Amyloid beta-protein (Abeta)1-40 protects neurons from damage induced by Abeta1-42 in culture and in rat brain

Previously, we found that amyloid beta-protein (Abeta)1-42 exhibits neurotoxicity, while Abeta1-40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal-mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Abeta1-40 against Abeta1-42-induced neurotoxicity in culture and in vivo. Neuronal death was induced by Abeta1-42 at concentrations higher than 2 microm, which was prevented by concurrent treatment with Abeta1-40 in a dose-dependent manner. However, metal chelators did not prevent Abeta1-42-induced neuronal death. Circular dichroism spectroscopy showed that Abeta1-40 inhibited the beta-sheet transformation of Abeta1-42. Thioflavin-T assay and electron microscopy analysis revealed that Abeta1-40 inhibited the fibril formation of Abeta1-42. In contrast, Abeta1-16, Abeta25-35, and Abeta40-1 did not inhibit the fibril formation of Abeta1-42 nor prevent Abeta1-42-induced neuronal death. Abeta1-42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Abeta1-40. These results indicate that Abeta1-40 protects neurons from Abeta1-42-induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the beta-sheet transformation and fibril formation of Abeta1-42. Our data suggest a mechanism by which elevated Abeta1-42/Abeta1-40 ratio accelerates the development of Alzheimer's disease (AD) in familial AD.     

Puerarin protects PC12 cells against beta-amyloid-induced cell injury

beta-Amyloid protein (Abeta), a major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of reactive oxygen species (ROS) and induction of apoptosis. In this study, the protective effect of puerarin, an isoflavone purified from the radix of the Chinese herb Pueraria lobata, on Abeta-induced rat pheochromocytoma (PC12) cultures was investigated. Although exposure of PC12 cells to 50 microM Abeta25-35 caused significant viability loss and apoptotic rate increase, pretreatment of the cells with puerarin for 24h reduced the viability loss and apoptotic rate. Puerarin (1 microM) significantly inhibited Abeta25-35-induced apoptosis of PC12 cells. Preincubation of the cell with puerarin also restored the ROS and mitochondrial membrane potential levels that had been altered as a result of Abeta25-35 treatment. Puerarin was also found to increase the Bcl-2/Bax ratio and reduce caspase-3 activation. These results suggest that puerarin could attenuate Abeta25-35-induced PC12 cell injure and apoptosis and could also promote the survival of PC12 cells. Therefore, puerarin may act as an intracellular ROS scavenger, and its antioxidant properties may protect against Abeta25-35-induced cell injury.     

Effect of the Alzheimer amyloid fragment Abeta(25-35) on Akt/PKB kinase and survival of PC12 cells

The phosphatidylinositol 3 kinase (PI3K)-Akt/PKB pathway protects neurons from apoptosis caused by diverse stress stimuli. However, its protective role against the amyloid beta peptide (Abeta), a major constituent of Alzheimer's disease plaques, has not been studied. We investigated the effect of the Abeta-derived Abeta(25-35) peptide on apoptosis and on the Akt survival pathway in PC12 cells. Cells submitted to micromolar concentrations of Abeta(25-35) exhibited increased production of reactive oxygen species (ROS) and morphological alterations consistent with apoptosis. Akt1 was activated shortly after incubation with Abeta(25-35) and Abeta(1-40) with a kinetics different to that of nerve-derived growth factor. Akt1 activation was blocked by the PI3K inhibitor wortmannin. We tested the hypothesis that Akt1 might modify the vulnerability of neural cells to apoptosis induced by Abeta(25-35). Overexpression of an active version of Akt1 attenuated the apoptotic effect of Abeta(25-35) as determined by flow cytometry. Moreover, PC12 cells overexpressing a membrane-targeted N-myristylated fusion protein of enhanced green fluorescence protein (EGFP) and mouse Akt1 exhibited lower levels of ROS than control EGFP-transfected cells. The present findings demonstrate that Akt1 is activated in response to Abeta(25-35) in a PI3K-dependent manner and that active Akt1 protects PC12 cells against the pro-apoptotic action of this peptide.     

Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents Abeta(25-35)-induced reduction in BPRP in PC12 cells

Several lines of evidence support that beta-amyloid (Abeta)-induced neurotoxicity is mediated through the generation of reactive oxygen species (ROS) and elevation of intracellular calcium. Salvianolic acid B (Sal B), the major and most active anti-oxidant from Salvia miltiorrhiza, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the effects of Sal B against beta-amyloid peptide 25-35 (Abeta(25-35))-induced neurotoxicity, focused mainly on the neurotoxic effects of Abeta(25-35) and the neuroprotective effects of Sal B on the expression of brain-pancreas relative protein (BPRP), which is a new protein and mainly expressed in brain and pancreas. Following exposure of PC12 cells to 20 microM Abeta(25-35), a marked reduction in the expression of BPRP was observed, accompanied with decreased cell viability and increased cell apoptosis, as well as increased ROS production and calcium influx. Treatment of the PC12 cells with Sal B significantly reversed the expression of BPRP and cell viability while it decreased ROS production and intracellular calcium. These data indicate that Abeta(25-35) decreases the expression of BPRP via enhanced formation of intracellular ROS and increased intracellular calcium, and that Sal B, as an anti-oxidant, protects against Abeta(25-35)-induced reduction in expression of BPRP through its effects on suppressing the production of ROS, calcium flux, and apoptosis. However, the role(s) of BPRP in AD and the definite mechanisms by which Sal B protects against Abeta(25-35)-induced reduction in the expression of BPRP require further study.     

Catechin and epicatechin from Smilacis chinae rhizome protect cultured rat cortical neurons against amyloid beta protein (25-35)-induced neurotoxicity through inhibition of cytosolic calcium elevation

We previously reported that the Smilacis chinae rhizome inhibits amyloid beta protein (25-35) (Abeta (25-35))-induced neurotoxicity in cultured rat cortical neurons. Here, we isolated catechin and epicatechin from S. chinae rhizome and also studied their neuroprotective effects on Abeta (25-35)-induced neurotoxicity in cultured rat cortical neurons. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced neuronal cell death at a concentration of 10 microM, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Catechin and epicatechin also inhibited glutamate release into medium induced by 10 microM Abeta (25-35), which was measured by HPLC, generation of reactive oxygen species (ROS) and activation of caspase-3. These results suggest that catechin and epicatechin prevent Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity. Furthermore, these effects of catechin and epicatechin may be associated with the neuroprotective effect of the S. chinae rhizome.     

Phosphatidylinositol and PI-4-monophosphate recover amyloid beta protein-induced inhibition of Cl- -ATPase activity

The neuronal Cl- -ATPase/pump is a candidate for an outwardly directed active Cl- transport system, which requires phosphatidylinositol-4-monophosphate (PI4P) for its optimal activity. We previously reported that low concentrations (1-10 nM) of amyloid beta proteins (Abetas, Abeta1-42, Abeta25-35), the neurotoxic peptides in Alzheimer's disease, reduced Cl- -ATPase activity in cultured rat hippocampal neurons without any changes in the activities of Na+/K+-ATPase or anion-insensitive Mg(2+)-ATPase, and decreased PI, PIP, and PIP2 levels in neuronal plasma membranes (Journal of Neurochemistry 2001, 78, 569-579). In this study, we examined the effects of exogenously applied PI and PI4P on the Abeta25-35-induced changes in Cl- -ATPase activity, the intracellular concentration of Cl- ([Cl- ]i), and glutamate neurotoxicity using primary cultured rat hippocampal neurons. The Abeta decreased Cl- -ATPase activity to 47% of control and increased [Cl- ]i in hippocampal pyramidal cell-like neurons to a level 3 times higher than the control. The addition of PI (50-750 nM) or PI4P (50-150 nM) dose-dependently blocked the inhibitory effects of Abeta on Cl- -ATPase activity. High doses of PI (750 nM) and PI4P (100-150 nM) reduced Na+/K+-ATPase activity to 41% and 35% of control, respectively, but this inhibition was attenuated by the co-application of phosphatidylserine (PS, 1 micro M). PI or PI4P (75 nM each) reversed the Abeta-induced increase in [Cl-]i. In the Abeta-exposed culture, stimulation by glutamate (10 micro M, 10 min) resulted in an increase in DNA fragmentation and decreases in cell viability. Addition of PI or PI4P prevented the Abeta-induced aggravation of glutamate neurotoxicity. Thus, PI and PI4P were demonstrated to prevent Abeta-induced decreases in Cl- -ATPase activity and increases in neuronal [Cl- ]i in parallel with the attenuation of Abeta-induced aggravation of glutamate neurotoxicity.     

