Amyloid beta proteins inhibit Cl(-)-ATPase activity in cultured rat hippocampal neurons

Cl(-)-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl(-) transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid beta proteins (Abetas), on the Cl(-)-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid beta proteins (1-40, 1-42 and 25-35) concentration-dependently (1-100 nM) and time-dependently (from 1 h to 6 day) decreased Cl(-)-ATPase activity and elevated intracellular Cl(-) concentrations ([Cl(-)]i), Abeta25-35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Abeta-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 microM wortmannin or 20 microM quercetin, but not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 microM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Abeta-treated cells as compared with each control. In the Abeta-exposed culture, but not in control, stimulation by 10 microM glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Abeta-treatment on the neurotoxicity of glutamate. Thus, Abetas reduce neuronal Cl(-)-ATPase activity, resulting in an increase in [Cl(-)]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD.     

Pathophysiological concentrations of amyloid beta proteins directly inhibit rat brain and recombinant human type II phosphatidylinositol 4-kinase activity

We previously found that pathophysiological concentrations (< or = 10 nm) of an amyloid beta protein (Abeta25-35) reduced the plasma membrane phosphatidylinositol monophosphate level in cultured rat hippocampal neurons with a decrease in phosphatidylinositol 4-monophosphate-dependent Cl- -ATPase activity. As this suggested an inhibitory effect of Abeta25-35 on plasma membrane phosphatidylinositol 4-kinase (PI4K) activity, in vitro effects of Abetas on PI4K activity was examined using rat brain subcellular fractions and recombinant human type II PI4K (PI4KII). Abeta25-35 (10 nm) inhibited PI4KII activity, but neither PI 3-kinase (PI3K) nor type III PI4K (PI4KIII) activity, in microsomal fractions, while 100 nm Abeta25-35 inhibited PI3K activity in mitochondrial fractions. In plasma membrane-rich fractions, Abetas (> 0.5 nm) dose-dependently inhibited PI4KII activity, the maximal inhibition to 77-87% of control being reached around 10 nm of Abetas without significant changes in apparent Km values for ATP and PI, suggesting non-competitive inhibition by Abetas. The inhibition by 10 nm Abeta25-35 was reversible. In recombinant human PI4KIIalpha, inhibition profiles of Abetas were similar to those in rat brain plasma membranes. Therefore, pathophysiological concentrations of Abetas directly and reversibly inhibited plasma membrane PI4KII activity, suggesting that plasma membrane PI4KII is a target of Abetas in the pathogenesis of Alzheimer's disease.     

Overexpression of glutathione peroxidase increases the resistance of neuronal cells to Abeta-mediated neurotoxicity

Senile plaques are neuropathological manifestations in Alzheimer's disease (AD) and are composed mainly of extracellular deposits of amyloid beta-peptide (Abeta). Various data suggest that the accumulation of Abeta may contribute to neuronal degeneration and that Abeta neurotoxicity could be mediated by oxygen free radicals. Removal of free radicals by antioxidant scavengers or enzymes was found to protect neuronal cells in culture from Abeta toxicity. However, the nature of the free radicals involved is still unclear. In this study, we investigated whether the neuronal overexpression of glutathione peroxidase (GPx), the major hydrogen peroxide (H2O2)-de-grading enzyme in neurons, could increase their survival in a cellular model of Abeta-induced neurotoxicity. We infected pheochromocytoma (PC12) cells and rat embryonic cultured cortical neurons with an adenoviral vector encoding GPx (Ad-GPx) prior to exposure to toxic concentrations of Abeta(25-35) or (1-40). Both PC12 and cortical Ad-GPx-infected cells were significantly more resistant to Abeta-induced injury. These data strengthen the hypothesis of a role of H2O2 in the mechanism of Abeta toxicity and highlight the potential of Ad-GPx to reduce Abeta-induced damage to neurons. These findings may have applications in gene therapy for AD.     

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.     

