Amyloid β-protein (Aβ)-containing astrocytes are located preferentially near N-terminal-truncated Aβ deposits in the human entorhinal cortex

The deposition of the amyloid β-protein (Aβ) is a pathological hallmark of Alzheimer’s disease (AD). Aβ is a peptide consisting of 39–43 amino acids and is derived by β- and γ-secretase cleavage from the Aβ protein precursor (AβPP). An N-terminal-truncated form of Aβ can occur following α- and γ-secretase cleavage of AβPP. Fleecy amyloid is a recently identified distinct type of Aβ deposits occurring in the internal layers (pri-α, pri-β and pri-γ) of the human entorhinal cortex. Fleecy amyloid consists exclusively of N-terminal-truncated Aβ and is a transient form of Aβ deposits, which disappears in late-stage β-amyloidosis. In this study, the entorhinal cortex of 15 cases with AD-related pathology was used to examine astrocytes in the vicinity of N-terminal-truncated Aβ in fleecy amyloid of the layers pri-α, pri-β, and pri-γ in comparison to astrocytes in the vicinity of full-length Aβ in layers pre-β and pre-γ. Immunohistochemistry was performed with antibodies directed against AβPP, Aβ₄₀, Aβ₄₂, Aβ_17–24, Aβ_1–17 and Aβ_8–17 as well as by double-labeling with antibodies directed against Aβ_17–24, Aβ₄₂, and glial fibrillary acid protein (GFAP). A large number of GFAP-positive astrocytes containing N-terminal-truncated Aβ fragments appeared in the vicinity of N-terminal-truncated Aβ, whereas Aβ-containing astrocytes were rarely seen in the vicinity of full-length Aβ. These results suggest that N-terminal-truncated Aβ peptide may be cleared preferentially from the extracellular space by astrocytic uptake and processing. Such an astroglial uptake of N-terminal-truncated Aβ may account for the transient nature of fleecy amyloid and point to the use of N-terminal truncation of Aβ in potential therapeutic strategies aimed at preventing the brain from amassing full-length Aβ deposits. Received: 20 August 1999 / Revised, accepted: 14 March 2000     

Intraneuronal Aβ immunoreactivity is not a predictor of brain amyloidosis-β or neurofibrillary degeneration

Amyloid β (Aβ) immunoreactivity in neurons was examined in brains of 32 control subjects, 31 people with Down syndrome, and 36 patients with sporadic Alzheimer’s disease to determine if intraneuronal Aβ immunoreactivity is an early manifestation of Alzheimer-type pathology leading to fibrillar plaque formation and/or neurofibrillary degeneration. The appearance of Aβ immunoreactivity in neurons in infants and stable neuron-type specific Aβ immunoreactivity in a majority of brain structures during late childhood, adulthood, and normal aging does not support this hypothesis. The absence or detection of only traces of reaction with antibodies against 4–13 aa and 8–17 aa of Aβ in neurons indicated that intraneuronal Aβ was mainly a product of α- and γ-secretases (Aβ_17–40/42). The presence of N-terminally truncated Aβ_17–40 and Aβ_17–42 in the control brains was confirmed by Western blotting and the identity of Aβ_17–40 was confirmed by mass spectrometry. The prevalence of products of α- and γ -secretases in neurons and β- and γ-secretases in plaques argues against major contribution of Aβ-immunopositive material detected in neuronal soma to amyloid deposit in plaques. The strongest intraneuronal Aβ_17–42 immunoreactivity was observed in structures with low susceptibility to fibrillar Aβ deposition, neurofibrillary degeneration, and neuronal loss compared to areas more vulnerable to Alzheimer-type pathology. These observations indicate that the intraneuronal Aβ immunoreactivity detected in this study is not a predictor of brain amyloidosis or neurofibrillary degeneration. The constant level of Aβ immunoreactivity in structures free from neuronal pathology during essentially the entire life span suggests that intraneuronal amino-terminally truncated Aβ represents a product of normal neuronal metabolism. This study was supported in part by funds from the New York State Office of Mental Retardation and Developmental Disabilities and grants from the National Institutes of Health (The National Institute of Child Health and Human Development R01 HD43960 and PO1 HD35897; and the National Institute of Aging P30 AG08051, AG03051, and PO1 AG11531).     

