secretase mediated conversion of the amyloid ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2009 | 111 | 1369–1382

doi: 10.1111/j.1471-4159.2009.06420.x

*Molecular Neurodegeneration Group, Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University Mainz, Mainz, Germany Molecular Neuropathology Group, Department of Neuropathology, Heinrich Heine University, Du¨sseldorf, Germany àInstitute of Neuropathology, Heinrich Heine University, Du¨sseldorf, Germany §Department of Neurosciences, University of California, San Diego, La Jolla, California, USA ¶Department of Neurology, Clinical Neuroscience, University of Bonn, Bonn, Germany

Abstract The Swedish mutation within the amyloid precursor protein (APP) causes early-onset Alzheimer’s disease due to increased cleavage of APP by BACE1. While b-secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild-type APP occurs mainly in endocytic compartments. However, we show that liberation of Ab from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall b-secretase cleaved soluble APP released from APPswe secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and

Cerebral accumulation of the 4 kDa amyloid b-peptide (Ab) in senile plaques is one of the hallmarks of Alzheimer’s disease (AD). The release of the Ab peptide from the amyloid precursor protein (APP) [for review, see Selkoe (2001)] is a complex process involving sequential cleavage by two proteases termed b- and c-secretase. The initial bsecretase cleavage by BACE1 (Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999) generates the N-terminal soluble domain [b-secretase cleaved soluble APP released from APPswe (APPsb)] and a membrane-tethered C-terminal fragment (bCTF or C99). Subsequently, Ab is released into the luminal space by c-secretase cleavage within the transmembrane domain of APP (Sastre et al. 2001; Weidemann et al. 2002). However, only a small percentage of APP gets cleaved by b-secretase. A larger portion of APP gets alternatively shedded within the Ab peptide by a-secretase

substantially decreased Ab secretion. We point out that asecretase activity-mediated conversion of C99 to C83 is the main cause of this Ab reduction. Furthermore, we demonstrate that a-secretase cleavage of C99 even contributes to the reduction of Ab secretion of internalization deficient wildtype APP. Therefore, inhibition of a-secretase cleavage increased Ab secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells. Keywords: Alzheimer’s disease, Amyloid precursor protein, Amyloid-beta-peptide, trafficking, C99, a-secretase cleavage. J. Neurochem. (2009) 111, 1369–1382.

Received June 26, 2009, revised manuscript received September 10, 2009, accepted September 11, 2009. Address correspondence and reprint requests to: Claus U. Pietrzik, Institute of Physiological Chemistry and Pathobiochemistry, Molecular Neurodegeneration, Johannes-Gutenberg-University Mainz, Duesbergweg 6, D-55099 Mainz, Germany. E-mail: [email protected] Abbreviations used: Ab, amyloid b-peptide; APP, amyloid precursor protein; AD, Alzheimer’s disease; CHO, Chinese hamster ovary; WT, wild-type; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; APPs, N-terminal soluble fragment of APP; APPswe, ‘‘Swedish’’ mutant APP; APPwt, wild-type APP; APPsbswe, b-secretase cleaved soluble APP released from APPswe; APPsbwt, b-secretase cleaved soluble APP released from APPwt; APPsa, soluble APPs released after a-secretase cleavage; CTF, C-terminal fragment; bCTF, C99, C-terminal fragment after b-secretase cleavage; aCTF, C83, C-terminal fragment after a-secretase cleavage; AICD, APP intracellular domain; ELISA, enzyme-linked immunoabsorbent assay; PBS, phosphate-buffered saline.

2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1369–1382

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1370 | S. Ja¨ger et al.

(Esch et al. 1990; Sisodia 1992). The a-secretase activity has been shown to be associated with the ADAM (a disintegrin and metalloprotease) family members 9, 10 and 17 (Buxbaum et al. 1998; Koike et al. 1999; Lammich et al. 1999). This a-secretase-dependent processing of APP liberates the longer APPsa and a shorter CTF, termed aCTF or C83. Even after intensive investigations of the last 15 years, precise locations of cleavage events and cellular compartments in which the Ab production occurs remain under debate. The constitutively active metalloproteases that function as a-secretases (Buxbaum et al. 1998; Koike et al. 1999; Lammich et al. 1999) appear to shed APP at or near the cell surface (Sambamurti et al. 1992; Sisodia 1992; De Strooper et al. 1993). While early investigations associated b-secretase activity with slightly acidic compartments such as the trans-Golgi-network or endosomes (Haass et al. 1992, 1993; Koo and Squazzo 1994; Thinakaran et al. 1996), the discovery of BACE1 verified its localization to these compartments (Hussain et al. 1999; Vassar et al. 1999; Huse et al. 2000). The second essential event of Ab production involves the cleavage of the CTFs by c-secretase (Edbauer et al. 2003). Some Ab production has been localized to all possible compartments of the secretory pathway (Cook et al. 1997; Hartmann et al. 1997; Chyung and Selkoe 2003; Chyung et al. 2005), but several investigations suggest that c-secretase activity resides at the plasma membrane or within endocytic compartments (Chyung and Selkoe 2003; Chyung et al. 2005; Kaether et al. 2006). It has been suggested that wild-type APP (APPwt) and Swedish APP (APPswe) utilize different cellular mechanisms for Ab generation. On the one hand, it has been shown that Ab generation from APPwt occurs in endocytic compartments after internalization from the cell surface, although no direct evidence has been reported for b-secretase cleavage of APPwt in endocytic compartments (Haass et al. 1993; Koo and Squazzo 1994; Perez et al. 1999). On the other hand, BACE cleavage of APPswe occurs late in the secretory pathway, most likely in Golgi-derived secretory vesicles (Haass et al. 1995; Thinakaran et al. 1996), and it has been suggested that C-terminal truncation of APPswe including the internalization motif leaves Ab production unaffected (Citron et al. 1992, 1995). Therefore, it has been hypothesized that b-secretase as well as csecretase cleavage of APPswe takes place in the secretory pathway. In contradiction to the model of APPswe processing, it was recently shown that in vivo inhibition of dynaminmediated endocytosis reduced Ab secretion in APPswe overexpressing Tg2576 mice (Hsiao et al. 1996) up to 70% (Cirrito et al. 2008). Additionally, an investigation of the localization of c-secretase activity by a C99-GFP fusion protein revealed that c-secretase does not cleave C99 in the endoplasmic reticulum (ER), the Golgi/trans-Golgi network,

