Neprilysin-sensitive synapse-associated amyloid-beta peptide oligomers impair neuronal plasticity and cognitive function

A subtle but chronic alteration in metabolic balance between amyloid-beta peptide (Abeta) anabolic and catabolic activities is thought to cause Abeta accumulation, leading to a decade-long pathological cascade of Alzheimer disease. However, it is still unclear whether a reduction of the catabolic activity of Abeta in the brain causes neuronal dysfunction in vivo. In the present study, to clarify a possible connection between a reduction in neprilysin activity and impairment of synaptic and cognitive functions, we cross-bred amyloid precursor protein (APP) transgenic mice (APP23) with neprilysin-deficient mice and biochemically and immunoelectron-microscopically analyzed Abeta accumulation in the brain. We also examined hippocampal synaptic plasticity using an in vivo recording technique and cognitive function using a battery of learning and memory behavior tests, including Y-maze, novel-object recognition, Morris water maze, and contextual fear conditioning tests at the age of 13-16 weeks. We present direct experimental evidence that reduced activity of neprilysin, the major Abeta-degrading enzyme, in the brain elevates oligomeric forms of Abeta at the synapses and leads to impaired hippocampal synaptic plasticity and cognitive function before the appearance of amyloid plaque load. Thus, reduced neprilysin activity appears to be a causative event that is at least partly responsible for the memory-associated symptoms of Alzheimer disease. This supports the idea that a strategy to reduce Abeta oligomers in the brain by up-regulating neprilysin activity would contribute to alleviation of these symptoms.     

Clearance of extracellular and cell-associated amyloid beta peptide through viral expression of neprilysin in primary neurons.

Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite constantly anabolized and catabolized in the brain. We previously demonstrated that neprilysin is the major Abeta-degrading enzyme in vivo. To investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating Abeta levels, we expressed neprilysin in primary cortical neurons using a Sindbis viral vector and examined the effect on Abeta metabolism. The corresponding recombinant protein, expressed in the cell bodies and processes, exhibited thiorphan-sensitive endopeptidase activity, whereas a mutant neprilysin with an amino acid substitution in the active site did not show any such activity. Expression of the wild-type neprilysin, but not the mutant, led to significant decreases in both the Abeta40 and 42 levels in the culture media in a dose-dependent manner. Moreover, neprilysin expression also resulted in reducing cell-associated Abeta, which could be more neurotoxic than extracellular Abeta. These results indicate that the manipulation of neprilysin activity in neurons, the major source of Abeta in the brain, would be a relevant strategy for controlling the Abeta levels and thus the Abeta-associated pathology in brain tissues.     

Effects of neprilysin chimeric proteins targeted to subcellular compartments on amyloid beta peptide clearance in primary neurons

Neprilysin (NEP) is a rate-limiting amyloid beta peptide (Abeta)-degrading enzyme in the brain. We demonstrated previously that overexpression of neprilysin in primary cortical neurons remarkably decreased not only extracellular but also intracellular Abeta levels. To investigate the subcellular compartments where neprilysin degrades Abeta most efficiently, we expressed neprilysin chimeric proteins containing various subcellular compartment-targeting domains in neurons. Sec12-NEP, beta-galactoside alpha2,6-sialyltransferase-NEP, transferrin receptor-NEP, and growth-associated protein 43-NEP were successfully sorted to the endoplasmic reticulum, trans-Golgi network, early/recycling endosomes, and lipid rafts, respectively. We found that intracellularly, wild-type neprilysin and all the chimeras showed equivalent Abeta40-degrading activities. Abeta40 was more effectively cleared than Abeta42, and this tendency was greater for intracellular Abeta than for extracellular Abeta. Wild-type and trans-Golgi network-targeted ST-NEP cleared more intracellular Abeta42 than the other chimeras. Wild-type neprilysin cleared extracellular Abeta more effectively than any of the chimeras, among which endoplasmic reticulum-targeted Sec12-NEP was the least effective. These observations indicate that different intracellular compartments may be involved in the metabolism of distinct pools of Abeta (Abeta40 and Abeta42) to be retained or recycled intracellularly and to be secreted extracellularly, and that the endogenous targeting signal in wild-type neprilysin is well optimized for the overall neuronal clearance of Abeta.     

