COMMUNICATION
The Biological and Pathological Function of the Presenilin-1 Delta Exon 9 Mutation Is Independent of Its Defect to Undergo Proteolytic Processing*

Harald SteinerDagger , Helmut Romig§, Melissa G. Grim, Uwe Philipp§, Brigitte PesoldDagger , Martin Citronparallel , Ralf Baumeister**, and Christian HaassDagger **

From the Dagger  Central Institute of Mental Health, Department of Molecular Biology, J5, 68159 Mannheim, Germany, § Boehringer Ingelheim KG, CNS Research, 55216 Ingelheim, Germany,  Genzentrum, Feodor-Lynen-Str.25, 81377 Munich, Germany, and parallel  Amgen Inc., Thousand Oaks, California 91320-1789

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

The two homologous presenilins are key factors for the generation of amyloid beta -peptide (Abeta ), since Alzheimer's disease (AD)-associated mutations enhance the production of the pathologically relevant 42-amino acid Abeta (Abeta 42), and a gene knockout of presenilin-1 (PS1) significantly inhibits total Abeta production. Presenilins undergo proteolytic processing within the domain encoded by exon 9, a process that may be closely related to their biological and pathological activity. An AD-associated mutation within the PS1 gene deletes exon 9 (PS1Delta exon9) due to a splicing error and results in the accumulation of the uncleaved full-length protein. We now demonstrate the unexpected finding that the pathological activity of PS1Delta exon9 is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) occurring at the aberrant exon 8/10 splice junction. Mutagenizing the cysteine residue at position 290 to the original serine residue completely inhibits the pathological activity in regard to the elevated production of Abeta 42. Like PS1Delta exon9, the resulting presenilin variant (PS1Delta exon9 C290S) accumulates as an uncleaved protein and fully replaces endogenous presenilin fragments. Moreover, PS1Delta exon9 C290S exhibits a significantly increased biological activity in a highly sensitive in vivo assay as compared with the AD-associated mutation. Therefore not only the increased Abeta 42 production but also the decreased biological function of PS1Delta exon9 is due to a point mutation and independent of the lack of proteolytic processing.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Early onset Alzheimer's disease (AD)1 can occur due to mutations within the genes encoding the beta -amyloid precursor protein (beta APP) and the two presenilins, PS1 and PS2 (Refs. 1-3; summarized in Refs. 4 and 5). The mutations increase the generation of the 42-amino acid version of Abeta (Abeta 42), which aggregates more readily (6) and is therefore preferentially deposited in senile plaques (5). PS proteins are also involved in the physiological production of Abeta , since the lack of PS1 expression in PS1-/- mice results in a dramatically reduced Abeta production (7). All but one PS mutations are point mutations affecting conserved amino acids (4, 5). However, due to a splicing error, the PS1Delta exon9 mutation results in the deletion of the domain encoded by exon 9 (8). This domain contains the cleavage site for proteolytic processing (9, 10), and therefore PS1Delta exon9 accumulates as an uncleaved protein (9). Since proteolytic processing is highly regulated (9, 11, 12) and appears to be altered by PS mutations (Refs. 13-15; summarized in Ref. 16), the lack of proteolytic processing caused by the exon 9 deletion was expected to be responsible for its pathological activity. We now demonstrate the unexpected finding that the pathological function of PS1Delta exon9 as well as its reduced biological activity is independent of its lack to undergo proteolytic processing, but is rather due to a point mutation (S290C) that is the result of the aberrant exon 8/10 splice junction.

    EXPERIMENTAL PROCEDURES

Cell Culture and Cell Lines-- K293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 200 µg/ml G418 (to select for beta APP expression) and 200 µg/ml zeocin (to select for presenilin expression). K293 cells stably expressing PS1Delta exon9 C290S were generated by transfection of K293 cells stably expressing beta APP containing the Swedish mutation (17). K293 cells stably transfected with Swedish beta APP695, wt PS1, and PS1Delta exon9 were described previously (18).

