Amyloid precursor protein and membrane phospholipids in primary cortical neurons increase with development, or after exposure to nerve growth factor or Abeta(1-40)

We examined the relationships between membrane phospholipid levels, the secretion and expression of the amyloid precursor protein (APP), and the responses of both to nerve growth factor (NGF), Abeta(1-40) or Abeta(40-1) in developing cortical neurons cultured from rat embryos. Neuronal membrane phospholipid levels per cell, and phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine increased individually between the first and seventh days of culturing. The amounts of APP holoprotein and APP mRNAs in the cells, as well as the amounts of soluble APP (APPs) secreted by them, also increased during neuronal development in vitro. The increases in APPs exceeded the increases in APP which, in turn, exceed those in phospholipid levels. The levels of APP holoprotein, but not of phospholipids, increased when neurons were grown in a choline-free medium, suggesting that increases in APP are not sufficient to stimulate changes in membrane phospholipids. Treatment of neuron cultures for four days with NGF or Abeta(1-40), but not with Abeta(40-1), dose-dependently increased membrane phospholipids, tau and GAP-43, as well as APP holoprotein and secreted APPs. These results indicate that agents, like NGF or Abeta(1-40), which enhance membrane phospholipid levels may promote neurite formation, APP expression and APPs secretion in primary neuronal cultures.     

Intravascular infusions of soluble β-amyloid compromise the blood–brain barrier, activate CNS glial cells and induce peripheral hemorrhage

Vascular wall levels of soluble β-amyloid_1–40 (Aβ_1–40) are elevated in Alzheimer's disease (AD). Moreover, plasma Aβ levels are increased in familial AD, as well as in some cases of sporadic AD. To determine the histopathologic and behavioral consequences of elevated vascular Aβ levels, Aβ_1–40 (50 μg in distilled water) or vehicle was intravenously infused twice daily into 3-month old male Sprague–Dawley rats for 2 weeks. Intravenous Aβ infusions impaired blood–brain barrier integrity, as indicated by substantial perivascular and parenchyma IgG immunostaining within the brain. Also evident in Aβ-infused animals was an increase in GFAP immunostaining around cerebral blood vessels, and an enhancement of OX-42 microglial immunostaining in brain white matter. Gross pulmonary hemorrhage was noted in most Aβ-infused animals. All the observed changes occurred in the absence of Congo red birefringence. No significant cognitive deficits were present in Aβ-infused animals during water maze acquisition and retention testing, which was conducted during the second week of treatment. These results indicate that circulating Aβ can: (1) induce vessel dysfunction/damage in both the brain and the periphery without complex Aβ fibril formation/deposition, and (2) induce an activation of brain astrocytes and microglia. Taken together, our results suggest that if circulating Aβ is elevated in AD, it is likely to have a pathophysiologic role.     

Apolipoprotein E and β-amyloid levels in the hippocampus and frontal cortex of Alzheimer's disease subjects are disease-related and apolipoprotein E genotype dependent

The ε4 allele of apolipoprotein E (apoE) is associated with increased risk for the development of Alzheimer's disease (AD), possibly due to interactions with the β-amyloid (Aβ) protein. The mechanism by which these two proteins are linked to AD is still unclear. To further assess their potential relationship with the disease, we have determined levels of apoE and Aβ isoforms from three brain regions of neuropathologically confirmed AD and non-AD tissue. In two brain regions affected by AD neuropathology, the hippocampus and frontal cortex, apoE levels were found to be decreased while Aβ_1–40 levels were increased. Levels of apoE were unchanged in AD cerebellum. Furthermore, levels of apoE and Aβ_1–40 were found to be apoE genotype dependent, with lowest levels of apoE and highest levels of Aβ_1–40 occurring in ε4 allele carriers. These results suggest that reduction in apoE levels may give rise to increased deposition of amyloid peptides in AD brain.     