or in secretory vesicles, but that AICD is generated at the cell surface or early endosomes (Kaether et al. 2006). To investigate the cause of this data discrepancy we have carefully examined processing of APPwt, APPswe and C99 in stably over-expressing cells to identify potential cellular compartments involved in APP processing. Interestingly, we found that b-secretase cleavage of APPwt and APPswe occur to a great extent in different cellular compartments. While b-secretase cleavage of APPwt is nearly entirely dependent on endocytic compartments, the b-secretase cleavage of APPswe is almost independent of a functional internalization signal and, therefore, occurs mainly in the late secretory pathway. We demonstrate that a-secretase shedding of C99 plays a crucial role in Ab reduction and that the conversion of C99 to C83 limits Ab secretion in APPwt and C99 over-expressing cells.

Experimental procedures Plasmid construction The plasmids pLHCX-APP695wt, pLHCX-APP770wt and the corresponding pLHCX-APP695swe, pLHCX-APP770swe were used in the generation of different APP expression constructs. To delete the internalization motif NPTY, a two-step PCR mutagenesis strategy with two overlapping primers encoding the mutation was applied. As the epitope of the antibody CT15, raised against the Cterminal end of APP, encloses the NPTY domain, the APP constructs were tagged with a myc-epitope at the extreme carboxyl terminus of APP in the pcDNA3.1 myc-his plasmid. The a-secretase cleavage inhibition mutation F615P (APP695 numbering) was introduced by a two-step PCR strategy. The C99 construct was a kind gift from S. Lichtenthaler (Lichtenthaler et al. 1999). The NPTY deletion construct was constructed by using a two-step PCR mutagenesis strategy. Both constructs were amplified by PCR and subcloned into pLHCX either directly or by replacing APP695 in pLHCX-APP695swemyc to generate a myc-tagged C99 expression construct. The a-secretase cleavage inhibition mutation F38P was introduced by a two-step PCR strategy. All constructs were verified by sequencing. Cell lines All Chinese hamster ovary (CHOK1) cell lines were grown in aminimum essential medium (a-MEM) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate and 100 units/mL penicillin/ streptomycin. All APP constructs were inserted into the retroviral expression vector pLHCX. For generation of stably expressing cells, virus production was induced by double transfection of pLHCX with pVSV-G at a 1 : 1 ratio into the packaging cell line GP2-293 (Clontech, Mountain View, CA, USA) using the calcium phosphate transfection method. The medium was changed after 48 h and viruses were collected for another 24 h. After infection with recombinant viruses in the presence of 5 lg/mL Polybrene for 24 h, stable CHOK1 cells were selected with 150 lg/mL Hygromycin (Invitrogen, Carlsbad, CA, USA). Stable pools with comparable expression levels were analyzed in further experiments.