Construction of the cDNA Encoding PS1Delta Exon9 C290S-- The cDNA encoding PS1Delta exon9 C290S was constructed by mutagenizing the cysteine at codon 290 of the PS1Delta exon9 cDNA (8) to serine in a two-step PCR procedure. The following primers were designed: first PCR, PS1-187-F (5'-CCGAATTCAAGAAAGAACCTCAA-3') and Delta E9-C290S-R (5'-GTGACTCCCTTTCTGTGGAGGAGTAAATGAGAGC-3'); second PCR, Delta E9-C290S-F (5'-GCTCTCATTTACTCCTCCACAGAAAGGGAGTCAC-3') and PS1-STOP-R (5'-CGCCTCGAGGCAAATATGCTAGATATA-3').

After gel purification the PCR products were mixed and subjected to a final PCR with primers PS1-187-F and PS1-STOP-R. The resulting PCR product was digested with EcoRI/XhoI and cloned into the pcDNA3.1/Zeo(+) expression vector (Invitrogen). The cDNA was sequenced to verify successful mutagenesis.

Antibodies-- The polyclonal antibodies against amino acids 263-407 of PS1 (3027) and amino acids 297-356 of PS2 (3711) were described previously (19, 20). The monoclonal antibodies to the PS1 and PS2 loop were raised against fusion proteins containing amino acids 263-407 (BI.3D7) or 297-356 (BI.HF5C).

Metabolic Labeling and Immunoprecipitation of PS-- Stably transfected K293 cell lines were grown to confluence in 10-cm dishes. After starvation for 1 h in 4 ml of methionine- and serum-free MEM (MEM supplemented with 1% L-glutamine and 1% penicillin/streptomycin) cells were metabolically labeled with 700 µCi of [35S]methionine (Promix, Amersham Pharmacia Biotech) in 4 ml of methionine- and serum-free MEM for 1 h. Cell extracts were prepared and subjected to immunoprecipitation of PS as described (12). PS immunoprecipitates were solubilized in sample buffer containing 4 M urea for 10 min at 65 °C, separated on SDS-urea gels, and analyzed by fluorography (19).

Combined Immunoprecipitation/Western Blotting-- Stably transfected K293 cell lines were grown to confluence. Cell extracts were prepared and subjected to immunoprecipitation as described (12). Following gel electrophoresis, immunoprecipitated proteins were identified by immunoblotting (12). Bound antibodies were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).

Analysis of Abeta 40 and Abeta 42-- Culture medium (2 ml) was collected from confluent K293 cells grown in six-well dishes for 24 h. The medium was assayed for Abeta 40 and Abeta 42 using a previously described ELISA assay (12). Abeta peptides were immunoprecipitated using antibody 6E10 (Senetek) according to established protocols (21).

Transgenic Lines of Caenorhabditis elegans and Rescue Assays-- Expression constructs were generated as described previously (22). Transgenic lines were established by microinjection of plasmid DNA mixtures into the C. elegans germ line to create extrachromosomal arrays as described previously (22). All plasmids used in this study were injected at a concentration of 20 ng/µl into sel-12(ar171) or sel-12(ar171) unc-1(e538) hermaphrodites along with ttx-3:GFP as a cotransformation marker. Successful transformation was monitored by the expression of GFP in the AIY interneurons of F1 and F2 generation animals. Four independent lines from the progeny of F2 generation animals were established.

As the sel-12(ar171) animals never lay eggs (23), rescue of the sel-12 defect can be quantified by scoring egg laying behavior in transgenic animals (22, 24). Consequently, for each transgenic line, we examined 50 transgenic animals for their ability to lay eggs and determined their brood size. The number of eggs laid by individual transgenic animals was counted and placed into four categories: "Egl+++," robust egg laying, more than 30 eggs laid (wt phenotype); "Egl++," 15-30 eggs laid; "Egl+," 5-15 eggs laid; "Egl-," no eggs laid.

