PTEN, Akt, and GSK3beta signalling in rat primary cortical neuronal cultures following tumor necrosis factor-alpha and trans-4-hydroxy-2-nonenal treatments.

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.

Involvement of glutaredoxin-1 and thioredoxin-1 in beta-amyloid toxicity and Alzheimer's disease.

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.

Flow cytometry as a method for studying effects of stressors on primary rat neurons.

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.

Distribution of presenilin 1 and 2 and their relation to Notch receptors and ligands in human embryonic/foetal central nervous system.

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.

Apolipoprotein E and beta-amyloid (1-42) regulation of glycogen synthase kinase-3beta.

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.

Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells expressing a familial Alzheimer's disease presenilin-1 mutation.

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.

Loss of stimulatory effect of guanosine triphosphate on [(35)S]GTPgammaS binding correlates with Alzheimer's disease neurofibrillary pathology in entorhinal cortex and CA1 hippocampal subfield.

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.

Akt activity in presenilin 1 wild-type and mutation transfected human SH-SY5Y neuroblastoma cells after serum deprivation and high glucose stress.

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.

Caspase cleavage of exon 9 deleted presenilin-1 is an early event in apoptosis induced by calcium ionophore A 23187 in SH-SY5Y neuroblastoma cells.

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.

Regulation of apolipoprotein E secretion in rat primary hippocampal astrocyte cultures.

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.

Apolipoprotein E isoform-specific disruption of phosphoinositide hydrolysis: protection by estrogen and glutathione.

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.

Receptor-G-protein signalling in Alzheimer's 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.

Dysfunctional intracellular calcium homoeostasis: a central cause of neurodegeneration in Alzheimer's disease.

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.

Expression of presenilin-1 and Notch-1 receptor in human embryonic CNS.

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.

Effects of apolipoprotein E (apoE) isoforms, beta-amyloid (Abeta) and apoE/Abeta complexes on protein kinase C-alpha (PKC-alpha) translocation and amyloid precursor protein (APP) processing in human SH-SY5Y neuroblastoma cells and fibroblasts.

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.

Beta-VLDL protects against A beta(1-42) and apoE toxicity in human SH-SY5Y neuroblastoma cells.

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.

Apolipoprotein E: a major piece in the Alzheimer's disease puzzle.

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.

Alzheimer's disease presenilin-1 exon 9 deletion and L250S mutations sensitize SH-SY5Y neuroblastoma cells to hyperosmotic stress-induced apoptosis.

Mutations in the presenilin-1 (PS1) and presenilin-2 (PS2) genes account for the majority of early-onset familial Alzheimer's disease cases. Recent studies suggest that presenilin gene mutations predispose cells to apoptosis by mechanisms involving altered calcium homeostasis and oxidative damage. In the present study, we determined whether PS1 mutations also sensitize cells to hyperosmotic stress-induced apoptosis. For this, we established SH-SY5Y neuroblastoma cell lines stably transfected with wild-type PS1 or either the PS1 exon 9 deletion (deltaE9) or PS1 L250S mutants. Cultured cells were exposed to an overnight (17 h) serum deprivation, followed by a 30 min treatment with either 20 mM glucose, 10 nM insulin-like growth factor-1 or 20 mM glucose + 10 nM insulin-like growth factor-1. Cells were then cultured for a further 3, 6 or 24 h and stained for apoptotic condensed nuclei using propidium iodide. Confirmation that cells were undergoing an active apoptotic process was achieved by labelling of DNA strand breaks using the terminal dUTP nick end labelling (TUNEL) technique. We also determined cell viability using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Propidium iodide staining revealed that all cell lines and controls showed an increased number of apoptotic cells appearing with condensed nuclei at 24 h compared with 6 h and 3 h. High glucose-induced hyperosmotic stress resulted in significantly more apoptotic cells in the PS1 deltaE9 and PS1 L250S mutation cell lines at 24 h, compared with the wild-type PS1 lines (P < 0.001, ANOVA for both comparisons). Mean values (+/-S.D.) for the percentage number of apoptotic cells at 24 h following high glucose treatment were 16.1 +/- 3.5%, 26.7 +/- 5.5% and 31.0 +/- 5.7% for the wild-type PS1, PS1 deltaE9 and PS1 L250S lines, respectively. The pro-apoptotic effects of high glucose treatment were reversed by 10 nM insulin-like growth factor-1, although to a lesser extent in the mutation cell lines (5.8 +/- 2.4%, 15.2 +/- 7.3% and 13.2 +/- 2.0% for the wild-type PS1, PS1 deltaE9 (P < 0.01 for comparison with wild-type PS1) and PS1 L250S (P < 0.01 for comparison with wild-type PS1) transfected lines, respectively. TUNEL labelling of cells at 24 h following treatment gave essentially the same results pattern as obtained using propidium iodide. The percentage number of apoptotic cells with DNA strand breaks (means +/- S.D.) following high glucose treatment was 15.4 +/- 2.6% for the wild-type PS1, 26.8 +/- 3.2% for the PS1 deltaE9 (P < 0.001 for comparison with wild-type PS1) and 29.7 +/- 6.1% for the PS1 L250S transfected lines (P < 0.001 for comparison with wild-type PS1). The PS1 deltaE9 and PS1 L250S transfected lines also showed a higher number of apoptotic cells with DNA strand breaks at 24 h following high glucose plus insulin-like growth factor-1 treatment (11.4 +/- 2.0% and 14.3 +/- 2.8%, respectively), compared with values for the wild-type PS1 lines (8.5 +/- 2.4%). These differences were significant (P < 0.01) for the comparison of wild-type PS1 and PS1 L250S, but not PS1 deltaE9 lines. The mutation-related increases in number of apoptotic cells at 24 h following high glucose treatment were not accompanied by significant differences in cell viability at this time-point. Our results indicate that PS1 mutations predispose to hyperosmotic stress-induced apoptosis and that the anti-apoptotic effects of insulin-like growth factor-1 are compromised by these mutations. Perturbations of insulin-like growth factor-1 signalling may be involved in PS1 mutation-related apoptotic neuronal cell death in Alzheimer's disease.

