Glycogen synthase kinase-3␤ (GSK-3␤) is a critical activator of neuronal apoptosis induced by a diverse array of neurotoxic insults.However, the downstream substrates of GSK-3␤ that ultimately induce neuronal death are unknown. Here, we show that GSK-3␤ phosphorylates and regulates the activity of Bax, a pro-apoptotic Bcl-2 family member that stimulates the intrinsic (mitochondrial) death pathway by eliciting cytochrome c release from mitochondria. In cerebellar granule neurons undergoing apoptosis, inhibition of GSK-3␤ suppressed both the mitochondrial translocation of an expressed green fluorescent protein (GFP)-Bax ␣ fusion protein and the conformational activation of endogenous Bax. GSK-3␤ directly phosphorylated Bax ␣ on Ser163, a residue found within a species-conserved, putative GSK-3␤ phosphorylation motif. Coexpression of GFP-Bax ␣ with a constitutively active mutant of GSK-3␤, GSK-3␤(Ser9Ala), enhanced the in vivo phosphorylation of wild-type Bax ␣ , but not a Ser163Ala mutant of Bax ␣ , in transfected human embryonic kidney 293 (HEK293) cells. Moreover, cotransfection with constitutively active GSK-3␤ promoted the localization of Bax ␣ to mitochondria and induced apoptosis in both transfected HEK293 cells and cerebellar granule neurons. In contrast, neither a Ser163Ala point mutant of Bax ␣ nor a naturally occurring splice variant that lacks 13 amino acids encompassing Ser163 (Bax ) were driven to mitochondria in HEK293 cells coexpressing constitutively active GSK-3␤. In a similar manner, either mutation or deletion of the identified GSK-3␤ phosphorylation motif prevented the localization of Bax to mitochondria in cerebellar granule neurons undergoing apoptosis. Our results indicate that GSK-3␤ exerts some of its pro-apoptotic effects in neurons by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.

Cerebellar granule neurons (CGNs) require depolarization for their survival in culture. When deprived of this stimulus, CGNs die via an intrinsic apoptotic cascade involving Bim induction, Bax translocation, cytochrome c release, and caspase-9 and -3 activation. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis; however, the precise role of mPTP opening in neuronal apoptosis is presently unclear. Here, we show that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria. A C-terminal (a9 helix) GFP-Bax point mutant (T182A) that constitutively localizes to mitochondria circumvents the requirement for mPTP opening and is entirely sufficient to induce CGN apoptosis. Collectively, these data indicate that the major role of mPTP opening in CGN apoptosis is to trigger Bax translocation to mitochondria, ultimately leading to cytochrome c release and caspase activation.

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Leukotrienes are mediators of inflammation that belong to a family of lipids derived from arachidonic acid by the action of 5-lipoxygenase. Leukotrienes have been detected in the central nervous system in association with different pathological events, but little is known about their biosynthesis or function in the brain. When rat neurons and glial cells in primary culture were stimulated with the calcium ionophore, no significant biosynthesis of leukotrienes was detected using liquid chromatography/mass spectrometry (LC/MS) techniques. However, when exogenous LTA 4 was added to these cultured cells, both neurons and glia were able to synthesize LTC 4 . Activated neutrophils are known to supply LTA 4 to other cells for transcellular biosynthesis of cysteinyl-leukotrienes.Since neutrophils can infiltrate brain tissue after stroke or traumatic brain injury, we examined whether neutrophils play a similar role in the central nervous system. When peripheral blood neutrophils were co-cultured with rat neurons, glia cells, and then stimulated with calcium ionophore, a robust production of LTC 4 , LTD 4 , and LTE 4 was observed, revealing that neurons and glia can participate in the transcellular mechanism of leukotriene biosynthesis. The formation of LTC 4 through this mechanism may be relevant in the genesis and progression of the inflammatory response as a result of brain injury.