Soybean-derived phosphatidylinositol inhibits in vivo low concentrations of amyloid beta protein-induced degeneration of hippocampal neurons in V337M human tau-expressing mice

In our previous reports using primary cultured rat hippocampal neurons, pathophysiological concentrations (< or =10 nM) of amyloid beta proteins (Abetas) showed neurotoxicity via a phosphatidylinositol metabolism disorder, and soybean-derived phosphatidylinositol protected the neurons against the Abeta's neurotoxicity. In the present study, such a neurotoxic effect of Abeta and a neuroprotective effect of phosphatidylinositol were examined in vivo using transgenic mice expressing V337 M human tau. Intrahippocampal CA1 injection of 1.5 mul of 100 nM or 1 microM Abeta25-35 increased the number of degenerating neurons with an apoptotic feature in bilateral hippocampal CA1, CA2, CA3 and dentate gyrus regions in 1 month, demonstrating an in vivo neurotoxic effect of Abeta at lower concentrations after diffusion. Intrahippocampal co-injection or intracerebroventricular administration of 1.5 microl of 500 nM phosphatidylinositol prevented the Abeta25-35-induced neuronal degeneration in all the hippocampal regions, while co-injection of another acidic phospholipid, phosphatidylserine (1.5 microl, 500 nM) with Abeta25-35 showed no protective effects. Thus, exogenously applied phosphatidylinositol appeared to minimize the toxic effects of Abeta in vivo. These results suggest that soybean-derived phosphatidylinositol may be effective in the treatment of Alzheimer's disease.     

Microglia enhance beta-amyloid peptide-induced toxicity in cortical and mesencephalic neurons by producing reactive oxygen species

The purpose of this study was to assess and compare the toxicity of beta-amyloid (Abeta) on primary cortical and mesencephalic neurons cultured with and without microglia in order to determine the mechanism underlying microglia-mediated Abeta-induced neurotoxicity. Incubation of cortical or mesencephalic neuron-enriched and mixed neuron-glia cultures with Abeta(1-42) over the concentration range 0.1-6.0 microm caused concentration-dependent neurotoxicity. High concentrations of Abeta (6.0 microm for cortex and 1.5-2.0 microm for mesencephalon) directly injured neurons in neuron-enriched cultures. In contrast, lower concentrations of Abeta (1.0-3.0 microm for cortex and 0.25-1.0 microm for mesencephalon) caused significant neurotoxicity in mixed neuron-glia cultures, but not in neuron- enriched cultures. Several lines of evidence indicated that microglia mediated the potentiated neurotoxicity of Abeta, including the observations that low concentrations of Abeta activated microglia morphologically in neuron-glia cultures and that addition of microglia to cortical neuron-glia cultures enhanced Abeta-induced neurotoxicity. To search for the mechanism underlying the microglia-mediated effects, several proinflammatory factors were examined in neuron-glia cultures. Low doses of Abeta significantly increased the production of superoxide anions, but not of tumor necrosis factor-alpha, interleukin-1beta or nitric oxide. Catalase and superoxide dismutase significantly protected neurons from Abeta toxicity in the presence of microglia. Inhibition of NADPH oxidase activity by diphenyleneiodonium also prevented Abeta-induced neurotoxicity in neuron-glia mixed cultures. The role of NADPH oxidase-generated superoxide in mediating Abeta-induced neurotoxicity was further substantiated by a study which showed that Abeta caused less of a decrease in dopamine uptake in mesencephalic neuron-glia cultures from NADPH oxidase-deficient mutant mice than in that from wild-type controls. This study demonstrates that one of the mechanisms by which microglia can enhance the neurotoxicity of Abeta is via the production of reactive oxygen species.     