Nicergoline stimulates protein kinase C mediated alpha-secretase processing of the amyloid precursor protein in cultured human neuroblastoma SH-SY5Y cells.

We investigated the ability of the antidementia agents, nicergoline, aniracetam and hydergine to stimulate PKC mediated alpha-secretase amyloid precursor protein (APP) processing in cultured human neuroblastoma SH-SY5Y cells. Western immunoblotting of cell conditioned media using the Mabs 22C11 and 6E10 revealed the presence of 2 bands with molecular mass of 90 and 120 kDa, corresponding to possible alternatively glycosylated forms of secreted APP (APPs). Short-term (30 min and 2 h) treatment of cells with nicergoline gave an increased intensity of both bands, compared to non-treated cells. Maximal nicergoline effects, of the order of 150-200% over basal APPs release, were seen at concentrations between 1 and 10 microM. Under the same condition, 1 microM PdBu, used as a positive control, gave 500-1000% increases of basal APPs release. In contrast, aniracetam and hydergine, did not show any effect on APPs secretion. 2 h treatment with nicergoline had no effect on cellular full-length APP levels, as determined by immunoblotting of cell extracts with 22C11 and CT15 antibodies. Immunoblotting with PKC isoform specific antibodies of soluble and membrane fractions prepared from 2 h treated cells, showed that nicergoline (50 microM) and PdBu (1 microM) both induced translocation of PKC alpha, gamma and epsilon, but not PKC beta. The involvement of PKC in mediating nicergoline stimulated APPs release was also studied using specific inhibitors. 1 microM calphostin C, a broad range PKC inhibitor, significantly reduced both PdBu (1 microM) and nicergoline (10 microM) induced APPs release. In contrast, Go6976 (1 microM), a selective PKC alpha and beta1 inhibitor, as well as the cAMP-dependent protein kinase inhibitor, H89 (1 microM) were without effect. These results indicate that nicergoline can modulate alpha-secretase APP processing by a PKC dependent mechanism that is likely to involve the gamma and epsilon isoforms of this enzyme.

Distribution of active glycogen synthase kinase 3beta (GSK-3beta) in brains staged for Alzheimer disease neurofibrillary changes.

Accumulation of paired helical filaments (PHFs) in neurofibrillary tangles, neuropil threads, and dystrophic neurites is one of the major neuropathological hallmarks of Alzheimer disease (AD). The principal protein subunit of PHFs is the abnormally hyperphosphorylated tau. Glycogen synthase kinase 3beta (GSK-3beta) is one of the candidate kinases involved in PHF-tau formation. To play a role in PHF-tau formation, it would be expected that GSK-3beta is active in tangle bearing neurons. In the present study, we investigated the regional and intracellular distributions of active and inactive forms of GSK-3beta in brains staged for neurofibrillary changes. We found that neurons with tangle-like inclusions positive for active, but not inactive, GSK-3beta appear initially in the Pre-alpha layer of the entorhinal cortex and extend to other brain regions, coincident with the sequence of the development of neurofibrillary changes. Active, but not inactive, GSK-3beta was found to initially accumulate in the cytoplasm of pretangle neurons. These data provide direct in situ evidence that is consistent with the involvement of GSK-3beta in PHF-tau formation.