Molecular Dissection of Domains in Mutant Presenilin 2 That Mediate Overproduction of Amyloidogenic Forms of Amyloid beta  Peptides
INABILITY OF TRUNCATED FORMS OF PS2 WITH FAMILIAL ALZHEIMER'S DISEASE MUTATION TO INCREASE SECRETION OF Abeta 42*

Taisuke TomitaDagger , Shinya TokuhiroDagger , Tadafumi HashimotoDagger , Keiko AibaDagger , Takaomi C. Saido§, Kei Maruyama, and Takeshi IwatsuboDagger parallel

From the Dagger  Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan, the § Laboratory for Proteolytic Neuroscience, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan, and the  Laboratory of Neurochemistry, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Mutations in presenilin (PS) 1 or PS2 genes account for the majority of early-onset familial Alzheimer's disease, and these mutations have been shown to increase production of species of amyloid beta  peptide (Abeta ) ending at residue 42, i.e. the most amyloidogenic form of Abeta . To gain insight into the molecular mechanisms whereby mutant PS induces overproduction of Abeta 42, we constructed cDNAs encoding mutant and/or truncated forms of PS2 and examined the secretion of Abeta 42 from COS or neuro2a cells transfected with these genes. Cells expressing full-length PS2 harboring both N141I and M239V mutations in the same polypeptide induced overproduction of Abeta 42, although the levels of Abeta 42 were comparable with those in cells engineered to express PS2 with one or the other of these PS2 mutations. In contrast, cells engineered to express partially truncated PS2 (eliminating the COOH-terminal third of PS2 while retaining the endoproteolytic NH2-terminal fragment) and harboring a N141I mutation, as well as cells expressing COOH-terminal fragments of PS2, did not overproduce Abeta 42, and the levels of Abeta 42 were comparable with those in cells that expressed full-length, wild-type PS2 or fragments thereof. These data indicate that: (i) the Abeta 42-promoting effects of mutant PS2 proteins reach the maximum level with a given single amino acid substitution (i.e. N141I or M239V); and (ii) the expression of full-length mutant PS2 is required for the overproduction of Abeta 42. Hence, cooperative interactions of NH2- and COOH-terminal fragments generated from full-length mutant PS2 may be important for the overproduction of Abeta 42 that may underlie familial Alzheimer's disease.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Alzheimer's disease (AD)1 is a progressive, dementing, neurological disorder characterized pathologically by an extensive neuronal loss in the cerebral cortex as well as a massive deposition of amyloid beta  peptides (Abeta ) as senile plaques and in the walls of blood vessels (1). A subset of early-onset AD is inherited as an autosomal dominant trait, and presenilin (PS) genes were identified as the major causative genes for these early-onset familial AD (FAD). PS1 gene, which is linked to the majority of early-onset FAD located on chromosome 14 (2), and PS2 gene (3), which is responsible for a subtype of FAD linked to chromosome 1, encode homologous polytopic membrane proteins that predominantly localize to endoplasmic reticulum (4-6) and span the membrane 8 times (7). More than 40 missense mutations (8), as well as an exon 10 deletion (9) in PS1 and two missense mutations of PS2 (3, 10), thus far have been identified in pedigrees of FAD.

The physiological function of PS proteins is unknown, although recent data from studies in Caenorhabditis elegans (11, 12) and PS1 gene knock-out mice (13, 14) indicate that PS1 may play some role in Notch signaling. The mechanisms whereby mutations in PS1 or PS2 genes cause AD also remain elusive, but several lines of evidence suggest that they may lead to AD by promoting beta -amyloid deposition. Amino acid substitutions, as well as an exon 10 deletion, of PS1 (15-17) and PS2 (17, 18) have been shown to increase the secretion of a species of Abeta ending at residue 42 (Abeta 42), i.e. the most amyloidogenic form of Abeta (19-21). Recent findings that the secretion of Abeta from primary neurons cultured from brains of mice that lack PS1 is decreased, despite the normal level of full-length beta -amyloid precursor protein (beta APP) or the amyloidogenic COOH-terminal fragment thereof, argue for the notion that PS is an important co-factor for the proteolytic processing of beta APP at the COOH terminus of Abeta termed gamma -cleavage (22). However, the mechanisms whereby mutant PS proteins affect gamma -cleavage and lead to the increased production of Abeta 42 is unknown. Some investigators have shown the direct association of PS and beta APP in cultured cells (23, 24), whereas others have not (25). Thus, one may speculate a direct or indirect "chaperone"-like effect of mutant PS or effects on intracellular vesicular trafficking to increase the susceptibility of beta APP to be cleaved at position 42.

Several questions arise from these observations. The mechanistic effects of a given single amino acid substitution in mutant PS protein leading to AD are not yet understood. Previous data showing that either of the two known PS2 mutations, i.e. the Volga German mutation that substitutes Asn-141 for Ile (N141I) or the Italian mutation causing a Met-239 to Val (M239V) mutation, considerably increase the percentage of secreted Abeta 42 (17, 18, 26), suggesting that a single amino acid substitution on PS2 may lead to a significant change in the protein folding and/or interaction with other proteins of PS2 compared with those with wild-type PS2. To gain insights into the nature of pathogenic effects caused by PS2 mutations, we first examined whether a mutant PS2 molecule harboring both the N141I and M239V mutations would increase the overproduction of Abeta 42, compared with singly mutated PS2 with one or the other of these mutations.

