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.     

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.     

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.     

Human platelet calcium mobilisation in response to beta-amyloid (25-35): buffer dependency and unchanged response in Alzheimer's disease

In the present study, the effects of beta-amyloid (25-35) (Abeta (25-35)) upon calcium signalling by the human platelet has been investigated. When assays were conducted using HEPES buffers, Abeta (25-35), but not the inactive peptide Abeta (35-25), produced a robust increase in intracellular calcium that remained after removal of extracellular calcium but was abolished by the phospholipase C inhibitor U-73122. There was no significant difference between the calcium response to Abeta (25-35) in platelets from patients with Alzheimer's disease and from age-matched controls. In contrast to the robust effects on calcium mobilisation in HEPES buffers, very little calcium response to Abeta (25-35) was seen when Krebs (pH 7.8) buffer was used.     

Pituitary adenylate cyclase-activating polypeptide inhibits caspase-3 activity but does not protect cerebellar granule neurons against beta-amyloid (25-35)-induced apoptosis

The beta-amyloid (Abeta) peptide Abeta25-35 provokes apoptosis of cerebellar granule cells through activation of caspase-3 while the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) promotes granule cell survival by inhibiting caspase-3 activation through the intrinsic apoptotic pathway. The aim of the present study was to determine whether PACAP could prevent Abeta25-35 neurotoxicity by inhibiting caspase-3 activity. A 24-h exposure of cultured cerebellar granule cells to Abeta25-35 induced shrinkage of cell bodies, neurite retraction and alteration of mitochondrial activity. Administration of graded concentrations (10-80 microM) of Abeta25-35 induced a dose-related decrease of the number of living cells, and the neurotoxic effect was highly significant after a 24-h exposure to 80 microM Abeta25-35. Exposure of cerebellar granule cells to Abeta25-35 markedly enhanced caspase-3 but not caspase-9 activity. Co-incubation with 1 microM PACAP significantly reduced Abeta25-35-evoked caspase-3 activation. In contrast, PACAP did not prevent the deleterious effects of Abeta25-35 on mitochondrial potential and granule cell survival. Taken together, these data suggest that caspase-3 activation is not the main pathway activated by Abeta25-35 that leads to granule cell death. The results also demonstrate that PACAP cannot be considered as a potent neuroprotective factor against Abeta25-35-induced apoptosis in cerebellar granule neurons.     

Inhibition of beta-amyloid-induced neurotoxicity by imidazopyridoindoles derived from a synthetic combinatorial library

Alzheimer's disease is a progressive neurodegenerative disorder characterized by the deposit of amyloid fibrils in the brain that result from the self-aggregative polymerization of the beta-amyloid peptide (Abeta). Evidence of a direct correlation between the ability of Abeta to form stable aggregates in aqueous solution and its neurotoxicity has been reported. The cytotoxic effects of Abeta have been attributed to the aggregation properties of a domain corresponding to the peptide fragment Abeta25-35. In an effort to generate novel inhibitors of Abeta neurotoxicity and/or aggregation, a mixture-based synthetic combinatorial library composed of 23 375 imidazopyridoindoles was generated and screened for inhibition of Abeta25-35 neurotoxicity toward the rat pheochromocytoma PC-12 cell line. The effect of the identified lead compounds on Abeta25-35 aggregation was then evaluated by means of circular dichroism (CD) and thioflavin-T fluorescence spectroscopy. Their activity against Abeta1-42 neurotoxicity toward the PC-12 cell line was also determined. The most active imidazopyridoindoles inhibited both Abeta25-35 and Abeta1-42 neurotoxicity in the low- to mid-micromolar range. Furthermore, inhibition of the random coil to beta-sheet transition and self-aggregation of Abeta25-35 was observed by CD and fluorescence spectroscopy, supporting the relationship between inhibition of the Abeta aggregation process and neurotoxicity.     

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.     

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.     

