Acetyl-L-carnitine-induced up-regulation of heat shock proteins protects cortical neurons against amyloid-beta peptide 1-42-mediated oxidative stress and neurotoxicity: implications for Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory and cognition and by senile plaques and neurofibrillary tangles in brain. Amyloid-beta peptide, particularly the 42-amino-acid peptide (Abeta(1-42)), is a principal component of senile plaques and is thought to be central to the pathogenesis of the disease. The AD brain is under significant oxidative stress, and Abeta(1-42) peptide is known to cause oxidative stress in vitro and in vivo. Acetyl-L-carnitine (ALCAR) is an endogenous mitochondrial membrane compound that helps to maintain mitochondrial bioenergetics and lowers the increased oxidative stress associated with aging. Glutathione (GSH) is an important endogenous antioxidant, and its levels have been shown to decrease with aging. Administration of ALCAR increases cellular levels of GSH in rat astrocytes. In the current study, we investigated whether ALCAR plays a protective role in cortical neuronal cells against Abeta(1-42)-mediated oxidative stress and neurotoxicity. Decreased cell survival in neuronal cultures treated with Abeta(1-42) correlated with an increase in protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (4-hydroxy-2-nonenal) formation. Pretreatment of primary cortical neuronal cultures with ALCAR significantly attenuated Abeta(1-42)-induced cytotoxicity, protein oxidation, lipid peroxidation, and apoptosis in a dose-dependent manner. Addition of ALCAR to neurons also led to an elevated cellular GSH and heat shock proteins (HSPs) levels compared with untreated control cells. Our results suggest that ALCAR exerts protective effects against Abeta(1-42) toxicity and oxidative stress in part by up-regulating the levels of GSH and HSPs. This evidence supports the pharmacological potential of acetyl carnitine in the management of Abeta(1-42)-induced oxidative stress and neurotoxicity. Therefore, ALCAR may be useful as a possible therapeutic strategy for patients with AD.     

Protective effect of D609 against amyloid-beta1-42-induced oxidative modification of neuronal proteins: redox proteomics study

Oxidative stress has been implicated in the pathophysiology of a number of diseases, including neurodegenerative disorders such as Alzheimer's disease (AD), a neurodegenerative disorder associated with cognitive decline and enhanced oxidative stress. Amyloid-beta peptide(1-42) (Abeta(1-42)), one of the main component of senile plaques, can induce in vitro and in vivo oxidative damage to neuronal cells through its ability to produce free radicals. The aim of this study was to investigate the protective effect of the xanthate D609 on Abeta(1-42)-induced protein oxidation by using a redox proteomics approach. D609 was recently found to be a free radical scavenger and antioxidant. In the present study, rat primary neuronal cells were pretreated with 50 microM of D609, followed by incubation with 10 microM Abeta(1-42) for 24 hr. In the cells treated with Abeta(1-42) alone, four proteins that were significantly oxidized were identified: glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, malate dehydrogenase, and 14-3-3 zeta. Pretreatment of neuronal cultures with D609 prior to Abeta(1-42) protected all the identified oxidized proteins in the present study against Abeta(1-42)-mediated protein oxidation. Therefore, D609 may ameliorate the Abeta(1-42)-induced oxidative modification. We discuss the implications of these Abeta(1-42)-mediated oxidatively modified proteins for AD pathology and for potential therapeutic intervention in this dementing disorder.     

Amyloid-beta down-regulates XIAP expression in human SH-SY5Y neuroblastoma cells

Recent observations suggest that amyloid-beta (Abeta), a major constituent of senile plaques, induces apoptosis in cultured neuronal cells. However, the concentration of Abeta that leads to neuronal cell death is much higher (10-25 microM) than that in the cerebrospinal fluid of normal controls or AD patients (nM order). As reported here, we found that subtoxic concentrations (100-500 nM) of Abeta(1-42) can down-regulate the expression of the X-linked inhibitor of apoptosis (XIAP) in human SH-SY5Y neuroblastoma cells, and that vulnerability to oxidative stress caused by Abeta(1-42) is attenuated by over-expression of XIAP. These results suggest that down-regulation of XIAP expression in response to subtoxic, more physiological concentrations (100-500 nM) of Abeta(1-42) increases vulnerability to oxidative stress.     

