Presenilin 1 is involved in the maturation of beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1)

One of the pathologic hallmarks of Alzheimer's disease is the excessive deposition of beta-amyloid peptides (Abeta) in senile plaques. Abeta is generated when beta-amyloid precursor protein (APP) is cleaved sequentially by beta-secretase, identified as beta-site APP-cleaving enzyme 1 (BACE1), and gamma-secretase, a putative enzymatic complex containing presenilin 1 (PS1). However, functional interaction between PS1 and BACE1 has never been known. In addition to this classical role in the generation of Abeta peptides, it has also been proposed that PS1 affects the intracellular trafficking and maturation of selected membrane proteins. We show that the levels of exogenous and endogenous mature BACE1 expressed in presenilin-deficient mouse embryonic fibroblasts (PS-/-MEFs) were reduced significantly compared to those in wild-type MEFs. Moreover, the levels of mature BACE1 were increased in human neuroblastoma cell line, SH-SY5Y, stably expressing wild-type PS1, compared to native cells. Conversely, the maturation of BACE1 was compromised under the stable expression of dominant-negative mutant PS1 overexpression. Immunoprecipitation assay showed that PS1 preferably interacts with proBACE1 rather than mature BACE1, indicating that PS1 can be directly involved in the maturation process of BACE1. Further, endogenous PS1 was immunoprecipitated with endogenous BACE1 in SH-SY5Y cells and mouse brain tissue. We conclude that PS1 is directly involved in the maturation of BACE1, thus possibly functioning as a regulator of both beta- and gamma-secretase in Abeta generation.     

Beta-amyloid peptide binding protein does not couple to G protein in a heterologous Xenopus expression system

Alzheimer's disease is a neurodegenerative disorder related to the formation of protein aggregates. beta-Amyloid protein (A beta), generated by enzymatic cleavage of amyloid precursor protein (APP), can cause such aggregation, and these aggregates may cause neuronal cell death by inducing apoptosis. However, A beta-induced intracellular signaling pathways involved in the neuronal death are not well understood. Recently it was shown that A beta aggregates induce neuronal cell death via beta-amyloid peptide-binding protein (BBP), a receptor for A beta in BBP-transfected cells, which is known to be sensitive to pertussis toxin, a G alpha(i/o) family inhibitor. However, the actual coupling of BBP to the pertussis-sensitive G protein was not demonstrated. In this study, we performed electrophysiological recordings using the two-electrode voltage-clamp technique to test whether human or Drosophila BBPs, singly or in combination with APP, are coupled to a specific type of G protein. Our results suggest that BBP is not directly coupled to G alpha(i/o), G alpha(s), or G alpha(q) proteins and that BBP may need a component other than APP to exert its toxic effect in concert with A beta.     

BACE1 interacts with lipid raft proteins

A neuropathological hallmark of Alzheimer's disease is the presence of amyloid plaques in the brain. Amyloid-beta peptide (Abeta) is the major constituent of the plaques and is generated by proteolytic cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. Growing evidence shows that lipid rafts are critically involved in regulating the Abeta generation. In support of this, APP, Abeta, and presenilins have been found in lipid rafts. Although cholesterol plays a crucial role in maintaining lipid rafts, functions of other components in the generation of Abeta are unknown. Caveolins (CAVs) and flotillins (FLOTs) are principal proteins related to lipid rafts and have been suggested to be involved in APP processing. Here, we report that FLOT-1 binds to BACE1 (beta-site APP cleaving enzyme 1) and that overexpression of CAV-1 or FLOT-1 results in recruiting BACE1 into lipid rafts and influence on beta-secretase activity in cultured cells. Our results show that both CAV-1 and FLOT-1 may modulate beta-secretase activity by interacting with BACE1.     

