Is gamma-secretase a multienzyme complex for membrane protein degradation? Models and speculations

The beta- and gamma-secretases cleave the amyloid protein precursor (APP) to release the amyloid protein (Abeta). While the beta-secretase has now been identified, the gamma-secretase remains an enigma. A number of mutations in the presenilins (PS) and APP have been shown to alter the cleavage specificity of gamma-secretase. However, the relationship between PS and gamma-secretase remains unclear. This article presents some models of gamma-secretase and suggests that the simplest interpretation of current data is that gamma-secretase is a complex of several proteases located in the lumen of secretory vesicles.     

BACE1 suppression by RNA interference in primary cortical neurons

Extracellular deposition of amyloid-beta (Abeta) aggregates in the brain represents one of the histopathological hallmarks of Alzheimer's disease (AD). Abeta peptides are generated from proteolysis of the amyloid precursor proteins (APPs) by beta- and gamma-secretases. Beta-secretase (BACE1) is a type I integral membrane glycoprotein that can cleave APP first to generate C-terminal 99- or 89-amino acid membrane-bound fragments containing the N terminus of Abeta peptides (betaCTF). As BACE1 cleavage is an essential step for Abeta generation, it is proposed as a key therapeutic target for treating AD. In this study, we show that small interfering RNA (siRNA) specifically targeted to BACE1 can suppress BACE1 (but not BACE2) protein expression in different cell systems. Furthermore, BACE1 siRNA reduced APP betaCTF and Abeta production in primary cortical neurons derived from both wild-type and transgenic mice harboring the Swedish APP mutant. The subcellular distribution of APP and presenilin-1 did not appear to differ in BACE1 suppressed cells. Importantly, pretreating neurons with BACE1 siRNA reduced the neurotoxicity induced by H2O2 oxidative stress. Our results indicate that BACE1 siRNA specifically impacts on beta-cleavage of APP and may be a potential therapeutic approach for treating AD.     

Effects of peptides derived from BACE1 catalytic domain on APP processing

One of the hallmarks of Alzheimer's disease (AD) is the deposition of beta-amyloid (Abeta) peptides in neuritic plaques. Abeta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. beta-APP cleaving enzyme-1 (BACE1) has been shown to be the major beta-secretase and is a primary therapeutic target for AD. We report here novel BACE1 inhibitory peptidomimetics, which are derived from catalytic domains of BACE1 themselves, instead of APP cleavage sites and are structurally modified by myristoylation in N-terminus for efficient cell permeability. The peptides not only inhibited the formation of APPbeta (a soluble N-terminal fragment of APP cleaved by beta-secretase), but also significantly reduced Abeta40 production. Our results suggest a new approach for identifying inhibitory agents for the treatment of AD.     

Visualizing interaction of proteins relevant to Alzheimer's disease in intact cells

To understand normal function of memory studying models of pathological memory decline is essential. The most common form of dementia leading to memory decline is Alzheimer's disease (AD), which is characterized by the presence of neurofibrillary tangles and amyloid plaques in the affected brain regions. Altered production of amyloid beta (Abeta) through sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases seems to be a central event in the molecular pathogenesis of the disease. Thus, the study of the complex interplay of proteins that are involved in or modify Abeta production is very important to gain insight into the pathogenesis of AD. Here, we describe the use of Fluorescence lifetime imaging microscopy (FLIM), a Fluorescence resonance energy transfer (FRET)-based method, to visualize protein-protein-interaction in intact cells, which has proven to be a valuable method in AD research.     

Biochemical and kinetic characterization of BACE1: investigation into the putative species-specificity for beta- and beta'-cleavage sites by human and murine BACE1

Beta-amyloid peptides (Abeta) are produced by a sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The lack of Abeta production in beta-APP cleaving enzyme (BACE1)(-/-) mice suggests that BACE1 is the principal beta-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1(-/-) mice supports cleavage of APP at the canonical beta-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as beta', resulting in Abeta peptides starting at Glu11. The apparent inability of human BACE1 to make this beta'-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the beta'-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the beta- and beta'-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the beta- and beta'-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated beta'-cleavage site. It does not appear to be a significant species specificity to this cleavage.     

