Aβ-induced Cell Toxicity Research Articles

Neuroprotective Effect of Curcumin-Loaded RGD Peptide-PEGylated Nanoliposomes.

Curcumin is known for its anti-inflammatory, neuroprotective, and antioxidant properties, but its use in biological applications is hindered by its sensitivity to light, oxygen, and temperature. Furthermore, due to its low water solubility, curcumin has a poor pharmacokinetic profile and bioavailability. In this study, we evaluated the potential application of curcumin as a neuroprotective agent encapsulated in RGD peptide-PEGylated nanoliposomes developed from salmon-derived lecithin. Salmon lecithin, rich in polyunsaturated fatty acids, was used to formulate empty or curcumin-loaded nanoliposomes. Transmission electron microscopy, dynamic light scattering, and nanoparticle tracking analysis characterizations indicated that the marine-derived peptide-PEGylated nanoliposomes were spherical in shape, nanometric in size, and with an overall negative charge. Cytotoxicity tests of curcumin-loaded nanoliposomes revealed an improved tolerance of neurons to curcumin as compared to free curcumin. Wild-type SH-SY5Y were treated for 24 h with curcumin-loaded nanoliposomes, followed by 24 h incubation with conditioned media of SH-SY5Y expressing the Swedish mutation of APP containing a high ratio of Aβ40/42 peptides. Our results revealed significantly lower Aβ-induced cell toxicity in cells pre-treated with RGD peptide-PEGylated curcumin-loaded nanoliposomes, as compared to controls. Thus, our data highlight the potential use of salmon lecithin-derived RGD peptide PEGylated nanoliposomes for the efficient drug delivery of curcumin as a neuroprotective agent.

MiR-29c-3p Attenuates β-Amyloid-Induced Neurotoxicity in Alzheimer’s Disease Through Regulating β-Site Amyloid Precursor Protein-Cleaving Enzyme 1

The aberrantly expressed microRNAs (miRNAs) including miR-29c-3p have been reported in the brains of Alzheimer's disease (AD) patients in recent researches. Nevertheless, the functional role and underlying molecular mechanism of miR-29c-3p in AD pathogenesis are still not well elucidated. The purpose of this study was to examine whether miR-29c-3p regulated β-Ameyloid (Aβ)-induced neurotoxicity by targeting β-site amyloid precursor protein-cleaving enzyme 1 (BACE1). The expressions of miR 29c 3p and BACE1 mRNA and protein levels in Aβ-treated PC12 cellular AD model were examined by qRT-PCR and western blot analyses. Luciferase reporter assay verified the potential target of miR 29c 3p. Cell viability, apoptosis, and caspase-3 activity in PC12 cells were detected by the MTT assay, flow cytometry, and caspase-3 activity assay, respectively. Our results indicated that miR-29c-3p downregulation and BACE1 upregulation existed in the cellular AD model of PC12 cells. Moreover, miR-29c-3p directly inhibited BACE1 expression. miR-29c-3p overexpression and BACE1 knockdown strengthened Aβ-induced cell apoptosis, and caspase-3 activity in PC12 cells, which was partially eliminated by over-expression of BACE1. Conversely, BACE1 knockdown reversed the miR-29c-3p inhibition- mediated inhibitory effect on Aβ-induced cell toxicity, apoptosis, and caspase-3 activity in PC12 cells. Considering, miR-29c-3p attenuated Aβ-induced neurotoxicity through targeting BACE1 in an cellular AD model of PC12, providing a potential therapeutic target for AD treatment.

Cross-seeding between Aβ and SEVI indicates a pathogenic link and gender difference between alzheimer diseases and AIDS.

Amyloid-β (Aβ) and semen-derived enhancer of viral infection (SEVI) are considered as the two causative proteins for central pathogenic cause of Alzheimer’s disease (AD) and HIV/AIDS, respectively. Separately, Aβ-AD and SEVI-HIV/AIDS systems have been studied extensively both in fundamental research and in clinical trials. Despite significant differences between Aβ-AD and SEVI-HIV/AIDS systems, they share some commonalities on amyloid and antimicrobial characteristics between Aβ and SEVI, there are apparent overlaps in dysfunctional neurological symptoms between AD and HIV/AIDS. Few studies have reported a potential pathological link between Aβ-AD and SEVI-HIV/AIDS at a protein level. Here, we demonstrate the cross-seeding interactions between Aβ and SEVI proteins using in vitro and in vivo approaches. Cross-seeding of SEVI with Aβ enabled to completely prevent Aβ aggregation at sub-stoichiometric concentrations, disaggregate preformed Aβ fibrils, reduce Aβ-induced cell toxicity, and attenuate Aβ-accumulated paralysis in transgenic AD C. elegans. This work describes a potential crosstalk between AD and HIV/AIDS via the cross-seeding between Aβ and SEVI, identifies SEVI as Aβ inhibitor for possible treatment or prevention of AD, and explains the role of SEVI in the gender difference in AD.

