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A major hallmark of Alzheimer's disease (AD) is the deposition of amyloid-β (Aβ) peptides in amyloid plaques. Aβ peptides are produced by sequential cleavage of the amyloid precursor protein by the β amyloid cleaving enzyme (BACE) and the γ-secretase (γ-sec) complex. Pharmacological treatments that decrease brain levels of in particular the toxic Aβ42 peptide are thought to be promising approaches for AD disease modification. Potent and selective BACE1 inhibitors as well as γ-sec modulators (GSMs) have been designed. Pharmacological intervention of secretase function is not without risks of either on- or off-target adverse effects. One way of improving the therapeutic window could be to combine treatment on multiple targets, using smaller individual doses and thereby minimizing adverse effect liability. We show that combined treatment of primary cortical neurons with a BACE1 inhibitor and a GSM gives an additive effect on Aβ42 level change compared with the individual treatments. We extend this finding to C57BL/6 mice, where the combined treatment results in reduction of brain Aβ42 levels reflecting the sum of the individual treatment efficacies. These results show that pharmacological targeting of two amyloid precursor protein processing steps is feasible without negatively interfering with the mechanism of action on individual targets. We conclude that targeting Aβ production by combining a BACE inhibitor and a GSM could be a viable approach for therapeutic intervention in AD modification., (© 2014 FEBS.)

Recent reports have implicated tau-tubulin kinase 1 (TTBK1) in the pathological phosphorylation of tau that occurs in Alzheimer's disease (AD). The present study was undertaken to provide an extensive characterization of TTBK1 mRNA and protein expression in human brain from AD cases and non-demented controls so as to better understand the disease relevance of this novel kinase. In situ hybridization and immunohistochemistry revealed abundant expression of TTBK1 in the somatodendritic compartment of cortical and hippocampal neurons of both AD cases and controls. TTBK1 immunoreactivity appeared to vary with the level of phospho-tau staining, and was strong in the somatodendritic compartment of apparently healthy hippocampal neurons as well as in pre-tangle neurons where it co-localized with diffuse phospho-Ser422 tau staining. Ser422 was confirmed as a TTBK1 substrate in vitro, and an antibody towards the site, in addition to labeling AT8-positive neurofibrillary tangles (NFTs), neuritic plaques and neuropil threads, also labeled a small population of neurons that were unlabeled with AT8. These data suggest a role for TTBK1 in pre-tangle formation prior to the formation of fibrillar tau and strengthen the idea that tau is phosphorylated at Ser422 at an early/intermediate stage in NFT formation., (© 2012 International Society of Neuropathology.)

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.

Mutations in parkin cause autosomal recessive forms of Parkinson's disease (PD), with an early age of onset and similar pathological phenotype to the idiopathic disease. Parkin has been identified as an E3 ubiquitin ligase that mediates different types of ubiquitination, which has made the search for substrates an intriguing possibility to identify pathological mechanisms linked to PD. In this study, we present PLCgamma1 as a novel substrate for parkin. This association was found in non-transfected human neuroblastoma SH-SY5Y cells as well as in stable cell lines expressing parkin WT and familial mutants R42P and G328E. Analysis of cortical, striatal and nigral human brain homogenates revealed that the interaction between parkin and PLCgamma1 is consistent throughout these regions, suggesting that the interaction is likely to have a physiological relevance for humans. Unlike many of the previously identified substrates, we could also show that the steady-state levels of PLCgamma1 is significantly higher in parkin KO mice and lower in parkin WT human neuroblastoma cells, suggesting that parkin ubiquitination of PLCgamma1 is required for proteasomal degradation. In line with this idea, we show that the ability to ubiquitinate PLCgamma1 in vitro differs significantly between WT and familial mutant parkin. In this study, we demonstrate that parkin interacts with PLCgamma1, affecting PLCgamma1 steady state protein levels in human and murine models with manipulated parkin function and expression levels. This finding could be of relevance for finding novel pathogenic mechanisms leading to PD.

Mutations in the E3 ubiquitin ligase parkin cause early-onset, autosomal-recessive juvenile parkinsonism (AJRP), presumably as a result of a lack of function that alters the level, activity, aggregation or localization of its substrates. Recently, we have reported that phospholipase Cgamma1 is a substrate for parkin. In this article, we show that parkin mutants and siRNA parkin knockdown cells possess enhanced levels of phospholipase Cgamma1 phosphorylation, basal phosphoinositide hydrolysis and intracellular Ca2+ concentration. The protein levels of Ca2+-regulated protein kinase Calpha were decreased in AJRP parkin mutant cells. Neomycin and dantrolene both decreased the intracellular Ca2+ levels in parkin mutants in comparison with those seen in wild-type parkin cells, suggesting that the differences were a consequence of altered phospholipase C activity. The protection of wild-type parkin against 6-hydroxydopamine (6OHDA) toxicity was also established in ARJP mutants on pretreatment with dantrolene, implying that a balancing Ca2+ release from ryanodine-sensitive stores decreases the toxic effects of 6OHDA. Our findings suggest that parkin is an important factor for maintaining Ca2+ homeostasis and that parkin deficiency leads to a phospholipase C-dependent increase in intracellular Ca2+ levels, which make cells more vulnerable to neurotoxins, such as 6OHDA.

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

Presenilin (PS1 and PS2) mutations cause early-onset familial Alzheimer's disease (AD). In addition to affecting beta-amyloid precursor protein (APP) processing and Abeta generation, PSs regulate a number of signaling pathways. We previously showed that PSs regulate both phospholipase C (PLC) and protein kinase C (PKC) alpha and gamma activities. We also reported that PS double knockout mouse embryonic fibroblasts (MEFs) have reduced levels of PKCalpha and enhanced levels of PKCdelta. Here, we determined whether the PS modulation of PLC/PKC has consequences for extracellular regulated kinase (Erk) signaling. Erk has been suggested to be important in AD pathology by modulating APP processing and tau phosphorylation. We found that knocking out PS1 or PS2 alone resulted in increased Erk activity and that this effect could be reversed by the PKCalpha inhibitor Gö6976. We also found that Erk activity following either PLC or PKC stimulation was significantly lower in PS double knockout cells and that treatment with the PKC activator phorbol 12,13-dibutyrate (PdBu) down-regulated total-Erk levels in all cells except PS double knockouts. These results demonstrate that PSs regulate Erk activity through a PKCalpha dependent pathway and that disruption of PLC/PKC signaling in the absence of both PS1 and PS2 results in lower downstream activation of Erk.

Presenilin proteins, mutated forms of which cause early onset familial Alzheimer's disease, are capable of modulating various cell signal transduction pathways, the most extensively studied of which has been intracellular calcium signalling. Disease causing presenilin mutations can potentiate inositol(1,4,5)trisphosphate (InsP3) mediated endoplasmic reticulum release due to calcium overload in this organelle, as well as attenuate capacitative calcium entry. Our own studies have shown a novel function for presenilins that involves regulation of acetylcholine muscarinic receptor-stimulated phospholipase C upstream of InsP3 regulated calcium release. This article reviews the mechanisms by which presenilins modulate intracellular calcium signalling and the role that deregulated calcium homeostasis could play in the pathogenesis of Alzheimer's disease.

