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.     

Presenilin 1 mediates retinoic acid-induced differentiation of SH-SY5Y cells through facilitation of Wnt signaling

Presenilin 1 interacts with beta-catenin, an essential component of the Wnt signaling pathway. To elucidate the role of presenilin 1-beta-catenin interaction in neuronal differentiation, we established SH-SY5Y cells stably expressing wild-type presenilin 1, P117L mutant presenilin 1, which is linked to the early-onset familial form of Alzheimer's disease, and D385A mutant presenilin 1, which has no aspartyl proteinase activity. We demonstrate that SH-SY5Y cells stably expressing D385A mutant presenilin 1 failed to differentiate in response to retinoic acid treatment. Retinoic acid caused an increase in nuclear beta-catenin levels in SH-SY5Y cells, which was followed by an increase in cyclin D1 protein levels. Abnormal cellular accumulation of beta-catenin was observed in D385A mutant transfected cells, whereas nuclear beta-catenin and cellular cyclin D1 levels failed to increase. Conversely, SH-SY5Y cells expressing the P117L mutant differentiated normally and showed increased nuclear beta-catenin and cellular cyclin D1 levels. These findings suggest that neuronal differentiation of SH-SY5Y cells involves the Wnt signaling pathway and that presenilin 1 plays a crucial role in Wnt signal transduction by regulating the nuclear translocation of beta-catenin.     

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.     

Beta-adrenergic and muscarinic cholinergic receptors: overlap in their genetic determination

Significant positive correlation between the ligand binding values for beta-adrenergic receptors and those for muscarinic cholinergic receptors was found in five inbred strains of mice (r = 0.84) and in different vertebrate species (r = 0.99). Comparative analysis of analogous receptor studies done on various brain regions in other laboratories revealed high positive correlation between regional binding values for these two receptors. In conjunction with the work presented here, an overlap in the genetic determination of beta-adrenergic and muscarinic cholinergic receptors is suggested.     

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.     

Identity and function of gamma-secretase

gamma-Secretase catalyzes intramembrane proteolysis of various type I membrane proteins, including the amyloid-beta precursor protein and the Notch receptor. Despite its importance in the pathogenesis of Alzheimer's disease and to normal development, this protease has eluded identification until only very recently. Four membrane proteins are now known to be members of the protease complex: presenilin, nicastrin, aph-1, and pen-2. Recent findings suggest that these four proteins are sufficient to reconstitute the active gamma-secretase complex and that together they mediate the cell surface signaling of a variety of receptors via intramembrane proteolysis.     

Donepezil, but not galantamine, blocks muscarinic receptor-mediated in vitro and in vivo responses

We have found that galantamine, but not donepezil, reversed isolation rearing‐induced deficits of prepulse inhibition (PPI) via an activation of muscarinic M1 receptors. To explain this difference, the present study examined the effects of these acetylcholinesterase inhibitors on muscarinic receptor‐mediated responses in in vitro and in vivo systems. Ca²⁺‐imaging study showed that donepezil, but not galantamine, blocked a muscarinic agonist carbachol‐induced increase in intracellular Ca²⁺ levels in SH‐SY5Y cells. Moreover, a microdialysis study showed that intraperitoneal administration of donepezil, but not galantamine, attenuated a preferential M1 receptor agonist Ndesmethylclozapine‐induced increase in dopamine release in mouse cerebral cortex. These results suggest that donepezil, but not galantamine, has an ability to block muscarinic receptor function and imply that the differential effects may be responsible for the difference in the effects on isolation rearing‐induced deficits of PPI between these drugs. Synapse, 2011. © 2011 Wiley‐Liss, Inc.     

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.     

Proteasome-mediated degradation of the C-terminus of the Alzheimer's disease beta-amyloid protein precursor: effect of C-terminal truncation on production of beta-amyloid protein

The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. Our previous studies have shown that the proteasome can cleave the C-terminal cytoplasmic domain of APP. To identify proteasome cleavage sites in APP, two peptides homologous to the C-terminus of APP were incubated with recombinant 20S proteasome. Cleavage of the peptides was monitored by reversed phase high-performance liquid chromatography and mass spectrometry. Proteasome cleaved the APP C-terminal peptides at several sites, including a region around the sequence YENPTY that interacts with several APP-binding proteins. To examine the effect of this cleavage on Abeta production, APP-CTFbeta and mutant forms of APP-CTFbeta terminating on either side of the YENPTY sequence were expressed in CHO cells. Truncation of APP-CTFbeta on the N-terminal side of the YENPTY sequence at residue 677 significantly decreased the amount of Abeta produced, whereas truncation on the C-terminal side of residue 690 had little effect. The results suggest that proteasomal cleavage of the cytosolic domain of APP at the YENPTY sequence decreases gamma-secretase processing, and consequently inhibits Abeta production. Therefore, the proteasome-dependent trafficking pathway of APP may be a valid therapeutic target for altering Abeta production in the Alzheimer's disease brain.     

