Activation of human macrophages by amyloid-beta is attenuated by astrocytes

In Alzheimer's disease, neuritic amyloid-beta plaques along with surrounding activated microglia and astrocytes are thought to play an important role in the inflammatory events leading to neurodegeneration. Studies have indicated that amyloid-beta can be directly neurotoxic by activating these glial cells to produce oxygen radicals and proinflammatory cytokines. This report shows that, using primary human monocyte-derived macrophages as model cells for microglia, amyloid-beta(1-42) stimulate these macrophages to the production of superoxide anions and TNF-alpha. In contrast, astrocytes do not produce both inflammatory mediators when stimulated with amyloid-beta(1-42). In cocultures with astrocytes and amyloid-beta(1-42)-stimulated macrophages, decreased levels of both superoxide anion and TNF-alpha were detected. These decreased levels of potential neurotoxins were due to binding of amyloid-beta(1-42) to astrocytes since FACScan analysis demonstrated binding of FITC-labeled amyloid-beta(1-42) to astrocytoma cells and pretreatment of astrocytes with amyloid-beta(1-16) prevented the decrease of superoxide anion in cocultures of human astrocytes and amyloid-beta(1-42)-stimulated macrophages. To elucidate an intracellular pathway involved in TNF-alpha secretion, the activation state of NF-kappaB was investigated in macrophages and astrocytoma cells after amyloid-beta(1-42) treatment. Interestingly, although activation of NF-kappaB could not be detected in amyloid-beta-stimulated macrophages, it was readily detected in astrocytoma cells. These results not only demonstrate that amyloid-beta stimulation of astrocytes and macrophages result in different intracellular pathway activation but also indicate that astrocytes attenuate the immune response of macrophages to amyloid-beta(1-42) by interfering with amyloid-beta(1-42) binding to macrophages.     

Peptidomic analyses of mouse astrocytic cell lines and rat primary cultured astrocytes

Astrocytes play an active role in the modulation of synaptic transmission by releasing cell-cell signaling molecules in response to various stimuli that evoke a Ca(2+) increase. We expand on recent studies of astrocyte intracellular and secreted proteins by examining the astrocyte peptidome in mouse astrocytic cell lines and rat primary cultured astrocytes, as well as those peptides secreted from mouse astrocytic cell lines in response to Ca(2+)-dependent stimulations. We identified 57 peptides derived from 24 proteins with LC-MS/MS and CE-MS/MS in the astrocytes. Among the secreted peptides, four peptides derived from elongation factor 1, macrophage migration inhibitory factor, peroxiredoxin-5, and galectin-1 were putatively identified by mass-matching to peptides confirmed to be found in astrocytes. Other peptides in the secretion study were mass-matched to those found in prior peptidomics analyses on mouse brain tissue. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion. Twenty-six peptides were observed in multiple stimulation experiments but not in controls and thus appear to be released in a Ca(2+)-dependent manner. These results can be used in future investigations to better understand stimulus-dependent mechanisms of astrocyte peptide secretion.     

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ_(1–40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3 × Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB.     

Bip/GRP78-induced production of cytokines and uptake of amyloid-beta(1-42) peptide in microglia

In the brains of Alzheimer's disease (AD) patients, fibrillar amyloid-beta peptides (Abeta) are markedly accumulated and the microglia associate with the amyloid plaques. However, the regulation of Abeta clearance is still unclear. In the present study, we examined the effect of a chaperone protein BiP/GRP78 on the microglial function. Exogenous addition of recombinant BiP/GRP78 induced the production of cytokines such as interleukin-6 and tumor necrosis factor-alpha, but heat treatment of this protein abolished the activity. Although Abeta(1-42) did not induce cytokine production, it was taken up by the microglia. In addition, the amount of Abeta(1-42) uptake and the number of microglia that phagocytosed Abeta(1-42) were markedly increased by BiP/GRP78. Exogenous BiP/GRP78 also translocated to the endoplasmic reticulum (ER). These results suggest that BiP/GRP78 stimulates Abeta clearance in the microglia, and that dysfunction in the ER may cause the accumulation of extracellular Abeta(1-42).     

