Article |
Address correspondence to Wim Annaert and Bart De Strooper, Center for Human Genetics, Neuronal Cell Biology Group, Herestraat 49, B-3000 Leuven, Belgium. Tel.: (32) 16-346-27. Fax: (32) 16-347-181. E-mail: ad{at}med.kuleuven.ac.be
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: presenilin 1; amyloid peptide; -secretase; ER retention; APP processing
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several lines of evidence have led to the hypothesis that PS is the -secretase. First,
-secretase cleavage of APP and Notch is inactivated partially in PS1-deficient cells and completely in PS1/PS2 doubledeficient cells (De Strooper et al., 1998, 1999; Song et al., 1999; Struhl and Greenwald, 1999; Herreman et al., 2000; Zhang et al., 2000). Moreover, mutagenesis of one or two aspartyl residues located in transmembrane domain 6 or 7 results in dominant negativelike effects on
-secretase cleavage, suggesting that these aspartates contribute to the active site of PS (Wolfe et al., 1999). Some similarities with the prepilin peptidases has been taken as further evidence that the PS are proteases (Steiner et al., 2000). However, the strongest argument in favor for this idea comes from the observation that several compounds that inhibit
-secretase bind specifically to PS (Esler et al., 2000; Li et al., 2000; Seiffert et al., 2000). Nevertheless, direct proof that the PS can hydrolyze peptide bounds is still lacking.
Remarkably, both Notch and APP apparently first need proteolytical trimming of their extracellular domain by TACE and -/ß-secretase, respectively, to become a substrate for PS/
-secretase (De Strooper et al., 1998; Brou et al., 2000; Mumm et al., 2000; Struhl and Adachi, 2000). These proteases are located downstream in the biosynthetic pathway and therefore both Notch and APP first have to leave the ER to become a substrate for PS/
-secretase. Despite significant advances in our understanding of
-secretase processing, it remains unclear how APP, after its cleavage by
- or ß-secretase, becomes exposed to PS. Moreover, Aß appears to be produced mainly in the endosomal compartment (Koo and Squazzo, 1994; Hartmann et al., 1997), whereas PS is abundantly present in the ER and intermediate compartment (Walter et al., 1996; Culvenor et al., 1997; Annaert et al., 1999; Kim et al., 2000). This problem can be summarized as "the spatial paradox" (Annaert and De Strooper, 1999) and we now tackle this problem further by analyzing the processing of a series of APP-trafficking mutants in primary cortical neurons derived from PS1+/+ or PS1-/- mice. We analyzed the consequences of restricting APP trafficking to certain subcellular compartments for the generation of total Aß and Aß42. Constructs tested: (a) APP with deleted cytosolic tail (APP-
ct); (b) APP with a double lysine ER retention motif in its cytoplasmic tail (APP-KK); (c) APP with a cytoplasmic tail from the LDL receptor (APP-LDL); (d) APP truncated at the ß-secretase cleavage site (APP-C99); and (e) APP-C99 with a double lysine (KK) motif. The APP-
ct is poorly reinternalized and stays for longer times at the cell surface (Tienari et al., 1996). The KK motif acts an ER-retrieval signal (Jackson et al., 1993; Gaynor et al., 1994) and therefore APP-KK colocalizes abundantly with endogenous PS1 (Peraus et al., 1997; Annaert et al., 1999). The APP-LDL recycles between the endosomal system and the cell surface (Annaert et al., 1999). APP-C99 is a direct substrate for
-secretase and after addition of the KK ER retention motif (APP-C99-KK) we anticipated creating an excellent substrate for PS1/
-secretase. Overall, our data confirm the spatial paradox in the PS-is-
-secretase hypothesis and provide evidence that the transfer of APP-C99 in a compartment downstream of the ER is needed in addition to processed PS1 to obtain
-secretase activity in primary cultures of neurons.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
In contrast to the other mutants, the levels of APPs and of the COOH-terminal ß-stubs generated from APP-KK are very low, confirming the low - and ß-secretase activity in the ER (Fig. 2, A and B). Accordingly, the total Aß (Fig. 2 B) and Aß42 (Fig. 2 C) secretion was also strongly reduced by 90%. However, when overexpressed in PS1-/- neurons, secretion of Aß peptides was inhibited to the same extent as observed for peptides derived from APP-WT (Fig. 2, B and C).