Small heat shock protein HspB8: its distribution in Alzheimer’s disease brains and its inhibition of amyloid-β protein aggregation and cerebrovascular amyloid-β toxicity

Alzheimer’s disease (AD) is characterized by pathological lesions, such as senile plaques (SPs) and cerebral amyloid angiopathy (CAA), both predominantly consisting of a proteolytic cleavage product of the amyloid-β precursor protein (APP), the amyloid-β peptide (Aβ). CAA is also the major pathological lesion in hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), caused by a mutation in the gene coding for the Aβ peptide. Several members of the small heat shock protein (sHsp) family, such as αB-crystallin, Hsp27, Hsp20 and HspB2, are associated with the pathological lesions of AD, and the direct interaction between sHsps and Aβ has been demonstrated in vitro. HspB8, also named Hsp22 of H11, is a recently discovered member of the sHsp family, which has chaperone activity and is observed in neuronal tissue. Furthermore, HspB8 affects protein aggregation, which has been shown by its ability to prevent formation of mutant huntingtin aggregates. The aim of this study was to investigate whether HspB8 is associated with the pathological lesions of AD and HCHWA-D and whether there are effects of HspB8 on Aβ aggregation and Aβ-mediated cytotoxicity. We observed the expression of HspB8 in classic SPs in AD brains. In addition, HspB8 was found in CAA in HCHWA-D brains, but not in AD brains. Direct interaction of HspB8 with Aβ_1–42, Aβ_1–40 and Aβ_1–40 with the Dutch mutation was demonstrated by surface plasmon resonance. Furthermore, co-incubation of HspB8 with D-Aβ_1–40 resulted in the complete inhibition of D-Aβ_1–40-mediated death of cerebrovascular cells, likely mediated by a reduction in both the β-sheet formation of D-Aβ_1–40 and its accumulation at the cell surface. In contrast, however, with Aβ_1–42, HspB8 neither affected β-sheet formation nor Aβ-mediated cell death. We conclude that HspB8 might play an important role in regulating Aβ aggregation and, therefore, the development of classic SPs in AD and CAA in HCHWA-D.     

Assessing multiple sclerosis activity: is the in vitro production of tumor necrosis factor-α, interleukins 2, 6, 4, and 10, and immunoglobulin G of value?

Tumor necrosis factor (TNF) α, interleukins (IL) 2, 4, 6, and 10, and IgG oligoclonal bands (IgG OB) in vitro production was assessed, after whole-blood stimulation with lipopolysaccharide or concanavalin A, in 61 patients presenting with relapsing-remitting, relapsing-progressive, or chronic progressive multiple sclerosis. Multiple sclerosis patients were receiving no treatment or azathioprine (AZA), cyclosporin, cyclophosphamide, subcutaneous interferon (IFN) β1a, or corticosteroids (CST). Statistical correlations significantly showed that: (a) AZA lowers TNF-α (P = 0.002) and increases IL-4 production (P = 0.0024), and IFN-β1a increases TNF-α and decreases IL-4 levels; (b) CST has a negative effect on TNF-α, IL-6, and IL-4 synthesis; and (c) AZA, IFN-β1a, and CST diminish IgG OB synthesis (P = 0.001). Although our study of the dynamics of TNF-α, IL-2, IL-4, IL-6, and IL-10 in vitro production generally found no statistically significant correlations (partly explained by the limited number of values in the various groups), IL-6 was shown to drop during the periods surrounding relapse (P = 0.05) in the absence of treatment, while TNF-α (P = 0.04) and IL-6 (P < 0.05) dropped before exacerbation in the presence of AZA. In vitro production of TNF-α was closely and positively correlated with that of IL-6, independently of clinical features. The enhanced production of IL-10 detected before or at relapse with AZA and IFN-β1a (trends) may interfere with initiation of the immune reaction and with the development of new CNS lesions. Some discrepancies with previously published results stress the difficulties in studying the state of stimulation of different populations of leukocytes by using a variety of in vitro stimuli and in establishing a correlation between mRNA studies and the amount of final or active protein produced. Received: 16 September 1997 Received in revised form: 21 May 1999 Accepted: 8 June 1999     