Beta-amyloid, neuronal death and Alzheimer's disease

Alzheimer's disease (AD) is a common neurodegenerative disease that affects cognitive function in the elderly. Large extracellular beta-amyloid (Abeta) plaques and tau-containing intraneuronal neurofibrillary tangles characterize AD from a histopathologic perspective. However, the severity of dementia in AD is more closely related to the degree of the associated neuronal and synaptic loss. It is not known how neurons die and synapses are lost in AD; the current review summarizes what is known about this issue. Most evidence indicates that amyloid precursor protein (APP) processing is central to the AD process. The Abeta in plaques is a metabolite of the APP that forms when an alternative (beta-secretase and then gamma-secretase) enzymatic pathway is utilized for processing. Mutations of the APP gene lead to AD by influencing APP metabolism. One leading theory is that the Abeta in plaques leads to AD because Abeta is directly toxic to the adjacent neurons. Other theories advance the notion that neuronal death is triggered by intracellular events that occur during APP processing or by extraneuronal preplaque Abeta oligomers. Some investigators speculate that in many cases there is a more general disorder of protein processing in neurons that leads to cell death. In the later models, Abeta plaques are a byproduct of the disease process, rather than the direct cause of neuronal death. A direct correlation between Abeta plaque burden and neuronal (or synaptic) loss should occur in AD if Abeta plaques cause AD through a direct toxic effect. However, histopathologic studies indicate that the correlation between Abeta plaque burden and neuronal (or synaptic) loss is poor. We conclude that APP processing and Abeta formation is important to the AD process, but that neuronal alterations that underlie symptoms of AD are not due exclusively to a direct toxic effect of the Abeta deposits that occur in plaques. A more general problem with protein processing, damage due to the neuron from accumulation of intraneuronal Abeta or extracellular, preplaque Abeta may also be important as underlying factors in the dementia of AD.     

Effect of mycelial extract of Clavicorona pyxidata on the production of amyloid beta-peptide and the inhibition of endogenous beta-secretase activity in vitro

Amyloid beta-peptide (Abeta), which is a product of the proteolytic effect of beta-secretase (BACE) on an amyloid precursor protein, is closely associated with Alzheimer's disease (AD) pathogenesis. There is sufficient evidence to suggest that a BACE inhibitor may reduce Abeta levels, thus decreasing the risk of AD. In a previous study, an extract of Clavicorona pyxidata DGUM 29005 mycelia was found to inhibit the production of a soluble beta-amyloid precursor protein (sbetaAPP), Abeta, and BACE in neuronal cell lines. We sought to determine whether this mycelial extract exerts the same effect in human rhabdomyosarcoma A-204 and rat pheochromocytoma PC-12 cells. We found that the production of Abeta decreased in a dose-dependent manner in the presence of the mycelial extract and that the concentration of Abeta never exceeded 50 microg/ml. The presence of sAPP was detected in every culture medium to which the mycelial extract had been added and its concentration remained the same, regardless of the concentration of the extract used. Endogenous beta-secretase activity in A-204 and PC-12 cellular homogenates also decreased in the presence of this extract. These cells, in culture, were not susceptible to the cytotoxic activity of the mycelial extract.     

