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

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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.