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Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder characterized pathologically by motor neuron degeneration and associated with aggregation of RNA-binding proteins. TATA-binding protein-associated factor 15 (TAF15) accumulates as cytoplasmic aggregates in neuronal cells, and clearance of these aggregates is considered a potential therapeutic strategy for ALS. However, the exact pathogenic mechanism of TAF15-induced neurotoxicity remains to be elucidated. Glycogen synthase kinase-3 (GSK-3) plays a critical role in the protection of ALS pathology. In the present study, we use a transgenic fly model over-expressing human TAF15 to study the protective effects of Shaggy/GSK3β on TAF15-induced neuronal toxicity in Drosophila brain. Transgenic flies were examined for locomotor activity and lithium treatment. The expression level and solubility of TAF15 were assessed with western blotting, whereas immunohistochemistry was used to assess TAF15 aggregation in Drosophila brain. We have revealed that Shaggy/GSK3β was abnormally activated in neurons of TAF15-expressing flies and its inhibition can suppress the defective phenotypes, thereby preventing retinal degeneration and locomotive activity caused by TAF15. We have also found that Shaggy/GSK3β inhibition in neuronal cells leads to a reduction in TAF15 levels. Indeed, the F-box proteins Slimb and archipelago genetically interact with TAF15 and control TAF15 protein level in Drosophila. Importantly, SCFslimb is a critical regulator for Shaggy/GSK3β-mediated suppression of TAF15-induced toxicity in Drosophila. The present study has provided an in vivo evidence supporting the molecular mechanism of GSK3β inhibition for protection against TAF15-linked proteinopathies.

Serotonin (5-HT) transporter (SERT) plays a crucial role in serotonergic transmission in the central nervous system, and any aberration causes serious mental illnesses. Nevertheless, the cellular mechanisms that regulate SERT function and trafficking are not entirely understood. Growing evidence suggests that several protein kinases act as modulators. Here, we delineate the molecular mechanisms by which glycogen synthase kinase-3s (GSK3s) regulates SERT. When mouse striatal synaptosomes were treated with the GSK3α/s inhibitor CHIR99021, we observed a significant increase in SERT function, Vmax , surface expression with a reduction in 5-HT Km and SERT phosphorylation. To further study how the SERT molecule is affected by GSK3α/s, we used HEK-293 cells as a heterologous expression system. As in striatal synaptosomes, CHIR99021 treatment of cells expressing wild-type hSERT (hSERT-WT) resulted in a time and dose-dependent elevation of hSERT function with a concomitant increase in the Vmax and surface transporters because of reduced internalization and enhanced membrane insertion; silencing GSK3α/s in these cells with siRNA also similarly affected hSERT. Converting putative GSK3α/s phosphorylation site serine at position 48 to alanine in hSERT (hSERT-S48A) completely abrogated the effects of both the inhibitor CHIR99021 and GSK3α/s siRNA. Substantiating these findings, over-expression of constitutively active GSK3s with hSERT-WT, but not with hSERT-S48A, reduced SERT function, Vmax , surface density, and enhanced transporter phosphorylation. Both hSERT-WT and hSERT-S48A were inhibited similarly by PKC activation or by inhibition of Akt, CaMKII, p38 MAPK, or Src kinase. These findings provide new evidence that GSK3s supports basal SERT function and trafficking via serine-48 phosphorylation.

Antibodies raised against defined phosphorylation sites of the microtubule-associated protein tau are widely used in scientific research and being applied in clinical assays. However, recent studies have revealed an alarming degree of non-specific binding found in these antibodies. In order to quantify and compare the specificity phospho-tau antibodies and other post-translational modification site-specific antibodies in general, a measure of specificity is urgently needed. Here, we report a robust flow cytometry assay using human embryonic kidney cells that enables the determination of a specificity parameter termed Φ, which measures the fraction of non-specific signal in antibody binding. We validate our assay using anti-tau antibodies with known specificity profiles, and apply it to measure the specificity of seven widely used phospho-tau antibodies (AT270, AT8, AT100, AT180, PHF-6, TG-3, and PHF-1) among others. We successfully determined the Φ values for all antibodies except AT100, which did not show detectable binding in our assay. Our results show that antibodies AT8, AT180, PHF-6, TG-3, and PHF-1 have Φ values near 1, which indicates no detectable non-specific binding. AT270 showed Φ value around 0.8, meaning that approximately 20% of the binding signal originates from non-specific binding. Further analyses using immunocytochemistry and western blotting confirmed the presence of non-specific binding of AT270 to non-tau proteins found in human embryonic kidney cells and the mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho-tau antibodies, and potentially for post-translational modification targeting antibodies in general. Cover Image for this issue: doi: 10.1111/jnc.14727.

Glycogen synthase kinase-3β (GSK3β) is a critical regulator of the balance between long-term depression and long-term potentiation which is essential for learning and memory. Our previous study demonstrated that GSK3β activity is highly induced during cocaine memory reactivation, and that reconsolidation of cocaine reward memory is attenuated by inhibition of GSK3β. NMDA receptors and protein phosphatase 1 (PP1) are activators of GSK3β. Thus, this study investigated the roles of NMDA receptor subtypes and PP1in the reconsolidation of cocaine contextual reward memory. Cocaine contextual memories were established and evaluated using cocaine conditioned place preference methods. The regulation of GSK3β activity in specific brain areas was assessed by measuring its phosphorylation state using immunoblot assays. Mice underwent cocaine place conditioning for 8 days and were tested for place preference on day 9. Twenty-four hours later, mice were briefly confined to the compartment previous paired with cocaine to reactivate cocaine-associated memories. Administration of the GluN2A- and GluN2B-NMDA receptor antagonists, NVP-AAM077 and ifenprodil, respectively, immediately following recall abrogated an established cocaine place preference, while preventing the activation of GSK3β in the amygdala, nucleus accumbens, and hippocampus during cocaine memory reactivation. PP1 inhibition with okadaic acid also blocked the activation of GSK3β and attenuated a previously established cocaine place preference. These findings suggest that the dephosphorylation of GSK3β that occurred upon activation of cocaine-associated reward memories may be initiated by the activation of PP1 during the induction of NMDA receptor-dependent reconsolidation of cocaine mnemonic traces. Moreover, the importance of NMDA receptors and PP1 in reconsolidation of cocaine memory makes them potential therapeutic targets in treatment of cocaine use disorder and prevention of relapse.

Myelin disruptions are frequently reported in human immunodeficiency virus (HIV)-infected individuals and can occur in the CNS very early in the disease process. Immature oligodendrocytes (OLs) are quite sensitive to toxic increases in [Ca2+ ]i caused by exposure to HIV-1 Tat (transactivator of transcription, a protein essential for HIV replication and gene expression), but sensitivity to Tat-induced [Ca2+ ]i is reduced in mature OLs. Tat exposure also increased the activity of Ca2+ /calmodulin-dependent kinase IIβ (CaMKIIβ), the major isoform of CaMKII expressed by OLs, in both immature and mature OLs. Since CaMKIIβ is reported to interact with glycogen synthase kinase 3β (GSK3β), and GSK3β activity is implicated in OL apoptosis as well as HIV neuropathology, we hypothesized that disparate effects of Tat on OL viability with maturity might be because of an altered balance of CaMKIIβ-GSK3β activities. Tat expression in vivo led to increased CaMKIIβ and GSK3β activity in multiple brain regions in transgenic mice. In vitro, immature murine OLs expressed higher levels of GSK3β, but much lower levels of CaMKIIβ, than did mature OLs. Exogenous Tat up-regulated GSK3β activity in immature, but not mature, OLs. Tat-induced death of immature OLs was rescued by the GSK3β inhibitors valproic acid or SB415286, supporting involvement of GSK3β signaling. Pharmacologically inhibiting CaMKIIβ increased GSK3β activity in Tat-treated OLs, and genetically knocking down CaMKIIβ promoted death in mature OL cultures treated with Tat. Together, these results suggest that the effects of Tat on OL viability are dependent on CaMKIIβ-GSK3β interactions, and that increasing CaMKIIβ activity is a potential approach for limiting OL/myelin injury with HIV infection.