Isolation and Structures of Two New Metabolites from Aspergillus ruber

Beta-amyloid (Aβ) has been suggested to induce neurotoxicity in Alzheimer’s disease. We evaluated the neuroprotective effects of delphinidin, an anthocyanidin commonly present in pigmented fruits and vegetables, against Aβ-induced toxicity. Aβ (25–35) significantly decreased the viability of PC12 cells, and this was accompanied by an increase in intracellular calcium levels and tau phosphorylation. However, treatment with delphinidin rescued PC12 cells from Aβ by attenuating the elevation of intracellular calcium levels and tau phosphorylation. Taken together, these results suggest that delphinidin protects PC12 cells against Aβ-induced toxicity by attenuating intracellular calcium influx and tau hyperphosphorylation.     

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.     

Protective effect of caffeic acid against beta-amyloid-induced neurotoxicity by the inhibition of calcium influx and tau phosphorylation

AimsThe progressive accumulation of beta-amyloid peptide (Aβ), in the form of senile plaques, has been recognized as one of the major causes of Alzheimer's disease (AD) pathology. Increased production of Aβ and the aggregation of Aβ to oligomers have been reported to trigger neurotoxicity, oxidative damage and inflammation. Furthermore, Aβ-induced tau hyperphosphorylation and neurotoxicity are downstream of Aβ. Therefore, we studied the possible neuroprotective effects of caffeic acid against Aβ-induced toxicity.Main methodsTreatment of PC12 cells with 10 μM Aβ (25–35) for 24 h significantly decreased the cell viability; this was accompanied by an increase in intracellular calcium levels and tau phosphorylation with GSK-3β (glycogen synthase kinase-3β) activation (phosphorylation).Key findingsHowever, pretreatment of the PC12 cells with 10 and 20 μg/ml of caffeic acid, for 1 h prior to Aβ, significantly reversed the Aβ-induced neurotoxicity by attenuating the elevation of intracellular calcium levels and tau phosphorylation.SignificanceTaken together, these results suggest that caffeic acid protected the PC12 cells against Aβ-induced toxicity. In addition, the neuroprotective mechanisms of caffeic acid against Aβ attenuated intracellular calcium influx and decreased tau phosphorylation by the reduction of GSK-3β activation.     

Abeta(25-35) and Abeta(1-40) act on different calcium channels in CA1 hippocampal neurons

The acute effects of beta-amyloid (25-35) and (1-40) on high voltage activated calcium channels were compared in CA1 pyramidal cells of adult mouse hippocampal slices using the whole-cell patch-clamp recording. Bath application of oligomeric beta-amyloid (25-35) reversibly increased the barium current (I(Ba)) to 1.61 (normalized amplitude), while oligomeric beta-amyloid (1-40) reversibly enhanced the I(Ba) to 1.74. Reverse-sequence beta-amyloid [(35-25) and (40-1)] had no effect. The effect of beta-amyloid (25-35) was blocked by nifedipine, a selective antagonist of L-type calcium channels. In contrast, the effect of beta-amyloid (1-40) was not blocked by nifedipine and I(Ba) was enhanced to 4.96. It is concluded that these oligomeric peptides may act through different types of calcium channels and/or receptors. The toxicity of Abeta(25-35) implicates a potentiation of L-type calcium channels while the one of Abeta(1-40) is related to an increase of non-L-type calcium channels, which may involve an increase in transmitter release.     

Beta-amyloid peptides stimulate endozepine biosynthesis in cultured rat astrocytes

Accumulation of beta-amyloid peptide (Abeta), which is a landmark of Alzheimer's disease, may alter astrocyte functions before any visible symptoms of the disease occur. Here, we examined the effects of Abeta on biosynthesis and release of diazepam-binding inhibitor (DBI), a polypeptide primarily expressed by astroglial cells in the CNS. Quantitative RT-PCR and specific radioimmunoassay demonstrated that aggregated Abeta(25-35), at concentrations up to 10(-4) m, induced a dose-dependent increase in DBI mRNA expression and DBI-related peptide release from cultured rat astrocytes. These effects were totally suppressed when aggregation of Abeta(25-35) was prevented by Congo red. Measurement of the number of living cells revealed that Abeta(25-35) induced a trophic rather than a toxic effect on astrocytes. Administration of cycloheximide blocked Abeta(25-35)-induced increase of DBI gene expression and endozepine accumulation in astrocytes, indicating that protein synthesis is required for DBI gene expression. Altogether, the present data suggest that Abeta-induced activation of endozepine biosynthesis and release may contribute to astrocyte proliferation associated with Alzheimer's disease.     