PS1 and PS2 have been shown to undergo endoproteolytic cleavage that yields a long NH2-terminal fragment (NTF) and a short COOH-terminal fragment (CTF) spanning the membrane 6 and 2 times, respectively (18, 27). These fragments are the predominant forms of PS1 or PS2 in cultured cells or brain tissues that do not overexpress PS (27). However, the relationship between cleavage and function of PS is not well understood. Next we sought to examine if the NTF or CTF forms of mutant PS2 alone are capable of promoting the secretion of Abeta 42. To this end, we expressed partially truncated forms of PS2 (eliminating the COOH-terminal third of PS2 while retaining the endoproteolytic NTF) harboring a N141I mutation, as well as CTFs of PS2 in cultured cells and examined the COOH-terminal properties of Abeta secreted from these cells.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction of Expression Plasmids-- A full-length cDNA encoding wild-type (WT) human PS2 was obtained by PCR from a normal human cDNA library, and the N141I or M239V PS2 mutations were introduced by the dU-template method as described previously (18). WT as well as mutant (mt) PS2 cDNAs were subcloned into pBluescript, and the coding region was then subcloned into a mammalian expression vector pcDNA3. The N141I/M239V double mutation was introduced by digesting the N141I mt PS2 cDNA in pBluescript with BamHI and BstXI and then inserting the resultant ~0.5-kilobase pair product between the BamHI-BstXI sites of M239V mt PS2 in pcDNA3.

cDNAs encoding COOH-terminally truncated WT or N141I mt PS2 (i.e. PS2/270stop, PS2/303stop, and PS2/388stop) were generated by PCR using Pfu polymerase (Stratagene), and the following oligonucleotides were used as PCR primers: 5'-CCGGGATCCAGACCTCTCTGCGGCCCCAAGT-3' as a sense primer, 5'-CATTCTCTCGAGCTATTTGGGACACAG-3' for PS2/270stop, 5'-AGCTCGAGCTAGCCAACCGTCCACAC-3' for PS2/303stop, and 5'-GGCTCGAGCTACGTGGTATTCCAGTC-3' for PS2/388stop as antisense primers, respectively. These primers were incubated with WT or N141I mt PS2 cDNAs in pcDNA3, and the PCR products were digested with BamHI and XhoI. Purified fragments were ligated into pcDNA3. cDNAs encoding varying lengths of PS2 COOH-terminal fragments (i.e. PS2/270ctf, PS2/304ctf, and PS2/344ctf) were similarly generated by PCR using the following primers: 5'-GGCACTCGAGTGTAAAACTATACAACTGC-3' as an antisense primer and 5'-CCGGATCCACCATGGGGCCTCTGAGA-3' for PS2/270ctf, 5'-CCGGATCCACCATGGCGAAGCTGGAC-3' for PS2/304ctf, and 5'-ACGGATCCATGAGTTTTGGGGAGCCT-3' for PS2/344ctf as sense primers. These primers were incubated with WT PS2 cDNA in pcDNA3, digested, and ligated into pcDNA3. Schematic depictions of truncated and/or mutated PS2 derivatives are shown in Fig. 1.

To express derivative polypeptides of PS2 fused to glutathione S-transferase (GST), cDNAs encoding the NH2-terminal (2-84) or the loop (301-361) portions of PS2 were amplified by PCR and subcloned into an Escherichia coli expression vector (pGEX-6P-1, Amersham Pharmacia Biotech). These ligations resulted in the fusion of PS2 sequences COOH-terminal and in-frame with GST.

All constructs were sequenced using Thermosequenase (Amersham Pharmacia Biotech) on an automated sequencer (Li-Cor, Lincoln, NE) as described (18).

Cell Culture and Transfection-- Monkey COS-1 cells or mouse neuro2a (N2a) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin at 37 °C in 5% CO2 atmosphere as described (18). Transient expression in COS-1 cells was performed by the DEAE-dextran method, and stable N2a cell lines were generated by transfecting the cDNAs in pcDNA3 vector using a mammalian transfection kit (Stratagene) or LipofectAMINE (Life Technologies, Inc.) and selection in Dulbecco's modified Eagle's medium containing G418 (Life Technologies, Inc.) at 400 or 500 µg/ml. Double transfection of COS-1 cells with cDNAs encoding PS2 or its derivatives in pcDNA3 and those encoding the COOH-terminal 100 amino acids (C100) of beta APP in p91023(E) was performed as described previously (18). Co-expression of NTF and CTF of PS2 was performed by transiently transfecting a cDNA encoding PS2/304ctf into N2a cells stably expressing WT or N141I mt PS2/303stop using LipofectAMINE according to the manufacturer's instructions.

Antibodies and Immunoblot Analysis-- Polyclonal antibodies were raised in rabbits using synthetic peptides conjugated to keyhole limpet hemocyanin or GST fusion proteins corresponding to the following predicted amino acid sequences of human PS2 as immunogens: anti-G2N2 against GST fused to amino acids 2-84 of PS2, anti-G2L against GST fused to amino acids 301-361 of PS2, and anti-PS2C2 against peptides corresponding to amino acids 443-448 of PS2. Anti-G2N2 and anti-PS2C2 antisera were further affinity-purified to their immunogens as described (28). Anti-PS2loop antiserum raised against GST fused to the loop domain of PS2 (29) was kindly provided by Dr. G. Thinakaran. The locations of immunogen peptides/protein fragments within PS2 are shown in Fig. 1.


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Fig. 1.   Schematic depictions of PS2 and truncated and/or mutated derivatives thereof. A, the location of the epitopes of antibodies used in this study (anti-G2N2, anti-G2L, anti-PS2loop, and anti-PS2C2) is shown by broken underlines. Arrowheads indicate the sites of standard (open arrowhead) and alternative (caspase-type (32); closed arrowhead) proteolytic processing. B, schematic representations of mutant or truncated forms of PS2 encoded by the cDNAs used in this study are shown. The names of cDNAs are indicated at the left of each bar, and squares with numbers represent putative TM domains. Small arrows on each bar show the location of amino acid substitutions linked to FAD mutations, and arrowheads between the TM 6/7 domains represent the sites of proteolytic processing shown in A.