Estrogen receptors have protective effects against ischemic brain injury. However, the molecular mechanisms underlying this phenomenon have yet to be well studied. Given that inhibition of glycogen synthase kinase (GSK3) can reduce cerebral ischemia/reperfusion injury, we hypothesized that estrogen receptors-mediated protective effects against ischemia-induced blood brain barrier (BBB) disruption involve inhibition of GSK3. Thus, we evaluated GSK3 expression in the brain of ovariectomized female rats, and examined the effects of intracerebroventricular pre-treatments of SB216763, GSK3 inhibitor, on BBB permeability following middle cerebral artery occlusion (MCAO). We also examined the role of specific estrogen receptor subtype in regulation of GSK3 expression and BBB permeability after MCAO. We found that ovariectomized female rats exhibited increased mRNA levels of estrogen receptor α (ERα) and estrogen receptor β (ERβ) and increased protein levels of GSK3β but not GSK3α in brain cortical areas. Furthermore, intracerebroventricular pre-treatments of SB216763 dose-dependently attenuated brain infarction volume, brain water contents, neurological deficits, and BBB disruption, and increased tight junction protein ZO-1 and occludin expression at 24 h following MCAO. Finally, activation of ERβ but not ERα dose-dependently decreased GSK3β expression at 24 h following MCAO. This was associated with increased tight junction protein expression and improved neurological scores. Thus, our study suggested that activation of ERβ may protect against brain ischemia-induced BBB disruption by inhibiting GSK3β-mediated signaling. This article is protected by copyright. All rights reserved.

Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. In this study we test how the axonal movement of a neuropeptide-containing dense-core vesicle (DCV) responds to alcohol stressors. We found that ethanol induces a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide-DCV while high doses inhibit bi-directional movement. This process required the presence of functional kinesin-1 motors as reduction in kinesin prevented the ethanol-induced stimulation of the anterograde movement of neuropeptide-DCV. Furthermore, expression of inactive glycogen synthase kinase 3 (GSK-3β) also prevented ethanol-induced stimulation of neuropeptide-DCV movement, similar to pharmacological inhibition of GSK-3β with lithium. Conversely, inhibition of PI3K/AKT signaling with wortmannin led to a partial prevention of ethanol-stimulated transport of neuropeptide-DCV. Taken together, we conclude that GSK-3β signaling mediates the stimulatory effects of ethanol. Therefore, our study provides new insight into the physiological response of the axonal movement of neuropeptide-DCV to exogenous stressors. Cover Image for this Issue: doi: 10.1111/jnc.14165.

The main purpose of this study was to evaluate whether donepezil, acetylcholinesterase inhibitor, shown to play a protective role through inhibiting glycogen synthesis kinase-3β (GSK-3β) activity, could also exert neuroprotective effects by stimulating protein phosphatase 2A (PP2A) activity in the amyloid-beta (Aβ)42-induced neuronal toxicity model of Alzheimer's disease. In Aβ42-induced toxic conditions, each PP2A and GSK-3β activity measured at different times showed time-dependent reverse pattern toward the direction of accelerating neuronal deaths with the passage of time. In addition, donepezil pre-treatment showed dose-dependent stepwise increase of neuronal viability and stimulation of PP2A activity. However, such effects on them were significantly reduced through the depletion of PP2A activity with either okadaic acid or PP2Ac siRNA. In spite of blocked PP2A activity in this Aβ42 insult, however, donepezil pretreatment showed additional significant recovering effect on neuronal viability when compared to the value without donepezil. Moreover, donepezil partially recovered its dephosphorylating effect on hyperphosphorylated tau induced by Aβ42. This observation led us to assume that additional mechanisms of donepezil, including its inhibitory effect on GSK-3β activity and/or the activation role of nicotinic acetylcholine receptors (nAChRs), might be involved. Taken together, our results suggest that the neuroprotective effects of donepezil against Aβ42-induced neurotoxicity are mediated through activation of PP2A, but its additional mechanisms including regulation of GSK-3β and nAChRs activity would partially contribute to its effects. We investigated neuroprotective mechanisms of donepezil against Aβ42 toxicity: Donepezil increased neuronal viability with reduced p-tau by enhancing PP2A activity. Despite of blocked PP2A activity, donepezil showed additional recovering effect on neuronal viability, which findings led us to assume that additional mechanisms of donepezil including its inhibitory effect on GSK-3β activity and activating role of nicotinic AChRs might be involved.

Glycogen synthase kinase 3β (GSK3β), which is abundantly present in the brain, is known to contribute to psychomotor stimulant-induced locomotor behaviors. However, most studies have been focused in showing that GSK3β is able to attenuate psychomotor stimulants-induced hyperactivity by increasing its phosphorylation levels in the nucleus accumbens (NAcc). So, here we examined in the opposite direction about the effects of decreased phosphorylation of GSK3β in the NAcc core on both basal and cocaine-induced locomotor activity by a bilateral microinjection into this site of an artificially synthesized peptide, S9 (0.5 or 5.0 μg/μL), which contains sequences around N-terminal serine 9 residue of GSK3β. We found that decreased levels of GSK3β phosphorylation in the NAcc core enhance cocaine-induced hyper-locomotor activity, while leaving basal locomotor activity unchanged. This is the first demonstration, to our knowledge, that the selective decrease of GSK3β phosphorylation levels in the NAcc core may contribute positively to cocaine-induced locomotor activity, while this is not sufficient for the generation of locomotor behavior by itself without cocaine. Taken together, these findings importantly suggest that GSK3β may need other molecular targets which are co-activated (or deactivated) by psychomotor stimulants like cocaine to contribute to generation of locomotor behaviors.

D2/D3 dopamine receptors (D2R/D3R) agonists regulate Akt, but their effects display a complex time-course. In addition, the respective roles of D2R and D3R are not defined and downstream targets remain poorly characterized, especially in vivo. These issues were addressed here for D3R. Systemic administration of quinelorane, a D2R/D3R agonist, transiently increased phosphorylation of Akt and GSK-3β in rat nucleus accumbens and dorsal striatum with maximal effects 10 min after injection. Akt activation was associated with phosphorylation of several effectors of the mammalian target of rapamycin complex 1 (mTORC1): p70S6 kinase, ribosomal protein-S6 (Ser240/244), and eukaryotic initiation factor-4E binding protein-1. The action of quinelorane was antagonized by a D2/D3R antagonist, raclopride, and the selective D3R antagonist S33084, inactive by themselves. Furthermore, no effect of quinerolane was seen in knock-out mice lacking D3R. In drd1a-EGFP transgenic mice, quinelorane activated Akt/GSK-3β in both neurons expressing and lacking D1 receptor. Thus, the stimulation of D3R transiently activates the Akt/GSK-3β pathway in the two populations of medium-size spiny neurons of the nucleus accumbens and dorsal striatum. This effect may contribute to the influence of D3R ligands on reward, cognition, and processes disrupted in schizophrenia, drug abuse, and Parkinson's disease.

Several epidemiological and preclinical studies suggest that non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX), reduce the risk of Alzheimer's disease (AD) and can lower β-amyloid (Aβ) production and inhibit neuroinflammation. However, follow-up clinical trials, mostly using selective cyclooxygenase (COX)-2 inhibitors, failed to show any beneficial effect in AD patients with mild to severe cognitive deficits. Recent data indicated that COX-1, classically viewed as the homeostatic isoform, is localized in microglia and is actively involved in brain injury induced by pro-inflammatory stimuli including Aβ, lipopolysaccharide, and interleukins. We hypothesized that neuroinflammation is critical for disease progression and selective COX-1 inhibition, rather than COX-2 inhibition, can reduce neuroinflammation and AD pathology. Here, we show that treatment of 20-month-old triple transgenic AD (3 × Tg-AD) mice with the COX-1 selective inhibitor SC-560 improved spatial learning and memory, and reduced amyloid deposits and tau hyperphosphorylation. SC-560 also reduced glial activation and brain expression of inflammatory markers in 3 × Tg-AD mice, and switched the activated microglia phenotype promoting their phagocytic ability. The present findings are the first to demonstrate that selective COX-1 inhibition reduces neuroinflammation, neuropathology, and improves cognitive function in 3 × Tg-AD mice. Thus, selective COX-1 inhibition should be further investigated as a potential therapeutic approach for AD.

