Glycogen synthase kinase-3ß (GSK3ß) expression in a mouse model of Alzheimer's disease: a light and electron microscopy study.

Glycogen synthase kinase-3ß (GSK3ß) activity has been previously linked to Alzheimer's disease (AD) by its phosphorylation of tau and activation by amyloid. GSK3ß intracellular distribution is important in regulating its activity by restricting access to compartment-specific substrates. This study investigated regional and intracellular distribution of GSK3ß in a mouse model of AD, a bigenic mouse with combined amyloid and tau pathology (BiAT), and controls (FVB). At two different ages, the entire rostrocaudal extent of each brain was examined. Young (6-months-old) FVB and BiAT mice did not differ in GSK3ß expression and localization. In old (13-month-old) BiAT mice, neurons showed increased GSK3ß expression only in AD-relevant brain regions as compared with modest staining in region- and age-matched controls. Two regions with the most robust changes between FVB and BiAT mice, the amygdala and piriform cortex, were quantified at the light microscopic level. In both regions, the density of darkly labeled neurons was significantly greater in the old BiAT mice vs. the old FVB mice. Electron microscopy of the piriform cortex showed neuronal GSK3ß labeling in the rough endoplasmic reticulum, on ribosomes, and on microtubules in dendrites in both strains of mice. In old BiAT mice, GSK3ß labeling was qualitatively more robust compared to age-matched controls, and GSK3ß also appeared in neurofibrillary tangles. In conclusion, GSK3ß expression was increased in specific intracellular locations and was found in tangles in old BiAT mice, suggesting that GSK3ß overexpression in specific brain areas may be intrinsic to AD pathology.

Dkk1 stabilizes Wnt co-receptor LRP6: implication for Wnt ligand-induced LRP6 down-regulation.

BACKGROUND: The low density lipoprotein receptor-related protein-6 (LRP6) is an essential co-receptor for canonical Wnt signaling. Dickkopf 1 (Dkk1), a major secreted Wnt signaling antagonist, binds to LRP6 with high affinity and prevents the Frizzled-Wnt-LRP6 complex formation in response to Wnts. Previous studies have demonstrated that Dkk1 promotes LRP6 internalization and degradation when it forms a ternary complex with the cell surface receptor Kremen. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that transfected Dkk1 induces LRP6 accumulation while inhibiting Wnt/LRP6 signaling. Treatment with Dkk1-conditioned medium or recombinant Dkk1 protein stabilized LRP6 with a prolonged half-life and induces LRP6 accumulation both at the cell surface and in endosomes. We also demonstrated that Kremen2 co-expression abrogated the effect of Dkk1 on LRP6 accumulation, indicating that the effect of Kremen2 is dominant over Dkk1 regulation of LRP6. Furthermore, we found that Wnt3A treatment induces LRP6 down-regulation, an effect paralleled with a Wnt/LRP6 signaling decay, and that Dkk1 treatment blocked Wnt3A-induced LRP6 down-regulation. Finally, we found that LRP6 turnover was blocked by an inhibitor of caveolae-mediated endocytosis. CONCLUSIONS/SIGNIFICANCE: Our results reveal a novel role for Dkk1 in preventing Wnt ligand-induced LRP6 down-regulation and contribute significantly to our understanding of Dkk1 function in Wnt/LRP6 signaling.

Mitochondrial viability in mouse and human postmortem brain.

Neuronal function in the brain requires energy in the form of ATP, and mitochondria are canonically associated with ATP production in neurons. The electrochemical gradient, which underlies the mitochondrial transmembrane potential (DeltaPsi(mem)), is harnessed for ATP generation. Here we show that DeltaPsi(mem) and ATP-production can be engaged in mitochondria isolated from human brains up to 8.5 h postmortem. Also, a time course of postmortem intervals from 0 to 24 h using mitochondria isolated from mouse cortex reveals that DeltaPsi(mem) in mitochondria can be reconstituted beyond 10 h postmortem. It was found that complex I of the mitochondrial electron transport chain was affected adversely with increasing postmortem intervals. Mitochondria isolated from postmortem mouse brains maintain the ability to produce ATP, but rates of production decreased with longer postmortem intervals. Furthermore, we show that postmortem brain mitochondria retain their DeltaPsi(mem) and ATP-production capacities following cryopreservation. Our finding that DeltaPsi(mem) and ATP-generating capacity can be reinitiated in brain mitochondria hours after death indicates that human postmortem brains can be an abundant source of viable mitochondria to study metabolic processes in health and disease. It is also possible to archive these mitochondria for future studies.

Light and electron microscopy study of glycogen synthase kinase-3beta in the mouse brain.

