂后的神经元达到峰值。在我们的神经系统特异性条件敲除小鼠中，Mcl-1或Bcl-xL的表达在胚胎7.5天（E7.5）时在神经干细胞中丢失，然而，直到E10开始神经发生时才观察到凋亡。Mcl-1基因敲除导致鼻咽癌分化过程中的细胞凋亡，而Bcl-xL基因敲除导致未成熟神经元神经发生末期的细胞凋亡。这表明Mcl-1和Bcl-xL不是NPC增殖生存所必需的，但在NPC分化时是必需的。相反，在npc中过表达Mcl-1或Bcl-xL可诱导细胞周期提前退出和分化。我的假设是Mcl-1和Bcl-xL具有双重作用，既促进细胞周期调节，又促进细胞存活，并且这些作用是相互关联的。具体来说，m期需要Mcl-1来促进NPC细胞周期退出，一旦细胞退出，则需要Bcl-xL来防止重新进入细胞周期。为了进一步表征这些相互关联的过程，我建议进行三组实验：1)确定Mcl-1如何促进NPC细胞周期退出和存活；2)确定Bcl-xL如何促进未成熟神经元的分化和细胞存活；3)确定Mcl-1和Bcl-xL在神经发生过程中的表达调控。***虽然已知Mcl-1和Bcl-xL是增殖癌细胞的细胞周期调节因子，但本研究将证明Bcl-2蛋白在细胞周期调节中的作用与其在细胞凋亡中的作用是如何相互关联的。本研究项目的发现将为Bcl-2蛋白在发育性神经发生中的生理作用提供重要的新见解。

Mammals have the most evolutionally advanced nervous system and its development is tightly regulated by cell proliferation, survival and death. The long term goal of my research program is to understand the roles of the Bcl-2 family in development of the mammalian nervous system. The canonical role of the Bcl-2 family is to regulate apoptosis, an active form of cell death. Recently however, Bcl-2 family members have been identified in roles distinct from apoptosis including mitochondrial dynamics, autophagy, calcium regulation and the cell cycle. In the short term, I propose to demonstrate that the anti-apoptotic roles of Bcl-2 proteins, Mcl-1 and Bcl-xL are linked to novel roles in cell cycle regulation during developmental neurogenesis. ***The nervous system arises from neural precursor cells (NPCs), a diverse proliferating cell population consisting of neural stem cells and progenitor cells. During neurogenesis, NPCs exit the cell cycle and differentiate into post mitotic neurons. Dysregulation of the cell cycle defaults to an apoptotic cell death. Expression of anti-apoptotic protein Mcl-1 peaks during M-phase prior to cell cycle exit or re-entry, while expression of anti-apoptotic protein Bcl-xL peaks in post mitotic neurons. In our nervous system-specific conditional knockout mice, expression of Mcl-1 or Bcl-xL is lost by embryonic day 7.5 (E7.5) in neural stem cells, however, apoptosis is not observed until the onset of neurogenesis at E10. Gene knockout of Mcl-1 results in apoptosis during NPC differentiation, whereas knockout of Bcl-xL causes apoptosis at the end of neurogenesis in immature neurons. This shows that Mcl-1 and Bcl-xL are not required for proliferating NPC survival but are required when NPCs differentiate. In contrast, overexpression of either Mcl-1 or Bcl-xL in NPCs induces premature cell cycle exit and differentiation. My hypothesis is that Mcl-1 and Bcl-xL have dual roles, promoting cell cycle regulation as well as cell survival, and these roles are inter-connected. Specifically, Mcl-1 is required during M-phase to promote NPC cell cycle exit and, once cells have exited, Bcl-xL is required to prevent re-entry into the cell cycle. To further characterize these interconnected processes, I propose to undertake three sets of experiments to: 1) identify how Mcl-1 promotes NPC cell cycle exit and survival; 2) identify how Bcl-xL promotes differentiation and cell survival in immature neurons; and 3) determine how Mcl-1 and Bcl-xL expression is regulated in neurogenesis.***Although Mcl-1 and Bcl-xL are known cell cycle regulators of proliferating cancer cells, this study will demonstrate how the roles of Bcl-2 proteins in cell cycle regulation are interconnected with their roles in apoptosis. The findings from this research program will provide important new insights into the physiological roles of Bcl-2 proteins during developmental neurogenesis.