The C-terminal domain of tetanus toxin (Hc-TeTx) has been suggested to act as a neuroprotective agent by activating signaling pathways related to neurotrophins and also to exert anti-apoptotic effects. Here, we show the beneficial properties of the recombinant protein Hc-TeTx to protect spinal motoneurons against excitotoxic damage. In vitro spinal cord organotypic cultures were used to assess acute glutamate excitotoxic damage. Our results indicate that Hc-TeTx treatment improves motoneuron survival within a short therapeutical window (the first 2 h post-injury). Within this interval, we found that p44/p42 MAP kinase (ERK1/2) and glycogen synthase kinase-3 (GSK3β) signaling pathways play a crucial role in the neuroprotective effect. Moreover, we demonstrated that Hc-TeTx treatment initiate autophagy which is ERK1/2- and GSK3β-dependent. These findings suggest a possible therapeutical tool to improve motoneuron survival immediately after excitotoxic insults or during the secondary injury phase that occurs after spinal cord trauma.

Muscle fiber degeneration in sporadic inclusion-body myositis (s-IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid-beta (Abeta)-precursor protein 751 (AbetaPP751), Abeta, phosphorylated tau, and other 'Alzheimer-characteristic' proteins. Proteasome inhibition is an important component of the s-IBM pathogenesis. In brains of Alzheimer's disease (AD) patients and AD transgenic-mouse models, phosphorylation of neuronal AbetaPP695 (p-AbetaPP) on Thr668 (equivalent to T724 of AbetaPP751) is considered detrimental because it increases generation of cytotoxic Abeta and induces tau phosphorylation. Activated glycogen synthase kinase3beta (GSK3beta) is involved in phosphorylation of both AbetaPP and tau. Lithium, an inhibitor of GSK3beta, was reported to reduce levels of both the total AbetaPP and p-AbetaPP in AD animal models. In relation to s-IBM, we now show for the first time that (1) In AbetaPP-overexpressing cultured human muscle fibers (human muscle culture IBM model: (a) proteasome inhibition significantly increases GSK3beta activity and AbetaPP phosphorylation, (b) treatment with lithium decreases (i) phosphorylated-AbetaPP, (ii) total amount of AbetaPP, (iii) Abeta oligomers, and (iv) GSK3beta activity; and (c) lithium improves proteasome function. (2) In biopsied s-IBM muscle fibers, GSK3beta is significantly activated and AbetaPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3beta inhibitors, might benefit s-IBM patients.

In this study, we provide evidence that the muscarinic M1 receptor targets NF-E2-related factor-2 (Nrf2), a transcription factor that regulates the expression of genes containing antioxidant response elements (AREs) in their promoters and that collectively constitute the phase II antioxidant response. In hippocampal primary and cerebellar granule neuron cultures expressing endogenous M1 receptor, carbachol increased the levels of a prototypical phase II antioxidant enzyme, heme oxygenase-1. Moreover, in a heterologous system, based on lentiviral expression of M1 receptor in PC12 pheochromocytoma cells, we found that M1 increased total and nuclear Nrf2 protein levels and heme oxygenase-1 messenger RNA and protein levels. Luciferase reporter constructs for AREs and the use of two inhibitors of protein kinase C (PKC), chelerythrine and 2-aminoethyl diphenylborinate, or transfection with relevant expression vectors allowed us to identify Galphaq, phospholipase C-beta and the classical PKC-gamma isoenzyme, as responsible for the regulation of Nrf2. A PKC-insensitive Nrf2S40A single-point mutant partially channeled M1 signaling to AREs, therefore suggesting the participation of additional intermediates. Inhibition of glycogen synthase kinase-3beta (GSK-3beta) augmented M1-dependent activation of AREs while a PKC-insensitive mutant of GSK-3beta (GSK-3beta-Delta9) blocked this effect and prevented M1-induced accumulation of Nrf2 in the nucleus. Our results demonstrate a previously unidentified role of the Galphaq/phospholipase C-beta/PKC/GSK-3beta axis in regulation of Nrf2 by M1. Such role provides additional conceptual support for the use of cholinemimetics in the treatment of pathologies that, like Alzheimer's disease, require a reinforcement of the cell antioxidant capacity.

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine -phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro. The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.

Loss of retinal ganglion cells occurs in a variety of pathological conditions, including central retinal artery occlusion, diabetes and glaucoma. Using an experimental model of retinal ischemia induced by transiently raise the intraocular pressure (IOP), In this study, we report the original observation that ischemic retinal ganglion cells death is associated with the transient deactivation of the pro-survival kinase Akt and activation of GSK-3beta followed, during reperfusion, by a longer lasting, PI3K-dependent, activation of Akt and phosphorylation of GSK-3beta. Under these experimental conditions, retinal ischemia induced the expression of Bad, a pro-apoptotic protein, member of the Bcl-2 family. The detrimental effects yielded by the ischemic stimulus were minimized by intravitreal administration of the NMDA receptor antagonist, MK801, that reduced the expression of Bad and significantly increased Akt phosphorylation. In conclusion, our present results contribute to unravel the mechanisms underlying retinal damage by high IOP-induced transient ischemia in rat. In addition, these data implicate the pro-survival PI3K/Akt pathway and the observed reduced expression of Bad in the neuroprotection afforded by MK801.

The novel protein kinase C-beta inhibitor enzastaurin (ENZA) induced apoptosis in LNT-229 and T98G cells whereas A172 cells were resistant. Further, ENZA reduced proliferation in glioblastoma-initiating cells T 269 and T 323 but did not induce apoptosis. ENZA-induced apoptosis involved cleavage of caspases 3, 8, and 9 and led to mitochondrial cytochrome c release and was strongly suppressed by the broad spectrum caspase inhibitor zVAD-fmk but only slightly by the expression of the viral caspase 1/8 inhibitor cytokine response modifier-A. ENZA did not reduce the phosphorylation of protein kinase B (Akt), but of p70 S6 kinase and of its substrate S6 protein in T98G cells. Inhibition of the phosphatidylinositol 3 kinase signaling pathway did not restore sensitivity of A172 cells towards ENZA, and constitutively active Akt did not protect LNT-229 and T98G cells from ENZA-induced apoptosis. Dephosphorylation of glycogen synthase kinase 3beta, a biomarker of ENZA action, and cell death induction by ENZA were separately regulated. Inhibition or activation of Akt only weakly modulated ENZA-induced dephosphorylation of glycogen synthase kinase 3beta. In ENZA-resistant A172 cells, apoptosis ligand 2 (Apo2L.0)-induced cleavage of caspases 3, 8, and 9 was increased by ENZA, resulting in synergistic activity of ENZA and Apo2L.0.