a-secretase processing of C99 limits Ab production | 1371

Antibodies The following antibodies were used: the polyclonal antibody 63D and the monoclonal antibodies 1G7 and 5A3, binding to the Nterminal ectodomain of APP, have been described before (Koo et al. 1996; Leuchtenberger et al. 2008). The polyclonal APPsbwt and APPsbswe specific antibodies 192wt and 192swe, respectively, were kindly provided by Dale Schenk [Elan Pharmaceuticals, South San Francisco, CA, USA (Seubert et al. 1993; Haass et al. 1995)]. Two different antibodies detecting the Ab sequence aminoterminal to the a-secretase cleavage site were used: the monoclonal antibody 26D6 recognizing residues 1-12 of the human Ab sequence has been described before (Pietrzik et al. 2002), while the second monoclonal antibody IC16 recognizing residues 1–16 of the human Ab sequence was manufactured during the course of this study. Synthetic human Ab peptide 1–16 was cross-linked to keyhole limpet hemocyanin (KLH; Eurogentec, Seraing, Belgium) and used, in combination with RIBI Adjuvant (Sigma, St Louis, MO, USA), to immunize mice with an ablated PrnP gene (Bueler et al. 1992). Hybridoma cells were generated by standard procedures and screened for recognition of HSA cross-linked to Ab 1-16 by ELISA. In a second screen, recognition of synthetic Ab 1–42 on a standardized dot blot procedure was confirmed and the epitope mapped to Ab 1–8 using a peptide array spanning the entire sequence of Ab (Jerini Peptide Technologies, Berlin, Germany). The polyclonal antibody CT15 against the last 15 C-terminal amino acids of human APP has been described before (Pietrzik et al. 2002) and the monoclonal antibody 9E10 recognizing the myc-epitope was purchased from Santa Cruz (Santa Cruz, CA, USA). Western blotting Cells were seeded in 60 mm dishes at identical density. To detect all forms of APPs and secreted Ab, the media was changed 48 h after seeding and collected for additional 24 h. Cells were washed once in ice-cold phosphate-buffered saline (PBS) and then scraped into PBS. After lysis in Nonidet P-40 buffer [1% Nonidet P-40 (Sigma, Taufkirchen, Germany), 150 mM NaCl, 0.02% Sodium Azide, 50 mM Tris, pH 7.4] containing 1· complete protease inhibitor mixture (Roche, Indianapolis, IN, USA) for 20 min at 4C, the lysate solution was centrifuged at 4C for 20 min at 21 000 g in a microcentrifuge. The soluble fraction of the extracts was removed to new tubes. All media samples were centrifuged for 10 min at 4C to remove particulate materials and were stored at )80C. Equal amounts of total protein, determined by using the BCA protein Assay (Pierce Chemicals, Rockford, IL, USA), were used for lysate analysis, while media samples were normalized to total protein content in the corresponding cell extract. Full-length APP in lysates and APPs in media were separated on 10% Tris–Glycine gels and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA), while APP C-terminal fragments in lysates and Ab in media were fractionated on 12% Bis-Tris gels and blotted onto polyvinylidene difluoride membranes (Millipore). Western blot analysis was performed with the indicated antibodies and enhanced chemiluminescent substrate (Pierce or Millipore) by using the LAS-3000mini (Fujifilm, Du¨sseldorf, Germany). Individual bands of APP and its fragments were measured in 3–6 experiments for quantification by MultiGauge software (Fujifilm). Data were corrected for background and normalized to full-length APP expression for each cell line.

Drug studies To alkalinize intracellular vesicles, the media was replaced 48 h after seeding with fresh media containing 10 mM NH4Cl. For csecretase inhibition cells were treated with 2.5 lM L-685 458 (Bachem, Bubendorf, Switzerland), 2.5 lM DAPT or with Me2SO vehicle. The media was conditioned for 24 h and the cells were lysed as described above. Surface-biotinylation To examine cell surface APP, cell surface biotinylation was performed following the manufacturers protocol. In brief, cells were grown in 60 mm dishes to 95% confluency and rinsed three times with ice-cold PBS. Cell surface proteins were biotinylated with 0.5 mg/mL Sulfo-NHS-LC-LC-Biotin (Pierce) in ice-cold PBS for 40 min at 4C. The biotin solution was exchanged once after 20 min. Cells were washed four times with ice-cold PBS containing 100 mM glycine. Cells were scraped off the plate and lysed in NP40 buffer. Equal amounts of proteins were incubated with Neutravidin Agarose resin (Pierce) to recover biotinylated proteins. The yield was dissociated by boiling in loading-dye, separated on 4–12% NuPAGE gels and transferred to polyvinylidene difluoride membranes. Cell surface APP was detected with the myc-epitope antibody 9E10.

Results In order to investigate possible differences in APP processing between APPwt and APPswe, we generated CHOKI cells stably expressing equal amounts of transgenic protein. In addition to the full-length APP constructs, we generated endocytosis deficient APP695wt and APPswe mutants by deleting the cytoplasmic NPTY-motif, a consensus sequence for coated pit-mediated re-internalization of cell surface proteins (Chen et al. 1990). Since our antibody CT15 recognizes the NPTY domain, the APP expression constructs were tagged with a myc-peptide to allow the detection of all CTFs. All stably over-expressing cell lines were generated with retroviral vectors for each mutant and used as pools after selection to eliminate clonal effects. Comparison of the metabolism of wild-type APP695myc with APP lacking the endocytosis motif (APP695DNPTYmyc) confirmed the previously published findings that APPsa and APPs secretion were substantially increased by deletion of the internalization motif NPTY (Fig. 1a, panels 2 and 3) (Perez et al. 1999). While a-secretase-mediated shedding of APP was enhanced in endocytosis deficient APP695DNPTYmyc expressing cells, b-secretase cleavage was strongly diminished (Fig. 1a, panel 4). This decrease in APPsbwt secretion was accompanied by a decline in C99 levels (Fig. 1a, panel 5) and a decrease in Ab generation (Fig. 1a, panel 6). These results indicate that most of the bsecretase cleavage of APPwt and consequently APPsbwt generation occurs in endocytic compartments. Therefore, prevention of APPwt internalization strongly decreases Ab generation mainly through reduction in b-secretase cleavage.