Presenilins (PSs) are involved in processing several proteins such as the amyloid precursor protein (APP), as well as in pathways for cell death and survival. We previously showed that some familial Alzheimer's disease PS mutations cause increased basal and acetylcholine muscarinic receptor-stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha and PKCgamma activations were significantly lower in PS1 and PS2 double knockout MEFs after PLC stimulation. Protein levels of PKCalpha and PKCgamma were lower in PS1 and PS2 double knockout MEFs. In contrast, PKCdelta levels were significantly elevated in PS1 and PS2 double knockout as well as in PS1 knockout MEFs. Also, PKCdelta levels were lowered after transfection of PS1 into PS1 knockout or PS double knockout MEFs. Using APP knockout MEFs we showed that the expression of PKCalpha, but not the other PKC isoforms is partially dependent on APP and can be regulated by APP intracellular domain (AICD). These results show that PLC and PKC activations are modulated by PS and also that PSs differentially regulate the expression of PKC isoforms by both APP/AICD-dependent and independent mechanisms.

Increasing evidence indicates that mitochondrial alterations contribute to the neuronal death in Alzheimer's disease (AD). Presenilin 1 (PS1) and Presenilin 2 (PS2) mutations have been shown to sensitize cells to apoptosis by mechanisms suggested to involve impaired mitochondrial function. We have previously detected active gamma-secretase complexes in mitochondria. We investigated the impact of PS/gamma-secretase on mitochondrial function using mouse embryonal fibroblasts derived from wild-type, PS1-/-, PS2-/- and PS double knock-out (PSKO) embryos. Measurements of mitochondrial membrane potential (DeltaPsim) showed a higher percentage of fully functional mitochondria in PS1-/- and PSwt as compared to PS2-/- and PSKO cells. This result was evident both in whole cell preparations and in isolated mitochondria. Interestingly, pre-treatment of isolated mitochondria with the gamma-secretase inhibitor L-685,458 resulted in a decreased population of mitochondria with high DeltaPsim in PSwt and PS1-/- cells, indicating that PS2/gamma-secretase activity can modify DeltaPsim. PS2-/- cells showed a significantly lower basal respiratory rate as compared to other cell lines. However, all cell lines demonstrated competent bioenergetic function. These data point toward a specific role of PS2/gamma-secretase activity for proper mitochondrial function and indicate interplay between PS1 and PS2 in mitochondrial functionality.

Strong evidence indicates oxidative stress in the pathogenesis of Alzheimer's disease (AD). Amyloid beta (Abeta) has been implicated in both oxidative stress mechanisms and in neuronal apoptosis. Glutaredoxin-1 (GRX1) and thioredoxin-1 (TRX1) are antioxidants that can inhibit apoptosis signal-regulating kinase (ASK1). We examined levels of GRX1 and TRX1 in AD brain as well as their effects on Abeta neurotoxicity. We show an increase in GRX1 and a decrease in neuronal TRX1 in AD brains. Using SH-SY5Y cells, we demonstrate that Abeta causes an oxidation of both GRX1 and TRX1, and nuclear export of Daxx, a protein downstream of ASK1. Abeta toxicity was inhibited by insulin-like growth factor-I (IGF-I) and by overexpressing GRX1 or TRX1. Thus, Abeta neurotoxicity might be mediated by oxidation of GRX1 or TRX1 and subsequent activation of the ASK1 cascade. Deregulation of GRX1 and TRX1 antioxidant systems could be important events in AD pathogenesis.

PTEN is a dual phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway important for cell survival. We determined effects of the inflammation and oxidative stresses of tumor necrosis factor-alpha (TNFalpha) and trans-4-hydroxy-2-nonenal (HNE), respectively, on PTEN, Akt, and GSK3beta signalling in rat primary cortical neurons. The inhibitors bisperoxovanadium [bpV(Pic)] and LY294002 were also used to determine PTEN and PI3K involvement in TNFalpha and HNE modulation of neuronal cell death. PTEN inhibition with bpV(Pic) alone did not affect Ser(473)Akt or Ser(9)GSK3beta phosphorylation. Instead, effects of this inhibitor were manifest when it was used together with TNFalpha and to a lesser extent with HNE. TNFalpha together with PTEN inhibition increased phosphorylation of Ser(473)Akt and Ser(9)GSK3beta. TNFalpha and HNE both gave decreased numbers of viable and increased numbers of early apoptotic neurons. PTEN inhibition partially reversed the toxic effect of TNFalpha as shown by an increased number of viable and a decreased number of early apoptotic neurons. All effects were reversed by PI3K inhibition. HNE together with inhibition of PTEN gave increased Ser(473)Akt but not Ser(9)GSK3beta phosphorylation and no effects on the number of viable or early apoptotic cells. In conclusion, PTEN inhibition gives a mild reversal of TNFalpha- but not HNE-induced cell death via the PI3K pathway.

The hallmarks of Alzheimer's disease include extracellular plaques primarily consisting of amyloid-beta peptide and intracellular neurofibrillary tangles composed of highly phosphorylated tau protein. We report that exposure of organotypic hippocampal cultures to synthetic amyloid-beta peptide(25-35) (50 microM, 96 h) causes neurodegeneration concomitant with a significant increase in tau phosphorylation at the Ser epitope (+60%). Furthermore, the level of active glycogen synthase kinase-3beta (GSK-3beta [pTyr]) was increased (+55%) after amyloid-beta peptide(25-35) exposure. These findings support the role of amyloid-beta peptide as a mediator of tau phosphorylation and demonstrate the usefulness of organotypic cultures for investigating the link between amyloid-beta peptide-induced neurotoxicity and tau phosphorylation. Our results also confirm that amyloid-beta peptide induces activation of glycogen synthase kinase-3beta.

Neurofibrillary tangles (NFT) of hyperphosphorylated tau protein are a major pathological hallmark of Alzheimer's disease (AD). One of the tau phosphorylating kinases with pathological relevance in AD has been suggested to be the cyclin-dependent kinase 5 (Cdk5). The proposed mechanism leading to pathological Cdk5 activity is through induced cleavage of p35 to a proteolytic product, p25. To further study activation of Cdk5 and its role in tau phosphorylation in vitro, we used differentiated SH-SY5Y cells treated with neurotoxic stimuli or transfected with p25. We show that glutamate increased tau phosphorylation, concomitant with an increased Cdk5 activity achieved by upregulation of Cdk5 and p35 protein levels. Treatment with the calcium ionophore A23187 generated the calpain cleaved p25 fragment but only in toxic conditions that caused dephosphorylation and loss of tau. When p25 was transfected to the cells, increased tau phosphorylation was achieved. However, application of the Cdk5 inhibitor Roscovitine did not result in inhibition of tau phosphorylation possibly due to activation of extracellular regulated kinase 1/2 (Erk1/2), which also is capable of phosphorylating tau. Cdk5 and Erk1/2 kinases share some common substrates but impact of their cross talk on tau phosphorylation has not previously been demonstrated. We also show that p25 is degraded via the proteasome in Roscovitine treated cells.

Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy.

Phospholipid changes occur in brain regions affected by Alzheimer disease (AD), including a marked reduction in plasmalogens, which could diminish brain function either by directly altering signaling events or by bulk membrane effects. However, model systems for studying the dynamics of lipid biosynthesis in AD are lacking. To determine if fibroblasts bearing the Swedish amyloid precursor protein (swAPP) mutation are a useful model to study the mechanism(s) associated with altered phospholipid biosynthesis in AD, we examined the steady-state phospholipid mass and composition of fibroblasts, including plasmalogens. We found a 15% increase in total phospholipid mass, accounted for by a 24% increase in the combined total of phosphatidylethanolamine and plasmanylethanolamine mass and a 19% increase in the combined total of phosphatidylcholine (PtdCho) and plasmanycholine (PakCho) mass in the swAPP mutant bearing fibroblasts. Cholesterol mass was unchanged in these cells. The changes in phospholipid mass did not alter the cellular molar composition of the phospholipids nor the cholesterol to phospholipid ratio. While plasmalogen mass was not altered, the ratio of choline plasmalogen (PlsCho) mass to PtdCho+PakCho mass was decreased 16% and there was a 14% reduction in the proportion of PlsCho as a percent of total phospholipids in the swAPP mutant bearing fibroblasts. This change in choline plasmalogen is consistent with the reported decreases in plasmalogen proportions in affected regions of AD brain, suggesting that these cells may serve as a useful model to determine the mechanism underlying changes in plasmalogen biosynthesis in AD brain.