The two‐hydrophobic domain tertiary structure of reticulon proteins is critical for modulation of β‐secretase BACE1

β‐Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is a membrane‐bound protease that is essential for the production of β‐amyloid protein (Aβ). Given the crucial role of Aβ accumulation in Alzheimer's disease (AD), inhibition of BACE1 activity may represent a feasible therapeutic strategy in the treatment of AD. Recently, we and others identified reticulon 3 (RTN3) and reticulon 4‐B/C (RTN4‐B/C or Nogo‐B/C) as membrane proteins that interact with BACE1 and inhibit its ability to produce Aβ. In this study, we employed various mutants of RTN3 and RTN4‐C and C. elegans RTN to investigate the molecular mechanisms by which RTNs regulate BACE1. We found that RTN3 mutants lacking the N‐terminal or C‐terminal or loop domain as well as a RTN4‐C mutant lacking the C‐terminal domain bound to BACE1 comparably to wild‐type RTN3 and RTN4‐C. Furthermore, overexpression of wild‐type RTN3, RTN4‐C, and these RTN mutants similarly reduced Aβ40 and Aβ42 secretion by cells expressing Swedish mutant APP. C. elegans RTN, which has low homology to human RTNs, also interacted with BACE1 and inhibited Aβ secretion. In contrast, two RTN3 mutants containing deletions of the first or second potential transmembrane domains and an RTN3 swap mutant of the second transmembrane domain bound BACE1 but failed to inhibit Aβ secretion. Collectively, these results suggest that the two‐transmembrane‐domain tertiary structure of RTN proteins is critical for the ability of RTNs to modulate BACE1 activity, whereas N‐terminal, C‐terminal and loop regions are not essential for this function. © 2009 Wiley‐Liss, Inc.     

Beta-secretase BACE1 is differentially controlled through muscarinic acetylcholine receptor signaling

The beta-amyloid peptides derived by proteolytic cleavage from the amyloid precursor protein (APP) play a major role in the pathogenesis of Alzheimer's disease (AD) by forming aggregated, fibrillary complexes that have been shown to be neurotoxic. The beta-site APP-cleaving enzyme (BACE1) has been identified as the key enzyme leading to beta-amyloid formation, and cholinergic mechanisms have been shown to control APP processing. The present study sought to determine whether BACE1 expression is controlled by muscarinic acetylcholine receptor (mAChR) subtypes in the neuroblastoma cell line SK-SH-SY5Y. Stimulation of cells with the M1/M3-selective mAChR agonist talsaclidine for 1 hr resulted in a dose-dependent increase in BACE1 expression up to twofold over basal levels. Similar effects of BACE1 up-regulation were observed when protein kinase C was directly activated by phorbol esters. However, when the MAP kinases MEK/ERK were inhibited, BACE1 expression was no longer up-regulated by the activation of M1-mAChR. In contrast, BACE1 expression was suppressed by stimulation of M2-mediated pathways via selective M2-agonist binding or direct activation of adenylate cyclase with forskolin, an effect that was prevented by inhibiting protein kinase A. These results may explain the observed deterioration of AD patients after initial improvements with AChE inhibitor or M1-mAChR agonist treatment.     

Cytoplasmic domain of the beta-amyloid protein precursor of Alzheimer's disease: function, regulation of proteolysis, and implications for drug development

The beta-amyloid protein precursor (APP) has been extensively studied for its role in amyloid production and the pathogenesis of Alzheimer's disease (AD). However, little is known about the normal function of APP and its biological interactions. In this Mini-Review, the role of the cytoplasmic domain of APP in APP trafficking and proteolysis is described. These studies suggest that proteins that bind to the cytoplasmic domain may be important targets for drug development in AD.     

Differential binding and activation of caspase-3 in cultured hippocampal neurons by assembly forms of A beta 1-42