Beta-amyloid increases somatostatin expression in cultured cortical neurons

In beta-amyloid (Abeta)-induced neurotoxicity, activation of the NMDA receptor, increased Ca2+ and oxidative stress are intimately associated with neuronal cell death as normally seen in NMDA-induced neurotoxicity. We have recently shown selective sparing of somatostatin (SST)-positive neurons and increased SST expression in NMDA agonist-induced neurotoxicity. Accordingly, the present study was undertaken to determine the effect of Abeta25-35-induced neurotoxicity on the expression of SST in cultured cortical neurons. Cultured cortical cells were exposed to Abeta25-35 and processed to determine the cellular content and release of SST into medium by radioimmunoassay and SST mRNA by RT-PCR. Abeta25-35 induces neuronal cell death in a concentration- and time-dependent fashion, increases SST mRNA synthesis and induces an augmentation in the cellular content of SST. No significant changes were seen on SST release at any concentration of Abeta25-35 after 24 h of treatment. However, Abeta25-35 induces a significant increase of SST release into medium only after 12 h in comparison with other time points. Most significantly, SST-positive neurons are selectively spared in the presence of a lower concentration of Abeta25-35, whereas, in the presence of higher concentrations of Abeta25-35 for extended time periods, SST-positive neurons decrease gradually. Furthermore, Abeta25-35 induces apoptosis at lower concentrations (5 and 10 micromol/L) and necrosis at higher concentrations (20 and 40 micromol/L). Consistent with the increased accumulation of SST, these data suggest that Abeta25-35 impairs cell membrane permeability. Selective sparing of SST-positive neurons at lower concentrations of Abeta25-35 at early time points directly correlates with the pathophysiology of Alzheimer's disease.     

Genistein ameliorates beta-amyloid peptide (25-35)-induced hippocampal neuronal apoptosis

beta-Amyloid protein (Abeta), a major component of senile plaques of Alzheimer's disease (AD) brain, causes elevation of the intracellular free Ca2+ level and the production of robust free radicals, both of which contribute greatly to the AD-associated cascade including severe neuronal loss in the hippocampus. Genistein, the most active molecule of soy isoflavones, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the neuroprotective effect of genistein against Abeta25-35-induced apoptosis in cultured hippocampal neurons, as well as the underlying mechanism. Abeta25-35-induced apoptosis, characterized by decreased cell viability, neuronal DNA condensation, and fragmentation, is associated with an increase in intracellular free Ca2+ level, the accumulation of reactive oxygen species (ROS), and the activation of caspase-3. All these phenotypes induced by Abeta25-35 are reversed by genistein. Our results further show that at the nanomolar (100 nM) level, genistein protects neurons from Abeta25-35-induced damage largely via the estrogen receptor-mediated pathway, and at the micromolar (40 microM) level, the neuroprotective effect of genistein is mediated mainly by its antioxidative properties. Our data suggest that genistein attenuates neuronal apoptosis induced by Abeta25-35 via various mechanisms.     