    RESULTS

The PS1Delta exon9 mutation changes a G to a T at the splice site for exon 9. It therefore destroys the minimal required consensus sequence for the splice acceptor site (8), which then results in an aberrant deletion of the domain encoded by exon 9 (Ref. 8; Fig. 1A). However, the mutation also changes codon 290 at the exon 8/10 splice junction from a serine to a cysteine (Ref. 8; Fig. 1A). We therefore have investigated if the single amino acid exchange or the deletion of the domain required for proteolytic processing is responsible for the pathological activity of the PS1Delta exon9 mutation in regard to Abeta 42 generation. For this purpose we mutagenized amino acid 290 of PS1Delta exon9 to a serine (PS1Delta exon9 C290S), thus correcting the point mutation (Fig. 1A). cDNAs encoding PS1Delta exon9 and PS1Delta exon9 C290S were stably transfected into kidney 293 cells overexpressing beta APP containing the Swedish mutation. This cell line was used previously to determine the pathological effect of PS mutations on Abeta production (12, 18). Untransfected K293 cells expressing endogenous PS, as well as cell lines overexpressing PS1Delta exon9 and PS1Delta exon9 C290S were pulse-labeled with [35S]methionine for 1 h, and cell lysates were immunoprecipitated with antibody 3027 to the large loop of PS1. Both PS1Delta exon9 and PS1Delta exon9 C290S accumulated as full-length proteins (Fig. 1B). As reported previously (9, 10) very little endogenous full-length PS1 could be detected in the untransfected cell line.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   Generation and expression of PS1Delta exon9 and PS1Delta exon9 C290S. A, schematic representation of the genomic structure (exon 8 to exon 10) of wt PS1, PS1Delta exon9 and the amino acid sequence at the splice junctions of exons 8, 9, and 10. The nucleotide exchange (G to T) in the PS1 gene identified in the DNA of patients with the PS1Delta exon9-linked mutation results in an amino acid exchange at the exon 8/10 splice junction, which changes codon 290 from a serine to a cysteine (bold letters). B, K293 cells expressing endogenous presenilins (control) as well as K293 cells stably overexpressing PS1Delta exon9, or PS1Delta exon9 C290S were pulse labeled with [35S]methionine for 1 h, and cell lysates were immunoprecipitated with antibody 3027 (19) to the large loop of PS1. Full-length PS (fl-PS) proteins accumulate in the cell lines expressing PS1Delta exon9 and PS1Delta exon9 C290S but not in cells expressing endogenous presenilins. The asterisk indicates dimeric forms of PS1 (19). C and D, stable expression of PS1Delta exon9 and PS1Delta exon9 C290S inhibits formation of endogenous PS1 (C) and PS2 C-terminal fragments (CTF) (D). Cell lysates from K293 cells and cell lines overexpressing PS1Delta exon9 and PS1Delta exon9 C290S were immunoprecipitated with the previously described antibodies (see "Experimental Procedures") specific to the large loop of PS1 (3027; Ref. 19) and PS2 (3711; Ref. 20) and detected by immunoblotting using the monoclonal antibodies BI.3D7 and BI.HF5C.

It has been shown before that overexpression of PS proteins results in the replacement of endogenous PS fragments and the accumulation of PS1Delta exon9 prevents the formation of stable PS fragments (9, 11, 12). To prove that overexpression of PS1Delta exon9 C290S still allows the reduction of endogenous PS fragments, cell lysates from unlabeled cells were immunoprecipitated with antibodies specific to the PS1 or PS2 loop domain (see "Experimental Procedures"), and PS fragments were identified with the PS1/PS2-specific monoclonal antibodies BI.3D7 and BI.HF5C (see "Experimental Procedures"). Whereas cell lines expressing endogenous presenilins produced PS1 as well as PS2 C-terminal fragments (Fig. 1, C and D), overexpression of PS1Delta exon9 and PS1Delta exon9 C290S inhibited the formation of endogenous PS1 and PS2 fragments (Fig. 1, C and D). This demonstrates that PS1Delta exon9 C290S, like PS1Delta exon9, fully replaced presenilins derived from the endogenous genes and also proves that PS1Delta exon9 C290S does not undergo proteolytic processing but rather accumulates as an uncleaved full-length protein.