Ca2+‐dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid β‐treated astrocytes and in a model of Alzheimer's disease

Neurotoxic effects of amyloid β peptides are mediated through deregulation of intracellular Ca²⁺ homeostasis and signaling, but relatively little is known about amyloid β modulation of Ca²⁺ homeostasis and its pathological influence on glia. Here, we found that amyloid β oligomers caused a cytoplasmic Ca²⁺ increase in cultured astrocytes, which was reduced by inhibitors of PLC and ER Ca²⁺ release. Furthermore, amyloid β peptides triggered increased expression of glial fibrillary acidic protein (GFAP), as well as oxidative and ER stress, as indicated by eIF2α phosphorylation and overexpression of chaperone GRP78. These effects were decreased by ryanodine and 2APB, inhibitors of ryanodine receptors and InsP₃ receptors, respectively, in both primary cultured astrocytes and organotypic cultures of hippocampus and entorhinal cortex. Importantly, intracerebroventricular injection of amyloid β oligomers triggered overexpression of GFAP and GRP78 in astrocytes of the hippocampal dentate gyrus. These data were validated in a triple‐transgenic mouse model of Alzheimer's disease (AD). Overexpression of GFAP and GRP78 in the hippocampal astrocytes correlated with the amyloid β oligomer load in 12‐month‐old mice, suggesting that this parameter drives astrocytic ER stress and astrogliosis in vivo. Together, these results provide evidence that amyloid β oligomers disrupt ER Ca²⁺ homeostasis, which induces ER stress that leads to astrogliosis; this mechanism may be relevant to AD pathophysiology.     

Apolipoprotein J (clusterin) activates rodent microglia in vivo and in vitro

Apolipoprotein J (apoJ; also known as clusterin and sulfated glycoprotein (SGP)-2) is associated with senile plaques in degenerating regions of Alzheimer's disease brains, where activated microglia are also prominent. We show a functional link between apoJ and activated microglia by demonstrating that exogenous apoJ activates rodent microglia in vivo and in vitro. Intracerebroventricular infusion of purified human plasma apoJ ( approximately 4 microg over 28 days) activated parenchymal microglia to a phenotype characterized by enlarged cell bodies and processes (phosphotyrosine immunostaining). In vitro, primary rat microglia were also activated by apoJ, with changes in morphology and induction of major histocompatibility complex class II (MHCII) antigen. ApoJ increased the secretion of reactive nitrogen intermediates in a dose-dependent manner (EC(50) 112 nm), which was completely blocked by aminoguanidine (AG), a nitric oxide synthase inhibitor. However, AG did not block the increased secretion of tumor necrosis factor-alpha by apoJ (EC(50) 55 nm). Microglial activation by apoJ was also blocked by an anti-apoJ monoclonal antibody (G7), and by chemical cleavage of apoJ with 2-nitro-5-thiocyanobenzoate. The mitogen-activated protein kinase kinase and protein kinase C inhibitors PD98059 and H7 inhibited apoJ-mediated induction of reactive nitrogen intermediate secretion from cultured microglia. As a functional measure, apoJ-activated microglia secreted neurotoxic agents in a microglia-neuron co-culture model. We hypothesize that ApoJ contributes to chronic inflammation and neurotoxicity through direct effects on microglia.     

Adult mouse astrocytes degrade amyloid-beta in vitro and in situ

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by excessive deposition of amyloid-beta (Abeta) peptides in the brain. One of the earliest neuropathological changes in AD is the accumulation of astrocytes at sites of Abeta deposition, but the cause or significance of this cellular response is unclear. Here we show that cultured adult mouse astrocytes migrate in response to monocyte chemoattractant protein-1 (MCP-1), a chemokine present in AD lesions, and cease migration upon interaction with immobilized Abeta(1-42). We also show that astrocytes bind and degrade Abeta(1-42). Astrocytes plated on Abeta-laden brain sections from a mouse model of AD associate with the Abeta deposits and reduce overall Abeta levels in these sections. Our results suggest a novel mechanism for the accumulation of astrocytes around Abeta deposits, indicate a direct role for astrocytes in degradation of Abeta and implicate deficits in astroglial clearance of Abeta in the pathogenesis of AD. Treatments that increase removal of Abeta by astrocytes may therefore be a critical mechanism to reduce the neurodegeneration associated with AD.     

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress

Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy, and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus, whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes.     

