Subcellular Compartment and Molecular Subdomain of beta -Amyloid Precursor Protein Relevant to the Abeta 42-promoting Effects of Alzheimer Mutant Presenilin 2*

Hiroshi IwataDagger §, Taisuke TomitaDagger , Kei Maruyama, and Takeshi IwatsuboDagger ||

From the Dagger  Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo Bunkyoku, Tokyo 113-0033, Japan and the  Department of Pharmacology, Saitama Medical School, Moroyama, Saitama 350-0495, Japan

Received for publication, August 31, 2000, and in revised form, February 23, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Increased production of amyloid beta  peptides ending at position 42 (Abeta 42) is one of the pathogenic phenotypes caused by mutant forms of presenilins (PS) linked to familial Alzheimer's disease. To identify the subcellular compartment(s) in which familial Alzheimer's disease mutant PS2 (mt PS2) affects the gamma -cleavage of beta APP to increase Abeta 42, we co-expressed the C-terminal 99-amino acid fragment of beta APP (C100) tagged with sorting signals to the endoplasmic reticulum (C100/ER) or to the trans-Golgi network (C100/TGN) together with mt PS2 in N2a cells. C100/TGN co-transfected with mt PS2 increased levels or ratios of intracellular as well as secreted Abeta 42 at similar levels to those with C100 without signals (C100/WT), whereas C100/ER yielded a negligible level of Abeta , which was not affected by co-transfection of mt PS2. To identify the molecular subdomain of beta APP required for the effects of mt PS2, we next co-expressed C100 variously truncated at the C-terminal cytoplasmic domain together with mt PS2. All types of C-terminally truncated C100 variants including that lacking the entire cytoplasmic domain yielded the secreted form of Abeta at levels comparable with those from C100/WT, and co-transfection of mt PS2 increased the secretion of Abeta 42. These results suggest that (i) late intracellular compartments including TGN are the major sites in which Abeta 42 is produced and up-regulated by mt PS2 and that (ii) the anterior half of C100 lacking the entire cytoplasmic domain is sufficient for the overproduction of Abeta 42 caused by mt PS2.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease (AD)1 is a progressive dementing disorder characterized pathologically by a massive loss of cortical neurons and an accumulation of two types of fibrillar lesions: i.e. amyloid deposits composed of amyloid beta  peptides (Abeta ) and tau-rich paired helical filaments (1). Abeta is produced from beta -amyloid precursor proteins (beta APP) through sequential cleavages by proteases originally termed beta - and gamma -secretases (1, 2); beta -secretase has recently been identified as a novel aspartyl protease, BACE (3-5). Deposition of beta -amyloid is considered to be closely related to the pathogenesis of AD because (i) deposition of Abeta is a neuropathological change relatively specific to AD; (ii) the diffuse type of senile plaque composed of highly aggregable Abeta 42 species (6, 7), as opposed to Abeta 40 that comprises the major portion of the secreted form of Abeta (8, 9), is the initial lesion of AD pathology; and (iii) mutations in genes coding for beta APP (10-14) or presenilin 1 (PS1) (15) or 2 (PS2) (16) are linked to some pedigrees of autosomal dominantly inherited familial AD (FAD), and these mutations increase the production of Abeta 42 species (12-14, 17-20). Mutations in PS genes that code for multipass integral membrane proteins account for the majority of early onset FAD. Studies in knockout mice or invertebrates demonstrated that PS is involved in gamma -cleavage of beta APP (2, 21, 22) as well as in site 3 cleavage of the Notch receptor (2, 23-25), both of which occur within the membrane or at the junction with cytoplasm, although it has not been clear if PS is a co-factor for gamma -cleavage or if PS is identical to gamma -secretase. However, recent data showing that transition state analogue gamma -secretase inhibitors directly and exclusively bound fragment forms of PS strongly support the hypothesis that PS represents the catalytic subunits of gamma -secretase (26-28).

Although there is ample evidence that mutations in PS genes increase the production of Abeta 42 (29-32), the intracellular compartment(s) in which mutant forms of PS interact with beta APP and promote gamma -cleavage at the Abeta 42 position has not been clearly identified. Generation of intracellular Abeta 42 has been shown to occur in endoplasmic reticulum (ER) of cultured neurons (33, 34) or in human embryonic kidney 293 cells (35), whereas the trans-Golgi network (TGN) (36) or endocytic pathway (37, 38) also are implicated in the generation of secretable Abeta 42. Although the ER localization of PS dovetails with the former data, others have suggested that Golgi may be related to the abnormal effect of mt PS1 to increase Abeta 42 (39, 40). Furthermore, subdomains in beta APP proteins that are required for this interaction with mt PS to increase production of Abeta 42 have not been definitively identified. In this study, we studied the intracellular compartment and intramolecular subdomain of beta APP that are relevant to the abnormal effects of mutant PS2 to affect gamma -cleavage and increase production of Abeta 42. For these purposes, we expressed modified forms of a C-terminal fragment of beta APP tagged with targeting signals to specific compartments or harboring deletion of defined cytoplasmic subdomains. We show here that TGN and other late intracellular compartments are the major sites where mt PS2 up-regulates Abeta 42 production and that the cytoplasmic domain of beta APP is dispensable for the overproduction of Abeta 42 caused by mt PS2.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Expression Plasmids-- A cDNA encoding the C- terminal 99 amino acids of beta APP fused to a signal peptide for rat preproenkephalin cDNA (C100) was previously described (32, 41). C100 peptides tagged with sorting signals to ER or TGN were generated using a C100 cDNA constructed in mammalian vector p91023 as a template. Briefly, oligonucleotides encoding the rat preproenkephalin signal peptide were used as a sense polymerase chain reaction primer: 5'-TTTAAGCTTCCACCATGGCGCAGTTCCTG-3'. The following oligonucleotides encoding the last four C-terminal amino acid residues of C100 (i.e. QMQN) followed by the signal sequences KKLN (for ER) or SDYQRL (for TGN) (42) were used as antisense polymerase chain reaction primers: 5'-CCCGGATCCCTAATTCAGATTATTGTTCTGCATCTG-3' for C100/ER, 5'-CCCGGATCCCTAGAGCCGCTGATAATCGAAGTTCTGCATCTG-3' for C100/TGN, and 5'-AAAGGATCCCTAGTTCTGCATCTG-3' for C100 (without signal motif). Amplification of cDNAs was performed using PfuTurbo DNA polymerase (Strategene). Amplified DNA fragments were digested with HindIII and BamHI and ligated into a pcDNA3.1-Hygro vector (Invitrogen). beta APP695 tagged with KKLN or SDYQRL motifs were constructed by ligating the EcoRI/XbaI fragments of tagged C100 with those of beta APP695 cDNA in pcDNA3.

cDNAs encoding C-terminally truncated C100 (i.e. C100/stop68, C100/stop56, and C100/stop52) were generated by polymerase chain reaction, using the following oligonucleotides as polymerase chain reaction primers: 5'-TTTAAGCTTCCACCATGGCGCAGTTCCTG-3' as a sense primer, 5'-TTTTCTAGACTAGTCAACCTCCAC-3' as an antisense primer for C100/stop68, 5'-CCCTCTAGACTACTGTTTCTTCTT-3' as an antisense primer for C100/stop56, and 5'-CCCTCTAGACTACAGCATCACCAA-3' as an antisense primer for C100/stop52. C-terminally truncated C100 peptides were amplified by polymerase chain reaction and ligated into pcDNA3.1-Hygro vector similarly as with C-terminally tagged C100. C-terminally truncated beta APP695 cDNAs were constructed by ligating the EcoRI/XbaI fragments of tagged C100 with those of beta APP695 cDNA in pcDNA3.