In order to investigate the pathological activity of the mutant PS derivatives on Abeta production, control cells as well as cells overexpressing PS1Delta exon9 and PS1Delta exon9 C290S were metabolically labeled with [35S]methionine, and Abeta 40 and Abeta 42 peptides were immunoprecipitated from the conditioned medium using antibody 6E10. This antibody is raised to Abeta 1-17 and therefore recognizes Abeta 40 as well as Abeta 42 (see "Experimental Procedures"). Isolated Abeta peptides were then separated on a previously described gel system, which allows the specific resolution of Abeta 40 and Abeta 42 (25). As shown in Fig. 2A, cells overexpressing PS1Delta exon9 secreted elevated levels of Abeta 42 as compared with control cells. In contrast, the cell line stably transfected with PS1Delta exon9 C290S produced significantly lower amounts of Abeta 42 as cells expressing the Alzheimer's disease-associated PS1Delta exon9 mutation. This suggests that the pathological activity of the PS1Delta exon9 mutation is due to the point mutation generated at the aberrant splice junction. In order to quantitate Abeta 42 and Abeta 40 production, we used a previously described specific ELISA (12). In this assay, two highly specific monoclonal antibodies are used for the discrimination of both peptide species (12). As reported before (12, 18, 26), the PS1Delta exon9 mutation results in an approximately 3-fold increase of the Abeta 42/Abeta total ratio (Fig. 2B). In contrast, the cell line stably overexpressing PS1Delta exon9 C290S showed no increased Abeta 42/Abeta total ratio (Fig. 2B). Therefore using two different approaches we can show that the pathological activity of PS1Delta exon9 is independent of its lack of proteolytic processing but is rather caused by the single amino acid change at the aberrant splice junction.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.   The pathological activity of PS1Delta exon9 is due to a point mutation at codon 290. A, Abeta production of K293 cells expressing endogenous presenilins (control (Ctrl.)) and cell lines overexpressing PS1Delta exon9 or PS1Delta exon9 C290S. Conditioned medium from metabolically labeled kidney 293 cells expressing the indicated presenilin variants was immunoprecipitated with antibody 6E10. Abeta 40 and Abeta 42 were separated on a previously described gel system, which allows the specific resolution of both species (25). As reported before, Abeta 42 migrates faster as Abeta 40 (25). Note that cells expressing PS1Delta exon9 produced increased amounts of Abeta 42, whereas cells expressing PS1Delta exon9 C290S do not show such an increase. Two individual immunoprecipitations of each cell lysate are shown. B, quantitation of the Abeta 42 and Abeta 40 concentrations in conditioned medium of kidney 293 cells expressing the indicated presenilin variants using a previously described highly specific ELISA (12). Expression of PS1Delta exon9 but not the expression of PS1Delta exon9 C290S results in a 3-fold increase of Abeta 42 production. Identical results were observed with independent cell clones.

Having demonstrated that PS1Delta exon9 C290S is pathologically inactive, we also wanted to test if this protein is sufficient to rescue a lin-12-mediated signaling defect that is a result of a mutant PS homologue (sel-12) in Caenorhabditis elegans (23). As reported previously, transgenic expression of human presenilins rescues the phenotype caused by mutations of sel-12 (22, 24). In contrast, transgenic expression of the PS1Delta exon9 variant in C. elegans resulted in an incomplete rescue of the sel-12 mutant phenotype, as indicated by a significantly reduced brood size and reduced egg laying (Refs. 22 and 24; see also Table I). In order to assess the in vivo function of PS1Delta exon9 C290S, we tested its ability to rescue the putative sel-12 null allele ar171 (23). Four independent transgenic strains expressing PS1Delta exon9 C290S displayed robust egg laying (Table I). Based on both numbers of laid progeny and numbers of eggs in utero, the phenotype of these strains is almost indistinguishable of that of wild type worms (Table I). These results demonstrate that PS1Delta exon9 C290S rescues the egg laying phenotype of the sel-12(ar171) mutant animals significantly better than PS1Delta exon9 (Table I). Therefore, the reduced biological activity of PS1Delta exon9 is also due to a single point mutation and completely independent of the lack of proteolytic processing.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Rescue of the sel-12 egg laying defect by human PS genes expressed from the sel-12 promoter
For 50 transgenic animals each, the numbers of progeny were counted and grouped in the following categories: +++, over 30 progeny laid by individual animal; ++, 15-30 progeny laid; +, 5-15 progeny laid; -, no progeny laid. The sel-12(ar171) strains carried an additional unc-1(e538) marker that did not affect egg laying or rescuing frequency. It was backcrossed several times from the strain originally published (23).