Impairment of protein synthesis is an early effect of amyloid-β in neurons

Amyloid β-peptides (Aβ) play a key role in the development of Alzheimer’s disease (AD). The response of neurons to the administration of Aβ in experimental animal models of AD remains poorly studied. We investigated the early effect of Aβ_25–35 in neurons of the CA1 field of the hippocampus after intraventricular or intrahippocampal injections. We used various microscopy methods, including light microscopy combined with immunohistochemistry, as well as fluorescent and electron microscopy. The ratio of red and green fluorescence, K _α, after staining of cells with acridine orange (AO) reflects the state of rRNA in ribosomes and correlates with the fraction of active polyribosomes in the total number of ribosomes in the cytoplasm, which allows one to estimate the intensity of protein synthesis. One day after intrahippocampal injection of Aβ_25–35, the K _α value decreased to 55% in morphologically undamaged neurons. Fourteen days after the treatment, most of the neurons exhibited signs of degeneration and contained Aβ deposits. Fourteen days after intraventricular administration of Aβ_25–35, the K _α value decreased to 35% in morphologically undamaged neurons. In these neurons Aβ deposits were not observed. Ultrastructural analysis revealed a substantial increase in number of heavy polyribosomes, which was probably a cause of the decreased K _α value. Thus, the early effect of Aβ on CA1 pyramidal neurons of the hippocampus is the impairment of protein synthesis due to the formation of an increased number of heavy polyribosomes in the cytoplasm. Subsequent neurodegeneration is probably a result of the accumulation of endogenous Aβ in the cytoplasm.     

The role of cytokines in Guillain–Barré syndrome

Cytokines play an important role in the pathogenesis of autoimmune diseases including Guillain–Barré syndrome (GBS) and its animal model experimental autoimmune neuritis (EAN). In this article, we reviewed the current knowledge of the role of cytokines such as TNF-α, IFN-γ, IL-1β, IL-6, IL-12, IL-18, IL-23, IL-17, IL-10, IL-4 and chemokines in GBS and EAN as unraveled by studies both in the clinic and the laboratory. However, these studies occasionally yield conflicting results, highlighting the complex role that cytokines play in the disease process. Efforts to modulate cytokine function in GBS and other autoimmune disease have shown efficiency indicating that cytokines are important therapeutic targets.     

Circulating beta amyloid protein is elevated in patients with acute ischemic stroke

Summary. Recent clinical and experimental studies suggest that ischemic strokes may play an important role in the pathogenesis of Alzheimer’s disease (AD). Beta amyloid (Aβ), a major component of senile plaque in AD, is known to be derived from ischemic brain or activated platelets. We prospectively enrolled 62 patients with acute ischemic stroke and 27 age-matched controls. The serum Aβ and P-selectin levels were determined using the Sandwich-ELISA. We divided ischemic strokes into subgroups according to the clinical syndrome, pathogenesis, and infarct size, and compared the Aβ level between each subgroup. The Aβ1–40 level was markedly elevated in ischemic stroke patients, as compared to controls (140.2 ± 54.0 vs 88.44 ± 34.96 pg/ml, p<0.001). Cardioembolic and larger artery atherosclerotic infarcts had higher Aβ1–40 level than small vessel disease (p = 0.001). Both infarct size and the initial NIHSS score had significantly positive correlations with the serum level of Aβ1–40 (r = 0.539, p<0.001 and r = 0.425, p = 0.001, respectively). However, the P-selectin level was not significantly correlated with serum Aβ1–40. Our data suggest that elevated circulating Aβ1–40 in ischemic stroke patients may be derived from brain as a consequence of ischemic insults.     

Insulysin

That the zinc metalloendopeptidase insulysin (insulin-degrading enzyme IDE) is a major β-amyloid (Aβ) peptide-degrading enzyme in vivo is shown by the higher Aβ peptide levels in the brain of an insulysin-deficient mouse. Insulysin was shown to initially cleave Aβ_1–40 and Aβ_1–42 at His¹³-Gln¹⁴, His¹⁴-Gln¹⁵, and Phe¹⁹-Phe²⁰. The insulysin-dependent cleavage of Aβ prevents both the neurotoxic effects of the peptide as well as the ability of Aβ to deposit onto synthetic amyloid plaques. The kinetics of the reaction of insulysin with the synthetic peptide substrate Abz-G-G-F-L-R-K-H-G-Q-EDDnp displays allosteric properties indicative of a regulated enzyme. Small peptide substrates increase the activity of insulysin toward the hydrolysis of Aβ_1–40 without affecting the activity of the enzyme toward insulin. These studies indicate that insulysin is a target for drug development in which small-molecule peptide analogs can be used to increase the rate of Aβ clearance without affecting insulin levels.     