Cells were lysed in 2% SDS sample buffer and briefly sonicated. The samples were separated by SDS-PAGE without prior heating, transferred to polyvinylidene difluoride membrane (Millipore), and probed with each of the anti-PS antibodies as described (18). The immunoblots were developed using an ECL system (Amersham Pharmacia Biotech).

Immunofluorescence Microscopy-- Transiently transfected COS-1 cells were cultured on glass coverslips. Cells were fixed by incubation in phosphate-buffered saline (PBS) containing 4% paraformaldehyde at room temperature for 30 min, permeabilized, and blocked with PBS-TB (10 mM phosphate buffer, pH 7.4, 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 against PS2 for 2 h and fluorescein isothiocyanate- or Texas Red-conjugated secondary antibodies in PBS-TB for 1 h, mounted in PermaFlour Aqueous Mounting Medium (IMMUNON), and viewed with a confocal laser scanning microscope (Fluoview, Olympus, Tokyo) as described (30).

Quantitation of Abeta by Two-site ELISAs-- Two-site ELISAs that specifically detect the COOH terminus of Abeta were used. BNT77 raised against human Abeta 11-28, which recognizes full-length as well as NH2-terminally truncated Abeta , was used as a capture antibody; BNT77 binds human as well as rodent-type Abeta but does not react with the 3-kDa fragment (p3) beginning at the Leu-17 residue of Abeta . BA27 and BC05, monoclonal antibodies that specifically recognize the COOH termini of Abeta 40 and Abeta 42, respectively, were conjugated with horseradish peroxidase and used as detector antibodies. The specificity and sensitivity of these ELISAs have been characterized previously (18, 31). Culture media were collected after appropriate incubation periods (60 h in COS-1 and 12 or 24 h in N2a cells) and subjected to BNT77/BA27 or BC05 ELISAs as described (18).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of Full-length PS2 Harboring N141I, M239V, or Both of the Mutations in Cultured Cells and Effects on Abeta Secretion-- We transfected cDNAs encoding WT or N141I, M239V single mt as well as N141I/M239V double mt PS2 cDNAs transiently into COS-1 cells or stably into N2a cells and analyzed the cell lysates by Western blots using anti-PS2 antibodies. As we have previously observed (18), when we transfected cells with cDNAs encoding WT or N141I mt PS2, 50-55-kDa polypeptides corresponding to full-length (fl) PS2, 35-40-kDa NTFs migrating as a doublet as well as a ~19-kDa CTF were detected in COS-1 cells (data not shown, but see Fig. 4 showing identical patterns of processing of PS2 in COS-1 cells); this ~19-kDa CTF was presumed to be produced by caspase-3-like proteolytic activities (32) because its generation was inhibited by a caspase-3 inhibitor, DEVD-CHO.2 In addition, a small amount of a ~23-kDa CTF was detected. In N2a cells, 50-55-kDa fl PS2 (Fig. 2, arrow), 35-kDa NTF (Fig. 2, closed arrowhead), as well as a 23-kDa CTF (Fig. 2, open arrowhead) were detected. However, the amounts or ratios of NH2- and COOH-terminal fragments were similar in M239V or N141I/M239V double mt PS2 compared with N141I mt or WT PS2 both in COS-1 and N2a cells (Fig. 2).


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Fig. 2.   Expression and metabolism of PS2 in N2a cells stably transfected with single or double mutant PS2 genes. Western blot analysis of stable N2a cells transfected with WT or mt PS2 cDNAs. Cell lysates (20 µg of protein) from N2a cells transfected with an empty pcDNA3 vector or with wild-type, N141I mt, M239V mt, or N141I/M239V double mt PS2 cDNAs were fractionated by SDS-PAGE and analyzed by immunoblotting with anti-G2N2 or anti-PS2loop antibodies. Full-length PS2 proteins are marked by arrows, NTFs by closed arrowheads, and CTFs by open arrowheads. The names of the transfected cDNA constructs are indicated at the top of each lane. Molecular mass standards are shown in kilodaltons.

We then quantitated the levels of Abeta 40 and Abeta 42 secreted from cells transfected with these mt PS2 cDNAs. The percentages of Abeta 42 secreted from COS-1 cells doubly transfected with beta APP C100 and N141I or M239V mt PS2 were elevated to almost similar levels (~30%) by 2.2-fold compared with those from cells with WT PS2 and beta APP C100 (average of Abeta 42, 13.8%), and the absolute levels of secreted Abeta 42 were increased by 2.3 (N141I) and 1.7 (M239V) times, respectively, compared with WT PS2. When COS-1 cells were transfected with N141I/M239V double mutated PS2 and beta APP C100, the Abeta 42 was 31.4%, and the Abeta 42 level also was similar to those in cells with singly mutated PS2 (Fig. 3A). Similar results were obtained in COS-1 cells doubly transfected with full-length WT or Swedish-type mutant (i.e. 595/596 KM-NL) beta APP together with mt PS2 (data not shown).


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Fig. 3.   Secreted Abeta 40 and Abeta 42 from cells expressing WT or single or double mutant PS2 genes. Levels of Abeta x-40 and Abeta x-42 secreted from COS-1 cells doubly transfected with beta APP C100 and PS2 genes (A) or N2a cells stably transfected with PS2 genes or an empty vector (B) quantitated by two-site ELISAs are shown. Mean values ± S.E. in four independent experiments in A and two independent experiments in B are shown. Transfected PS2 cDNAs are indicated below the columns; PS2WT, wild-type PS2; PS2N141I, N141I mutant PS2; PS2M239V, M239V mutant PS2; and PS2N141I/M239V, N141I and M239V double mutant PS2.