PAR-4 is involved in regulation of beta-secretase cleavage of the Alzheimer amyloid precursor protein

Mounting evidence indicates that aberrant production and aggregation of amyloid beta-peptide (Abeta)-(1-42) play a central role in the pathogenesis of Alzheimer disease (AD). Abeta is produced when amyloid precursor protein (APP) is cleaved by beta- and gamma-secretases at the N and C termini of the Abeta domain, respectively. The beta-secretase is membrane-bound aspartyl protease, most commonly known as BACE1. Because BACE1 cleaves APP at the N terminus of the Abeta domain, it catalyzes the first step in Abeta generation. PAR-4 (prostate apoptosis response-4) is a leucine zipper protein that was initially identified to be associated with neuronal degeneration and aberrant Abeta production in models of AD. We now report that the C-terminal domain of PAR-4 is necessary for forming a complex with the cytosolic tail of BACE1 in co-immunoprecipitation assays and in vitro pull-down experiments. Overexpression of PAR-4 significantly increased, whereas silencing of PAR-4 expression by RNA interference significantly decreased, beta-secretase cleavage of APP. These results suggest that PAR-4 may be directly involved in regulating the APP cleavage activity of BACE1. Because the increased BACE1 activity observed in AD patients does not seem to arise from genetic mutations or polymorphisms in BACE1, the identification of PAR-4 as an endogenous regulator of BACE1 activity may have significant implications for developing novel therapeutic strategies for AD.     

Imipramine and citalopram facilitate amyloid precursor protein secretion in vitro

Comorbid depression of Alzheimer's disease (AD) is a common mood disorder in the elderly and a broad spectrum of antidepressants have been used for its treatment. Abeta peptides and other derivatives of the amyloid precursor protein (APP) have been implicated as central to the pathogenesis of AD. However, the functional relationship of APP and its proteolytic derivatives to antidepressant therapy is not known. In this study, Western blotting was used to test the ability of the tricyclic antidepressant (TCA) imipramine or the selective serotonin reuptake inhibitor (SSRI) citalopram to change the release of APP and the protein kinase C (PKC) content. Both antidepressants increased APP secretion in primary rat neuronal cultures. Imipramine or citalopram enhanced the level of secreted APP by 3.2- or 3.4-fold, respectively. Increases in PKC level were observed only after imipramine treatment. These in vitro data suggest that both TCA and SSRI are able to interfere with the APP metabolism. Imipramine promotes the non-amyloidogenic route of APP processing via stimulatory effects on PKC. We propose that PKC is not involved in the mechanism underlying the effects of citalopram on the APP metabolism. Since the secreted APP is not further available for the pathological cleavage of beta- and gamma-secretases, antidepressant medication might be beneficial in AD therapy.     

GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Abeta42

Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (Abeta) peptides with varying lengths. Particularly Abeta42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Abeta42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Abeta species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Abeta42, leave the level of Abeta40 unaffected, but increase Abeta38 and shorter Abeta species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that gamma-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Abeta42 production.     

Suppression of the caspase cleavage of beta-amyloid precursor protein by its cytoplasmic phosphorylation

beta-Amyloid precursor protein (APP) is a type I transmembrane protein. Its cleavages by beta- and gamma-secretases yield beta-amyloid, which is the main constituent of senile plaques in Alzheimer's disease (AD). In apoptotic cells and AD brains, APP is alternatively cleaved by caspases in the cytoplasmic region after the Asp664 residue (with respect to the numbering conversion for the APP695 isoform). Caspase-cleaved fragments of APP are cytotoxic and have been implicated in AD pathogenesis; however, the mechanisms regulating the cleavage have not been studied. APP is constitutively phosphorylated at Thr668 in brain. In the present study, we demonstrate that APP phosphorylated at Thr668 is less vulnerable to cytoplasmic cleavage by caspase-3 and caspase-8. This suggests that APP phosphorylation suppresses the generation of caspase-cleaved fragments of APP in the brain and that perturbation of this phosphorylation may be involved in APP-mediated neurotoxicity.     

beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide

Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.     

Proteomic identification of specifically carbonylated brain proteins in APP(NLh)/APP(NLh) × PS-1(P264L)/PS-1(P264L) human double mutant knock-in mice model of Alzheimer disease as a function of age

Alzheimer disease (AD) is the most common type of dementia and is characterized pathologically by the presence of neurofibrillary tangles (NFTs), senile plaques (SPs), and loss of synapses. The main component of SP is amyloid-beta peptide (Aβ), a 39 to 43 amino acid peptide, generated by the proteolytic cleavage of amyloid precursor protein (APP) by the action of beta- and gamma-secretases. The presenilins (PS) are components of the γ-secretase, which contains the protease active center. Mutations in PS enhance the production of the Aβ42 peptide. To date, more than 160 mutations in PS1 have been identified. Many PS mutations increase the production of the β-secretase-mediated C-terminal (CT) 99 amino acid-long fragment (CT99), which is subsequently cleaved by γ-secretase to yield Aβ peptides. Aβ has been proposed to induce oxidative stress and neurotoxicity. Previous studies from our laboratory and others showed an age-dependent increase in oxidative stress markers, loss of lipid asymmetry, and Aβ production and amyloid deposition in the brain of APP/PS1 mice. In the present study, we used APP (NLh)/APP(NLh) × PS-1(P246L)/PS-1(P246L) human double mutant knock-in APP/PS-1 mice to identify specific targets of brain protein carbonylation in an age-dependent manner. We found a number of proteins that are oxidatively modified in APP/PS1 mice compared to age-matched controls. The relevance of the identified proteins to the progression and pathogenesis of AD is discussed.     

