Identification of a Novel Aspartic Protease (Asp 2) as β-Secretase

The Alzheimer's disease β-amyloid peptide (Aβ) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of β- and then γ-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of β-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal β-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the β-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Aβ. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.     

Presenilin/γ-Secretase Activity Is Located in Acidic Compartments of Live Neurons

Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of β-amyloid (Aβ) peptides. However, exactly where within the neuron γ-secretase processes APP C99 to generate Aβ and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly “visualize” the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aβ from C99, we also show that intracellular Aβ is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aβ being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.SIGNIFICANCE STATEMENT Alzheimer''s disease (AD) genetics and histopathology highlight the importance of amyloid precursor protein (APP) processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly “visualize” that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular β-amyloid (Aβ) is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aβ in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.     

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.     

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.     

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.     

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.     

HIF-1alpha inhibition ameliorates neonatal brain injury in a rat pup hypoxic-ischemic model

In this study, we tested if caspase-3 inhibition decreased ischemia-induced Aβ elevation by reducing β-secretase (BACE1) activity. Changes in caspase-3, Aβ and BACE1 levels were detected in rat striatum on different days after middle cerebral artery occlusion using immunostaining. We found that the positive labeled cells of activated caspase-3, Aβ, and BACE1 were significantly and time-dependently increased in the ipsilateral striatum. The results of Western blotting and RT-PCR showed that caspase-3 inhibitor Z-DEVD-FMK reduced BACE1 mRNA and protein levels, and inhibited its protease activity, thereby decreasing the amount of APP C99 and Aβ in ischemic brains. Moreover, Z-DEVD-FMK reduced BACE1 and GFAP double-labeled cells, but not GFAP protein levels or GFAP-labeled cells, in the ipsilateral striatum. Thus, we demonstrated that caspase-3 inhibition attenuated ischemia-induced Aβ formation by reducing BACE1 production and activity. This finding provides a therapeutic strategy for preventing Aβ accumulation and reducing the risk of neurodegeneration after stroke.     

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.     

Presenilin Is Essential for ApoE Secretion,a Novel Role of Presenilin Involved in Alzheimer's Disease Pathogenesis

Alzheimer''s disease (AD) is a debilitating dementia characterized by progressive memory loss and aggregation of amyloid-β (Aβ) protein into amyloid plaques in patient brains. Mutations in presenilin (PS) lead to abnormal generation of Aβ, which is the major cause of familial AD (FAD), and apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) onset. However, whether dysfunction of PS is involved in the pathogenesis of SAD is largely unknown. We found that ApoE secretion was completely abolished in PS-deficient cells and markedly decreased by inhibition of γ-secretase activity. Blockade of γ-secretase activity by a γ-secretase inhibitor, DAPT, decreased ApoE secretion, suggesting an important role of γ-secretase activity in ApoE secretion. Reduced ApoE secretion is also observed in nicastrin-deficient cells with reduced γ-secretase activity. PS deficiency enhanced nuclear translocation of ApoE and binding of ApoE to importin α4, a nuclear transport receptor. Moreover, the expression of PS mutants in PS-deficient cells suppressed the restoration effects on ApoE secretion compared with the expression of wild-type PS. Plasma ApoE levels were lower in FAD patients carrying PS1 mutations compared with normal control subjects. Our findings suggest a novel role of PS contributing to the pathogenesis of SAD by regulating ApoE secretion.SIGNIFICANCE STATEMENT Familial AD (FAD) typically results from mutations in the genes encoding amyloid precursor protein, presenilin 1 (PS1), or PS2. Many PS mutants have been found to exert impaired γ-secretase activity and increased amyloid-β 42 (Aβ42)/Aβ40 ratio, which induce early amyloid deposition and FAD. On the other hand, apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) and contributes to AD pathogenesis because it has reduced Aβ clearance capability compared with ApoE3 and ApoE2. FAD and SAD have long been considered to be caused by these two independent mechanisms; however, for the first time, we demonstrated that PS is essential for ApoE secretion and PS mutants affected ApoE secretion in vitro and in human samples, suggesting a novel mechanism by which PS is also involved in SAD pathogenesis.     

