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.

BackgroundHyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies.MethodsGenetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A “click” chemistry labeling method is developed for the detection of O-GlcNAcylated tau.ResultsSubstantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity.ConclusionThe present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer’s disease and other neurodegenerative tauopathies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-017-0181-0) contains supplementary material, which is available to authorized users.

Apoptosis plays a crucial role in brain development by ensuring that only appropriately growing, migrating, and synapse-forming neurons and their associated glial cells survive. This process involves an intimate relationship between cell-cell interactions and developmental cues and is further impacted by environmental stress during neurogenesis and disease. Oligodendrocytes (OLs), the major myelin-forming cells in the central nervous system, largely form after this wave of neurogenesis but also show a selective vulnerability to cell death stimuli depending on their stage of development. This can affect not only embryonic and early postnatal brain formation but also the response to demyelinating pathologies. In the present review, we discuss the stagespecific sensitivity of OL lineage cells to damage-induced death and how this might impact myelin survival and regeneration during injury or disease. Apoptosis is a major form of programmed cell death required for the normal development of metazoan tissues, including the brain. 1 During embryonic development of the brain, many more cells than ultimately needed are generated and then selection occurs, resulting in the apoptotic depletion of the surplus cells. The importance of the apoptotic pathway in early brain development has been demonstrated by the targeted deletion in mice of the death-specific cysteine proteases caspase-3 or caspase-9, or of the co-activator Apaf-1, all of which cause severe brain overgrowth and perinatal death. 2,3 In the adult brain, 90% of the cells belong to the glial lineage, which includes oligodendrocytes (OLs), astrocytes and microglia. The glia ensures proper development, function and repair of the neuronal network. This is possible through continuous cross-talk between the glia and neurons mediated by neurotransmitters, cytokines, growth and trophic factor secretion and signaling in a reciprocal manner. [4][5][6][7] In the central nervous system (CNS), OLs are responsible for axon myelination, which insulates the electrical signals transmitted between neurons. OL and neuron development is tightly regulated and the myelin sheath is constructed only when OLs reach maturity and neurons have grown appropriately. In response to injury or during the course of neurological diseases, the neuron-glia network can be replenished to some extent but the degree of repair is dependent on the developmental stage of the OL. This complexity is compounded by the differential sensitivity of OL lineage cells to apoptotic stimuli. In the present review, we will first briefly examine the stages of differentiation of OL cells and then discuss several diseases that are impacted by OL apoptosis, noting how the stage of cell differentiation governs the sensitivity to apoptosis. Defined Stages of OL DevelopmentOligodendrocyte development can be divided into four distinct stages according to the temporal expression of cell surface markers and morphology (Table 1). 8 In the first stage, OL progenitor cells (OPCs) originate from the neuroepithelium of the ventricul...

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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