Metabolites of the β-amyloid precursor protein generated by β-secretase localise to the trans-golgi network and late endosome in 293 cells

Deposition of β-amyloid occurs in the brains of all sufferers of Alzheimer's disease. β-amyloid is proteolytically derived from the β-amyloid precursor protein by as yet unidentified enzymes termed secretases. We have generated and characterised antisera to the carboxy-terminal domain and β-secretase cleavage site of the Alzheimer's amyloid precursor protein. The β-secretase cleavage event occurs at the extreme N-terminus of the β-amyloid peptide. Our antiserum to the N-terminus of the β-amyloid peptide (NTβ4) specifically recognises β-secretase cleaved species as opposed to intact βAPP. NTβ4 specifically immunoprecipitates a 13 kDa fragment of βAPP (p13) which is potentially amyloidogenic. We have used these anti-sera in confocal laser scanning immunofluorescence microscopy to localise the intracellular location of potentially amyloidogenic βAPP processing fragments such as p13. Using a number of marker antisera of known intracellular location, we have defined the major location of βAPP fragments possessing the Asp-1 N-terminus of β-amyloid as the trans-Golgi network or late endosome on the basis of colocalisation with a monoclonal antibody to the cation-independent mannose-6-phosphate receptor. The colocalisation was further investigated using brefeldin A which demonstrated that the p13 fragment and mannose-6-phosphate receptor are trafficked by alternative pathways from the trans-Golgi network. © 1996 Wiley-Liss, Inc.     

β-amyloid peptide decreases membrane fluidity

The β-amyloid peptide-25–35 (βA25–35) decreases the fluidity of mouse brain membranes in a concentration-depending fashion. First effects were already seen at a βA25–35 concentration of 100 nmol/1. β-Amyloid peptide (1–40) was similarly active. βA25–35 also decreases the fluidity of human lymphocyte membranes and of membranes from the cortex, hippocampus, striatum, and cerebellum of the rat, although the effects in the rat cerebellum are only weak. Scrambled βA25–35 when investigated under similar conditions showed no effects on membrane fluidity. It is suggest that the effect on cellular calcium-signalling but also the neurotoxic properties of β-amyloid might be the result of its concentration depending effects on membrane properties.     

Testosterone attenuates β-amyloid toxicity in cultured hippocampal neurons

Accumulating evidence suggests that testosterone has neurotrophic and perhaps neuroprotective actions. Thus, age-related depletion of testosterone may increase the brain’s vulnerability to Alzheimer’s disease and related disorders. To begin investigating this issue, cultured neurons were exposed to the Alzheimer-related insult β-amyloid in the presence of testosterone. β-Amyloid neurotoxicity was significantly reduced by testosterone via a rapid, estrogen-independent mechanism. These data may provide additional insight into the treatment of age-related neurodegenerative disorders.     

Co-expression of β-amyloid precursor protein (βAPP) and apolipoprotein E in cell culture: analysis of βAPP processing

Apolipoprotein E (ApoE) is the major genetic risk factor for Alzheimer's disease (AD). The ApoE4 allele is associated with earlier disease onset and greater cerebral deposition of the amyloid beta peptide (Aβ), the major constituent of senile (amyloid) plaques. The molecular mechanism underlying these effects of ApoE4 remains unclear; ApoE alleles could have different influences on Aβ production, extracellular aggregation, or clearance. Because the missense mutations on chromosomes 14 and 21 that cause familial forms of AD appear to lead to increased secretion of Aβ, it is important to determine whether ApoE4 has a similar effect. Here, we have examined the effects of all three ApoE alleles on the processing of βAPP and the secretion of Aβ in intact cells. We established neural (HS683 human glioma) and non-neural (Chinese hamster ovary) cell culture systems that constitutively secrete both ApoE and Aβ at concentrations like those in human cerebrospinal fluid. βAPP metabolites, generated in the presence of each ApoE allele, were analysed and quantified by two methods: immunoprecipitation and phosphorimaging, and ELISA. We detected no consistent allele-specific effects of ApoE on βAPP processing in either cell type. Our data suggest that the higher amyloid burden found in AD subjects expressing ApoE4 is not due to increased amyloidogenic processing of βAPP, in contrast to findings in AD linked to chromosome 14 or 21. These co-expressing cell lines will be useful in the further search for the effects of ApoE on Aβ aggregation or clearance under physiologically relevant conditions.     

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.     