Suppressed expression of nicotinic acetylcholine receptors by nanomolar β-amyloid peptides in PC12 cells

Summary. A line of evidence has shown that a link between the common pathological features of β-amyloid peptide (Aβ) deposition and cholinergic degeneration observed in Alzheimer's disease (AD) may exist, however, no experimental evidence has shown that exposure to Aβ can decrease expression of nicotinic acetylcholine receptors (nAChRs), which have been shown to play roles in brain cognitive functions. Here, we report that treatment with Aβ_1–40 and Aβ_25–35 at nanomolar concentrations significantly decreased the [³H]epibatidine and [¹²⁵I]α-bungarotoxin binding sites, the protein and mRNA levels of nAChR α3, α7 and β2 subunits in PC12 cells. Aβ_1–40 and Aβ_25–35 at the concentrations used in the treatment study neither bound to nAChRs nor induced apoptosis, but significantly inhibited the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide) reduction. These data suggest that the decreased biosynthesis of nAChRs induced by Aβ may be attributable partially to perturbances of some intracellular signal transduction pathways. The results presented in this study lead to a hypothesis that Aβ can degenerate nAChRs early in the course of AD before the formation of abundant Aβ fibrils. Received May 3, 2001; accepted August 23, 2001     

Overexpression of amyloid precursor protein induces susceptibility to oxidative stress in human neuroblastoma SH-SY5Y cells

Summary. In Alzheimer’s disease amyloid β peptide (Aβ) produced from amyloid precursor protein (APP) is considered to induce cell death. To clarify the molecular mechanism underlying Aβ neurotoxicity, we established the cell line overexpressing wild or mutant (His684Arg) APP in human SH-SY5Y cells. This paper presents that overexpression of wild-APP in the cells (SH/w-APP) increased the levels of APP and Aβ_1–40 but not Aβ_1–42, and reduced Bcl-2 level and proteasome activity with increased susceptibility to oxidative stress. The intracellular levels of reactive oxygen species in SH/w-APP increased significantly by H₂O₂ treatment. The level of Bcl-2 protein, but not mRNA, was markedly decreased in SH/w-APP cells, which was inversely correlated with APP expression among subcloned SH/w-APP cells. These results indicate that increased expression of wild type APP renders neuronal cells more vulnerable to oxidative stress leading to cell death.     

Parkin Attenuates Wild-Type τ Modification in the Presence of β-Amyloid and α-Synuclein

Changes in tau (τ) metabolism comprise important pathological landmarks in the tauopathies with parkinsonism as well as Parkinson’s disease and Alzheimer’s disease. Mutations in the parkin gene are associated with Parkinson’s disease. Deposits of amyloid proteins, including Aβ and α-synuclein coexist in the brains of patients with dementia with Lewy bodies; however, it is not known how either of them interacts with τ to provoke neurofibrillary tangle formation across the tauopathies. Here, we show a role for parkin against τ pathology in the presence of intracellular Aβ or α-synuclein. Parkin attenuates four-repeat human τ, but not mutant P301L, hyperphosphorylation in the presence of intracellular Aβ_1–42, or α-synuclein and decreases GSK-3β activity in amyloid-stressed M17 human neuroblastoma cells. These data suggest that parkin may counteract the alteration of τ metabolism in certain neurodegenerative diseases with τ cytopathy and parkinsonism. An erratum to this article can be found at     

Amyloid β peptide 25–35 modulates hydrolysis of phosphoinositides by membrane phospholipase(s) C of adult brain cortex