The neuroprotective effects of phytoestrogens on amyloid beta protein-induced toxicity are mediated by abrogating the activation of caspase cascade in rat cortical neurons

Amyloid beta protein (Abeta) elicits a toxic effect on neurons in vitro and in vivo. In present study we attempt to elucidate the mechanism by which Abeta confers its neurotoxicity. The neuroprotective effects of phytoestrogens on Abeta-mediated toxicity were also investigated. Cortical neurons treated with 5 microm Abeta-(25-35) for 40 h decreased the cell viability by 45.5 +/- 4.6% concomitant with the appearance of apoptotic morphology. 50 microm kaempferol and apigenin decreased the Abeta-induced cell death by 81.5 +/- 9.4% and 49.2 +/- 9.9%, respectively. Abeta increased the activity of caspase 3 by 10.6-fold and to a lesser extent for caspase 2, 8, and 9. The Abeta-induced activation of caspase 3 and release of cytochrome c showed a biphasic pattern. Apigenin abrogated Abeta-induced cytochrome c release, and the activation of caspase cascade. Kaempferol showed a similar effect but to a less extent. Kaempferol was also capable of eliminating Abeta-induced accumulation of reactive oxygen species. These two events accounted for the remarkable effect of kaempferol on neuroprotection. Quercetin and probucol did not affect the Abeta-mediated neurotoxicity. However, they potentiated the protective effect of apigenin. Therefore, these results demonstrate that Abeta elicited activation of caspase cascades and reactive oxygen species accumulation, thereby causing neuronal death. The blockade of caspase activation conferred the major neuroprotective effect of phytoestrogens. The antioxidative activity of phytoestrogens also modulated their neuroprotective effects on Abeta-mediated toxicity.     

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.     

Bis(7)-tacrine attenuates beta amyloid-induced neuronal apoptosis by regulating L-type calcium channels

Beta amyloid protein (Abeta) and acetylcholinesterase (AChE) have been shown to be closely implicated in the pathogenesis of Alzheimer's disease. In the current study, we investigated the effects of bis(7)-tacrine, a novel dimeric AChE inhibitor, on Abeta-induced neurotoxicity in primary cortical neurons. Bis(7)-tacrine, but not other AChE inhibitors, elicited a marked reduction of both fibrillar and soluble oligomeric forms of Abeta-induced apoptosis as evidenced by chromatin condensation and DNA specific fragmentation. Both nicotinic and muscarinic receptor antagonists failed to block the effects of bis(7)-tacrine. Instead, nimodipine, a blocker of L-type voltage-dependent Ca2+ channels (VDCCs), attenuated Abeta neurotoxicity, whereas N-, P/Q- or R-type VDCCs blockers and ionotropic glutamate receptor antagonists did not. Fluorescence Ca2+ imaging assay revealed that, similar to nimodipine, bis(7)-tacrine reversed Abeta-triggered intracellular Ca2+ increase, which was mainly contributed by the extracellular Ca2+ instead of endoplasmic reticulum and mitochondria Ca2+. Concurrently, using whole cell patch-clamping technique, it was found that bis(7)-tacrine significantly reduced the augmentation of high voltage-activated inward calcium currents induced by Abeta. These results suggest that bis(7)-tacrine attenuates Abeta-induced neuronal apoptosis by regulating L-type VDCCs, offers a novel modality as to how the agent exerts neuroprotective effects.     

Synaptotoxicity in Alzheimer's disease: the Wnt signaling pathway as a molecular target

Recent evidence supports a role of the Wnt pathway in neurodegenerative disorders such as Alzheimer's disease (AD). A relationship between amyloid-beta-peptide (Abeta)-induced neurotoxicity and a decrease in the cytoplasmatic levels of beta-catenin has been proposed. Also, the inhibition of glycogen synthase kinase (GSK-3beta), a central modulator of the pathway, protects rat hippocampal neurons from Abeta-induced damage. Interestingly, during the progression of AD, it has been described that active GSK-3beta is found in neuronal cell bodies and neurites, co-localizing with pre-neurofibrillary tangles observed in disease brains. Since Abeta oligomers are associated with the post-synaptic region and we have found that the non-canonical Wnt signaling modulates PSD-95 and glutamate receptors, we propose that the synaptic target for Abeta oligomers in AD is the postsynaptic region and at the molecular level is the non-canonical Wnt signaling pathway. Altogether, our evidence suggests that a sustained loss of Wnt signaling function may be involved in the Abeta-dependent neurodegeneration observed in AD brains and that the activation of this signaling pathway could be of therapeutic interest in AD.     