Association of human,rat, and rabbit apolipoprotein E with β-amyloid

In humans, apolipoprotein E (apoE) has three major isoforms, E2 (Cys¹¹², Cys¹⁵⁸), E3 (Cys¹¹², Arg¹⁵⁸), and E4 (Arg¹¹², Arg¹⁵⁸). While ϵ4 is a genetic risk factor for Alzheimer's disease (AD), ϵ2 may protect against late-onset AD. Using native preparations of apoE from conditioned tissue culture media or plasma lipoproteins, we have previously shown that when equivalent amounts of apoE3 or E4 were incubated with β-amyloid (Aβ), apoE3 formed 20 times as much SDS-stable complex with the peptide as apoE4. This preferential binding of Aβ to apoE3 was abolished when apoE was purified by a process which includes delipidation and denaturation. Here we expand these observations to include Aβ binding to lipoprotein-associated and purified apoE2. Lipoproteins isolated from the plasma of individuals homozygous for either ϵ2 or ϵ3 were incubated with Aβ(1-40). SDS-stable complex formation was analyzed by a non-reducing gel shift assay, followed by immunoblotting with either Aβ or apoE antibodies. ApoE2:Aβ complex formation was comparable to apoE3:Aβ in both native and purified preparations of apoE. In addition, lipoprotein-associated rat apoE (Arg¹¹², Arg¹⁵⁸), like human apoE4, did not form complex with Aβ, while lipoprotein-associated rabbit apoE (Cys¹¹², Arg¹⁵⁸) did bind the peptide. These binding studies provide one possible explanation for protective effects of both apoE2 and E3 against the development of Alzheimer's disease. J. Neurosci. Res. 49:9–18, 1997. © 1997 Wiley-Liss Inc.     

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.     

Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: A role for transforming growth factor β

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces inducible nitric oxide (iNOS), nitric oxide (NO), and interleukin-1β (IL-1β) production in microglial cells. Earlier we demonstrated that endotoxin induced iNOS but not IL-1β expression in microglial cells is inhibited by the presence of astroglial cells. In the present paper we describe studies on the mechanism by which astroglial cells exert selective suppressive action on iNOS expression by microglial cells. Expression of iNOS and IL-1β was studied by single or double label immunocytochemical techniques and cell identification was performed with GSA-I-B₄-isolectin and an antibody against GFAP. Production of IL-1β and NO was determined by measurement of IL-1β and nitrite concentrations in cell lysates and the culture medium, respectively. TGFβ, a cytokine known to inhibit NO production by endotoxin challenged macrophages, was measured in culture medium of mixed glial cell cultures using a bioassay. Microglial, astroglial, and mixed glial cell cultures produced similar concentrations of TGFβ. The potential effect of TGFβ was studied by using immunoneutralizing antibodies against TGFβ1 and TGFβ2 on the induction of iNOS in microglial cells in the presence of astroglial cells. Incubation of the mixed glial cell culture with these TGFβ antibodies (3 μg/ml) markedly increased endotoxin-induced NO production and iNOS expression in microglial cells, whereas the production of IL-1β was not affected. The antibodies against TGFβ1 and TGFβ2 marginally increased NO production in pure microglial cell cultures, nonetheless in cultures of purified microglial cells recombinant TGFβ1 and TGFβ2 together with endotoxin inhibited NO production. We conclude that the presence of astroglial cells is essential for the inhibitory effect of TGFβ on NO production by microglial cells (possibly) by activation of TGFβ or by increasing the sensitivity of microglial cells for TGFβ. GLIA 19:190–198, 1997. © 1997 Wiley-Liss, Inc.     

β-amyloid binds to p75NTR and activates NFκB in human neuroblastoma cells

Amyloid β peptide (Aβ), a proteolytic fragment of the amyloid precursor protein (APP), is a major component of the plaques found in the brain of Alzheimer's disease (AD) patients. These plaques are thought to cause the observed loss of cholinergic neurons in the basal forebrain of AD patients. In these neurons, particularly those of the nucleus basalis of Meynert, an up-regulation of 75kD-neurotrophin receptor (p75^NTR), a nonselective neurotrophin receptor belonging to the death receptor family, has been reported. p75^NTR expression has been described to correlate with β-amyloid sensitivity in vivo and in vitro, suggesting a possible role for p75^NTR as a receptor for Aβ. Here we used a human neuroblastoma cell line to investigate the involvement of p75^NTR in Aβ-induced cell death. Aβ peptides were found to bind to p75^NTR resulting in activation of NFκB in a time- and dose-dependent manner. Blocking the interaction of Aβ with p75^NTR using NGF or inhibition of NFκB activation by curcumin or NFκB SN50 attenuated or abolished Aβ-induced apoptotic cell death. The present results suggest that p75^NTR might be a death receptor for Aβ, thus being a possible drug target for treatment of AD. J. Neurosci. Res. 54:798–804, 1998. © 1998 Wiley-Liss, Inc.     

