Mutations in APP have independent effects on Aβ and CTFγ generation

doi:10.1016/j.nbd.2004.04.018

Neurobiology of Disease

Volume 17, Issue 2, November 2004, Pages 205-218

https://doi.org/10.1016/j.nbd.2004.04.018 Get rights and content

Understanding the molecular mechanism of β-amyloid (Aβ) generation is crucial for Alzheimer's disease pathogenesis as well as for normal APP function. The transmembrane domain (TM) of APP appears to undergo presenilin-dependent γ-secretase cleavage at two topologically distinct sites: a site in the middle of the TM domain that is crucial for the generation of Aβ-peptides, and a site close to the cytoplasmic border (S3-like/ɛ site) of the TM domain that leads to production of the APP intracellular domain (CTFγ/AICD). We demonstrate that, in contrast to the unique effect of familial Alzheimer's disease (FAD) mutations in APP on Aβ42 production, some but not all FAD mutations also affect CTFγ generation. Furthermore, changes in total CTFγ levels do not correlate with either an increase or a decrease of any Aβ species, and inhibition of Aβ-peptide formation starting from position +1 (Aβ_1–x) does not affect CTFγ production. These results suggest that cleavage at the γ_40/42- and the S3-like sites can be dissociated, and that APP signaling and Aβ production are not tightly linked.

Introduction

Production of Aβ42 is believed to be a central event in the pathogenesis of Alzheimer's disease (AD) (Golde et al., 2000, Sambamurti et al., 2002, Selkoe, 2001). Almost all mutations in the three genes (β-amyloid precursor protein—APP, presenilin1—PS1 and presenilin 2—PS2) that have been found to cause familial Alzheimer's disease (FAD) increase the ratio of Aβ42 to Aβ40. However, it is still unclear how FAD mutations alter the cleavage specificity toward longer Aβ species or how under normal conditions the production of the shorter Aβ40 peptide is favored.

Aβ is generated from the β-amyloid precursor protein (APP) as a result of proteolytic processing through the β-secretase pathway (Esler and Wolfe, 2001). The N-terminus of Aβ is produced by β-secretase cleavage (BACE1) (Sinha et al., 1999, Vassar et al., 1999, Yan et al., 1999), while generation of the C-terminus of Aβ involves an unusual intramembranous cleavage by a multi-protein γ-secretase complex (De Strooper et al., 1998, Steiner et al., 1999). γ-Secretase cleavage seems not to be site restricted since it generates Aβ fragments of 37–43 amino acids in length (Wang et al., 1996). γ-Secretase cleavage depends critically on the presenilins (De Strooper et al., 1998, Steiner et al., 1999). Two aspartate residues in TM domains 6 and 7 of PS are essential for activity and could represent part of the γ-secretase catalytic center (Berezovska et al., 2000, Kimberly et al., 2000, Steiner et al., 1999, Wolfe et al., 1999). Other components of the γ-secretase complex have also been identified: nicastrin, Aph1, and Pen2 (De Strooper, 2003, Edbauer et al., 2003, Francis et al., 2002, Goutte et al., 2002, Levitan et al., 2001, Steiner et al., 2002, Yu et al., 2000). Indeed, recent in vitro studies have demonstrated that these four proteins are sufficient to reconstitute γ-secretase activity (Edbauer et al., 2003). While the β-secretase pathway is favored in neurons, processing of APP by the α-secretase pathway is predominant in all other cell types. α-Secretase cleaves APP within the Aβ sequence (between amino acids 16 and 17 of Aβ), this is followed by γ-secretase cleavage generating an N-terminally truncated non-amyloidogenic version of the Aβ peptide called p3. Recent in vivo and in vitro studies have identified a PS-dependent C-terminal fragment (CTFγ/AICD) that may mediate APP signaling in a manner analogous to the NICD fragment of Notch (Kimberly et al., 2001, Leissring et al., 2002). Indeed, it has been shown that CTFγ enters the nucleus when bound to Fe65 protein and that this complex can activate transcription of reporter genes (Cao and Sudhof, 2001). Thus, it seems that both Aβ and CTFγ production may be important for understanding APP biology.