Presenilins, β-amyloid precursor protein and the molecular basis of Alzheimer's disease

Progressive cerebral deposition of the 40- and 42-residue amyloid β-proteins is an early and invariant event in all forms of Alzheimer's disease (AD). Aβ proteins are generated from the β-amyloid precursor protein (APP) via two sequential cleavages by proteases designated β-secretase and γ-secretase and are constitutively secreted by essentially all cells throughout life. APP can undergo these cleavages during its secretory trafficking to the cell surface, yet much of Aβ appears to be generated after APP reaches the surface, i.e. in the endosomal pathway. Presenilin (PS) 1 and 2, homologous proteins with eight transmembrane (TM) domains, play a critical role in the γ-secretase cleavage of APP. Deletion of presenilin 1 (PS1) in mice markedly decreases Aβ production, whereas AD-causing PS1 mutations selectively increase Aβ₄₂ production, thereby markedly accelerating amyloid plaque formation, both in humans and transgenic mice. Small amounts of APP and PS can be co-immunoprecipitated from cells, suggesting a direct role of PS1 in the cleavage of APP by γ-secretase. We recently observed and mutated two unusual intramembranous aspartate residues in TM6 and TM7 of each presenilin, resulting in complete blockage of the γ-secretase cleavage of APP, with no detectable Aβ production by cells. Because our protease inhibitor studies suggest that γ-secretase is an aspartyl protease, we hypothesize that these two key aspartates serve as the active site of an unprecedented intramembranous aspartyl protease (i.e. γ-secretase). The recent discovery that Notch, a protein critical for cell fate determination during development, also undergoes an intramembranous cleavage mediated by PS suggests that presenilin may be a key regulatory enzyme for several vital proteolytic events. Thought of in this context, AD may arise late in the post-reproductive life of humans as an ancillary metabolic consequence of a proteolytic mechanism which confers strong evolutionary advantage. Specific and potent inhibitors of γ-secretase/presenilin or of the recently cloned β-secretase may well prove useful for the treatment and the prevention of AD.     

Homer2 and Homer3 interact with amyloid precursor protein and inhibit Abeta production

The study of Amyloid Precursor Protein (APP) processing has been the focus of considerable interest, since it leads to Aβ peptide generation, the main constituent of neuritic plaques found in brains of Alzheimer's disease patients. Therefore, the identification of novel APP binding partners that regulate Aβ peptide production represents a pharmaceutical target aiming at reducing Αβ pathology. In this study, we provide evidence that Homer2 and Homer3 but not Homer1 proteins interact specifically with APP. Their expression inhibits APP processing and reduces secretion of Aβ peptides. In addition, they decrease the levels of cell surface APP and inhibit maturation of APP and β-secretase (BACE1). The effects of Homer2 and Homer3 on APP trafficking to the cell surface and/or on APP and BACE1 maturation could be part of the mechanism by which the expression of these proteins leads to the significant reduction of Aβ peptide production.     

Cell-free generation of the notch1 intracellular domain (NICD) and APP-CTfgamma: evidence for distinct intramembranous "gamma-secretase" activities

PSEN1 and PSEN2 encode polytopic membrane proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2) that play an essential role in intramembranous (“γ-secretase”) proteolysis of selected type I membrane proteins, that include Notch1 and β-amyloid precursor protein (APP). In order to gain insights into biochemical mechanisms underlying γ-secretase processing of Notch1 and APP, we have developed a novel in vitro assay in which γ-secretase-mediated generation of S3/NICD and APP-CTFγ can be readily detected in isolated membrane fractions derived from immortalized PS1+/− mouse embryonic fibroblasts; production of the APP and Notch1 derivatives are inhibited by a highly selective and potent γ-secretase inhibitor, L-685,458, with a IC₅₀ of ∼50 pM. In membranes prepared from PS1-deficient fibroblasts, we detected APP-CTFγ, albeit at low levels. Unexpectedly, and despite the presence of endogenous PS2 in membranes prepared from PS1-deficient fibroblasts, production of the Notch derivatives, S3/NICD, was nearly undetectable in these reactions. Moreover, S3/NICD production is neither detected in detergent-solubilized membrane preparations from PS1-deficient cells, nor in reactions containing PS1-containing membranes that were co-solubilized with membranes from PS −/− cells expressing a chimeric Notch 1 species. These findings strongly suggest that the factors responsible for intramembranous, γ-secretase proteolysis of APP and Notch1 are neither equivalent, nor exchangeable.     

