Caspase-dependent Apoptosis Induced by Thapsigargin was Prevented by Glycogen Synthase Kinase-3 Inhibitors in Cultured Rat Cortical Neurons

Calcium ion is essential for cellular functions including signal transduction. Uncontrolled calcium stress has been linked causally to a variety of neurodegenerative diseases. Thapsigargin, which inhibits Ca²⁺-ATPase in the endoplasmic reticulum (ER) and blocks the sequestration of calcium by the ER, induced apoptotic cell death (chromatin condensation and nuclear fragmentation) accompanied by GRP78 protein expression and caspase-3 activation in rat fetal cortical neurons (days in vitro 9–10). Blockade of N-methyl-d-aspartate (NMDA) receptors with NMDA antagonists induced apoptosis without GRP78 protein expression. Apoptosis accompanied both caspase-9 and caspase-3 activation. We then examined whether GSK-3 is involved in thapsigargin-induced cell death by using GSK-3 inhibitors. We assayed the effects of selective GSK-3 inhibitors, SB216763, alsterpaullone and 1-azakenpaullone, on thapsigargin-induced apoptosis. These inhibitors completely protected cells from thapsigargin-induced apoptosis. In addition, GSK-3 inhibitors inhibited caspase-9 and caspase-3 activation accompanied by thapsigargin-induced apoptosis. These results suggest that thapsigargin induces caspase-dependent apoptosis mediated through GSK-3β activation in rat cortical neurons.     

Colchicine-Induced Apoptosis Was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

The purpose of this study was to examine whether glycogen synthase kinase-3 (GSK-3) is involved in colchicine-induced cell death in PC12 cells by using GSK inhibitors. Colchicine increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation or fragmentation. GSK-3 inhibitors such as alsterpaullone, SB216763, and AR-A014418 prevented colchicine-induced cell death and caspase-3 activation. These results suggest that colchicine induces caspase-dependent apoptotic cell death and that GSK-3 activation is involved in cell death in PC12 cells.     

Chelation of Extracellular Calcium-Induced Cell Death was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

Calcium ion is a secondary messenger that mediates a variety of physiological responses of neurons, including cell survival responses. To determine the role of calcium in regulating neuronal survival and death, we examined whether chelation of extracellular calcium with EGTA induces caspase-dependent apoptotic cell death and whether glycogen synthase kinase-3 is involved in EGTA-induced cell death in PC12 cells. EGTA increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation and fragmentation accompanied by caspase activation. EGTA increased GRP78 protein expression, suggesting that EGTA induces ER stress. Glycogen synthase kinase-3 inhibitors prevented EGTA-induced apoptosis. In addition, nerve growth factor and insulin growth factor-I completely blocked EGTA-induced cell death. Moreover, caspase-3 activation was inhibited by glycogen synthase kinase-3 inhibitors. These results suggest that chelation of extracellular calcium with EGTA induces caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.     

Differential Cellular Phosphorylation of Neurofilament Heavy Side-Arms by Glycogen Synthase Kinase-3 and Cyclin-Dependent Kinase-5

Abstract: To investigate the cellular mechanisms regulating neurofilament-heavy subunit (NF-H) side-arm phosphorylation, we studied the ability of three putative neurofilament kinases, glycogen synthase kinase-3 (GSK-3)α, GSK-3β, and cyclin-dependent kinase-5 (cdk-5), to phosphorylate NF-H in transfected cells. We analysed NF-H phosphorylation by using a panel of phosphorylation-dependent antibodies and also by monitoring the electrophoretic mobility of the transfected NF-H on sodium dodecyl sulphate-polyacrylamide gel electrophoresis because this is known to be affected by side-arm phosphorylation. Our results demonstrate that whereas GSK-3α, GSK-3β, and cdk-5 will all phosphorylate NF-H, they generate different antibody reactivity profiles. GSK-3α and GSK-3β induce a partial retardation of a proportion of the transfected NF-H, but only cdk-5 alters the rate of electrophoretic migration to that of NF-H from brain. We conclude that cdk-5 and GSK-3 phosphorylate different residues or sets of residues within NF-H sidearms in cells. We further show that cdk-5 is active in both the CNS and the PNS but that this activity is not dependent on expression of its activator, p35. This suggests that there are other activators of cdk-5.     