To circumvent the need for "preactivation" of APP-KK by - or ß-secretase, we generated APP-C99-KK, corresponding to APP truncated at the ß-secretase site (Lichtenthaler et al., 1999) and containing the KK-ER retention motif. When expressed in neurons, APP-C99-KK migrated with the same apparent molecular weight (10 kD) as the ß-stub coming from APP-WT or the APP-C99 without KK motif (Fig. 4
A; Lichtenthaler et al., 1999). APP-C99, like APP-WT, can be processed by
-secretase as reflected by the generation of
-APPCTF (Fig. 4 A). APP-C99-KK, in contrast, is either not processed or processed very little by
- or ß-secretase, confirming the specific retention of APP-C99-KK in the ER and cis-Golgi region. In line with the prediction that APP-C99 does not require
- or ß-secretase cleavage to become a substrate for
-secretase, an estimated sevenfold increase in total Aß secretion (Fig. 4 A) is observed. This effect is also observed with Aß42, as detected by ELISA (Fig. 4 B). The
-secretase cleavage of APP-C99 is strongly inhibited in the absence of PS1 (Fig. 4 C). Most surprisingly, however, was the observation that APP-C99-KK, which was also expected to be a substrate for PS1-
-secretase because it is retained in the ER, turned out to yield little if any Aß peptides (Fig. 4, A and B). It is unlikely that the KK motif directly interfered with the recognition of C99 by the
-secretase complex, since the cytoplasmic tail of APP can be removed without affecting
-secretase cleavage (Fig. 3). Moreover, C99KK becomes a substrate for
-secretase under conditions specified below. Alternatively, the low Aß secretion from neurons expressing APP-C99-KK might be explained by intracellular retention of newly formed Aß peptides. Again, this could be ruled out, as no increased amounts of Aß could be immunoprecipitated from cell extracts (result not shown, but see Fig. 7
below).
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
APP-LDL expression selectively increased Aß40 secretion, as predicted from its preferential localization in the endosomes (Koo and Squazzo, 1994; Peraus et al., 1997; Perez et al., 1999). Again, in the absence of PS1, Aß generation from APP-LDL strongly decreased, demonstrating that -secretase processing of APP in the endosomes depends on PS1. It should be noted that we have not been able to detect any endogenous PS1 immunoreactivity at the cell surface or in the endosomes of the neuronal cells used in the current study (Annaert et al., 1999).
We next investigated whether we could increase -secretase processing of APP in the ER and the intermediate compartment, which contain the bulk of PS1 immunoreactivity in these neurons. Although several authors have provided evidence that the ER is a production site of this peptide, in particular Aß42 (Chyung et al., 1997; Cook et al., 1997; Hartmann et al., 1997; Wild-Bode et al., 1997), we and others have not been able to confirm these observations (Annaert et al., 1999; Iwata et al., 2001; Maltese et al., 2001). As shown in Fig. 2, a severe decrease in both Aß40 and Aß42 secretion is observed when APP is retained in the ER by means of the double lysine retention motif (APP-KK). Since the retention of APP in the ER implies that cleavage by
- and ß-secretase (residing in the TGN, cell surface, and endosomes) becomes strongly compromised, and since proteolytic trimming of the APP ectodomain is a prerequisite for further
-secretase processing (Struhl and Adachi, 2000), it seems logical that the APP-KK mutation results in a strongly decreased
-secretase cleavage and inhibition of Aß generation. It is by consequence difficult to interpret the data obtained with APP-KK in regard to
-secretase sensitivity. Therefore, we generated the APP-C99 construct, which is a direct substrate for
-secretase (Lichtenthaler et al., 1999), and added to it the double lysine ER retention motif. As shown in Fig. 5, this construct is indeed retained in the ER, where it codistributes abundantly with PS1. Although we expected increased Aß production, no significant amount of Aß could be precipitated from the wild-type neurons (Fig. 4; Iwata et al., 2001; Maltese et al., 2001). This result was even more significant, as neurons expressing APP-C99 secreted about seven times more total Aß, or specifically Aß42, than those expressing APP-WT. The possibility that the Aß generated from APP-C99-KK is retained in the cells was ruled out, since no significant accumulation of cells associated Aß could be demonstrated (Fig. 7 A, lane 9, and B, lanes 2 and 5)The possibility that the KK motif directly interfered with
-secretase cleavage was ruled out by two observations. First, it is clear from the results with APP-
ct that the cytoplasmic domain of APP is not involved in the processing of APP by
-secretase. Second, treatment of the cell cultures with BFA could restore the processing of C99-KK, clearly demonstrating that the inhibitory effect of the KK mutations is a trans, and not a cis phenomenon (Fig. 7). It should be pointed out here that PS1 in the ER is already proteolytically processed by the "presenilinase" and present as PS1-CTF and PS1-NTF (Annaert et al., 1999), thus in the putative active conformation. It follows from these data that the simple colocalization of a substrate (APP-C99-KK) with its putative protease (PS1) is not sufficient for proteolytic cleavage.