Pronounced neurodegeneration of hippocampal pyramidal neurons has been shown in Alzheimer's disease. The aim of this study was to establish an organotypic in vitro model for investigating effects of the amyloid beta (Abeta)-peptide on pyramidal neuron degeneration, glial cell activation and tau phosphorylation. Tissue cultures in a quasi-monolayer were obtained using roller-drum incubation of hippocampal slices from neonatal Sprague Dawley rats. Neuronal populations identified included N-methyl-D-aspartate (NMDA-R1) receptor immunoreactive pyramidal neurons, and neurons immunopositive for glutamic acid decarboxylase-65 (GAD65) or gamma amino butyric acid (GABA). Many neurons expressed phosphorylated tau as shown by pS(396), AD2 and PHF-tau immunostaining. Astrocytes, microglial cells and macrophages were also identified. The Abeta(25-35) peptide formed fibrillar networks within 2 days as demonstrated by electron microscopy. In the presence of the neurotoxic Abeta(25-35) peptide, but not Abeta(35-25), deposits developed in the tissue that were stainable with Thioflavine T and Congo red and showed the characteristic birefringence of Abeta plaques. Following Abeta(25-35) exposure, neurodegenerative cells were observed with Fluoro-Jade B staining. Further characterization of pyramidal neurons immunopositive for NMDA-R1 showed a decrease of cell number in the immediate surrounding of Abeta(25-35) deposits in a time- and concentration-dependent fashion. Similar effects on pyramidal neurons were obtained following exposure to the full-length, Abeta(1-40) peptide. Also, a loss of neuronal processes was seen with GAD65, but not GABA, immunohistochemistry after exposure to Abeta(25-35). Abeta(25-35)-exposed neurons immunopositive for phospho-tau showed degenerating, bent and often fragmented processes. Astrocytes showed increased GFAP-positive reactivity after Abeta(25-35) exposure and formation of large networks of processes. No obvious effect on microglial cells and macrophages could be seen after the Abeta(25-35) exposure. The developed in vitro system may constitute a useful tool for screening novel drugs against Abeta-induced alterations of tau and degeneration of hippocampal neurons.

Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) is a dual (protein tyrosine and lipid) phosphatase one of the functions of which is to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol-3,4-biphosphate thereby inhibiting phosphoinositide-dependent kinase activation of the cell survival kinase Akt. Akt activity is up regulated in Alzheimer's disease (AD) brain in parallel to the progression of neurofibrillary pathology. The present study determined whether altered expression of PTEN occurs in Alzheimer's disease brain. Western immunoblotting revealed no significant changes of PTEN protein levels in nuclear and membrane fractions of medial temporal cortex from a series of Alzheimer's disease and control cases. Similarly, no changes in PTEN protein levels, as determined by dot-blotting, were seen in temporal cortex homogenates from a separate series of Alzheimer's disease and control brains. A small but significant decrease in the levels of Ser(380) p-PTEN was seen in homogenates of Alzheimer's disease temporal cortex. Immunohistochemistry revealed PTEN immunoreactivity in a number of brain structures including neurons, capillaries and structures resembling oligodendrocytes and astrocytes. The majority of temporal cortex pyramidal neurons (93-100%) were PTEN immunopositive. The Alzheimer's disease cases had significantly lower numbers of total ( approximately 12% loss, P<0.02) and PTEN immunopositive ( approximately 15% loss, P<0.01) pyramidal neurons as compared to the control cases.

The mechanisms associated with cell death have been an important focus for neurobiology research. In the present study, the methodology of flow cytometry was used to optimize quantification of the toxic effects of tumor necrosis factor-alpha (TNF-alpha), trans-4-hydroxy-2-nonenal (4-HNE), and aged amyloid-beta (Abeta1-42) on rat primary cortical neurons. The fluorescent dyes annexin V-FITC and propidium iodide (PI) were used to identify populations of viable, early apoptotic, necrotic and late apoptotic cells by flow cytometry. Prior to exposure, the primary cultures showed 83% cell viability. Flow cytometry following labeling of cells with a specific neuronal marker, TUJ-1, revealed 82% pure neuronal populations, whereas approximately 7% were astrocytic as shown by glial fibrillary acidic protein positivity. Exposure of primary cultures to TNF-alpha, 4-HNE, and aged Abeta1-42 gave an increased number of early apoptotic cells. We show that flow cytometry is a suitable method for quantifying effects of different stressors on neurons in primary cultures. This technique could be useful for screening and testing of pharmacological compounds relevant to neurodegenerative disorders., (Copyright (c) 2005 Wiley-Liss, Inc.)

Zinc levels are increased in brain areas severely affected by Alzheimer's disease (AD) pathologies. Zinc has both protective and neurotoxic properties and can stimulate both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Several kinases related to these pathways including protein kinase B (PKB), p70 S6 kinase (p70S6K), and extracellular signal-regulated kinase 1/2 (ERK1/2) are known cell survival factors and are overactivated in neurons bearing neurofibrillary tangles (NFTs) in AD. The present study aimed to determine whether anti-apoptotic effects of zinc are mediated via these signaling pathways. Zinc was used to treat SH-SY5Y neuroblastoma cells and effects investigated in relation to PKB, p70S6K, and ERK1/2 in the absence and presence of the pro-apoptotic agent staurosporine (STS). Cell damage was evaluated by measuring levels of DNA fragmentation as well as the WST-1 assay for cell viability. Results indicated that: (1) treatment with high doses of zinc (>/=400 microM) for short time periods (2 h reversed an increased DNA fragmentation due to U0126 inhibition of ERK1/2; (3) increased DNA fragmentation due to STS could be protected against by 100 microM zinc; (4) the protective effects of 100 microM zinc on STS-induced DNA fragmentation could be partially reversed by U0126. These results indicate that a zinc-induced anti-apoptotic response in SH-SY5Y cells likely occurs through ERK1/2.

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.

Abeta (beta-amyloid) peptides are found aggregated in the cortical amyloid plaques associated with Alzheimer's disease neuropathology. Inhibition of the proteasome alters the amount of Abeta produced from APP (amyloid precursor protein) by various cell lines in vitro. Proteasome activity is altered during aging, a major risk factor for Alzheimer's disease. In the present study, a human neuroblastoma cell line expressing the C-terminal 100 residues of APP (SH-SY5Y-SPA4CT) was used to determine the effect of proteasome inhibition, by lactacystin and Bz-LLL-COCHO (benzoyl-Leu-Leu-Leu-glyoxal), on APP processing at the gamma-secretase site. Proteasome inhibition caused a significant increase in Abeta peptide levels in medium conditioned by SH-SY5Y-SPA4CT cells, and was also associated with increased cell death. APP is a substrate of the apoptosis-associated caspase 3 protease, and we therefore investigated whether the increased Abeta levels could reflect caspase activation. We report that caspase activation was not required for proteasome-inhibitor-mediated effects on APP (SPA4CT) processing. Cleavage of Ac-DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin), a caspase substrate, was reduced following exposure of SH-SY5Y-SPA4CT cells to lactacystin, and co-treatment of cells with lactacystin and a caspase inhibitor [Z-DEVD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone)] resulted in higher Abeta levels in medium, augmenting those seen with lactacystin alone. This study indicated that proteasome inhibition could increase APP processing specifically at the gamma-secretase site, and increase release of Abeta, in the absence of caspase activation. This indicates that the decline in proteasome function associated with aging would contribute to increased Abeta levels.

Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mitochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzheimer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familial forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active gamma-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the beta-amyloid precursor protein (beta-APP) generating the amyloid beta-peptide and the beta-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. gamma-secretase activity in isolated mitochondria was demonstrated using C83 (alpha-secretase-cleaved C-terminal 83-residue beta-APP fragment from BD8 cells lacking presenilin and thus gamma-secretase activity) or recombinant C100-Flag (C-terminal 100-residue beta-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the gamma-secretase inhibitors l-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of gamma-secretase activity to mitochondria.

Notch signaling in vertebrates is mediated by four Notch receptors (Notch-1, -2, -3, and -4) that are activated by interacting with at least five different Notch ligands, Jagged-1, Jagged-2, Delta-1, -2, and -3. Recent studies have shown that the gamma-secretase-like intramembranous cleavage of Notch receptors to release their cytoplasmic signaling domains requires the presenilin (PS) proteins 1 and 2 (PS1 and PS2). Here, we used immunohistochemistry to compare the distribution of all four Notch receptor proteins and three ligands in the context of co-localization with PS1 and PS2 in first trimester human central nervous system (CNS). In addition, we investigated Notch receptors and ligands expression by Western blotting. The study was performed on the forebrain and spinal cord of human embryonic/foetal CNS (5-11 gestational weeks). Results showed a divergent distribution of the different Notch receptor proteins with only Notch-1 being co-localized with PS1 and PS2. Notch-2 was only seen occasionally within the developing cortex and spinal cord. Notch-3 expression was restricted to neuroepithelial cells of the spinal cord and endothelial cells in blood vessels of both developing cerebral cortex and spinal cord. The weak, punctate staining of Notch-4 in the neuroepithelium of the spinal cord could not be confirmed with Western blotting. Neither Notch-2, nor -3 showed overlap with either PS1 or PS2 immunoreactivity. The ligand Jagged-1 was found sporadically in the neuroepithelial cell layer in cerebral cortex of the earlier stages of development and of the spinal cord during the first trimester while Jagged-2 was not detected. Jagged-1 and Jagged-2 immunoreactivities were not found in the 9-11-week cortex. No co-distribution of Jagged-1 and PS1 or PS2 was found. Delta-1 ligand expression was detected in neuroepithelial cells of the ventricular zone of the cerebral cortex, and also in maturating neurons in the cortical plate and ventral horns of the developing spinal cord. The presence of Notch-1, Delta-1 and Jagged-1 in the neuroepithelium of developing CNS indicates that Notch signaling in proliferating human progenitor cells only involves these two receptor ligands and that cleavage of Notch-1 is mediated both by PS1 and PS2. The strong immunoreactivity of Notch-1, Delta-1 and PS1 in the cortical plate and in maturating neurons of the spinal cord also suggests that these proteins may regulate the maturation processes of post-mitotic neurons. The pronounced PS1 immunoreactivity in neurites in the hindbrain and spinal cord without detectable expression of any Notch receptor or ligand suggests that a possible role for PS1 in neurite growth involves either gamma-secretase-mediated cleavage of other substrates or gamma-secretase-independent mechanisms.

The paired helical filaments of highly phosphorylated tau protein are the main components of neurofibrillary tangles (NFT) in Alzheimer's disease (AD). Protein kinases including glycogen synthase kinase 3 beta (GSK3beta), cyclin-dependent kinase 5 (Cdk5), and c-Jun N-terminal kinase (JNK) have been implicated in NFT formation making the use of selective kinase inhibitors an attractive treatment possibility in AD. When sequentially treated with retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), the human neuroblastoma SH-SY5Y differentiates to neuron-like cells. We found that coincident with morphologically evident neurite outgrowth, both the content and phosphorylation state of tau increased in RA-BDNF differentiated SH-SY5Y cells. Tau phosphorylation increased at all the examined sites ser-199, ser-202, thr-205, ser-396, and ser-404, all of which are hyperphosphorylated in AD brain. We also investigated whether GSK3beta, Cdk5 or JNK was involved in tau phosphorylation in the differentiated SH-SY5Y cells. We found that GSK3beta contributed most and that Cdk5 made a minor contribution. JNK was not involved in tau phosphorylation in this system. The GSK3beta-inhibitor, lithium, inhibited tau phosphorylation in a concentration-dependent manner and with good reproducibility, which enables ranking of substances in this cell model. RA-BDNF differentiated SH-SY5Y cells could serve as a suitable model for studying the mechanisms of tau phosphorylation and for screening potential GSK3beta inhibitors.

Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.

Enzyme activities of the serine/threonine kinase Akt were compared in mid-temporal and mid-frontal cortices from Alzheimer's disease cases and matched controls. Activities (GSK-3alpha/beta fusion protein phosphorylation by immunoprecipitated Akt) were significantly increased in temporal cortex soluble fractions from Alzheimer's disease compared with non-disease controls and positive disease controls with another neurodegenerative disease. Temporal cortex soluble fraction Akt activities positively correlated with Braak staging for neurofibrillary changes. Frontal cortex soluble fraction activities were significantly reduced in positive disease compared with Alzheimer's disease cases and non-disease controls. Strong Ser Akt immunoreactivity was seen in Alzheimer's disease pyramidal neurons likely undergoing degeneration and in reactive astroglia. Non-disease and positive disease controls showed moderate Ser Akt immunostaining of occasional pyramidal neurons.

Familial Alzheimer's disease (FAD) presenilin 1 (PS1) mutations give enhanced calcium responses upon different stimuli, attenuated capacitative calcium entry, an increased sensitivity of cells to undergo apoptosis, and increased gamma-secretase activity. We previously showed that the FAD mutation causing an exon 9 deletion in PS1 results in enhanced basal phospholipase C (PLC) activity (Cedazo-Minguez, A., Popescu, B. O., Ankarcrona, M., Nishimura, T., and Cowburn, R. F. (2002) J. Biol. Chem. 277, 36646-36655). To further elucidate the mechanisms by which PS1 interferes with PLC-calcium signaling, we studied the effect of two other FAD PS1 mutants (M146V and L250S) and two dominant negative PS1 mutants (D257A and D385N) on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular calcium concentrations ([Ca2+]i) in SH-SY5Y neuroblastoma cells. We found a significant increase in basal PI hydrolysis in PS1 M146V cells but not in PS1 L250S cells. Both PS1 M146V and PS1 L250S cells showed a significant increase in carbachol-induced [Ca2+]i as compared with nontransfected or wild type PS1 transfected cells. The elevated carbachol-induced [Ca2+]i signals were reversed by the PLC inhibitor neomycin, the ryanodine receptor antagonist dantrolene, the general aspartyl protease inhibitor pepstatin A, and the specific gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. The cells expressing either PS1 D257A or PS1 D385N had attenuated carbachol-stimulated PI hydrolysis and [Ca2+]i responses. In nontransfected or PS1 wild type transfected cells, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester and pepstatin A also attenuated both carbachol-stimulated PI hydrolysis and [Ca2+]i responses to levels found in PS1 D257A or PS1 D385N dominant negative cells. Our findings suggest that PS1 can regulate PLC activity and that this function is gamma-secretase activity-dependent.