Pro-opiocortin fragments in human and rat brain: β-endorphin and α-MSH are the predominant peptides

The ‘pro-opiocortin’ fragments, β-lipotropin, β-endorphin, ACTH and α-MSH, were estimated in discrete areas of rat and human brain and pituitaries by means of radioimmunoassay in combination with gelfiltration. These peptides exihibited parallel patterns of distribution, but with β-endorphin and α-MSH predominant in the brain of rat and man, and, in contrast, their respective precursors, β-LPH and ACTH predominant in the adenohypophysis of rat and man. These data may be indicative of important differences in post-translational processing of ‘pro-opiocortin’ between these contrasting tissues.     

The effect of age on the response of the rat brains to continuous β-amyloid infusion

Young (3 months old) and aging (18–21 months old) rats were infused intracerebroventricularly with β-amyloid (1–40; 4.2 nmol) for 14 days. In both age groups, β-amyloid led to deficits in water-maze and decreased choline acetyltransferase activity and somatostatin levels. Cortical substance P levels also decreased whereas neuropeptide Y levels remained unaltered. There were no significant age dependent differences among these neurochemicals except a decrease in hippocampal neuropeptide Y levels in the aging group. It is concluded that young and aging rat brains respond similarly to β-amyloid infusion.     

Enhanced expression of ERα in astrocytes modifies the response of cortical neurons to β-amyloid toxicity

Estrogen receptor α (ERα) is over-expressed in reactive glia under conditions of neuronal damage. To elucidate the functional significance of ERα overexpression, an in vitro model of reactive astrocytes with enhanced expression of ERα was obtained by growth in G5 culture supplement. Exposure of cortical neurons to β-amyloid in the presence of either conditioned medium from reactive astrocytes previously treated with 17β-estradiol (17βE2) or transferring of 17βE2-pretreated astrocytes, caused a greater neuroprotective effect compared to the respective control conditions, although reactive glia resulted being per se neuroprotective. Blockade of ERα overexpression by the ER antagonist ICI182,780 was not successful as ICI182,780 behaved as an agonist. However, complete prevention of 17βE2 effect by ICI182,780 produced an increased sensitivity of neurons to β-amyloid toxicity. A similar effect was observed when ERα knock-down was induced by siRNA. It is suggested that increased ERα expression in reactive glia may have a role in limiting neuronal damage.     

Apolipoprotein E deposition and astrogliosis are associated with maturation of β-amyloid plaques in βAPPswe transgenic mouse: Implications for the pathogenesis of Alzheimer’s disease

A transgenic mouse expressing the human β-amyloid precursor protein with the ‘Swedish’ mutation, Tg2576, was used to investigate the mechanism of β-amyloid (Aβ) deposition. Previously, we have reported that the major species of Aβ in the amyloid plaques of Tg2576 mice are Aβ_1-40 and Aβ_1-42. Moreover, Aβ_1-42 deposition precedes Aβ_1-40 deposition, while Aβ_1-40 accumulates in the central part of the plaques later in the pathogenic process. Those data indicate that Aβ deposits in Tg2576 mice have similar characteristics to those in Alzheimer’s disease. In the present study, to understand more fully the amyloid deposition mechanism implicating Alzheimer’s disease pathogenesis, we examined immunohistochemically the distributions of apolipoprotein E (apoE) and Aβ in amyloid plaques of aged Tg2576 mouse brains. Our findings suggest that Aβ_1-42 deposition precedes apoE deposition, and that Aβ_1-40 deposition follows apoE deposition during plaque maturation. We next examined the relationship between apoE and astrogliosis associated with amyloid plaques using a double-immunofluorescence method. Extracellular apoE deposits were always associated with reactive astrocytes whose processes showed enhancement of apoE-immunoreactivity. Taken together, the characteristics of amyloid plaques in Tg2576 mice are similar to those in Alzheimer’s disease with respect to apoE and astrogliosis. Furthermore, apoE deposition and astrogliosis may be necessary for amyloid plaque maturation.     