Glycogen synthase kinase-3beta (GSK3beta) is highly abundant in the brain. Various biochemical analyses have indicated that GSK3beta is localized to different intracellular compartments within brain cells. However, ultrastructural visualization of this kinase in various brain regions and in different brain cell types has not been reported. The goal of the present study was to examine GSK3beta distribution and subcellular localization in the brain using immunohistochemistry combined with light and electron microscopy. Initial examination by light microscopy revealed that GSK3beta is expressed in brain neurons and their dendrites throughout all the rostrocaudal extent of the adult mouse brain, and abundant GSK3beta staining was found in the cortex, hippocampus, basal ganglia, the cerebellum, and some brainstem nuclei. Examination by transmission electron microscopy revealed highly specific subcellular localization of GSK3beta in neurons and astrocytes. At the subcellular level, GSK3beta was present in the rough endoplasmic reticulum, free ribosomes, and mitochondria of neurons and astrocytes. In addition GSK3beta was also present in dendrites and dendritic spines, with some postsynaptic densities clearly labeled for GSK3beta. Phosphorylation at serine-9 of GSK3beta (pSer9GSK3beta) reduces kinase activity. pSer9GSK3beta labeling was present in all brain regions, but the pattern of staining was clearly different, with an abundance of labeling in microglia cells in all regions analyzed and much less neuronal staining in the subcortical regions. At the subcellular level pSer9GSK3beta labeling was located in the endoplasmic reticulum, free ribosomes and in some of the nuclei. Overall, in normal brains constitutively active GSK3beta is predominantly present in neurons while pSer9GSK3beta is more evident in resting microglia cells. This visual assessment of GSK3beta localization within the subcellular structures of various brain cells may help in understanding the diverse role of GSK3beta signaling in the brain.

The basal flux of Akt in the mitochondria is mediated by heat shock protein 90.

Akt is a known client protein of heat shock protein 90 (HSP90). We have found that HSP90 is responsible for Akt accumulation in the mitochondria in unstimulated cells. Treatment of SH-SY5Y neuroblastoma cells and human embryonic kidney cells with the HSP90 inhibitors novobiocin and geldanamycin caused substantial decreases in the level of Akt in the mitochondria without affecting the level of Akt in the cytosol. Moreover, intracerebroventricular injection of novobiocin into mice brains decreased Akt levels in cortical mitochondria. Knockdown of HSP90 expression with short interfering RNA also caused a significant decrease in Akt levels in the mitochondria without affecting total Akt levels. Using a mitochondrial import assay it was found that Akt is transported into the mitochondria. Furthermore, it was found that the mitochondrial import of Akt was independent of Akt activation as both an unmodified Akt and constitutively active mutant Akt; both readily accumulated in the mitochondria in an HSP90-dependent manner. Interestingly, incubation of isolated mitochondria with constitutively active Akt caused visible alterations in mitochondrial morphology, including pronounced remodeling of the mitochondrial matrix. This effect was blocked when Akt was mostly excluded from the mitochondria with novobiocin treatment. These results indicate that the level of Akt in the mitochondria is dependent on HSP90 chaperoning activity and that Akt import can cause dynamic changes in mitochondrial configuration.

Unregulated mitochondrial GSK3beta activity results in NADH: ubiquinone oxidoreductase deficiency.

GSK3beta is prominent for its role in apoptosis signaling and has been shown to be involved in Parkinson's disease (PD) pathogenesis. The overall effects of GSK3beta activity on cell fate are well-established, but the effects of mitochondrial GSK3beta activity on mitochondrial function and cell fate are unknown. Here we selectively expressed constitutively active GSK3beta within the mitochondria and found that this enhanced the apoptosis signaling activated by the PD-mimetic NADH:ubiquinone oxidoreductase (complex I) inhibitors 1-methyl-4-phenylpyridinium ion (MPP+) and rotenone. Additionally, expression of GSK3beta in the mitochondria itself caused a significant decrease in complex I activity and ATP production. Increased mitochondrial a GSK3beta activity also increased reactive oxygen species production and perturbed the mitochondrial morphology. Conversely, chemical inhibitors of GSK3beta inhibited MPP+- and rotenone-induced apoptosis, and attenuated the mitochondrial GSK3beta-mediated impairment in complex I. These results indicate that unregulated mitochondrial GSK3beta activity can mimic some of the mitochondrial insufficiencies found in PD pathology.

The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels.

The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3alpha and Ser9 of GSK3beta. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3beta, but not GSK3alpha. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels.

Akt1 is dynamically modified with O-GlcNAc following treatments with PUGNAc and insulin-like growth factor-1.