Minocycline or iNOS inhibition block 3-nitrotyrosine increases and blood–brain barrier leakiness in amyloid beta-peptide-injected rat hippocampus

This work has examined levels of 3-nitrotyrosine (3-NT, a marker for peroxynitrite formation) and intactness of blood–brain barrier (BBB) in amyloid beta-peptide (Aβ_1–42)-injected rat hippocampus. Immunohistochemical analysis demonstrated 3-NT immunoreactivity in microglia/macrophages and astrocytes were significantly increased at 7 days post-Aβ_1–42 injection. Administration of the broad spectrum anti-inflammatory agent minocycline or the selective iNOS inhibitor 1400W markedly reduced 3-NT levels. Double immunofluorescence staining showed that 3-NT was prominently expressed in microglia/macrophages and astrocytes located in proximity to blood vessels. Additionally, Aβ_1–42 injection caused a marked increase in permeability of the BBB to immunoglobulin G (IgG); both minocycline and 1400W were highly effective in decreasing the leakiness of the BBB. Our results suggest the involvement of glial-derived reactive nitrogen species in mediating increased BBB permeability in Aβ_1–42 injected rat hippocampus.     

S-Adenosylhomocysteine increases β-amyloid formation in BV-2 microglial cells by increased expressions of β-amyloid precursor protein and presenilin 1 and by hypomethylation of these gene promoters

S-Adenosylhomocysteine (SAH) has been implicated as a risk factor for neurodegenerative diseases such as Alzheimer's disease. As SAH is a potent inhibitor of all cellular methyltransferases, we herein examined the hypothesis that SAH may increase the formation of amyloid β-peptide (Aβ) in BV-2 mouse microglial cells through hypomethylation of presenilin 1 protein (PS1) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1), both of which cleave Aβ precursor protein (APP) to form Aβ. The results showed that SAH increased Aβ protein formation in a concentration-dependent manner (10–500 nM), and this effect of SAH was accompanied by significantly increased expression of APP and PS1 proteins, although SAH only significantly increased the expression of BACE1 at the highest concentration used (500 nM). SAH (500 nM) markedly induced hypomethylation of APP and PS1 gene promoters. Incubation of cells with 5′-azc (20 μM), also an inhibitor of DNA methyltransferases enhanced Aβ protein expression and APP and PS1 gene promoters hypomethylation. By contrast, pre-incubation of cells with betaine (1.0 mM), 30 min followed by incubation with SAH (500 nM) or 5′-azc (20 μM) for 24 h markedly prevented the expression of Aβ protein (by 50%, P < 0.05) and the gene promoter hypomethylation of APP and PS1. Taken together, this study demonstrates that SAH increases the production of Aβ in BV-2 cells possibly by increased expression of APP and induction of hypomethylation of APP and PS1 gene promoters.     

Distribution of β-amyloid and amyloid precursor protein in the brain of spawning (senescent) salmon: a natural, brain-aging model

Brain amyloid precursor protein (APP), a normal constituent of neurons, glial cells and cerebrospinal fluid, has several proposed functions (e.g., in neuronal growth and survival). It appears, however, that altered processing of APP is an initial or downstream step in the neuropathology of brain aging, Alzheimer's disease (AD), and Down's syndrome (DS). Some studies suggest that proteolytic cleavage of APP, producing β-amyloid (Aβ_1–42), could have neurotoxic or neuroprotective effects. In this study, we utilized antibodies to human APP₆₉₅ and Aβ_1–42, and Congo red staining, to search for amyloid deposition in the brain of semelparous spawning kokanee salmon (Oncorhynchus nerka kennerlyi). Intracellular APP₆₉₅ immunoreactivity (APP-ir) was observed in brain regions involved in gustation (glomerulosus complex), olfaction (putative hippocampus, olfactory bulb), vision (optic tectum), the stress response (nucleus preopticus and nucleus lateralis tuberis), reproductive behavior (nucleus preopticus magnocellularis, nucleus preopticus periventricularis, ventral telencephalon), and coordination (cerebellum). Intra- and extra-neuronal Aβ_1–42 immunoreactivity (Aβ-ir) were present in all APP-ir regions except the nucleus lateralis tuberis and Purkinje cells of the cerebellum (coordination). Thus, the relationship between APP and Aβ deposition during brain aging could shed light on the processing of APP into Aβ, neurodegeneration, and possible protection of neurons that are functioning in spawning but senescent salmon. Pacific salmon, with their predictable and synchronized life history, could provide research options not available with the existing models for studies of brain aging and amyloidosis.     