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C. Cholinergic pathways are important in learning and memory, and deficits in cholinergic transmission have been implicated in Alzheimer’s disease (AD). AD is also associated with increased β-amyloid plaques. In the present study, we have investigated the effect of the amyloid β (Aβ)synthetic peptide homologous to residue 25–35 of Aβ in nonaggregated and aggregated forms on the degradation of inositol phospholipids. Synaptic plasma membranes (SPM) and the cytosolic fraction from rat brain cortex served as a source of enzymes. The studies were carried out with radioactive inositol phospholipids in the presence of endogenous and 2 mM CaCl₂. The enzyme(s) activity was evaluated by determination of the product formation of [³H]inositol-1-phosphate (IP₁) or [³H]inositol-1,4,5-trisphosphate (IP₃). Results show that the PI-PLC activity was significantly higher in cytosol compared to SPM, and this enzyme was stimulated by 2 mM CaCl₂, but not by GTPγS or carbachol, a cholinergic receptor agonist. Activity of the SPM-bound PIP₂-PLC was similar to that in cytosol and was not activated by 2 mM CaCl₂. The SPM PIP₂-PLC was significantly stimulated by GTPγS together with the cholinergic agonist, carbachol. Fresh-water-soluble Aβ 25–35 activated PI-PLC in SPM markedly by two- to threefold, but this effect was absent in the presence of 2 mM CaCl₂. Moreover, Aβ 25–35 had no effect on basal PIP₂-PLC activity and cytosolic PI-PLC and PIP₂-PLC. The aggregated form of Aβ 25–35 significantly inhibited PIP₂-PLC only in the presence of endogenous CaCl₂. It also inhibited the carbachol and GTP(γ)S-stimulated PIP₂-PLC. Our findings show that depending on the aggregation state and Ca²⁺ concentration, Aβ modulates phosphoinositide degradation differently and exclusively in brain synaptic plasma membranes. Our data suggested that aggregated Aβ peptide may be responsible for the significant impairment of phosphoinositide signaling found in brain membranes during AD.     

Circulating B7-H3(CD276) Elevations in Cerebrospinal Fluid and Plasma of Children with Bacterial Meningitis

The objective of this study was to analyze the clinical significance of cerebrospinal fluid (CSF) and plasma concentrations of B7-H3, tumor necrosis factor-alpha (TNF-α), gamma interferon (IFN-γ), and interleukin-17 (IL-17) in bacterial and aseptic meningitis in children. The participants were six children with bacterial meningitis, 16 with aseptic meningitis, and 12 control subjects. All participants were between 2 months and 12 years of age on admission. Cytokines determination was performed by enzyme-linked immunosorbent assay technique. CSF and plasma-circulating B7-H3 were significantly higher in the bacterial meningitis group as compared with the aseptic group (p = 0.001) and the control group (p = 0.000 and p = 0.001 respectively). However, CSF and plasma-circulating B7-H3 in aseptic meningitis were not significantly higher than control group (p = 0.071 and p = 0.72 respectively).CSF and plasma-circulating TNF-α were significantly higher in the bacterial meningitis group as compared with the aseptic group (p = 0.004 and p < 0.0001 respectively) and control group (p = 0.004 and p < 0.0001 respectively). Similarly, we did not observe significant elevated TNF-α levels in CSF and plasma in aseptic group compared with control group (p = 0.03 and p = 0.12 respectively). IFN-γ levels in CSF and plasma were undetectable in control group, and we did not find statistical significances in both of CSF and plasma between the elevated IFN-γ level in bacterial meningitis group and aseptic meningitis group(p = 0.055 and p = 0.095 respectively) CSF and plasma levels of IL-17 were undetectable in all subjects. There were correlations between B7-H3 and TNF-α, IFN-γ (r = 0.875, p = 0.000; r = −0.693, p = 0.000, respectively) in CSF in meningitis subjects. In plasma, levels of B7-H3 in bacterial meningitis on admission correlated positively with TNF-α (r = 0.968, p = 0.002), and white blood cell counts (r = 0.973, p = 0.001). Detectable CSF levels of B7-H3, TNF-α, and IFN-γ on admission were not associated significantly with any of CSF characteristics. Additionally, CSF and plasma levels of B7-H3 decreased remarkably after treatment. Altogether, our data indicated that circulating B7-H3 and TNF-α levels in the CSF and plasma were useful markers for distinguishing bacterial from aseptic meningitis, and Circulating B7-H3 was demonstrated to be useful in evaluating the intensity of the infectious inflammatory process in the central nervous system in children. An erratum to this article can be found at     

Advances in the cellular and molecular biology of the beta-amyloid protein in Alzheimer's disease