Cell Culture, Transfection, and Caspase Inhibitor Treatment-- Mouse neuro2a (N2a) neuroblastoma cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere as described (32, 43, 44). Transient transfection of C100 cDNAs into N2a cells and co-expression of C100 cDNAs in N2a cells stably expressing human PS2 cDNAs (43) were performed using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. Expression of transfected proteins was enhanced by treatment with 10 mM butyric acid for 24 h prior to harvesting cells or culture supernatants. For the inhibition of caspase activities, cells were treated with a 100 µM concentration of a pancaspase inhibitor, zVAD-fmk, for 24 h prior to analysis by Western blotting.

Immunoblot Analysis of beta APP or PS2 Derivatives and Cell-associated Abeta -- Cells were lysed in 2% SDS sample buffer and briefly sonicated. Samples were separated by SDS-polyacrylamide gel electrophoresis using a Tris-Tricine gel system, transferred to polyvinylidene difluoride membrane (Millipore Corp.), and probed with monoclonal antibodies BAN50 (specific for human Abeta 1-16) for the detection of C100 derivatives. A rabbit polyclonal antibody anti-G2N4 raised against a recombinant protein corresponding to the N-terminal residues 2-59 of human PS2 was used to probe PS2 and its derivatives. For the detection of Abeta in RIPA-soluble fractions of cell lysates, samples were initially lysed in RIPA (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate), and the supernatants after centrifugation at 15,000 × g for 5 min were immunoprecipitated by BAN50 using protein G-agarose and then analyzed by immunoblotting with BA27, BC05, or BAN50, using previously described procedures (44, 45, 47). Extraction of cell-associated Abeta by formic acid was performed as described (46). Briefly, cell pellets confluently grown in two 10-cm dishes were solubilized by ultrasonication followed by incubation in 100 µl of 70% formic acid at room temperature for 30 min. Supernatants after centrifugation at 100,000 × g for 20 min were desiccated and then solubilized in 100 µl of SDS sample buffer. Samples containing Abeta were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and reacted with antibodies after boiling (44, 45, 47). The immunoblots were developed using an ECL system (Amersham Pharmacia Biotech) or Immunostar (Wako Pure Chemicals).

Immunofluoresence Microscopy-- Transiently transfected N2a cells were cultured on glass coverslips for 48 h. Cells were fixed by incubation with phosphate-buffered saline (10 mM phosphate buffer, pH 7.4) containing 4% paraformaldehyde for 30 min at room temperature and then permeabilized and blocked with PBS-TB (phosphate-buffered saline containing 150 mM NaCl, 0.1% Triton X-100 and 3% bovine serum albumin) for 30 min at room temperature. Coverslips were then incubated with primary antibodies (a rabbit polyclonal antibody, C4, against the C terminus of beta APP (48) and monoclonal antibodies specific for BiP or adaptin-gamma ) for 2 h followed by an incubation with a mixture of fluorescein isothiocyanate-conjugated anti-rabbit IgG and Texas Red-conjugated anti-mouse IgG antibodies in PBS-TB for 1 h and then mounted in PermaFlour aqueous mounting medium (Immunon) and viewed with a confocal microscope (Fluoroview, Olympus, Tokyo) as described (43). BAN50 and fluorescein isothiocyanate-conjugated secondary antibody were used for single immunofluorescence detection of C100 derivatives.

Quantitation of Abeta by Two-site ELISAs-- Two-site ELISAs that specifically detect the C terminus of Abeta were used as described. BAN50, which was used as a capture antibody, binds only human beta APP or Abeta and does not cross-react with rodent Abeta or with an N-terminally truncated fragment (e.g. p3). BA27 and BC05 that specifically recognize the C terminus of Abeta 40 and Abeta 42, respectively, were conjugated with horseradish peroxidase and used as detector antibodies. Culture medium was collected after an appropriate incubation period (48 h) and subjected to BAN50/BA27 or BAN50/BC05 ELISAs as described (32, 43, 44). Cell-associated Abeta was quantitated after solubilization in 1% Nonidet P-40. ELISA data were statistically analyzed by analysis of variance using StatView-J.4.11.

Subcellular Fractionation and Differential Extraction-- Subcellular fractionation was performed using iodixanol as medium according to the previously described method (39) with some modifications. N2a cells stably expressing either WT PS2 or N141I mt PS2 and transiently transfected with C100 cDNAs were grown on three 10-cm dishes. Cells were scraped in TS and resuspended in 4 ml of homogenization buffer (10 mM HEPES (pH 7.4), 1 mM EDTA, 0.25 M sucrose, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 µg/ml pepstatin A, and 0.25 mM phenylmethylsulfonyl fluoride). Cells were disrupted by a Polytron homogenizer (Hitachi) at power level 2 for 10 s, and nuclei and large cell debris were pelleted by centrifugation at 1500 × g for 10 min. The postnuclear supernatants were centrifuged for 1 h at 65,000 × g. The resultant vesicle pellets (i.e. microsomal fractions) were resuspended in 0.8 ml of homogenization buffer. Gradients were set up in 13-ml Beckman SW41 centrifuge tubes by diluting iodixanol with homogenization buffer: 2.5%, 1 ml; 5%, 2 ml; 7.5%, 2 ml; 10%, 2 ml; 12.5%, 0.5 ml; 15%, 2 ml; 17.5%, 0.5 ml; 20%, 0.5 ml; 30%, 0.3 ml (iodixanol concentration/volume). The resuspended vesicle fractions were loaded on the top of the gradients and centrifuged in a SW41 rotor at 40,000 × rpm for 2.5 h. The resulting gradients were collected in 1-ml fractions. For the differential extraction of C-terminally truncated C100, microsomal fractions of N2a cells transiently expressing C100/WT, C100/stop68, C100/stop56, and C100/stop52 prepared as above were extracted by 0.5 M Na2CO3 (pH 11.0) or 1% Triton X-100, and solubilized proteins in supernatants after centrifugation at 100,000 × g for 15 min and insoluble pellets were analyzed by Western blotting with BAN50.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of FAD Mutant PS2 on Abeta Production from beta APP C100 Targeted to ER or TGN-- To identify the intracellular compartments where Abeta , especially Abeta 42, is generated and destined to be secreted, we transiently expressed cDNAs coding for the C-terminal 99 amino acids of human beta APP harboring a signal peptide at the N terminus (C100) or C100 tagged with sorting signals for retention to ER (C100/ER) or for recycling to TGN (C100/TGN) tagged at the C terminus in mouse N2a cells (Fig. 1, A-C). C100, C100/ER, or C100/TGN was expressed as a major ~13-kDa and a minor ~8-kDa polypeptide on immunoblots, the latter corresponding to fragments cleaved by caspases (see below), and the banding patterns were similar between C100 with and without sorting signals (Fig. 1B). Immunocytochemistry by C4 (against the cytoplasmic tail of beta APP (48)) combined with anti-BiP antibody that specifically reacts with a KDEL sequence (ER marker) or anti-adaptin-gamma antibody (TGN marker) showed retention of C100/ER in a meshwork like pattern overlapping with an immunolabeling with the ER marker (Fig. 1C, top left), whereas immunoreactive pattern for C100/TGN completely overlapped with that for adaptin-gamma (Fig. 1C, middle right), suggesting proper localization of C100 variants at the intended sites. C100/WT showed a combined ER and TGN localization (Fig. 1C, lower panels). We then quantitated Abeta -(1-40) and Abeta -(1-42) secreted from N2a cells expressing C100 variants by two-site ELISAs using BAN50 as a capture antibody, which specifically detects human Abeta but not endogenous murine Abeta (Fig. 1D). Cells transfected with C100/TGN secreted ~2000 pM Abeta -(1-40) and ~200 pM Abeta -(1-42), which were at comparable levels with those secreted from cells expressing C100/WT. In contrast, C100/ER did not secrete detectable levels of Abeta -(1-40) or Abeta -(1-42) as in N2a cells transfected with an empty vector. These results suggested that TGN would be the major intracellular site in which gamma -cleavage to yield Abeta -(1-40) as well as Abeta -(1-42) that are destined for secretion takes place.