Role of glycine-33 and methionine-35 in Alzheimer's amyloid beta-peptide 1-42-associated oxidative stress and neurotoxicity

Recent theoretical calculations predicted that Gly33 of one molecule of amyloid beta-peptide (1-42) (Abeta(1-42)) is attacked by a putative sulfur-based free radical of methionine residue 35 of an adjacent peptide. This would lead to a carbon-centered free radical on Gly33 that would immediately bind oxygen to form a peroxyl free radical. Such peroxyl free radicals could contribute to the reported Abeta(1-42)-induced lipid peroxidation, protein oxidation, and neurotoxicity, all of which are prevented by the chain-breaking antioxidant vitamin E. In the theoretical calculations, it was shown that no other amino acid, only Gly, could undergo such a reaction. To test this prediction we studied the effects of substitution of Gly33 of Abeta(1-42) on protein oxidation and neurotoxicity of hippocampal neurons and free radical formation in synaptosomes and in solution. Gly33 of Abeta(1-42) was substituted by Val (Abeta(1-42G33V)). The substituted peptide showed almost no neuronal toxicity compared to the native Abeta(1-42) as well as significantly lowered levels of oxidized proteins. In addition, synaptosomes subjected to Abeta(1-42G33V) showed considerably lower dichlorofluorescein-dependent fluorescence - a measure of reactive oxygen species (ROS) - in comparison to native Abeta(1-42) treatment. The ability of the peptides to generate ROS was also evaluated by electron paramagnetic resonance (EPR) spin trapping methods using the ultrapure spin trap N-tert-butyl-alpha-phenylnitrone (PBN). While Abeta(1-42) gave a strong mixture of four- and six-line PBN-derived spectra, the intensity of the EPR signal generated by Abeta(1-42G33V) was far less. Finally, the ability of the peptides to form fibrils was evaluated by electron microscopy. Abeta(1-42G33V) does not form fibrils nearly as well as Abeta(1-42) after 48 h of incubation. The results suggest that Gly33 may be a possible site of free radical propagation processes that are initiated on Met35 of Abeta(1-42) and that contribute to the peptide's toxicity in Alzheimer's disease brain.     

Enhanced G-protein activation by a mixture of Abeta(25-35), Abeta(1-40/42) and zinc

Beta-amyloid peptides (Abetas) bind to several G-protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Abeta(1-42), Abeta(1-40), Abeta(25-35) and their mixtures on [(35)S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Abetas alone induced a concentration-dependent activation of G-proteins (IC50 approximately 10(-6) m), while aggregated Abeta fibrils only affected GTP binding at concentrations above 10(-5) m. Mixing Abeta(25-35) with Abeta(1-42) or Abeta(1-40) induced a several-fold increase in GTP-binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Abeta(1-40) and Abeta(1-42) or highly aggregated Abetas. Zinc had no effect on Abeta(1-40/42) but strongly potentiated the Abeta(25-35) or the mixed peptides-induced GTP-binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.     

Effects of amyloid peptides on A-type K+ currents of Drosophila larval cholinergic neurons

Accumulation of amyloid (Abeta) peptides has been suggested to be the primary event in Alzheimer's disease. In neurons, K+ channels regulate a number of processes, including setting the resting potential, keeping action potentials short, timing interspike intervals, synaptic plasticity, and cell death. In particular, A-type K+ channels have been implicated in the onset of LTP in mammalian neurons, which is thought to underlie learning and memory. A number of studies have shown that Abeta peptides alter the properties of K+ currents in mammalian neurons. We set out to determine the effects of Abeta peptides on the neuronal A-type K+ channels of Drosophila. Treatment of cells for 18 h with 1 microM Abeta1-42 altered the kinetics of the A-type K+ current, shifting steady-state inactivation to more depolarized potentials and increasing the rate of recovery from inactivation. It also caused a decrease in neuronal viability. Thus it seems that alteration in the properties of the A-type K+ current is a prelude to the amyloid-induced death of neurons. This alteration in the properties of the A-type K+ current may provide a basis for the early memory impairment that was observed prior to neurodegeneration in a recent study of a transgenic Drosophila melanogaster line over-expressing the human Abeta1-42 peptide.     