We conclude that Aß production, including Aß42, can not occur directly in the ER and requires at least one factor derived from a post-ER compartment. This conclusion is corroborated by two important pieces of evidence from the current work. First, APP-C99, which is a good substrate for -secretase, is mainly localized in a PS1-, BIP-, and ERGIC53-negative subcellular compartment (Fig. 6). Although the characterization of this compartment requires further experimentation, this result suggests that at least the proteolytic cleavage of the APP-C99 substrate occurs in a non-ER compartment. The second, more important argument comes from the experiments using BFA. BFA is a drug that causes the redistribution of post-ER compartments into the ER (Lippincott-Schwartz et al., 1989). Treatment with this drug was sufficient to restore partially
-secretase processing of APP-C99-KK proving that at least one non-ER component is needed to activate
-secretase in the ER (Fig. 7). BFA treatment results in
-secretase processing of APP at position Aß42, something which could possibly be anticipated based on current knowledge (Chyung et al., 1997; Cook et al., 1997; Hartmann et al., 1997; Wild-Bode et al., 1997). Surprisingly, however, cleavage at position Aß40 is also observed (Fig. 7 B), which is believed to occur mainly in the late compartments of the secretory and in the endosomal limbs of the subcellular trafficking pathways (Koo and Squazzo, 1994; Hartmann et al., 1997; Tienari et al., 1997).
Summarizing, our data indicate that efficient -secretase cleavage of APP can occur in compartments that contain little, if any, PS1, and that no or little
-secretase activity is observed in compartments where abundant (maturated) PS1 is residing. This is a surprising result since most of the data currently available imply that PS1 is closely involved in
-secretase type enzymatic activity (De Strooper et al., 1998, 1999; Steiner et al., 1999, 2000; Struhl and Greenwald, 1999; Wolfe et al., 1999; Esler et al., 2000; Herreman et al., 2000; Li et al., 2000; Zhang et al., 2000). The spatial paradox between PS1 localization and
-secretase activity can be explained by two hypotheses: (a) the minute amounts of PS1 in postcis-Golgi compartments are the active enzymes. This implies that in addition to activation by presenilinase, additional proteins from these compartments are needed to make PS1 enzymatically active; and (b) PS1 is actually not the
-secretase but acts, probably in concert with other proteins in the ER and intermediate compartment, to dispatch APP or
-secretase to the subcellular compartment where cleavage will occur. In this hypothesis, BFA treatment results in the relocalization of the catalytic subunit(s) of
-secretase to the ER.
In support for the first hypothesis, evidence for the presence of minute amounts of PS1 in other compartments than the ER has been provided. In some studies, the specificity of the antibodies used can be questioned (Dewji and Singer, 1997), but in polarized cell types like MDCK cells (Georgakopoulos et al., 1999) or in specialized cell membranes like growth cones or lamellipodia (Schwarzman et al., 1999), some PS1 seems to be located near or at the cell surface. Ray et al. (1999) provided evidence that PS1 can travel in complex with Notch to the cell surface, albeit that this conclusion was based on a single type of experimental approach, i.e., cell surface biotinylation. Furthermore, the hypothesis that PS travels together with its substrates from the ER to the cell surface and becomes activated there is probably not tenable if one takes into account that the relative amount of PS1 to its substrates is extremely low (Thinakaran et al., 1998). If PS1 needs to accompany every substrate that it will cleave from the ER to the cell surface, it becomes, given the limited amounts of PS available in any cell type, difficult to understand why strong overexpression of APP does not saturate the system and does not lead to inefficient -secretase processing.
Although it remains impossible to exclude that minute amounts of PS1 in the endosomes or at the cell surface are the active enzymes, it is clear that this hypothesis raises several new questions that need to be addressed. In addition, the function of the "inactive" pool of PS1 in the ER also remains to be explained. From the BFA experiments it follows that at least one other component of a non-ER compartment is needed to obtain activated PS1. This component is not the elusive protease called "presenilinase" responsible for PS maturation, and most probably not nicastrin, since this protein is present in the ER (Yu et al., 2000).
