An oestrogen membrane receptor participates in estradiol actions for the prevention of amyloid-beta peptide1-40-induced toxicity in septal-derived cholinergic SN56 cells

Although oestrogen [17 beta-estradiol (E2)]-related neuroprotection has been demonstrated in different models, the involvement of non-classical oestrogen receptors (ERs) remains unexplored. Using the SN56 cholinergic cell line, we present evidence indicating that an ER associated with the plasma membrane participates in oestrogen-dependent inhibition of cell death induced by amyloid-beta peptide (A beta) toxicity. Similarly to E2 alone, a 15-min exposure to estradiol-horseradish peroxidase (E-HRP) significantly reduced A beta-induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC-20 antibody directed to a region neighbouring the ligand-binding domain of ER alpha. Using confocal microscopy on unpermeabilized SN56 cells exposed to MC-20 antibody, we identified a protein at the plasma membrane level. Western blot analysis of purified SN56 cell membrane fractions using MC-20 antibody revealed the presence of one band with the same electrophoretic mobility as intracellular ER alpha. Using conjugated forms of the steroid, E-HRP and E2 conjugated to bovine serum albumin-FITC, we demonstrated by confocal microscopy that SN56 cells contain surface binding sites for E2. Binding of both conjugates was blocked by pre-incubation with E2 and decreased by either ICI 182,780 or MC-20 antibody in a concentration-dependent manner. Thus, a membrane-related ER that shares some structural homologies with ER alpha may participate in oestrogen-mediated neuroprotection.     

Plasma membrane oestrogen receptor mediates neuroprotection against beta-amyloid toxicity through activation of Raf-1/MEK/ERK cascade in septal-derived cholinergic SN56 cells

Rapid oestrogen neuroprotection against beta-amyloid peptide (Abeta)-induced toxicity, a main feature of Alzheimer's disease, may be partially initiated at the plasma membrane. However, the mechanism by which this oestrogen effect occurs is unknown. In a septal murine cell line (SN56), we observed that short exposures to either 17beta-oestradiol (E2) or membrane impermeant E2 bound to horseradish peroxidase (E-HRP) induced a biphasic stimulation of extracellular-signal regulated protein kinase (ERK1/2) phosphorylation, with peak inductions detected around 4-8 min in the early phase and a second maximum around 8 h after treatment. ERK1/2 phosphorylation was abolished by ERK1/2 kinase (MEK) inhibitors PD98059 and U0126. Interestingly, PD98059 was also shown to block rapid E2-related prevention of death in cells exposed to Abeta fragment 1-40 (Abeta1-40) for 24 h. In contrast, no neuroprotective effects were obtained when MEK inhibitor was used to selectively abolish the late phosphorylation phase. Furthermore, both ERK1/2 activation and E2-associated protection were blocked by an inhibitor of Raf-1 kinase. Raf-1 may be involved in these effects because oestrogen caused the rapid serine 338 (Ser338) phosphorylation of this protein. In addition, the oestrogen receptor (ER) antagonist ICI 182,780 was also observed to block ERK1/2 phosphorylation. We propose a novel mechanism in SN56 cells by which rapid effects of oestrogen leading to neuroprotection are signalled through Raf-1/MEK/ERK1/2 pathway, possibly by activation of a membrane-related ER.     

Neuron-specific phosphorylation of Alzheimer's beta-amyloid precursor protein by cyclin-dependent kinase 5

The mature form of Alzheimer's beta-amyloid precursor protein (APP) is phosphorylated specifically at Thr(668) in neurons. In mature neurons, phosphorylated APP is detected in neurites, with dephosphorylated APP being found mostly in the cell body. In vitro, active cyclin-dependent kinase 5 (Cdk5) phosphorylated the cytoplasmic domain of APP at Thr(668). Treatment of mature neurons with an antisense oligonucleotide to Cdk5 suppressed Cdk5 expression and significantly diminished the level of phosphorylated APP. The expression of APP was unaffected in antisense-treated neurons. These results indicate that in neurons APP is phosphorylated by Cdk5, and that this may play a role in its localization.     

