Abstract
Developing sympathetic neurons die by apoptosis when deprived of NGF. BIM, a BH3-only member of the BCL-2 family, is induced after NGF withdrawal in these cells and contributes to NGF withdrawal–induced death. Here, we have investigated the involvement of the Forkhead box, class O (FOXO) subfamily of Forkhead transcription factors in the regulation of BIM expression by NGF. We find that overexpression of FOXO transcription factors induces BIM expression and promotes death of sympathetic neurons in a BIM-dependent manner. In addition, we find that FKHRL1 (FOXO3a) directly activates the bim promoter via two conserved FOXO binding sites and that mutation of these sites abolishes bim promoter activation after NGF withdrawal. Finally, we show that FOXO activity contributes to the NGF deprivation–induced death of sympathetic neurons.
Highlights
Programmed cell death (PCD) accounts for the death of approximately half of all neurons generated during embryogenesis and is essential for the correct innervation of target tissues and formation of neuronal networks during neural development (Oppenheim, 1991; Yuan and Yankner, 2000)
Using reverse transcription PCR (RT-PCR), we found that the same treatment resulted in a more robust induction of bim mRNA to a level that is ف60% of that seen for NGF withdrawal (Fig. 1 B)
We found that FKHRL1 is expressed in sympathetic neurons and that phosphorylation at one of the critical phosphorylation sites (Thr32) substantially decreases after NGF withdrawal (Fig. 2 A)
Summary
Programmed cell death (PCD) accounts for the death of approximately half of all neurons generated during embryogenesis and is essential for the correct innervation of target tissues and formation of neuronal networks during neural development (Oppenheim, 1991; Yuan and Yankner, 2000). A well-studied model of neuronal PCD is provided by sympathetic neurons, which depend on NGF for survival during early postnatal life. Developing sympathetic neurons undergo apoptosis when deprived of NGF in culture, providing a useful in vitro system for studying the molecular mechanisms of neuronal PCD (Deshmukh and Johnson, 1997). NGF deprivation activates the intrinsic pathway of apoptosis in sympathetic neurons (Putcha et al, 2002). This involves the release of cytochrome c from the mitochondria into the cytosol which is regulated by members of the BCL-2 family through their ability to influence mitochondrial integrity (Hengartner, 2000). NGF withdrawal–induced apoptosis of sympathetic neurons can be blocked by inhibitors of RNA and protein synthesis (Martin et al, 1988), suggesting that
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