PrP C as a Transducer of Physiological and Pathological Signals.

After the discovery of prion phenomenon, the physiological role of the cellular prion protein (PrPC) remained elusive. In the past decades, molecular and cellular analysis has shed some light regarding interactions and functions of PrPC in health and disease. PrPC, which is located mainly at the plasma membrane of neuronal cells attached by a glycosylphosphatidylinositol (GPI) anchor, can act as a receptor or transducer from external signaling. Although the precise role of PrPC remains elusive, a variety of functions have been proposed for this protein, namely, neuronal excitability and viability. Although many issues must be solved to clearly define the role of PrPC, its connection to the central nervous system (CNS) and to several misfolding-associated diseases makes PrPC an interesting pharmacological target. In a physiological context, several reports have proposed that PrPC modulates synaptic transmission, interacting with various proteins, namely, ion pumps, channels, and metabotropic receptors. PrPC has also been implicated in the pathophysiological cell signaling induced by β-amyloid peptide that leads to synaptic dysfunction in the context of Alzheimer’s disease (AD), as a mediator of Aβ-induced cell toxicity. Additionally, it has been implicated in other proteinopathies as well. In this review, we aimed to analyze the role of PrPC as a transducer of physiological and pathological signaling.

Novel Donepezil-Arylsulfonamide Hybrids as Multitarget-Directed Ligands for Potential Treatment of Alzheimer's Disease.

Alzheimer’s disease (AD) is one of the most devastating neurodegenerative disorders, characterized by multiple pathological features. Therefore, multi-target drug discovery has been one of the most active fields searching for new effective anti-AD therapies. Herein, a series of hybrid compounds are reported which were designed and developed by combining an aryl-sulfonamide function with a benzyl-piperidine moiety, the pharmacophore of donepezil (a current anti-AD acetylcholinesterase AChE inhibitor drug) or its benzyl-piperazine analogue. The in vitro results indicate that some of these hybrids achieve optimized activity towards two main AD targets, by displaying excellent AChE inhibitory potencies, as well as the capability to prevent amyloid-β (Aβ) aggregation. Some of these hybrids also prevented Aβ-induced cell toxicity. Significantly, drug-like properties were predicted, including for blood-brain permeability. Compound 9 emerged as a promising multi-target lead compound (AChE inhibition (IC50 1.6 μM); Aβ aggregation inhibition 60.7%). Overall, this family of hybrids is worthy of further exploration, due to the wide biological activity of sulfonamides.

Design, Synthesis, and In Vitro Evaluation of Hydroxybenzimidazole-Donepezil Analogues as Multitarget-Directed Ligands for the Treatment of Alzheimer's Disease.

A series of multi-target-directed ligands (MTDLs), obtained by attachment of a hydroxyphenylbenzimidazole (BIM) unit to donepezil (DNP) active mimetic moiety (benzyl-piperidine/-piperazine) was designed, synthesized, and evaluated as potential anti-Alzheimer’s disease (AD) drugs in terms of biological activity (inhibition of acetylcholinesterase (AChE) and β–amyloid (Aβ) aggregation), metal chelation, and neuroprotection capacity. Among the DNP-BIM hybrids studied herein, the structural isomerization did not significantly improve the biological properties, while some substitutions, namely fluorine atom in each moiety or the methoxy group in the benzyl ring, evidenced higher cholinergic AChE activity. All the compounds are able to chelate Cu and Zn metal ions through their bidentate BIM moieties, but compound 5, containing a three-dentate chelating unit, is the strongest Cu(II) chelator. Concerning the viability on neuroblastoma cells, compounds 9 and 10 displayed the highest reduction of Aβ-induced cell toxicity. In silico calculations of some pharmacokinetic descriptors indicate that all the compounds but the nitro derivatives have good potential oral-bioavailability. Overall, it can be concluded that most of the studied DNP-BIM conjugates showed quite good anti-AD properties, therefore deserving to be considered in further studies with the aim of understanding and treating AD.