Phosphorylation of Neurofilament Heavy-Chain Side-Arm Fragments by Cyclin-Dependent Kinase-5 and Glycogen Synthase Kinase-3α in Transfected Cells

Abstract: The side-arm domain of neurofilament heavy-chain (NF-H) is heavily phosphorylated in axons. Much of this phosphate is located within a multiphosphorylation repeat (MPR) domain situated toward the carboxy terminus of the molecule. The MPR domain contains the repeat motif KSP of which there are two broad categories, KSPXX and KSPXK. In mouse NF-H, the KSPXK repeats are situated toward the latter part of the MPR domain. We have expressed in mammalian cells fragments of mouse NF-H side-arm containing all of the MPR domain, the latter part of the MPR domain containing the KSPXK repeats, and the complementary amino-terminal part of the MPR domain, which contains the KSPXX repeats. By cotransfecting these fragments with the neurofilament kinases cyclin-dependent kinase-5 (cdk-5)/p35 and glycogen synthase kinase-3α (GSK-3α), we show that cdk-5 induces cellular phosphorylation of the KSPXK-containing fragment of NF-H. Using the transfected fragments, we also map the epitopes for several commonly utilised NF-H monoclonal antibodies and describe the effects that phosphorylation by cdk-5 and GSK-3α have on their reactivities.     

HIV-1 Tat-Mediated Activation of Glycogen Synthase Kinase-3β Contributes to Tat-Mediated Neurotoxicity

Human immunodeficiency virus type 1 (HIV-1) Tat induces neuronal apoptosis. To examine the mechanism(s) that contribute to this process, we studied Tat's effects on glycogen synthase kinase-3beta (GSK-3beta), an enzyme that has been implicated in the regulation of apoptosis. Addition of Tat to rat cerebellar granule neurons resulted in an increase in GSK-3beta activity, which was not associated with a change in protein expression and could be abolished by the addition of an inhibitor of GSK-3beta (lithium). Lithium also enhanced neuronal survival following exposure to Tat. Coprecipitation experiments revealed that Tat can associate with GSK-3beta, but direct addition of Tat to purified GSK-3beta had no effect on enzyme activity, suggesting that Tat's effects might be mediated indirectly. As the activation of platelet activating factor (PAF) receptors is critical for the induction of neuronal death by several candidate HIV-1 neurotoxins, we determined whether PAF can also activate GSK-3beta. Application of PAF to neuronal cultures activated GSK-3beta, and coincubation with lithium ameliorated PAF-induced neuronal apoptosis. These findings are consistent with the existence of one or more pathways that can lead to GSK-3beta activation in neurons, and they suggest that the dysregulation of this enzyme could contribute to HIV-induced neuronal apoptosis.     

糖原合酶激酶-3，一个信号通路的重要调控因子

糖原合酶激酶-3 (glycogen synthase kinase-3,GSK-3) 是一种多功能的丝氨酸／苏氨酸蛋白激酶,在蛋白质合成、信号传递、细胞增殖、细胞分化、神经功能、肿瘤形成及胚胎发育等众多细胞进程中均扮演重要的角色.GSK-3 能够使多种底物发生磷酸化,并参与胰岛素、Wnt及Hedgehog 等多个信号通路的调控. GSK-3抑制剂在信号通路中能有效地抑制病理情况下GSK-3活性的异常增高,达到治疗的目的.GSK-3的抑制剂将作为一种潜在的药物对治疗糖尿病、阿尔海默茨症、肿瘤等疾病发挥效用.     

Glycogen Synthase Kinase-3 (GSK3): Inflammation, Diseases, and Therapeutics

Deciphering what governs inflammation and its effects on tissues is vital for understanding many pathologies. The recent discovery that glycogen synthase kinase-3 (GSK3) promotes inflammation reveals a new component of its well-documented actions in several prevalent diseases which involve inflammation, including mood disorders, Alzheimer’s disease, diabetes, and cancer. Involvement in such disparate conditions stems from the widespread influences of GSK3 on many cellular functions, with this review focusing on its regulation of inflammatory processes. GSK3 promotes the production of inflammatory molecules and cell migration, which together make GSK3 a powerful regulator of inflammation, while GSK3 inhibition provides protection from inflammatory conditions in animal models. The involvement of GSK3 and inflammation in these diseases are highlighted. Thus, GSK3 may contribute not only to primary pathologies in these diseases, but also to the associated inflammation, suggesting that GSK3 inhibitors may have multiple effects influencing these conditions. Special issue dedicated to John P. Blass.     