Huperzine A attenuates amyloid beta-peptide fragment 25-35-induced apoptosis in rat cortical neurons via inhibiting reactive oxygen species formation and caspase-3 activation.

Huperzine A, a novel Lycopodium alkaloid originally discovered in the Chinese herb Qian Ceng Ta (Huperzia serrata), is a reversible, potent, and selective acetylcholinesterase (AChE) inhibitor and has been extensively used for the treatment of Alzheimer's disease (AD) in China. The present studies were designed to investigate effects of huperzine A on amyloid beta-peptide fragment 25-35 (Abeta25-35)-induced neuronal apoptosis and potential mechanisms in primary cultured rat cortical neurons. After exposure of the cells to Abeta25-35 (20 microM), apoptotic cell death was observed as evidenced by a significant decrease in cell viability, alteration of neuronal morphology, and DNA fragmentation. Pretreatment of the cells with huperzine A (0.01-10 microM) prior to Abeta25-35 exposure significantly elevated the cell survival and reduced Abeta25-35-induced nuclei fragmentation. Reactive oxygen species (ROS)-based fluorescence, caspase-3-like fluorogenic cleavage, and Western blot analysis demonstrated that huperzine A reduced Abeta25-35-induced ROS formation in a dose-dependent manner, and 1 microM of huperzine A attenuated Abeta25-35-induced caspase-3 activity at 6, 12, 24, and 48 hr posttreatment. Our results provide the first direct evidence that huperzine A protects neurons against Abeta25-35-induced apoptosis via the inhibition of ROS formation and caspase-3 activity.     

Fas and Fas ligand are associated with neuritic degeneration in the AD brain and participate in beta-amyloid-induced neuronal death

It has recently been suggested that neuronal cell death in response to many brain insults may be mediated by the upregulation of tumor necrosis factor receptor (TNFR) family members and their ligands. In the present study, we investigated whether the expression of the TNFR family death domain receptor, Fas, and its ligand, FasL, is altered in association with neuropathology and activated caspase markers in Alzheimer disease (AD) brain, and Abeta-induced neuronal cell death in vitro. To evaluate this hypothesis, we examined Fas and FasL expression in AD and control brain, and Abeta-treated primary neurons, using immunocytochemistry and Western blots. Neurons in both AD brain and Abeta-treated cultures exhibited FasL upregulation and changes in immunoreactivity for Fas receptor. Further, FasL expression was remarkably elevated in senile plaques and neurofilament-positive dystrophic neurites, and in association with caspase activation and neuritic apoptosis in AD brain. Based on these and previous data regarding protection of primary neuronal cultures from Abeta(1-42)-induced apoptosis by blockade of Fas-associated death domain signaling, we also tested the hypothesis that dynamic regulation of Fas and FasL may contribute to Abeta-mediated neuronal cell death. Accordingly, neuronal cultures derived from mice carrying inactivating mutations in Fas (Faslpr) or FasL (Fasgld) exhibited protection from Abeta(1-42)-induced cell death. These findings suggest that Fas-FasL interactions may contribute to mechanisms of neuronal loss and neuritic degeneration in AD.     

Protection against beta-amyloid peptide-induced memory impairment with long-term administration of extract of Angelica gigas or decursinol in mice

We investigated the effect of long-term oral administration of ethanolic extract of Angelica gigas Nakai (Umbelliferae) (EAG) or decursinol, a coumarin isolated from A. gigas, on beta-amyloid peptide 1-42 (Abeta(1-42))-induced memory impairment in mice. Mice were allowed free access to drinking water (control) or water containing different concentrations of EAG. After 4 weeks, Abeta(1-42) (410 pmol) was administered via intracerebroventricular injection. Pretreatment of mice with EAG (0.1%) for 4 weeks significantly blocked the Abeta(1-42)-induced impairment in passive avoidance performance. Next, mice were fed with chow mixed with various doses of decursinol for 4 weeks before intracerebroventricular injection of Abeta(1-42) (410 pmol). Pretreatment of mice with decursinol (0.001%, 0.002%, and 0.004%) for 4 weeks significantly attenuated the Abeta(1-42)-induced impairment in passive avoidance performance. Decursinol (0.004%) also significantly blunted the Abeta(1-42)-induced decrease in alternation behavior (spatial working memory) in the Y-maze test without change in general locomotor activity. These findings suggest that EAG or decursinol may have preventive effect against memory impairment related with Abeta of Alzheimer's disease.     