The second hypothesis, that PS1 is needed for the correct trafficking of -secretase and its substrates, finds some theoretical support in the analogy with the sterol regulatory elementbinding protein (SREBP)SCREB cleavageactivating protein (SCAP) complex and the regulation of the proteolytical cleavage of SREBP (Brown et al., 2000). SCAP is, like PS1, an ER resident, multitransmembrane domaincontaining protein and regulates the trafficking of the membrane-bound transcription factor SREBP to post-ER compartments. Upon cholesterol depletion, SREBP travels to the cis-Golgi, where a site 1 protease cleaves SREBP in its luminal domain. The remaining NH2-terminal, membrane-bound fragment then becomes a substrate for a site 2 protease that cleaves in the transmembrane domain of SREBP (Rawson et al., 1997; Nohturfft et al., 1999). The coupling of vesicular protein transport and proteolysis provides a stringent control on the activation of the system and it could be envisaged that PS1, like SCAP, provides a similar control on APP (and Notch) processing. However, it should be pointed out that in case of SREBP processing, SCAP regulates its luminal cleavage and that the site 2 intramembraneous cleavage occurs by default. In case of PS1, its role is limited to the regulation of the intramembraneous
-secretase cleavage (De Strooper et al., 1998).
In conclusion, our data indicate a complex relationship between APP trafficking and its processing by -secretase. Moreover, they directly question the exact role of PS1 in
-secretase processing and demonstrate that at least one cofactor (in the first hypothesis) or the protease (in the second hypothesis) is located in a compartment downstream of the ER. In the future, reconstitution of
-secretase processing of APP, by mixing extracts of purified ER fractions and post-ER compartments, should allow us to further define at a molecular level this highly intriguing proteolytic system.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Semliki forest virus (SFV) constructs
The following modifications were made to the cDNA coding for human APP695 (Fig. 1): (a) deletion of the last 43 amino acids of the cytoplasmic tail (APP-ct) as described in Tienari et al. (1996); (b) addition of a di-lysine motif (QM mutated to KK, APP-KK) by site-directed mutagenesis (Stratagene); (c) exchange of its cytoplasmic domain with that of the LDL receptor (APP-LDL); (d) full deletion of the APP ectodomain until the Asp1 amino acid of the amyloid peptide region (ß-secretase site). An additional AspAla (DA) was cloned by site-directed mutagenesis (Stratagene) between the last amino acid of the signal sequence (Ala) and the first of the Aß region (Lichtenthaler et al., 1999) (APP-C99). Site-directed mutagenesis was used to add the di-lysine motif (APP-C99-KK). Recombinant SFV containing the APP mutants were generated as described previously (De Strooper et al., 1995).
Transduction and metabolic labeling of neuronal cell cultures
Recombinant SFV was diluted 10-fold in neurobasal medium and added to the culture dishes. After a 1-h incubation at 37°C, fresh neurobasal medium was added. After 2 h, cells were metabolically labeled in methionine-free MEM (GIBCO BRL) supplemented with B27 and 100 µCi/ml [35S]methionine (ICN Biomedicals). 10 µM BFA (Epicentre) was added when indicated. After 4 h, conditioned medium and cells were recovered. Cells were lysed in IP buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS) and cleared by centrifugation. Labeled protein was immunoprecipitated using 25 µl protein G Sepharose and specific antibodies as indicated below and in Fig. 1. Immunoprecipitates were finally solubilized in 25 µl Nu-Page sample buffer (Invitrogen) and electrophoresed on 412% or 10% acrylamide NuPage Bis-Tris gels under reducing conditions and MES in the running buffer (Invitrogen). Results were analyzed using a PhosphorImager (Molecular Dynamics) and ImagQuaNT4.1. All data were normalized to the value obtained for the corresponding APP holoform to normalize for variations between culture dishes. The following antibodies were used (Fig. 1). Rabbit pAb B7 recognizes the first 17 amino acids of Aß, B11 the last 20 amino acids of the APP COOH-terminal domain (De Strooper et al., 1995), goat pAb 207 the APP ectodomain (provided by Dr. M. Savage, Cephalon, Inc., West Chester, PA), mAb 4G8 (Senetek) amino acids 17 to 24 of Aß, and mab W0-2 the NH2-terminal region of Aß (provided by Dr. Tobias Hartmann and Konrad Beyreuther, University of Heidelberg, Heidelberg, Germany) (Ida et al., 1996). Antibodies FCA40 and FCA42 specifically precipitate Aß peptides ending at residue 40 (40) or at 42 (
42) and were provided by Dr. F. Checler (Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France) (Barelli et al., 1997).
Quantification of the Aß42 peptide by ELISA
Levels of Aß42 in conditioned media and cell lysates were quantified by a sandwich ELISA test (De Strooper et al., 1998), according to published protocols (Vanderstichele et al., 2000). In brief, samples were dried by speed vacuum (Savant), resuspended in 300 µl of sample diluent, and incubated on 96-well ELISA plates precoated with mAb 3D6 against Aß. After washing, samples were incubated with a biotin-labeled anti-Aß42 antibody mAb 21F12 that only recognizes the final two amino acids of the Aß42 sequence, followed by streptavidine-HRP. After adding the HRP substrate, samples were measured spectrophotometrically using a Victor2 (Wallac) with a 450-nm filter. The Aß42 concentration in the samples was calculated based on the Aß42 standards sigmoid curve equation, and using Prism 3.0 (GraphPad Software).