In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase-3β

Abstract: The two pathological lesions found in the brains of Alzheimer's disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule-associated protein τ. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of β-amyloid protein, a 4-kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline-directed protein kinases, glycogen synthase kinase-3β (GSK-3β) and mitogen-activated protein kinase (MAPK), were able to hyperphosphorylate τ, GSK-3β but not MAPK phosphorylated recombinant APP_cyt. The sole site of phosphorylation in APP_cyt by GSK-3β was determined by phosphoamino acid analysis and phosphorylation of APP_cyt mutant peptides to be Thr⁷⁴³ (numbering as for APP₇₇₀). This site was confirmed by endoproteinase Glu-C digestion of APP_cyt and peptide sequencing. The ability of GSK-3β to phosphorylate APP_cyt and τ provides a putative link between the two lesions and indicates a critical role of GSK-3β in the pathogenesis of Alzheimer's disease.     

Glycogen Synthase Kinase-3 Interaction Domain Enhances Phosphorylation of SARS-CoV-2 Nucleocapsid Protein

A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3β constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3β binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3β similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3β. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.     

Calmodulin Modulates Mitogen-Activated Protein Kinase Activation in Response to Membrane Depolarization in PC12 Cells

Abstract: In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca²⁺ influx through voltage-gated Ca²⁺ channels, which is able to stimulate extracellular-regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca²⁺ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K⁺ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca²⁺ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K⁺ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca²⁺ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.     

抑制GSK-3β活性促进NF-κB诱导的儿童急性淋巴细胞白血病细胞凋亡

核转录因子-κB(NF-κB)是维持急性淋巴细胞白血病(ALL)细胞生存的关键因子.近年来发现,糖原合成酶激酶-3β(GSK-3β)可以正性调控NF-κB的活性.本研究通过抑制GSK-3β活性初步探讨ALL细胞中GSK-3β在NF-κB诱导细胞凋亡中的作用机制.收集ALL患儿骨髓单个核细胞,采用免疫荧光细胞化学方法检测到ALL细胞核内GSK-3β有明显聚集.体外培养ALL细胞后经GSK-3β抑制剂氯化锂(LiCl)和SB216763处理,采用Western印迹和EMSA检测发现,ALL细胞核内GSK-3β表达下降,而NF-κBP65蛋白无明显变化,但是其活性明显降低.同时RT-PCR结果显示,NF-κB下游抗凋亡基因存活素(survivin)的表达随之下降,AnnexinV-PE/7-AAD双染流式细胞仪检测结果证实,ALL细胞凋亡明显增加(P0.01).该结果表明,抑制GSK-3β活性可以下调NF-κB的转录活性,并通过下调抗凋亡基因存活素的表达而促进ALL细胞的凋亡.     

Glycogen metabolism in tissues from a mouse model of Lafora disease

Laforin, encoded by the EPM2A gene, by sequence is a member of the dual specificity protein phosphatase family. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons, muscle and other tissues. Glycogen metabolizing enzymes were analyzed in a transgenic mouse over-expressing a dominant negative form of laforin that accumulates Lafora bodies in several tissues. Skeletal muscle glycogen was increased 2-fold as was the total glycogen synthase protein. However, the -/+glucose-6-P activity of glycogen synthase was decreased from 0.29 to 0.16. Branching enzyme activity was increased by 30%. Glycogen phosphorylase activity was unchanged. In whole brain, no differences in glycogen synthase or branching enzyme activities were found. Although there were significant differences in enzyme activities in muscle, the results do not support the hypothesis that Lafora body formation is caused by a major change in the balance between glycogen elongation and branching activities.     

GSK-3在阿尔茨海默病样细胞骨架蛋白过度磷酸化中的作用(英)