CEPO (carbamylated erythropoietin)-Fc protects hippocampal cells in culture against beta amyloid-induced apoptosis: considering Akt/GSK-3β and ERK signaling pathways.

The tissue-protective properties of erythropoietin (EPO) have been described in several neurodegenerative diseases models, but erythrocytosis following EPO treatment may lead to deleterious effects. Carbamylated erythropoietin, an EPO derivative lacking hematopoietic side effects, has shown protective properties in some studies. However, it is not known if CEPO protects primary hippocampal cells against Aβ25-35 toxicity. The present study aimed to investigate the effect of CEPO-Fc on biochemical alterations in Akt, GSK-3β, and ERK signaling and cell death induced by Aβ25-35 in isolated hippocampal cell culture. The embryonic hippocampal cells were obtained from 18-19 day rat embryos. The cells were exposed with Aβ25-35 (20 µM) in the absence or presence of CEPO-Fc (1 or 5 IU) and PI3k and ERK inhibitors. CEPO-Fc at the dose of 5 IU significantly prevented the cell loss and caspase-3 cleavage caused by Aβ25-35. Additionally, CEPO-Fc noticeably reversed Aβ mediated decrement of Akt and GSK-3β phosphorylation. With exposure to LY294002, PI3 kinase inhibitor, Akt phosphorylation diminished and CEPO-Fc protective effects disappeared. Furthermore, while CEPO-Fc nullified Aβ-induced increment of phospho-ERK, inhibition of ERK activity by PD98059, had no effect on Aβ25-35-mediated caspase-3 cleavage and cell toxicity. These results imply that protective effects of CEPO-Fc seem to be mainly exerted through the PI3K/Akt pathway rather than ERK signaling. This study suggested that CEPO-Fc prevents Aβ-induced cell toxicity as well as Akt/GSK-3β and ERK alterations in isolated hippocampal cells. These findings might provide a new perspective on CEPO-Fc protective properties as a prospective remedial factor for neurodegenerative diseases like AD.

Donepezil-based hybrids as multifunctional anti-Alzheimer's disease chelating agents: Effect of positional isomerization

The intricate and multifactorial nature of Alzheimer's disease (AD) requires the development of compounds able to hit different pathophysiological targets, such as cholinergic dysfunction, deposits of amyloid beta (Aβ) peptide and metal dyshomeostasis. In order to continue the search for new anti-AD drugs, a design strategy was once more followed based on repositioning donepezil (DNP) drug, by ortho-attaching a benzylpiperidine mimetic of DNP moiety to a hydroxyphenyl-benzimidazole (BIM) chelating unit (compound 1). Herein, compound 1 and a positional isomer 2 are compared in terms of their potential multiple properties: both present good acetylcholinesterase (AChE) inhibition (low μmolar range) and are moderate/good inhibitors of Aβ self- and Cu-mediated aggregation, the inhibition process being mainly due to ligand intercalation between the β-sheets of the fibrils; compound 1 has a higher chelating capacity towards Cu2+ and Zn2+ (pCu = 14.3, pZn = 6.4, pH 7.4, CL/CM = 10, CM = 10−6 M) than 2 (pCu = 10.7, pZn = 6.3), attributed to its ability to establish a tridentate (N,O,O) coordination to the metal ion. Both compounds are eligible as drug candidates for oral administration but compound 1 shows improved neuroprotective role by completely preventing Aβ-induced cell toxicity.

MiR-16 attenuates β-amyloid-induced neurotoxicity through targeting β-site amyloid precursor protein-cleaving enzyme 1 in an Alzheimer's disease cell model.