Amyloid-beta (A beta) peptides, the primary constituents of amyloid plaques in the brain in Alzheimer's disease (AD), may cause AD, but how they do so is not clear. A beta peptides spontaneously aggregate, or self-assemble, to generate several distinct macromolecular and morphological forms that can differ significantly in their effects on cells. We have compared different assembly forms of A beta(1-42) (A beta 42) for their ability to trigger apoptosis in cultured hippocampal neurons at a submicromolar concentration and for their binding to such neurons. Fibrillar A beta 42 caused both morphological changes indicative of apoptosis and specific activation of caspase-3, a characteristic marker of neurodegeneration in AD, in hippocampal neurons, whereas other preparations tested did not do so under the same conditions. More aggregated forms of A beta 42, including both fibrils and a mixture of assembly forms termed A beta-derived diffusible ligands (ADDLs), bound to neurons much more extensively and at lower concentrations than preparations that contained smaller forms. Fibrillar A beta 42, in particular, bound to neurons at concentrations as low as 1 nM. Colocalization studies showed that fibrillar A beta 42 bound almost exclusively at nonsynaptic sites. These results show differences between assembly forms of A beta 42 in the ability to trigger apoptotic signaling in CNS neurons, and they directly demonstrate differences between assembly forms in the binding to CNS neurons, a possible first step in the pathogenesis of AD. These results suggest that fibrillar A beta 42 contributes to the pathogenesis of AD.     

Binding and uptake of Aβ1‐42 by primary human astrocytes in vitro

Clearance of the amyloid‐β peptide (Aβ) as a remedy for Alzheimer's disease (AD) is a major target in on‐going clinical trials. In vitro studies confirmed that Aβ is taken up by rodent astrocytes, but knowledge on human astrocyte‐mediated Aβ clearance is sparse. Therefore, by means of flow cytometry and confocal laser scanning microscopy (CLSM), we evaluated the binding and internalization of Aβ1‐42 by primary human fetal astrocytes and adult astrocytes, isolated from nondemented subjects (n = 8) and AD subjects (n = 6). Furthermore, we analyzed whether α1‐antichymotrypsin (ACT), which is found in amyloid plaques and can influence Aβ fibrillogenesis, affects the Aβ uptake by human astrocytes. Upon over night exposure of astrocytes to FAM‐labeled Aβ1‐42 (10 μM) preparations, (80.7 ± 17.7)% fetal and (52.9 ± 20.9)% adult Aβ‐positive astrocytes (P = 0.018) were observed. No significant difference was found in Aβ1‐42 uptake between AD and non‐AD astrocytes, and no influence of ApoE genotype on Aβ1‐42 uptake was observed in any group. There was no difference in the percentage of Aβ‐positive cells upon exposure to Aβ1‐42 (10 μM) combined with ACT (1,000:1, 100:1, and 10:1 molar ratio), versus Aβ1‐42 alone. CLSM revealed binding of Aβ1‐42 to the cellular surfaces and cellular internalization of smaller Aβ1‐42 fragments. Under these conditions, there was no increase in cellular release of the proinflammatory chemokine monocyte‐chemoattractant protein 1, as compared with nontreated control astrocytes. Thus, primary human astrocytes derived from different sources can bind and internalize Aβ1‐42, and fetal astrocytes were more efficient in Aβ1‐42 uptake than adult astrocytes. © 2008 Wiley‐Liss, Inc.     

Cysteine proteases are the major beta-secretase in the regulated secretory pathway that provides most of the beta-amyloid in Alzheimer's disease: role of BACE 1 in the constitutive secretory pathway

This article focuses on beta-amyloid (Abeta) peptide production and secretion in the regulated secretory pathway and how this process relates to accumulation of toxic Abeta in Alzheimer's disease. New findings are presented demonstrating that most of the Abeta is produced and secreted, in an activity-dependent manner, through the regulated secretory pathway in neurons. Only a minor portion of cellular Abeta is secreted via the basal, constitutive secretory pathway. Therefore, regulated secretory vesicles contain the primary beta-secretases that are responsible for producing the majority of secreted Abeta. Investigation of beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells demonstrated that cysteine proteases account for the majority of the beta-secretase activity. BACE 1 is present in regulated secretory vesicles but provides only a small percentage of the beta-secretase activity. Moreover, the cysteine protease activities prefer to cleave the wild-type beta-secretase site, which is relevant to the majority of AD cases. In contrast, BACE 1 prefers to cleave the Swedish mutant beta-secretase site that is expressed in a minor percentage of the AD population. These new findings lead to a unifying hypothesis in which cysteine proteases are the major beta-secretases for the production of Abeta in the major regulated secretory pathway and BACE 1 is the beta-secretase responsible for Abeta production in the minor constitutive secretory pathway. These results indicate that inhibition of multiple proteases may be needed to decrease Abeta production as a therapeutic strategy for Alzheimer's disease.     