Glycogen synthase kinase-3beta (GSK-3beta) is implicated in regulating apoptosis and tau protein hyperphosphorylation in Alzheimer's disease (AD). We investigated the effects of two key AD molecules, namely apoE (E3 and E4 isoforms) and beta-amyloid (Abeta) 1-42 on GSK-3beta and its major upstream regulators, intracellular calcium and protein kinases C and B (PKC and PKB) in human SH-SY5Y neuroblastoma cells. ApoE3 induced a mild, transient, Ca2+-independent and early activation of GSK-3beta. ApoE4 effects were biphasic, with an early strong GSK-3beta activation that was partially dependent on extracellular Ca2+, followed by a GSK-3beta inactivation. ApoE4 also activated PKC-alpha and PKB possibly giving the subsequent GSK-3beta inhibition. Abeta(1-42) effects were also biphasic with a strong activation dependent partially on extracellular Ca2+ followed by an inactivation. Abeta(1-42) induced an early and potent activation of PKC-alpha and a late decrease of PKB activity. ApoE4 and Abeta(1-42) were more toxic than apoE3 as shown by MTT reduction assays and generation of activated caspase-3. ApoE4 and Abeta(1-42)-induced early activation of GSK-3beta could lead to apoptosis and tau hyperphosphorylation. A late inhibition of GSK-3beta through activation of upstream kinases likely compensates the effects of apoE4 and Abeta(1-42) on GSK-3beta, the unbalanced regulation of which may contribute to AD pathology.

In Alzheimer's disease (AD) brain the activity of protein phosphatase (PP)-2A is compromised and that of the extracellular signal-regulated protein kinase (ERK1/2) of the mitogen-activated protein kinase (MAPK) family, which can phosphorylate tau, is up-regulated. We investigated whether a decrease in PP-2A activity could underlie the activation of these kinases and the abnormal hyperphosphorylation of tau. Rat brain slices, 400-microm-thick, kept under metabolically active conditions in oxygenated (95% O(2), 5% CO(2)) artificial CSF were treated with 1.0 micromol/L okadaic acid (OA) for 1 hour at 33 degrees C. Under this condition, PP-2A activity was decreased to approximately 35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, we observed a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase both immunohistochemically and by Western blots using phosphorylation-dependent antibodies against these kinases. Treatment of 6-microm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation/activation of these kinases. Hyperphosphorylation of tau at several abnormal hyperphosphorylation sites was also observed, as seen in AD brain. These results suggest 1) that PP-2A down-regulates ERK1/2, MEK1/2, and p70 S6 kinase activities through dephosphorylation at the serine/threonine residues of these kinases, and 2) that in AD brain the decrease in PP-2A activity could have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase, and the abnormal hyperphosphorylation of tau both via an increase in its phosphorylation and a decrease in its dephosphorylation.

G-protein alpha-subunits (Galphao, Galphai, Galphas, Galphaq) and adenylyl cyclase (AC) I and II isoforms were quantified in hippocampus and entorhinal cortex from 22 cases staged for Alzheimer's disease (AD) pathologies according to Braak and Braak. Hippocampal Galphai levels declined significantly with neurofibrillary staging, whereas AC I levels in this region increased. Significant amyloid stage-related reductions of Galphai were seen in both the hippocampus and entorhinal cortex. The hippocampus also showed a significant reduction of Galphao with amyloid staging. It is concluded that levels of inhibitory G-protein subunits Galphao, and in particular Galphai, decrease in parallel to the extent of AD pathology.

The ribosomal S6 protein kinase p70 S6 kinase is known for its role in modulating cell-cycle progression, cell size, and cell survival. In response to mitogen stimulation, p70 S6 kinase activation up-regulates ribosomal biosynthesis and enhances the translational capacity of the cell. In Alzheimer's disease (AD), there is a marked increase in total tau protein in the form of abnormally hyperphosphorylated tau (PHF-tau) in neurons with neurofibrillary tangles (NFTs). In the present study, we investigated whether p70 S6 kinase activation is associated with PHF-tau accumulation in AD. By immunohistochemistry, we found that the levels of phosphorylated p70 S6 kinase (at Thr389 or at Thr421/Ser424) were increased in accordance with the progressive sequence of neurofibrillary changes according to Braak's criteria. Confocal microscopy showed that in AD brain, phosphorylated p70 S6 kinase appeared especially in neurons that are known to later develop NFTs. This pattern of neurons showed dot-like structures of phosphorylated p70 S6 kinase and hyperphosphorylated tau, which partially correlated with rab5 (endosome marker), lamp-1 (lysosome marker), and ubiquitin (ubiquitin-proteasomal system marker). By indirect enzyme-linked immunosorbent assay, phosphorylated p70 S6 kinase (Thr389 or Thr421/Ser424), total tau, and PHF-tau were found to be significantly increased in AD brain as compared to control cases. The levels of total p70 S6 kinase and p70 S6 kinase phosphorylated at Thr421/Ser424 showed significant correlations with the levels of both total tau and PHF-tau. Regression analyses revealed a significant dependence of total tau or PHF-tau on p70 S6 kinase phosphorylated at Thr421/Ser424 rather than at Thr389. The levels of ribosomal protein S6 as well as the levels of markers for the proteolytic system were also significantly increased in AD as compared to control brain. Using a SH-SY5Y neuroblastoma cell model, we found that 100 micro mol/L zinc sulfate could induce p70 S6 kinase phosphorylation and activation, in particular at Thr421/Ser424. This up-regulation of the activated kinase resulted in an increased expression and phosphorylation of tau. Pretreatment of cells with rapamycin (an inhibitor of FRAP/mTOR which is the immediate upstream kinase of the p70 S6 kinase) attenuated the effects induced by zinc. In primary cultured neurons of rat cortical cortex, zinc sulfate treatment could repeat p70 S6 kinase phosphorylation and activation at Thr421/Ser424, followed by increased expression and phosphorylation of tau. Taken together, these data suggest that activated p70 S6 kinase could mediate an up-regulation of tau translation. The partial co-localization of phosphorylated p70 S6 kinase with rab5, lamp-1 and ubiquitin, or PHF-tau with ubiquitin suggests that the activated proteolytic system might not be sufficient to degrade the over-produced and over-phosphorylated tau protein. A p70 S6 kinase modulated up-regulation of tau translation might contribute to PHF-tau accumulation in neurons with neurofibrillary changes.

Protein kinase B (PKB) is an important intermediate in the phosphatidylinositol-3 kinase signaling cascade that acts to phosphorylate glycogen synthase kinase-3 (GSK-3) at its serine 9 residue, thereby inactivating it. Activated GSK-3 has been previously shown to be preferentially associated with neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brain. In the present study, we performed immunohistochemistry with an antibody to the active form of PKB in brains with different stages of neurofibrillary degeneration. We found that the amount of activated PKB (p-Thr308) increased in correlation to the progressive sequence of AT8 immunoreactivity and neurofibrillary changes assessed according to Braak's criteria. By confocal microscopy, activated PKB (p-Thr308) was found to appear in particular in neurons that are known to later develop NFTs in AD. Western blotting showed that activated PKB was increased by more than 50% in the 16,000- g supernatants of AD brains as compared with normal aged and Huntington's disease controls. This increase in PKB levels corresponded with a several-fold increase in the levels of total tau and abnormally hyperphosphorylated tau at the Tau-1 site. These studies suggest the involvement of PKB/GSK-3 signaling in Alzheimer neurofibrillary degeneration.