Confocal microscopy
Hippocampal neurons grown on poly-L-lysinecoated glass coverslips in the presence of a glial feeder layer (Goslin and Banker, 1991; De Strooper et al., 1995) were transduced with SFV-APP-C99 or -C99KK. 4 h after transduction, cycloheximide (100 µg/ml) was added to block further protein synthesis. After 6 h, neurons were fixed in 120 mM phosphate buffer containing 4% paraformaldehyde (pH 7.3) and 4% sucrose, permeabilized in ice-cold methanol and acetone, and immunostained (Annaert et al., 1999). Recombinant APP-C99 was detected with pAb 6687 (gift of Dr. C. Haass, University of München, Germany) or mAb 3D6 (gift of Dr. Seubert, Elan Pharmaceuticals, San Francisco, CA). mAbs to BIP were purchased from Sigma. PAb B17.2 was used to detect endogenous PS1 (Annaert et al., 1999). The intermediate compartment and Golgi region were identified using anti-ERGIC-53, provided by Dr. J. Saraste (University of Bergen, Norway), and anti-GM130 (Transduction Laboratories), respectively. Alexa 488 and Alexa 546conjugated secondary antibodies were from Molecular Probes. BioRad MRC1024 confocal microscope and Adobe Photoshop® 5.2 were used for final processing (Annaert et al., 1999).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was financially supported by the F.W.O.-Vlaanderen, the K.U. Leuven, the Flanders interuniversity Institute for Biotechnology, and the Bayer Alzheimer Disease Research Network (BARN). P. Cupers and W. Annaert are holders of a Fonds voor Wetenschappelijik Onderzoek postdoctoral fellowship.
Submitted: 11 April 2001
Revised: 27 June 2001
Accepted: 4 July 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Annaert, W., and B. De Strooper. 1999. Presenilins: molecular switches between proteolysis and signal transduction. Trends Neurosci. 22:439443.[Medline]
Annaert, W.G., L. Levesque, K. Craessaerts, I. Dierinck, G. Snellings, D. Westaway, P.S. George-Hyslop, B. Cordell, P. Fraser, and B. De Strooper. 1999. Presenilin 1 controls -secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons. J. Cell Biol. 147:277294.
Barelli, H., A. Lebeau, J. Vizzanova, P. Delaere, N. Chevalier, C. Drouot, P. Marambaud, K. Ancolio, J.D. Buxbaum, O. Khorkova, et al. 1997. Characterization of new polyclonal antibodies specific for 40 and 42 amino acid long amyloid ß peptides: their use to examine the cell biology of presenilins and the immunohistochemistry of sporadic Alzheimer's disease and cerebral amyloid angiopathy cases. Mol. Med. 3: 695707.[Medline]
Brou, C., F. Logeat, N. Gupta, C. Bessia, O. LeBail, J.R. Doedens, A. Cumano, P. Roux, R.A. Black, and A. Israel. 2000. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol. Cell. 5:207216.[Medline]
Brown, M.S., J. Ye, R.B. Rawson, and J.L. Goldstein. 2000. Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell. 100:391398.[Medline]
Buxbaum, J.D., K.N. Liu, Y. Luo, J.L. Slack, K.L. Stocking, J.J. Peschon, R.S. Johnson, B.J. Castner, D.P. Cerretti, and R.A. Black. 1998. Evidence that tumor necrosis factor converting enzyme is involved in regulated
-secretase cleavage of the Alzheimer amyloid protein precursor. J. Biol. Chem. 273:2776527767.
Chyung, A.S.C., B.D. Greenberg, D.G. Cook, R.W. Doms, and V.M. Lee. 1997. Novel ß-secretase cleavage of ß-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells. J. Cell Biol. 138:671680.
Cook, D.G., M.S. Forman, J.C. Sung, S. Leight, D.L. Kolson, T. Iwatsubo, V.M. Lee, and R.W. Doms. 1997. Alzheimer's A ß(1-42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nat. Med. 3:10211023.[Medline]
Creemers, J.W., D.I. Dominguez, E. Plets, L. Serneels, N.A. Taylor, G. Multhaup, K. Craessaerts, W. Annaert, and B. De Strooper. 2000. Processing of ß-secretase (Bace) by furin and other members of the proprotein convertase family. J. Biol. Chem. 8:8.