Oxidant injury activates the neuroprotective pathway represented by phosphatidylinositol 3 kinase (PI3K) and Akt. However, the final outcome of oxidant exposure is often associated with neuronal death. This study was aimed to identify the molecular mechanism responsible for loss of tolerance to an oxidative environment. In N2A neuroblasts, serum and H2O2 exhibited different kinetics of regulation for the Ser/Thr kinases Akt and glycogen synthase kinase 3β (GSK-3β) and for the transcription factor Nrf2, which governs redox homeostasis. Thus, H2O2 rapidly activated Akt, inhibited GSK-3β, and directed the transcription factor Nrf2 to the nucleus, but after 4 h Akt was inactive, GSK-3β was active and Nrf2 was more cytosolic than nuclear. Inhibition of the PI3K/Akt pathway by LY294002, impeded the short-term effect of H2O2 on nuclear translocation of Nrf2. GSK-3β activation (inhibiting PI3K/Akt) or direct GSK-3β inhibition in cerebellar granule neurons resulted in respective nuclear exclusion and nuclear accumulation of Nrf2. Moreover, in these neurons, nuclear accumulation of Nrf2 correlated with increased heme oxygenase-1 expression. Over-expression of the kinase active GSK-3β (Δ9) mutant, induced Nrf2 cytoplasmic localization and inhibited Nrf2 transcriptional activity towards an antioxidant-response-element luciferase reporter. Moreover, GSK-3β (Δ9) sensitized N2A neuroblasts to H2O 2-induced oxidative stress and cell death. This study identifies GSK-3β, a kinase known to participate in neurodegeneration, as a fundamental element in the down-regulation of the antioxidant cell defense elicited by Nrf2 after oxidant injury and provides a mechanism to explain the loss of oxidant tolerance that happens under persistent oxidant exposure such as those found in several neuropathologies. © 2007 The Authors., This work was supported by grants from Fundación de Investigación Médica Mutua Madrileña (2005), and SAF2004-02039 of the Spanish Ministry of Education.

3-methyl-6-chloro-7,8-hydroxy-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959), a selective agonist for the putative phosphatidylinositol (PI)-linked dopamine receptor (DAR), has been shown to possess potent anti-Parkinson disease effects but produces less dyskinesia and motor fluctuation that are frequently observed in Parkinson disease drug therapies. The present study was designed to detect the neuroprotection of SKF83959 and its potential mechanism for the effect in cultured rat cortical cells. The presence of SKF83959 with a dose range of 0.1-30 micromol/L improved H2O2-reduced cell viability in a dose-dependent manner. The anti-apoptotic action of SKF83959 was partially abolished by pre-application of the D1 antagonist SCH23390 (30 micromol/L) and the PI 3-kinase (PI 3-K) inhibitor LY294002 but not by the MEK1/2 inhibitor PD98059 (30 micromol/L). Moreover, SKF83959 treatment significantly inhibited H2O2-activated glycogen synthase kinase-3beta (GSK-3beta) which was associated with the drug's neuroprotective effect, but this inhibition was attenuated by SCH23390 and a selective PI 3-K inhibitor. Moreover, the application of either SKF83959 or a pharmacological inhibitor of GSK-3beta attenuated the inhibition by H2O2 on the expression of inducible NO synthase and production of NO. This indicates that D1-like receptor, presumably PI-linked D1 receptor, -mediated alteration of PI 3-K/Akt/GSK-3beta pathway is involved in the neuroprotection by SKF83959. In addition, SKF83959 also effectively decreased the level of the lipid peroxidation and increased the activity of GSH-peroxidase altered by H2O2. These results suggest that SKF83959 exerts its neuroprotective effect through both receptor-dependent and independent mechanisms: Inhibition of GSK-3beta and consequently increasing the expression of inducible NO synthase via putative PI-linked DAR; and its anti-oxidative activity which is independent of DAR.

To quantitatively measure tau aggregation in situ, we established a cell model system using a split green fluorescence protein (GFP) complementation assay. In this assay the more aggregated the protein of interest the lower the GFP fluorescence. Tau microtubule-binding domain constructs, whose aggregation characteristics have been described previously (Khlistunova et al. 2006), were used to validate the assay. The aggregation-prone construct exhibited the lowest GFP intensity whereas the aggregation-resistant construct showed the highest GFP intensity. To examine the role of glycogen synthase kinase 3beta (GSK3beta) activity and caspase 3 cleavage on tau aggregation, GFP complementation of full length (T4), caspase-cleaved (T4C3), and pseudophosphorylated at S396/S404 (T4-2EC) tau was examined in the presence of an active or a kinase-dead GSK3beta. Extensive phosphorylation of T4 by GSK3beta resulted in increased GFP intensity. T4C3 showed neither efficient phosphorylation nor a significant GFP intensity change by GSK3beta. The GFP intensity of T4-2EC was significantly reduced by GSK3beta accompanying its presence in the sarkosyl-insoluble fraction, thus demonstrating that T4-2EC was partitioning into aggregates. This indicates that if the majority of tau is phosphorylated at S396/S404, in combination with increased GSK3beta activity, tau aggregation is favored. These data demonstrate that split GFP complementation may be a valuable approach to determine the aggregation process in living cells.

In cultured bovine adrenal chromaffin cells, 48 h-treatment with 20 mmol/L LiCl, 1 mmol/L valproic acid, 30 micromol/L SB216763, 30 micromol/L SB415286, or 100 nmol/L insulin, a condition that inhibits constitutive active glycogen synthase kinase-3 (GSK-3), decreased cell surface (125)I-insulin binding capacity by approximately 39%, without altering the K(d) value; LiCl, SB216763 or insulin decreased insulin receptor (IR) and IR precursor levels, attenuating insulin-induced Tyr-autophosphorylation of IR. LiCl increased inhibitory Ser9-phosphorylation of GSK-3beta at 6 h, decreasing (125)I-insulin binding at 24 h. SB216763-induced (125)I-insulin binding reduction (IC(50) = 3 micromol/L) was preceded by beta-catenin level increase by SB216763 (EC(50) = 11 micromol/L), a hallmark of GSK-3 inhibition. Insulin-induced rapid (> 1 min) Ser9-phosphorylation of GSK-3beta (Nemoto et al. 2006) was followed by approximately 48% decrease of IR level. LiCl did not stimulate endocytosis, nor proteolysis of IR. LiCl destabilized IR mRNA (t(1/2) = 9.3 vs. 6.5 h), decreasing IR mRNA level by approximately 47%, without altering IR gene transcription. Decreases of (125)I-insulin binding and IR level, as well as increased Ser9-phosphorylation of GSK-3beta were restored to the control levels by washing the test compound-treated cells. Thus, GSK-3beta regulates IR level via controlling IR mRNA stability.

Although there is substantial evidence supporting the neuroprotective efficacy of basic fibroblast growth factor (bFGF) in the rodent retina there is no consensus to date as to the protective mechanism involved. We hypothesise that bFGF can assert its neuroprotective effects directly on mouse photoreceptors transduced via the activation of specific intracellular signalling pathways. In mouse photoreceptor-derived 661W cells, bFGF promoted a rapid inactivation of glycogen synthase kinase 3beta (GSK3beta) by phosphorylation at Ser9. The effects of bFGF on GSK3beta were dependent on protein kinase A (PKA) activation, as inhibition of this pathway blocked inactivation. Furthermore, bFGF protection against oxidative stress was dependent on PKA inactivation of GSK3beta as PKA inhibition attenuated bFGF-induced protection. Furthermore, transfection of cells with mutant dominant negative GSK3betaS9A that cannot be phosphorylated on Ser9 also abrogated neuroprotection. Activation of the transcription factor cAMP-response element binding protein (CREB) and subsequent up-regulation of Bcl-2 in response to bFGF was also dependent on PKA as inhibition with H-89 attenuated increased pCREB levels and Bcl-2 expression. These results indicate that the protective efficacy of bFGF in mouse photoreceptors involves PKA-dependent inactivation of GSK3beta and subsequent up-regulation of Bcl-2 via CREB activation.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a member of low-density lipoprotein receptor (LDLR) family which cooperates with Frizzled receptors to transduce the canonical Wnt signal. As a critical component of the canonical Wnt pathway, LRP6 is essential for appropriate brain development, however, the mechanism by which LRP6 facilitates Wnt canonical signaling has not been fully elucidated. Interestingly, LRP6 which lacks its extracellular domain can constitutively activate TCF/LEF and potentiate the Wnt signal. Further, the free cytosolic tail of LRP6 interacts directly with glycogen synthase kinase (GSK3) and inhibits GSK3's activity in the Wnt canonical pathway which results in increased TCF/LEF activation. However, whether these truncated forms of LRP6 are physiologically relevant is unclear. Recent studies have shown that other members of the LDLR family undergo gamma-secretase dependent regulated intramembrane proteolysis (RIP). Using independent experimental approaches, we show that LRP6 also undergoes RIP. The extracellular domain of LRP6 is shed and released into the surrounding milieu and the cytoplasmic tail is cleaved by gamma-secretase-like activity to release the intracellular domain. Furthermore, protein kinase C, Wnt 3a and Dickkopf-1 modulate this process. These findings suggest a novel mechanism for LRP6 in Wnt signaling: induction of ectodomain shedding of LRP6, followed by the gamma-secretase involved proteolytic releasing its intracellular domain (ICD) which then binds to GSK3 inhibiting its activity and thus activates the canonical Wnt signaling pathway.