Effects of HNE-modification induced by Aβ on neprilysin expression and activity in SH-SY5Y cells

The cerebral accumulation of β-amyloid (Aβ) is a consistent feature of and likely contributor to the development of Alzheimer's disease. In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism also plays an important role in Aβ accumulation. We have previously shown that neprilysin (NEP), the major protease which cleaves Aβ in vivo , is modified by 4-hydroxy-nonenal (HNE) adducts in the brain of Alzheimer's disease patients. To determine if these changes affected Aβ, SH-SY5Y cells were treated with HNE or Aβ, and then NEP mRNA, protein levels, HNE adducted NEP, NEP activity and secreted Aβ levels were determined. Intracellular NEP developed HNE adducts after 24 h of HNE treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. HNE-modified NEP showed decreased catalytic activity, which was associated with elevations in Aβ1–40 in SH-SY5Y and H4 APP695wt cells. Incubation of cells with Aβ1–42 also induced HNE adduction of NEP. In an apparent compensatory response, Aβ-treated cells showed increased NEP mRNA and protein expression. Despite elevations in NEP protein, the activity was significantly lower compared with the NEP protein level. This study demonstrates that NEP can be inactivated by HNE-adduction, which is associated with, at least partly, reduced Aβ cleavage and enhanced Aβ accumulation.     

Apolipoprotein E and beta-amyloid (1-42) regulation of glycogen synthase kinase-3beta

Glycogen synthase kinase-3beta (GSK-3beta) is implicated in regulating apoptosis and tau protein hyperphosphorylation in Alzheimer's disease (AD). We investigated the effects of two key AD molecules, namely apoE (E3 and E4 isoforms) and beta-amyloid (Abeta) 1-42 on GSK-3beta and its major upstream regulators, intracellular calcium and protein kinases C and B (PKC and PKB) in human SH-SY5Y neuroblastoma cells. ApoE3 induced a mild, transient, Ca2+-independent and early activation of GSK-3beta. ApoE4 effects were biphasic, with an early strong GSK-3beta activation that was partially dependent on extracellular Ca2+, followed by a GSK-3beta inactivation. ApoE4 also activated PKC-alpha and PKB possibly giving the subsequent GSK-3beta inhibition. Abeta(1-42) effects were also biphasic with a strong activation dependent partially on extracellular Ca2+ followed by an inactivation. Abeta(1-42) induced an early and potent activation of PKC-alpha and a late decrease of PKB activity. ApoE4 and Abeta(1-42) were more toxic than apoE3 as shown by MTT reduction assays and generation of activated caspase-3. ApoE4 and Abeta(1-42)-induced early activation of GSK-3beta could lead to apoptosis and tau hyperphosphorylation. A late inhibition of GSK-3beta through activation of upstream kinases likely compensates the effects of apoE4 and Abeta(1-42) on GSK-3beta, the unbalanced regulation of which may contribute to AD pathology.     

Toxicity of Pyroglutaminated Amyloid β-Peptides 3(pE)-40 and -42 Is Similar to That of Aβ1-40 and -42

An N-terminal truncated isoform of the amyloid beta-peptide (A beta) that begins with a pyroglutamate (pE) residue at position 3 [A beta3(pE)-42] is the predominant isoform found in senile plaques. Based upon previous in vitro studies regarding A beta N-terminal truncated isoforms, it has been hypothesized that A beta3(pE)-x isoforms may aggregate more rapidly and become more toxic than corresponding Abeta1-x peptides. However, the toxicity and aggregation properties of A beta3(pE)-42 and A beta3(pE)-40 have not previously been examined. After initial solubilization and 1-week preaggregation of each peptide at 37 degrees C and pH 7.4, the toxicity of 5-50 microM A beta3(pE)-42 was similar to that of A beta1-42. Moreover, the toxicity of A beta3(pE)-40 paralleled that induced by A beta1-40 in both 1 day in vitro (DIV) cortical and 7 DIV hippocampal cells. Circular dichroism spectra did not reveal major differences in secondary structure between aged A beta1-42, A beta3(pE)-42, A beta3(pE)-40, and A beta1-40 or freshly solubilized forms of these peptides. Overall, the data indicate that the loss of the two N-terminal amino acids and the cyclization of glutamate at position 3 do not alter the extracellular toxicity of A beta.     

Effects of the monomeric, oligomeric, and fibrillar Abeta42 peptides on the proliferation and differentiation of adult neural stem cells from subventricular zone

The incidence of amyloid plaques, composed mainly of beta-amyloid peptides (Abeta), does not correlate well with the severity of neurodegeneration in patients with Alzheimer's disease (AD). The effects of Abeta(42) on neurons or neural stem cells (NSCs) in terms of the aggregated form remain controversial. We prepared three forms of oligomeric, fibrillar, and monomeric Abeta(42) peptides and investigated their effects on the proliferation and neural differentiation of adult NSCs, according to the degree of aggregation or concentration. A low micromolar concentration (1 micromol/L) of oligomeric Abeta(42) increased the proliferation of adult NSCs remarkably in a neurosphere assay. It also enhanced the neuronal differentiation of adult NSCs and their ability to migrate. These results provide us with valuable information regarding the effects of Abeta(42) on NSCs in the brains of patients with AD.     