Amyloid beta-peptide (1-42)-induced oxidative stress and neurotoxicity: implications for neurodegeneration in Alzheimer's disease brain. A review

Oxidative stress, manifested by protein oxidation, lipid peroxidation, DNA oxidation and 3-nitrotyrosine formation, among other indices, is observed in Alzheimer's disease (AD) brain. Amyloid beta-peptide (1-42) [Abeta(1-42)] may be central to the pathogenesis of AD. Our laboratory and others have implicated Abeta(1-42)-induced free radical oxidative stress in the neurodegeneration observed in AD brain. This paper reviews some of these studies from our laboratory. Recently, we showed both in-vitro and in-vivo that methionine residue 35 (Met-35) of Abeta(1-42) was critical to its oxidative stress and neurotoxic properties. Because the C-terminal region of Abeta(1-42) is helical, and invoking the i + 4 rule of helices, we hypothesized that the carboxyl oxygen of lle-31, known to be within a van der Waals distance of the S atom of Met-35, would interact with the latter. This interaction could alter the susceptibility for oxidation of Met-35, i.e. free radical formation. Consistent with this hypothesis, substitution of lle-31 by the helix-breaking amino acid, proline, completely abrogated the oxidative stress and neurotoxic properties of Abeta(1-42). Removal of the Met-35 residue from the lipid bilayer by substitution of the negatively charged Asp for Gly-37 abrogated oxidative stress and neurotoxic properties of Abeta(1-42). The free radical scavenger vitamin E prevented A(beta (1-42)-induced ROS formation, protein oxidation, lipid peroxidation, and neurotoxicity in hippocampal neurons, consistent with our model for Abeta-associated free radical oxidative stress induced neurodegeneration in AD. ApoE, allele 4, is a risk factor for AD. Synaptosomes from apoE knock-out mice are more vulnerable to Abeta-induced oxidative stress (protein oxidation, lipid peroxidation, and ROS generation) than are those from wild-type mice. We also studied synaptosomes from allele-specific human apoE knock-in mice. Brain membranes from human apoE4 mice have greater vulnerability to Abeta(1-42)-induced oxidative stress than brain membranes from apoE2 or E3, assessed by the same indices, consistent with the notion of a coupling of the oxidative environment in AD brain and increased risk of developing this disorder. Using immunoprecipitation of proteins from AD and control brain obtained no longer than 4h PMI, selective oxidized proteins were identified in the AD brain. Creatine kinase (CK) and beta-actin have increased carbonyl groups, an index of protein oxidation, and Glt-1, the principal glutamate transporter, has increased binding of the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE). Abeta inhibits CK and causes lipid peroxidation, leading to HNE formation. Implications of these findings relate to decreased energy utilization, altered assembly of cytoskeletal proteins, and increased excitotoxicity to neurons by glutamate, all reported for AD. Other oxidatively modified proteins have been identified in AD brain by proteomics analysis, and these oxidatively-modified proteins may be related to increased excitotoxicity (glutamine synthetase), aberrant proteasomal degradation of damaged or aggregated proteins (ubiquitin C-terminal hydrolase L-1), altered energy production (alpha-enolase), and diminished growth cone elongation and directionality (dihydropyrimindase-related protein 2). Taken together, these studies outlined above suggest that Met-35 is key to the oxidative stress and neurotoxic properties of Abeta(1-42) and may help explain the apoE allele dependence on risk for AD, some of the functional and structural alterations in AD brain, and strongly support a causative role of Abeta(1-42)-induced oxidative stress and neurodegeneration in AD.     