The aberrant deposition of β-amyloid (Aβ) is closely linked to the pathogenesis and development of Alzheimer's disease (AD). MiR-16 was abnormally downregulated and may be related to the development of AD. However, the functional role and molecular mechanism of miR-16 in AD pathogenesis are still not well elucidated. The expressions of miR-16 and β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) mRNA and protein levels in AD brain tissues and Aβ-treated PC12 cellular AD model were examined by qRT-PCR and western blot analyses. Luciferase reporter assay was used to verify the potential target of miR-16. The cell viability, apoptosis, and caspase-3 activity in PC12 cells were determined by the MTT assay, flow cytometry analysis, and caspase-3 activity assay, respectively. Downregulation of miR-16 and upregulation of BACE1 existed in AD tissues and the cellular AD model of PC12. In addition, miR-16 directly suppressed BACE1 expression. Moreover, miR-16 overexpression and BACE1 knockdown facilitated Aβ-induced cell toxicity, apoptosis, and caspase-3 activity in N2a cells, which was partially eliminated by overexpression of BACE1. In contrast, BACE1 knockdown reversed the miR-16 inhibition-mediated inhibitory effect on Aβ-induced cell toxicity, apoptosis, and caspase-3 activity in PC12 cells. Collectively, miR-16 attenuated Aβ-induced neurotoxicity through targeting BACE1 in an Aβ insult cellular AD model, providing a potential therapeutic target for AD treatment.

Irisin Exerts Neuroprotective Effects on Cultured Neurons by Regulating Astrocytes

Neurons suffer detrimental effects from β-amyloid toxicity in Alzheimer's disease. The exercise hormone, irisin, is found to induce a neuroprotective gene program and facilitates the beneficial effects on cognitive function. But no effort is made to test its direct protective effects on neurons against the Aβ-induced cell toxicity so far. In the present study, we investigated whether irisin could protect neurons against Aβ- (25–35) induced cell damage and explored the possible underlying mechanisms. Primary cell cultures of astrocytes and neurons were established. Conditioned medium from astrocyte was collected for the treatment and biochemistry assay study. To explore the protein expression changes, Western blot and ELISA assays were used in these in vitro cell culture models. Exposure of hippocampal neurons to 10 μM Aβ (25–35) caused significant reduction on cell viability, and the toxic effect was not significantly reduced by the coadministration of irisin. However, pretreated astrocyte-conditioned medium with irisin for 12 hours notably protected the neurons from the toxicity of Aβ. Also, we found that irisin could attenuate the release of IL-6 and IL-1β from cultured astrocytes and decrease the expression level of COX-2 and phosphorylation of AKT. Last, we found that irisin could reduce NFκB activation in astrocyte exposed to Aβ by preventing the phosphorylation and the loss of IκBα. Our finding may provide novel evidence for the future application of irisin in the treatment of Alzheimer's disease and the memory dysfunction in diabetes mellitus.

Quetiapine prevents Aβ25-35-induced cell death in cultured neuron by enhancing brain-derived neurotrophic factor release from astrocyte.

β-Amyloid (Aβ)-induced neuronal toxicity is an early event in the pathogenesis of Alzheimer's disease. Quetiapine (QTP) is an atypical antipsychotic drug that has neuroprotectant properties, but little is known about its direct protective effects on neurons against the Aβ-induced cell toxicity. In the present study, we investigated the neuroprotective effects of QTP on Aβ25-35-induced cell death and the possible underlying mechanisms in primary cultures of neurons. Exposure of cortical neurons to 10 μM or more Aβ25-35 caused significant viability loss in a MTT assay, and the toxic effects were not significantly prevented by the simultaneous coadministration of QTP. However, pretreated astrocyte conditioned medium (ACM) with QTP (ACMQTP) for 24 h markedly protected the neurons against the amyloid-induced cell loss. Furthermore, we revealed that QTP increased both the release of brain-derived neurotrophic factor from cultured astrocytes and the phosphorylation of extracellular signal-regulated kinase after 24 h of treatment, which might be responsible for its protective effects on neurons. Consistent with the aforementioned findings, the protective effects of ACM on neurons could potentially be abolished by the extracellular signal-regulated kinase inhibitor and tropomyosin receptor kinase B receptor blocker. In conclusion, our data demonstrated that QTP exerted its neuroprotective effects against amyloid toxicity by enhancing the brain-derived neurotrophic factor release from astrocytes.

Identification of a New Function of Cardiovascular Disease Drug 3-Morpholinosydnonimine Hydrochloride as an Amyloid-β Aggregation Inhibitor.