Transforming growth factor beta2 autocrinally mediates neuronal cell death induced by amyloid-beta

Amyloid beta (Abeta), the major component of the senile plaques of Alzheimer's disease, is implicated in neuronal cell death. We have found that Abeta42, a neurotoxic form of Abeta peptide, induces both neuronal and glial expression of TGFbeta2. We have further demonstrated that the addition into culture media of neutralizing antibody to TGFbeta2 or a large amount of the recombinant soluble amyloid precursor protein alpha, the extracellular domain of amyloid precursor protein (APP) generated by alpha secretase, suppresses death in primary cortical neurons (PCNs) induced by Abeta42 in vitro. Combined with the finding in our recent study indicating that TGFbeta2 is a neuronal cell death-inducing ligand for APP, it is suggested that TGFbeta2 is an autocrinal mediator for Abeta42-induced death in PCNs.     

beta-Amyloid and its binding protein in the hippocampus of diabetic mice: effect of APP17 peptide.

The objective of this study was to investigate whether changes in Abeta and ERAB exist in the brain of diabetic mice, and to observe the effects of APP17 peptide. The numbers of neurons stained by APP17 peptide Abeta1-40 Abeta1-42 Abeta1-16 and ERAB antibodies in the brain of diabetic mice was increased compared with normal mice. Staining in APP17 peptide-protected mice was similar to normal mice. We conclude that increased Abeta1-42 and ERAB is an important cause of neuronal degeneration in diabetic encephalopathy. APP17 peptide retards neuronal degeneration by regulating the metabolism of Abeta.     

IL-8 enhancement of amyloid-beta (Abeta 1-42)-induced expression and production of pro-inflammatory cytokines and COX-2 in cultured human microglia

The effects of the chemokine IL-8 on amyloid beta peptide (Abeta(1-42))-induced responses in cultured human microglia were investigated using RT-PCR, ELISA and immunocytochemistry. Abeta(1-42) (5 microM) applied for 8 h induced the expression and increased the production of the pro-inflammatory cytokines IL-6, IL-1beta, TNF-alpha, the inducible enzyme COX-2 and chemokine IL-8. Microglial treatment with IL-8 added (at 100 ng/mL) with Abeta(1-42) led to enhancement in both expression and production of all of these pro-inflammatory factors compared with peptide alone. Stimulation with IL-8 itself was effective in increasing microglial expression of pro-inflammatory cytokines, IL-8 and COX-2, however, had no effect on protein levels of all these factors. The expression of the anti-inflammatory cytokines IL-10 and TGFbeta(1) remained unchanged from basal levels with stimulation using either Abeta(1-42), IL-8 or the peptide together with IL-8. The actions of IL-8 to potentiate Abeta(1-42)-induced inflammatory mediators may have particular relevance to Alzheimer disease brain which exhibits elevated levels of the chemokine.     

Role of potassium channels in Abeta(1-40)-activated apoptotic pathway in cultured cortical neurons

Potassium channel dysfunction has been implicated in Alzheimer's disease (AD). In the present study, by using potassium channel blocker tetraethylammonium (TEA), we investigated the relationship between the enhancement of potassium currents and the alteration of apoptotic cascade in the neuronal apoptotic model induced by beta-amyloid peptide 1-40(Abeta(1-40)). Cortical neurons exposed to Abeta(1-40) 5 muM developed a specific increase in the delayed rectifier potassium current (I(K)), but not the transient outward potassium currents (I(A)), before the appearance of neuronal apoptosis. Abeta(1-40) induced various apoptotic features such as chromatin condensation, a decrease in the amount of Bcl-2 protein, an increase in the amount of Bax protein, cytochrome c release from mitochondria, and caspase-3 activation. Potassium channel blocker 5 mM TEA attenuated Abeta(1-40)-induced neuronal death and prevented the alterations of all above mentioned apoptotic indicators. The study indicates that I(K) enhancement might play an important role in certain form of programmed cell death induced by beta-amyloid peptide (Abeta). Increased potassium channel activity might trigger the activation of apoptosis cascade in Abeta(1-40)-treated rat cortical neurons.     