The abnormal hyperphosphorylation of tau in Alzheimer's disease (AD) has been proposed to involve the extracellular-signal-regulated protein kinase (ERK) of the mitogen-activated protein (MAP) kinase family. ERK is phosphorylated and thereby activated by MAP kinase kinase (MEK). In the present study, we determined the intracellular and regional distribution of the active forms of both MEK1/2 and ERK1/2, i.e. p-MEK1/2 and p-ERK1/2 in the entorhinal, hippocampal, and temporal cortices of 49 brains staged for neurofibrillary changes according to Braak and Braak's protocol. We found that p-MEK1/2 and p-ERK1/2 were present in the initial stages of neurofibrillary degeneration in the projecting neurons in the transentorhinal region, and extended into other brain regions co-incident with the progressive sequence of neurofibrillary changes up to and including Braak stage VI. It appeared that the accumulation of p-MEK1/2 and p-ERK1/2 was initiated in the cytoplasm of pretangle neurons in varying size granules, which grew into large aggregates co-existing with the progressive development of neurofibrillary tangles. Accumulation of p-MEK1/2 and p-ERK1/2 was found in cases with stages I-III neurofibrillary degeneration, which were devoid of amyloid deposition. These data provide direct in situ evidence consistent with the possible involvement of MAP kinase pathway in the hyperphosphorylation of tau and the presence of this lesion before deposition of beta-amyloid in AD.

The major component of Alzheimer's disease (AD) neurofibrillary tangles (NFTs) is abnormally hyperphosphorylated tau aggregated as paired helical filaments (PHFs). Cell division cycle (cdc) 2 kinase is one of the main candidate kinases that phosphorylates normal tau in vitro at several sites seen in PHF-tau. Using brains staged according to Braak and Braak criteria, we investigated the role of cdc2 in neurofibrillary changes in the hippocampal formation, and the entorhinal and temporal cortices. Neurons with tangle-like inclusions positive for active cdc2 were found to appear first in the Pre-alpha layer of the entorhinal cortex, and then extend to other brain regions co-incident with the progressive sequence of neurofibrillary changes. This predictable progressive pattern is not associated with amyloid. The intraneuronal accumulation of active cdc2 appeared to precede the deposition of PHF-tau phosphorylated at Ser 202/Thr 205 sites. These data are consistent with the notion that cdc2 might be involved in the abnormal hyperphosphorylation of tau and consequently aggregation of tau into PHF at an early stage and that increased cdc2 activity is not consequent to the deposition of beta-amyloid in AD brain.

We studied effects of the familial Alzheimer's disease presenilin 1 (PS1) exon 9 deletion (PS1-DeltaE9) mutation on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular Ca(2+) concentrations ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. We demonstrate that PS1-DeltaE9 cells have an enhanced basal PI hydrolysis and [Ca(2+)](i) as compared with both wild type PS1 (PS1-WT) and nontransfected (NT) cells. Both were reversed by the phospholipase C (PLC) inhibitor neomycin. The PS1-DeltaE9-related high basal [Ca(2+)](i) was also reversed by xestospongin C confirming that this effect was inositol trisphosphate receptor-mediated. Carbachol gave a greater stimulation of [Ca(2+)](i) in PS1-DeltaE9 cells that took longer to return to basal as compared with responses seen in NT and PS1-WT cells. This long tail-off effect seen in PS1-DeltaE9 cells after carbachol stimulation was reversed by xestospongin C and dantrolene, suggesting that it was mediated by inositol trisphosphate receptor and ryanodine receptor amplification of Ca(2+). Ruthenium red only reduced carbachol peak elevations of [Ca(2+)](i) in NT and PS1-WT cells and not in PS1-DeltaE9 cells. No significant between cell type differences were seen for basal and carbachol-stimulated [Ca(2+)](i) with either ryanodine or the endoplasmic reticulum Ca(2+) ATPase inhibitor cyclopiazonic acid. Immunostaining experiments revealed that for all the cell types PS1 is present at the plasma membrane and co-localizes with N-cadherin, a component of the cell-cell adhesion complex. Immunoblotting of cell extracts for PLC-beta1 showed that, compared with NT and PS1-WT cells, the PS1-DeltaE9 transfectants gave a relative increase in levels of the calpain generated N-terminal fragment (100 kDa) over full-length (150 kDa) PLC-beta1. Our results suggest that the PS1-DeltaE9 mutation causes upstream changes in PI signaling with enhanced basal PLC activity as a primary effect that leads to a higher [Ca(2+)](i). This may provide a novel mechanism by which the PS1-DeltaE9 mutation sensitizes cells to apoptotic stimuli and enhanced amyloid beta generation.

Presenilins are involved in the proteolytic production of Alzheimer's amyloid peptides, but are also known to regulate Ca(2+) homeostasis in various cells types. In the present study, we examined intracellular Ca(2+) stores coupled to muscarinic receptors and capacitative Ca(2+) entry (CCE) in the human neuroblastoma SH-SY5Y cell line, and how these were modulated by over-expression of either wild-type presenilin 1 (PS1wt) or a mutant form of presenilin 1 (PS1 deltaE9) which predisposes to early-onset Alzheimer's disease. Ca(2+) stores discharged by application of 100 microM muscarine (in Ca(2+)-free perfusate) in PS1wt and PS1 DeltaE9 cells were significantly larger than those in control cells, as determined using Fura-2 microfluorimetry. Subsequent CCE, observed in the absence of muscarine when Ca(2+) was re-admitted to the perfusate, was unaffected in PS1wt cells, but significantly suppressed in PS1 deltaE9 cells. However, when Ca(2+) stores were fully depleted with thapsigargin, CCE was similar in all three cell groups. Western blots confirmed increased levels of PS1 in the transfected cells, but also demonstrated that the proportion of intact PS1 in the PS1 deltaE9 cells was far greater than in the other two cell groups. This study represents the first report of modulation of both Ca(2+) stores and CCE in a human, neurone-derived cell line, and indicates a distinct effect of the PS1 mutation deltaE9 over wild-type PS1., (Copyright 2002 Elsevier Science B.V.)

Mutations in presenilin 1 (PS1) are the major cause of autosomal dominant Alzheimer's disease. We have measured the voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y using whole-cell patch-clamp. When cells were stably transfected to over-express PS1, no change in K+ current was observed. However, over-expression of a deletion mutation (deltaE9) in PS1 led to a decreased K+ current. These changes were channel specific since no change in the Na+ current could be observed in the same cells. Confocal microscopy revealed that the K(V)3.1 K+ channel subunit had a diminished plasma membrane distribution when the deltaE9 over-expressing cells were compared to control cells. Intracellular retention of Kv3.1 is consistent with the notion that PS1 can modulate the activity and trafficking of ion channels in central neurones and implicates a compromise in electrical signalling as an underlying factor in the pathogenesis of familial Alzheimer's disease., (Copyright 2002 Lippincott Williams & Wilkins.)

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G-proteins) couple many different cell surface receptor types to intracellular effector mechanisms. Uncoupling between receptors and G-proteins and between G-proteins and adenylyl cyclase (AC) and phospholipase C (PLC) has been described for Alzheimer's disease (AD) brain. However, there is little information on whether altered G-protein signaling in AD is just an end-stage phenomenon or is important for the progression of disease pathology. Here we used [(35)S]GTPgammaS autoradiography to study G-protein distribution in sections of entorhinal cortex and hippocampus from 23 cases staged for neurofibrillary changes and amyloid deposits according to Braak and Braak (Acta Neuropathol. [1991] 82:239-259). We also studied the effects of GTP, which has been found to increase [(35)S]GTPgammaS binding in an Mg(2+)-dependent manner. Results show that the ability of GTP (3 microM) to stimulate [(35)S]GTPgammaS binding declined significantly with staging for neurofibrillary changes in the entorhinal cortex (P < 0.05, ANOVA) and CA1 subfield of the hippocampus (P < 0.05, ANOVA). No significant changes were seen for [(35)S]GTPgammaS binding in the absence of GTP. Our results suggest a decrease in G-protein GTP hydrolysis, which correlates with the progression of AD neurofibrillary changes, in the regions most affected by this pathology. These alterations appear to occur prior to stages corresponding to clinical disease and could lead to an impaired regulation of several signaling systems in AD brain., (Copyright 2002 Wiley-Liss, Inc.)