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Fig. 1.   Expression of beta APP C100 harboring sorting signals to ER or TGN and Abeta secretion in N2a cells. A, schematic depiction of C100 tagged with sorting signals to ER (C100/ER) or trans-Golgi network (C100/TGN). C100/WT is C100 without a sorting signal. The shaded area represents Abeta flanked by cleavage sites for beta - and gamma -secretases (arrows). B, Western blot analysis of C100 harboring sorting signals expressed in N2a cells with BAN50. An arrow indicates the holoprotein of C100. mock, cells expressing an empty vector alone. Molecular markers are shown in kilodaltons. C, double fluorescence immunocytochemistry of N2a cells expressing C100/ER, C100/TGN, or C100/WT labeled by C4 (probe for C100; green) and anti-BiP (ER marker, left lane; red) or anti-adaptin-gamma (Adgamma , TGN marker, right lane; red) and viewed with a confocal microscope. Areas visualized in yellow represent co-localization of C100 and ER or TGN markers. Transfected cDNAs and primary antibodies are shown to the left of and above the panels, respectively. Scale bar, 10 µm. D, levels of secreted Abeta -(1-40) (open column) and Abeta -(1-42) (closed column) quantitated by two-site ELISAs. Mean values ± S.E. in four independent experiments are shown. Transfected C100 cDNAs are shown below the columns.

We then expressed C100/ER, C100/TGN, or C100/WT in N2a cells that stably express WT or N141I or M239V FAD mt PS2 and examined the production of secreted or intracellular Abeta . N2a cells expressing WT PS2 transiently transfected with C100/WT or C100/TGN secreted similar levels of Abeta -(1-40) and Abeta -(1-42), whereas those with C100/ER did not secrete detectable levels of Abeta , in almost identical patterns to those observed in cells without exogenous PS2. In contrast, cells expressing mt PS2 transiently transfected with C100/WT or C100/TGN secreted larger amounts of Abeta -(1-42) compared with Abeta -(1-40), whereas those with C100/ER did not secrete detectable Abeta (Fig. 2A). These data suggest that TGN or later intracellular compartments, but not ER, are the intracellular site where mt PS2 affects gamma -cleavage of beta APP to promote secretion of Abeta 42 in N2a cells.


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Fig. 2.   Effects of FAD mutant PS2 on Abeta production from beta APP C100 targeted to ER or TGN. A, levels of Abeta -(1-40) (open column) and Abeta -(1-42) (closed column) secreted from N2a cells stably expressing WT PS2 (left), N141I (middle), or M239V (right) FAD mt PS2 and transiently transfected with C100 with targeting signals (ER, C100/ER; TGN, C100/TGN; wt, C100/WT) quantitated by two-site ELISAs. Mean values ± S.E. in four independent experiments are shown. Transfected C100 cDNAs are shown with lines below the columns. B, immunoprecipitation/Western blot analysis of RIPA-extractable cell-associated Abeta (Abeta -(1-40), middle panel; Abeta -(1-42), lower panel) and C100 (upper panel) in cells stably expressing WT (left), N141I (middle), or M239V (right) mt PS2 and transiently transfected with C100 with targeting signals. Transfected C100 cDNAs are shown in each lane. Proteins were first immunoprecipitated by BAN50 and then probed by Western blotting with BA27 (for Abeta -(1-40)), BC05 (for Abeta -(1-42)), or BAN50 (for C100), respectively. Molecular mass markers are shown in kilodaltons. C, Western blot analysis of formic acid-extracted Abeta 40 by BA27 (middle panel) and Abeta 42 by BC05 (lower panel) in cells overexpressing C100/ER (middle lane; ER), C100/WT (right lane; wt), or mock-transfected cells (left lane; mock). Expression of C100 in cell lysates probed by BAN50 is shown in upper panel. D, iodixanol density gradient fractionation of N2a cells stably expressing WT (upper two panels) or N141I FAD mt (middle two panels) PS2 transiently transfected with C100/WT. Each fraction was analyzed by Western blotting with anti-G2N4 (for full-length and N-terminal fragments of PS2), BAN50 (for C100), anti-adaptin-gamma (marker for TGN), or anti-BiP (marker for ER). The numbers of the fractions are shown above. Molecular mass markers are shown in kilodaltons. E, levels of cell-associated Abeta -(1-42) in N2a cell fractions stably expressing WT (open column) or N141I FAD mt (closed column) PS2 and transiently transfected with C100/WT quantitated by two-site ELISAs. Mean values ± S.E. of four independent experiments are shown. The numbers of the fractions are indicated below the columns in the order shown in D.