A critical role of TRPM2 channel in Aβ42‐induced microglial activation and generation of tumor necrosis factor‐α

Amyloid β (Aβ)‐induced neuroinflammation plays an important part in Alzheimer's disease (AD). Emerging evidence supports a role for the transient receptor potential melastatin‐related 2 (TRPM2) channel in Aβ‐induced neuroinflammation, but how Aβ induces TRPM2 channel activation and this relates to neuroinflammation remained poorly understood. We investigated the mechanisms by which Aβ₄₂ activates the TRPM2 channel in microglial cells and the relationships to microglial activation and generation of tumor necrosis factor‐α (TNF‐α), a key cytokine implicated in AD. Exposure to 10–300 nM Aβ₄₂ induced concentration‐dependent microglial activation and generation of TNF‐α that were ablated by genetically deleting (TRPM2 knockout ;TRPM2‐KO) or pharmacologically inhibiting the TRPM2 channel, revealing a critical role of this channel in Aβ₄₂‐induced microglial activation and generation of TNF‐α. Mechanistically, Aβ₄₂ activated the TRPM2 channel via stimulating generation of reactive oxygen species (ROS) and activation of poly(ADPR) polymerase‐1 (PARP‐1). Aβ₄₂‐induced generation of ROS and activation of PARP‐1 and TRPM2 channel were suppressed by inhibiting protein kinase C (PKC) and NADPH oxidases (NOX). Aβ₄₂‐induced activation of PARP‐1 and TRPM2 channel was also reduced by inhibiting PYK2 and MEK/ERK. Aβ₄₂‐induced activation of PARP‐1 was attenuated by TRPM2‐KO and moreover, the remaining PARP‐1 activity was eliminated by inhibiting PKC and NOX, but not PYK2 and MEK/ERK. Collectively, our results suggest that PKC/NOX‐mediated generation of ROS and subsequent activation of PARP‐1 play a role in Aβ₄₂‐induced TRPM2 channel activation and TRPM2‐dependent activation of the PYK2/MEK/ERK signalling pathway acts as a positive feedback to further facilitate activation of PARP‐1 and TRPM2 channel. These findings provide novel insights into the mechanisms underlying Aβ‐induced AD‐related neuroinflammation.     

Effects of β-endorphin and its fragments on inhibitory avoidance behavior in rats

The effects on retrieval of a one-trial learning inhibitory avoidance response of β-endorphin, α-endorphin, and γ-endorphin, given prior to test have been studied in rats. β-Endorphin (β-LPH_61–91) in a relatively low dose (1.5 μg sc. or 50 ng icv.) facilitated inhibitory avoidance behavior, while a higher dose (10 μg sc. or 100 ng icv.) caused bimodal changes (facilitation in 50% of the animals and attenuation in another 40%. Peripheral injection of γ-endorphin attenuated inhibitory avoidance behaviour in a dose-dependent manner. The C-terminus of β-endorphin (β-LPH_78–91) was ineffective. α-Endorphin facilitated inhibitory avoidance behavior in a dose-dependent manner. Naltrexone pretreatment antagonized the bimodal effect of β-endorphin: following pretreatment with the opiate antagonist the low latency component disappeared, but the facilitatory effect of the neuropeptide remained the same.It is suggested that β-endorphin carries more than one bit of behavioral information. Inherent activities either related or unrelated to naltrexone-sensitive opiate receptors as well as biotransformation into α- and γ-endorphin may contribute to the multiple behavioral effects of this neuropeptide.     

Carbachol blocks β-amyloid fragment 31–35-induced apoptosis in cultured cortical neurons

We reported previously that many neurodegenerative changes characteristic of apoptosis could be induced by a short fragment of β-amyloid protein, Aβ31–35, in cultured newborn mice cortical neurons, and that these changes were accompanied with alterations in expression of some genes. This study was designed to examine whether the apoptotic processes and related gene modulations in this model could be affected by coadministration of carbachol by electrophoretic analysis for DNA ladder formation and by RT-PCR assays for genomic modulation. The results showed that (1) simultaneous incubation with carbachol dose- and time-dependently blocked the specific DNA ladder formation induced by exposure to Aβ31–35 and (2) the Aβ31–35-induced downregulation of bcl-2 and upregulations of bax, p53, and c-fos genes were reversed or ameliorated by the coadministration of carbachol. It is proposed that Aβ31–35-induced apoptosis can be prevented by carbachol through mechanisms that modulate the expression of related genes.