Distinct mechanisms account for β-amyloid toxicity in PC12 and differentiated PC12 neuronal cells

Whether reactive oxygen species (ROS) mediate beta-amyloid (A beta) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by A beta(25-35). The viability of PC12 and dPC12 cells decreased by 30-40% after a 48-hour exposure to 20 microM A beta(25-35). Microscopic examination showed that A beta(25-35) induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 microM) and other antioxidants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of A beta(25-35). Since H(2)O(2) has been proposed to be involved in A beta toxicity, the effects of H(2)O(2) on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be significantly more resistant to H(2)O(2) than naive PC12 cells. These data suggest that ROS may mediate A beta(25-35) toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were elevated during the differentiation of PC12 cells, the baseline levels of ROS in these two model cell types may determine the intracellular mediators for A beta(25-35) toxicity. Therefore, the protective effects of antioxidants against A beta may depend upon the redox state of the cells.     

The correlation between neurotoxicity, aggregative ability and secondary structure studied by sequence truncated Abeta peptides

Aggregated beta-amyloid (Abeta) peptides are neurotoxic and cause neuronal death both in vitro and in vivo. Although the formation of a beta-sheet structure is usual required to form aggregates, the relationship between neurotoxicity and the Abeta sequence remains unclear. To explore the correlation between Abeta sequence, secondary structure, aggregative ability, and neurotoxicity, we utilized both full-length and fragment-truncated Abeta peptides. Using a combination of spectroscopic and cellular techniques, we demonstrated that neurotoxicity and aggregative ability are correlated while the relationship between these characteristics and secondary structure is not significant. The hydrophobic C-terminus, particularly the amino acids of 17-21, 25-35, and 41-42, is the main region responsible for neurotoxicity and aggregation. Deleting residues 17-21, 25-35 or 41-42 significantly reduced the toxicity. On the other hand, truncation of the peptides at either residues 22-24 or residues 36-40 had little effect on toxicity and aggregative ability. While the N-terminal residues 1-16 may not play a major role in neurotoxicity and aggregation, a lack of N-terminal fragment Abeta peptide, (e.g. Abeta17-35), does not display the neurotoxicity of either full-length or 17-21, 25-35 truncated Abeta peptides.     

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.     

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.     

2-Cyclopropylimino-3-methyl-1,3-thiazoline Hydrochloride Protects Against Beta-amyloid-induced Activation of the Apoptotic Cascade in Cultured Cortical Neurons

Aggregated β-amyloid, implicated in the pathogenesis of Alzheimer’s disease (AD), induces neurotoxicity by evoking a cascade of oxidative damage-dependent apoptosis in neurons. We investigated the molecular mechanisms underlying the protective effect of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride (KHG26377) against the beta-amyloid (Aβ_25–35)-induced primary cortical neuronal cell neurotoxicity. Treatment with KHG26377 attenuated the Aβ_25–35-induced apoptosis by decreasing the Bax/Bcl-2 ratio and suppressing the activation of caspase-3. A marked increase in calcium influx and in the level of reactive oxygen species together with a decrease in glutathione levels was found after Aβ_25–35 exposure; however, KHG26377 treatment reversed these changes in a concentration-dependent manner. In addition, KHG26377 significantly suppressed Aβ_25–35-induced toxicity concomitant with a reduction in the activation of extracellular signal-regulated kinases 1 and 2 and nuclear factor kappa B. The KHG26377-induced protection of neuronal cells against Aβ toxicity was also mediated by suppressing the expression of glycogen synthase kinase-3β, increasing the levels of β-catenin, and reducing the levels of phosphorylated tau. Our findings suggest that KHG26377 may modulate the neurotoxic effects of β-amyloid and provide a rationale for treatment of AD.     

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.