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.     

Amyloid beta-protein (Abeta)1-40 protects neurons from damage induced by Abeta1-42 in culture and in rat brain

Previously, we found that amyloid beta-protein (Abeta)1-42 exhibits neurotoxicity, while Abeta1-40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal-mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Abeta1-40 against Abeta1-42-induced neurotoxicity in culture and in vivo. Neuronal death was induced by Abeta1-42 at concentrations higher than 2 microm, which was prevented by concurrent treatment with Abeta1-40 in a dose-dependent manner. However, metal chelators did not prevent Abeta1-42-induced neuronal death. Circular dichroism spectroscopy showed that Abeta1-40 inhibited the beta-sheet transformation of Abeta1-42. Thioflavin-T assay and electron microscopy analysis revealed that Abeta1-40 inhibited the fibril formation of Abeta1-42. In contrast, Abeta1-16, Abeta25-35, and Abeta40-1 did not inhibit the fibril formation of Abeta1-42 nor prevent Abeta1-42-induced neuronal death. Abeta1-42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Abeta1-40. These results indicate that Abeta1-40 protects neurons from Abeta1-42-induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the beta-sheet transformation and fibril formation of Abeta1-42. Our data suggest a mechanism by which elevated Abeta1-42/Abeta1-40 ratio accelerates the development of Alzheimer's disease (AD) in familial AD.     

Oxidative stress induced by beta-amyloid peptide(1-42) is involved in the altered composition of cellular membrane lipids and the decreased expression of nicotinic receptors in human SH-SY5Y neuroblastoma cells

The neurotoxic effects and influence of beta-amyloid peptide (Aβ)_1–42 on membrane lipids and nicotinic acetylcholine receptors (nAChRs) in human SH-SY5Y neuroblastoma cells were investigated in parallel. Exposure of the cultured cells to varying concentrations of Aβ_1–42 evoked a significantly decrease in cellular reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide), together with enhanced lipid peroxidation and protein oxidation. Significant reductions in the total contents of phospholipid and unbiquinone-10, as well as in the levels of the 3 and 7 subunit proteins of nAChRs were detected in cells exposed to Aβ_1–42. In contrast, such treatment had no effect on the total cellular content of cholesterol. Among these alterations, increased lipid peroxidation and decreased levels of cellular phospholipids were most sensitive to Aβ_1–42, occurring at lower concentrations. In addition, when SH-SY5Y cells were pretreated with the antioxidant Vitamin E, prior to the addition of Aβ_1–42, these alterations in neurotoxicity, oxidative stress, composition of membrane lipids and expression of nAChRs were partially prevented. These findings suggest that stimulation of lipid peroxidation by Aβ may be involved in eliciting the alterations in membrane lipid composition and the reduced expression of nAChRs associated with the pathogenesis of AD.     

The effects of chronic estradiol treatment on working memory deficits induced by combined infusion of beta-amyloid (1-42) and ibotenic acid

Estrogen limits in vitro neuron death induced by application of beta-amyloid, the cytotoxic peptide linked to Alzheimer's disease. However, the ability of estrogen to protect neurons and preserve cognitive function in vivo following exposure to beta-amyloid has not been demonstrated. Our objective was to evaluate the potential of estrogen to reduce spatial working memory deficits in female rats induced by administration of a neurotoxic form of beta-amyloid in combination with the excitotoxin, ibotenic acid. The interaction of beta-amyloid with excitotoxic factors may underlie cognitive deficits associated with Alzheimer's disease. Therefore, to create an experimental model typical of early Alzheimer's disease a low dose of ibotenic acid was administered with beta-amyloid into the dorsal hippocampus. Ovariectomized rats were implanted subcutaneously with Silastic capsules that produce physiological levels of 17beta-estradiol 10 days before bilateral intrahippocampal injections of aggregated beta-amyloid (1-42) and ibotenic acid. Capsules remained in situ throughout behavioral testing. When tested 3-10 weeks after neurotoxin treatment, females without estrogen capsules exhibited delay-dependent impairments in working memory performance on a water maze and a radial arm maze. Females treated with estrogen and combined neurotoxins displayed working memory performance comparable to unlesioned females on both tasks. Neurotoxin treatment increased immunoreactivity for glial fibrillary acidic protein but this measure was unaffected by estradiol treatment indicating that estrogen did not limit glial proliferation. Results indicate that estrogen prevented deficits in spatial working memory induced by neurotoxin treatments intended to mimic the pathology of early Alzheimer's disease.     