We then examined the secretion of Abeta from stably transfected N2a cell lines expressing WT, N141I, M239V, or N141I/M239V mt PS2. As described previously (18), N2a cells expressing N141I mt PS2 secreted considerably increased amounts or percentages of Abeta 42 (71.6% of total Abeta and 4.2 times compared with those with WT PS2: 17.0%). N2a cells expressing M239V mt PS2 also secreted significantly increased levels (3.4 times compared with those with WT PS2) or percentage (mean, 58.3%) of Abeta 42. However, the secretion of Abeta 42 from cells expressing N141I/M239V double mutant PS2 were again similar to those with either of the single PS2 mutations in terms of the absolute levels (4.1 times compared with those with WT PS2) or percentages (mean, 59.3%) (Fig. 3B). The expression of endogenous beta APP was at similar levels between different N2a cell lines (data not shown).

Characterization of Truncated Forms of WT or N141I mt PS2 Proteins Expressed in Cultured Cells and Their Subcellular Localization-- To gain insights into the biological significance of endoproteolytic processing of PS2 and especially to examine whether the NTF or CTF of PS2 is biologically active, we then expressed truncated forms of WT or N141I mt PS2 in cultured cells and characterized their metabolism and subcellular localization. Two categories of cDNAs encoding truncated PS2 were used (see Fig. 1): (i) NTF constructs (WT or N141I mt) ending at residues 270, 303, or 388 (designated PS2/270stop, PS2/303stop, and PS2/388stop), retaining the NH2-terminal 6 (or 7) transmembrane (TM) domains and shorter than, close to, or longer than the predicted size of PS2 NTF, respectively (33, 34); accordingly, PS2/388stop retains the entire length of the loop region as well as the 7th TM domain of PS2; (ii) CTF constructs starting at residues 271, 304, or 344 (designated PS2/271ctf, PS2/304ctf, and PS2/344ctf), longer than, close to, or shorter than the predicted size of native CTF, respectively. PS2/344ctf is close to the size of the "ALG-3" fragment, which was found to inhibit cellular apoptosis (35).

In COS-1 cells, PS2/270stop, PS2/303stop, and PS2/388stop (WT or mt) were expressed as doublets migrating at 30-33, 32-35, and 45-50 kDa, respectively (Fig. 4A). PS2/271ctf, PS2/304ctf, and PS2/344ctf were expressed as ~27-, ~23- and ~16-kDa fragments, respectively (Fig. 4B). These polypeptides migrated at slightly slower positions than those estimated from their predicted sizes (PS2/270stop, 30.6 kDa; PS2/303stop, 37.2 kDa; PS2/388stop, 43.5 kDa; PS2/271ctf, 19.6 kDa; PS2/304ctf, 15.9 kDa; and PS2/344ctf, 11.3 kDa). In addition to the full-length transfected proteins, PS2/388stop yielded 35-40-kDa doublet proteins that were equivalent in size to the NTFs cleaved from fl PS2 (Fig. 4A, arrowhead); PS2/271ctf (Fig. 4B) also yielded proteolytic fragments of 23 (arrowhead) and 19 kDa (asterisk) in size, and the 19-kDa CTF was also observed with PS2/304ctf (Fig. 4B, asterisk). Immunopositive bands at higher molecular weight ranges relative to these polypeptides (Fig. 4, A and B) would presumably represent dimeric forms and/or aggregates of these proteins.


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Fig. 4.   Expression and metabolism of PS2 in transiently transfected COS-1 cells with cDNAs encoding truncated PS2. A, Western blot analysis of expression of WT or mt PS2 NTFs in transiently transfected COS-1 cells. Cell lysates (10 µg of protein) from COS-1 cells transfected with an empty pcDNA3 vector or with WT or N141I mt fl, WT or N141I mt 270stop, WT or N141I mt 303stop, and WT or N141I mt 388stop PS2 cDNAs were fractionated by SDS-PAGE and analyzed by immunoblotting with anti-G2N2 antibody. The positions of fl PS2 and NTFs are marked by arrows and arrowheads, respectively. B, Western blot analysis of expression of PS2 CTFs in transiently transfected COS-1 cells. Cell lysates (10 µg of protein) from COS-1 cells transfected with an empty pcDNA3 vector or with WT fl, 271ctf, 304ctf, or 344ctf PS2 cDNAs were fractionated by SDS-PAGE and analyzed by immunoblotting with anti-G2L or anti-PS2C2 antibodies. The positions of fl PS2, 23-kDa standard CTF, and 19-kDa alternative CTF are marked by arrows, arrowheads, and asterisks, respectively. The names of the transfected cDNA constructs are indicated at the top of each lane. Molecular mass standards are shown in kilodaltons.

In stable N2a cells, the expression patterns of PS2 derivatives were essentially similar to those in COS-1 cells with some differences (Fig. 5). Notably, 45-50-kDa polypeptides corresponding to PS2/388stop were barely processed to form 35-kDa NTF (Fig. 5A) that was present in cells with fl PS2 (Fig. 5A, arrowhead). PS2/271ctf (Fig. 5B) and PS2/304ctf (Fig. 5B) also did not produce proteolytic fragments of smaller sizes. The patterns of expression of endogenous beta APP were almost similar between these cell lines (data not shown).