Acute effect of beta amyloid on synchronized spontaneous Ca2+ oscillations in cultured hippocampal networks

The effects of beta amyloid (Abeta) on cytoplasmic Ca(2+) ([Ca(2+)](c)) have been studied extensively, but the current literature on this aspect is confusing. We reported that 20 microM Abeta(25-35) significantly inhibited the synchronized spontaneous cytoplasmic Ca(2+) transients immediately after application, whereas it had little effect on the baseline [Ca(2+)](c) concentration in neurons. Abeta(1-42) had a similar effect on the Ca(2+) transients as Abeta(25-35), while it increased baseline [Ca(2+)](c) concentration gradually. However, Abeta(1-40) had little effect on either Ca(2+) transients or baseline [Ca(2+)](c). Such differential effects of Abeta on Ca(2+) signals might explain, at least partially, the confusing observations from the previous studies and provide important therapeutic implications for preventing or reversing early neuron damage in Alzheimer's disease.     

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+-dependent apoptosis induced by amyloid beta-peptide in human neuroblastoma IMR32 cells

Amyloid beta-peptide (Abeta) plays a fundamental role in the pathogenesis of Alzheimer's disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid beta-peptide (Abeta) 1-42 (Met35) strongly affects the peptide's ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca(2+) homeostasis resulting in the activation of pro-apoptotic pathways has been proposed as a mechanism underlying Abeta toxicity. Therefore, we investigated correlations between the Met35 redox state, Abeta toxicity, and altered intracellular Ca(2+) signaling in human neuroblastoma IMR32 cells. Cells incubated for 6-24 h with 10 microM Abeta1-42 exhibited significantly increased KCl-induced Ca(2+) transient amplitudes and resting free Ca(2+) concentrations. Nifedipine-sensitive Ca(2+) current densities and Ca(v)1 channel expression were markedly enhanced by Abeta1-42. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42(Met35-Ox)). Cell pre-treatment with the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (1 microM) or the Ca(v)1 channel blocker nifedipine (5 microM) significantly attenuated Abeta1-42-induced apoptosis but had no effect on Abeta1-42(Met35-Ox) toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Abeta1-42 toxicity by rendering the peptide capable of disrupting intracellular Ca(2+) homeostasis and thereby provoking apoptotic cell death.     

Demonstration of an anti-oxidative stress mechanism of quetiapine: implications for the treatment of Alzheimer's disease

We have shown that quetiapine, a new antipsychotic drug, protects cultured cells against oxidative stress-related cytotoxicities induced by amyloid beta (Abeta)25-35, and that quetiapine prevents memory impairment and decreases Abeta plaques in the brains of amyloid precursor protein (APP)/presenilin-1 (PS-1) double-mutant mice. The aim of this study was to understand why quetiapine has these protective effects. Because the cytotoxicity of both Abeta(25-35) and Abeta(1-40) requires fibril formation, our first experiments determined the effect of quetiapine on Abeta(25-35) aggregation. Quetiapine inhibited Abeta(25-35) aggregation in cell-free aqueous solutions and blocked the fibrillar aggregation of Abeta(25-35), as observed under an electron microscope. We then investigated why quetiapine inhibits Abeta(25-35) aggregation. During the aggregation of Abeta(25-35), a hydroxyl radical (OH*) was released, which in turn amplified Abeta(25-35) aggregation. Quetiapine blocked OH*-induced Abeta(25-35) aggregation and scavenged the OH* produced in the Fenton system and in the Abeta(25-35) solution, as analyzed using electron paramagnetic resonance spectroscopy. Furthermore, new compounds formed by quetiapine and OH* were observed in MS analysis. Finally, we applied Abeta(25-35) to PC12 cells to observe the effect of quetiapine on living cells. Abeta(25-35) increased levels of intracellular reactive oxygen species and calcium in PC12 cells and caused cell death, but these toxic effects were prevented by quetiapine. These results demonstrate an anti-oxidative stress mechanism of quetiapine, which contributes to its protective effects observed in our previous studies and explains the effectiveness of this drug for Alzheimer's disease patients with psychiatric and behavioral complications.     