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.     

Transmembrane signaling through phospholipase C-beta in the developing human prefrontal cortex

To investigate changes in muscarinic receptor-stimulated phospholipase C-beta (PLC-beta) activity during brain development, we examined the functional coupling of each of the three major protein components of the phosphoinositide system (M1, M3, and M5 muscarinic receptor subtypes; Gq/11 proteins; PLC-beta1-4 isoforms) in membrane preparations from post-mortem human prefrontal cerebral cortex collected at several stages of prenatal and postnatal development. In human prenatal brain membranes, PLC was found to be present and could be activated by calcium, but the ability of guanosine-5'-o-3 thiotriphosphate (GTPgammaS) or carbachol (in the presence of GTPgammaS) to modulate prenatal PLC-beta was significantly weaker than that associated with postnatal PLC-beta. Western blot analysis revealed that the levels of Galphaq/11 did not change significantly during development. In contrast, dramatically higher levels of expression of PLC-beta1-4 isoforms and of M1, M3, and M5 muscarinic receptors were detected in the child vs. the fetal brain, a finding that might underlie the observed increased activity of PLC. Thus, inositol phosphate production may be more efficiently regulated by altering the amount of effectors (PLC-beta1-4) and receptors (M1,3,5 subtypes) than by altering the level of Galphaq/11 subunits. These results demonstrate that different PLC isoforms are expressed in the prefrontal cortex of the developing human brain in an age-specific manner, suggesting specific roles not only in synaptic transmission but also in the differentiation and maturation of neurons in the developing brain.     

Possible role of scavenger receptor SRCL in the clearance of amyloid-beta in Alzheimer's disease

Accumulation of beta-amyloid protein (Abeta) in the brain is a hallmark of Alzheimer's disease (AD), and Abeta-mediated pathogenesis could result from increased production of Abeta or insufficient Abeta clearance by microglia, astrocytes, or the vascular system. Cell-surface receptors, such as scavenger receptors, might play a critical role in the binding and clearing of Abeta; however, the responsible receptors have yet to be identified. We show that scavenger receptor with C-type lectin (SRCL), a member of the scavenger receptor family containing coiled-coil, collagen-like, and C-type lectin/carbohydrate recognition domains, is expressed in cultured astrocytes and microglia. In contrast to the low expression of SRCL in the wild-type mouse brain, in a double transgenic mouse model of AD (Tg-APP/PS1), immunohistochemistry showed that SRCL was markedly induced in Abeta-positive astrocytes and Abeta-positive vascular/perivascular cells, which are associated closely with cerebral amyloid angiopathy. In patients with AD, the distribution of SRCL was similar to that seen in the Tg-APP/PS1 temporal cortex. The presence of a large number of SRCL/Abeta double-positive particles in the intracellular compartments of reactive astrocytes and vascular/perivascular cells in Tg-APP/PS1 mice and AD patients suggests a role for SRCL in Abeta clearance. Moreover, CHO-K1 cells transfected with SRCL isoforms were found to bind fibrillar Abeta(1-42). These findings suggest that SRCL could be the receptor involved in the binding or clearing of Abeta by glial and vascular/perivascular cells in AD.     

Tobacco cembranoids block behavioral sensitization to nicotine and inhibit neuronal acetylcholine receptor function

Cembranoids are cyclic diterpenoids found in tobacco and in marine invertebrates. The present study established that tobacco cembranoids inhibit behavioral sensitization to nicotine in rats and block several types of nicotine acetylcholine receptors (AChRs). 1) At the behavioral level, rat locomotor activity induced by nicotine was significantly increased after seven daily nicotine injections. This sensitization to nicotine was blocked by mecamylamine (1 mg/kg) and by the cembranoids eunicin, eupalmerin acetate (EUAC), and (4R)-2,7,11-cembratriene-4-6-diol (4R), each at 6 mg/kg. None of these compounds modified locomotor activity of nonsensitized rats. 2) In cells expressing human AChRs, cembranoids blocked carbamoylcholine-induced (86)Rb(+) flux with IC(50) in the low micromolar range. The cell lines used were the SH-EP1-halpha4beta2 cell line heterologously expressing human alpha4beta2-AChR, the SH-SY5Y neuroblastoma line naturally expressing human ganglionic alpha3beta4-AChR, and the TE671/RD cell line naturally expressing embryonic muscle alpha1beta1gammadelta-AChR. The tobacco cembranoids tested were 4R and its diastereoisomer 4S, and marine cembranoids tested were EUAC and 12,13-bisepieupalmerin. 3) At the molecular level, tobacco (4R and 4S) and marine (EUAC) cembranoids blocked binding of the noncompetitive inhibitor [(3)H]tenocyclidine to AChR from Torpedo californica electric organ. IC(50) values were in the submicromolar to low-micromolar range, with 4R displaying an order of magnitude higher potency than its diastereoisomer, 4S.     