Astrocytic expression of the Alzheimer's disease beta-secretase (BACE1) is stimulus-dependent

The beta-site APP-cleaving enzyme (BACE1) is a prerequisite for the generation of beta-amyloid peptides, which give rise to cerebrovascular and parenchymal beta-amyloid deposits in the brain of Alzheimer's disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to beta-amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N-M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by beta-amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1-immunoreactive astrocytes in proximity to beta-amyloid plaques in the brains of aged Tg2576 mice and Alzheimer's disease patients.     

Functional reconstitution of gamma-secretase through coordinated expression of presenilin, nicastrin, Aph-1, and Pen-2

The gamma-secretase complex has emerged as an unusual membrane-bound aspartyl protease with the ability to cleave certain substrate proteins at peptide bonds believed to be buried within the hydrophobic environment of the lipid bilayer. This cleavage is responsible for a key biochemical step in signaling from several different cell-surface receptors, and it is also crucial in generating the neurotoxic amyloid peptides that are central to the pathogenesis of Alzheimer's disease. Active gamma-secretase is a multimeric protein complex consisting of at least four different proteins, presenilin, nicastrin, Aph-1, and Pen-2, with presenilin serving as the catalytically active core of the aspartyl protease. Presenilin itself undergoes endoproteolytic maturation, a process that is tightly regulated during the assembly and maturation of gamma-secretase, and that depends on the three cofactors nicastrin, Aph-1, and Pen-2. Recent studies have demonstrated that presenilin and its three cofactors are likely to be the major proteins needed for functional reconstitution of active gamma-secretase and have begun to elucidate the specific functions of the cofactors in the ordered assembly of gamma-secretase.     

Hippocampus of Ames dwarf mice is resistant to beta-amyloid-induced tau hyperphosphorylation and changes in apoptosis-regulatory protein levels

The Ames dwarf mouse has a long lifespan and is characterized by a marked resistance to cellular stress, an event that is implicated in the pathogenesis of many neurodegenerative disorders that are associated with aging, including Alzheimer's disease. However, very little is known on the extent to which the Ames dwarf mouse is protected against Alzheimer's disease. We have developed an organotypic slice system cultured from hippocampi of adult dwarf mice and examined deleterious effects of beta-amyloid (Abeta) peptide, a key pathogenic event in the course of Alzheimer's disease. We present the first evidence that long living Ames mice resist beta-amyloid toxicity. We demonstrate that organotypic slices from adult dwarf mice, but not their normal phenotype counterparts (wild type), are resistant to Abeta25-35-induced hyperphosphorylation of tau protein, reduction in levels of the antiapoptotic protein Bcl-2, increase in levels of the pro-apoptotic protein Bax, and activation of caspase 3. Moreover, incubation of organotypic sections with the GSK-3beta inhibitor SB216763 prevented tau phosphorylation but not alterations in levels of Bcl-2, Bax, and caspase-3. Because the hippocampus is a brain area that is severely affected in Alzheimer's disease, our study proposes that organotypic slices from hippocampi of adult Ames dwarf mice may constitute a model system for understanding endogenous factors that may confer protection against Abeta.     