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Fig. 5.   Expression and metabolism of PS2 in stably transfected N2a cells with cDNAs encoding truncated PS2. Western blot analysis of expression of WT or mt PS2 derivatives in stably transfected N2a cells is shown. Cell lysates (20 µg of protein) from N2a cells transfected with an empty pcDNA3 vector or with WT or N141I mt fl, WT or N141I mt 270stop, WT or N141 mt 303stop, WT or N141I mt 388stop, and 271ctf or 304ctf PS2 cDNAs were fractionated by SDS-PAGE and analyzed by immunoblotting with anti-G2N2 (A) or anti-G2L (B) antibodies. The positions of fl PS2 and NTFs (in A) or CTFs (in B) are marked by arrows and arrowheads, respectively. Note that PS2/303stop comigrates with the 35-kDa NTF in A and that PS2/304ctf comigrates with the 23-kDa standard CTF in B, respectively. The names of the transfected cDNA constructs are indicated at the top of each lane. Molecular mass standards are shown in kilodaltons.

Next we examined the subcellular localization of the PS2 NTFs or CTFs in COS-1 cells by immunofluorescence microscopy. Remarkably, all constructs encoding NTFs and CTFs of WT or N141I mt types of PS2 showed similar distribution in a fine meshlike pattern throughout the cytoplasm as well as dense immunostaining in the perikaryal areas, which corresponded to those with BiP, a marker for endoplasmic reticulum (Fig. 6). N2a stable cells also showed similar patterns of ER localization of PS2 derivatives (data not shown).


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Fig. 6.   Immunofluorescence localization of truncated PS2 derivatives expressed in COS-1 cells. COS-1 cells transfected with WT fl (A and B), N141I mt fl PS2 (C), WT 270stop (D), WT 303stop (E), WT 388stop (F), N141I mt 270stop (G), N141I mt 303stop (H), N141I mt 388stop (I), 271ctf (J), 304ctf (K), or 344ctf (L) cDNAs were immunostained with appropriate anti-PS2 antibodies (A and C-L; the primary antibodies used are indicated in each panel) or doubly with an anti-BiP monoclonal antibody (B) and observed with a confocal microscope after labeling with fluorescein-conjugated anti-rabbit IgG secondary antibody (in A and B, together with Texas Red-conjugated anti-mouse IgG antibody). Scale bar, 10 µm.

Characterization of Abeta Secreted from Cells Expressing Truncated Forms of WT or N141I mt PS2-- We then quantitated the levels and percentages of Abeta 40 and Abeta 42 secreted from cells expressing truncated forms of N141I mt or WT PS2. In COS-1 cells doubly transfected with beta APP C100 and each of the three types of truncated mt PS2, the Abeta 42 in total Abeta was ~10% in all, and they were similar to those in cells with corresponding forms of truncated WT PS2, whereas Abeta 42 comprised 22.3% of total Abeta in cells expressing fl mt PS2, which was 1.7 times relative to that in cells expressing fl WT PS2 (13.5%). However, the total levels of Abeta were increased by ~2-fold in cells with truncated mt PS2 compared with those with truncated WT PS2. When CTFs of PS2 were transfected together with beta APP C100, the Abeta 42 ranged between 11.9 and 14.6%, which also was similar to cells with fl WT PS2 (Fig. 7A). Similar results were obtained in COS-1 cells doubly transfected with WT or Swedish-type mutant (i.e. 595/596 KM-NL) beta APP together with these PS2 derivatives (data not shown).


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Fig. 7.   Secreted Abeta 40 and Abeta 42 from cells expressing cDNAs encoding WT or mt truncated PS2 derivatives. Levels of Abeta x-40 and Abeta x-42 secreted from COS-1 cells doubly transfected with beta APP C100 and truncated PS2 genes (A) or N2a cells stably transfected with truncated PS2 genes (B) or co-transfected with NTF as well as CTF of PS2 (C) quantitated by two-site ELISAs. Mean values ± S.E. in four (A, B) or three (C) independent experiments are shown. Transfected cDNAs encoding PS2 derivatives are indicated below the columns; in C, stably expressed cDNAs for NTF and transiently transfected cDNAs for CTF (with "+") are shown in the lower and upper lanes, respectively. Vector, empty pcDNA3 vector.

In N2a cells stably expressing three types of truncated forms of N141I mt PS2, the percentage of Abeta 42 that comprised the total Abeta ranged between 14.2 and 18.0%, which was similar to those in cells with truncated (12.3-16.5%) or fl WT PS2 (20.5%), and the absolute amounts of secreted Abeta were at similar levels between cells expressing truncated WT or mt PS2 (Fig. 7B). This was in sharp contrast to the marked increase in the percentage or level of Abeta 42 from cells with fl mt PS2 (52.1%). The levels as well as percentages of Abeta 42 secreted from N2a cells expressing PS2 CTFs (10.1-14.2%) also were similar to those with fl WT PS2 or mock-transfected cells (Fig. 7B).

To examine if coexpression of mt PS2 NTF together with CTF reconstitutes overproduction of Abeta 42, we transiently transfected PS2/304ctf in N2a cells stably expressing WT or mt PS2/303stop. Upon co-transfection of PS2/304ctf, the total levels of secreted Abeta were decreased by ~50% both in WT and mt PS2/303stop stable cells relative to those in mock-transfected cells, whereas the Abeta 42 remained unchanged both in cells expressing WT (9.7% in double transfection versus 11.3% in mock transfection) and mt (11.1% in double transfection versus 10.3% in mock transfection) PS2/303stop (Fig. 7C).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In this study, we have clearly shown that (i) full-length PS2 harboring both N141I and M239V mutations in the same polypeptide induced overproduction of Abeta 42 at similar levels to those in cells expressing PS2 with one or the other of these PS2 mutations (i.e. N141I or M239V); (ii) NTFs or CTFs of PS2 expressed in cells predominantly localize in ER; and (iii) cells expressing partially truncated PS2 (eliminating the COOH-terminal third of PS2 while retaining the endoproteolytic NH2-terminal fragment) and harboring a N141I mutation, as well as cells expressing COOH-terminal fragments of PS2, did not overproduce Abeta 42, and the levels or percentages of Abeta 42 were comparable with those in cells that expressed full-length and wild-type PS2 as well as fragments thereof.