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Fig. 1 Deletion of the NPTY domain in APP695wt and APP695swe diminishes Ab secretion by different mechanisms. (a) Comparison between CHOKI cells stably over-expressing a C-terminal myc-tagged APP695wt (APP695myc) with an APPwt internalization deficient mutant (APP695DNPTYmyc). (b) Comparison between CHOKI cells stably over-expressing a myc-tagged APPswe (APP695swemyc) with an APPswe internalization deficient mutant (APP695sweDNPTYmyc).

Cell media, conditioned for 24 h, and cell lysates were resolved by SDS-PAGE, followed by western blotting. Note that APPsbwt was strongly diminished in APP695DNPTYmyc cells, while APPsbswe secretion was almost unaffected in APPsweDNPTYmyc cells. Surprisingly, this deletion caused a substantial reduction in C99 in APP695wt as well as in APP695swe cells, which subsequently resulted in robust reduction in Ab secretion.

Studies of the Swedish familial Alzheimer’s disease mutation (K595N/M596L) of human APP revealed that this mutation enhances Ab production through increased b-secretase cleavage (Citron et al. 1992; Felsenstein et al. 1994; Citron et al. 1995) which generates increased levels of C99 in APPswe over-expressing cells compared with APPwt cells. Similar to APPwt, disruption of the endocytosis signal NPTY in APPswe (APP695sweDNPTYmyc) substantially increased total APPs secretion through boosted APPsa secretion (Fig. 1b, panels 2 and 3). In contrast to wild-type APP695myc, the b-secretase cleavage of APP695swemyc remained almost unaffected after deletion of the NPTY-motif (Fig. 1b, panel 4). These data confirmed that b-secretase shedding of APPswe occurs primarily late in the secretory pathway (Haass et al. 1995). Unexpectedly, we found that the C99 levels were decreased in CHOKI cells expressing APP695sweDNPTYmyc (Fig. 1b, panel 5). Consequently, the disruption of the NPTY domain resulted in a strong attenuation of Ab secretion (Fig. 1b, panel 6). To exclude an APP isoform-specific effect, we generated cells stably over-expressing APP770swemyc or APP770sweDNPTYmyc (Fig. 2). Comparison of cell pools expressing the APP770 isoform showed results similar to those observed for the APP695 one. Quantification of several experiments revealed an approximate four-fold increase in APPsa secretion (Fig. 2, panel 3; Supp. Fig. 1a), while

APPsbswe secretion was not significantly altered in APP770sweDNPTYmyc expressing cells (Fig. 2, panel 4; Fig. S1b). Despite similar amounts of APPsbswe, C99 levels were reduced by more than 50% (Fig. 2, panel 5; Fig. S1c). Concurrently C83 was increased in APP770sweDNPTYmyc expressing cells (Fig. 2, panel 5; Fig. S1c). Consistent with the diminished C99 levels, Ab secretion was decreased by more than 50% in APP770sweDNPTYmyc cells (Fig. 2, panel 6; Fig. S1d). Point mutations within the YENPTY domain of APP695swemyc led to an increase in both APPs and APPsa secretion while APPsbswe production was almost unaffected. At the same time C99 levels and Ab secretion were strongly decreased in comparison to APP695swemyc over-expressing cells (data not shown). In order to address the Ab-species specific alterations, we next examined the levels of Ab40 and Ab42. Both Ab40 and Ab42 secretion were reduced by around 50% in cells expressing APP695swemyc containing different point mutations in the YENPTY-motif (Fig. S1e). We finally confirmed that deletion of the NPTY-motif and the point mutations of the YENPTY abolish the internalization of APPswe. Cell surface biotinylation of the cells overexpressing APP770swemyc and APP770sweDNPTYmyc revealed that the deletion of the NPTY-motif caused a strong increase in mature APP at the cell surface (Fig. 3a). Similarly, different point mutations of the YENPTY-motif



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Fig. 2 Reduction in levels of C99 from APP770sweDNPTYmyc decreases Ab secretion despite of unaltered b-secretase cleavage. Comparison of CHOKI cells stably over-expressing a full-length Cterminal myc-tagged APP770swe (APP770swemyc) with the NPTY deletion mutant (APP770sweDNPTYmyc). Lysates and the 24 h conditioned media were immunoblotted with the indicated antibodies.

also caused an accumulation of APP695swemyc at the plasma membrane (Fig. 3b). These results indicate that the decrease in Ab generation is due to the impairment of APP internalization and the accumulation of APP at the plasma membrane. They demonstrate that secretion of APPsbswe derived from APPswe is independent of its endocytosis motif and, therefore, occurs mainly late in the secretory pathway. C99 levels and Ab generation do not correlate with the APPsbswe secretion, as both were decreased by more than 50% in endocytosis deficient mutants. Taking into account similar levels of secreted APPsbswe in both APPswemyc and APPsweDNPXYmyc expressing cells, we anticipated similar levels of C99 generated in these cells. In contrast, the decrease in Ab secretion in the endocytosis deficient cells more or less correlated with the surprising reduction in C99. We hypothesized that C99 derived from APPswe with any mutation within the YENPTY-motif is metabolized faster than C99 from cells expressing APPswe without any mutation. Due to previous evidence of APP degradation taking place in lysosomes, we tried to stabilize C99 by treatment with NH4Cl, a weak base that is taken up by cells and which neutralizes acidic organelles such as lysosomes. Neutralization causes an inhibition of lysosomal acid-dependent hydrolases. Treatment with lysosomal inhibitors is known to accumulate APP-CTFs (Caporaso