Protective effects of insulin-like growth factor-I on the somatostatinergic system in the temporal cortex of beta-amyloid-treated rats

Insulin-like growth factor-I (IGF-I) has protective effects against beta-amyloid (Abeta)-induced neuronal cell death. Because alterations of the somatostatinergic system have been described in Alzheimer's disease, we investigated the effects of the Abeta peptide and the possible protective role of IGF-I on the somatostatinergic system of the rat temporal cortex and on cell death and phosphorylated (p)-Akt levels in this area. Abeta25-35 was administered intracerebroventricularly to male rats via an osmotic minipump over 14 days (300 pmol/day). Another group received a subcutaneous IGF-I infusion (50 microg/kg/day), concomitant with Abeta25-35 administration, whereas a third group received IGF-I alone. Abeta25-35 significantly decreased the somatostatin (SRIF)-like immunoreactive content and the SRIF receptor density, as a result of a decrease in the levels of the SRIF receptor subtype 2. The inhibitory effect of SRIF on adenylyl cyclase activity was significantly lower after Abeta25-35 infusion, whereas the levels of the inhibitory G protein subunit Gialpha1, Gialpha2 or Gialpha3 were unaltered. Cell death was increased and p-Akt levels decreased in Abeta25-35-treated animals. IGF-I administration increased immunoreactive IGF-I levels in the temporal cortex and restored all parameters affected by Abeta25-35 to baseline values. These findings suggest that IGF-I prevents the deleterious effect of Abeta25-35 on the somatostatinergic system.     

Vinpocetine attenuates the metabolic dysfunction induced by amyloid beta-peptides in PC12 cells

The cytoprotective effect of vinpocetine [14-ethoxycarbonyl-(3alpha, 16alpha-ethyl)-14,15-eburnamine] was investigated on PC12 cells treated with the amyloid beta-peptides (Abeta) for 24 hours. Vinpocetine was shown to protect cells from the inhibition in redox status induced by exposure to Abeta25-35 and Abeta1-40, the maximal protection being achieved at a vinpocetine concentration of 40 microM. At this concentration, vinpocetine blocked the inhibition of the mitochondrial respiratory chain complexes II-III and IV and completely abolished the depletion of pyruvate levels induced by toxic concentrations of Abeta peptides. Furthermore, the accumulation of ROS in cells exposed to Abeta25-35 and Abeta1-40 evaluated using the fluorescent probe 2',7'-dichlorofluorescin (DCF), was reduced in the presence of 40 microM vinpocetine. Taken together, the data presented herein demonstrate that vinpocetine protects cells from Abeta toxicity, preventing the generation of oxidative stress due to the excessive accumulation of ROS. This study suggests that vinpocetine can exert neuroprotective properties which might be of importance and contribute to its clinical efficacy in the treatment of Alzheimer's disease or other neurodegenerative disorders in which oxidative stress is involved.     

Protective effect of melatonin on beta-amyloid-induced apoptosis in rat astroglioma C6 cells and its mechanism

Astrocytosis is a common feature of amyloid plaques. The Abeta-astrocyte interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration in Alzheimer disease (AD). The regulation of astrocyte apoptosis is essential to physiological and pathological processes in the CNS. Melatonin is a potent antioxidant and free radical scavenger. Previously, we showed that melatonin alleviated the learning and memory deficits in the APP 695 transgenic mouse model of AD. In this study, the importance of melatonin in the management of Abeta-induced apoptosis in an astrocyte-like cell is discussed. We found that rat astroglioma C6 cells treated with Abeta25-35 or Abeta1-42 undergo apoptosis and that melatonin pretreatment at 10(-5), 10(-6), and 10(-7) M significantly attenuates Abeta25-35- or Abeta1-42-induced apoptosis. The antiapoptotic effects of melatonin were extremely reproducible and corroborated by multiple quantitative methods, including an MTT cell viability assay, Hoechst 33342 nuclei staining, DNA fragmentation analysis, and flow cytometric analysis. In addition, melatonin effectively suppressed Abeta1-42-induced nitric oxide formation, remarkably prevented Abeta1-40-induced intracellular calcium overload, and significantly alleviated Abeta1-40-induced membrane rigidity. Our results demonstrate that, in addition to the beneficial effects of providing direct antioxidant protection to neurons, melatonin may enhance neuroprotection against Abeta-induced neurotoxicity by promoting the survival of glial cells.     