Beta-amyloid (1-42)-induced learning and memory deficits in mice: involvement of oxidative burdens in the hippocampus and cerebral cortex

We have demonstrated that oxidative stress is involved, at least in part, in beta-amyloid protein (Abeta)-induced neurotoxicity in vivo [Eur. J. Neurosci. 1999;11:83-90; Neuroscience 2003;119:399-419]. However, mechanistic links between oxidative stress and memory loss in response to Abeta remain elusive. In the present study, we examined whether oxidative stress contributes to the memory deficits induced by intracerebroventricular injection of Abeta (1-42) in mice. Abeta (1-42)-induced memory impairments were observed, as measured by the water maze and passive avoidance tests, although these impairments were not found in Abeta (40-1)-treated mice. Treatment with antioxidant alpha-tocopherol significantly prevented memory impairment induced by Abeta (1-42). Increased activities of the cytosolic Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and mitochondrial Mn-superoxide dismutase (Mn-SOD) were observed in the hippocampus and cerebral cortex of Abeta (1-42)-treated animals, as compared with Abeta (40-1)-treated mice. The induction of Cu,Zn-SOD was more pronounced than that of Mn-SOD after Abeta (1-42) insult. However, the concomitant induction of glutathione peroxidase (GPX) in response to significant increases in SOD activity was not seen in animals treated with Abeta (1-42). Furthermore, glutathione reductase (GRX) activity was only increased at 2h after Abeta (1-42) injection. Production of malondialdehyde (lipid peroxidation) and protein carbonyl (protein oxidation) remained elevated at 10 days post-Abeta (1-42), but the antioxidant alpha-tocopherol significantly prevented these oxidative stresses. Therefore, our results suggest that the oxidative stress contributes to the Abeta (1-42)-induced learning and memory deficits in mice.     

In vitro model of neurotoxicity of Abeta 1-42 and neuroprotection by a pentapeptide: irreversible events during the first hour

The cell biology of Alzheimer's disease (AD) is characterized mainly by the neurodegeneration caused by the beta-amyloid (Abeta) peptides and by the formation of neurofibrillary tangles. The initial events of neurodegeneration in the brain tissue include synaptic dysfunction and axonopathy. Abeta-induced axonopathy and neurite degeneration were studied in vitro on differentiated human-derived neurotypic SH-SY5Y cells. Different methods were used to investigate the mechanism of action of aggregated Abeta on neuroblastoma cells. Abeta 1-42 aggregated for 1 h induced irreversible changes in the neurite morphology. Change of tau hyperphosphorylation and cell viability (cytoplasmic redox state and active membrane uptake) was irreversible during the first hour after the addition of Abeta 1-42 to the cells. These rapid events indicate that Abeta might induce neurodegeneration even at an early stage of Abeta-cell contact. A novel pentapeptide LPYFD-amide, an analog of Soto's LPFFD, significantly decreased neurite degeneration, tau aggregation, and cell viability reduction induced by Abeta 1-42.     

α7 nicotinic acetylcholine receptor expression in Alzheimer's disease

The brains of people with Alzheimer's disease (AD) display several characteristic pathological features, including deposits (plaques) of beta-amyloid 1-42 (Abeta1-42), intraneuronal accumulations (tangles) of hyperphosphorylated tau, degeneration of the basal forebrain cholinergic pathway, and gliosis. Abeta1-42 plaques develop in specific brain regions, including hippocampus and cortex, as well as in the vasculature. Abeta1-42 might promote neurodegeneration through the induction of free radicals and disruption of Ca2+ homeostasis, giving rise to the symptoms of AD. Abeta1-42 interacts with the alpha7 subtype of the nicotinic acetylcholine receptor (alpha7 nAChR), which is widely expressed throughout the central and peripheral nervous systems, as well as in several nonneuronal loci, such as epithelial cells, lymphoid tissues, and peripheral blood lymphocytes. Western blot and autoradiographic analyses indicate that the alpha7 nAChR subunit protein is up-regulated in human brain samples from Alzheimer patients, as well as in animal models of AD (Dineley et al., 2001; Bednar et al., 2002), and might be involved in nicotine-mediated reduction of Abeta1-42 deposition (Hellstrom et al., 2004), although the nature of this relationship remains ill-defined. We have undertaken a semiquantitative histological evaluation of alpha7 nAChR expression in a mouse model of AD pathology, as well as a comparison of alpha7 nAChR levels in lymphocytes from AD patients and control subjects.     