beta-Amyloid increases dendritic Ca2+ influx by inhibiting the A-type K+ current in hippocampal CA1 pyramidal neurons

Accumulation of the beta-amyloid peptide (Abeta) is a primary event in the pathogenesis of Alzheimer's disease (AD). However, the mechanisms by which Abeta mediates neurotoxicity and initiates the degenerative processes of AD are still not clear. Recent evidence shows that voltage-gated K+ channels may be involved in Abeta-induced neurodegenerative processes. In particular, a transient A-type K+ current, with a linear increase in its density with distance from soma to distal dendrites in hippocampal CA1 pyramidal neurons, has been shown to contribute to dendritic membrane excitability. Here, I report that Abeta (1-42) inhibits the dendritic A-type K+ current in hippocampal CA1 pyramidal neurons, and this inhibition causes increases in back-propagating dendritic action potential amplitude and associated Ca2+ influx. These results suggest that the persistent inhibition of the A-type K+ current resulting from deposition of Abeta in dendritic arborization will induce a sustained increase in dendritic Ca2+ influx and lead to loss of Ca2+ homeostasis. This may be a component of the events that cause synaptic failure and initiate neuronal degenerative processes in the hippocampus.     

Comparative study of endoplasmic reticulum stress-induced neuronal death in rat cultured hippocampal and cerebellar granule neurons

In this study, experiments were performed to characterize further the pathways responsible for neuronal death induced by endoplasmic reticulum (ER) stress in cultured hippocampal neurons (HPN) and cerebellar granule neurons (CGN) using tunicamycin (TM) and amyloid beta-peptide (Abeta). Exposure of HPN to Abeta or TM resulted in a time-dependent increase in the expression of 78-kDa glucose-regulated protein (GRP78) and caspase-12, an ER-resident caspase. In contrast, in CGN, although a drastic increase in the expression of GRP78 was found as was the case in HPN, no up-regulation of caspase-12 was detected. These results were consistent with immunohistochemical results that there were far lower number of caspase-12-positive cells in the cerebellum than in the cerebral cortex and hippocampus, and that caspase-12-positive cells were not identified in the external granule cell layer of the cerebellum of P7 rats. In CGN, a significant increase in the expression of C/EBP homologous protein (CHOP) protein was detected after exposure to Abeta or TM, whereas no such an increase in the protein expression was observed in HPN. In addition, S-allyl-L-cysteine (SAC), an organosulfur compound purified from aged garlic extract, protected neurons against TM-induced neurotoxicity in HPN but not in CGN, as in the case of Abeta-induced neurotoxicity. These results suggest that the pathway responsible for neuronal death induced by Abeta and TM in HPN differs from that in CGN, and that a caspase-12-dependent pathway is involved in HPN while a CHOP-dependent pathway is involved in CGN in ER stress-induced neuronal death.     

Acetyl-Ile-Gly-Leu protects neurons from Aβ1–42 induced toxicity in vitro and in V337M human tau-expressing mice

AimsWe previously reported that the neurotoxicity of amyloid β protein (Aβ_1–42, 10 nM) was blocked by an Aβ-derived tripeptide, Aβ_32–34 (Ile-Gly-Leu, IGL), suggesting that IGL may be a lead compound in the design of Aβ antagonists. In the present study, three stable forms of IGL peptide with acetylation of its N-terminal and/or amidation of its C-terminal (acetyl-IGL, IGL-NH₂ and acetyl-IGL-NH₂) were synthesized and examined for their effects on Aβ-induced neurotoxicity.Main methodsPhosphatidylinositol 4-kinase type II (PI4KII) activity was measured using recombinant human PI4KIIα kinase and cell viability was assessed in primary cultured hippocampal neurons. To test effects in vivo, 1.5 μl of 100 nM Aβ and/or 100 nM acetyl-IGL was injected into the hippocampal CA1 region of right hemisphere in transgenic mice expressing V337M human tau protein. Four weeks later, behavior performance in the Morris water maze was tested and after another 2 weeks, sections of brain were prepared for immunohistochemistry.Key findingsAmong the three modified tripeptides, acetyl-IGL attenuated the Aβ-induced inhibition of PI4KII activity as well as enhancement of glutamate neurotoxicity in primary cultured rat hippocampal neurons. Injection of Aβ into the hippocampus of mice impaired spatial memory and increased the number of degenerating neurons in bilateral hippocampal regions. Co-injection of acetyl-IGL prevented the learning impairment as well as the neuronal degeneration induced by Aβ.SignificanceThese results suggest that a modified tripeptide, acetyl-IGL, may be effective in the treatment of Alzheimer's disease.     