Culvenor, J.G., F. Maher, G. Evin, F. Malchiodi-Albedi, R. Cappai, J.R. Underwood, J.B. Davis, E.H. Karran, G.W. Roberts, K. Beyreuther, and C.L. Masters. 1997. Alzheimer's disease-associated presenilin 1 in neuronal cells: evidence for localization to the endoplasmic reticulum-Golgi intermediate compartment. J. Neurosci. Res. 49:719731.[Medline]
De Strooper, B., M. Simons, G. Multhaup, F. Van Leuven, K. Beyreuther, and C.G. Dotti. 1995. Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence. EMBO J. 14:49324938.[Abstract]
De Strooper, B., P. Saftig, K. Craessaerts, H. Vanderstichele, G. Guhde, W. Annaert, K. Von Figura, and F. Van Leuven. 1998. Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature. 391:387390.[Medline]
De Strooper, B., W. Annaert, P. Cupers, P. Saftig, K. Craessaerts, J.S. Mumm, E.H. Schroeter, V. Schrijvers, M.S. Wolfe, W.J. Ray, A. Goate, and R. Kopan. 1999. A presenilin-1-dependent -secretase-like protease mediates release of Notch intracellular domain. Nature. 398:518522.[Medline]
Dewji, N.N., and S.J. Singer. 1997. Cell surface expression of the Alzheimer disease-related presenilin proteins. Proc. Natl. Acad. Sci. USA. 94:99269931.
Esler, W.P., W.T. Kimberly, B.L. Ostaszewski, T.S. Diehl, C.L. Moore, J.Y. Tsai, T. Rahmati, W. Xia, D.J. Selkoe, and M.S. Wolfe. 2000. Transition-state analogue inhibitors of -secretase bind directly to presenilin-1. Nat. Cell Biol. 2:428434.[Medline]
Gaynor, E.C., S. te Heesen, T.R. Graham, M. Aebi, and S.D. Emr. 1994. Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast. J. Cell Biol. 127:653665.[Abstract]
Georgakopoulos, A., P. Marambaud, S. Efthimiopoulos, J. Shioi, W. Cui, H.C. Li, M. Schutte, R. Gordon, G.R. Holstein, G. Martinelli, P. Mehta, V.L. Friedrich, Jr., and N.K. Robakis. 1999. Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts. Mol. Cell. 4:893902.[Medline]
Goslin, K., and G. Banker. 1991. Rat hippocampal neurons in low-density culture. Culturing Nerve Cells. G. Banker and K. Goslin, editors. MIT Press, Cambridge, MA. 251281.
Hartmann, T., S.C. Bieger, B. Bruhl, P.J. Tienari, N. Ida, D. Allsop, G.W. Roberts, C.L. Masters, C.G. Dotti, K. Unsicker, and K. Beyreuther. 1997. Distinct sites of intracellular production for Alzheimer's disease A ß40/42 amyloid peptides. Nat. Med. 3:10161020.[Medline]
Herreman, A., L. Serneels, W. Annaert, D. Collen, L. Schoonjans, and B. De Strooper. 2000. Total inactivation of -secretase activity in presenilin-deficient embryonic stem cells. Nat. Cell Biol. 2:461462.[Medline]
Ida, N., T. Hartmann, J. Pantel, J. Schroder, R. Zerfass, H. Forstl, R. Sandbrink, C.L. Masters, and K. Beyreuther. 1996. Analysis of heterogeneous A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay. J. Biol. Chem. 271:2290822914.
Iwata, H., T. Tomita, K. Maruyama, and T. Iwatsubo. 2001. Subcellular compartment and molecular subdomain of ß-amyloid precursor protein relevant to the Abeta 42-promoting effects of Alzheimer mutant presenilin 2. J. Biol. Chem. 216:2167821685.
Jackson, M.R., T. Nilsson, and P.A. Peterson. 1993. Retrieval of transmembrane proteins to the endoplasmic reticulum. J. Cell Biol. 121:317333.[Abstract]
Kim, S.H., J.J. Lah, G. Thinakaran, A. Levey, and S.S. Sisodia. 2000. Subcellular localization of presenilins: association with a unique membrane pool in cultured cells. Neurobiol. Dis. 7:99117.[Medline]
Koike, H., S. Tomioka, H. Sorimachi, T.C. Saido, K. Maruyama, A. Okuyama, A. Fujisawa-Sehara, S. Ohno, K. Suzuki, and S. Ishiura. 1999. Membrane-anchored metalloprotease MDC9 has an -secretase activity responsible for processing the amyloid precursor protein. Biochem. J. 343:371375.[Medline]
Koo, E.H., and S.L. Squazzo. 1994. Evidence that production and release of amyloid ß-protein involves the endocytic pathway. J. Biol. Chem. 269:1738617389.