The Ser/Thr kinase Akt1 is activated by growth factors subsequent to its phosphorylation on Thr308 and Ser473. In the present study, Akt1 was found to be constitutively modified with O-GlcNAc. Treatment of SH-SY5Y cells with O(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), which inhibits the enzymatic removal of O-GlcNAc from proteins, increased cytosolic O-GlcNAc-Akt1 levels. Treatment of cells with insulin-like growth factor-1 (IGF-1) also increased O-GlcNAc-Akt1 levels and increased Akt1 phosphorylation. PUGNAc treatment did not attenuate IGF-1 induced Akt1 phosphorylation. These results indicate that Akt1 can be simultaneously modified with O-GlcNAc and phosphorylated. However, PUGNAc induced the nuclear accumulation of Akt1 suggesting that the O-GlcNAc-modification on Akt1 may play a role in Akt1 nuclear localization.

In vivo regulation of GSK3 phosphorylation by cholinergic and NMDA receptors.

Glycogen synthase kinase-3 (GSK3), which is inhibited by serine-phosphorylation, is involved in the neuropathology of Alzheimer's disease (AD). We tested if the two therapeutic strategies used for AD, inhibition of acetylcholinesterase and of N-methyl-D-aspartate (NMDA) receptors, modulate the phosphorylation state of the two isoforms of GSK3 in mouse brain. Large, rapid increases in the levels of phospho-Ser21-GSK3alpha and phospho-Ser9-GSK3beta occurred in mouse hippocampus, cerebral cortex, and striatum after treatment of mice with the muscarinic agonist pilocarpine or the acetylcholinesterase inhibitor physostigmine. Treatment with memantine, an NMDA receptor antagonist, also increased the serine-phosphorylation of both GSK3 isoforms in mouse brain. Co-administration of physostigmine and memantine increased serine-phosphorylated GSK3 levels equally to that achieved by either agent alone, indicating that the actions of these two drugs converge on overlapping pools of GSK3. Thus, drugs in each class of therapeutic agents used for AD have the common property of increasing the regulatory serine-phosphorylation of GSK3 within common pools of the enzyme.

Regulation of FOXO3a by brain-derived neurotrophic factor in differentiated human SH-SY5Y neuroblastoma cells.

FOXO3a is a ubiquitously expressed mammalian forkhead transcription factor with a high expression level in adult brain. The activity of FOXO3a is inhibited by growth factors through activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, which phosphorylates FOXO3a and decreases the level of FOXO3a in the nucleus. In the present study, we examined the regulation of FOXO3a by brain-derived neurotrophic factor (BDNF) in retinoic acid (RA)-differentiated human SH-SY5Y neuroblastoma cells. BDNF caused a rapid and time-dependent decrease of nuclear FOXO3a with a corresponding increase of cytosolic FOXO3a. The rate of the BDNF-induced nuclear/cytosolic redistribution was consistent with the time course of BDNF-induced threonine32-phosphorylation of FOXO3a, and was mediated by the PI3K/Akt signaling pathway. Active FOXO3a rapidly increased the level of Bcl-2-interacting mediator (bim) in differentiated SH-SY5Y cells, and BDNF decreased the FOXO3a-induced increase of bim through activation of both PI3K/Akt and Erk signaling pathways. Thapsigargin, an endoplasmic reticulum (ER) stress-inducing agent, significantly decreased threonine32-phosphorylation of FOXO3a, and increased nuclear and decreased cytosolic FOXO3a, suggesting that thapsigargin activates FOXO3a. Treatment with BDNF completely reversed and blocked the thapsigargin-induced dephosphorylation and nuclear accumulation of FOXO3a. In addition, protein phosphatase 1/2A inhibitors increased threonine32-phosphorylation of FOXO3a, decreased nuclear FOXO3a, and blocked thapsigargin-induced activity of FOXO3a. The regulatory effect of BDNF on FOXO3a and its target genes may play a significant role in the BDNF-mediated neuronal survival, differentiation, and plasticity.

Glycogen synthase kinase-3 beta is highly activated in nuclei and mitochondria.

Glycogen synthase kinase-3 beta (GSK3 beta) is located predominantly in the cytosol, but also is in nuclei and mitochondria. In SH-SY5Y cells, primary cortical neurons, and mouse brain, the portion of active GSK3 beta (not phosphorylated on serine-9) was 5- to 8-fold greater in nuclei and mitochondria than in cytosol. Correspondingly greater GSK3 beta activities were measured in nuclei and mitochondria compared with cytosol. Stimulation of apoptotic signaling by treatment with camptothecin or thapsigargin activated GSK3 beta in the nucleus and mitochondria, but not the cytosol, whereas inhibition of GSK3 beta by lithium treatment affected all three pools of GSK3 beta. Thus, the nuclei and mitochondria contain disproportionately high levels of active GSK3 beta, which is selectively further activated by some apoptotic stimuli.

Glycogen synthase kinase-3beta (GSK3beta) binds to and promotes the actions of p53.