The influence of denervation on β-amyloid protein precursor and α1-antichymotrypsin in mouse skeletal muscle

A serpin, α₁-antichymotrypsin (α₁-ACT), and Kunitz inhibitor containing forms of the β-amyloid precursor protein (βAPP) may be important components of the balance between serine proteases and inhibitors in the nervous system. In the current report we studied whether axotomy affected the localization of βAPP and α₁-ACT in adult mouse muscle. Immunocytochemical experiments indicated that βAPP was present in normal muscle both at neuromuscular junctions and within intramuscular nerves. α₁-ACT was also present at neuromuscular junctions, on the perineurium of nerves and endothelial cell surfaces. Following axotomy, both βAPP and α₁-ACT disappeared from intramuscular nerves simultaneously. However, at the neuromuscular junction α₁-ACT decreased more rapidly with βAPP lingering before disappearing.     

The β-secretase, BACE

Evidence suggests that the β-amyloid peptide (Aβ) is central to the pathophysiology of Alzheimer’s disease (AD). Amyloid plaques, primarily composed of Aβ, progressively develop in the brains of AD patients, and mutations in three genes (APP, PS1, and PS2) cause early onset familial AD (FAD) by directly increasing synthesis of the toxic, plaque-promoting Aβ42 peptide. Given the strong association between Aβ and AD, therapeutic strategies to lower the concentration of Aβ in the brain should prove beneficial for the treatment of AD. One such strategy would involve inhibiting the enzymes that generate Aβ. Aβ is a product of catabolism of the large TypeI membrane protein, amyloid precursor protein (APP). Two proteases, called β- and γ-secretase, mediate the endoproteolysis of APP to liberate the Aβ peptide. For over a decade, the molecular identities of these proteases were unknown. Recently, the γ-secretase has been tentatively identified as the presenilin proteins, PS1 and PS2, and the identity of the β-secretase has been shown to be the novel transmembrane aspartic protease, β-site APP cleaving enzyme 1 (BACE1; also called Asp2 and memapsin2). BACE2, a novel protease homologous to BACE1, was also identified, and together the two enzymes define a new family of transmembrane aspartic proteases. BACE1 exhibits all the properties of the β-secretase, and as the key rate-limiting enzyme that initiates the formation of Aβ, BACE1 is an attractive drug target for AD. Here, I review the identification and initial characterization of BACE1 and BACE2, and summarize our current understanding of BACE1 post-translational processing and intracellular trafficking. In addition, I discuss recent studies of BACE1 knockout mice and the BACE1 X-ray structure, and relate implications for BACE1 drug development.     

Cellular signaling roles of TGFβ, TNFα and βAPP in brain injury responses and Alzheimer's disease

β-Amyloid precursor protein (βAPP), transforming growth factor β (TGFβ), and tumor necrosis factor-α (TNFα) are remarkably pleiotropic neural cytokines/neurotrophic factors that orchestrate intricate injury-related cellular and molecular interactions. The links between these three factors include: their responses to injury; their interactive effects on astrocytes, microglia and neurons; their ability to induce cytoprotective responses in neurons; and their association with cytopathological alterations in Alzheimer's disease. Astrocytes and microglia each produce and respond to TGFβ and TNFα in characteristic ways when the brain is injured. TGFβ, TNFα and secreted forms of βAPP (sAPP) can protect neurons against excitotoxic, metabolic and oxidative insults and may thereby serve neuroprotective roles. On the other hand, under certain conditions TNFα and the fibrillogenic amyloid β-peptide (Aβ) derivative of βAPP can promote damage of neuronal and glial cells, and may play roles in neurodegenerative disorders. Studies of genetically manipulated mice in which TGFβ, TNFα or βAPP ligand or receptor levels are altered suggest important roles for each factor in cellular responses to brain injury and indicate that mediators of neural injury responses also have the potential to enhance amyloidogenesis and/or to interfere with neuroregeneration if expressed at abnormal levels or modified by strategic point mutations. Recent studies have elucidated signal transduction pathways of TGFβ (serine/threonine kinase cascades), TNFα (p55 receptor linked to a sphingomyelin-ceramide-NFκB pathway), and secreted forms of βAPP (sAPP; receptor guanylate cyclase-cGMP-cGMP-dependent kinase-K⁺ channel activation). Knowledge of these signaling pathways is revealing novel molecular targets on which to focus neuroprotective therapeutic strategies in disorders ranging from stroke to Alzheimer's disease.     