    DISCUSSION

Our results demonstrate that the pathological effect of the PS1Delta exon9 mutation is independent of its lack of proteolytic cleavage and the deletion of the exon 9-encoded domain. Reverting the cysteine residue generated by the exon 8/10 splice junction at codon 290 back to its wt residue (serine) still inhibits its proteolytic processing, causes an accumulation of the uncleaved protein, and prevents formation of endogenous PS fragments. Although the artificially generated PS1Delta exon9 C290S variant behaves like PS1Delta exon9 in regard to the characteristic biochemical features described above, it does not allow pathological overproduction of Abeta 42. Moreover, PS1Delta exon9 C290S regains full biological activity in a very sensitive in vivo assay system. This demonstrates that not only the pathological overproduction of Abeta 42 but also the reduced biological function is due to the single amino acid exchange. Therefore, similar to all other known PS mutations, the pathological effect of the PS1Delta exon9 mutation is due to a rather subtle amino acid exchange at a single highly conserved codon whereas the large deletion of the complete domain encoded by exon 9 does not affect the biological and pathological function. However, consistent with our previous results (27), we suggest that PS molecules lacking the domain encoded by exon 9 mimic a proteolytically processed and biologically active PS complex and therefore rescue the phenotype of the mutant nematode. Consequently, the rescuing activity of PS1Delta exon9 is significantly enhanced when the pathologically relevant point mutation at codon 290 is reverted to the wt residue.

It remains to be shown if the mutation at codon 290 is also pathologically active within the full-length protein like all other AD-associated point mutations. It may however be possible that the mutation only exhibits a pathological activity if it is aberrantly flanked by the domains encoded by exons 8 and 10. Structural changes that may specifically occur in the PS1Delta exon9 molecule (12, 19) might be responsible for the pathological activity of this very unusual mutation.

Based on our results it would be interesting to investigate if the point mutations in the PS genes require the endoproteolytic cleavage for their pathological activity. Therefore artificial PS molecules should be generated containing an AD associated mutation as well as the smallest possible alteration of the sequence at the cleavage site which would inhibit PS processing. However, such mutations might be very difficult to generate since PS can be cleaved at multiple sites (10, 12) and PS molecules containing larger deletions at the cleavage sites might mimic a proteolytically processed PS molecule (27).

    ACKNOWLEDGEMENTS

We thank Roland Donhauser for injecting PS constructs into C. elegans and Drs. Bernard Lakowski and Helmut Jacobsen for critical discussion.

    Note Added in Proof

While this manuscript was in press, we found that expression of PS1Delta exon9 C290S containing a FAD-associated mutation (M146L) results in elevated Abeta 42 production, in further support of the findings presented here.

    FOOTNOTES

* This work was supported by grants from the Verum Foundation (Abeta 42 generation) and the Deutsche Forschungsgemeinschaft (DFG) (to C. H. and R. B.).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.

** To whom correspondence should be addressed: Central Institute of Mental Health, Dept. of Molecular Biology, J5, 68159 Mannheim, Germany. Tel.: 49-621-1703-884; Fax: 49-621-23429; E-mail: Haass{at}as200.zi-mannheim.de (for C. H.) or Genzentrum, Feodor-Lynen-Str.25, 81377 Munich, Germany. Tel.: 49-89-7401-7347; Fax: 49-89-7401-7314; E-mail: bmeister{at}LMB.uni-muenchen.de (for R. B., for correspondence regarding C. elegans).