To verify the intracellular production of Abeta -(1-40) and Abeta -(1-42) from C100 with or without sorting signal motifs, we analyzed RIPA-extracted lysates of N2a cells expressing C100/WT, C100/ER, or C100/TGN together with WT or N141I mt PS2 by Western blotting with antibodies to beta APP (i.e. BAN50 against the Abeta N terminus) or with those against C termini of Abeta after immunoprecipitation with BAN50 (Fig. 2B). C100/WT, C100/ER, or C100/TGN were expressed as ~13-kDa as well as ~8-kDa polypeptides as observed without co-expression of PS2. In N2a cells co-expressing WT PS2, C100/TGN yielded a ~4-kDa polypeptide positive for Abeta 40 as well as an equally intense Abeta 42-positive ~4-kDa polypeptide, in a similar pattern to those observed with C100/WT. In N2a cells expressing N141I or M239V mt PS2, however, C100/WT and C100/TGN yielded comparable levels of Abeta 42-positive 4-kDa bands, whereas only trace amounts of Abeta 40-positive bands were detected in cell lysates, despite robust expression of C100 and its derivatives. In contrast, detectable levels of Abeta 40 or Abeta 42-positive polypeptides were not observed in Western blots of cell lysates expressing C100/ER together with WT or N141I or M239V mt PS2. To confirm the lack of detectable cell-associated Abeta in cells expressing C100/ER, we solubilized N2a cells expressing C100/ER or C100/WT in 70% formic acid and analyzed the extracted proteins by Western blotting with BA27 and BC05. Comparable levels of Abeta 40-positive and Abeta 42-positive ~4-kDa proteins were detected in cells expressing C100/WT, whereas no Abeta -positive bands were detectable in cells expressing C100/ER despite robust expression of C100/ER holoprotein (Fig. 2C), suggesting that the levels of formic acid-extractable ER-associated Abeta are very low, if any, in our N2a cells overexpressing C100 derivatives.

We next analyzed the relationship between the subcellular localization of PS2 and the intracellular production site of Abeta 42. To this end, we expressed C100/WT together with WT or N141I mt PS2, separated the cells by iodixanol density fractionation, and analyzed the fractions by Western blotting (Fig. 2D). N-terminal fragments of PS2 were chiefly distributed in fractions 4-10, which overlapped with those positive for a TGN marker (adaptin-gamma ; fractions 3-10). In contrast, the distribution of full-length PS2 (fractions 11 and 12) was limited to those positive for an ER marker (i.e. BiP; fractions 10-12). The distribution patterns of PS2 and its derivatives were similar between WT and N141I mt PS2. ELISA quantitation of cell fraction-associated Abeta 42 showed that cells expressing N141I mt PS2 harbored elevated levels of Abeta -(1-42) in fractions 8 and 9, which corresponded to those positive for PS2 N-terminal fragment as well as for a TGN marker, supporting the notion that TGN is the site in which mt PS2 affects gamma -cleavage of C100 to promote Abeta 42 production (Fig. 2E).

To examine if the conclusions drawn from experiments using tagged C100 above are applicable to full-length beta APP, we transiently co-expressed human beta APP tagged with KKLN (beta APP/ER) or SDYQRL (beta APP/TGN) or without the tags (beta APP/WT) in N2a cells that stably express WT or N141I or M239V FAD mt PS2 and examined the production of secreted Abeta . N2a cells expressing WT PS2 transiently transfected with beta APP/WT or beta APP/TGN secreted similar levels of Abeta -(1-40) and Abeta -(1-42), whereas those with beta APP/ER did not secrete detectable levels of Abeta (Fig. 3A, left), and the results were similar to those in cells expressing C100 and its derivatives. In contrast, cells expressing N141I or M239V mt PS2 transiently transfected with beta APP/WT or beta APP/TGN secreted increased levels of Abeta -(1-42), whereas those with beta APP/ER again did not secrete detectable Abeta (Fig. 3A, middle and right). To examine whether the lack of ER-associated Abeta observed in experiments based on C100 is reproducible with full-length beta APP in our N2a system, we solubilized N2a cells expressing beta APP/ER or beta APP/WT in 70% formic acid and analyzed the extracted proteins by Western blotting with BA27 and BC05. Comparable levels of Abeta 40-positive and Abeta 42-positive ~4-kDa polypeptides were detected in cells expressing beta APP/WT, whereas no Abeta -positive bands were detectable in cells expressing beta APP/ER despite comparable levels of expression of beta APP/ER and beta APP/WT (Fig. 3B). Taken together, it was strongly suggested that late intracellular compartments including TGN, but not ER, are the major intracellular site of Abeta production and of mt PS2 effects on gamma -cleavage of beta APP to promote secretion of Abeta 42 in N2a cells.


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Fig. 3.   Abeta production in N2a cells expressing full-length beta APP tagged with sorting signals. A, levels of Abeta -(1-40) (open column) and Abeta -(1-42) (closed column) secreted from N2a cells stably expressing WT PS2 (left), N141I (middle), or M239V (right) FAD mt PS2 and transiently transfected with full-length beta APP with targeting signals (ER, beta APP/ER; TGN, beta APP/TGN; wt, beta APP/WT) quantitated by two-site ELISAs. Mean values ± S.E. in four independent experiments are shown. Transfected C100 cDNAs are shown by lines below the columns. B, Western blot analysis of formic acid-extracted Abeta 40 (middle panel) by BA27 and Abeta 42 (lower panel) by BC05 in cells overexpressing beta APP/ER (middle lane; ER), beta APP/WT (right lane; wt), or mock-transfected cells (left lane; mock). Expression of full-length beta APP in cell lysates probed by BAN50 is shown in the upper panel. Molecular mass markers are shown in kilodaltons.

Expression of C-terminally Truncated C100 and Effects of Co-expression of mt PS2 on Abeta Secretion-- Since C100 was fully susceptible to gamma -cleavage as well as to the Abeta 42-promoting effect of mt PS2, we next examined if the C-terminal cytoplasmic domain of beta APP, which has been implicated in a number of functions including intermolecular association, caspase cleavage, and endocytosis, is required for the abnormal function of mt PS2. For this purpose, we constructed following C100 derivatives truncated at various positions within the cytoplasmic domain: C100/stop68 truncated at Asp68 (numbering starting from residue Asp1 of Abeta ) documented as a caspase-3 cleavage site (49), C100/stop56 retaining the KKKQ sequence flanking the membrane that is attributed to membrane anchoring (50), and C100/stop52 that lacks the entire cytoplasmic domain (Fig. 4A).


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Fig. 4.   Expression of beta APP C100 truncated at the cytoplasmic domain and Abeta secretion in N2a cells. A, schematic depiction of C100 truncated at the cytoplasmic domain. C100/stop68 is truncated at Asp68 (starting from Asp1 of the Abeta sequence), which is inferred as the caspase-3 cleavage site; C100/stop56 retains the membrane-flanking four amino acid residues KKKQ; and C100/stop52 lacks the entire cytoplasmic domain. B, Western blot analysis of C-terminally truncated C100 transiently expressed in N2a cells with BAN50. An asterisk shows the co-migration of an ~8-kDa polypeptide derived from C100/WT with C100/stop68. Molecular mass markers are shown in kilodaltons, and the names of transfected cDNAs are indicated above each lane. C, inhibition of the generation of ~8-kDa band (*) from C100/WT by a caspase inhibitor zVAD-fmk. -, N2a cells transfected with C100/WT without zVAD-fmk treatment. zVAD, N2a cells transfected with C100/WT treated with 100 mM zVAD-fmk for 24 h. D, differential extraction of C-terminally truncated C100 by Na2CO3 or Triton X-100. Microsomal fractions of N2a cells transiently expressed C100/WT (wt), C100/stop68 (stop68), C100/stop56 (stop56), C100/stop52 (stop52) were extracted by 0.5 M Na2CO3 (pH 11.0) or 1% Triton X-100, and solubilized proteins (S) and insoluble pellets (P) were analyzed by Western blotting with BAN50. E, immunofluorescence localization of C-terminally truncated C100 in N2a cells revealed by BAN50. Scale bar, 10 µm. F, levels of Abeta -(1-40) (open column) and Abeta -(1-42) (closed column) secreted from transiently transfected N2a cells quantitated by two-site ELISAs. Note that C100/stop52 yielded significantly reduced levels of Abeta -(1-42) (*, p < 0.01 by analysis of variance). Mean values ± S.E. in four independent experiments are shown. Transfected C100 cDNAs are shown below each column.