Although CTFγ is a C-terminal product of γ-secretase cleavage, it is not a direct product of cleavage at the γ_40/42 site(s) but is generated by an additional cleavage event within the TM domain of APP that resembles the S3 cleavage site of Notch1 (Figs. 1A and B) (Gu et al., 2001, Sastre et al., 2001, Yu et al., 2001). The S3-like cleavage site of APP (also termed γ₄₉ or ɛ site) has a similar primary sequence to the S3 site in Notch1 (Val at the P1′ position of the cleavage), is located close to the cytoplasmic side of the membrane, is presenilin-dependent, and is sensitive to γ-secretase inhibitors (Sastre et al., 2001, Yu et al., 2001, Weidemann et al., 2002). The major CTFγ fragment contains 50 amino acids (CTFγ50) (Sastre et al., 2001, Weidemann et al., 2002), although minor fragments of CTFγ48/49/51 have also been reported (Gu et al., 2001, Yu et al., 2001). Intramembranous cleavage at two topologically distinct sites (one in the middle of the TM domain that generates Aβ-like peptides and the other close to the cytoplasmic border that liberates intracellular domains-ICDs) has also been reported for other γ-secretase substrates: Notch1 and CD44 (Fig. 1A) (Kimberly et al., 2003, Lammich et al., 2002, Okochi et al., 2002).

The generation of CTFγ50 presents a puzzle since neither Aβ49, the N-terminal species left after γ-secretase cleavage at the S3-like site, nor CTFγ57/59 fragments, that would be generated by cleavage at the γ_40/42 sites, have been detected. Several alternative mechanisms of Aβ and CTFγ generation have been suggested: cleavage at the γ_40/42 site followed by aminopeptidase activity to generate CTFγ50, cleavage at the S3-like site followed by carboxypeptidase activity to generate Aβ40/42, or cleavage by γ-secretase at both the γ_40/42 and S3-like site. Alternatively, γ_40/42 and S3-like cleavage could be mediated by different enzyme activities that are both presenilin-dependent.

Since the γ_40/42 site(s) and the S3-like site lie within the transmembrane domain of APP, we tested whether these cleavage events could affect each other and whether there could be a relationship between Aβ and CTFγ production. In addition, since all FAD mutations in the TM domain of APP lie close to or between the γ₄₂ and S3-like site (T714–L723P) (Kwok et al., 2000), we investigated whether these FAD mutations also affect CTFγ production. We show that some familial Alzheimer's disease (FAD) mutations in APP that lie between these two cleavage sites can directly affect both γ_40/42 cleavage and S3-like cleavage. Furthermore, we demonstrate that formation of CTFγ and Aβ can be dissociated: total CTFγ levels do not correlate with either an increase or decrease in Aβ-peptides and inhibition of Aβ-peptide formation by β-secretase cleavage site mutation does not affect CTFγ generation. These results support the notion that the presenilin-dependent dual intramembranous cleavages of APP are two separate events, thus APP signaling and Aβ production are not tightly linked. Our findings have implications for understanding the mechanism and the role of the PS-dependent dual intramembranous cleavage of APP and other γ-secretase substrates.

Section snippets

Cell culture and transfection

Human embryonic kidney 293 (HEK 293) cells were obtained from the ATCC (Rockville, MD) and PS1^−/−PS2^−/− mouse embryonic fibroblasts were provided by Dr. Bart De Strooper. Cells were grown in DMEM (GibcoBRL, Grand Island, NY) containing 10% fetal bovine serum (GibcoBRL), 2 mM l-glutamine, 100 μg/ml penicillin and streptomycin under a 5% CO₂ atmosphere. Cells were transiently transfected using Fugene™ 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) and harvested 40–48 h