The recent discovery of direct interactions between two important regulators of cell fate, the tumor suppressor p53 and glycogen synthase kinase-3beta (GSK3beta), led us to examine the mechanism and outcomes of this interaction. Two regions of p53 were identified that regulate its binding to GSK3beta. Deletion of the p53 activation domain-1 (AD1), but not mutations that prevent MDM2 binding through the AD1 domain, enhanced GSK3beta binding to p53, indicating that the AD1 domain interferes with p53 binding to GSK3beta. Deletion of the p53 basic domain (BD) abrogated GSK3beta binding, and a ten amino acid region within the C-terminal BD domain was identified as necessary for binding to GSK3beta. GSK3beta activity was not required for p53 binding, but inhibition of GSK3beta stabilized the association, suggesting a transient interaction during which active GSK3beta promotes actions of p53. This regulatory role of GSK3beta was demonstrated by large reductions of p53-induced increases in the levels of MDM2, p21, and Bax when GSK3beta was inhibited. Besides promoting p53-mediated transcription, GSK3beta also contributed to mitochondrial p53 apoptotic signaling. After DNA damage, mitochondrial GSK3beta co-immunoprecipitated with p53 and was activated, and inhibition of GSK3beta blocked cytochrome c release and caspase-3 activation. Thus, GSK3beta interacts with p53 in both the nucleus and mitochondria and promotes its actions at both sites.

Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3-kinase activation.

We describe here a new component of the phosphatidylinositol 3-kinase/Akt signaling pathway that directly impacts mitochondria. Akt (protein kinase B) was shown for the first time to be localized in mitochondria, where it was found to reside in the matrix and the inner and outer membranes, and the level of mitochondrial Akt was very dynamically regulated. Stimulation of a variety of cell types with insulin-like growth factor-1, insulin, or stress (induced by heat shock), induced translocation of Akt to the mitochondria within only several minutes of stimulation, causing increases of nearly eight- to 12-fold, and the mitochondrial Akt was in its phosphorylated, active state. Two mitochondrial proteins were identified to be phosphorylated following stimulation of mitochondrial Akt, the beta-subunit of ATP synthase and glycogen synthase kinase-3beta. The finding that mitochondrial glycogen synthase kinase-3beta was rapidly and substantially modified by Ser9 phosphorylation, which inhibits its activity, following translocation of Akt to the mitochondria is the first evidence for a regulatory mechanism affecting mitochondrial glycogen synthase kinase-3beta. These results demonstrate that signals emanating from plasma membrane receptors or generated by stress rapidly modulate Akt and glycogen synthase kinase-3beta in mitochondria.

Glycogen synthase kinase-3beta, mood stabilizers, and neuroprotection.

Glycogen synthase kinase-3beta (GSK-3beta) is a central component in many critical intracellular signaling mechanisms. These include the phosphatidylinositol 3-kinase/Akt cell survival pathway, which inhibits GSK-3beta activity. GSK-3beta itself inhibits the activation of several transcription factors, which are important cell survival factors, such as heat shock factor 1. These factors likely contribute to the recent revelation that GSK-3beta is a pro-apoptotic enzyme. Recently, lithium has been identified as a selective and direct inhibitor of GSK-3beta. Based on these findings, we have proposed that part of the neuroprotectant properties of lithium is due to its ability to inhibit GSK-3beta, and thus block the facilitation of apoptosis produced by GSK-3beta. Since several anticonvulsants recently have been shown to be effective mood stabilizers, we examined if these agents are capable of protecting cells from GSK-3beta-facilitated apoptosis. In addition to lithium, both valproic acid and lamotrigine, but not carbamazepine, provided protection from GSK-3beta-facilitated apoptosis in human neuroblastoma SH-SY5Y cells. These results demonstrate that several drugs therapeutic for bipolar disorder can provide neuroprotection by inhibiting the pro-apoptotic effects of GSK-3beta, providing new evidence that dysregulation of GSK-3beta may contribute to the pathophysiology of bipolar disorder.

Direct, activating interaction between glycogen synthase kinase-3beta and p53 after DNA damage.

Glycogen synthase kinase-3beta (GSK3beta) is a central figure in Wnt signaling, in which its activity is controlled by regulatory binding proteins. Here we show that binding proteins outside the Wnt pathway also control the activity of GSK3beta. DNA damage induced by camptothecin, which activates the tumor suppressor p53, was found to activate GSK3beta. This activation occurred by a phosphorylation-independent mechanism involving direct binding of GSK3beta to p53, which was confined to the nucleus where p53 is localized, and mutated p53 (R175H) bound but did not activate GSK3beta. Activation of GSK3 promoted responses to p53 including increases in p21 levels and caspase-3 activity. Thus, after DNA damage there is a direct interaction between p53 and GSK3beta, and these proteins act in concert to regulate cellular responses to DNA damage.