When transfected transiently in N2a cells, each C100 derivative was expressed as ~6-13-kDa polypeptides of corresponding sizes on Western blots. Notably, C100/stop68 co-migrated with the ~8-kDa band derived from C100 (Fig. 4B), and the ~8-kDa band diminished upon treatment of cells expressing C100 with a caspase inhibitor zVAD-fmk (Fig. 4C), suggesting that the latter polypeptide is cleaved by caspase from C100. Every C100 derivative was solubilized by 1% Triton X-100, but not by Na2CO3 (pH 11.0), suggesting that all types of C-terminally truncated C100 were inserted into membranes (Fig. 4D). By immunocytochemistry with BAN50 that reacts with the extracellular portion of C100, C100/wt, or C100/stop68, -56, or -52 showed similar meshwork-like staining patterns accentuated in perinuclear areas, suggesting the ER/Golgi localization of these C100 derivatives (Fig. 4E). We then quantitated Abeta secreted from N2a cells transiently transfected with these C100 derivatives (Fig. 4F). All C-terminally deleted C100 derivatives showed robust secretion of Abeta -(1-40) at similar levels ranging from 1500 to 1800 pM. C100/stop68 and C100/stop56 produced similar levels of Abeta -(1-42), comprising ~11-13% that of total Abeta (Abeta -(1-40) + Abeta -(1-42)). However, cells expressing C100/stop52 secreted significantly lower levels of Abeta -(1-42), which comprised only ~3.8% that of total Abeta (*, p < 0.01 by analysis of variance).

We then transiently transfected cDNAs coding for C-terminally deleted C100 in N2a cells stably expressing WT or N141I or M239V mt PS2 and quantitated Abeta -(1-40) and Abeta -(1-42) secreted into culture media. In cells expressing WT PS2, levels of secreted Abeta -(1-40) were similar among all C100 derivatives, and C100/stop52 yielded significantly reduced levels or ratios (~3%) of Abeta -(1-42) compared with those from other C100 derivatives, as observed in cells without transfection of PS2 (Fig. 5A; *, p = 0.011 in ratios by analysis of variance). In contrast, co-transfection of FAD mt PS2 increased the percentage of secreted Abeta -(1-42) as a fraction of total Abeta to ~60% (N141I) or ~50% (M239V) with all types of C-terminally deleted C100, except that co-expression of C100/stop52 and M239V mt PS2 yielded ~20% of Abeta 42, although this was still significantly higher than that with WT PS2 (Fig. 5A). To confirm that the cytoplasmic domain of beta APP is dispensable for gamma -cleavage and mt PS2 effect on Abeta 42 generation on a full-length beta APP basis, we next co-expressed C-terminally deleted full-length beta APP in N2a cells stably expressing WT or N141I or M239V mt PS2 and quantitated Abeta -(1-40) and Abeta -(1-42) in culture media (Fig. 5B). Upon cotransfection with WT PS2, the levels of total secreted Abeta were significantly lower in cells expressing C-terminally truncated beta APP compared with that with full-length beta APP presumably due to lack of endocytosis as previously reported (51), whereas there was a uniform increase in the secretion of Abeta 42 in cells stably expressing N141I or M239V mt PS2. These data suggest that the anterior half of the C100 (i.e. Abeta sequence plus the following intramembranous portion of beta APP) is sufficient for the full effect of mt PS2 to increase secretion of Abeta -(1-42).


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Fig. 5.   Secretion of Abeta -(1-40) and Abeta -(1-42) from N2a cells stably expressing WT or FAD mt PS2 and transiently transfected with C-terminally truncated C100 or beta APP. Levels of secreted Abeta -(1-40) (open columns) and Abeta -(1-42) (closed columns) from N2a cells stably expressing WT (left columns), N141I (middle columns), or M239V (right columns) FAD mt PS2 and transiently transfected with C-terminally truncated 100 (A) or beta APP (B) quantitated by two-site ELISAs. Mean ± S.E. in four independent experiments are shown in A and B.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we showed that (i) TGN is one of the major intracellular sites in which a secretable pool of Abeta 42 is produced and up-regulated by the abnormal function of FAD mt PS2, (ii) lack of the entire cytoplasmic domain of C100 selectively decreases the production of Abeta 42, and (iii) the anterior half of C100 lacking the entire cytoplasmic domain is sufficient for the abnormal function of mt PS2 to increase production of Abeta 42.

The intracellular site of Abeta 42 generation in relation to PS function has been a matter of controversy (38). Here we showed that C100 targeted to TGN (C100/TGN) yielded similar levels of intracellular as well as secreted forms of Abeta -(1-42) and Abeta -(1-40), compared with those derived from C100 without sorting signals, whereas C100 targeted to ER (C100/ER) did not produce detectable levels of intracellular as well as secreted Abeta . Moreover, FAD-linked N141I and M239V mt PS2 fully increased the production of Abeta -(1-42) from C100/TGN at an extent comparable with that for C100 without signals, whereas production of intracellular as well as secreted Abeta -(1-42) was not up-regulated by co-expression of C100/ER. Use of C100 that does not require beta -cleavage, which is presumed to occur in the post-Golgi compartments (52), to trigger gamma -cleavage enabled us to directly address the intracellular site where gamma -cleavage takes place and is affected by the abnormal effects of mt PS2. It has been reported that cultured neurons (33, 34) as well as human embryonic kidney 293 cells (35) produce Abeta -(1-42) in ER upon overexpression of beta APP. These findings were in good agreement with the predominant ER localization of PS, which has been implicated in gamma -cleavage. However, it was subsequently shown that the ER-associated Abeta 42 was not directly secreted and was considered to comprise a distinct pool from the secreted Abeta (53). The reason for our failure to detect ER-associated Abeta in our cell system is not clear at present; however, the following possibilities could be considered to explain these discrepancies: (i) full-length Abeta may be detected in ER only by extremely high level expression of APP (e.g. by Semliki Forest virus infection) (33), and modest levels of overexpression of beta APP or C100 fail to produce detectable levels of Abeta ; (ii) in N2a cells, small amounts of Abeta 42 truncated at the N terminus, but not full-length species, have been detected in ER (36, 47), which escaped our detection system specific for human full-length Abeta ; and (iii) ER-derived Abeta is present at a relatively small amount compared with those in late compartments, the former being lower than the detection limit of our highly sensitive immunoblot assay.