Lammich, S., E. Kojro, R. Postina, S. Gilbert, R. Pfeiffer, M. Jasionowski, C. Haass, and F. Fahrenholz. 1999. Constitutive and regulated -secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease. Proc. Natl. Acad. Sci. USA. 96:39223927.
Li, Y.M., M. Xu, M.T. Lai, Q. Huang, J.L. Castro, J. DiMuzio-Mower, T. Harrison, C. Lellis, A. Nadin, J.G. Neduvelil, et al. 2000. Photoactivated -secretase inhibitors directed to the active site covalently label presenilin 1. Nature. 405:689694.[Medline]
Lichtenthaler, S.F., G. Multhaup, C.L. Masters, and K. Beyreuther. 1999. A novel substrate for analyzing Alzheimer's disease -secretase. FEBS Lett. 453:288292.[Medline]
Lippincott-Schwartz, J., L.C. Yuan, J.S. Bonifacino, and R.D. Klausner. 1989. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 56:801813.[Medline]
Maltese, W.A., S. Wilson, Y. Tan, S. Suomensaari, S. Sinha, R. Barbour, and L. McConlogue. 2001. Retention of the Alzheimer's amyloid precursor protein fragment C99 in the endoplasmic reticulum prevents formation of amyloid ß-peptide. J. Biol. Chem. 23:2026720279.
Mumm, J.S., E.H. Schroeter, M.T. Saxena, A. Griesemer, X. Tian, D.J. Pan, W.J. Ray, and R. Kopan. 2000. A ligand-induced extracellular cleavage regulates -secretase-like proteolytic activation of Notch1. Mol. Cell. 5:197206.[Medline]
Naruse, S., G. Thinakaran, J.J. Luo, J.W. Kusiak, T. Tomita, T. Iwatsubo, X. Qian, D.D. Ginty, D.L. Price, D.R. Borchelt, P.C. Wong, and S.S. Sisodia. 1998. Effects of PS1 deficiency on membrane protein trafficking in neurons. Neuron. 21:12131221.[Medline]
Nohturfft, A., R.A. DeBose-Boyd, S. Scheek, J.L. Goldstein, and M.S. Brown. 1999. Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi. Proc. Natl. Acad. Sci. USA. 96:1123511240.
Peraus, G.C., C.L. Masters, and K. Beyreuther. 1997. Late compartments of amyloid precursor protein transport in SY5Y cells are involved in ß-amyloid secretion. J. Neurosci. 17:77147724.
Perez, R.G., S. Soriano, J.D. Hayes, B. Ostaszewski, W. Xia, D.J. Selkoe, X. Chen, G.B. Stokin, and E.H. Koo. 1999. Mutagenesis identifies new signals for ß-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including aß42. J. Biol. Chem. 274:1885118856.
Rawson, R.B., N.G. Zelenski, D. Nijhawan, J. Ye, J. Sakai, M.T. Hasan, T.Y. Chang, M.S. Brown, and J.L. Goldstein. 1997. Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs. Mol. Cell. 1:4757.[Medline]
Ray, W.J., M. Yao, J. Mumm, E.H. Schroeter, P. Saftig, M. Wolfe, D.J. Selkoe, R. Kopan, and A.M. Goate. 1999. Cell surface presenilin-1 participates in the -secretase-like proteolysis of notch. J. Biol. Chem. 274:3680136807.
Schwarzman, A.L., N. Singh, M. Tsiper, L. Gregori, A. Dranovsky, M.P. Vitek, C.G. Glabe, P.H. St George-Hyslop, and D. Goldgaber. 1999. Endogenous presenilin 1 redistributes to the surface of lamellipodia upon adhesion of jurkat cells to a collagen matrix. Proc. Natl. Acad. Sci. USA. 96:79327937.
Seiffert, D., J.D. Bradley, C.M. Rominger, D.H. Rominger, F. Yang, J.E. Meredith, Jr., Q. Wang, A.H. Roach, L.A. Thompson, S.M. Spitz, et al. 2000. Presenilin-1 and -2 are molecular targets for -secretase inhibitors. J. Biol. Chem. 275:3408634091.