In vivo CTFγ assay

To enable analysis of CTFγ production in vivo (in the cell), we designed APP constructs containing a 6myc tag at their C-terminus. The C99+6myc or APPsw+6myc constructs were transiently cotransfected in PS1/2 KO cells with either wild type PS1, PS1-ΔE9 (a functional FAD-PS1 variant that does not go endoproteolysis) or a loss of function mutant (PS1-D257A) that has previously been shown to abolish Aβ and NICD formation. Processing of 6myc-tagged C99 (Fig. 2A) and APPsw (not shown) was analyzed

Discussion

Presenilin-dependent dual intramembranous cleavage has been described for several γ-secretase substrates: APP, Notch1, and CD44 (Gu et al., 2001, Kimberly et al., 2003, Lammich et al., 2002, Okochi et al., 2002, Sastre et al., 2001). One cleavage (in the middle of the TM domain) results in secretion of a hydrophobic peptide (Aβ/Nβ/CD44β) while the second cut (close to the cytoplasmic border) liberates the intracellular domain-ICD (AICD/NICD/CD44-ICD) for putative nuclear signaling. This finding

Acknowledgments

We thank Dr. R. Kopan for the pCS2+6MT vector, and for critical review of the manuscript and Drs. D. Selkoe, P. Seubert and P. Mathews for antibodies. We thank Anne Brunkan, Yonghua Pan, and Ma. Xenia U. Garcia for technical advice and Dr. Krista Moulder and K. Alexander Smith for help in confocal microscopy. S.H. is supported by a Fulbright Postdoctoral fellowship, The John Douglas French Alzheimer's Foundation and the Daniel Weinstock Research Fund for Alzheimer disease. This work was

