Inhibition of ATF-3 expression by B-Raf mediates the neuroprotective action of GW5074

GW5074 a brain-permeable 3′ substituted indolone, protects neurons from death in culture and in an in vivo paradigm of neurodegeneration. Using low potassium (LK) induced apoptosis of cerebellar granule neurons, we report here that the protective action of GW5074 is mediated through the activation of B-Raf. Over-expression of a kinase-dead form of B-Raf blocks the ability of GW5074 to neuroprotect, whereas over-expression of active forms of B-Raf protect even in the absence of GW5074. Although mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK) are activated by GW5074, pharmacological inhibition of MEK-ERK signaling by U0126 or PD98059 does not reduce neuroprotection suggesting that B-Raf signals through a non-canonical signaling pathway. GeneChip microarray analyses identified activating transcription factor-3 (ATF-3) as a gene whose expression is induced by LK but that is negatively regulated by GW5074. Forced inhibition of ATF-3 expression using siRNA protects neurons against LK-induced apoptosis, whereas the over-expression of ATF-3 blocks GW5074-mediated neuroprotection. Not unexpectedly, expression of active B-Raf inhibits the apoptosis-associated increase in ATF-3 expression. We extended our work to include three other 3′ substituted indolones - a commercially available inhibitor of RNA-dependent protein kinase and two novel compounds designated as SK4 and SK6. Like GW5074, RNA-dependent protein kinase inhibitor, SK4, and SK6 all inhibited c-Raf in vitro but activated B-Raf in neuronal cultures. All four compounds also inhibited ATF-3 expression. Taken together our results indicate that all four indolones mediate neuroprotection by a common mechanism which involves B-Raf activation, and that a downstream target of B-Raf is ATF-3.