Caspase cleavage of the amyloid precursor protein modulates amyloid beta-protein toxicity

The amyloid beta-protein precursor (APP) is proteolytically cleaved to generate the amyloid beta-protein (Abeta), the principal constituent of senile plaques found in Alzheimer's disease (AD). In addition, Abeta in its oligomeric and fibrillar forms have been hypothesized to induce neuronal toxicity. We and others have previously shown that APP can be cleaved by caspases at the C-terminus to generate a potentially cytotoxic peptide termed C31. Furthermore, this cleavage event and caspase activation were increased in the brains of AD, but not control, cases. In this study, we show that in cultured cells, Abeta induces caspase cleavage of APP in the C-terminus and that the subsequent generation of C31 contributes to the apoptotic cell death associated with Abeta. Interestingly, both Abeta toxicity and C31 pathway are dependent on the presence of APP. Both APP-dependent Abeta toxicity and C31-induced apoptotic cell death involve apical or initiator caspases-8 and -9. Our results suggest that Abeta-mediated toxicity initiates a cascade of events that includes caspase activation and APP cleavage. These findings link C31 generation and its potential cell death activity to Abeta cytotoxicity, the leading mechanism proposed for neuronal death in AD.     

Neprylisin decreases uniformly in Alzheimer's disease and in normal aging

The proteolysis of beta-amyloid (Abeta) requires neprylisin, an enzyme that has been shown as reduced in Alzheimer's disease (AD). We investigated whether a decrease in neprilysin levels contributes to the accumulation of amyloid deposits not only in AD but also in the normal aging. We analyzed neprilysin mRNA and protein levels in cerebral cortex from 10 cognitively normal elderly subjects with amyloid plaques (NA), 10 cases of AD, and 10 control cases free of amyloid plaques. We found a significant decrease in neprilysin mRNA levels in both AD and NA compared to control cases. Thereby, the defect of neprilysin appears to correlate with Abeta deposition but not with degeneration and dementia.     

Alternative, non-secretase processing of Alzheimer's beta-amyloid precursor protein during apoptosis by caspase-6 and -8.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. Although the pathogenesis of AD is unknown, it is widely accepted that AD is caused by extracellular accumulation of a neurotoxic peptide, known as Abeta. Mutations in the beta-amyloid precursor protein (APP), from which Abeta arises by proteolysis, are associated with some forms of familial AD (FAD) and result in increased Abeta production. Two other FAD genes, presenilin-1 and -2, have also been shown to regulate Abeta production; however, studies examining the biological role of these FAD genes suggest an alternative theory for the pathogenesis of AD. In fact, all three genes have been shown to regulate programmed cell death, hinting at the possibility that dysregulation of apoptosis plays a primary role in causing neuronal loss in AD. In an attempt to reconcile these two hypotheses, we investigated APP processing during apoptosis and found that APP is processed by the cell death proteases caspase-6 and -8. APP is cleaved by caspases in the intracellular portion of the protein, in a site distinct from those processed by secretases. Moreover, it represents a general effect of apoptosis, because it occurs during cell death induced by several stimuli both in T cells and in neuronal cells.     

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.     

Ferulic acid ethyl ester protects neurons against amyloid beta- peptide(1-42)-induced oxidative stress and neurotoxicity: relationship to antioxidant activity

Alzheimer's disease (AD) is neuropathologically characterized by depositions of extracellular amyloid and intracellular neurofibrillary tangles, associated with loss of neurons in the brain. Amyloid beta-peptide (Abeta) is the major component of senile plaques and is considered to have a causal role in the development and progress of AD. Several lines of evidence suggest that enhanced oxidative stress and inflammation play important roles in the pathogenesis or progression of AD. The present study aimed to investigate the protective effects of ethyl-4-hydroxy-3-methoxycinnamic acid (FAEE), a phenolic compound which shows antioxidant and anti-inflammatory activity, on Abeta(1-42)-induced oxidative stress and neurotoxicity. We hypothesized that the structure of FAEE would facilitate radical scavenging and may induce protective proteins. Abeta(1-42) decreases cell viability, which was correlated with increased free radical formation, protein oxidation (protein carbonyl, 3-nitrotyrosine), lipid peroxidation (4-hydroxy-2-trans-nonenal) and inducible nitric oxide synthase. Pre-treatment of primary hippocampal cultures with FAEE significantly attenuated Abeta(1-42)-induced cytotoxicity, intracellular reactive oxygen species accumulation, protein oxidation, lipid peroxidation and induction of inducible nitric oxide synthase. Treatment of neurons with Abeta(1-42) increases levels of heme oxygenase-1 and heat shock protein 72. Consistent with a cellular stress response to the Abeta(1-42)-induced oxidative stress, FAEE treatment increases the levels of heme oxygenase-1 and heat shock protein 72, which may be regulated by oxidative stresses in a coordinated manner and play a pivotal role in the cytoprotection of neuronal cells against Abeta(1-42)-induced toxicity. These results suggest that FAEE exerts protective effects against Abeta(1-42) toxicity by modulating oxidative stress directly and by inducing protective genes. These findings suggest that FAEE could potentially be of importance for the treatment of AD and other oxidative stress-related diseases.     

