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.     

A novel member of the leucine-rich repeat superfamily induced in rat astrocytes by beta-amyloid.

beta-Amyloid (Abeta) deposition and senile plaque-associated astrocytes are common neuropathological features of Alzheimer's disease (AD). Although the molecular mechanisms by which Abeta contributes to the progression of neuropathologic changes have not been established entirely, there is little doubt that the association of Abeta with astrocytes, the predominant cell type in brain, has significant influence on exacerbation of the disease. In an effort to identify key molecules involved in AD, we investigated Abeta-responsive genes using rat astrocytes. In this study, we identified a novel Abeta-induced rat gene, designated as Lib, encoding a type I transmembrane protein with an extracellular domain that contains fifteen leucine-rich repeats (LRRs). Human counterpart of rat Lib is located on chromosome 3q29 and human Lib mRNA found in particularly placenta. Lib mRNA levels in rat C6 astrocytoma cells can be increased by pro-inflammatory cytokines and the rat Lib-transfected cells express Lib protein on the cell surfaces. Lib appears to be a member of the LRR superfamily which is involved in cell-cell and/or -extracellular matrix interactions including adhesion or target recognition in neuroinflammatory states.     

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.     

22R-Hydroxycholesterol protects neuronal cells from beta-amyloid-induced cytotoxicity by binding to beta-amyloid peptide

22R-hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, was found at lower levels in Alzheimer's disease (AD) hippocampus and frontal cortex tissue specimens compared to age-matched controls. beta-Amyloid (Abeta) peptide has been shown to be neurotoxic and its presence in brain has been linked to AD pathology. 22R-hydroxycholesterol was found to protect, in a dose-dependent manner, against Abeta-induced rat sympathetic nerve pheochromocytoma (PC12) and differentiated human Ntera2/D1 teratocarcinoma (NT2N) neuron cell death. Other steroids tested were either inactive or acted on rodent neurons only. The effect of 22R-hydroxycholesterol was found to be stereospecific because its enantiomer 22S-hydroxycholesterol failed to protect the neurons from Abeta-induced cell death. Moreover, the effect of 22R-hydroxycholesterol was specific for Abeta-induced cell death because it did not protect against glutamate-induced neurotoxicity. The neuroprotective effect of 22R-hydroxycholesterol was seen when using Abeta1-42 but not the Abeta25-35 peptide. To investigate the mechanism of action of 22R-hydroxycholesterol we examined the direct binding of this steroid to Abeta using a novel cholesterol-protein binding blot assay. Using this method the direct specific binding, under native conditions, of 22R-hydroxycholesterol to Abeta1-42 and Abeta17-40, but not Abeta25-35, was observed. These data suggest that 22R-hydroxycholesterol binds to Abeta and the formed 22R-hydroxycholesterol/Abeta complex is not toxic to rodent and human neurons. We propose that 22R-hydroxycholesterol offers a new means of neuroprotection against Abeta toxicity by inactivating the peptide.     

Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the alpha7, alpha4 and beta2 subunits in response to nanomolar concentrations of the beta-amyloid peptide(1-42)

Neuronal nicotinic acetylcholine receptors (nAChRs) are thought to be involved in the pathogenesis of Alzheimer's disease (AD). Interestingly, in the brains of patients with this disease, losses of several subtypes of nAChRs on neurons have been reported, while an increase in alpha7 nAChRs was recently detected in the astrocytes. However, little is presently known about the expressions of individual subunits of nAChR on rat astrocytes in primary culture or the possible influence of exposure to beta-amyloid peptide (Abeta), a neuropathological hallmark of AD, on this expression. Thus, in the present investigation the levels of individual nAChR subunits on primary rat astrocytes and the possible direct influence of Abetas on the receptors were examined by RT-PCR, Western blotting, monitoring intracellular free calcium and immunohistochemistry. The alpha4, alpha7, beta2 and beta3 subunits and related calcium channel responses were found in these cells, whereas neither alpha2 nor alpha3 could be detected. Elevation in the levels of alpha7, alpha4 and beta2 mRNAs and proteins were observed in astrocytes exposed to 0.1-100nM Abeta(1-42). In contrast, incubation with 1muM Abeta(1-42) or Abeta(35-25) did not affect these levels. We propose that the enhanced expression of alpha7, alpha4 and beta2 nAChRs by astrocytes stimulated directly by nanomolar concentrations of Abeta(1-42) might be related to ongoing defensive or compensative mechanisms.     