Clusterin is an ATP-independent chaperone with very broad substrate specificity that stabilizes stressed proteins in a folding-competent state

We recently reported that the ubiquitous, secreted protein clusterin has chaperone activity in vitro [Humphreys et al. (1999) J. Biol. Chem. 274, 6875-6881]. In this study, we demonstrate that clusterin (i) inhibits stress-induced precipitation of a very broad range of structurally divergent protein substrates, (ii) binds irreversibly via an ATP-independent mechanism to stressed proteins to form solubilized high molecular weight complexes, (iii) lacks detectable ATPase activity, (iv) when acting alone, does not effect refolding of stressed proteins in vitro, and (v) stabilizes stressed proteins in a state competent for refolding by heat shock protein 70 (HSP70). Furthermore, we show that, at physiological levels, clusterin inhibits stress-induced precipitation of proteins in undiluted human serum. Clusterin represents the first identified secreted mammalian chaperone. However, reports from others suggest that, at least under stress conditions, clusterin may be retained within cells to exert a protective effect. Regardless of the topological site(s) of action, the demonstration that clusterin can stabilize stressed proteins in a refolding-competent state suggests that, during stresses, the action of clusterin may inhibit rapid and irreversible protein precipitation and produce a reservoir of inactive but stabilized molecules from which other refolding chaperones can subsequently salvage functional proteins.     

A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells

Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of βIII-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages.These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors.     

Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.     

Endoplasmic reticulum chaperones inhibit the production of amyloid-beta peptides

Abeta (amyloid-beta peptides) generated by proteolysis of APP (beta-amyloid precursor protein), play an important role in the pathogenesis of AD (Alzheimer's disease). ER (endoplasmic reticulum) chaperones, such as GRP78 (glucose-regulated protein 78), make a major contribution to protein quality control in the ER. In the present study, we examined the effect of overexpression of various ER chaperones on the production of Abeta in cultured cells, which produce a mutant type of APP (APPsw). Overexpression of GRP78 or inhibition of its basal expression, decreased and increased respectively the level of Abeta40 and Abeta42 in conditioned medium. Co-expression of GRP78's co-chaperones ERdj3 or ERdj4 stimulated this inhibitory effect of GRP78. In the case of the other ER chaperones, overexpression of some (150 kDa oxygen-regulated protein and calnexin) but not others (GRP94 and calreticulin) suppressed the production of Abeta. These results indicate that certain ER chaperones are effective suppressors of Abeta production and that non-toxic inducers of ER chaperones may be therapeutically beneficial for AD treatment. GRP78 was co-immunoprecipitated with APP and overexpression of GRP78 inhibited the maturation of APP, suggesting that GRP78 binds directly to APP and inhibits its maturation, resulting in suppression of the proteolysis of APP. On the other hand, overproduction of APPsw or addition of synthetic Abeta42 caused up-regulation of the mRNA of various ER chaperones in cells. Furthermore, in the cortex and hippocampus of transgenic mice expressing APPsw, the mRNA of some ER chaperones was up-regulated in comparison with wild-type mice. We consider that this up-regulation is a cellular protective response against Abeta.     

Unique lipoproteins secreted by primary astrocytes from wild type, apoE (-/-), and human apoE transgenic mice.

Composition of central nervous system lipoproteins affects the metabolism of lipoprotein constituents within the brain. The epsilon4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease via an unknown mechanism(s). As glia are the primary central nervous system cell type that synthesize apoE, we characterized lipoproteins secreted by astrocytes from wild type (WT), apoE (-/-), and apoE transgenic mice expressing human apoE3 or apoE4 in a mouse apoE (-/-) background. Nondenaturing size exclusion chromatography demonstrates that WT, apoE3, and apoE4 astrocytes secrete particles the size of plasma high density lipoprotein (HDL) composed of phospholipid, free cholesterol, and protein, primarily apoE and apoJ. However, the lipid:apoE ratio of particles containing human apoE is significantly lower than WT. ApoE localizes across HDL-like particle sizes. ApoJ localizes to the smallest HDL-like particles. ApoE (-/-) astrocytes secrete little phospholipid or free cholesterol despite comparable apoJ expression, suggesting that apoE is required for normal secretion of astrocyte lipoproteins. Further, particles were not detected in apoE (-/-) samples by electron microscopy. Nondenaturing immunoprecipitation experiments indicate that apoE and apoJ reside predominantly on distinct particles. These studies suggest that apoE expression influences the unique structure of astrocyte lipoproteins, a process further modified by apoE species.     