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.     

Abeta 42-induced increase in neprilysin is associated with prevention of amyloid plaque formation in vivo

Brain beta-amyloid plaques are principal targets for the development of treatments designed to slow the progression of Alzheimer's disease. Intracranial injections of synthetic beta-amyloid peptide (Abeta(42)) in transgenic mice expressing the Alzheimer's disease-causing Swedish APP double mutations increased neuronal levels of neprilysin, a metalloendopeptidase that degrades Abeta(42) in vivo, on mRNA and protein level. This increase was associated with significant reductions in brain levels of Abeta and with almost complete prevention of amyloid plaque formation throughout the brain. In addition, astrogliosis normally associated with amyloidosis was significantly reduced. Our results suggest that up-regulation of neprilysin in brain may represent an opportunity to reduce or prevent amyloid plaque formation in vivo.     

Neprilysin degrades both amyloid beta peptides 1-40 and 1-42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases

To identify the amyloid beta peptide (Abeta) 1-42-degrading enzyme whose activity is inhibited by thiorphan and phosphoramidon in vivo, we searched for neprilysin (NEP) homologues and cloned neprilysin-like peptidase (NEPLP) alpha, NEPLP beta, and NEPLP gamma cDNAs. We expressed NEP, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PEX), NEPLPs, and damage-induced neuronal endopeptidase (DINE) in 293 cells as 95- to 125-kDa proteins and found that the enzymatic activities of PEX, NEPLP alpha, and NEPLP beta, as well as those of NEP and DINE, were sensitive to thiorphan and phosphoramidon. Among the peptidases tested, NEP degraded both synthetic and cell-secreted Abeta1-40 and Abeta1-42 most rapidly and efficiently. PEX degraded cold Abeta1-40 and NEPLP alpha degraded both cold Abeta1-40 and Abeta1-42, although the rates and the extents of the digestion were slower and less efficient than those exhibited by NEP. These data suggest that, among the endopeptidases whose activities are sensitive to thiorphan and phosphoramidon, NEP is the most potent Abeta-degrading enzyme in vivo. Therefore, manipulating the activity of NEP would be a useful approach in regulating Abeta levels in the brain.     

Acyl peptide hydrolase, a serine proteinase isolated from conditioned medium of neuroblastoma cells, degrades the amyloid-beta peptide

Considerable evidence indicates that the amyloid-beta (Abeta) peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Abeta, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Abeta of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl peptide hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Abeta-degrading enzyme, and we show that the degradation of Abeta by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Abeta peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.     

Effects of Hypoxia and Oxidative Stress on Expression of Neprilysin in Human Neuroblastoma Cells and Rat Cortical Neurones and Astrocytes

Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression. Special issue dedicated to Dr. Moussa Youdim.     

Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.     

Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor

Alzheimer's disease (AD) is characterized by overproduction of beta amyloid peptides in the brain with progressive loss of neuronal cells. The 42-aa form of the beta amyloid peptide (Abeta(42)) is implied as a major causative factor, because it is toxic to neurons and elicits inflammatory responses in the brain by activating microglial cells. Despite the overproduction of Abeta(42), AD brain tissue also generates protective factor(s) that may antagonize the neurodestructive effect of Abeta(42). Humanin is a gene cloned from an apparently normal region of an AD brain and encodes a 24-aa peptide. Both secreted and synthetic Humanin peptides protect neuronal cells from damage by Abeta(42), and the effect of Humanin may involve putative cellular receptor(s). To elucidate the molecular identity of such receptor(s), we examined the activity of synthetic Humanin on various cells and found that Humanin induced chemotaxis of mononuclear phagocytes by using a human G protein-coupled formylpeptide receptor-like-1 (FPRL1) and its murine counterpart FPR2. Coincidentally, FPRL1 and FPR2 are also functional receptors used by Abeta(42) to chemoattract and activate phagocytic cells. Humanin reduced the aggregation and fibrillary formation by suppressing the effect of Abeta(42) on mononuclear phagocytes. In neuroblast cells, Humanin and Abeta(42) both activated FPRL1; however, only Abeta(42) caused apoptotic death of the cells, and its cytopathic effect was blocked by Humanin. We conclude that Humanin shares human FPRL1 and mouse FPR2 with Abeta(42) and suggest that Humanin may exert its neuroprotective effects by competitively inhibiting the access of FPRL1 to Abeta(42).     

APP processing and synaptic function

A large body of evidence has implicated Abeta peptides and other derivatives of the amyloid precursor protein (APP) as central to the pathogenesis of Alzheimer's disease (AD). However, the functional relationship of APP and its proteolytic derivatives to neuronal electrophysiology is not known. Here, we show that neuronal activity modulates the formation and secretion of Abeta peptides in hippocampal slice neurons that overexpress APP. In turn, Abeta selectively depresses excitatory synaptic transmission onto neurons that overexpress APP, as well as nearby neurons that do not. This depression depends on NMDA-R activity and can be reversed by blockade of neuronal activity. Synaptic depression from excessive Abeta could contribute to cognitive decline during early AD. In addition, we propose that activity-dependent modulation of endogenous Abeta production may normally participate in a negative feedback that could keep neuronal hyperactivity in check. Disruption of this feedback system could contribute to disease progression in AD.     

Selective cytotoxicity of intracellular amyloid beta peptide1-42 through p53 and Bax in cultured primary human neurons

Extracellular amyloid beta peptides (Abetas) have long been thought to be a primary cause of Alzheimer's disease (AD). Now, detection of intracellular neuronal Abeta1--42 accumulation before extracellular Abeta deposits questions the relevance of intracellular peptides in AD. In the present study, we directly address whether intracellular Abeta is toxic to human neurons. Microinjections of Abeta1--42 peptide or a cDNA-expressing cytosolic Abeta1--42 rapidly induces cell death of primary human neurons. In contrast, Abeta1--40, Abeta40--1, or Abeta42--1 peptides, and cDNAs expressing cytosolic Abeta1--40 or secreted Abeta1--42 and Abeta1--40, are not toxic. As little as a 1-pM concentration or 1500 molecules/cell of Abeta1--42 peptides is neurotoxic. The nonfibrillized and fibrillized Abeta1--42 peptides are equally toxic. In contrast, Abeta1--42 peptides are not toxic to human primary astrocytes, neuronal, and nonneuronal cell lines. Inhibition of de novo protein synthesis protects against Abeta1--42 toxicity, indicating that programmed cell death is involved. Bcl-2, Bax-neutralizing antibodies, cDNA expression of a p53R273H dominant negative mutant, and caspase inhibitors prevent Abeta1--42-mediated human neuronal cell death. Taken together, our data directly demonstrate that intracellular Abeta1--42 is selectively cytotoxic to human neurons through the p53--Bax cell death pathway.