Amyloid-beta peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway

Amyloid-beta peptide (Abeta) accumulation in senile plaques, a pathological hallmark of Alzheimer's disease (AD), has been implicated in neuronal degeneration. We have recently demonstrated that Abeta induced oligodendrocyte (OLG) apoptosis, suggesting a role in white matter pathology in AD. Here, we explore the molecular mechanisms involved in Abeta-induced OLG death, examining the potential role of ceramide, a known apoptogenic mediator. Both Abeta and ceramide induced OLG death. In addition, Abeta activated neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase, resulting in increased ceramide generation. Blocking ceramide degradation with N-oleoyl-ethanolamine exacerbated Abeta cytotoxicity; and addition of bacterial sphingomyelinase (mimicking cellular nSMase activity) induced OLG death. Furthermore, nSMase inhibition by 3-O-methyl-sphingomyelin or by gene knockdown using antisense oligonucleotides attenuated Abeta-induced OLG death. Glutathione (GSH) precursors inhibited Abeta activation of nSMase and prevented OLG death, whereas GSH depletors increased nSMase activity and Abeta-induced death. These results suggest that Abeta induces OLG death by activating the nSMase-ceramide cascade via an oxidative mechanism.     

Overexpression of superoxide dismutase 1 protects against beta-amyloid peptide toxicity: effect of estrogen and copper chelators

Beta-amyloid peptides (Abeta) are major constituents of senile plaques in Alzheimer's disease (AD) brain and contribute to neurodegeneration, operating through activation of apoptotic pathways. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. Estrogen is thought to play a protective role against neurodegeneration through a variety of mechanisms including scavenging of reactive oxygen species (ROS). In this study, we have challenged with Abeta, either in the presence or in the absence of 17beta-estradiol, differentiated human neuroblastoma SH-SY5Y cells (named line SH) and the same line overexpressing anti-oxidant enzyme superoxide dismutase 1 (SOD1; named line WT). We have observed that: (1) WT cells are less susceptible than SH cells to Abeta insult; (2) caspase-3, but not caspase-1, is involved in Abeta-induced apoptosis in this system; (3) estrogen protects both lines, without significantly affecting SOD activity; and (4) copper chelators prevent Abeta-induced toxicity. Our results further support the notion that anti-oxidant therapy might be beneficial in the treatment of AD by preventing activation of selected apoptotic pathways.     

Calcium signals induced by amyloid beta peptide and their consequences in neurons and astrocytes in culture

In Alzheimer's disease, amyloid beta (Abeta) peptide is deposited in neuritic plaques in the brain. The Abeta peptide 1-42 or the fragment 25-35 are neurotoxic. We here review our recent explorations of the mechanisms of Abeta toxicity in hippocampal cultures. Abeta had no effect on intracellular calcium in neurons but caused striking changes in nearby astrocytes. The [Ca(2+)](c) signals started approximately 5-15 min after Abeta application and consisted of sporadic [Ca(2+)](c) pulses. These were entirely dependent on extracellular Ca(2+), independent of ER Ca(2+) stores and resulted from Ca(2+) influx, probably through Abeta-induced membrane channels. The Ca(2+) signals were closely associated with transient, episodic acidification which may reflect displacement of protons from binding sites or Ca(2+)/2H(+) exchange. Abeta caused an increased rate of generation of reactive oxygen species (ROS), also seen in astrocytes and not in neurons. The increased ROS generation was blocked by inhibitors of the NADPH oxidase, strongly suggesting that this enzyme, normally associated with immune cells, is expressed in astrocytes. ROS generation was also Ca(2+)-dependent, suggesting that Abeta activation of the enzyme may be secondary to the increase in [Ca(2+)](c). Abeta caused delayed neuronal death despite the fact that all responses were seen only in astrocytes. Neurons could not be protected by glutamate receptor antagonists, but were rescued by inhibition of the NADPH oxidase, by antioxidants and by increasing glutathione. These data suggest that Abeta causes Ca(2+)-dependent oxidative stress by activating an astrocytic NADPH oxidase, and that neuronal death follows through a failure of antioxidant support.     

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.     