Activation of inward rectifier K+ channels by hypoxia in rabbit coronary arterial smooth muscle cells

We examined the effects of acute hypoxia on Ba2+-sensitive inward rectifier K+ (K(IR)) current in rabbit coronary arterial smooth muscle cells. The amplitudes of K(IR) current was definitely higher in the cells from small-diameter (<100 microm) coronary arterial smooth muscle cells (SCASMC, -12.8 +/- 1.3 pA/pF at -140 mV) than those in large-diameter coronary arterial smooth muscle cells (>200 microm, LCASMC, -1.5 +/- 0.1 pA pF(-1)). Western blot analysis confirmed that Kir2.1 protein was expressed in SCASMC but not LCASMC. Hypoxia activated much more KIR currents in symmetrical 140 K+. This effect was blocked by the adenylyl cyclase inhibitor SQ-22536 (10 microM) and mimicked by forskolin (10 microM) and dibutyryl-cAMP (500 microM). The production of cAMP in SCASMC increased 5.7-fold after 6 min of hypoxia. Hypoxia-induced increase in KIR currents was abolished by the PKA inhibitors, Rp-8-(4-chlorophenylthio)-cAMPs (10 microM) and KT-5720 (1 microM). The inhibition of G protein with GDPbetaS (1 mM) partially reduced (approximately 50%) the hypoxia-induced increase in KIR currents. In Langendorff-perfused rabbit hearts, hypoxia increased coronary blood flow, an effect that was inhibited by Ba2+. In summary, hypoxia augments the KIR currents in SCASMC via cAMP- and PKA-dependent signaling cascades, which might, at least partly, explain the hypoxia-induced coronary vasodilation.     

Green tea catechins prevent cognitive deficits caused by Abeta1-40 in rats

Amyloid beta peptide (Abeta)-induced oxidative stress is involved in the pathogenesis of Alzheimer's disease (AD). In contrast, green tea catechins confer potent antioxidative defense to brain neurons. Therefore, we examined whether long-term administration of green tea catechins [Polyphenon E (PE): 63% of epigallocatechin-3-gallate, 11% of epicatechin, 6% of (-)-epigallocatechin and 6% of (-)-epicatechin-gallate] prevents cognitive impairment in an animal model of AD, rats infused with Abeta(1-40) into the cerebral ventricle. Five-week-old male Wistar rats fed with an MF diet were randomly divided into two groups: 0.0% PE (rats administered with water only) and 0.5% PE (rats administered with 5 g/L of PE). Twenty weeks after the PE administration, the 0.0% PE group was divided into the Vehicle group (rats infused with the solvent used for dissolving Abeta) and the Abeta(1-40)-infused rat group (Abeta group), whereas the 0.5% PE group was divided into the PE+Vehicle group (PE-preadministered vehicle-infused rats) and the PE+Abeta group (PE-preadministered Abeta-infused rats). Abeta(1-40) or vehicle was infused into the cerebral ventricle using a mini osmotic pump. Behavioral changes in the rats were assessed by an eight-arm radial maze. PE administration for 26 weeks significantly decreased the Abeta-induced increase in the number of reference and working memory errors, with a concomitant reduction of hippocampal lipid peroxide (LPO; 40%) and cortico-hippocampal reactive oxygen species (ROS; 42% and 50%, respectively). Significantly reduced levels of LPO in the plasma (24%) and hippocampus (25%) as well as those of ROS in the hippocampus (23%) and cortex (41%) were found in the PE+Vehicle group as compared with the Vehicle group. Furthermore, rats with preadministered PE had higher ferric-reducing antioxidation power of plasma as compared with the Vehicle group. Our results suggest that long-term administration of green tea catechins provides effective prophylactic benefits against Abeta-induced cognitive impairment by increasing antioxidative defenses.