Lactacystin decreases amyloid-beta peptide production by inhibiting beta-secretase activity

The human amyloid precursor protein (APP) is processed by the nonamyloidogenic and the amyloidogenic catabolic pathways. The sequential cleavage of APP by the beta- and gamma-secretase activities, known as the amyloidogenic processing of APP, leads to the formation of the amyloid-beta peptide (Abeta). Abeta is the main constituent of the amyloid core of senile plaques, a typical hallmark of Alzheimer's disease. In addition to secretases, other cellular proteolytic activities, like the proteasome, might participate in the metabolism of APP. We investigated the consequence of proteasome inhibition on the amyloidogenic processing of human APP. CHO cells and primary cultures of rat cortical neurons expressing human APP or a protein corresponding to its beta-cleaved C-terminal fragment (C99) were treated with lactacystin, an irreversible inhibitor of the chymotrypsin-like activity of the proteasome. Lactacystin significantly decreased the level of Abeta produced from APP in both cellular models, whereas the production of Abeta from C99 was not affected. Lactacystin did not inhibit gamma-secretase activity but was found to inhibit the beta-cleavage of APP, leading to a proportional decrease in Abeta production. Although lactacystin did not inhibit the catalytic activity of recombinant BACE1, a decrease in neuronal beta-secretase activity was measured after treatment with lactacystin.     

Intracellular sAPP retention in response to Aβ is mapped to cytoskeleton‐associated structures

Amyloid β (Aβ) contributes to neurodegeneration in Alzheimer's disease and provides a close association between molecular events and pathology, although the underlying molecular mechanisms are unclear. In the work described here, Aβ did not induce amyloid precursor protein (APP) expression, but APP processing/trafficking was markedly affected. In COS‐7 cells, Aβ provokes retention of intracellular sAPPα (isAPPα). Intracellular holo‐APP levels remain unchanged, and extracellular total sAPP increases, although extracellular sAPPα alone was not altered significantly. In primary neuronal cultures and PC12 cells, isAPP also increased, but this was mirrored by a decrease in extracellular total sAPP. The isAPP retention was particularly associated with the cytoskeletal fraction. The retention “per se” occurred in vesicular‐like densities, negative for a C‐terminal antibody and strongly positive for the 6E10 antibody, clearly showing abnormal intracellular accumulation of sAPPα in response to Aβ. Our data support a dynamic model for intracellular retention of sAPPα as an early response to Aβ exposure. Particularly noteworthy was the observation that removal of Aβ reversed the isAPP accumulation. Mechanistically, these findings disclose an attractive physiological response, revealing the capacity of cells to deal with adverse effects induced by Aβ. © 2008 Wiley‐Liss, Inc.     

Overexpression of amyloid precursor protein is associated with degeneration, decreased viability, and increased damage caused by neurotoxins (prostaglandins A1 and E2, hydrogen peroxide, and nitric oxide) in differentiated neuroblastoma cells

Inflammatory reactions are considered one of the important etiologic factors in the pathogenesis of Alzheimer's disease (AD). Prostaglandins such as PGE2 and PGA1 and free radicals are some of the agents released during inflammatory reactions, and they are neurotoxic. The mechanisms of their action are not well understood. Increased levels of beta-amyloid fragments (Abeta40 and Abeta42), generated through cleavage of amyloid precursor protein (APP), oxidative stress, and proteasome inhibition, are also associated with neurodegeneration in AD brains. Therefore, we investigated the effect of PGs and oxidative stress on the degeneration and viability of cyclic AMP-induced differentiated NB cells overexpressing wild-type APP (NBP2-PN46) under the control of the CMV promotor in comparison with differentiated vector (NBP2-PN1) or parent (NBP2) control cells. Results showed that differentiated NBP2-PN46 cells exhibited enhanced spontaneous degeneration and decreased viability in comparison with differentiated control cells, without changing the level of Abeta40 and Abeta42. PGA1 or PGE2 treatment of differentiated cells caused increased degeneration and reduced viability in all three cell lines. These effects of PGs are not due to alterations in the levels of vector-derived APP mRNA or human APP holoprotein, secreted levels of Abeta40 and Abeta42, or proteasome activity. H2O2 or SIN-1 (an NO donor) treatment did not change vector-derived APP mRNA levels, but H2O2 reduced the level of human APP protein more than SIN-1. Furthermore, SIN-1 increased the secreted level of Abeta40, but not of Abeta42, whereas H2O2 had no effect on the level of secreted Abeta fragments. Both H2O2 and SIN-1 inhibited proteasome activity in the intact cells. The failure of neurotoxins to alter APP mRNA levels could be due to the fact that they do not affect CMV promoter activity. These results suggest that the mechanisms of action of PGs on neurodegeneration are different from those of H2O2 and SIN-1 and that the mechanisms of neurotoxicity of H2O2 and SIN-1 are, at least in part, different from each other.     