神经原纤维缠结是阿尔茨海默病（Alzheimer disease, AD）的特征性病理改变.蛋白激酶和蛋白磷酸酯酶失衡可导致骨架蛋白的异常过度磷酸化,而异常过度磷酸化的tau 和神经丝 (neurofilament, NF) 是神经原纤维缠结的组成部分.在众多激酶中,糖原合酶激酶-3（glycogen synthase kinase-3,GSK-3）可能是AD神经退行性变起重要作用.为深入探讨GSK-3在AD样神经退行性变中的作用,以磷酯酰肌醇三磷酸激酶（phosphatidylinositol 3-kinase,PI3K）的特异性抑制剂渥曼青霉素（wortmannin,WT）处理野生型鼠成神经瘤细胞株（wild type mouse neuroblastoma cell lines, N2a wt）,系统观察WT处理N2a wt不同时间点（1 h、3 h、6 h）细胞代谢率、细胞形态、细胞骨架蛋白tau和NF的磷酸化状态改变以及细胞的命运,并分析了GSK-3活性与上述参数改变之间的相关性.结果发现：1 μmol/L WT处理细胞1 h,GSK-3活性与未经WT处理的对照组相比明显增高,并伴有Ser9磷酸化的GSK-3水平的降低; NF磷酸化程度增强,tau在Ser198/Ser199/Ser202位点的磷酸化增强. 1 μmol/L WT处理细胞3 h,GSK-3活性与对照组和处理1 h 组相比明显下降,NF磷酸化程度较1 h降低,但仍高于正常水平.1 μmol/L WT处理细胞6 h,细胞形态、GSK-3活性、Ser9磷酸化形式的GSK-3β的表达、NF磷酸化程度与对照组相比均无明显改变.WT呈剂量依赖性降低细胞代谢率.1 μmol/L WT处理细胞1 h和3 h导致细胞变圆,突起变短甚至消失.1 μmol/L WT处理细胞1 h,用TUNEL法和电子显微镜技术未观察到细胞凋亡.研究结果提示：在N2a细胞中过度激活GSK-3可导致神经细丝和tau蛋白的AD样过度磷酸化,从而引起神经细胞的AD样退行性变.     

Neurotrophic Factors Prevent Ceramide-Induced Apoptosis Downstream of c-Jun N-Terminal Kinase Activation in PC12 Cells

Abstract: Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala- dl -Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.     

Localization and Developmental Changes of τ Protein Kinase I/Glycogen Synthase Kinase-3β in Rat Brain

Abstract: τ protein kinase I (TPKI) purified from bovine brain extract has been shown to phosphorylate τ and to form paired helical filament (PHF) epitopes and was found recently to be identical to glycogen synthase kinase-3β (GSK-3β). Before elucidating a role of TPKI/GSK-3β in PHF formation, it is necessary to investigate the normal function of the enzyme. To study the distribution and developmental changes of the enzyme, specific polyclonal antibodies were prepared against TPKI and GSK-3α. Immunoblot analysis demonstrated that TPKI was nearly specifically localized in the brain of adult rats. The level of TPKI in the rat brain was high at gestational day 18, peaked on postnatal day 8, and then decreased rapidly to a low level, which was sustained up to 2 years. Immunohistochemistry indicated primarily neuronal localization of TPKI. Growing axons were stained most intensely in the developing cerebellum, but the immunoreactivity became restricted to the gray matter in the mature tissue. Parallel fibers had a high level of TPKI and also stained intensely for τ. These findings indicate that τ is one of the physiological substrates of TPKI and suggest that the enzyme plays an important role in the growth of axons during development of the brain.     

Complex I Inhibitors Induce Dose-Dependent Apoptosis in PC12 Cells: Relevance to Parkinson's Disease

Abstract: The mode of cell death in Parkinson's disease (PD) substantia nigra is uncertain. However, evidence is accumulating that certain of the biochemical abnormalities present in PD nigra at the time of death may precipitate apoptosis. We have investigated the mode of death induced by complex I inhibition of dopaminergic cell cultures, and our results suggest that both 1-methyl-4-phenylpyridinium and rotenone cause apoptosis at low concentrations and necrosis at high concentrations. This dose-dependent shift in the mode of cell death induced by these mitochondrial toxins may have important implications for the mechanism of neuronal cell death in PD.     

Induction of Nitric Oxide Synthase and Nitric Oxide-Mediated Apoptosis in Neuronal PC12 Cells After Stimulation with Tumor Necrosis FActor-α/Lipopolysaccharide

Abstract: Exposure of neuronal PC12 cells, differentiated by nerve growth factor, to tumor necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differntiated PC12 cells was associated with cell death characterized by features of apoptosis, The NOS inhibitors N -monomethylarginine, aminoguanidine, and 2-amino-5,6-dihydro-6-methyl-4 H -1,3-thiazine HCl prevented TNF-α/LPS-induced cell death and DNA fragmentation, suggesting that the TNF-α/LPS-induced cell death is mediated by iNOS-derived NO. This hypothesis is supported by the finding that addition of l -arginine, which serves as a precursor and limiting factor of enzyme-derived NO production, potentiated TNF-α/LPS-induced loss of viability.