Fibrillar amyloid-beta peptides activate microglia via TLR2: implications for Alzheimer's disease

Microglial activation is an important pathological component in brains of patients with Alzheimer's disease (AD), and fibrillar amyloid-beta (Abeta) peptides play an important role in microglial activation in AD. However, mechanisms by which Abeta peptides induce the activation of microglia are poorly understood. The present study underlines the importance of TLR2 in mediating Abeta peptide-induced activation of microglia. Fibrillar Abeta1-42 peptides induced the expression of inducible NO synthase, proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-6), and integrin markers (CD11b, CD11c, and CD68) in mouse primary microglia and BV-2 microglial cells. However, either antisense knockdown of TLR2 or functional blocking Abs against TLR2 suppressed Abeta1-42-induced expression of proinflammatory molecules and integrin markers in microglia. Abeta1-42 peptides were also unable to induce the expression of proinflammatory molecules and increase the expression of CD11b in microglia isolated from TLR2(-/-) mice. Finally, the inability of Abeta1-42 peptides to induce the expression of inducible NO synthase and to stimulate the expression of CD11b in vivo in the cortex of TLR2(-/-) mice highlights the importance of TLR2 in Abeta-induced microglial activation. In addition, ligation of TLR2 alone was also sufficient to induce microglial activation. Consistent to the importance of MyD88 in mediating the function of various TLRs, antisense knockdown of MyD88 also inhibited Abeta1-42 peptide-induced expression of proinflammatory molecules. Taken together, these studies delineate a novel role of TLR2 signaling pathway in mediating fibrillar Abeta peptide-induced activation of microglia.     

Amyloid beta peptide increases DP5 expression via activation of neutral sphingomyelinase and JNK in oligodendrocytes

There is growing recognition that white matter pathology is a common feature in Alzheimer's disease. We have previously reported that the amyloid beta peptide (Abeta) induces apoptosis in oligodendrocytes (OLG), via activation of neutral sphingomyelinase (nSMase) and resultant generation of ceramide. In the current study, we report that both Abeta and ceramide increased expression of the proapoptotic protein DP5/Hrk (DP5), and release of cytochrome C from mitochondria to cytoplasm in OLGs. We provide evidence that the Jun N-terminal kinase (JNK) signaling pathway mediates Abeta- and ceramide-induced apoptosis: Both Abeta and ceramide activated JNK phosphorylation, and subsequent AP-1 DNA binding activity; JNK siRNA decreased AP-1 DNA binding, DP5 expression and reduced cell death. Furthermore, inhibition of nSMase attenuated Abeta-induced JNK phosphorylation, AP-1 DNA binding activity, DP5 expression, and cytochrome C release. Collectively, these results suggest that Abeta-induced apoptosis involves the sequential activation of nSMase with ceramide generation, JNK activation, AP-1 DNA binding, and DP5 expression.     

Humanin rescues human cerebrovascular smooth muscle cells from Abeta-induced toxicity

Cerebral amyloid beta-protein (Abeta) angiopathy (CAA) is a key pathological feature of Alzheimer's disease (AD) and related disorders. We have used human cerebrovascular smooth muscle (HCSM) cells as an in vitro model system to investigate the pathogenic mechanisms of the pathology of CAA. It was previously demonstrated that certain pathogenic forms of Abeta induce several pathologic responses in these cells, including fibril assembly at the cell surface, increased levels of Abeta precursor, degradation of HCSM cell alpha-actin and cell death. The recently discovered novel rescue factor humanin (HN) was shown to protect neuronal cells in culture from various AD-relevant insults including treatment with Abeta. In this report we investigated whether the HN peptide could rescue HCSM cells from Abeta-induced toxicity. We found that treatment of HCSM cells with 10 microm HN prevented pathogenic Abeta-induced HCSM cell death using a fluorescent cell viability assay, and degradation of HCSM alpha-actin was diminished shown by quantitative immunoblotting. However, Abeta deposition and fibril formation at the cell surface and increased levels of cell-associated AbetaPP were not affected by treatment with HN as demonstrated by a thioflavin T fluorescence assay and immunochemical methods, respectively. These results suggest that the protective effects of HN occur downstream of these cell surface molecular events. This is the first demonstration of a rescue factor for HCSM cells from Abeta-mediated cell death as well as being the first report to show that neuronal cells and HCSM cells may share a common downstream mechanism in the Abeta-induced cell death pathway.     