Inhibition of amyloid-β-induced cell death in human brain pericytes in vitro

Amyloid-β protein (Aβ) deposition in the cerebral vascular walls is one of the key features of Alzheimer’s disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Aβ_1–40 carrying the ‘Dutch’ mutation (HCHWA-D Aβ_1–40) induces pronounced degeneration of cultured human brain pericytes. In this study, we aimed to identify inhibitors of Aβ-induced toxicity in human brain pericytes. The toxic effect of HCHWA-D Aβ_1–40 on human brain pericytes was inhibited by co-incubation with catalase, but not with superoxide dismutase, glutathione or vitamin E analogue Trolox. Catalase interacts with Aβ, both in cell cultures and in cell-free assays, and has a prominent effect on the amount and conformational state of Aβ binding to the cell surface of human brain pericytes. This activity of catalase is likely based on its ability to bind and slowly degrade Aβ and not by its usual capacity to convert hydrogen peroxide. Our data confirm that assembly of Aβ at the cell surface of human brain pericytes is a crucial step in Aβ-induced cellular degeneration of human brain pericytes. Inhibition of fibril formation at the cell surface could be an important factor in therapy aimed at reducing cerebral amyloid angiopathy.     

Alzheimer's β-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40

Accumulating evidence suggests that β-amyloid (Aβ)-induced inflammatory reactions may partially drive the pathogenesis of Alzheimer's disease (AD). Recent data also implicate similar inflammatory processes in cerebral amyloid angiopathy (CAA). To evaluate the roles of Aβ in the inflammatory processes in vascular tissues, we have tested the ability of Aβ to trigger inflammatory responses in cultured human vascular cells. We found that stimulation with Aβ dose-dependently increased the expression of CD40, and secretion of interferon-γ (IFN-γ) and interleukin-1β (IL-1β) in endothelial cells. Aβ also induced expression of IFN-γ receptor (IFN-γR) both in endothelial and smooth muscle cells. Characterization of the Aβ-induced inflammatory responses in the vascular cells showed that the ligation of CD40 further increased cytokine production and/or the expression of IFN-γR. Moreover, IL-1β and IFN-γ synergistically increased the Aβ-induced expression of CD40 and IFN-γR. We have recently found that Aβ induces expression of adhesion molecules, and that cytokine production and interaction of CD40–CD40 ligand (CD40L) further increase the Aβ-induced expression of adhesion molecules in these same cells. These results suggest that Aβ can function as an inflammatory stimulator to activate vascular cells and induces an auto-amplified inflammatory molecular cascade, through interactions among adhesion molecules, CD40–CD40L and cytokines. Additionally, Aβ_1–42, the more pathologic form of Aβ, induces much stronger effects in endothelial cells than in smooth muscle cells, while the reverse is true for Aβ_1–40. Collectively, these findings support the hypothesis that the Aβ-induced inflammatory responses in vascular cells may play a significant role in the pathogenesis of CAA and AD.     

Residues at P2-P1 positions of - and ζ-cleavage sites are important in formation of β-amyloid peptide

Most of the Alzheimer's disease (AD)-linked mutations in amyloid precursor protein (APP), which cause abnormal production of β-amyloid (Aβ), are localized at the major β-secretase-and γ-secretase cleavage sites. In this study, using an APP-knockout mouse neuronal cell line, our data demonstrated that at the P2-P1 positions of the -cleavage site at Aβ49 and the ζ-cleavage site at Aβ46, aromatic amino acids caused a strong reduction in total Aβ. On the other hand, residues with a long side chain caused a decrease in Aβ₄₀ and a concomitant increase in Aβ₄₂ and Aβ₃₈. These findings indicate that the structures of the substituting residues at these key positions strongly determine the efficiency and preference of γ-secretase-mediated APP processing, which determines the ratio of different secreted Aβ species, a crucial factor in the disease development. Our findings provide new insight into the mechanisms of γ-secretase-mediated APP processing and, specifically, into why most AD-linked APP mutations are localized at major γ-secretase cleavage sites. This information may contribute to the development of methods of prevention and treatment of Alzheimer's disease aimed at modulating γ-secretase activity.     

Application of quantitative LC–MS surrogate peptide methodology in the analysis of the amyloid beta peptide (Aβ) biosynthetic intermediate protein APP–βCTF