Proteomic identification of proteins oxidized by Abeta(1-42) in synaptosomes: implications for Alzheimer's disease

Protein oxidation has been implicated in Alzheimer's disease (AD) and can lead to loss of protein function, abnormal protein turnover, interference with cell cycle, imbalance of cellular redox potential, and eventually cell death. Recent proteomics work in our laboratory has identified specifically oxidized proteins in AD brain such as: creatine kinase BB, glutamine synthase, ubiquitin carboxy-terminal hydrolase L-1, dihydropyrimidase-related protein 2, alpha-enolase, and heat shock cognate 71, indicating that a number of cellular mechanisms are affected including energy metabolism, excitotoxicity and/or synaptic plasticity, protein turnover, and neuronal communication. Synapse loss is known to be an early pathological event in AD, and incubation of synaptosomes with amyloid beta peptide 1-42 (Abeta 1-42) leads to the formation of protein carbonyls. In order to test the involvement of Abeta(1-42) in the oxidation of proteins in AD brain, we utilized two-dimensional gel electrophoresis, immunochemical detection of protein carbonyls, and mass spectrometry to identify proteins from synaptosomes isolated from Mongolian gerbils. Abeta(1-42) treatment leads to oxidatively modified proteins, consistent with the notion that Abeta(1-42)-induced oxidative stress plays an important role in neurodegeneration in AD brain. In this study, we identified beta-actin, glial fibrillary acidic protein, and dihydropyrimidinase-related protein-2 as significantly oxidized in synaptosomes treated with Abeta(1-42). Additionally, H+-transporting two-sector ATPase, syntaxin binding protein 1, glutamate dehydrogenase, gamma-actin, and elongation factor Tu were identified as increasingly carbonylated. These results are discussed with respect to their potential involvement in the pathogenesis of AD.     

Effects of Abeta1-42 fibrils and of the tetrapeptide Pr-IIGL on the phosphorylation state of the tau-protein and on the alpha7 nicotinic acetylcholine receptor in vitro

In order to investigate the possible links connecting beta-amyloid (Abeta) accumulation, tau-hyperphosphorylation and nicotinic receptor expression, rat embryonic primary hippocampal cultures were incubated with amyloidogenic peptides. Exposure to 0.5 microm fibrillar Abeta(1-42) for 3 days caused retraction of dendrites, shrinkage of cell bodies and a decrease in the expression of microtubule-associated proteins 2b (MAP2b), without affecting the total number of neurons and their viability. No impact on the tau-phosphorylation sites Ser-202, Thr231/Ser235, Ser262 and Ser396/Ser404 was found. The total number of homomeric alpha7-nicotinic receptors (alpha7-nAChRs) and their affinity for [(125)I]alpha-bungarotoxin remained unaltered. Upon incubation with the putatively protective tetrapeptide propionyl-isoleucine-isoleucine-glycine-leucine (Pr-IIGL), an analogue of the region [31-34] of Abeta, cell bodies were swollen in the region of the apical dendrite. These morphological alterations, different from those elicited by Abeta(1-42), did not involve MAP2 expression changes. In contrast to Abeta(1-42), Pr-IIGL caused a massive hyperphosphorylation of the tau-protein at Ser-202 and at Ser396/Ser404. The total number of homomeric alpha7-nAChRs and their affinity for [(125)I]alpha-bungarotoxin were unaffected. In conclusion, the present results show a toxic effect of Abeta(1-42) on the cytoskeletal structure at concentrations normally present in the brains of Alzheimer's disease patients, but raise some doubts about the role of Abeta(1-42) fibrils as a direct trigger of tau-hyperphosphorylation. The tetrapeptide Pr-IIGL cannot be considered protective with regard to cell morphology. Although it prevents the Abeta(1-42)-induced retraction of dendrites, it exhibits other toxic properties. The homomeric alpha7-nAChRs were not affected either by Abeta(1-42) incubation or by Pr-IIGL-induced tau-hyperphosphorylation.     