Protective effects of S-nitrosoglutathione against amyloid beta-peptide neurotoxicity

Amyloid beta-peptide (Abeta) is a major constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. We have previously demonstrated ceramide production secondary to Abeta-induced activation of neutral sphingomyelinase (nSMase) in cerebral endothelial cells and oligodendrocytes, which may contribute to cellular injury during progression of AD. In this study, we first established the "Abeta --> nSMase --> ceramide --> free radical --> cell death" pathway in primary cultures of fetal rat cortical neurons. We also provided experimental evidence showing that S-nitrosoglutathione (GSNO), a potent endogenous antioxidant derived from the interaction between nitric oxide (NO) and glutathione, caused dose-dependent protective effects against Abeta/ceramide neurotoxicity via inhibition of caspase activation and production of reactive oxygen species (ROS). This GSNO-mediated neuroprotection appeared to involve activation of cGMP-dependent protein kinase (PKG), phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated kinase (ERK). Activation of the cGMP/PKG pathway induced expression of thioredoxin and Bcl-2 that were beneficial to cortical neurons in antagonizing Abeta/ceramide toxicity. Consistently, exogenous application of thioredoxin exerted remarkable neuroprotective efficacy in our experimental paradigm. Results derived from the present study establish a neuroprotective role of GSNO, an endogenous NO carrier, against Abeta toxicity via multiple signaling pathways.     

Cellular cofactors potentiating induction of stress and cytotoxicity by amyloid beta-peptide

Insights into factors underlying causes of familial Alzheimer's disease (AD), such as mutant forms of beta-amyloid precursor protein and presenilins, and those conferring increased risk of sporadic AD, such as isoforms of apolipoprotein E and polymorphisms of alpha2-macroglobulin, have been rapidly emerging. However, mechanisms through which amyloid beta-peptide (Abeta), the fibrillogenic peptide most closely associated with neurotoxicity in AD, exerts its effects on cellular targets have only been more generally outlined. Late in the course of AD, when Abeta fibrils are abundant, non-specific interactions of amyloid with cellular elements are likely to induce broad cytotoxicity. However, early in AD, when concentrations of Abeta are much lower and extracellular deposits are infrequent, mechanisms underlying cellular dysfunction have not been clearly defined. The key issue in elucidating the means through which Abeta perturbs cellular properties early in AD is the possibility that protective therapy at such times may prevent cytotoxicity at a point when damage is still reversible. This brief review focusses on two cellular cofactors for Abeta-induced cellular perturbation: the cell surface immunoglobulin superfamily molecule RAGE (receptor for advanced glycation endproducts) and ABAD (Abeta binding alcohol dehydrogenase). Although final proof for the involvement of these cofactors in cellular dysfunction in AD must await the results of further in vivo experiments, their increased expression in AD brain, as well as other evidence described below, suggests the possibility of specific pathways for Abeta-induced cellular perturbation which could provide future therapeutic targets.     

The amyloid beta peptide abeta (25-35) induces apoptosis independent of p53

Apoptosis of neuronal cells apparently plays a role in Alzheimer's disease (AD). The amyloid beta (Abeta) peptide derived from beta-amyloid precursor protein is found in AD brain in vivo and can induce apoptosis in vitro. While p53 accumulates in cells of AD brain, it is not known if p53 plays an active role in Abeta-induced apoptosis. We show here that inactivation of p53 in two experimental cell lines, either by expression of the papillomavirus E6 protein or by a shift to restrictive temperature, does not affect apoptosis induction by Abeta (25-35), indicating that Abeta induces apoptosis in a p53-independent manner.     

Protective effect of new S-acylglutathione derivatives against amyloid-induced oxidative stress

Recent data support the role of oxidative stress in the pathogenesis of Alzheimer disease (AD). In particular, glutathione (GSH) metabolism is altered and its levels are decreased in affected brain regions and peripheral cells from AD patients and in experimental models of AD. In the past decade, interest in the protective effects of various antioxidants aimed at increasing intracellular GSH content has been growing. Because much experimental evidence suggests a possible protective role of unsaturated fatty acids in age-related diseases, we designed the synthesis of new S-acylglutathione (acyl-SG) thioesters. S-Lauroylglutathione (lauroyl-SG) and S-palmitoleoylglutathione (palmitoleoyl-SG) were easily internalized into the cells and they significantly reduced Abeta42-induced oxidative stress in human neurotypic SH-SY5Y cells. In particular, acyl-SG thioesters can prevent the impairment of intracellular ROS scavengers, intracellular ROS accumulation, lipid peroxidation, and apoptotic pathway activation. Palmitoleoyl-SG seemed more effective in cellular protection against Abeta-induced oxidative damage than lauroyl-SG, suggesting a valuable role for the monounsaturated fatty acid. In this study, we demonstrate that acyl-SG derivatives completely avoid the sharp lipoperoxidation in primary fibroblasts from familial AD patients occurring after exposure to Abeta42 aggregates. Hence, we put forward these derivatives as new antioxidant compounds which could be excellent candidates for therapeutic treatment of AD and other oxidative stress-related diseases.     

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.