Selkoe, D.J. 1999. Translating cell biology into therapeutic advances in Alzheimer's disease. Nature. 399:A23A31.[Medline]
Sisodia, S.S. 1992. ß-amyloid precursor protein cleavage by a membrane-bound protease. Proc. Natl. Acad. Sci. USA. 89:60756079.[Abstract]
Song, W., P. Nadeau, M. Yuan, X. Yang, J. Shen, and B.A. Yankner. 1999. Proteolytic release and nuclear translocation of Notch-1 are induced by presenilin-1 and impaired by pathogenic presenilin-1 mutations. Proc. Natl. Acad. Sci. USA. 96:69596963.
Steiner, H., K. Duff, A. Capell, H. Romig, M.G. Grim, S. Lincoln, J. Hardy, X. Yu, M. Picciano, K. Fechteler, et al. 1999. A loss of function mutation of presenilin-2 interferes with amyloid ß-peptide production and notch signaling. J. Biol. Chem. 274:2866928673.
Steiner, H., M. Kostka, H. Romig, G. Basset, B. Pesold, J. Hardy, A. Capell, L. Meyn, M.L. Grim, R. Baumeister, K. Fechteler, and C. Haass. 2000. Glycine 384 is required for presenilin-1 function and is conserved in bacterial polytopic aspartyl proteases. Nat. Cell Biol. 2:848851.[Medline]
Struhl, G., and A. Adachi. 2000. Requirements for presenilin-dependent cleavage of notch and other transmembrane proteins. Mol. Cell. 6:625636.[Medline]
Struhl, G., and I. Greenwald. 1999. Presenilin is required for activity and nuclear access of Notch in Drosophila. Nature. 398:522525.[Medline]
Thinakaran, G., J.B. Regard, C.M. Bouton, C.L. Harris, D.L. Price, D.R. Borchelt, and S.S. Sisodia. 1998. Stable association of presenilin derivatives and absence of presenilin interactions with APP. Neurobiol. Dis. 4:438453.[Medline]
Tienari, P.J., B. De Strooper, E. Ikonen, M. Simons, A. Weidemann, C. Czech, T. Hartmann, N. Ida, G. Multhaup, C.L. Masters, F. Van Leuven, K. Beyreuther, and C.G. Dotti. 1996. The ß-amyloid domain is essential for axonal sorting of amyloid precursor protein. EMBO J. 15:52185229.[Abstract]
Tienari, P.J., N. Ida, E. Ikonen, M. Simons, A. Weidemann, G. Multhaup, C.L. Masters, C.G. Dotti, and K. Beyreuther. 1997. Intracellular and secreted Alzheimer ß-amyloid species are generated by distinct mechanisms in cultured hippocampal neurons. Proc. Natl. Acad. Sci. USA. 94:41254130.
Vanderstichele, H., E. Van Kerschaver, C. Hesse, P. Davidsson, M.A. Buyse, N. Andreasen, L. Minthon, A. Wallin, K. Blennow, and E. Vanmechelen. 2000. Standardization of measurement of ß-amyloid(1-42) in cerebrospinal fluid and plasma. Amyloid. 7:245258.[Medline]
Vassar, R., B.D. Bennett, S. Babu-Khan, S. Kahn, E.A. Mendiaz, P. Denis, D.B. Teplow, S. Ross, P. Amarante, R. Loeloff, et al. 1999. ß-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 286:735741.
Walter, J., A. Capell, J. Grunberg, B. Pesold, A. Schindzielorz, R. Prior, M.B. Podlisny, P. Fraser, P.S. Hyslop, D.J. Selkoe, and C. Haass. 1996. The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum. Mol. Med. 2:673691.[Medline]
Wild-Bode, C., T. Yamazaki, A. Capell, U. Leimer, H. Steiner, Y. Ihara, and C. Haass. 1997. Intracellular generation and accumulation of amyloid ß-peptide terminating at amino acid 42. J. Biol. Chem. 272:1608516088.
Wolfe, M.S., W. Xia, B.L. Ostaszewski, T.S. Diehl, W.T. Kimberly, and D.J. Selkoe. 1999. Two transmembrane aspartates in presenilin-1 required for presenelin endoproteolysis and -secretase activity. Nature. 398:513517.[Medline]
Yu, G., M. Nishimura, S. Arawaka, D. Levitan, L. Zhang, A. Tandon, Y.Q. Song, E. Rogaeva, F. Chen, T. Kawarai, et al. 2000. Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and ßAPP processing. Nature. 407:4854.[Medline]
Zhang, Z., P. Nadeau, W. Song, D. Donoviel, M. Yuan, A. Bernstein, and B.A. Yankner. 2000. Presenilins are required for -secretase cleavage of ß-APP and transmembrane cleavage of Notch-1. Nat. Cell Biol. 2:463465.[Medline]