Apolipoprotein J/clusterin prevents a progressive glomerulopathy of aging

Apoliprotein J (apoJ)/clusterin has attracted considerable interest based on its inducibility in multiple injury processes and accumulation at sites of remodeling, regression, and degeneration. We therefore sought to investigate apoJ/clusterin's role in kidney aging, as this may reveal the accumulated effects of diminished protection. Aging mice deficient in apoJ/clusterin developed a progressive glomerulopathy characterized by the deposition of immune complexes in the mesangium. Up to 75% of glomeruli in apoJ/clusterin-deficient mice exhibited moderate to severe mesangial lesions by 21 months of age. Wild-type and hemizygous mice exhibited little or no glomerular pathology. In the apoJ/clusterin-deficient mice, immune complexes of immunoglobulin G (IgG), IgM, IgA, and in some cases C1q, C3, and C9 were detectable as early as 4 weeks of age. Electron microscopy revealed the accumulation of electron-dense material in the mesangial matrix and age-dependent formation of intramesangial tubulo-fibrillary structures. Even the most extensively damaged glomeruli showed no evidence of inflammation or necrosis. In young apoJ/clusterin-deficient animals, the development of immune complex lesions was accelerated by unilateral nephrectomy-induced hyperfiltration. Injected immune complexes localized to the mesangium of apoJ/clusterin-deficient but not wild-type mice. These results establish a protective role of apoJ/clusterin against chronic glomerular kidney disease and support the hypothesis that apoJ/clusterin modifies immune complex metabolism and disposal.     

Design and evaluation of a 6-mer amyloid-beta protein derived phage display library for molecular targeting of amyloid plaques in Alzheimer's disease: Comparison with two cyclic heptapeptides derived from a randomized phage display library

Amyloid plaques are the main molecular hallmark of Alzheimer's disease. Specific carriers are needed for molecular imaging and for specific drug delivery. In order to identify new low molecular weight amyloid plaque-specific ligands, the phage display technology was used to design short peptides that bind specifically to amyloid-beta protein, which is the principal component of amyloid plaques. For this purpose, a phage display library was designed from the amino acid sequence of amyloid-beta 1-42. Then, the diversity was increased by soft oligonucleotide-directed mutagenesis. This library was screened against amyloid-beta 1-42 and several phage clones were isolated. Their genomes were sequenced to identify the displayed peptides and their dissociation constants for amyloid-beta 1-42 binding were evaluated by ELISA. The two best peptides, which are derived from the C-terminus hydrophobic domain of amyloid-beta 1-42 that forms a beta-strand in amyloid fibers, were synthesized and biotinylated. After confirming their binding affinity for amyloid-beta 1-42 by ELISA, the specific interaction with amyloid plaques was validated by immunohistochemistry on brain sections harvested from a mouse model of Alzheimer's disease. The thioflavin T aggregation assay has furthermore shown that our peptides are able to inhibit the amyloid fiber formation. They are not toxic for neurons, and some of them are able to cross the blood-brain barrier after grafting to a magnetic resonance imaging contrast agent. To conclude, these peptides have high potential for molecular targeting of amyloid plaques, either as carriers of molecular imaging and therapeutic compounds or as amyloid fiber disrupting agents.     

Purification and characterization of astrocyte-secreted apolipoprotein E and J-containing lipoproteins from wild-type and human apoE transgenic mice