Altered emotional behaviors and the expression of 5-HT1A and M1 muscarinic receptors in micro-opioid receptor knockout mice

Anxiety and depression alterations have been reported in micro-opioid receptor knockout mice after exon 2 disruption. However, emotional behaviors, such as novelty and emergence responses have not been reported in micro-opioid receptor knockout mice due to the disruptions of exon 2 and 3. Here, we report that mu-opioid receptor knockout mice, with deletion of exon 2 and 3, display significant emotional behavior changes; they showed less anxiety in the elevated plus maze and emergence tests, reduced response to novel stimuli in the novelty test, and less depressive-like behavior in the forced-swim test. Analysis of the compensatory mechanism in mu-opioid receptor knockout mice revealed that the M1 mRNA levels were reduced in the cortex, caudate putamen, nucleus accumbens, and hippocampus, and that M1 receptor levels were reduced in the nucleus accumbens, CA1, and the dentate gyrus of the hippocampus, versus the wild-type. However, 5-HT1A receptor levels were significantly elevated in the cerebral cortex and in the hypothalamus of mu-opioid receptor knockout mice versus the wild-type. These aberrant emotional behavioral phenotypes are possibly related to M1 and 5-HT1A receptor alterations in the micro-opioid receptor knockout mice. Overall, our study suggests that micro-opioid receptor may play a role in the modification of emotional responses to novelty, anxiety, and depression.     

Direct autoradiographic determination of M1 and M2 muscarinic acetylcholine receptor distribution in the rat brain: Relation to cholinergic nuclei and projections

The autoradiographic distributions of receptors with high affinity for [3H]oxotremorine-M (the M2 receptor) and [3H]pirenzepine (the M1 receptor) were studied in the rat brain. M1 receptors were seen in highest density only in telencephalic structures: cerebral cortex (layers I-II), hippocampus, dentate gyrus, medial and basolateral amygdala, nucleus accumbens and caudate/putamen. M2 receptors were detected throughout the brain, with highest levels observed in cerebral cortical layers III and V, forebrain cholinergic nuclei, caudate/putamen, various thalamic areas, inferior and superior colliculus, interpeduncular and pontine nuclei, brainstem cholinergic nuclei and cervical spinal cord regions. M2 receptors were found to be good markers for cholinergic cell groups and the majority of cholinergic projection areas, whereas M1 receptors were only found in a large sub-group of telencephalic cholinergic projection areas, and the pattern of distribution of receptors in these areas differed from that of M2 receptors. Scatchard analysis of [3H]oxotremorine-M binding to inferior collicular slices revealed one site with a dissociation constant (Kd) of 1.9 nM and a receptor density (Bmax) of 1.4 pmol/mg protein. Our data support the hypothesis that M1 and M2 receptors are physically distinct sub-types of the muscarinic acetylcholine receptor.     

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.     

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.     

Cerebrolysin decreases amyloid-beta production by regulating amyloid protein precursor maturation in a transgenic model of Alzheimer's disease

Cerebrolysin is a peptide mixture with neurotrophic effects that might reduce the neurodegenerative pathology in Alzheimer's disease (AD). We have previously shown in an amyloid protein precursor (APP) transgenic (tg) mouse model of AD-like neuropathology that Cerebrolysin ameliorates behavioral deficits, is neuroprotective, and decreases amyloid burden; however, the mechanisms involved are not completely clear. Cerebrolysin might reduce amyloid deposition by regulating amyloid-beta (Abeta) degradation or by modulating APP expression, maturation, or processing. To investigate these possibilities, APP tg mice were treated for 6 months with Cerebrolysin and analyzed in the water maze, followed by RNA, immunoblot, and confocal microscopy analysis of full-length (FL) APP and its fragments, beta-secretase (BACE1), and Abeta-degrading enzymes [neprilysin (Nep) and insulin-degrading enzyme (IDE)]. Consistent with previous studies, Cerebrolysin ameliorated the performance deficits in the spatial learning portion of the water maze and reduced the synaptic pathology and amyloid burden in the brains of APP tg mice. These effects were associated with reduced levels of FL APP and APP C-terminal fragments, but levels of BACE1, Notch1, Nep, and IDE were unchanged. In contrast, levels of active cyclin-dependent kinase-5 (CDK5) and glycogen synthase kinase-3beta [GSK-3beta; but not stress-activated protein kinase-1 (SAPK1)], kinases that phosphorylate APP, were reduced. Furthermore, Cerebrolysin reduced the levels of phosphorylated APP and the accumulation of APP in the neuritic processes. Taken together, these results suggest that Cerebrolysin might reduce AD-like pathology in the APP tg mice by regulating APP maturation and transport to sites where Abeta protein is generated. This study clarifies the mechanisms through which Cerebrolysin might reduce Abeta production and deposition in AD and further supports the importance of this compound in the potential treatment of early AD.