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT) gene family comprises three human homologs that phosphorylate and inactivate glycogen synthase kinase 3beta (GSK3beta). Studies have reported the genetic association of AKT1 with schizophrenia. Additionally, decreased AKT1 protein expression and the reduced phosphorylation of GSK3beta were reported in this disease, leading to a new theory of attenuated AKT1-GSK3beta signaling in schizophrenia pathogenesis. We have evaluated this theory by performing both genetic and protein expression analyses. A family based association test of AKT1 did not show association with schizophrenia in Japanese subjects. The expression levels of total AKT, AKT1 and phosphorylated GSK3beta detected in the schizophrenic brains from two different brain banks also failed to support the theory. In addition, no attenuated AKT-GSK3beta signaling was observed in the lymphocytes from Japanese schizophrenics, contrasting with previous findings. Importantly, we found that the level of phosphorylated GSK3beta at Ser9 tended to be inversely correlated with postmortem intervals, and that the phosphorylation levels of AKT were inversely correlated with brain pH, issues not assessed in the previous study. These data introduce a note of caution when estimating the phosphorylation levels of GSK3beta and AKT in postmortem brains. Collectively, this study failed to support reduced signaling of the AKT-GSK3beta molecular cascade in schizophrenia.

In this study we evaluated UCN-01, a small molecule that inhibits protein kinases by interacting with the ATP-binding site, as a potential anti-cancer agent for neuroblastoma. UCN-01 was effective at inducing apoptosis in six neuroblastoma cell lines with diverse cellular and genetic phenotypes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assays, detection of active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) confirmed that UCN-01 induced apoptosis. Cell cycle analysis determined that the UCN-01 treated cells accumulated in S phase by 16 h. Unlike vinblastine and docetaxel that increased survivin expression, UCN-01 treatment did not increase X-linked inhibitor of apoptosis protein (XIAP) and survivin levels. Analysis of specific phosphoepitopes on chk1/2, Akt, and GSK3beta following UCN-01 treatment determined that there was no significant change in phospho-chk1/2. However, there was decreased immunoreactivity at Ser473 and Thr308 of Akt and Ser9 of GSK3beta by 4 h indicating that the Akt survival pathway and downstream signalling was compromised. Thus, UCN-01 was effective at inducing apoptosis in neuroblastoma cell lines.

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein that consists of PDZ domains, a guanylate kinase domain, and WW domains. It interacts with N-methyl-d-aspartate receptor subunits, neuroligin, and beta-catenin. Here, we identified Axin as a novel binding partner of S-SCAM. Axin was co-immunoprecipitated with S-SCAM from rat brain, detected in the post-synaptic density fraction in rat brain subcellular fractionation, and partially co-localized with S-SCAM in neurons. The guanylate kinase domain of S-SCAM directly bound to the GSK3beta-binding region of Axin. S-SCAM formed a complex with beta-catenin and Axin, but competed with GSK3beta for Axin-binding. Thereby, S-SCAM inhibited the Axin-mediated phosphorylation of beta-catenin by GSK3beta.

Glycogen synthase kinase-3 beta (GSK-3β) dysfunction may play an essential role in the pathogenesis of psychiatric, metabolic, neurodegenerative diseases, in which oxidative stress exists concurrently. Some studies have shown that GSK-3β activity is up-regulated under oxidative stress. This study evaluated how oxidative stress regulates GSK-3β activity in human embryonic kidney 293 (HEK293)/Tau cells treated with hydrogen peroxide (H₂O₂). Here, we show that H₂O₂ induced an obvious increase of GSK-3β activity. Surprisingly, H₂O₂ dramatically increased phosphorylation of GSK-3β at Ser9, an inactive form of GSK-3β,while there were no changes of phosphorylation of GSK-3β at Tyr216. Moreover, H₂O₂ led to a transient [Ca²⁺](i) elevation, and simultaneously increased the truncation of GSK-3β into two fragments of 40 kDa and 30 kDa, whereas inhibition of calpain decreased the truncation and recovered the activity of GSK-3β. Furthermore, tau was hyperphosphorylated at Ser396, Ser404, and Thr231, three most common GSK-3β targeted sites after 100 μM H₂O₂ administration in HEK293/Tau cells, whereas inhibition of calpain blocked the tau phosphorylation. In addition, we found that there were no obvious changes of Cyclin-dependent kinase 5 (CDK5) expression (responsible for tau phosphorylation) and of p35 cleavage, the regulatory subunit of CDK5 in H₂O₂-treated HEK293/Tau cells. In conclusion, Ca²⁺-dependent calpain activation leads to GSK-3β truncation, which counteracts the inhibitory effect of Ser9 phosphorylation, up-regulates GSK-3β activity, and phosphorylates tau in H₂O₂-treated HEK293/Tau cells.

Neuronal activity shapes the dendritic arbour; however, most of the molecular players in this process remain to be identified. We observed that depolarization-induced neuronal activity causes an increase in the phosphorylation of glycogen synthase kinase-3 (GSK-3)α/β on Ser21/9 in cerebellar granule neurons. Using several approaches, including gene knockdown and GSK-3α/β(S21A/S21A/S9A/S9A) double knockin mice, we demonstrated that both GSK-3β and GSK-3α mediate activity-dependent dendritic growth and that Ser21/9 phosphorylation of GSK-3α/β plays an important role in this process. Collapsin response mediator protein-2 (CRMP-2), which is crucial for axon development, is phosphorylated at Thr514 and inactivated by GSK-3. We found CRMP-2 was located mainly in the dendrites of cerebellar granule neurons, in contrast to the axonal distribution in hippocampal neurons. Over-expression of CRMP-2 promoted and knockdown of CRMP-2 impaired dendritic growth, suggesting that CRMP-2 is necessary and sufficient for activity-dependent dendritic development. Furthermore, silencing CRMP-2 completely blocked the dendritic growth-promoting effects of GSK-3 knockdown, and expression of Thr514 nonphosphorylated form of CRMP-2 counteracted the inhibitory effect of constitutively active GSK-3. This data indicate that CRMP-2 functions downstream of GSK-3. Together, these findings identify a novel GSK-3/CRMP-2 pathway that connects neuronal activity to dendritic growth.

The peroxisome proliferator-activated receptor gamma(PPARc) agonists thiazolidinediones (TZDs) are prescribed for the treatment of type 2 diabetes mellitus. Furthermore, it has been reported that TZDs have a beneficial effect on neurodegenerative disorders, such as Alzheimer’s disease. However, the molecular mechanisms underlying this effect are not fully understood. Here, we investigated whether and how troglitazone, a parent TZD drug, inhibits tau phosphorylation.Treatment with troglitazone decreased tau-Thr231 phosphorylation and p35, the specific activator of cyclin-dependent kinase 5 (CDK5), in a dose- and time-dependent manner. Troglitazone also decreased CDK5 enzymatic activity, and ectopic expression of p25, the cleaved and more active form of p35, restored the troglitazone-induced decrease in tau-Thr231 phosphorylation. Treatment with either MG-132, a reversible proteasome inhibitor, or lactacystin, a specific and irreversible 26S proteasome inhibitor, significantly reversed the observed inhibitory effects of troglitazone. However, GW9662, a specific and irreversible PPARc antagonist, did not alter the observed inhibitory effects. Similar results were also found when other TZD drugs, pioglitazone and rosiglitazone, were used. Treatment with various inhibitors revealed that troglitazone-induced inhibitions of tau-Thr231 phosphorylation and p35 expression were not mediated by glycogen synthase kinase 3b, protein kinase A, and protein phosphatase 2A signaling pathways.Finally, we also found that the same observed inhibitory effects of troglitazone hold true for the use of primary cortical neurons. Taken together, we demonstrated that TZDs repressed tau-Thr231 phosphorylation via the inhibition of CDK5 activity, which was mediated by the proteasomal degradation of p35 and a PPARc-independent signaling pathway.