The nature of the structural or functional changes of the polytopic membrane protein PS2 caused by the two known mutations is not fully understood at present. Regarding the N141I Volga German PS2 mutation, a PS1 mutation at the homologous site (N135D) was reported (36), and these homologous residues in PS1 and PS2 are located at the NH2-terminal flank (designated N-cap position) of the second transmembrane (TM2) domain, which is believed to be important in the accurate positioning of the transmembrane alpha -helix structure (37). Another PS2 mutation of the Italian type (M239V) is situated within the TM5 domain; a PS1 mutation linked to FAD at the homologous site (M233T) also was documented (38), and substitution of Met for Val was observed in multiple residues in the TM2 domain of PS1 (i.e. M139V and M146V) (8), suggesting that Met to Val substitution may cause some common structural changes in the TM domains of PS1 or PS2. Our observation that the N141I/M239V double mutation did not have additive effects on the increase in the levels or percentages of secreted Abeta 42 suggests that Abeta 42-promoting capacities of mt PS2 proteins reach the maximum level with a given single amino acid substitution (i.e. N141I or M239V). This contrasts with the recent observation that the Abeta 42-promoting effects of M146L/L286V double mutant PS1 were additive (39) and also with the clinical observation that FAD patients with PS1 mutations develop AD at a uniformly early age, whereas the age of onset in Volga German families with the N141I PS2 mutation is variable and relatively late (40). The reason for these discrepancies is not clear at present. However, one should consider the differences in the protein levels of endogenous PS1 versus PS2 in the brains of FAD patients (10, 18). For example, it may be that the changes in Abeta 42-promoting effects of PS2 caused by a given single mutation per molecule is stronger than those with mt PS1, whereas the overall pathogenic effects of mt PS1 become more intense than those of mt PS2 because the total amount of PS1 proteins in neurons or brain tissues is higher than that of PS2.

NTFs or CTFs of PS2 of various sizes predominantly localized to ER. Recently, it has been shown that the NH2-terminal 166 residues, but not 138 residues, of PS2 are sufficient for the ER targeting, suggesting that the initial two transmembrane domains are necessary for ER localization (41). Our findings confirmed these observations with respect to the NTFs and further extended these data by showing that CTFs of PS2, including those corresponding to the COOH-terminal 103 amino acids (ALG-3) also localize to ER. Although the precise orientation of the membrane insertion of these CTFs is yet to be determined, the occurrence of "caspase-type" cleavage (32) of these CTFs in similar patterns to those observed in cells expressing fl PS2 suggests that the NH2-terminal portions (i.e. loop region of PS2) of these CTFs are properly oriented to the cytoplasmic side. Moreover, these CTFs harbor two transmembrane domains (i.e. TM7 and -8). Taken together, the COOH-terminal region may harbor other ER-targeting signal sequences besides those in the NH2-terminal region, or alternatively, the presence of multiple (i.e. more than two) TM domains, but not particular subregions, of PS2 may determine its ER localization.

The most unexpected, yet intriguing, finding in this study was that cells expressing COOH-terminally truncated N141I mt PS2 that are equivalent to or longer than the endoproteolytic NH2-terminal fragment did not overproduce Abeta 42. This was surprising because most of the PS proteins in native cells or tissues (including brains) exist as NTF and CTF forms, and the NTFs contain six of the eight TM domains of PS molecules. Recently, it has been suggested that the levels of PS within cells are strictly regulated by competition for limiting cellular factors (29). Moreover, it was shown that NTF and CTF of PS1 or PS2 remain noncovalently bound to each other after cleavage forming a very stable complex (25, 42, 43) and that they may form a 100-150-kDa molecular mass complex (43). Our finding that NTF of mt PS2 or CTF alone does not promote Abeta 42 overproduction supports the notion that the stable complex forms of PS NTF and CTF constitute the functional units under biological as well as pathological conditions. An alternative possibility would be that the nascent, full-length form of PS is functional and the cleavage at loop domain is a switch-off phenomenon. However, the observations that full-length PS is short-lived (44) and not readily incorporated into the stable complex (29, 43) and that full-length PS is rare in native cells (27) render this possibility rather unlikely. The COOH-terminally truncated forms of PS1 (29) or PS2 (i.e. PS2/388stop in this study), which contain the proteolytic cleavage sites, were not cleaved, and the truncated PS1 did not influence the stable complex formation in stably transfected N2a cells (29). Taken together with our results that mt PS2/388stop did not promote overproduction of Abeta 42, it is highly conceivable that the condition under which nascent PS proteins are stabilized and properly cleaved to produce NTF and CTF and form a stoichiometric stable complex is the prerequisite for the normal or pathological function of PS (29, 44). Our data that co-expression of mt PS2/303stop together with PS2/304ctf did not reconstitute overproduction of Abeta 42 further support the notion that the heterodimeric complex of NTF and CTF of PS derived from a full-length PS molecule is required for the pathological effects of PS mutations.