Huperzine A alleviates synaptic deficits and modulates amyloidogenic and nonamyloidogenic pathways in APPswe/PS1dE9 transgenic mice

Huperzine A (HupA) is a potent acetylcholinesterase inhibitor (AChEI) used in the treatment of Alzheimer's disease (AD). Recently, HupA was shown to be active in modulating the nonamyloidogenic metabolism of β-amyloid precursor protein (APP) in APP-transfected human embryonic kidney cell line (HEK293swe). However, in vivo research concerning the mechanism of HupA in APP transgenic mice has not yet been fully elucidated. The present study indicates that the loss of dendritic spine density and synaptotagmin levels in the brain of APPswe/presenilin-1 (PS1) transgenic mice was significantly ameliorated by chronic HupA treatment and provides evidence that this neuroprotection was associated with reduced amyloid plaque burden and oligomeric β-amyloid (Aβ) levels in the cortex and hippocampus of APPswe/PS1dE9 transgenic mice. Our findings further demonstrate that the amelioration effect of HupA on Aβ deposits may be mediated, at least in part, by regulation of the compromised expression of a disintegrin and metalloprotease 10 (ADAM10) and excessive membrane trafficking of β-site APP cleavage enzyme 1 (BACE1) in these transgenic mice. In addition, extracellular signal-regulated kinases 1/2 (Erk1/2) phosphorylation may also be partially involved in the effect of HupA on APP processing. In conclusion, our work for the first time demonstrates the neuroprotective effect of HupA on synaptic deficits in APPswe/PS1dE9 transgenic mice and further clarifies the potential pharmacological targets for this protective effect, in which modulation of nonamyloidogenic and amyloidogenic APP processing pathways may be both involved. These findings may provide adequate evidence for the clinical and experimental benefits gained from HupA treatment.     

Tyrosine phosphatase STEP61 negatively regulates amyloid β‐mediated ERK/CREB signaling pathways via α7 nicotinic acetylcholine receptors

Striatal‐enriched phosphatase 61 (STEP₆₁) plays an essential role in synaptic plasticity and has recently been implicated in neurodegenerative disease. Here we characterized a possible role of STEP₆₁ in Alzheimer's disease (AD) pathology using a mouse model of AD (Tg‐APPswe/PSEN1dE9, APP/PS1 mice) and an in vitro model of AD [cortical neurons treated with amyloid β (Aβ)_1–42 peptides]. Our data indicate age‐related elevation of STEP₆₁ levels and the proportion of dephosphorylated STEP₆₁ (active STEP₆₁) in wild‐type mice, which was enhanced in APP/PS1 mice. Furthermore, the increased STEP₆₁ levels and active STEP₆₁ were observed in the hippocampus and cortex from 12‐month‐old APP/PS1 mice and in Aβ_1–42‐treated cortical neurons. An α7 nicotinic acetylcholine receptors (nAChRs) antagonist, α‐bungarotoxin (BTX), inhibited the Aβ_1–42‐induced increase of STEP₆₁ expression and activation. In addition, extracellular signal‐regulated kinase 1/2 (ERK1/2) and cAMP response element binding (CREB) were impaired in Aβ_1–42‐treated cortical neurons, and knockdown of STEP₆₁ enhanced the activation of ERK1/2 and CREB. Collectively, these findings indicate two alternate pathological pathways effecting STEP₆₁ regulation in AD. First, Aβ regulating STEP₆₁ activity is mediated by Aβ binding to α7 nAChRs. Second, STEP₆₁ negatively regulates Aβ‐mediated ERK/CREB pathway, an important signaling cascade involved in memory formation. © 2013 Wiley Periodicals, Inc.