Fig. 3 Mutations within the YENPTY-motif of APPswe cause an accumulation of mature APPswe at the cell surface. (a) APP770swemyc or APP770sweDNPTYmyc over-expressing CHOKI cells were surface biotinylated and the cells lysates were immunoprecipitated with Neutravidin Agarose resin. 20 lg of the lysate and the recovered biotinylated proteins were resolved by SDS-PAGE and APP was detected with the antibody 9E10. (b) Point mutations within the GYENPTY domain of APP695swemyc were stably expressed in CHOKI cells. Cells were surfaced biotinylated as described in (a) and surface APP was detected with 9E10 antibody.

et al. 1992; Perez et al. 1996). While treatment of CHOKI cells stably over-expressing APP695swemyc or APP695sweDNPTYmyc for 24 h with NH4Cl did not affect APPs and APPsa generation (Fig. 4a, panels 2 and 3), it partially inhibited APPsbswe secretion (Fig. 4a, panel 4), which caused a minor decrease in Ab secretion (Fig. 4a; panel 7). This is in line with an acidic pH-optimum of BACE1 (Vassar et al. 1999). Detection of APP-CTFs revealed that NH4Cl treatment strongly increased C83 levels, while C99 remained almost unaffected (Fig. 4a; panels 5 and 6). Additionally, stabilized C83 levels were higher in APPswemyc than in APP695sweDNPTYmyc cells. This indicates that C83 in APP695sweDNPTYmyc cells is less efficiently transported from the surface to lysosomal compartments than in APPswemyc cells. These results demonstrate that reduction in C99 levels in APPsweDNPTYmyc cells is not caused by accelerated lysosomal degradation of C99. We next examined whether a C99 in APP695sweDNPTYmyc cells might be a better target for c-secretase cleavage, but that the generated Ab is not efficiently secreted.



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Fig. 4 The decrease in C99 levels of NPTY deletion mutants of Swedish APP is not caused by lysosomal degradation or enhanced c-secretase processing. (a) CHOKI cells stably over-expressing APP695swemyc or APP695sweDNPTYmyc were treated with 10 mM NH4Cl to neutralize intracellular acidic compartments. APP processing was examined with the indicated antibodies. (b) c-secretase inhibition was carried out by L-685 458 compound (2.5 lM) and APP processing was compared between the CHOKI cells expressing the full-length APP695swemyc and APP695sweDNPTYmyc.

c-secretase inhibition did not affect APP levels or the levels of APPs, APPsa or APPsbswe (Fig. 4b, panels 2–4). As expected, Ab secretion was completely abolished in L-685 458 treated cells (Fig. 4b, panel 7). Consistent with

previous reports, c-secretase inhibition caused accumulation of C83 as well as C99, whereas C83 was the predominantly stabilized form (Fig. 4b; panels 5 and 6). While C99 was strongly elevated in APPswemyc cells after c-secretase inhibition, we could only detect a minor increase in C99 in APP695sweDNPTYmyc expressing cells. As the difference in C99 between APP695swemyc and APP695sweDNPTYmyc expressing cells was not leveled out by c-secretase inhibition, these results indicate that the decrease in C99 levels in cells expressing the endocytosis mutant of APP695swe was not caused by an excess in c-secretase cleavage. These results brought another possibility into our focus. C83 generated in the APP695sweDNPTYmyc expressing cells was surprisingly less stabilized than C83 generated in the APP695swemyc cells after NH4Cl treatment. This indicated to us that internalization of cell surface CTFs into endosomal–lysosomal compartments is equally dependent on the functional endocytosis motif as full-length APP. Reduced internalization of C83 from the cell surface would therefore explain the decreased lysosomal accumulation of C83 after pH neutralization by NH4Cl treatment. In contrast, retention of C99 at the cell surface would probably label these CTFs as a-secretase targets and, therefore, cause a further conversion of C99 to C83. To investigate this assumption, we compared CHOKI cells stably over-expressing wild-type APP695myc or Swedish mutant APP695swemyc with or without the deletion of the NPTY-motif. Additionally, we introduced the F615P mutation, which was shown to inhibit a-secretase cleavage, into APP695wtDNPTYmyc and APP695sweDNPTYmyc (Fig. 5a, for quantifications see Fig. S2a–d) (Sisodia 1992; Haass et al. 1994). While disruption of the APP internalization domain (APP695DNPTYmyc and APP695sweDNPTYmyc, respectively) substantially increased the APPsa and the APPs secretion, additional inhibition of the a-secretase cleavage by the F615P mutation (APP695DNPTY-F615P-myc and APP695sweDNPTY-F615P-myc, respectively) blocked the increase in the total APPs as well as in the APPsa secretion observed in the internalization deficient mutants (Fig. 5a, panels 2 and 3). Once again, the levels of secreted APPsbwt and APPsbswe revealed differences in the cleavage pattern between APP695wt and APP695swe. Deletion of the NPTYmotif in wild-type APP695myc strongly diminished APPsbwt secretion (Fig. 5a, panel 4, Fig. 2a), while the same mutation did not affect APPsbswe secretion of APPswemyc cells (Fig. 5a, panel 5, Fig. 2b). Additional inhibition of asecretase cleavage in APPwt as well as APPswe did not alter APPsb secretion compared with the NPTY deletion mutants (Fig. 5a, panels 4 and 5, Fig. 2a and b). This shows that b-secretase cleavage can occur both in the secretory pathway and in endocytic compartments. Additionally, these results indicate that the extent of b-secretase cleavage in these compartments diverges for APPwt and APPswe.