What is the nature of the limiting factor(s) that determines the integrity of the functional complex of PS molecules? Our finding that mt PS2/388stop, which comprises the NH2-terminal 87% of the entire length of PS2 as well as 7 of the 8 putative TM domains, failed to overproduce Abeta 42 strongly suggests that some critical subdomain in the COOH-terminal portion of PS2, or an as yet unidentified binding protein(s) that interacts with this domain, should play key roles in the formation of functional complex of PS. An alternative possibility would be that the integrity of the whole PS molecule, not particular subdomains, is required for the formation of the complex. Further efforts to define this subdomain of PS2 or identify binding proteins that are essential for the function as well as stabilization of PS should facilitate our understanding of the mechanisms whereby mt PS proteins influence the gamma -cleavage of beta APP to overproduce Abeta 42, thereby leading to AD.

    ACKNOWLEDGEMENTS

We thank A. Koyama and N. Takasugi for skillful technical assistance, G. Thinakaran for anti-PS2loop antiserum, J. Q. Trojanowski for helpful comments, D. J. Selkoe for making available preprints of manuscripts in press, and Takeda Chemical Industries for continuous support.

    FOOTNOTES

* This work was supported by grants-in-aid from the Ministry of Health and Welfare, Japan, and CREST of Japan Science and Technology Corporation.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.

parallel To whom correspondence should be addressed: Dept. of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo Bunkyoku Tokyo 113-0033, Japan. Tel.: 81-3-5689-7255; Fax: 81-3-5689-7255; E-mail: iwatsubo{at}mol.f.u-tokyo.ac.jp.

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta  peptide; beta APP, beta -amyloid precursor protein; CTF, carboxyl-terminal fragment; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; N2a, mouse neuro2a neuroblastoma; NTF, amino-terminal fragment; PS, presenilin; TM, transmembrane; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferaseFAD, familial Alzheimer's diseaseWT, wild typemt, mutantfl, full-lengthPBS, phosphate-buffered salineAbeta 42, species of Abeta ending at residue 42PCR, polymerase chain reaction.