The cerebral endothelium can be a vital source of signaling factors such as brain-derived neurotrophic factor that defends the neuronal parenchyma against stress and injury. But the underlying mechanisms remain to be fully defined. Here, we use cell models to ask how vascular neuroprotection is sustained. Human brain endothelial cells were grown in culture, and conditioned media were transferred to primary rat cortical neurons. Brain endothelial cell-conditioned media activated neuronal Akt signaling and protected neurons against hypoxia and oxygen-glucose deprivation. Blockade of Akt phosphorylation with the PI3-kinase inhibitor LY294002 negated this vascular neuroprotective effect. Upstream of Akt signaling, the brain-derived neurotrophic factor receptor TrkB (neurotrophic tyrosine kinase receptor, type 2) was involved because depletion with TrkB/Fc eliminated the ability of endothelial-conditioned media to protect neurons against hypoxia. Downstream of Akt signaling, activation of GSK-3β (glycogen synthase kinase 3 beta), caspase 9, caspase 3 and Bad pathways were detected. Taken together, these findings suggest that the molecular basis for vascular neuroprotection involves TrkB-Akt signaling that ameliorates neuronal apoptosis. Further investigation of these mechanisms may reveal new approaches for augmenting endogenous vascular neuroprotection in stroke, brain injury, and neurodegeneration.

Higher plasma urate level is reported to be associated with a reduced risk and slower progression of Parkinson's disease (PD). In this study, we explored the effects of urate on dopaminergic neurons in nigrostriatal pathway in the 6-hydroxydopamine (6-OHDA) unilaterally lesioned rats. Uric acid (UA), when given twice daily at 200 mg/kg intraperitoneally for 10 consecutive days, elevated urate (the anionic form of UA) in plasma and striatum by 55% and 36.8%, respectively, as compared with vehicle group. This regimen of UA was found to ameliorate the behavioral deficits, dopaminergic neuron loss as well as dopamine depletion in the nigrostriatal system. Moreover, UA administration was capable of increasing glutathione level and superoxide dismutase activity while decreasing malondialdehyde accumulation in striatum. In addition, the phosphorylation of both protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) in the lesioned striata of 6-OHDA-lesioned rats was dramatically reduced as compared with sham-operated rats. This reduction was attenuated in the Parkinsonian rats receiving UA treatment. Similarly, in vitro findings showed that UA alleviated the decrease in Akt activation and the increase in GSK3β activity caused by 6-OHDA. Furthermore, neuroprotection by urate and its regulation on GSK3β phosphorylation at Ser9 was found to be abolished in the presence of PI3K inhibitor. Therefore, our findings demonstrated that urate was able to protect dopaminergic neurons in rat nigrostriatal pathway against the neurotoxicity of 6-OHDA, and showed that its beneficial effects may be related to its regulation on Akt/GSK3β signaling.

J. Neurochem. (2012) 122, 308–320. Abstract Cortical dopamine (DA) modulation of the gamma-amino butyric acid (GABA) system is closely associated with cognitive function and psychiatric disorders. We recently reported that the glycogen synthase kinase 3β (GSK-3β) pathway is required for hyperdopamine/D2 receptor-mediated inhibition of NMDA receptors in the prefrontal cortex. Here we explore whether or not GSK-3β is also involved in dopaminergic modulation of GABAA receptor-mediated inhibitory transmission. We confirmed that DA induces a dose-dependent, bidirectional regulatory effect on inhibitory postsynaptic currents (IPSCs) in prefrontal neurons. The modulatory effects of DA were differentially affected by co-application of GSK-3β inhibitors and different doses of DA. GSK-3β inhibitors completely blocked high-dose (20 μM) DA-induced depressive effects on IPSCs but exhibited limited effects on the facilitating regulation of IPSC in low-dose DA (200 nM). We also confirmed that surface expressions of GABAA receptor β2/3 subunits were significantly decreased by DA applied in cultured prefrontal neurons and in vivo administration of DA reuptake inhibitor. These effects were blocked by prior administration of GSK-3β inhibitors. We explored DA-mediated regulation of GABAA receptor trafficking and exhibited the participation of brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) or dynamin-dependent trafficking of GABAA receptors. Together, these data suggest that DA may act through different signaling pathways to affect synaptic inhibition, depending on the concentration. The GSK-3β signaling pathway is involved in DA-induced decrease in BIG2-dependent insertion and an increase in the dynamin-dependent internalization of GABAA receptors, which results in suppression of inhibitory synaptic transmission.

Type I interferons (IFNs) have been shown to act on neurons and to cause neuronal damage through mechanisms not completely defined. Here, we investigated the effects of type I IFNs on brain-derived neurotrophic factor (BDNF)-induced TrkB receptor signaling and neurotrophic activity. In retinoic acid-treated human SH-SY5Y neuroblastoma cells and mouse primary cortical neurons, long-term exposure to IFNs curtailed BDNF-induced activation of phosphatidylinositol 3-kinase, phospholipase Cγ and extracellular-regulated kinases 1 and 2 signaling. Moreover, IFN-β inhibited BDNF-induced cell survival, neurite outgrowth, and expression of neuronal markers, such as neurofilament proteins, growth-associated protein-43 and glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR1. The IFN inhibitory effects were associated with down-regulation of TrkB and inhibition of TrkB autophosphorylation. In SH-SY5Y cells, blockade of either Janus kinase with pyridone 6 or signal transducer and activator of transcription (STAT) 1 with siRNA transfection attenuated IFN-β-induced TrkB down-regulation. Quantitative real time RT-PCR indicated that IFN-β significantly reduced TrkB mRNA levels. Moreover, blockade of protein kinase R counteracted IFN-β-induced inhibition of TrkB expression and signaling. These data indicate that in neuronal cells IFNs negatively regulate BDNF signaling and neurotrophic activity through inhibition of TrkB activation and Janus kinase/Signal transducer and activator of transcription-dependent down-regulation of TrkB.

Several metabolic neuroimaging studies have indicated that bipolar patients with mania exhibit alterations in metabolic activity, suggesting that perturbations in corticolimbic function contribute to the functional deficits associated with the disease. Because pharmacological stimulation of K(v)7 channel function has shown anti-manic like efficacy in the D-amphetamine and chlordiazepoxide (AMPH+CDP) induced hyperactivity mouse model of mania, we addressed whether this effect of K(v)7 channels could be associated with changes in cerebral [¹⁴C]2-deoxyglucose (2-DG) uptake, a surrogate marker of brain metabolic activity. Acute administration of the Kv7 channel modulators, retigabine (pan K(v)7.2-K(v)7.5 channel opener) and ICA-27243 (K(v)7.2/K(v)7.3 channel-preferring opener) reduced 2-DG uptake in several mouse forebrain structures with a brain regional signature similar to the mood stabilizers, lithium and valproate. Combined administration of AMPH+CDP enhanced 2-DG uptake in the striatum, cortex and thalamus, and both retigabine and ICA-27243 fully prevented this stimulatory effect of AMPH+CDP. In addition, both K(v)7 channel openers dose-dependently increased phospho-serine-9 levels of GSK3β in the prefrontal cortex and hippocampus, a common molecular mechanism shared by anti-manic drugs. In combination, these data emphasize the potential of K(v)7 channel openers in the treatment of bipolar disorder, and suggest that heteromeric K(v)7.2/K(v)7.3 channels may present a novel anti-manic therapeutic target.