Molecular and cellular mechanisms for Alzheimer's disease: understanding APP metabolism

Alzheimer's disease (AD) is the most common neurodegenerative disease associated with aging. One important pathologic feature of AD is the formation of extracellular senile plaques in the brain, whose major components are small peptides called beta-amyloid (Abeta) that are derived from beta-amyloid precursor protein (APP) through sequential cleavages by beta-secretase and gamma-secretase. Because of the critical role of Abeta in the pathogenesis of AD, unraveling the cellular and molecular events underlying APP/Abeta metabolism has been and remains, of paramount importance to AD research. In this article we will focus on the regulation of APP metabolism leading to Abeta generation. We will review current knowledge of the secretases (alpha-, beta-, and gamma-secretases) involved in APP processing and various molecular and cellular mechanisms underlying intracellular trafficking of APP, which is a highly regulated process and whose disturbance has direct impacts on the production of Abeta.     

Are presenilins intramembrane-cleaving proteases? Implications for the molecular mechanism of Alzheimer's disease.

The amyloid-beta protein (Abeta) is strongly implicated in the pathogenesis of Alzheimer's disease. The final step in the production of Abeta from the amyloid precursor protein (APP) is proteolysis by the unidentified gamma-secretases. This cleavage event is unusual in that it apparently occurs within the transmembrane region of the substrate. Studies with substrate-based inhibitors together with molecular modeling and mutagenesis of the gamma-secretase cleavage site of APP suggest that gamma-secretases are aspartyl proteases that catalyze a novel intramembranous proteolysis. This proteolysis requires the presenilins, proteins with eight transmembrane domains that are mutated in most cases of autosomal dominant familial Alzheimer's disease. Two conserved transmembrane aspartates in presenilins are essential for gamma-secretase activity, suggesting that presenilins themselves are gamma-secretases. Moreover, presenilins also mediate the apparently intramembranous cleavage of the Notch receptor, an event critical for Notch signaling and embryonic development. Thus, if presenilins are gamma-secretases, then they are also likely the proteases that cleave Notch within its transmembrane domain. Another protease, S2P, involved in the processing of the sterol regulatory element binding protein, is also a multipass integral membrane protein which cleaves within or very close to the transmembrane region of its substrate. Thus, presenilins and S2P appear to be members of a new type of polytopic protease with an intramembranous active site.     

COPI-mediated retrograde transport is required for efficient gamma-secretase cleavage of the amyloid precursor protein

Sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases results in the production of beta-amyloid peptide, which is a key determinant in Alzheimer's disease. Since several putative locations for gamma-secretase cleavage have been identified along the secretory pathway, trafficking of APP may be of importance for beta-amyloid peptide production. Here we have studied the role of retrograde transport in APP processing. We found that APP interacts with the beta subunit of the coatomer protein I (COPI) complex, which is involved in retrograde transport. In line with a role of retrograde trafficking in APP transport, inhibition of COPI-dependent transport altered APP trafficking, decreased APP cell surface expression, and coincided with a profound reduction in gamma-secretase cleavage. These results suggest that COPI-dependent retrograde transport is important for APP processing and influences production of beta-amyloid peptide.     

The cytosolic domain of APP and its possible role in the pathogenesis of Alzheimer's disease

APP is a type I membrane protein of unknown function, whose proteolytic processing, driven by beta- and gamma-secretases, generates the beta-amyloid peptides, one of the hallmarks of the pathogenesis of Alzheimer's disease. The short cytosolic domain of APP is the center of a complex network of protein-protein interactions. This network appears to play a crucial role in the regulation of the APP processing and in turn in the generation of the amyloid peptides, thus suggesting candidate targets for new therapeutic approaches. Furthermore, some possible functions of APP could just emerge from the study of this cytodomain and its partners.     

The amyloid beta-protein precursor of Alzheimer's disease is degraded extracellularly by a Kunitz protease inhibitor domain-sensitive trypsin-like serine protease in cultures of chick sympathetic neurons.