Vascular endothelial growth factor (VEGF) affects processing of amyloid precursor protein and β-amyloidogenesis in brain slice cultures derived from transgenic Tg2576 mouse brain

The up-regulation of the angiogenic vascular endothelial growth factor (VEGF) in brains of Alzheimer patients in close relationship to β-amyloid (Aβ) plaques, suggests a link of VEGF action and processing of the amyloid precursor protein (APP). To reveal whether VEGF may affect APP processing, brain slices derived from 17-month-old transgenic Tg2576 mice were exposed with 1 ng/ml VEGF for 6, 24, and 72 h, followed by assessing cytosolic and membrane-bound APP expression, level of both soluble and fibrillar Aβ-peptides, as well as activities of α- and β-secretases in brain slice tissue preparations.Treatment of brain slices with VEGF did not significantly affect the expression level of APP, regardless of the exposure time studied. In contrast, VEGF exposure of brain slices for 6 h reduced the formation of soluble, SDS extractable Aβ(1–40) and Aβ(1–42) as compared to brain slice cultures incubated in the absence of any drug, while the fibrillar Aβ peptides did not change significantly. This effect was less pronounced 24 h after VEGF exposure, but was no longer detectable when brain slices were exposed by VEGF for 72 h, which indicates an adaptive response to chronic VEGF exposure. The VEGF-mediated reduction in Aβ formation was accompanied by a transient decrease in β-secretase activity peaking 6 h after VEGF exposure. To reveal whether the VEGF-induced changes in soluble Aβ-level may be due to actions of VEGF on Aβ fibrillogenesis, the fibrillar status of Aβ was examined using the thioflavin-T binding assay. Incubation of Aβ preparations obtained from Tg2576 mouse brain cortex, in the presence of VEGF slightly decreased the fibrillar content with increasing incubation time up to 72 h. The data demonstrate that VEGF may affect APP processing, at least in vitro, suggesting a role of VEGF in the pathogenesis of Alzheimer's disease.     

The role of β- and α-adrenoceptors in the regulation of the stages of the sleep-waking cycle in the cat

The effects of β-adrenergic drugs alone and in combination with α-adrenergic drugs on the stages of the sleep-waking cycle were studied in adult cats. Polygraphic sleep recordings of 16 h showed that prenalterol (20 and 40 mg/kg i.p.), a β₁-adrenoceptor-stimulating drug increased paradoxical sleep (PS) in a dose-related manner during 4–12 h. Salbutamol (40 mg/kg), a β₂-adrenoceptor-stimulating drug, decreased PS during the first 4 h. Metoprolol (10 and 50 mg/kg), a relatively selective β₁-adrenoceptor blocking drug, increased drowsy waking during the first 4 h. The larger dose also tended to decrease PS. Already at the lower dose metoprolol partially antagonized the PS increase produced by prazosin, an α₁-adrenoceptor blocking drug. Propranolol (5 mg/kg), a β₁-andβ₂-adrenoceptor blocking drug, which alone decreases PS, antagonized the PS increase induced by phentolamine, an α₁-andα₂-adrenoceptor drug. Atenolol (5 mg/kg), a poorly lipid-soluble β-adrenoceptor blocking drug, failed to counteract phentolamine in increasing PS. Metoprolol (10 and 50 mg/kg) and propranolol (5 mg/kg) clearly potentiated the increase in drowsy waking and decrease in deep slow wave sleep and PS induced by clonidine (0.01 mg/kg), an α₂-adrenoceptor-stimulating drug.The results support the involvement of β-adrenoceptors in the regulation of the sleep-waking cycle. A high level of β-adrenergic activity may facilitate the production of PS. A low level of β-adrenergic activity, especially in combination with a high level of α₂-adrenergic activity, may facilitate the production of drowsy waking. Central α₁-andβ₁-adrenoceptors may mediate opposite functions in the regulation of PS.     

Retinoic Acid-Elicited RARα/RXRα Signaling Attenuates Aβ Production by Directly Inhibiting γ-Secretase-Mediated Cleavage of Amyloid Precursor Protein

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