The majority of early-onset familial Alzheimer disease cases are caused by mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations have been hypothesised to cause Alzheimer disease either by altering amyloid precursor protein metabolism or by increasing the vulnerability of neurons to undergo death by apoptosis. We showed previously that PS1 exon 9 deletion (PS1 DeltaE9) and L250S mutations predispose SH-SY5Y neuroblastoma cells to high glucose stress-induced apoptosis and that the anti-apoptotic effect of insulin-like growth factor I (IGF-I) is compromised by these mutations. The present study investigates whether the susceptibility of PS1 mutation transfected SH-SY5Y cells to undergo apoptosis is likely due to a downregulation of Akt/protein kinase B (Akt), a key intermediate in the phosphatidylinositol 3 (PI3)-kinase arm of the IGF-I signaling pathway. We used two methods to determine the regulation of Akt in response to the pro-apoptotic stimuli of serum deprivation and high glucose stress, as well as treatment with IGF-I. We also looked at the phosphorylatiom state of GSK-3beta at Ser9. Using a kinase assay with immunoprecipitated Akt, we detected an increased Akt activity in PS1 L250S cells at 1 hr after the combination of 20 mM glucose plus 10 nM IGF-I, when compared to the other cell types. This effect, however, was transient in that no mutation related differences were seen at either 6- or 24-hr post-treatment. Immunoblotting for Phospho-Akt as a ratio of total Akt, as well as for GSK-3beta phosphorylated at Ser9 revealed no apparent between cell type and treatment differences. This data strongly indicates that PS1 wt and mutant cells show no major differences in the pattern of Akt regulation after exposure to the pro-apoptotic stimuli of either serum deprivation or high glucose stress, or treatment with IGF-I. It is suggested that another component of IGF-I signaling is likely disrupted in these cells to increase their vulnerability to undergo death by apoptosis., (Copyright 2001 Wiley-Liss, Inc.)

Presenilins (PSs) are mutated in a majority of familial Alzheimer disease (FAD) cases. Mutated PSs may cause FAD by a number of pro-apoptotic mechanisms, or by regulating gamma-secretase activity, a protease involved in beta-amyloid precursor protein processing to the neurotoxic beta-amyloid peptide. Besides their normal endoproteolytic processing, PSs are substrates for caspases, being cleaved to alternative N-terminal and C-terminal fragments. So far little is known about the role of PSs cleavage in the apoptotic machinery. Here, we used SH-SY5Y neuroblastoma cells stably transfected with wild-type or exon 9 deleted presenilin 1 (PS1) in a time-course study after the exposure to the calcium ionophore A23187. During and after exposure to A 23187, intracellular calcium levels were higher in exon 9 deleted PS1 cells as compared with non-transfected and wild-type PS1 transfected cells. Cell death and the enrichment of apoptotic cells after A23187 exposure were increased by overexpression of exon 9 deleted PS1 as compared with the control cell lines. Wild-type PS1 cells were compared with exon 9 deleted PS1 cells and the temporal relationship between PS1 and other caspase substrates cleavages was analyzed. Exon 9 deleted PS1 cells exhibited a higher caspase-3 activation and a greater cleavage of PS1 and poly(ADP-ribose) polymerase (PARP) compared with wild-type PS1 cells. Exon 9 deleted PS1 cleavage occurred earlier than other caspase substrate cleavages (i.e., PARP and gelsolin), simultaneous with minimum detectable caspase-3 activation. Therefore, alternative cleavage of PS1 may play an important role for the regulation of the proteolytic cascade activated during apoptosis., (Copyright 2001 Wiley-Liss, Inc.)

The mechanism(s) by which the E4 isoform of apolipoprotein E (apoE4) influences Alzheimer's disease (AD) are not fully known. We report that apoE4, but not apoE3, disrupts carbachol-stimulated phosphoinositide (PI) hydrolysis in SH-SY5Y neuroblastoma cells. Carbachol responses were also disrupted by beta-amyloid (Abeta) (1-42) and apoE4/Abeta(1-42) complexes, but not by apoE3/Abeta(1-42). Glutathione and estrogen protected against apoE4 and Abeta(1-42) effects, as well as those of H(2)O(2). Estrogen protection was partially blocked by wortmannin, suggesting the involvement of phosphatidylinositol 3-kinase. An apoE4-induced disruption of acetylcholine muscarinic receptor-mediated signalling may explain the lower effectiveness of cholinergic replacement treatments in apoE4 AD patients. Also, the beneficial effect of estrogen in AD may be partially due to its ability to protect against apoE4- and Abeta(1-42)-mediated disruption of PI hydrolysis.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple etiologies. The presence of the E4 isoform of apolipoprotein E (apoE) has been shown to increase the risk and to decrease the age of onset for AD and is the major susceptibility factor known for the disease. ApoE4 has been shown to intensify all the biochemical disturbances characteristic of AD, including beta amyloid (Abeta) deposition, tangle formation, neuronal cell death, oxidative stress, synaptic plasticity and dysfunctions of lipid homeostasis and cholinergic signalling. In contrast, other apoE isoforms are protective. Here we review and discuss these major hypotheses of the apoE4-AD association.

We investigated the effects of different apolipoprotein E (apoE) isoforms, Abeta (1-42), and apoE/Abeta complexes on PKC-alpha translocation and APP processing in human SH-SY5Y neuroblastoma cells and fibroblasts. Treatment of cells with either 10 nM apoE3 or apoE4, 10 microM Abeta (1-42), or apoE/Abeta complexes induced significant translocation of PKC-alpha in both cell types. Effects were seen using both human recombinant apoE and apoE loaded into beta-very low density lipoprotein (beta-VLDL) particles. Time course (5-24 h) studies of APP processing revealed that some conditions induced transient or moderate increases in the secretion of proteins detected by 22C11. In contrast, the secretion of alpha-secretase cleaved APP was either not modified or transiently decreased, as determined by immunoblotting with the antibody 6E10. These results suggest that apoE, Abeta (1-42) and apoE/Abeta complexes can modulate PKC activity but do not have major consequences for APP processing. These effects could contribute to the reported PKC alterations seen in AD. However, it is unlikely that the contribution of different apoE isoforms to AD pathology occurs via effects on APP processing.

The toxic effects of beta-amyloid (A beta) (1-42), apolipoprotein E (apoE) isoforms, and apoE/A beta complexes were studied in human SH-SY5Y neuroblastoma cells and fibroblasts using MTT reduction. In SH-SY5Y cells, A beta(1-42) gave time-dependent toxicity over 2-48 h, which was reduced by co-incubation with rabbit beta-very low density lipoproteins (beta-VLDL). Human recombinant apoE3 and E4 isoforms were also toxic by themselves and also potentiated A beta effects when used alone, but not when associated with beta-VLDL. None of the treatments were toxic to human fibroblasts. These results suggest that beta-VLDL has a protective role on A beta-induced neurotoxicity and that the status of apoE or the conformation of lipoprotein containing apoE particles may be important for determining the contribution of apoE to neurodegeneration.

The principal protein component of paired helical filaments (PHFs) in Alzheimer disease is abnormally hyperphosphorylated tau (PHF-tau). The stress activated protein kinases JNK and p38 have been shown to phosphorylate tau at some sites only seen in PHF-tau. If JNK and p38 are involved in the abnormal hyperphosphorylation of tau, they should be activated in neurons undergoing neurofibrillary degeneration. In the present study, we determined the intracellular and regional distribution of the active forms of JNK and p38 kinase in entorhinal, hippocampal, and temporal cortices of brains staged for neurofibrillary changes according to Braak and Braak. Neurons with tangle-like inclusions positive for active forms of JNK and p38 kinase were found to appear first in the Pre-alpha layer of the entorhinal cortex, and then extend into other brain regions co-incident with the progressive sequence of neurofibrillary changes. The intraneuronal accumulation of active forms of JNK and p38 kinase apeared to precede the deposition of amyloid in the extracellular space. These data indicate that increased activation of the stress related kinases JNK and p38 occurs very early in the disease and might be involved in the intraneuronal protein phosphorylation/dephosphorylation imbalance that leads to neurofibrillary degeneration in Alzheimer disease.