Mechanism of membrane depolarization caused by the Alzheimer Abeta1-42 peptide

We report a novel observation that the neurotoxic Alzheimer peptide Abeta1-42, when pre-incubated, causes a dramatic and lasting membrane depolarization in differentiated human hNT neuronal cells and in rodent PC12 cells in a concentration-dependent manner. This phenomenon involves activation of the metabotropic glutamate receptor, mGluR(1). Abeta-induced membrane depolarization in PC12 cells is sensitive to mGluR(1) antagonists and to pertussis and cholera toxins, indicating the involvement of particular G-proteins. The effect is different from the known ability of aggregated Abeta1-42 to cause a calcium influx. Since mGluR(1) agonists mimic the Abeta effect, we deduce that in this cell system glutamate can control the membrane potential and thereby the excitability of its target neurons. We propose that Abeta-induced membrane depolarization described here leads in Alzheimer's disease to hyperexcitability of affected neurons and is a crucially important molecular mechanism for beta-amyloid toxicity and cognitive dysfunction in the disease.     

Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis

Synaptic vesicles (SVs) are retrieved by clathrin-mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP-2 complex directly interacts with phosphatidylinositol 4-phosphate 5-kinase gamma661 (PIP5Kgamma661), the major PI(4,5)P2-producing enzyme in the brain. The beta2 subunit of AP-2 was found to bind to the C-terminal tail of PIP5Kgamma661 and cause PIP5Kgamma661 activation. The interaction is regulated by PIP5Kgamma661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kgamma661 C-terminal region in hippocampal neurons suppresses depolarization-dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kgamma661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.     

L-glutamate suppresses amyloid beta-protein-induced stellation of cultured rat cortical astrocytes

Alzheimer's amyloid beta-protein (Abeta) has been reported to potentiate glutamate toxicity in neurons, but very little is known about interaction between Abeta and glutamate in astrocytes. Therefore, in the present study, we investigated the effects of Abeta and glutamate on morphology of astrocytes. Cultured rat cortical astrocytes exhibited polygonal morphology in the absence of stimulation and differentiated into process-bearing stellate cells following exposure to Abeta (20 microM). L-Glutamate (30-1,000 microM) had no effect on astrocyte morphology in the absence of stimulation but strongly suppressed Abeta-induced stellation. The suppressive effect of L-glutamate on Abeta-induced stellation was not mimicked by glutamate receptor agonists and not blocked by glutamate receptor antagonists. In contrast, the suppressive effect of L-glutamate was mimicked by D- and L-aspartate and transportable glutamate uptake inhibitors. These results suggest that Abeta-induced astrocyte stellation is suppressed by a mechanism related to glutamate transporters.     

Small molecule, non-peptide p75 ligands inhibit Abeta-induced neurodegeneration and synaptic impairment

The p75 neurotrophin receptor (p75(NTR)) is expressed by neurons particularly vulnerable in Alzheimer's disease (AD). We tested the hypothesis that non-peptide, small molecule p75(NTR) ligands found to promote survival signaling might prevent Abeta-induced degeneration and synaptic dysfunction. These ligands inhibited Abeta-induced neuritic dystrophy, death of cultured neurons and Abeta-induced death of pyramidal neurons in hippocampal slice cultures. Moreover, ligands inhibited Abeta-induced activation of molecules involved in AD pathology including calpain/cdk5, GSK3beta and c-Jun, and tau phosphorylation, and prevented Abeta-induced inactivation of AKT and CREB. Finally, a p75(NTR) ligand blocked Abeta-induced hippocampal LTP impairment. These studies support an extensive intersection between p75(NTR) signaling and Abeta pathogenic mechanisms, and introduce a class of specific small molecule ligands with the unique ability to block multiple fundamental AD-related signaling pathways, reverse synaptic impairment and inhibit Abeta-induced neuronal dystrophy and death.     