An area of current research in Alzheimer's disease (AD) is the biosynthetic pathway of amyloid beta peptides (Aβ) via consecutive proteolytic cleavages of the amyloid beta precursor protein (APP) by BACE and γ-secretase enzymes. APP is first cleaved by BACE to form a C-terminal fragment APP–βCTF, or also called C99, which then undergoes further cleavage by γ-secretase to form Aβ. Inhibitors of γ-secretase have been observed to yield a so-called ‘Aβ rise’ phenomenon whereby low inhibitor concentrations result in an increase in Aβ levels while high inhibitor concentrations result in lower Aβ levels. A previous report from our labs indicated that this phenomenon was related to ratios of APP–βCTF substrate relative to γ-secretase enzyme. A quantitative Western blot analysis was used with a recombinant C100 protein as calibration standards to assess the relationship of APP–βCTF, γ-secretase enzyme and various inhibitors resulting in the ‘Aβ rise’. An on-line liquid chromatography mass spectrometry (LC–MS) method employing the ‘surrogate peptide’ methodology was developed to accurately quantify the recombinant C100 used in the Western blot analyses. The surrogate peptide approach utilizes tryptic digestion of the protein to stoichiometrically yield a unique peptide fragment, in this case ^C100Aβ17–28 (LVFFAEDVGSNK) that can be readily detected by LC–MS. The absolute quantitative assessment of C100 was accomplished using synthetic Aβ17–28 to generate calibration curves over a 0.001–1 μM range and 15N isotopically labeled Aβ1–40 as the internal standard for enzymatic digestion and its proteolytic peptide [15N]-Aβ17–28 for the analysis.     

X11α haploinsufficiency enhances Aβ amyloid deposition in Alzheimer's disease transgenic mice

The neuronal adaptor protein X11α/mint-1/APBA-1 binds to the cytoplasmic domain of the amyloid precursor protein (APP) to modulate its trafficking and metabolism. We investigated the consequences of reducing X11α in a mouse model of Alzheimer's disease (AD). We crossed hAPPswe/PS-1ΔE9 transgenic (AD tg) mice with X11α heterozygous knockout mice in which X11α expression is reduced by approximately 50%. The APP C-terminal fragments C99 and C83, as well as soluble Aβ40 and Aβ42, were increased significantly in brain of X11α haploinsufficient mice. Aβ/amyloid plaque burden also increased significantly in the hippocampus and cortex of one year old AD tg/X11α (+/−) mice compared to AD tg mice. In contrast, the levels of sAPPα and sAPPβ were not altered significantly in AD tg/X11α (+/−) mice. The increased neuropathological indices of AD in mice expressing reduced X11α suggest a normal suppressor role for X11α on CNS Aβ/amyloid deposition.     

Activation of microglial cells by PrP and β-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels

The prion protein (PrP) and the amyloid β (Aβ) precursor protein (APP) are two normal proteins constitutively synthesised in human brain. An altered form of PrP accumulates in Creutzfeldt–Jakob disease, while Aβ is involved in the pathogenesis of Alzheimer's disease. Synthetic fragments of both proteins, PrP106–126 and β25–35 (β25–35), have been demonstrated to induce neurodegeneration and microglia activation. This study was undertaken to compare PrP106–126 and β25–35 capability of activating human resting microglial cells. Our results show that both peptides are able to induce microglial activation and to elicit an increase in [Ca²⁺]_i levels in cells loaded with calcium-green 1. Inhibitors of L-type voltage-sensitive calcium channels (verapamil, nifedipine and diltiazem) prevented the increase in [Ca²⁺]_i concentration as observed after treatment with PrP106–126 and β25–35, thus indicating a transmembrane calcium influx through these channels. In addition, verapamil abolished the proliferative effect of both PrP106–126 and β25–35.     

Aβ25–35 induces rapid lysis of red blood cells: contrast with Aβ1–42 and examination of underlying mechanisms

Amyloid β-peptide (Aβ) is produced by many different cell types and circulates in blood and cerebrospinal fluid in a soluble form. In Alzheimer's disease (AD), Aβ forms insoluble fibrillar aggregates that accumulate in association with cells of the brain parenchyma and vasculature. Both full-length Aβ (Aβ1–40/42) and the Aβ25–35 fragment can damage and kill neurons by a mechanism that may involve oxidative stress and disruption of calcium homeostasis. Circulating blood cells are exposed to soluble Aβ1–40/42 and may also be exposed to Aβ aggregates associated with the luminal surfaces of cerebral microvessels. We therefore examined the effects of Aβ25–35 and Aβ1–42 on human red blood cells (RBCs) and report that Aβ25–35, in contrast to Aβ1–42, induces rapid (10–60 min) lysis of RBCs. The mechanism of RBC lysis by Aβ25–35 involved ion channel formation and calcium influx, but did not involve oxidative stress because antioxidants did not prevent cell lysis. In contrast, Aβ1–42 induced a delayed (4–24 h) damage to RBCs which was attenuated by antioxidants. The damaging effects of both Aβ25–35 and Aβ1–42 towards RBCs were completely prevented by Congo red indicating a requirement for peptide fibril formation. Aβ1–42 induced membrane lipid peroxidation in RBC, and basal levels of lipid peroxidation in RBCs from AD patients were significantly greater than in age-matched controls, suggesting a possible role for Aβ1–42 in previously reported alterations in RBCs from AD patients.     