Based on radioligand binding studies, it has long been assumed that the neurochemical pathology of Alzheimer's disease (AD) does not involve widespread changes in post-synaptic neurotransmitter function. However, more recent studies suggest that receptor function in AD may be compromised due to disrupted post-receptor signal transduction, in particular that mediated by the G-protein regulated phosphoinositide hydrolysis and adenylate cyclase (AC) pathways. The phosphoinositide hydrolysis pathway has been shown to be altered at a number of levels in AD post-mortem brains, including impaired agonist and G-protein regulation of phospholipase C, decreased protein kinase C (PKC) levels and activity, and a reduced number of receptor sites for the second messenger, Ins(1,4,5)P3. Of these, loss of Ins(1,4,5)P3 receptors and PKC in the entorhinal cortex and hippocampus correlates with AD-related neurofibrillary changes, as staged according to Braak's protocol. Disregulation of the phosphoinositide hydrolysis pathway may therefore have consequences for the progression of AD pathology. In contrast to the extensive pattern of disruption seen with the phosphoinositide hydrolysis pathway, changes to AC signalling in AD appear more circumscribed. Disruptions include a lesion at the level of Gs-protein stimulation of AC and, at least in the hippocampus, reduced enzyme activities in response to forskolin stimulation. Of these, the latter change has been shown to precede neurofibrillary changes. Apart from a loss of calcium/calmodulin sensitive AC isoforms, other components of this signalling pathway, including G-protein levels, Gi-protein mediated inhibition and protein kinase A levels and activity, remain relatively preserved in the disorder.

Apolipoprotein E isoforms may have differential effects on a number of pathological processes underlying Alzheimer's disease. Recent studies suggest that the amount, rather than the type, of apolipoprotein E may also be an important determinant for Alzheimer's disease. Therefore, understanding the regulated synthesis of apolipoprotein E is important for determining its role in Alzheimer's disease. We show here that in rat primary hippocampal astrocyte cultures, dibutyryl-cAMP increased apolipoprotein E secretion with time in a dose-dependent manner (to 177% at 48 h) and that retinoic acid potentiated this effect (to 298% at 48 h). Dibutyryl-cAMP also gave a rapid, albeit transient, increase of apolipoprotein E mRNA expression (to 200% at 1 h). In contrast, the protein kinase C activator phorbol 12-myristate 13-acetate decreased both apolipoprotein E secretion (to 59% at 48 h) and mRNA expression (to 22% at 1 h). Phorbol 12-myristate 13-acetate also reversed the effects of dibutyryl-cAMP. Apolipoprotein E secretion was also modulated by receptor agonists for the adenylyl cyclase/cAMP pathway. Isoproterenol (50 nM, a beta-adrenoceptor agonist) enhanced, while clonidine (250 nM, an alpha2-adrenoceptor agonist) decreased, secreted apolipoprotein E. We also analysed the effects of agonists for the phospholipase C/protein kinase C pathway. Arterenol (1 microM, an alpha1-adrenoceptor agonist) and serotonin (2.5 microM) enhanced, whereas carbachol (10 microM, an acetylcholine muscarinic receptor agonist) decreased secreted apolipoprotein E. The effects of these non-selective receptor agonists were modest, probably due to effects on different signalling pathways. Arterenol also potentiated the isoproterenol-mediated increase. We also show that phorbol 12-myristate 13-acetate and dibutyryl-cAMP have opposite effects on nerve growth factor, as compared to apolipoprotein E, secretion, suggesting that the results obtained were unlikely to be due to a general effect on protein synthesis. We conclude that astrocyte apolipoprotein E production can be regulated by factors that affect cAMP intracellular concentration or activate protein kinase C. Alterations in these signalling pathways in Alzheimer's disease brain may have consequences for apolipoprotein E secretion in this disorder.

The clinical symptoms of all forms of Alzheimer's disease (AD) result from a slowly progressive neurodegeneration that is associated with the excessive deposition of beta-amyloid (A beta) in plaques and in the cerebrovasculature, and the formation of intraneuronal neurofibrillary tangles, which are composed primarily of abnormally hyperphosphorylated tau protein. The sequence of cellular events that cause this pathology and neurodegeneration is unknown. It is, however, most probably linked to neuronal signal transduction systems that become misregulated in the brains of certain individuals, causing excessive A beta to be formed and/or deposited, tau to become aggregated and hyperphosphorylated and neurons to degenerate. We hypothesize that a progressive alteration in the ability of neurons to regulate intracellular calcium, particularly at the level of the endoplasmic reticulum, is a crucial signal transduction event that is linked strongly to the initiation and development of AD pathology. In this chapter we will discuss the key findings that lend support to this hypothesis.

In vitro studies have shown that the Alzheimer's disease-related presenilin-1 protein can mediate Notch-1 receptor cleavage during signalling. In the present study, we compared the distribution of presenilin-1 and Notch-1 receptor immunoreactivities in human embryonic CNS tissue during the first trimester of development. Our aim was to gain insight into whether these proteins are likely to interact functionally during human fetal brain development. CNS material was obtained from routine abortions, cryosectioned and studied by means of immunohistochemistry with antibodies to presenilin-1 and Notch-1. At very early stages of embryonic development (four to five gestational weeks) intensive presenilin-1 immunoreactivity could be seen predominantly in neurites in the ventral horn of the spinal cord, where it overlapped with 200-kDa neurofilament immunoreactivity. Presenilin-1 immunoreactivity was also seen in neuroblasts of the ventricular zone of the tel- and mesencephalon, as well as of the brainstem. Notch-1 receptor appeared in neuronal and ependymal cells throughout the CNS. Seven- to eight-week CNS tissue showed similar patterns of presenilin-1 and Notch-1 receptor expression in the spinal cord and cerebral cortex as was seen at five weeks. Both proteins were localised in the neuroepithelial cell layer lining the ventricles, as well as in the cortical plate layer, where immunoreactivity was seen in the cell bodies. In addition, presenilin-1 immunoreactivity was seen in thin neurites in the subplate of the developing cortex. At 10 weeks, presenilin-1 immunoreactivity was reduced in the spinal cord. These results show that, although presenilin-1 and Notch-1 receptor are localised to the same differentiating cell populations in the human cerebral cortex, making a direct interaction possible, these proteins are otherwise confined to different neurons or neuronal compartments, suggesting a role for presenilin-1 during early CNS differentiation that does not involve Notch-1 receptor processing. Double staining for presenilin-1 in the endoplasmic reticulum and presenilin-1 in the Golgi showed overlap to some extent in investigated CNS regions, but not in neurites. This suggests that presenilin-1 function during neurogenesis is not exclusively correlated to protein processing within the endoplasmic reticulum and Golgi, but that presenilin-1 may also be involved in other processes, such as axonal and dendritic outgrowth or synaptic formation. In summary, our findings provide supportive evidence that the presenilin-1 protein is involved in the development and maturation of the human fetal CNS. The presence of presenilin-1 immunoreactivity in both the cell bodies and neurites of developing neurons strongly suggests divergent mechanisms of function for presenilin-1 during human brain development. These may include interactions with any of the Notch receptor proteins, as well as Notch-independent mechanisms.