Our data indicating that TGN harbored elevated levels of "secretable" Abeta 42 upon co-expression of mt PS2 strongly support the view that the active form of PS (i.e. endoproteolytic fragments that are stabilized (44) and form a high molecular weight complex (54)) resides in Golgi/TGN as well as in additional late intracellular compartments and that mt PS1 up-regulates production and secretion of Abeta 42 in these compartments (39, 40). In this case, a relatively small amount of "active" presenilin complex may be sufficient for the generation of Abeta in the late compartments. Notwithstanding the present data, the problem of the "spatial paradox" (38) between the localization of PS and gamma -secretase activities has not been completely clarified. Further careful studies on the intracellular distribution of presenilin complex and gamma -cleavage activities for the processing of beta APP as well as Notch in different types of cells will be needed.

We and others have shown that co-expression of C100 that lacks the majority of the extracellular domain of beta APP with FAD-associated mt PS1 (40) or PS2 (32) is sufficient to induce overproduction of Abeta 42. To examine whether the cytoplasmic domain of beta APP is required for the abnormal effect of mt PS, we co-expressed C-terminally truncated forms of C100 and evaluated the secretion of Abeta . Unexpectedly, we found that the expression of C100 lacking the entire cytoplasmic domain (C100/stop52), with or without co-expression of WT PS2, dramatically reduced the secretion of Abeta 42, although the total levels of Abeta secretion were not significantly altered. Similar results were obtained also in COS cells (data not shown). The mechanism whereby gamma -secretase differentially cleaves Abeta 40 and Abeta 42 within the transmembrane segment of beta APP is not well understood. However, accumulating data suggest that gamma -cleavage occurs in a position-dependent manner within the membranous portion, irrespective of the amino acid sequences (54, 55). The lack of the cytoplasmic domain including the KKKQ motif at the membrane-flanking portion, which is presumed to work as a membrane anchor (50), may destabilize the positioning of the transmembrane domain of beta APP, thereby leading to predominant cleavage at the Abeta 40 position. Moreover, C100 ending at the putative caspase-3 cleavage site (C100/stop68) did not change the level or proportion of secreted Abeta . It has been shown that beta APP truncated at the caspase-3 cleavage site increased the secretion of Abeta 40 (49). The reason for this discrepancy is unknown, but it is possible that the caspase-cleaved beta APP may promote beta -cleavage (49), thereby increasing Abeta 40 secretion.

Finally, we have shown that the cytoplasmic domain of C100 is dispensable for the abnormal effects of mt PS2 to increase production of Abeta 42. This domain is implicated in a number of beta APP functions including interaction with a number of binding proteins (i.e. FE65 (57) or X11 (58, 59)) as well as endocytosis (51), all of which are known to alter Abeta production (60). However, co-transfection of mt PS2 fully increased the secretion of Abeta 42 from all types of C100 truncated at the cytoplasmic domain. It has recently been suggested that PS serves as a gamma -secretase harboring two intramembranous aspartates in TM6 and TM7 domains as a catalytic center (61). Taken together with our present data, shift of gamma -cleavage from the predominant Abeta 40 position to a more pathogenic Abeta 42 position caused by the abnormal gain-of-function of FAD mt PS may require solely the intramembranous interaction between the TM domains of beta APP and PS. Further analysis on the molecular mechanism whereby mt PS leads to increased production of Abeta 42 should facilitate the understanding of the pathogenesis of AD as well as of the unusual but important proteolytic mechanism recently referred to as regulated intramembrane endoproteolysis (62).

    ACKNOWLEDGEMENTS

We thank N. Takasugi for anti-G2N4 antibody and Takeda Chemical Industries for continuous support.

    FOOTNOTES

* This work was supported by Grants-in-Aid from the Ministry of Health and Welfare, the Ministry of Education, Science, Culture and Sports, CREST of Japan Science and Technology Corporation, and RIKEN, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ A Research Fellow of the Japan Society for the Promotion of Science.

|| To whom correspondence should be addressed. Tel.: 81-3-5841-4877; Fax: 81-3-5841-4708; E-mail: iwatsubo@mol.f.u-tokyo.ac.jp.

Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M007989200

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta  peptide; beta APP, beta -amyloid precursor protein(s); C100, C-terminal 99-amino acid fragment of beta APP; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; FAD, familial Alzheimer's disease; N2a, mouse Neuro2a neuroblastoma; PS, presenilin(s); TGN, trans-Golgi network; mt, mutant; RIPA, radioimmune precipitation assay buffer; WT, wild type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp- (OMe)-fluoromethylketone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Selkoe, D. J. (1999) Nature 399 (Suppl. 6738), A23-A31[CrossRef][Medline] [Order article via Infotrieve]
2. Haass, C., and De Strooper, B. (1999) Science 286, 916-919[Abstract/Free Full Text]
3. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) Science 286, 735-741[Abstract/Free Full Text]
4. Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M., Dovey, H. F., Frigon, N., Hong, J., Jacobson-Croak, K., Jewett, N., Keim, P., Knops, J., Lieberburg, I., Power, M., Tan, H., Tatsuno, G., Tung, J., Schenk, D., Seubert, P., Suomensaari, S. M., Wang, S., Walker, D., Zhao, J., McConlogue, L., and John, V. (1999) Nature 402, 537-540[CrossRef][Medline] [Order article via Infotrieve]
5. Capell, A., Steiner, H., Willem, M., Kaiser, H., Meyer, C., Walter, J., Lammich, S., Multhaup, G., and Haass, C. (2000) J. Biol. Chem. 275, 30849-30854[Abstract/Free Full Text]
6. Iwatsubo, T., Odaka, A., Suzuki, N., Mizusawa, H., Nukina, N., and Ihara, Y. (1994) Neuron 13, 45-53[Medline] [Order article via Infotrieve]
7. Iwatsubo, T., Mann, D. M. A., Odaka, A., Suzuki, N., and Ihara, Y. (1995) Ann. Neurol. 37, 294-299[Medline] [Order article via Infotrieve]
8. Haass, C., Schlossmacher, M. G., Hung, A.-Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E.-H., Schenk, D., Teplow, D. B., and Selkoe, D. J. (1992) Nature 359, 322-325[CrossRef][Medline] [Order article via Infotrieve]
9. Shoji, M., Golde, T. E., Ghiso, J., Cheung, T. T., Estus, S., Shaffer, L. M., Cai, X.-D., McKay, D. M., Tintner, R., Frangione, B., and Younkin, S. G. (1992) Science 258, 126-129[Medline] [Order article via Infotrieve]
10. Goate, A., Chartier-Harlin, M. C., Mullan, M., Brown, J., Crawford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L., Mant, R., Newton, P., Rooke, K., Talbot, C., Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M., and Hardy, J. (1991) Nature 349, 704-706[CrossRef][Medline] [Order article via Infotrieve]
11. Mullan, M., Crawford, F., Axelman, K., Houlden, H., Lilius, L., Winblad, B., and Lannfelt, L. (1992) Nat. Genet. 1, 345-347[Medline] [Order article via Infotrieve]
12. Eckman, C. B., Mehta, N. D., Crook, R., Perez-tur, J., Prihar, G., Pfeiffer, E., Graff-Radford, N., Hinder, P., Yager, D., Zenk, B., Refolo, L. M., Prada, C. M., Younkin, S. G., Hutton, M., and Hardy, J. (1997) Hum. Mol. Genet. 6, 2087-2089[Abstract/Free Full Text]
13. Ancolio, K., Dumanchin, C., Barelli, H., Warter, J. M., Brice, A., Campion, D., Frebourg, T., and Checler, F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4119-4124[Abstract/Free Full Text]
14. Kwok, J. B., Li, Q.-X., Hallupp, M., Whyte, S., Ames, D., Beyreuther, K., Masters, C. L., and Schofield, P. R. (2000) Ann. Neurol. 47, 249-253[CrossRef][Medline] [Order article via Infotrieve]
15. Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Ikeda, M., Chi, H., Lin, C., Li, G., Holman, K., Tsuda, T., Mar, L., Foncin, J.-F., Bruni, A. C., Montesi, M. P., Sorbi, S., Rainero, I., Pinessi, L., Nee, L., Chumakov, I., Pollen, D., Brookes, A., Sanseau, P., Polinsky, R. J., Wasco, W., Da Silva, H. A. R., Haines, J. L., Pericak-Vance, M. A., Tanzi, R. E., Roses, A. D., Fraser, P. E., Rommens, J. M., and St. George-Hyslop, P. H. (1995) Nature 375, 754-760[CrossRef][Medline] [Order article via Infotrieve]
16. Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D. M., Oshima, J., Pettingell, W. H., Yu, C., Jondro, P. D., Schmidt, S. D., Wang, K., Crowley, A. C., Fu, Y.-H., Guenette, S. Y., Galas, D., Nemens, E., Wijsman, E. M., Bird, T. D., Schellenberg, G. D., and Tanzi, R. E. (1995) Science 269, 973-977[Medline] [Order article via Infotrieve]
17. Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A.-Y., Seubert, P., Vigo-Pelfrey, C., Lieberburg, I., and Selkoe, D. J. (1992) Nature 360, 672-674[CrossRef][Medline] [Order article via Infotrieve]
18. Cai, X.-D., Golde, T. E., and Younkin, S. G. (1993) Science 259, 514-516[Medline] [Order article via Infotrieve]
19. Suzuki, N., Cheung, T. T., Cai, X.-D., Odaka, A., Otvos, L., Jr., Eckman, C., Golde, T. E., and Younkin, S. G. (1994) Science 264, 1336-1340[Medline] [Order article via Infotrieve]
20. Maruyama, K., Tomita, T., Shinozaki, K., Kume, H., Asada, H., Saido, T. C., Ishiura, S., Iwatsubo, T., and Obata, K. (1996) Biochem. Biophys. Res. Commun. 227, 730-735[CrossRef][Medline] [Order article via Infotrieve]
21. De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhde, G., Annaert, W., Von Figura, K., and Van Leuven, F. (1998) Nature 391, 387-390[CrossRef][Medline] [Order article via Infotrieve]
22. Naruse, S., Thinakaran, G., Luo, J. J., Kusiak, J. W., Tomita, T., Iwatsubo, T., Qian, X., Ginty, D. D., Price, D. L., Borchelt, D. R., Wong, P. C., and Sisodia, S. S. (1998) Neuron 21, 1213-1221[Medline] [Order article via Infotrieve]
23. De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray, W. J., Goate, A., and Kopan, R. (1999) Nature 398, 518-522[CrossRef][Medline] [Order article via Infotrieve]
24. Struhl, G., and Greenwald, I. (1999) Nature 398, 522-525[CrossRef][Medline] [Order article via Infotrieve]
25. Steiner, H., Duff, K., Capell, A., Romig, H., Grim, M. G., Lincoln, S., Hardy, J., Yu, X., Picciano, M., Fechteler, K., Citron, M., Kopan, R., Pesold, B., Keck, S., Baader, M., Tomita, T., Iwatsubo, T., Baumeister, R., and Haass, C. (1999) J. Biol. Chem. 274, 28669-28673[Abstract/Free Full Text]
26. Li, Y.-M., Xu, M., Lai, M. T., Huang, Q., Castro, J. L., DiMuzio-Mower, J., Harrison, T., Lellis, C., Nadin, A., Neduvelil, J. G., Register, R. B., Sardana, M. K., Shearman, M. S., Smith, A. L., Shi, X. P., Yin, K. C., Shafer, J. A., and Gardell, S. J. (2000) Nature 405, 689-694[CrossRef][Medline] [Order article via Infotrieve]
27. Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Diehl, T. S., Moore, C. L., Tsai, J. Y., Rahmati, T., Xia, W., Selkoe, D. J., and Wolfe, M. S. (2000) Nat. Cell Biol. 2, 428-434[CrossRef][Medline] [Order article via Infotrieve]