Overexpression of amyloid precursor protein is associated with degeneration, decreased viability, and increased damage caused by neurotoxins (prostaglandins A1 and E2, hydrogen peroxide, and nitric oxide) in differentiated neuroblastoma cells

Inflammatory reactions are considered one of the important etiologic factors in the pathogenesis of Alzheimer's disease (AD). Prostaglandins such as PGE2 and PGA1 and free radicals are some of the agents released during inflammatory reactions, and they are neurotoxic. The mechanisms of their action are not well understood. Increased levels of beta-amyloid fragments (Abeta40 and Abeta42), generated through cleavage of amyloid precursor protein (APP), oxidative stress, and proteasome inhibition, are also associated with neurodegeneration in AD brains. Therefore, we investigated the effect of PGs and oxidative stress on the degeneration and viability of cyclic AMP-induced differentiated NB cells overexpressing wild-type APP (NBP2-PN46) under the control of the CMV promotor in comparison with differentiated vector (NBP2-PN1) or parent (NBP2) control cells. Results showed that differentiated NBP2-PN46 cells exhibited enhanced spontaneous degeneration and decreased viability in comparison with differentiated control cells, without changing the level of Abeta40 and Abeta42. PGA1 or PGE2 treatment of differentiated cells caused increased degeneration and reduced viability in all three cell lines. These effects of PGs are not due to alterations in the levels of vector-derived APP mRNA or human APP holoprotein, secreted levels of Abeta40 and Abeta42, or proteasome activity. H2O2 or SIN-1 (an NO donor) treatment did not change vector-derived APP mRNA levels, but H2O2 reduced the level of human APP protein more than SIN-1. Furthermore, SIN-1 increased the secreted level of Abeta40, but not of Abeta42, whereas H2O2 had no effect on the level of secreted Abeta fragments. Both H2O2 and SIN-1 inhibited proteasome activity in the intact cells. The failure of neurotoxins to alter APP mRNA levels could be due to the fact that they do not affect CMV promoter activity. These results suggest that the mechanisms of action of PGs on neurodegeneration are different from those of H2O2 and SIN-1 and that the mechanisms of neurotoxicity of H2O2 and SIN-1 are, at least in part, different from each other.     

CSF biomarkers in the evaluation of idiopathic normal pressure hydrocephalus

BACKGROUND - To evaluate cerebrospinal fluid (CSF) markers for neuronal degeneration and demyelination in idiopathic normal pressure hydrocephalus (INPH), subcortical arteriosclerotic encephalopathy (SAE), and neurologically healthy subjects. METHODS - Lumbar CSF concentrations of sulfatide, neurofilament protein light (NFL), total-tau (T-tau), hyperphosphorylated tau (P-tau), and beta-amyloid(1-42) (Abeta42) were analyzed in 62 INPH patients, 26 SAE patients, and 23 neurologically healthy controls. In INPH patients, samples before and after shunt surgery were analysed. RESULTS - The CSF concentration of NFL was elevated in INPH and SAE compared with the controls, and levels of T-tau, P-tau, and Abeta42 were lower in INPH compared with SAE and controls. No difference was seen for sulfatide. All markers except Abeta42 were significantly elevated after shunt surgery. CONCLUSIONS - The most striking finding was the power of the combined pattern of NFL, P-tau, and Abeta42 in distinguishing between the clinical diagnoses of INPH, SAE, and neurologically healthy elderly.     