Alzheimer’s disease (AD) is a progressive senile dementia characterized by deposition of a 4 kDa peptide of 39–42 residues known as amyloid beta-peptide (Aβ) in the form of senile plaques and the microtubule associated protein tau as paired helical filaments. Genetic studies have identified mutations in the Aβ precursor protein (APP) as the key triggers for the pathogenesis of AD. Other genes such as presenilins 1 and 2 (PS1/2) and apolipoprotein E (APOE) also play a critical role in increased Aβ deposition. Several biochemical and molecular studies using transfected cells and transgenic animals point to mechanisms by which Aβ is generated and aggregated to trigger the neurodegeneration that may cause AD. Three important enzymes collectively known as “secretases” participate in APP processing. An enzymatic activity, β-secretase, cleaves APP on the amino side of Aβ producing a large secreted derivative, sAPPβ, and an Aβ-bearing membrane-associated C-terminal derivative, CTFβ, which is subsequently cleaved by the second activity, γ-secretase, to release Aβ. Alternatively, a third activity, α-secretase, cleaves APP within Aβ to the secreted derivative sAPPα and membrane-associated CTFα. The predominant secreted APP derivative is sAPPα in most cell-types. Most of the secreted Aβ is 40 residues long (Aβ40) although a small percentage is 42 residues in length (Aβ42). However, the longer Aβ42 aggregates more readily and was therefore considered to be the pathologically important form. Advances in our understanding of APP processing, trafficking, and turnover will pave the way for better drug discovery for the eventual treatment of AD. In addition, APP gene regulation and its interaction with other proteins may provide useful drug targets for AD. The emerging knowledge related to the normal function of APP will help in determining whether or not the AD associated changes in APP metabolism affect its function. The present review summarizes our current understanding of APP metabolism and function and their relationship to other proteins involved in AD.     

Formation of amyloid-β oligomers in brain vascular smooth muscle cells transiently exposed to iron-induced oxidative stress

Vascular smooth muscle cells are involved in deposition of amyloid in brain blood vessels. Accumulation of amyloid-β peptide (Aβ) in cultured brain vascular smooth muscle cells that overexpress human amyloid-β precursor protein (APP) Swedish, is strongly enhanced by exposure to iron ions. We studied cellular accumulation of Aβ and APP processing in vascular smooth muscle cells during recovery after exposure to ferrous ions using cells cultured from Tg2576 mice. The treatment with ferrous ions for 24 and 48 h significantly increased the intracellular levels of ferric, but not ferrous iron. The treatment led to cellular accumulation of C-terminal fragments of APP and to a decreased secretion of APP, Aβ1–40, and Aβ1–42, all of which were quickly normalized in iron-free culture conditions. These effects of iron were neutralized by α-tocopherol, suggesting the role of oxygen reactive species in altered APP processing. Formation of abundant Aβ oligomers, mainly Aβ1–40 tetramers and pentamers, were detected in iron-treated cells, particularly during subsequent culture in iron-free media for up to 72 h. The data suggest that transient increases in local availability of iron in brain blood vessel walls in vivo, e.g., after microhemorhages, may trigger Aβ oligomerization.     

Amyloid-β Induces a Caspase-mediated Cleavage of P2X4 to Promote Purinotoxicity

Overproduction of the β-amyloid fragment 1–42 (Aβ_1–42) is thought to contribute to synaptic dysfunction and neuronal death in Alzheimer’s disease. Mounting evidence suggests that purinergic receptors play critical roles in synaptic plasticity and neuronal survival, but the potential involvement of these receptors in Aβ_1–42-induced synaptic dysfunction and neuronal death has not been addressed. Here we report that Aβ_1–42 promoted accumulation of the calcium-permeable purinergic receptor P2X4 in neurons. We also report evidence that Aβ_1–42 induced a caspase-3-mediated cleavage of the receptor that slowed channel closure times and prevented agonist-induced internalization of the receptor. Molecular interference to reduce the expression of P2X4 in primary rodent neurons attenuated Aβ_1–42-induced neuronal death while induced expression of P2X4 in a neuronal cell line that does not normally express P2-receptors enhanced the toxic effect of Aβ_1–42. Together these findings suggest that Aβ_1–42-induced synaptic dysfunction and neuronal death may involve perturbations in P2X4 purinergic receptors.