Cardiovascular disease (CVD) and Alzheimer’s disease (AD) have a mutual cause-and-effect relationship, and they share some common risk factors. Although numerous Food and Drug Administration (FDA)-approved drugs have been developed for CVD treatment, no drugs are clinically available for AD treatment. Given the common disease-causing factors and links between the two diseases and the well-demonstrated drugs for CVD, we propose to re-examine the new potential of the existing CVD drugs as amyloid-β (Aβ) inhibitors. 3-Morpholinosydnonimine hydrochloride (SIN-1) is an FDA-approved drug for inhibiting platelet aggregation in CVD. Herein, we examine the inhibition activity of SIN-1 on the aggregation and toxicity of Aβ1–42 using combined experimental and computational approaches. Collective experimental data from ThT, circular dichroism, and atomic force microscopy demonstrate that SIN-1 can effectively inhibit amyloid formation at every stage of Aβ aggregation by prolonging lag phase, slowing down aggregation rate, and reducing final fibril formation. The cell viability assay also shows that SIN-1 enables the protection of SH-SY5Y cells from Aβ-induced cell toxicity. Such an inhibition effect is attributed to interference with the structural transition of Aβ toward a β-sheet structure by SIN-1. Furthermore, molecular dynamic simulations confirm that SIN-1 preferentially binds to the C-terminal β-sheet grooves of an Aβ oligomer and consequently disrupts the β-sheet structure of Aβ and Aβ–Aβ association, explaining experimental observations. This work discovers a new function of SIN-1, making it a promising compound with dual protective roles in inhibiting both platelet and Aβ aggregations against CVD and AD.

Protective effect of centipedegrass against Aβ oligomerization and Aβ-mediated cell death in PC12 cells

Context: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the abnormal accumulation of β-amyloid (Aβ). Multiple Aβ-aggregated species have been identified, and neurotoxicity appears to be correlated with the amount of non-fibrillar oligomers. Potent inhibitors of Aβ oligomer formation or Aβ-induced cell toxicity have emerged as attractive means of therapeutic intervention. Eremochloa ophiuroide Hack. (Poaceae), also known as centipedegrass (CG), originates from China and South America and is reported to contain several C-glycosyl flavones and phenolic constituents.Objective: We investigated whether CG could suppress Aβ aggregation, BACE1 activity, and toxicity at neuronal cell.Materials and methods: The inhibitory effect of CG extracts toward aggregation of Aβ42 was investigated in the absence and presence of 50 µg/mL CG. We investigated the inhibitory effects of CG (0–5 µg/mL) on BACE1 using fluorescence resonance energy transfer (FRET)-based assay. The effects of CG (0–75 µg/mL) on Aβ42-induced neurotoxicity were examined in PC12 cells in the presence or absence of maysin and its derivatives of CG.Results: We isolated EA-CG fraction (70% MeOH fraction from EtOAc extracts) from methanol extracts of CG, which contained approximately 60% maysin and its derivatives. In the present studies, we found that several Aβ oligomeric forms such as the monomer, dimer, trimer, and highly aggregated oligomeric forms were remarkably inhibited in the presence of 50 µg/mL of EA-CG. EA-CG also inhibited BACE1 enzyme activity in a dose-dependent manner. EA-CG treatment generated approximately 50% or 85% inhibition to the control at the tested concentrations of 1 or 5 µg/mL, respectively. Moreover, the neurotoxicity induced by Aβ42 was significantly reduced by treatment of EA-CG, and the 75 µg/mL EA-CG treatment significantly increased cell viability up to 82.5%.Discussion and conclusion: These results suggested that the anti-Alzheimer’s effects of CG occurred through inhibition of neuronal cell death by intervening with oligomeric Aβ formation and reducing BACE1 activity. Maysin in CG could be an excellent therapeutic candidate for the prevention of AD.

A structurally simplified analogue of geldanamycin exhibits neuroprotective activity.

The syntheses of a structurally simplified geldanamycin analogue 2 and two related compounds are described. Compound 2 conferred cytoprotection and quenched ROS and lipid peroxidation in a dose-dependent manner in Friedreich's ataxia (FRDA) lymphocytes at low micromolar concentrations. It also prevented ROS-induced damage of cellular lipid membranes and maintained the mitochondrial membrane potential of FRDA lymphocytes. In addition, 2 did not inhibit Hsp90 when tested at micromolar concentrations, exhibited no cytotoxicity, and afforded neuroprotection to differentiated SH-SY5Y cells under conditions of Aβ-induced cell toxicity.

Tanshinones inhibit amyloid aggregation by amyloid-β peptide, disaggregate amyloid fibrils, and protect cultured cells.