The glial glutamate transporter, GLT-1, is oxidatively modified by 4-hydroxy-2-nonenal in the Alzheimer's disease brain: the role of Aβ1–42

Glutamate transporters are involved in the maintenance of synaptic glutamate concentrations. Because of its potential neurotoxicity, clearance of glutamate from the synaptic cleft may be critical for neuronal survival. Inhibition of glutamate uptake from the synapse has been implicated in several neurodegenerative disorders. In particular, glutamate uptake is inhibited in Alzheimer's disease (AD); however, the mechanism of decreased transporter activity is unknown. Oxidative damage in brain is implicated in models of neurodegeneration, as well as in AD. Glutamate transporters are inhibited by oxidative damage from reactive oxygen species and lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE). Therefore, we have investigated a possible connection between the oxidative damage and the decreased glutamate uptake known to occur in AD brain. Western blots of immunoprecipitated HNE-immunoreactive proteins from the inferior parietal lobule of AD and control brains suggest that HNE is conjugated to GLT-1 to a greater extent in the AD brain. A similar analysis of beta amyloid (Abeta)-treated synaptosomes shows for the first time that Abeta1-42 also increases HNE conjugation to the glutamate transporter. Together, our data provide a possible link between the oxidative damage and neurodegeneration in AD, and supports the role of excitotoxicity in the pathogenesis of this disorder. Furthermore, our data suggests that Abeta may be a possible causative agent in this cascade.     

Toll-like receptors 2 and 4 mediate Abeta(1-42) activation of the innate immune response in a human monocytic cell line

The primary molecules for mediating the innate immune response are the Toll-like family of receptors (TLRs). Recent work has established that amyloid-beta (Aβ) fibrils, the primary components of senile plaques in Alzheimer's disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP-1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Aβ(1–42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam₃CSK₄) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Aβ(1–42) species. Pre-treatment of the cells with 10 μg/mL of a TLR2-specific antibody blocked ∼50% of the cell response to fibrillar Aβ(1–42), completely blocked the Pam₃CSK₄ response, and had no effect on the LPS-induced response. A TLR4-specific antibody (10 μg/mL) blocked ∼35% of the cell response to fibrillar Aβ(1–42), completely blocked the LPS response, and had no effect on the Pam₃CSK₄ response. Polymyxin B abolished the LPS response with no effect on Aβ(1–42) ruling out bacterial contamination of the Aβ samples. Combination antibody pre-treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Aβ(1–42) response than the antibodies used alone. These data demonstrate that fibrillar Aβ(1–42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Aβ-induced tumor necrosis factor alpha production in a human monocytic cell line.     

Copper-dependent inhibition of cytochrome c oxidase by Abeta(1-42) requires reduced methionine at residue 35 of the Abeta peptide

By altering key amino acid residues of the Alzheimer's disease-associated amyloid-beta peptide, we investigated the mechanism through which amyloid-beta inhibits cytochrome c oxidase (EC 1.9.3.1). Native amyloid-beta inhibited cytochrome oxidase by up to 65%, and the level of inhibition was determined by the period of amyloid-beta ageing before the cytochrome oxidase assay. Substituting tyrosine-10 with alanine did not affect maximal enzyme inhibition, but the altered peptide required a longer period of ageing. By contrast, oxidizing the sulfur of methionine-35 to a sulfoxide, or substituting methionine-35 with valine, completely abrogated the peptide's inhibitory potential towards cytochrome oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the loss of inhibitory potential towards cytochrome oxidase with the methionine-35-altered peptides did not correlate with a substantially different distribution of amyloid-beta oligomeric species. Although the amyloid-beta-mediated inhibition of cytochrome oxidase was completely dependent on the presence of divalent Cu2+, it was not supported by monovalent Cu+, and experiments with catalase and H2O2 indicated that the mechanism of cytochrome oxidase inhibition does not involve amyloid-beta-mediated H2O2 production. We propose that amyloid-beta-mediated inhibition of cytochrome oxidase is dependent on the peptide's capacity to bind, then reduce Cu2+, and that it may involve the formation of a redox active amyloid-beta-methionine radical.     