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Fig. 5 a-Secretase activity causes reduction in C99 levels and Ab secretion of internalization deficient APP. (a) APP processing was examined in CHOK1 cells over-expressing either a APP695wt or APP695swe, with or without endocytosis motif, NPTY, and with or without a-secretase cleavage mutation, F615P. (b) Surface biotinylation of CHOKI cells stably over-expressing APP770swemyc or APP770sweDNPTYmyc was carried out to examine accumulation of mature APP770sweDNPTYmyc as well as CTFs at the cell surface.

b-secretase cleavage of APPswe is almost independent of the endocytosis motif and, therefore, occurs principally in the secretory pathway. In contrast, the same cleavage of APPwt depends largely on the internalization domain and, therefore, takes place mainly in the endocytic compartments.

Detection of CTFs derived from wild-type APP695myc revealed mainly C83, while C99 was hardly detectable. As expected, deletion of the NPTY domain increased C83 levels, while the low levels of C99 were decreased even further. In comparison to APP695DNPTYmyc expressing cells, the additional introduction of the a-secretase cleavage mutation, strongly reduced C83 levels in APP695DNPTYF615P-myc cells. Furthermore, the F615P mutation in wildtype APP695DNPTYmyc expressing cells caused a pattern of CTFs, which all migrated with a slower electrophoretic velocity relative to C83 in APP695DNPTYmyc cells (Fig. 5a, panel 6). In contrast, the NPTY deletion in APPswe resulted in an inversion of the APP-CTF ratio. While C99 levels were slightly higher than C83 levels in APP695swemyc expressing cells, the removal of the endocytosis motif caused a strong decrease in C99 and an increase in C83 in APP695sweDNPTYmyc expressing cells. The additional asecretase cleavage inhibition in APP695sweDNPTYmyc (APP695sweDNPTY-F615Pmyc) reduced the levels of C83 below the levels detected in APP695swemyc cells, while C99 levels were restored to similar amounts as found in APP695swemyc cells (Fig. 5a, panel 6). Overall Ab levels revealed a similar pattern in both APPwt and APPswe cells, except that they were several folds higher in APPswe cells than in APPwt cells (Fig. 5a, panels 7 and 8). Removal of the endocytosis motif strongly decreased Ab secretion of both APPwt and APPswe expressing cells (Fig. 5a, panels 7 and 8, Fig. 2c and d). In contrast, additional inhibition of a-secretase cleavage of the NPTY deletion constructs (APP695DNPTY-F615P-myc and APP695sweDNPTY-F615P-myc, respectively) restored Ab secretion to levels observed in cells expressing wild-type APP695myc or APP695swemyc, respectively (Fig. 5a, panels 7 and 8, Fig. 2c and d). These results indicate that asecretase-mediated conversion of C99 to C83 causes the reduced C99 levels and, hence, the diminished Ab secretion observed in APP695sweDNPTYmyc expressing cells. These results also indicated that mutations within the YENPTY domain of APPswe cause an increase in cell surface levels of full-length APP and CTFs. To verify this assumption we performed cell surface biotinylation experiments. As predicted, APP full-length levels were strongly increased in APP770sweDNPTYmyc cells compared to APP770swemyc expressing cells (Fig. 5b, upper panel). Additionally, cell surface CTFs were also found to be highly elevated in these cells (Fig. 5b, lower panel). Mutations within the YENPTY-motif therefore increase cell surface levels of full-length APP as well as APP-CTFs, whereby APP and C99 become targets of a-secretase cleavage. Our observations suggest that b-secretase cleavage of APPswe generates C99 in the secretory pathway and that these C99 fragments are subsequently transported to the cell surface. Retention of C99 at the cell surface partially



prevented Ab generation, suggesting that the c-secretase cleavage occurs at least in part in endocytic compartments. Conceptually, direct expression of an endocytosis deficient C99-construct should yield a similar effect. We, therefore, generated CHOKI cell lines stably over-expressing either C99 or C99 with a deletion of the NPTY-motif. Cells expressing C99DNPTY revealed a strong reduction of Ab secretion compared with C99 expressing cells (Fig. 6a). While Ab secretion was abrogated, C99 levels were increased by treatment of these cells with the c-secretase inhibitor L-685 458 (Fig. 6a). (a)

In order to investigate whether the decrease in Ab secretion in C99DNPTY expressing cells was caused by a-secretase cleavage of cell surface C99, we tried to stabilize the generated C83 fragments. To distinguish between exogenous and endogenous CTFs we used a C99 tagged with a myc-epitope. Similar to the non-tagged C99 cells, stable C99DNPTYmyc showed a strong reduction in Ab secretion in comparison to C99myc expressing cells (Fig. S2d, panel 3 and Fig. S2e). As expected, neutralization of acidic compartments with NH4Cl strongly elevated endogenous C83 levels (Fig. S2d, panel 2), whereas (b)