Inhibitory effects of long-term administration of ferulic acid on astrocyte activation induced by intracerebroventricular injection of beta-amyloid peptide (1-42) in mice

Accumulating evidence indicates that glial cells are actively involved in the pathogenesis of Alzheimer's disease. We recently reported protective effects of long-term administration of ferulic acid against learning and memory deficit induced by centrally administered beta-amyloid peptide (Abeta)1-42 in mice. In that report, we found that the Abeta1-42-induced increases in immunoreactivities of glial fibrillary acidic protein, the astrocyte marker, and interleukin(IL)-1beta in the hippocampus are also suppressed by pretreatment with ferulic acid. In the present study, we aimed to further characterize the effect of long-term administration of ferulic acid on the centrally administered Abeta1-42-induced activation of glial cells in mice. Mice were allowed free access to drinking water (control) or water containing ferulic acid (0.006%) for 4 weeks, and then Abeta1-42 (410 pmol) was administered via intracerebroventricular injection. Intracerebroventricularly injected Abeta1-42 induced an increase in immunoreactivities of endothelial nitric oxide synthase (eNOS) and 3-nitrotyrosine (3-NT) in the activated astrocytes in the hippocampus. Pretreatment of ferulic acid for 4 weeks prevented the Abeta1-42-induced increase in eNOS and 3-NT immunoreactivities. Administration of ferulic acid per se induced a transient and slight increase in eNOS immunoreactivity in the hippocampus on day 14, which returned to basal levels on day 28. Intracerebroventricularly injected Abeta1-42 also increased interleukin-1alpha(IL-1alpha) immunoreactivity in the hippocampus, which was also suppressed by pretreatment with ferulic acid. These results demonstrate that long-term administration of ferulic acid induces suppression of the centrallly injected Abeta1-42-induced activation of astrocytes which is suggested to underlie the protective effect of ferulic acid against Abeta1-42 toxicity in vivo.     

Gamma-glutamylcysteine ethyl ester protection of proteins from Abeta(1-42)-mediated oxidative stress in neuronal cell culture: a proteomics approach

Protein oxidation mediated by amyloid beta-peptide (1-42) (Abeta[1-42]) has been proposed to play a central role in the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder associated with aging and the loss of cognitive function. The specific mechanism by which Abeta(1-42), the primary component of the senile plaque and a pathologic hallmark of AD, contributes to the oxidative damage evident in AD brain is unknown. Moreover, the specific proteins that are vulnerable to oxidative damage induced by Abeta(1-42) are unknown. Identification of such proteins could contribute to our understanding of not only the role of Abeta(1-42) in the pathogenesis of AD, but also provide insight into the mechanisms of neurodegeneration at the protein level in AD. We report the proteomic identification of two proteins found to be oxidized significantly in neuronal cultures treated with Abeta(1-42): 14-3-3zeta and glyceraldehyde-3-phosphate dehydrogenase. We also report that pretreatment of neuronal cultures with gamma-glutamylcysteine ethyl ester, a compound that supplies the limiting substrate for the synthesis of glutathione and results in the upregulation of glutathione in neuronal cultures, protects both proteins against Abeta(1-42)-mediated protein oxidation.     

Lipopolysaccharide-induced-neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice

The present study was designed to examine whether brain inflammation caused by systemic administration of lipopolysaccharides (LPS) alters the expression/processing of amyloid precursor protein (APP) and increases the generation of amyloid beta peptide (Abeta). APPswe transgenic (Tg) mice were treated with either LPS or phosphate-buffered saline (PBS). In LPS-treated APPswe mice, Abeta1-40/42 was 3-fold and APP was 1.8-fold higher than those in PBS-treated mice (P < 0.05) by ELISA, Western blots and immunoprecipitation-mass spectrometry (IP-MS) ProteinChip analysis. Numbers of Abeta- and APP-immunoreactive neurons (Abeta(+) and APP(+) neurons) increased significantly in LPS-treated APPswe mice; APP(+) and Abeta(+) neurons in neocortex were associated with an increased number of F4/80-immunoreactive microglia (F4/80(+) microglia) in their anatomical environment. Our findings demonstrate that experimental neuroinflammation increases APP expression/processing and causes intracellular accumulation of Abeta. It remains to be seen whether such events can cause neuronal dysfunction/degeneration and, with time, lead to extracellular Abeta deposits, as they occur in Alzheimer's disease.