Insulin offers trophic support through receptors expressed widely on peripheral neurons. In this work, we studied whether peripheral sensory neurons demonstrate resistance to its trophic properties, a property relevant during type 2 diabetes mellitus or following supraphysiological therapy. Insulin receptors were not only localized to neuronal membranes and cytoplasm but also had a unique, previously unrecognized localization to neuronal nuclei. We confirmed that nanomolar doses increased neurite outgrowth of adult sensory neurons, but in response to micromolar doses of insulin, even following a brief 2-h exposure, survival and outgrowth of neurites were blunted. Neurons exposed to picomolar insulin concentrations in their media for 5 days had resistance to the impact of later nanomolar doses of insulin. Using a stripe assay seeded with insulin, neurites were more likely to reject higher doses of insulin. Insulin down-regulated mRNAs of the insulin receptor β subunit and up-regulated levels of GSK-3β, both potential mechanisms of insulin resistance, while down-regulating the protein expression of pAkt and pGSK-3β. Overall, these studies identify neuronal nuclear targeting of insulin and evidence for insulin-induced resistance to its trophic properties. The findings have implications for the understanding of the actions of insulin in the treatment of diabetes and neurological disorders.

J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07532.x Abstract Alzheimer’s disease (AD) is a neurodegenerative disorder leading to a progressive loss of cognitive function and is pathologically characterized by senile plaques and neurofibrillary tangles. Glycogen synthase kinase-3 (GSK-3) is involved in AD pathogenesis. GSK-3 is reported not only to phosphorylate tau, a major component of neurofibrillary tangles, but also to regulate the production of amyloid β, which is deposited in senile plaques. Therefore, pharmacological inhibition of GSK-3 is considered an attractive therapeutic approach. In this study, we report the pharmacological effects of a novel GSK-3 inhibitor, 2-methyl-5-(3-{4-[(S)-methylsulfinyl]phenyl}-1-benzofuran-5-yl)-1,3,4-oxadiazole (MMBO), which displays high selectivity for GSK-3 and brain penetration following oral administration. MMBO inhibited tau phosphorylation in primary neural cell culture and also in normal mouse brain. When administered to a transgenic mouse model of AD, MMBO significantly decreased hippocampal tau phosphorylation at GSK-3 sites. Additionally, chronic MMBO administration suppressed tau pathology as assessed by AT8-immunoreactivity without affecting amyloid β pathology. Finally, in behavioral assessments, MMBO significantly improved memory and cognitive deficits in the Y-maze and in novel object recognition tests in the transgenic AD mouse model. These results indicate that pharmacological GSK-3 inhibition ameliorates behavioral dysfunction with suppression of tau phosphorylation in an AD mouse model, and that MMBO might be beneficial for AD treatment.

Various environmental factors are known to influence the onset and progression of Alzheimer's disease (AD). Environmental enrichment was reported to improve cognitive performance in various Alzheimer's transgenic mice via an amyloid-related or unrelated mechanism. However, stress has been found to accelerate amyloid deposition and cognitive deficits in many AD models. The aim of this study was to determine whether environmental enrichment compensates for the effects of stress on disease progression in the Tg2576 mice, an established AD model. We housed Tg2576 mice under environmental enrichment, enrichment plus stress, stress, or control conditions at 3 months of age. In this study, we first report that environmental enrichment counteracts the effects of stress in terms of cognitive deficits, tau phosphorylation, neurogenesis, and neuronal proliferation during AD-like disease progression. These results strongly implicate the importance of environmental factors as a major modulator for the disease progression of AD.

Glycogen synthase kinase-3β (GSK-3β) plays a crucial role in memory deficits and tau hyperphosphorylation as seen in Alzheimer's disease, the most common dementia in the aged population. We reported that ventricular co-injection of wortmannin and GF-109203X (WT/GFX) can induce tau hyperphosophorylation and memory impairment of rats through activation of GSK-3 [Liu S. J., Zhang A. H., Li H. L., Wang Q., Deng H. M., Netzer W. J., Xu H. X. and Wang J. Z. (2003) J. Neurochem. 87, 1333]. In the present study, we found that feeding the rats with Acetyl-L-Carnitine (ALCAR, 50 mg/day·rat, per os) for 2 weeks rescued the WT/GFX-induced spatial memory retention impairment of the rats by antagonizing GSK-3β activation independent of Akt, PKCζ and Erk1/2. We also found that ALCAR arrested microtubule-associated protein tau hyperphosphorylation at multiple Alzheimer's disease sites in vivo and in vitro. Moreover, ALCAR enhanced the expression of several memory-associated proteins including c-Fos, synapsin I in rat hippocampus. These results suggest that ALCAR could ameliorate WT/GFX-induced spatial memory deficits through inhibition tau hyperphosphorylation and modulation of memory-associated proteins.

Exposure to cocaine-associated conditioned stimuli elicits craving and increases the probability of cocaine relapse in cocaine users even after extended periods of abstinence. Recent evidence indicates that cocaine seeking can be inhibited by disrupting the reconsolidation of the cocaine cue memories and that basolateral amygdala (BLA) neuronal activity plays a role in this effect. Previous studies demonstrated that glycogen synthase kinase 3β (GSK-3β) plays a role in the reconsolidation of fear memory. Here, we used a conditioned place preference procedure to examine the role of GSK-3β in the BLA in the reconsolidation of cocaine cue memories. GSK-3β activity in the BLA, but not central amygdala (CeA), in rats that acquired cocaine (10 mg/kg)-induced conditioned place preference increased after re-exposure to a previously cocaine-paired chamber (i.e., a memory reactivation procedure). Systemic injections of the GSK-3β inhibitor lithium chloride after memory reactivation impaired the reconsolidation of cocaine cue memories and inhibited subsequent cue-induced GSK-3β activity in the BLA. Basolateral amygdala, but not central amygdala, injections of SB216763, a selective inhibitor of GSK-3β, immediately after the reactivation of cocaine cue memories also disrupted cocaine cue memory reconsolidation and prevented cue-induced increases in GSK-3β activity in the BLA. The effect of SB216763 on the reconsolidation of cocaine cue memories lasted at least 2 weeks and was not recovered by a cocaine priming injection. These results indicate that GSK-3β activity in the BLA mediates the reconsolidation of cocaine cue memories.

As a ubiquitous serine/threonine protein kinase, glycogen synthase kinase 3β (GSK-3β) has been considered to be important in the synaptic plasticity that underlies dopamine-related behaviors and diseases. We recently found that GSK-3β activity in the nucleus accumbens (NAc) core is critically involved in cocaine-induced behavioral sensitization. The present study further explored the association between the changes in GSK-3β activity in the NAc and the chronic administration of methamphetamine. We also examined whether blocking GSK-3β activity in the NAc could alter the initiation and expression of methamphetamine (1 mg/kg, i.p.)-induced locomotor sensitization in rats using systemic administration of lithium chloride (LiCl, 100 mg/kg, i.p) and brain region-specific administration of the GSK-3β inhibitor SB216763 (1 ng/side). We found that GSK-3β activity increased in the NAc core, but not NAc shell, after chronic methamphetamine administration. The initiation and expression of methamphetamine-induced locomotor sensitization was attenuated by systemic administration of LiCl and direct infusion of SB216763 into the NAc core, but not NAc shell. These results indicate that GSK-3β activity in the NAc core mediates the initiation and expression of methamphetamine-induced locomotor sensitization, suggesting that GSK-3β may be a potential target for the treatment of psychostimulant addiction.