Overexpression of wild-type human APP in mice causes cognitive deficits and pathological features unrelated to Aβ levels

Transgenic mice expressing mutant human amyloid precursor protein (APP) develop an age-dependent amyloid pathology and memory deficits, but no overt neuronal loss. Here, in mice overexpressing wild-type human APP (hAPP_wt) we found an early memory impairment, particularly in the water maze and to a lesser extent in the object recognition task, but β-amyloid peptide (Aβ₄₂) was barely detectable in the hippocampus. In these mice, hAPP processing was basically non-amyloidogenic, with high levels of APP carboxy-terminal fragments, C83 and APP intracellular domain. A tau pathology with an early increase in the levels of phosphorylated tau in the hippocampus, a likely consequence of enhanced ERK1/2 activation, was also observed. Furthermore, these mice presented a loss of synapse-associated proteins: PSD95, AMPA and NMDA receptor subunits and phosphorylated CaMKII. Importantly, signs of neurodegeneration were found in the hippocampal CA1 subfield and in the entorhinal cortex that were associated to a marked loss of MAP2 immunoreactivity. Conversely, in mice expressing mutant hAPP, high levels of Aβ₄₂ were found in the hippocampus, but no signs of neurodegeneration were apparent. The results support the notion of Aβ-independent pathogenic pathways in Alzheimer's disease.     

Magnesium-L-threonate exhibited a neuroprotective effect against oxidative stress damage in HT22 cells and Alzheimer’s disease mouse model

BACKGROUNDOxidative stress results in the production of excess reactive oxygen species (ROS) and triggers hippocampal neuronal damage as well as occupies a key role in the pathological mechanisms of neurodegenerative disorders such as Alzheimer’s disease (AD). A recent study confirmed that magnesium had an inhibitory effect against oxidative stress-related malondialdehyde in vitro. However, whether Magnesium-L-threonate (MgT) is capable of suppressing oxidative stress damage in amyloid β (Aβ)_25-35-treated HT22 cells and the AD mouse model still remains to be investigated.AIMTo explore the neuroprotective effect of MgT against oxidative stress injury in vitro and in vivo, and investigate the mechanism.METHODSAβ_25-35-induced HT22 cells were preconditioned with MgT for 12 h. APPswe/PS1dE9 (APP/PS1) mice were orally administered with MgT daily for 3 mo. After MgT treatment, the viability of Aβ_25-35-treated HT22 cells was determined via conducting cell counting kit-8 test and the cognition of APP/PS1 mice was measured through the Morris Water Maze. Flow cytometry experiments were applied to assess the ROS levels of HT22 cells and measure the apoptosis rate of HT22 cells or hippocampal neurons. Expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), hypoxia-inducible factor (HIF)-1α, NADPH oxidase (NOX) 4, Aβ_1-42 and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway proteins was quantified by Western blot.RESULTS In vitro data confirmed that Aβ_25–35-induced HT22 cells had a significantly lower cell viability, higher ROS level and higher apoptosis rates compared with those of control cells (all P < 0.001). MgT prevented the Aβ_25-35-triggered oxidative stress damage by elevating viability and decreasing ROS formation and apoptosis of HT22 cells (all P < 0.001). APP/PS1 mice exhibited worse cognitive performance and higher apoptosis rate of hippocampal neurons than wild-type (WT) mice (all P < 0.01). Meanwhile, significant higher expression of Aβ_1-42 and NOX4 proteins was detected in APP/PS1 mice than those of WT mice (both P < 0.01). MgT also ameliorated the cognitive deficit, suppressed the apoptosis of hippocampal neuron and downregulated the expression of Aβ_1-42 and NOX4 proteins in APP/PS1 mouse (all P < 0.05). Moreover, MgT intervention significantly downregulated HIF-1α and Bax, upregulated Bcl-2 and activated the PI3K/Akt pathway both in vitro and in vivo (all P < 0.05).CONCLUSIONMgT exhibits neuroprotective effects against oxidative stress and hippocampal neuronal apoptosis in Aβ_25-35-treated HT22 cells and APP/PS1 mice.     