The 4 allele of apolipoprotein E (apoE) is a genetic risk factor for Alzheimer's disease (AD). In order to gain a better understanding of the molecular mechanisms by which apoE and possibly other apolipoproteins produced in the central nervous system (CNS) influence AD pathogenesis, we have purified and characterized the two most abundant apolipoproteins produced in the CNS, apoE and apoJ. We purified apoE and apoJ from primary cultures of mouse astrocytes, which were derived from transgenic mice expressing human apoE isoforms in the absence of mouse apoE. Utilizing antibody affinity columns, we were able to purify both human apoE3 and apoE4, as well as mouse apoJ-containing lipoproteins. Astrocyte-secreted human apoE was present in high density-like lipoproteins of three predominant sizes ranging from 8 to 15 nm in diameter. Mouse apoJ was in particles between 10 and 17 nm in diameter with a peak size range of 11 nm. ApoE and apoJ were in distinct lipoproteins. Utilization of quick-freeze, deep-etch electron microscopy revealed the apoE particles were discs while the apoJ particles were smaller and more irregular in appearance. The lipid composition of apoE particles was very different from those containing apoJ. ApoE-particles contained a similar mass of apoE and lipid, with cholesterol and phospholipid being about equal in mass per particle. ApoJ-particles were relatively lipid poor (three parts protein, one part lipid), with phospholipids being much more abundant than cholesterol. Detailed characterization of phospholipid composition by electrospray ionization mass spectrometry analysis revealed ethanolamine glycerophospholipids to be the most abundant phospholipid present in both apoE and apoJ particles. Analysis of cerebrospinal fluid from apoE3 and apoE4 transgenic mice revealed that human and mouse apoE were in particles the same size as those secreted by astrocytes. Further use of physiological preparations of CNS-derived lipoproteins may allow for a detailed understanding of the role of these molecules in the normal brain and in diseases such as AD.     

Astroglial regulation of apolipoprotein E expression in neuronal cells. Implications for Alzheimer's disease

Although apolipoprotein (apo) E is synthesized in the brain primarily by astrocytes, neurons in the central nervous system express apoE, albeit at lower levels than astrocytes, in response to various physiological and pathological conditions, including excitotoxic stress. To investigate how apoE expression is regulated in neurons, we transfected Neuro-2a cells with a 17-kilobase human apoE genomic DNA construct encoding apoE3 or apoE4 along with upstream and downstream regulatory elements. The baseline expression of apoE was low. However, conditioned medium from an astrocytic cell line (C6) or from apoE-null mouse primary astrocytes increased the expression of both isoforms by 3-4-fold at the mRNA level and by 4-10-fold at the protein level. These findings suggest that astrocytes secrete a factor or factors that regulate apoE expression in neuronal cells. The increased expression of apoE was almost completely abolished by incubating neurons with U0126, an inhibitor of extracellular signal-regulated kinase (Erk), suggesting that the Erk pathway controls astroglial regulation of apoE expression in neuronal cells. Human neuronal precursor NT2/D1 cells expressed apoE constitutively; however, after treatment of these cells with retinoic acid to induce differentiation, apoE expression diminished. Cultured mouse primary cortical and hippocampal neurons also expressed low levels of apoE. Astrocyte-conditioned medium rapidly up-regulated apoE expression in fully differentiated NT2 neurons and in cultured mouse primary cortical and hippocampal neurons. Thus, neuronal expression of apoE is regulated by a diffusible factor or factors released from astrocytes, and this regulation depends on the activity of the Erk kinase pathway in neurons.     

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.     

Radiofrequency radiation does not induce stress response in human T-lymphocytes and rat primary astrocytes

Heat shock proteins (HSPs) are rapidly induced by a variety of stressors, including heat shock, ethanol, heavy metals, UV, and gamma-radiation. Mitogen-activated protein kinases (MAPKs) are also involved in the stress transduction pathways in all eukaryotes. In this study, we attempted to determine whether radiofrequency (RF) radiation is able to induce a non-thermal stress response. Human T-lymphocyte Jurkat cells and rat primary astrocytes were exposed to 1763 MHz of RF radiation at an average specific absorption rate (SAR) of either 2 W/kg or 20 W/kg, for 30 min or 1 h. Temperature was completely controlled at 37 +/- 0.2 degrees C throughout the exposure period. The sham exposures were performed under exactly identical experimental conditions without exposure to RF radiation. We assessed alterations in the expression of HSPs and the activation of MAPKs in the RF-exposed cells. No detectable difference was observed in the expression levels of HSP90, HSP70, and HSP27. The phosphorylation status of MAPKs, extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal protein kinases (JNK1/2), or p38, did not change significantly. In order to determine whether RF radiation can promote the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on stress response, cells were exposed to RF radiation coupled with TPA treatment. When TPA alone was applied, the MAPKs were found to be phosphorylated in a dose-dependent manner. However, RF radiation did not result in any enhancement of TPA-induced MAPK phosphorylation. Neither TPA nor RF radiation exerted any detectable effect on the induction of HSPs. These results indicate that 1763 MHz RF radiation alone did not elicit any stress response, nor did it have any effect on TPA-induced MAPK phosphorylation, under our experimental conditions.