2 T. Tomita and T. Iwatsubo, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Selkoe, D. J. (1994) Annu. Rev. Neurosci. 17, 489-517[CrossRef][Medline] [Order article via Infotrieve]
  2. 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., DaSilva, 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]
  3. Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D. M., Oshima, J., Pettingell, W. H., Yu, C.-e., 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]
  4. Kovacs, D. M., Fausett, H. J., Page, K. J., Kim, T. W., Moir, R. D., Merriam, D. E., Hollister, R. D., Hallmark, O. G., Mancini, R., Felsenstein, K. M., Hyman, B. T., Tanzi, R. E., and Wasco, W. (1996) Nat. Med. 2, 224-229[Medline] [Order article via Infotrieve]
  5. Cook, D. G., Sung, J. C., Golde, T. E., Felsenstein, K. M., Wojczyk, B. S., Tanzi, R. E., Trojanowski, J. Q., Lee, V. M.-Y., and Doms, R. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9223-9228[Abstract/Free Full Text]
  6. Walter, J., Capell, A., Grünberg, J., Pesold, B., Schindzielorz, A., Prior, R., Podlisny, M. B., Fraser, P., St George-Hyslop, P., Selkoe, D. J., and Haass, C. (1996) Mol. Med. 2, 673-691[Medline] [Order article via Infotrieve]
  7. Doan, A., Thinakaran, G., Borchelt, D. R., Slunt, H. H., Ratovitsky, T., Podlisny, M., Selkoe, D. J., Seegar, M., Gandy, S. E., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 1023-1030[Medline] [Order article via Infotrieve]
  8. Hardy, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2095-2097[Free Full Text]
  9. Perez-Tur, J., Froelich, S., Prihar, G., Crook, R., Baker, M., Duff, K., Wragg, M., Busfield, F., Lendon, C., Clark, R. F., Roques, P., Fuldner, R. A., Johnston, J., Cowburn, R., Forsell, C., Axelman, K., Lilius, L., Houlden, H., Karran, E., Roberts, G. W., Rossor, M., Adams, M. D., Hardy, J., Goate, A., Lannfelt, L., and Hutton, M. (1995) Neuroreport 7, 297-301[Medline] [Order article via Infotrieve]
  10. Rogaev, E. I., Sherrington, R., Rogaeva, E. A., Levesque, G., Ikeda, M., Liang, Y., Chi, H., Lin, C., Holman, K., Tsuda, T., Mar, L., Sorbi, S., Nacmias, B., Piacentini, S., Amaducci, L., Chumakov, I., Cohen, D., Lannfelt, L., Fraser, P. E., Rommens, J. M., and St George-Hyslop, P. H. (1995) Nature 376, 775-778[CrossRef][Medline] [Order article via Infotrieve]
  11. Levitan, D., Doyle, T. G., Brousseau, D., Lee, M. K., Thinakaran, G., Slunt, H. H., Sisodia, S. S., and Greenwald, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14940-14944[Abstract/Free Full Text]
  12. Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes Function 1, 149-159[Medline] [Order article via Infotrieve]
  13. Wong, P. C., Zheng, H., Chen, H., Becher, M. W., Sirinathsinghji, D. J., Trumbauer, M. E., Chen, H. Y., Price, D. L., Van der Ploeg, L. H., and Sisodia, S. S. (1997) Nature 387, 288-292[CrossRef][Medline] [Order article via Infotrieve]
  14. Shen, J., Bronson, R. T., Chen, D. F., Xia, W., Selkoe, D. J., and Tonegawa, S. (1997) Cell 89, 629-639[Medline] [Order article via Infotrieve]
  15. 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]
  16. 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]
  17. 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]
  18. Tomita, T., Maruyama, K., Saido, T. C., Kume, H., Shinozaki, K., Tokuhiro, S., Capell, A., Walter, J., Grünberg, J., Haass, C., Iwatsubo, T., and Obata, K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2025-2030[Abstract/Free Full Text]
  19. Jarrett, J. T., Berger, E. P., and Lansbury, P. T., Jr. (1993) Biochemistry 32, 4693-4697[Medline] [Order article via Infotrieve]
  20. 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]
  21. Iwatsubo, T., Odaka, A., Suzuki, N., Mizusawa, H., Nukina, N., and Ihara, Y. (1994) Neuron 13, 45-53[Medline] [Order article via Infotrieve]
  22. 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]
  23. Weidemann, A., Paliga, K., Durrwang, U., Czech, C., Evin, G., Masters, C. L., and Beyreuther, K. (1997) Nat. Med. 3, 328-332[Medline] [Order article via Infotrieve]
  24. Xia, W., Zhang, J., Perez, R., Koo, E. H., and Selkoe, D. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8208-8213[Abstract/Free Full Text]
  25. Thinakaran, G., Regard, J. B., Bouton, C. M. L., Harris, C. L., Price, D. L., Borchelt, D. R., and Sisodia, S. S. (1998) Neurobiol. Dis. 4, 438-453[CrossRef][Medline] [Order article via Infotrieve]
  26. Xia, W., Zhang, J., Kholodenko, D., Citron, M., Podlisny, M. B., Teplow, D. B., Haass, C., Seubert, P., Koo, E. H., and Selkoe, D. J. (1997) J. Biol. Chem. 272, 7977-7982[Abstract/Free Full Text]
  27. Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190[Medline] [Order article via Infotrieve]
  28. Saido, T. C., Yokota, M., Maruyama, K., Yamao-Harigaya, W., Tani, E., Ihara, Y., and Kawashima, S. (1994) J. Biol. Chem. 269, 15253-15257[Abstract/Free Full Text]
  29. Thinakaran, G., Harris, C. L., Ratovitski, T., Davenport, F., Slunt, H. H., Price, D. L., Borchelt, D. R., and Sisodia, S. S. (1997) J. Biol. Chem. 272, 28415-28422[Abstract/Free Full Text]
  30. Baba, M., Nakajo, S., Tu, P.-H., Tomita, T., Nakaya, K., Lee, V. M.-Y., Trojanowski, J. Q., and Iwatsubo, T. (1998) Am. J. Pathol. 152, 879-884[Abstract]
  31. Asami-Odaka, A., Ishibashi, Y., Kikuchi, T., Kitada, C., and Suzuki, N. (1995) Biochemistry 34, 10272-10278[Medline] [Order article via Infotrieve]
  32. Kim, T.-W., Pettingell, W. H., Jung, Y. K., Kovacs, D. M., and Tanzi, R. E. (1997) Science 277, 373-376[Abstract/Free Full Text]
  33. Podlisny, M. B., Citron, M., Amarante, P., Sherrington, R., Xia, W., Zhang, J., Diehl, T., Levesque, G., Fraser, P., Haass, C., Koo, E. H., Seubert, P., St George-Hyslop, P., Teplow, D. B., and Selkoe, D. J. (1997) Neurobiol. Dis. 3, 325-337[CrossRef][Medline] [Order article via Infotrieve]
  34. Shirotani, K., Takahashi, K., Ozawa, K., Kunishita, T., and Tabira, T. (1997) Biochem. Biophys. Res. Commun. 240, 728-731[CrossRef][Medline] [Order article via Infotrieve]
  35. Vito, P., Lacana, E., and D'Adamio, L. (1996) Science 271, 521-525[Abstract]
  36. Crook, R., Ellis, R., Shanks, M., Thal, L. J., Perez-Tur, J., Baker, M., Hutton, M., Haltia, T., Hardy, J., and Galasko, D. (1997) Ann. Neurol. 42, 124-128[Medline] [Order article via Infotrieve]
  37. Richardson, J. S., and Richardson, D. C. (1988) Science 240, 1648-1652[Medline] [Order article via Infotrieve]
  38. Kwok, J. B., Taddei, K., Hallupp, M., Fisher, C., Brooks, W. S., Broe, G. A., Hardy, J., Fulham, M. J., Nicholson, G. A., Stell, R., St George-Hyslop, P. H., Fraser, P. E., Kakulas, B., Clarnette, R., Relkin, N., Gandy, S. E., Schofield, P. R., and Martins, R. N. (1997) Neuroreport 8, 1537-1542[Medline] [Order article via Infotrieve]
  39. Citron, M., Eckman, C. B., Diehl, T. S., Corcoran, C., Ostaszewski, B. L., Xia, W., Levesque, G., St George-Hyslop, P., Younkin, S. G. & Selkoe, D. J. (1998) Neurobiol. Dis., in press
  40. Mann, D. M. A., Iwatsubo, T., Nochlin, D., Sumi, S. M., Levy-Lahad, E., and Bird, T. D. (1997) Ann. Neurol. 41, 52-57[Medline] [Order article via Infotrieve]
  41. Janicki, S., and Monteiro, M. J. (1997) J. Cell Biol. 139, 485-495[Abstract/Free Full Text]
  42. Seeger, M., Nordstedt, C., Petanceska, S., Kovacs, D. M., Gouras, G. K., Hahne, S., Fraser, P., Levesque, L., Czernik, A. J., St George-Hyslop, P., Sisodia, S. S., Thinakaran, G., Tanzi, R. E., Greengard, P., and Gandy, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5090-5094[Abstract/Free Full Text]
  43. Capell, A., Grünberg, 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]
  44. Ratovitski, T., Slunt, H. H., Thinakaran, G., Price, D. L., Sisodia, S. S., and Borchelt, D. R. (1997) J. Biol. Chem. 272, 24536-24541[Abstract/Free Full Text]


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