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(e)

Fig. 6 Reduction in Ab secretion from C99 is caused by a-secretase mediated conversion of C99 to C83. (a) CHOKI cells were stably transfected with C99 or C99DNPTY and Ab secretion was examined with or without the treatment of cells with the c-secretase inhibitor L685 458 (2.5 lM). (b) Cells stably expressing C99myc or C99DNPTYmyc were treated with the c-secretase inhibitor L-685 458 (2.5 lM). C99, C83 and Ab were measured with the indicated antibodies. (c) An a-secretase cleavage inhibition mutation, F38P, was introduced into C99myc (C99-F38P-myc) and the C99 lacking the endocytosis motif

(C99DNPTY-F38P-myc). Ab generation was examined from the cell culture medium released from the corresponding cells. (d) Quantified total Ab data is presented. Cell culture medium was collected from C99myc, C99DNPTYmyc, C99-F38P-myc and C99DNPTY-F38P-myc expressing cells. Statistical analysis was performed by using one-way ANOVA and Dunett’s post tests. *Significance at p < 0.05, **Significance at p < 0.01. (e) Treatment of the cells used in D with the csecretase inhibitor DAPT was carried out and the levels of APP-C99 and APP-C83 were examined.



exogenous C99myc (Fig. S2d, panel 1) and Ab secretion were not affected (Fig. S2d, panel 3). But, surprisingly, we did not observe an increase in C83myc (Fig. S2d, panel 1). It is important to note that the antibody CT15 recognizes the NPTY epitope, therefore no exogenous signal of C99DNPTYmyc was detected (Fig. S2e, panel 2). We speculated that the low level of generated C83myc might be readily degraded by c-secretase. Therefore, in further experiments we blocked c-secretase cleavage by the transition state c-secretase inhibitor L-685 458, which completely abolished Ab secretion (Fig. 6b, panel 3). While exogenous C99myc levels were increased by c-secretase inhibition, a very strong exogenous C83myc signal appeared in C99DNPTYmyc as well as C99myc expressing cells (Fig. 6b, panel 1). Similarly, endogenous C83 levels were strongly elevated (Fig. 6b, panel 2). These results indicate that both C99 as well as C99DNPTY undergo conversion to C83. Noteworthy, the exogenous C83myc levels were higher in C99DNPTYmyc than in C99myc expressing cells after csecretase inhibition (Fig. 6b, panel 1). In contrast, C99myc was less stabilized in C99DNPTYmyc than in C99myc expressing cells (Fig. 6b, panel 1). This indicates that internalization deficient C99DNPTYmyc accumulates at the cell surface, the primary site of a-secretase activity. This causes an enforced conversion of C99DNPTYmyc to C83DNPTYmyc, thereby decreasing C99 while increasing C83 levels. Mutations of the YENPTY domain abolish APP endocytosis and therefore provoke amplified a-secretase shedding of full-length APP. Our results clearly show that this boosted asecretase activity diminishes Ab generation by shedding of C99DNPTYmyc fragments. Hence, we next examined whether the a-secretase cleavage also limits Ab secretion of endocytosis competent C99myc. Therefore, we additionally introduced the a-secretase inhibition mutation of APP, F615P, into C99myc and C99DNPTYmyc (labeled F38P accordingly to the amino acid numbering of the C99 construct). As before, the deletion of the internalization motif strongly reduced Ab secretion (Fig. 6c and d). In contrast, the C99-F38P-myc expressing cells revealed an increase in Ab generation when compared with C99myc cells. Additional introduction of the F38P mutation in C99DNPTYmyc (C99DNPTY-F38P-myc) not only prevented the reduction in Ab production seen in C99DNPTYmyc cells, but it even elevated the Ab secretion to levels observed in C99-F38P-myc expressing cells (Fig. 6c and d). To demonstrate that the increase in Ab secretion of the F38P mutants was caused by the reduced conversion of C99 to C83, we stabilized the CTFs by treating the cells with the c-secretase inhibitor DAPT. The abrogation of c-secretase cleavage caused a stronger accumulation of C83myc in C99DNPTYmyc than in C99myc expressing cells (Fig. 6e), demonstrating that impairment of APP internalization

enhances a-secretase-mediated conversion of C99myc to C83myc. In contrast, the F38P mutation in C99DNPTYmyc strongly diminished this conversion (Fig. 6e). In comparison with C99myc and C99DNPTYmyc expressing cells, csecretase inhibition resulted in decreased levels of C83myc and elevated C99myc levels in C99DNPTY-F38P-myc cells. Similarly, treatment of cells over-expressing C99-F38P-myc caused an increase in C99myc levels as well as a reduction in C83myc levels when compared with C99myc over-expressing cells (Fig. 6e). Altogether, these results further corroborated our hypothesis that a-secretase-mediated conversion of C99 to C83 is a regular process in the APP metabolism, which is of vital importance in limiting Ab generation.