The misfolding and aggregation of amyloid-β (Aβ) peptides into amyloid fibrils is regarded as one of the causative events in the pathogenesis of Alzheimer's disease (AD). Tanshinones extracted from Chinese herb Danshen (Salvia Miltiorrhiza Bunge) were traditionally used as anti-inflammation and cerebrovascular drugs due to their antioxidation and antiacetylcholinesterase effects. A number of studies have suggested that tanshinones could protect neuronal cells. In this work, we examine the inhibitory activity of tanshinone I (TS1) and tanshinone IIA (TS2), the two major components in the Danshen herb, on the aggregation and toxicity of Aβ1-42 using atomic force microscopy (AFM), thioflavin-T (ThT) fluorescence assay, cell viability assay, and molecular dynamics (MD) simulations. AFM and ThT results show that both TS1 and TS2 exhibit different inhibitory abilities to prevent unseeded amyloid fibril formation and to disaggregate preformed amyloid fibrils, in which TS1 shows better inhibitory potency than TS2. Live/dead assay further confirms that introduction of a very small amount of tanshinones enables protection of cultured SH-SY5Y cells against Aβ-induced cell toxicity. Comparative MD simulation results reveal a general tanshinone binding mode to prevent Aβ peptide association, showing that both TS1 and TS2 preferentially bind to a hydrophobic β-sheet groove formed by the C-terminal residues of I31-M35 and M35-V39 and several aromatic residues. Meanwhile, the differences in binding distribution, residues, sites, population, and affinity between TS1-Aβ and TS2-Aβ systems also interpret different inhibitory effects on Aβ aggregation as observed by in vitro experiments. More importantly, due to nonspecific binding mode of tanshinones, it is expected that tanshinones would have a general inhibitory efficacy of a wide range of amyloid peptides. These findings suggest that tanshinones, particularly TS1 compound, offer promising lead compounds with dual protective role in anti-inflammation and antiaggregation for further development of Aβ inhibitors to prevent and disaggregate amyloid formation.

H2S releasing aspirin protects amyloid beta induced cell toxicity in BV-2 microglial cells

β-Amyloid (Aβ) plaques are characteristic hallmarks of Alzheimer's disease. In the present study, we examined the neuroprotective effects of S-aspirin, a hydrogen sulfide (H(2)S)-releasing aspirin, on Aβ-induced cell toxicity. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that S-aspirin, but not aspirin, significantly increased cell viability in BV-2 microglial cells, indicating that S-aspirin may protect cells against injury via releasing H(2)S. S-aspirin at 2.5-10 μM significantly increased cell viability and decreased lactate dehydrogenase release in Aβ-treated BV-2 microglial cells. Western blotting analysis showed that S-aspirin suppressed the protein expression levels of cyclooxygenase-2 and growth arrest DNA damage (GADD). These data suggest that S-aspirin may protect microglial cells by inhibition of Aβ-induced inflammation and cell cycle re-entry. To study whether S-aspirin can protect mitochondria function, mitochondria membrane potential was measured with molecular probe JC-1. It was found that S-aspirin protected mitochondria from Aβ-induced loss of mitochondrial member potential. (ΔΨm). In addition, S-aspirin also prevented Aβ-induced activation of p38-mitogen activated protein kinase (MAPK). In conclusion, our results suggest that S-aspirin may protect microglial injury via inhibition of inflammation, prevention of mitochondria function, and stimulation of cell growth via stimulating p38-MAPK pathway. Our study may suggest that S-aspirin may have potential therapeutic value for the treatment of Alzheimer's disease.

Hydrogen Sulfide Protects Amyloid-β Induced Cell Toxicity in Microglia

Alzheimer's disease (AD) is pathologically characterized by the accumulation of senile plaques, containing activated microglia and amyloid-β peptides (Aβ). We found that aggregated Aβ1-40 peptide (25 μM, 24 h) significantly decreased viability of BV-2 microglial cells. This was concentration-dependently attenuated by NaHS (a hydrogen sulfide (H2S) donor, 25-500 μM). NaHS also significantly attenuated Aβ-induced LDH release and the up-regulation of protein expression of growth arrest DNA damage (GADD 153). These data suggest that H2S may attenuate Aβ-induced cell toxicity and cell cycle re-entry. Pretreatment with NaHS also suppressed the release of nitric oxide and the upregulation of inducible nitric oxide synthase. These effects were attenuated by exogenous application of NaHS or stimulation of endogenous generation of H2S with S-adenosyl-L-methionine, a cystathionine β synthase activator. NaHS also decreased the releases of TNF-α and suppressed the up-regulation of protein expression of cyclooxygenase 2, which were mimicked by blockade of p38 and JNK-MAPK. In addition, Aβ induced loss of mitochondrial member potential (ΔΨm) and activation of p38-, JNK-, and ERK-MAPKs. Application of NaHS attenuated these effects but failed to affect the activation of ERK. In conclusion, we demonstrated for the first time that H2S may protect cell against Aβ-induced cell injury by inhibition of inflammation, promotion of cell growth and preservation of mitochondrial function in a p38- and JNK-MAPK dependent manner. Our results suggest that H2S may have potential therapeutic value for treatment of AD.