28. Seiffert, D., Bradley, J. D., Rominger, C. M., Rominger, D. H., Yang, F., Meredith, J., Wang, Q., Roach, A. H., Thompson, L. A., Spitz, S. M., Higaki, J. N., Prakash, S. R., Combs, A. P., Copeland, R. A., Arneric, S. P., Hartig, P. R., Robertson, D. W., Cordell, B., Stern, A. M., Olson, R. E., and Zaczek, R. (2000) J. Biol. Chem. 275, 34086-34091[Abstract/Free Full Text]
29. Duff, K., Eckman, C., Zehr, C., Yu, X., Prada, C.-M., Perez-tur, J., Hutton, M., Buee, L., Harigaya, Y., Yager, D., Morgan, D., Gordon, M. N., Holcomb, L., Refolo, L., Zenk, B., Hardy, J., and Younkin, S. (1996) Nature 383, 710-713[CrossRef][Medline] [Order article via Infotrieve]
30. Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Davenport, F., Ratovitsky, T., Prada, C. M., Kim, G., Seekins, S., Yager, D., Slunt, H. H., Wang, R., Seeger, M., Levey, A. I., Gandy, S. E., Copeland, N. G., Jenkins, N. A., Price, D. L., Younkin, S. G., and Sisodia, S. S. (1996) Neuron 17, 1005-1013[Medline] [Order article via Infotrieve]
31. Citron, M., Westaway, D., Xia, W., Carlson, G., Diehl, T., Levesque, G., Johnson-Wood, K., Lee, M., Seubert, P., Davis, A., Kholodenko, D., Motter, R., Sherrington, R., Perry, B., Yao, H., Strome, R., Lieberburg, I., Rommens, J., Kim, S., Schenk, D., Fraser, P., St. George-Hyslop, P., and Selkoe, D. J. (1997) Nat. Med. 3, 67-72[Medline] [Order article via Infotrieve]
32. Tomita, T., Maruyama, K., Saido, T. C., Kume, H., Shinozaki, K., Tokuhiro, S., Capell, A., Walter, J., Grunberg, J., Haass, C., Iwatsubo, T., and Obata, K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2025-2030[Abstract/Free Full Text]
33. Cook, D. G., Forman, M., Sung, J. C., Leight, S., Kolson, D. L., Iwatsubo, T., Lee, V. M.-Y., and Doms, R. W. (1997) Nat. Med. 3, 1021-1023[Medline] [Order article via Infotrieve]
34. Hartmann, T., Bieger, S. C., Bruhl, B., Tienari, P. J., Ida, N., Allsop, D., Roberts, G. W., Masters, C. L., Dotti, C. G., Unsicker, K., and Beyreuther, K. (1997) Nat. Med. 3, 1016-1020[Medline] [Order article via Infotrieve]
35. Wild-Bode, C., Yamazaki, T., Capell, A., Leimer, U., Steiner, H., Ihara, Y., and Haass, C. (1997) J. Biol. Chem. 272, 16085-16088[Abstract/Free Full Text]
36. Greenfield, J. P., Tsai, J., Gouras, G. K., Hai, B., Thinakaran, G., Checler, F., Sisodia, S. S., Greengard, P., and Xu, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 742-747[Abstract/Free Full Text]
37. Soriano, S., Chyung, A. S., Chen, X., Stokin, G. B., Lee, V. M.-Y., and Koo, E.-H. (1999) J. Biol. Chem. 274, 32295-32300[Abstract/Free Full Text]
38. Annaert, W. G., Levesque, L., Craessaerts, K., Dierinck, I., Snellings, G., Westaway, D., St. George-Hyslop, P., Cordell, B., Fraser, P., and De Strooper, B. (1999) J. Cell Biol. 147, 277-294[Abstract/Free Full Text]
39. Xia, W., Zhang, J., Ostaszewski, B. L., Kimberly, W. T., Seubert, P., Koo, E. H., Shen, J., and Selkoe, D. J. (1998) Biochemistry 37, 16465-16471[CrossRef][Medline] [Order article via Infotrieve]
40. Sudoh, S., Hua, G., Kawamura, Y., Maruyama, K., Komano, H., and Yanagisawa, K. (2000) Eur. J. Biochem. 267, 2036-2045[Abstract/Free Full Text]
41. Maruyama, K., Terakado, K., Usami, M., and Yoshikawa, K. (1990) Nature 347, 566-569[CrossRef][Medline] [Order article via Infotrieve]
42. Peraus, G. C., Masters, C. L., and Beyreuther, K. (1997) J. Neurosci. 17, 7714-7724[Abstract/Free Full Text]
43. Tomita, T., Tokuhiro, S., Hashimoto, T., Aiba, K., Saido, T. C., Maruyama, K., and Iwatsubo, T. (1998) J. Biol. Chem. 273, 21153-21160[Abstract/Free Full Text]
44. Tomita, T., Takikawa, R., Koyama, A., Morohashi, Y., Takasugi, N., Saido, T. C., Maruyama, K., and Iwatsubo, T. (1999) J. Neurosci. 19, 10627-10634[Abstract/Free Full Text]
45. Ida, N., Hartmann, T., Pantel, J., Schroder, J., Zerfass, R., Forstl, H., Sandbrink, R., Masters, C. L., and Beyreuther, K. (1996) J. Biol. Chem. 271, 22908-22914[Abstract/Free Full Text]
46. Yamazaki, T., Chang, T. Y., Haass, C., and Ihara, Y. (2001) J. Biol. Chem. 276, 4454-4460[Abstract/Free Full Text]
47. Sudoh, S., Kawamura, Y., Sato, S., Wang, R., Saido, T. C., Oyama, F., Sakaki, Y., Komano, H., and Yanagisawa, K. (1998) J. Neurochem. 71, 1535-1543[Medline] [Order article via Infotrieve]
48. Tomita, T., Chang, T. Y., Kodama, T., and Iwatsubo, T. (1998) Neuroreport 5, 911-913
49. Gervais, F. G., Xu, D., Robertson, G. S., Vaillancourt, J. P., Zhu, Y., Huang, J., LeBlanc, A., Smith, D., Rigby, M., Shearman, M. S., Clarke, E. E., Zheng, H., Van Der Ploeg, L. H., Ruffolo, S. C., Thornberry, N. A., Xanthoudakis, S., Zamboni, R. J., Roy, S., and Nicholson, D. W. (1999) Cell 97, 395-406[Medline] [Order article via Infotrieve]
50. Usami, M., Yamao-Harigaya, W., and Maruyama, K. (1993) J. Neurochem. 61, 239-246[Medline] [Order article via Infotrieve]
51. Koo, E. H., and Squazzo, S. L. (1994) J. Biol. Chem. 269, 17386-17389[Abstract/Free Full Text]
52. Haass, C., Lemere, C. A., Capell, A., Citron, M., Seubert, P., Schenk, D., Lannfelt, L., and Selkoe, D. J. (1995) Nat. Med. 1, 1291-1296[Medline] [Order article via Infotrieve]
53. Skovronsky, D. M., Doms, R. W., and Lee, V. M.-Y. (1998) J. Cell Biol. 141, 1031-1039[Abstract/Free Full Text]
54. Capell, A., Grunberg, J., Pesold, B., Diehlmann, A., Citron, M., Nixon, R., Beyreuther, K., Selkoe, D. J., and Haass, C. (1998) J. Biol. Chem. 273, 3205-3211[Abstract/Free Full Text]
55. Lichtenthaler, S. F., Wang, R., Grimm, H., Uljon, S. N., Masters, C. L., and Beyreuther, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3053-3058[Abstract/Free Full Text]
56. Murphy, M. P., Hickman, L. J., Eckman, C. B., Uljon, S. N., Wang, R., and Golde, T. E. (1999) J. Biol. Chem. 274, 11914-11923[Abstract/Free Full Text]
57. Sabo, S. L., Lanier, L. M., Ikin, A. F., Khorkova, O., Sahasrabudhe, S., Greengard, P., and Buxbaum, J. D. (1999) J. Biol. Chem. 274, 7952-7957[Abstract/Free Full Text]
58. Borg, J. P., Yang, Y., De Taddeo-Borg, M., Margolis, B., and Turner, R. S. (1998) J. Biol. Chem. 273, 14761-14766[Abstract/Free Full Text]
59. Tomita, S., Ozaki, T., Taru, H., Oguchi, S., Takeda, S., Yagi, Y., Sakiyama, S., Kirino, Y., and Suzuki, T. (1999) J. Biol. Chem. 274, 2243-2254[Abstract/Free Full Text]
60. De Strooper, B., and Annaert, W. (2000) J. Cell Sci. 113, 1857-1870[Abstract/Free Full Text]
61. Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517[CrossRef][Medline] [Order article via Infotrieve]
62. Brown, M. S., Ye, J., Rawson, R. B., and Goldstein, J. L. (2000) Cell 100, 391-398[Medline] [Order article via Infotrieve]


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