The SIRT1 activator resveratrol protects SK-N-BE cells from oxidative stress and against toxicity caused by α-synuclein or amyloid-β (1-42) peptide

Human sirtuins are a family of seven conserved proteins (SIRT1-7). The most investigated is the silent mating type information regulation-2 homolog (SIRT1, NM_012238 ), which was associated with neuroprotection in models of polyglutamine toxicity or Alzheimer's disease (AD) and whose activation by the phytocompound resveratrol (RES) has been described. We have examined the neuroprotective role of RES in a cellular model of oxidative stress, a common feature of neurodegeneration. RES prevented toxicity triggered by hydrogen peroxide or 6-hydroxydopamine (6-OHDA). This action was likely mediated by SIRT1 activation, as the protection was lost in the presence of the SIRT1 inhibitor sirtinol and when SIRT1 expression was down-regulated by siRNA approach. RES was also able to protect SK-N-BE from the toxicity arising from two aggregation-prone proteins, the AD-involved amyloid-β (1-42) peptide (Aβ42) and the familiar Parkinson's disease linked α-synuclein(A30P) [α-syn(A30P)]. Alpha-syn(A30P) toxicity was restored by sirtinol addition, while a partial RES protective effect against Aβ42 was found even in presence of sirtinol, thus suggesting a direct RES effect on Aβ42 fibrils. We conclude that SIRT1 activation by RES can prevent in our neuroblastoma model the deleterious effects triggered by oxidative stress or α-syn(A30P) aggregation, while RES displayed a SIRT1-independent protective action against Aβ42.     

β-Amyloid-Induced Cell Toxicity: Enhancement of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-Dependent Cell Death

Abstract: In an attempt to understand the cause of neurodegeneration in Alzheimer's disease, the toxic effects of β-amyloid (Aβ) peptides have been widely studied. At high micromolar concentrations Aβ peptides have been demonstrated to be acutely toxic to various cell types. At submicromolar concentrations, Aβ peptides have been suggested to inhibit cellular metabolic activity, due to their inhibition of the ability of cells to metabolize the oxidoreductase substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Here we show, first, that MTT reduction surprisingly leads to a breakdown in PC12 cell membrane integrity and cell death, presumably through the formation of a crystalline formazan product, and, second, that pretreatment of PC12 cells with nanomolar concentrations of Aβ peptide, rather than inhibiting their metabolic activity, increases the susceptibility of these cells to the secondary toxic effect of formazan crystal formation. These results suggest that low nanomolar concentrations of Aβ render membranes more susceptible to damage by a secondary insult, in this case, MTT reduction. It is plausible that such an effect, when combined with additional risk factors, could contribute to the neurodegeneration that occurs in Alzheimer's disease.     

Enhanced G-protein activation by a mixture of Abeta(25-35), Abeta(1-40/42) and zinc

Beta-amyloid peptides (Abetas) bind to several G-protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Abeta(1-42), Abeta(1-40), Abeta(25-35) and their mixtures on [(35)S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Abetas alone induced a concentration-dependent activation of G-proteins (IC50 approximately 10(-6) m), while aggregated Abeta fibrils only affected GTP binding at concentrations above 10(-5) m. Mixing Abeta(25-35) with Abeta(1-42) or Abeta(1-40) induced a several-fold increase in GTP-binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Abeta(1-40) and Abeta(1-42) or highly aggregated Abetas. Zinc had no effect on Abeta(1-40/42) but strongly potentiated the Abeta(25-35) or the mixed peptides-induced GTP-binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.     

The Intracellular Component of Cellular 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction Is Specifically Inhibited by β-Amyloid Peptides

Abstract: In vitro cell culture model systems for investigating the biochemical mechanisms involved in the neurodegenerative actions of β-amyloid peptide (β-AP) have been established. Using rat pheochromocytoma PC12 or human epitheloid HeLa cell lines, submicromolar concentrations of the β-AP fragments β1–40, β1–39, and β25–35, but not β1–28, were found to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In both cell lines, the β-AP-sensitive component represented ∼70% of total cellular MTT reduction. When the reduction of a series of structurally related dyes was compared with that of MTT, the reduction of 3α-naphthyl-2-phenyl-5-(4-nitrophenyl)-2 H -tetrazolium chloride (NTV) was also found to be sensitive to β25–35, but that of seven other redox dyes was not. A property common to MTT and NTV is that they are both readily taken up into PC12 and HeLa cells and do not require an artificial electron coupling agent to be reduced. Microscopic analysis of MTT-formazan product formation in PC12 and HeLa cells following β25–35 treatment revealed that it was the intracellular component of the reduction of this dye that was abolished. These results support the hypothesis that the cellular reduction of MTT represents a specific indicator of the initial events underlying the mechanism of β-AP toxicity.