JNK regulates APP cleavage and degradation in a model of Alzheimer's disease

Secretion of Amyloid-beta peptide (Aβ) circulating oligomers and their aggregate forms derived by processing of beta-amyloid precursor protein (APP) are a key event in Alzheimer's disease (AD).We show that phosphorylation of APP on threonine 668 may play a role in APP metabolism in H4-APP^sw cell line, a degenerative AD model. We proved that JNK plays a fundamental role in this phosphorylation since its specific inhibition, with the JNK inhibitor peptide (D-JNKI1), induced APP degradation and prevented APP phosphorylation at T668. This results in a significant drop of βAPPs, Aβ fragments and Aβ circulating oligomers. Moreover the D-JNKI1 treatment produced a switch in the APP metabolism, since the peptide reduced the rate of the amyloidogenic processing in favour of the non-amyloidogenic one. All together our results suggest an important link between APP metabolism and the JNK pathway and contribute to shed light on the molecular signalling pathway of this disease indicating JNK as an innovative target for AD therapy.     

Discovery of a Novel Pharmacological and Structural Class of Gamma Secretase Modulators Derived from the Extract of Actaea racemosa

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Amyloid β-peptides inhibit Na/K-ATPase: tissue slices versus primary cultures

Aβ 1-40 (20 μM) has been reported to selectively inhibit Na⁺/K⁺-ATPase activity in rat primary hippocampal cultures after 2–6 days of exposure [10]. We expanded these studies to include Aβ’s effects on Na⁺/K⁺-ATPase activity in rat primary cortical cultures and hippocampal slices, and we correlated these effects with estimates of cell survival in rat brain primary cultures. Using optimized assay conditions, a 5-day exposure to 50 μM Aβ 25-35, 20 μM Aβ 1-40, and 20 μM Aβ 1-42 decreased Na⁺/K⁺-ATPase activity in rat primary cortical cultures 66%, 60%, and 22%, respectively. Aβ 25-35 (50 μM) at 24 h was the only condition that caused inhibition of Na⁺/K⁺-ATPase activity in the absence of cell death, defined as an extracellular shift in the localization of the cytoplasmic enzyme lactate dehydrogenase (LDH). We also found that hippocampal slices were sensitive to Aβ, exhibiting a 40–60% reduction in membrane Na⁺/K⁺-ATPase activity when exposed to 1–30 nM of Aβ 1-40 for 60 min. This inhibition was not readily reversible, as it withstood homogenization and repeated dilution and centrifugation. Additionally, this inhibition occurred only after amyloid incubation with intact hippocampal slices, not with disrupted membranes. The inhibition of Na⁺/K⁺-ATPase in brain slices by physiological, low nM concentrations of Aβ 1-40 is consistent with effects on neurotransmitter release and intrasynaptosomal calcium responses [4 and 7].     

Costimulatory Effects of Interferon-γ and Interleukin-1β or Tumor Necrosis Factor α on the Synthesis of Aβ1-40 and Aβ1-42 by Human Astrocytes

Chronic inflammation and astrocytosis are characteristic histopathological features of Alzheimer's Disease (AD). Astrocytes are one of the predominant cell types in the brain. In AD they are activated and produce inflammatory components such as complement components, acute phase proteins, and cytokines. In this study we analyzed the effect of cytokines on the production of amyloid β (Aβ) in the astrocytoma cell line U373 and in primary human astrocytes isolated postmortem from healthy aged persons as well as from patients with AD. Astrocytes did not produce Aβ in the absence of stimuli or following stimulation with IL-1β, TNFα, IL-6, and TGF-β1. Neither did combinations of TNFα and IL-1β, IL-6 or TGF-β1, or the coadministration of IFNγ and IL-6 or TGF-β1 induce Aβ production. In contrast, pronounced production of Aβ1-40 and Aβ1-42 was observed when primary astrocytes or astrocytoma cells were stimulated with combinations of IFNγ and TNFα or IFNγ and IL-1β. Induction of Aβ production was accompanied by decreased glycosylation of APP as well as by increased secretion of APPsβ. Our results suggest that astrocytes may be an important source of Aβ in the presence of certain combinations of inflammatory cytokines. IFNγ in combination with TNFα or IL-1β seems to trigger Aβ production by supporting β-secretase cleavage of the immature APP molecule.