A Kinetic Analysis of the Mechanism of β-Amyloid Induced G Protein Activation

β-Amyloid (Aβ) is the primary protein component of senile plaques found in Alzheimer's disease. In an aggregated (amyloid fibril, protofibril, or low molecular weight oligomer) state, Aβ has been consistently shown to be toxic to neurons, but the molecular mechanism of this toxicity is poorly understood. We have previously shown that Aβ activates a G i/o protein, and that inhibition of this specific G protein activation attenuated Aβ-induced cell toxicity. In the present study, we use a kinetic analysis to examine the mechanism of Aβ-induced G protein activation. Using synthetic Aβ(1–40) and phospholipid vesicles containing purified G 0α subunits, we examined the relationship between Aβ concentration, G 0α subunit concentration, GTP concentration and rate of GTP hydrolysis experimentally. We found that at low concentrations of Aβ (less than 10 μM), Aβ increased the rate of GTP hydrolysis over the rate of hydrolysis in the absence of peptide, however, at high concentrations of Aβ, significantly decreased rates of GTP hydrolysis were observed. We postulated several molecular level mechanisms for the observed rate behavior, from those mechanisms derived rate equations, and then tested the mechanisms against our experimental rate data. Based on our results, we identified a plausible mechanism for Aβ-induced G protein activation which is consistent with available experimental data. This work demonstrates the utility of an engineering approach to examining steps in the mechanism of Aβ-induced cell toxicity and could provide insight into our understanding of the mechanism of Alzheimer's disease.

Glutathione Cycle Impairment Mediates Aβ-induced Cell Toxicity

Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the action of amyloid beta-peptide (A beta). We observed that A beta 25-35 induced an increase in reactive oxygen species (ROS) in NT2 rho+ cells, leading to protein and lipid oxidation. This oxidative status was partially prevented by the antioxidants, vitamin E, reduced glutathione, and by melatonin. However, NT2 rho0 cells (that lack mitochondrial DNA) in the absence of A beta showed an increase in ROS production, lipid and protein oxidation, as compared with parental rho+ cells. Upon A beta 25-35 treatment, in rho+ cells, a decrease in glutathione reductase activity and in GSH levels was observed, whereas glutathione peroxidase activity was shown to be increased. In NT2 rho0 cells, in the absence of A beta, GSH levels were maintained, whereas glutathione reductase and peroxidase activities were increased. The exposure of A beta to rho0 cells did not induce any change in these parameters. We observed that melatonin prevented caspase activation and DNA fragmentation in rho+ cells treated with A beta. Considering the evidence presented, we argue that the glutathione cycle impairment is a key event in A beta-induced cell toxicity.

Reduction in cholesterol and sialic acid content protects cells from the toxic effects of beta-amyloid peptides.

beta-Amyloid (Abeta) is the primary protein component of senile plaques associated with Alzheimer's disease and has been implicated in the neurotoxicity associated with the disease. A variety of evidence points to the importance of Abeta-membrane interactions in the mechanism of Abeta neurotoxicity and indicates that cholesterol and gangliosides are particularly important for Abeta aggregation and binding to membranes. We investigated the effects of cholesterol and sialic acid depletion on Abeta-induced GTPase activity in cells, a step implicated in the mechanism of Abeta toxicity, and Abeta-induced cell toxicity. Cholesterol reduction and depletion of membrane-associated sialic acid residues both significantly reduced the Abeta-induced GTPase activity. In addition, cholesterol and membrane-associated sialic acid residue depletion or inhibition of cholesterol and ganglioside synthesis protected PC12 cells from Abeta-induced toxicity. These results indicate the importance of Abeta-membrane interactions in the mechanism of Abeta toxicity. In addition, these results suggest that control of cellular cholesterol and/or ganglioside content may prove useful in the prevention or treatment of Alzheimer's disease.