Effect of thrombin on survival of hippocampal neurons

The effect of thrombin on the rat hippocampal neurons death in model of neurotoxicity induced by hemoglobin or glutamate, was studied. Thrombin (10 nM) was shown to inhibit 100-mkM glutamate--or 10-mkM hemoglobin-induced apoptosis of the rat hippocampal neurons. With the aid of PAR1 (protease-activated receptor1) agonist peptide and PAR1 antagonist, the PAR1 was found to be necessary for protective action of thrombin in hippocampal neurons in models of neurotoxicity induced by hemoglobin or glutamate. Because the prolonged elevation [Ca2+] ib neurons is a critical part of neurodestructive processes in CNS, the effect of thrombin on Ca2+-homeostatis of neurons after its injury by the inducer of neuronal apoptosis: a synthetic agonist of the NMDA receptors N-methyl-D-aspartate (NMDA), was studied. We hypothesized that thrombin via receptors PAR may prove to be neuroprotective for the hippocampus. Thrombin was shown to stimulate via PAR1 a transient increase in [Ca2+] in neurons in a concentration-dependent manner. Thrombin (1 nM) decreased the [Ca2+] signal induced by activation of the NMDA-subtype of glutamate receptors. This thrombin effect may be one of the reasons of the protective action of thrombin in hippocampal neurons.     

Peroxisomal proliferation protects from beta-amyloid neurodegeneration

Alzheimer disease is a neurodegenerative process that leads to severe cognitive impairment as a consequence of selective death of neuronal populations. The molecular pathogenesis of Alzheimer disease involves the participation of the beta-amyloid peptide (Abeta) and oxidative stress. We report here that peroxisomal proliferation attenuated Abeta-dependent toxicity in hippocampal neurons. Pretreatment with Wy-14.463 (Wy), a peroxisome proliferator, prevent the neuronal cell death and neuritic network loss induced by the Abeta peptide. Moreover, the hippocampal neurons treated with this compound, showed an increase in the number of peroxisomes, with a concomitant increase in catalase activity. Additionally, we evaluate the Wy protective effect on beta-catenin levels, production of intracellular reactive oxygen species, cytoplasmic calcium uptake, and mitochondrial potential in hippocampal neurons exposed to H(2) O(2) and Abeta peptide. Results show that the peroxisomal proliferation prevents beta-catenin degradation, reactive oxygen species production, cytoplasmic calcium increase, and changes in mitochondrial viability. Our data suggest, for the first time, a direct link between peroxisomal proliferation and neuroprotection from Abeta-induced degenerative changes.     

Secreted Abeta does not mediate neurotoxicity by antibody-stimulated amyloid precursor protein

Antibodies against APP, a precursor of Abeta deposited in Alzheimer's disease brain, have been shown to cause neuronal death. Therefore, it is important to determine whether Abeta mediates antibody-induced neurotoxicity. When primary neurons were treated with anti-APP antibodies, Abeta40 and Abeta42 in the cultured media were undetectable by an assay capable of detecting 100 nM Abeta peptides. However, exogenously treated Abeta1-42 or Abeta1-43 required >3 microM to exert neurotoxicity, and 25 microM Abeta1-40 was not neurotoxic. Glutathione-ethyl-ester inhibited neuronal death by anti-APP antibody, but not death by Abeta1-42, whereas serum attenuated toxicity by Abeta1-42, but not by anti-APP antibody. Using immortalized neuronal cells, we specified the domain responsible for toxicity to be cytoplasmic His(657)-Lys(676), but not the Abeta1-42 region, of APP. This indicates that neuronal cell death by anti-APP antibody is not mediated by secreted Abeta.