The amyloid beta-protein precursor (APP) of Alzheimer's disease (AD) is cleaved either by alpha-secretase to generate an N-terminally secreted fragment, or by beta- and gamma-secretases to generate the beta-amyloid protein (Abeta). The accumulation of Abeta in the brain is an important step in the pathogenesis of AD. Alternative mRNA splicing can generate isoforms of APP which contain a Kunitz protease inhibitor (KPI) domain. However, little is known about the physiological function of this domain. In the present study, the metabolic turnover of APP was examined in cultured chick sympathetic neurons. APP was labelled by incubating neurons for 5 h with [35S]methionine and [35S]cysteine. Intracellular labelled APP decayed in a biphasic pattern suggesting that trafficking occurs through two metabolic compartments. The half-lives for APP in each compartment were 1.5 and 5.7 h, respectively. A small fraction (10%) of the total APP was secreted into the culture medium where it was degraded with a half-life of 9 h. Studies using specific protease inhibitors demonstrated that this extracellular breakdown was due to cleavage by a trypsin-like serine protease that was secreted into the culture medium. Significantly, this protease was inhibited by a recombinant isoform of APP (sAPP751), which contains a region homologous to the Kunitz protease inhibitor (KPI) domain. These results suggest that KPI forms of APP regulate extracellular cleavage of secreted APP by inhibiting the activity of a secreted APP-degrading protease.     

Biochemical and cell-based assays for characterization of BACE-1 inhibitors

The deposition of beta-amyloid peptides (A beta42 and A beta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). A beta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. BACE-1 has been shown to be the major beta-secretase and is a primary therapeutic target for AD. In this article, two novel assays for the characterization of BACE-1 inhibitors are reported. The first is a sensitive 96-well HPLC biochemical assay that uses a unique substrate containing an optimized peptide cleavage sequence, NFEV, spanning from the P2-P2' positions This substrate was processed by BACE-1 approximately 10 times more efficiently than was the widely used substrate containing the Swedish (NLDA) sequence. As a result, the concentration of the enzyme required for the assay can be as low as 100 pM, permitting the evaluation of inhibitors with subnanomolar potency. The assay has also been applied to related aspartyl proteases such as cathepsin D (Cat D) and BACE-2. The second assay is a homogeneous electrochemiluminescence assay for the evaluation of BACE-1 inhibition in cultured cells that assesses the level of secreted amyloid EV40_NF from HEK293T cells stably transfected with APP containing the novel NFEV sequence. To illustrate the use of these assays, the properties of a potent, cell-active BACE-1 inhibitor are described.     

Regulation of amyloid beta-protein precursor by phosphorylation and protein interactions

Amyloid beta-protein precursor (APP), a type I membrane protein, is cleaved by primary alpha-or beta-secretase and secondary gamma-secretase. Cleavage of APP by beta- and gamma-secretases generates amyloid beta-protein, the main constituent of the cerebrovascular amyloid that accompanies Alzheimer disease. The generation and aggregation of amyloid beta-protein in the brain are believed to be a primary cause of Alzheimer disease pathogenesis, and indeed, early onset Alzheimer disease is genetically linked to APP and also to presenilins 1 and 2, which are components of gamma-secretase. Proteolytic cleavage of APP has been investigated as a candidate target for Alzheimer disease therapy, but the mechanisms regulating APP metabolism are still unclear. APP is a type I membrane protein with a short cytoplasmic region consisting of 47 amino acids. Recent research has elucidated the significance of the cytoplasmic region in the metabolism, trafficking, and physiological function of APP. The structure and function of the APP cytoplasmic domain can be modified by phosphorylation and through interaction with cytoplasmic proteins. This minireview summarizes a large body of recent information on the regulation of APP by phosphorylation and protein interaction, along with some of the physiological functions of APP. Recent findings regarding the regulation of APP processing contribute to the development of novel drugs and/or therapies for Alzheimer disease.     

Secretion and differential localization of the proteolytic cleavage products Abeta40 and Abeta42 of the Alzheimer amyloid precursor protein in human fetal myogenic cells

Abeta peptides are major components of the amyloid plaques that characterize Alzheimer's disease. These peptides are proteolytic cleavage products of the amyloid precursor protein (APP) and are generated by beta- and gamma-secretases. Here we show by multiparameter immunofluorescence imaging in muscle cells that localization of the Abeta40 and Abeta42 cleavage products reveals different myocyte types in a three-dimensional culture system. These myocyte types are heterogeneous by selective intracellular concentration of either Abeta40 or Abeta42 in vesicular structures, whilst only the Abeta40 peptide is secreted as indicated by Western blot analysis. This cellular pattern of APP proteolysis and Abeta peptide secretion correlates with lack of L-APP mRNA splice isoforms. Differential secretion and intracellular accumulation of Abeta peptides is characteristic for the early myocyte development and might be related to cell fusion.