Neuroglobin (Ngb) is a recently identified member of hemoglobin family, distributed mainly in central and peripheral nervous systems. Recent studies suggest that Ngb can protect neural cells from β-amyloid-induced toxicity in Alzheimer disease (AD). Hyperphosphorylation of tau is another characterized pathological hallmark in the AD brains; however, it is not reported whether Ngb also affects tau phosphorylation. In this study, we found that the level of Ngb was significantly reduced in Tg2576 mice (a recognized mouse model of AD) and TgMAPt mice, and the level of Ngb was negatively correlated with tau phosphorylation. Over-expression of Ngb attenuates tau hyperphosphorylation at multiple AD-related sites induced by up-regulation of glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase. While Ngb activates Akt and thus inhibits GSK-3β, simultaneously inhibition of Akt abolishes the effects of Ngb on GSK-3β inhibition and tau hyperphosphorylation. Our data indicate that Ngb may attenuate tau hyperphosphorylation through activating Akt signaling pathway, implying a therapeutic target for AD.

Mammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3α and GSK3β, while a splice variant of GSK3β (GSK3β2), containing a 13 amino acid insert, is enriched in neurons. GSK3α and GSK3β deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, β-catenin, c-Myc, c-Jun and some residues on tau associated with Alzheimer's disease, is altered in cortical tissue lacking both isoforms of GSK3. This confirms that they are physiological targets for GSK3. However, deletion of each GSK3 isoform produces distinct substrate phosphorylation, indicating that each has a different spectrum of substrates (e.g. phosphorylation of Thr509, Thr514 and Ser518 of CRMP is not detectable in cortex lacking GSK3β, yet normal in cortex lacking GSK3α). Furthermore, the neuron-enriched GSK3β2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than GSK3β1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each GSK3β isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each GSK3β splice variant is phosphorylated to a similar extent at the regulatory sites, Ser9 and Tyr216, and exhibit identical sensitivities to the ATP competitive inhibitor CT99021, suggesting upstream regulation and ATP binding properties of GSK3β1 and GSK3β2 are similar.

Lesioned axons do not regenerate in the adult mammalian CNS, owing to the over-expression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3beta (GSK3beta) and extracellular-related kinase (ERK) 1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3beta and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: (i) cerebellar granule cells and (ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3beta inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Finally, these regenerative effects were corroborated in the lesioned entorhino-hippocampal pathway in NgR1-/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06581.x Abstract Glycogen synthase kinase-3 (GSK-3) has become an important target for the treatment of mood disorders and neurodegenerative disease. It comprises three enzymes, GSK-3α, β and the neuron-specific isoform, β2. GSK-3 regulates axon growth by phosphorylating microtubule-associated proteins including Tau. A genetic polymorphism that leads to an increase in the ratio of GSK-3β1 to GSK-3β2 interacts with Tau haplotypes to modify disease risk in Parkinson’s and Alzheimer’s disease. We have examined the roles of each isoform of GSK-3 in neurons. Silencing of GSK-3β2 inhibited retinoic acid-induced neurite outgrowth in SH-SY5Y neuroblastoma cells and axon growth in rat cortical neurons. Inhibition of neurite outgrowth was prevented by co-expression of GSK-3β2 but not by co-expression of GSK-3α or GSK-3β1. Ectopic expression GSK-3β2 enhanced the effects of retinoic acid on neurite length and induced neurite formation in the absence of retinoic acid. GSK-3β2 phosphorylated Tau at a subset of those sites phosphorylated by GSK-3β1. In addition, Axin, which regulates responses to Wnt signals, associated more readily with GSK-3β1 than with GSK-3β2. Our results suggest that GSK-3 inhibitors that target the Axin-binding site in GSK-3 will preserve the beneficial effects of GSK-3β2 on axon growth.

Nerve growth factor (NGF) promotes neurite outgrowth through regulating cytoskeletal organization and cell adhesion. These activities are modulated by protein phosphorylation. Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinase-like kinase that phosphorylates several transcription factors. Although NLK is known to be expressed at relatively high levels in the nervous system, its function is not well understood. We found that NGF promotes the translocation of NLK to PC12 cells' leading edges, and triggers NLK kinase activity in them. Activated NLK directly phosphorylates microtubule-associated protein-1B (MAP1B) and the focal adhesion adaptor protein, paxillin. Knockdown of NLK attenuates the phosphorylation of both paxillin and MAP1B and inhibits both the NGF-induced re-distribution of F-actin and neurite outgrowth. We also discovered that NLK is a LiCl- sensitive kinase. LiCl is known to block NGF-induced neurite outgrowth and the phosphorylation of MAP1 B and paxillin in PC12 cells. Therefore, the effects of LiCl are mediated in part by blocking NLK activity. These results suggest that NLK controls the dynamics of the cytoskeleton downstream of NGF signaling.

Glycogen synthase kinase 3beta (GSK-3beta) is a ubiquitous serine/threonine protein kinase involved in a number of signaling pathways. Previous studies have demonstrated a role for GSK-3beta in the synaptic plasticity underlying dopamine-associated behaviors and diseases. Drug sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. However, the role of GSK-3beta in cocaine-induced behavior sensitization has not been examined. The present study investigated the effects of chronic cocaine exposure on GSK-3beta activity in the nucleus accumbens (NAc) and determined whether changes in GSK-3beta activity in the NAc are associated with cocaine-induced locomotor sensitization. We also explored whether blockade of GSK-3beta activity in the NAc inhibits the initiation and expression of cocaine-induced locomotor sensitization in rats using systemic or brain region-specific administration of the GSK-3beta inhibitors lithium chloride (LiCl) and SB216763. GSK-3beta activity in the NAc core, but not NAc shell, increased after chronic cocaine (10 mg/kg, i.p.) administration. The initiation and expression of cocaine-induced locomotor sensitization was attenuated by systemic administration of LiCl (100 mg/kg, i.p.) or direct infusion of SB216763 (1 ng/side) into the NAc core, but not NAc shell. Collectively, these results indicate that GSK-3beta activity in the NAc core, but not NAc shell, mediates the initiation and expression of cocaine-induced locomotor sensitization, suggesting that GSK-3beta may be a potential target for the treatment of cocaine addiction.

This study examined the role of calcineurin, a major calcium-dependent protein phosphatase, in dephosphorylating Ser-9 and activating glycogen synthase kinase-3beta (GSK-3beta). Treatment with calcineurin inhibitors increased phosphorylation of GSK-3beta at Ser-9 in SH-SY5Y human neuroblastoma cells. The over-expression of a constitutively active calcineurin mutant, calcineurin A beta (1-401), led to a significant decrease in phosphorylation at Ser-9, an increase in the activity of GSK-3beta, and an increase in the phosphorylation of tau. K(m) of calcineurin for a GSK-3beta phosphopeptide was 469.3 microM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK-3beta were co-immunoprecipitated in neuron-derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK-3beta. We conclude that in vitro, calcineurin can dephosphorylate GSK-3beta at Ser-9 and form a stable complex with GSK-3beta, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK-3betain vivo.

The protective effects of insulin-like growth factor I on the somatostatin (SRIF) system in the temporal cortex after beta-amyloid (Abeta) injury may be mediated through its N-terminal tripeptide glycine-proline-glutamate (GPE). GPE is cleaved to cyclo[Pro-Gly] (cPG), a metabolite suggested to mediate in neuroprotective actions. We evaluated the effects of GPE and cPG in the temporal cortex of Abeta25-35-treated rats on SRIF and SRIF receptor protein and mRNA levels, adenylyl cyclase activity, cell death, Abeta25-35 accumulation, cytosolic calcium levels ([Ca(2+)](c)) and the intracellular signaling mechanisms involved. GPE and cPG did not change Abeta25-35 levels, but GPE partially restored SRIF and SRIF receptor 2 protein content and mRNA levels and protected against cell death after Abeta25-35 insult, which was coincident with Akt activation and glycogen synthase kinase 3beta inhibition. In addition, GPE displaced glutamate from NMDA receptors and blocked the glutamate induced rise in cytosolic calcium in isolated rat neurons and moderately increased Ca(2+) influx per se. Our findings suggest that GPE, but not its metabolite, mimics insulin-like growth factor I effects on the SRIF system through a mechanism independent of Abeta clearance that involves modulation of calcium and glycogen synthase kinase 3beta signaling.