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta -peptide; beta APP, beta amyloid precursor protein; PS, presenilin; wt, wild type; PCR, polymerase chain reaction; MEM, minimal essential medium; ELISA, enzyme-linked immunosorbent assay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. 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]
  2. 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]
  3. 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]
  4. Price, D., and Sisodia, S. (1998) Annu. Rev. Neurosci. 21, 479-505[CrossRef][Medline] [Order article via Infotrieve]
  5. Selkoe, D. J. (1996) J. Biol. Chem. 271, 18295-18298[Free Full Text]
  6. Jarret, J. T., and Lansbury, P. T., Jr. (1993) Cell 73, 1055-1058[Medline] [Order article via Infotrieve]
  7. 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]
  8. Perez-Tur, J., Froehlich, 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]
  9. 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]
  10. Podlisny, M., Citron, M., Amarante, P., Sherrington, R., Xia, W., Zhang, J., Diehl, T., Levesque, G., Fraser, P., Haass, C., Koo, E., Seubert, P., St. George-Hyslop, P., Teplow, D., and Selkoe, D. (1997) Neurobiol. Dis. 3, 325-337[CrossRef][Medline] [Order article via Infotrieve]
  11. 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]
  12. Steiner, H., Capell, A., Pesold, B., Citron, M., Kloetzel, P.-M., Selkoe, D., Romig, H., Mendla, K., and Haass, C. (1998) J. Biol. Chem. 273, 32322-32331[Abstract/Free Full Text]
  13. Lee, M. L., Borchelt, D. R., Kim, G., Thinakaran, G., Slunt, H., Ratovitski, T., Martin, L. J., Kittur, A., Gandy, S., Levey, A., Jenkins, N., Copeland, N., Price, D. L., and Sisodia, S. (1997) Nat. Med. 3, 756-760[Medline] [Order article via Infotrieve]
  14. Mercken, M., Takahashi, H., Honda, T., Sato, K., Murayama, M., Nakazato, Y., Noguchi, K., Imahori, K., and Takashima, A. (1996) FEBS Lett. 389, 297-303[CrossRef][Medline] [Order article via Infotrieve]
  15. Murayama, O., Honda, T., and Mercken, M. (1997) Neurosci. Lett. 229, 61-64[CrossRef][Medline] [Order article via Infotrieve]
  16. Grünberg, J., Capell, A., Leimer, U., Steiner, B., Steiner, H., Walter, J., and Haass, C. (1997) Alzheimer's Res. 3, 253-259
  17. Citron, M., Oltersdorf, T., Haass, C., McConlogue, 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. Citron, M., Westaway, D., Xia, W., Carlson, G., Diehl, T. S., 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. (1997) Nat. Med. 3, 67-72[Medline] [Order article via Infotrieve]
  19. Walter, J., Grünberg, J., Capell, A., Pesold, B., Schindzielorz, A., Citron, M., Mendla, K., St. George-Hyslop, P., Multhaup, G., Selkoe, D. J., and Haass, C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5349-5354[Abstract/Free Full Text]
  20. Walter, J., Grünberg, J., Schindzielorz, A., and Haass, C. (1998) Biochemistry 37, 5961-5967[CrossRef][Medline] [Order article via Infotrieve]
  21. Haass, C., Schlossmacher, M., Hung, A. Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B., Lieberburg, I., Koo, E., Schenk, D., Teplow, D., and Selkoe, D. J. (1992) Nature 359, 322-325[CrossRef][Medline] [Order article via Infotrieve]
  22. Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes Funct. 1, 149-159[Medline] [Order article via Infotrieve]
  23. Levitan, D., and Greenwald, I. (1995) Nature 377, 351-354[CrossRef][Medline] [Order article via Infotrieve]
  24. Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and Greenwald, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14940-14944[Abstract/Free Full Text]
  25. Klafki, H.-W., Wilfang, J., and Staufenbiel, M. (1996) Anal. Biochem. 237, 24-29[CrossRef][Medline] [Order article via Infotrieve]
  26. Borchelt, D., Thinakaran, G., Eckman, C., Lee, M., Davenport, F., Ratovitsky, T., Prada, C.-M., Kim, G., Seekins, S., Yager, D., Slunt, H., Wang, R., Seeger, M., Levey, A., Gandy, S., Copeland, N., Jenkins, N., Price, D., Younkin, S., and Sisodia, S. S. (1996) Neuron 17, 1005-10013[Medline] [Order article via Infotrieve]
  27. Capell, A., Grünberg, J., Pesold, B., Diehlmann, A., Citron, M., Nixon, R., Beyreuther, K., Selkoe, D., and Haass, C. (1998) J. Biol. Chem. 273, 3205-3211[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.