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For example, expression of several familial AD-linked PS1 variants elevate the production of Aβ42 but fully block AICD formation (44–46). Similarly, Hecimovic et al. (50) have reported that mutations within the APP transmembrane domain that elevate Aβ42 production have no effect on production of AICD. In this regard, He et al. (47) demonstrated that a γ-secretase-activating protein increases Aβ production but appears to reduce the generation of AICD.
The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid β (Aβ) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid β peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch.
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The finding that FAD mutations subtly alter APP processing to generate increased levels of Aβ42 has been a major foundation of the amyloid hypothesis and has been a widely repeated finding.7 However, only a few studies have examined the effects of PS1 mutations on AICD with varying results.21,32 In this study, we show that an FAD mutant form of PS1 increases Aβ42 and reduces AICD levels.
To understand the cleavage of the amyloid β protein (Aβ) precursor (APP) by γ-secretase and to determine its changes in a representative familial Alzheimer disease (FAD) mutation.
Transfected cells expressing wild-type and FAD mutant APP were analyzed for changes in the levels of the major secreted Aβ species and of the corresponding intracellular C-terminal APP fragments (APP intracellular domain, AICD) generated by γ-secretase, whereas radio-sequencing was used to precisely identify the resulting cleavage site(s).
The AICD fragment(s) generated by γ-secretase cleavage comigrated in gels with a 50-residue synthetic peptide used as control, which is smaller than the 59 and 57 residues predicted from Aβ ending at positions 40 (Aβ40) and 42 (Aβ42), respectively. In agreement with previous findings, an FAD mutant form of presenilin 1 (PS1-M139V) significantly increased the longer Aβ42 while showing trends toward reducing Aβ40. AICD levels were reduced by the mutation, suggesting that γ-secretase activity may be actually impaired by the mutation. Radiosequence analysis in cells expressing wild-type PS1 detected γ-secretase cleavage sites at the Aβ peptide bond L⁴⁹-V⁵⁰ to generate a 50-amino acid (aa) AICD fragment (AICD50) and the Aβ peptide bond T⁴⁸-L⁴⁹, generating an AICD of 51 aa (AICD51). No other cleavage sites were reliably detected.
Based on findings that the FAD mutation that increases Aβ42 also reduces AICD, we propose that γ-secretase activity is impaired by FAD mutations and predict that physiologic and environmental agents that inhibit γ-secretase will actually induce AD pathogenesis rather that prevent it. Furthermore, we propose that the cleavage site to generate AICD is naturally ragged and occurs predominantly at two sites 48 and 49 aa from the start of the Aβ sequence. Thus, end specific antibodies to these two sites will need to be generated to study the quantitative relationships between these two cleavages in sporadic AD and FAD.
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Cholesterol accumulation in Niemann–Pick type C disease (NPC) causes increased levels of the amyloid-precursor-protein C-terminal fragments (APP-CTFs) and intracellular amyloid-β peptide (Aβ), the two central molecules in Alzheimer's disease (AD) pathogenesis. We previously reported that cholesterol accumulation in NPC-cells leads to cholesterol-dependent increased APP processing by β-secretase (BACE1) and decreased APP expression at the cell surface (Malnar et al. Biochim Biophys Acta. 1802 (2010) 682–691.). We hypothesized that increased formation of APP-CTFs and Aβ in NPC disease is due to cholesterol-mediated altered endocytic trafficking of APP and/or BACE1. Here, we show that APP endocytosis is prerequisite for enhanced Aβ levels in NPC-cells. Moreover, we observed that NPC cells show cholesterol dependent sequestration and colocalization of APP and BACE1 within enlarged early/recycling endosomes which can lead to increased β-secretase processing of APP. We demonstrated that increased endocytic localization of APP in NPC-cells is likely due to both its increased internalization and its decreased recycling to the cell surface. Our findings suggest that increased cholesterol levels, such as in NPC disease and sporadic AD, may be the upstream effector that drives amyloidogenic APP processing characteristic for Alzheimer's disease by altering endocytic trafficking of APP and BACE1.
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Our results and the current understanding of membrane protein dynamics support this model, but further experiments are needed to rigorously test this hypothesis in more detail (41). The influence of mutations in the transmembrane domain of APP on γ and ϵ cleavage is well documented (28, 42–44). In an accompanying manuscript, Sagi et al, (49) show that the sequence of the transmembrane domain also alters the ability of GSMs to modulate γ cleavage.
γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ϵ, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1–42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1–40 to 1–33 without significant changes to ϵ cleavage. These results further support a model where ϵ cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.
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Familial Alzheimer's disease (AD) due to PSEN1 mutations provides an opportunity to examine AD biomarkers in persons in whom the diagnosis is certain. We describe a 55 year-old woman with clinically probable AD and a novel PSEN1 mutation who underwent genetic, clinical, biochemical and magnetic resonance and nuclear imaging assessments. We also describe neuropathological findings in her similarly affected brother. Neuropsychological testing confirmed deficits in memory, visuospatial and language function. CSF t-tau and p-tau181 were markedly elevated and Aβ₄₂ levels reduced. FDG-PET revealed hypometabolism in the left parietotemporal cortex. FDDNP-PET showed increased binding of tracer in medial temporal and parietal lobes and in the head of the caudate and anterior putamen bilaterally. Neuropathological examination of her brother showed the typical findings of AD and the striatum demonstrated amyloid pathology and marked neurofibrillary pathology beyond that typically seen in late-onset AD. A novel S212Y substitution in PSEN1 was present in the index patient and her affected brother but not in an older unaffected sister. An in vitro assay in which the S212Y mutation was introduced in cell culture confirmed that it was associated with increased production of Aβ₄₂. We describe biochemical, imaging, and neuropathological changes in a pedigree with a novel PSEN1 mutation. This allows us to validate the pathogenicity of this mutation and the indices used to assess AD.
Mutation analysis of the presenilin 1 N-terminal domain reveals a broad spectrum of γ-secretase activity toward amyloid precursor protein and other substrates
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In the second approach, we transiently expressed APP C99–6myc and assessed the levels AICD generated by γ-secretase cleavage of C99 between Leu49 and Val50, which is referred to as the ϵ-cleavage (42). There have been contrasting reports on the correlation between Aβ production (γ-cleavage) and AICD generation (ϵ-cleavage) (43–46). Therefore, we felt it is important to determine how LL1 mutations influenced ϵ-cleavage activity.
The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease β-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.

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Mutations in APP have independent effects on Aβ and CTFγ generation

Introduction

Section snippets

Cell culture and transfection

In vivo CTFγ assay

Discussion

Acknowledgments

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