Discussion Recently, it has been shown that endocytosis inhibition in Tg2576 mice, which over-express APP695 with the Swedish mutation (Hsiao et al. 1996), by a dynamin dominantnegative inhibitory peptide, decreased Ab levels released into the brain interstitial fluid (ISF) by 70% in a dose-dependent manner (Cirrito et al. 2008). These results oppose the long standing idea, that Ab which originates from APPswe is preferentially generated prior to the endocytosis of fulllength APP. Therefore, we set out to clarify this controversy and have reinvestigated the APP processing of wild-type and APPswe. Intriguingly, we found that a-secretase cleavage of C99 plays a crucial role in restricting Ab generation of wild-type as well as Swedish APP. It has been shown that generation of Ab in CHO cells expressing APPwt involves endocytosis of cell surfaceassociated APP via clathrin-coated pit-mediated internalization. While radiolabeled cell surface APPwt was metabolized to Ab after endocytosis, the removal of the APP endocytosis motif abolished Ab secretion (Citron et al. 1992; Haass et al. 1993; Koo and Squazzo 1994). We mutated the endocytosis consensus motif GYENPTY within the cytoplasmic APP tail, which is essential for APP endocytosis and trafficking (Lai et al. 1995; Perez et al. 1999), to continue along these investigations. A pivotal role of this domain is indicated by the fact that it is absolutely conserved across species in all APP family members (King et al. 2003). As shown before, the prevention of wild-type APP695 endocytosis increases APPsa as well as total APPs secretion. In addition, we demonstrate for the first time that retention of APPwt at the cell surface strongly diminishes general BACE cleavage and, thus, APPsbwt secretion. This indicates that the majority of bsecretase shedding of APPwt occurs in endocytic compartments. However, as we detected residual APPsbwt secretion of APPDNPTY deletion mutants, we conclude that some bsecretase cleavage occurs prior to the arrival of full-length APP at the cell surface. Consequently, Ab secretion was almost abrogated in APP695DNPTYmyc expressing cells (Figs 1a and 5a). This suggests that inhibition of APPwt



endocytosis diminishes Ab liberation through prevention of b-secretase cleavage in endocytic compartments. Ab42 levels correlated with Ab40 levels in cells expressing internalization incompetent APP mutants. This indicates that both Ab species released from APPwt are mainly generated in endocytic compartments (Perez et al. 1999). Previous investigations of APPswe expressing cells obtained inconsistent results. Transient expression of Cterminal truncated APPswe did not affect Ab generation. This indicated that APPswe is cleaved to Ab largely in the secretory pathway (Citron et al. 1992, 1995). Surface iodination revealed a greater than twofold increase in Ab secretion in cells expressing Swedish mutant APP as compared with APPwt (Perez et al. 1996), suggesting that a large fraction of APPswe gets processed in endocytic compartments. To investigate whether APP harboring the Swedish mutation would follow the same processing route as it was reported by Cirrito et al., we also mutated the YENPTYmotif of APPswe (Cirrito et al. 2008). Similar to APPwt, deletion of the NPTY domain of APPswe caused an accumulation of mature APPswe at the cell surface (Fig. 3a), leading to a strong increase in APPsa and total APPs generation (Fig. 1b, 2, 5a). This validates previous evidences for the NPTY-motif being essential in mediating effective endocytosis of APP. In contrast to APPwt, deletions of the NPTY domain of APP695swe and APP770swe (Fig. 1b, 2, 5a) as well as point mutations in the YENPTY domain caused almost no change in secretion of APPsbswe. This is in line with previous observations that b-secretase cleavage of APPswe occurs mainly in the late secretory pathway (Haass et al. 1995; Thinakaran et al. 1996). The b-secretase, BACE1, is primarily localized in the Golgi and the endosomes, whereas only small amounts are observed in the ER and lysosomes (Vassar et al. 1999). The investigations of APPwt as well as APPswe processing indicate that b-secretase cleavage can occur in the secretory pathway as well as in endocytic compartments. Notably, bsecretase cleavage of APPwt and APPswe proceed to a different extent in these compartments. While b-secretase cleavage of APPwt occurs primarily in endosomes, shedding of APPswe results mainly from the activity in Golgi-derived vesicles. In contrast to b-secretase cleavage, c-secretase-mediated release of Ab was diminished by more than 50% in cells stably expressing APPswe with mutations in the YENPTY domain (Figs 1b, 2, 5a). Therefore, we hypothesized that the reduced Ab release under these circumstances did not arise from altered b-secretase, but rather from reduced accessibility of C99 to c-secretase. Hence, we stably transfected cells with a C99 construct, thus simulating the APP fragment generated by b-secretase shedding and, therefore, bypassing BACE cleavage itself. Remarkably, Ab release from C99

was also partially dependent on the endocytosis motif NPTY (Fig. 6a–d). This indicated that c-secretase cleavage of APPswe occurs either in endocytic compartments or close to the cell surface. Subcellular localization of presenilins, the catalytic components of c-secretase (Haass 2004), suggested that c-secretase resides primarily or solely in the endoplasmic reticulum and the early Golgi compartment (Selkoe 2001). On the other hand, the catalytic activity of c-secretase was localized at the plasma membrane and to endosomes (Chyung and Selkoe 2003; Chyung et al. 2005; Kaether et al. 2006). In agreement with the later reports, our results indicate that c-secretase activity resides in endocytic compartments as well as at the cell surface. Therefore, the release of Ab from APPwt and APPswe largely depends on efficient transport into endocytic compartments. Ab liberation from APPwt mainly depends on the internalization of full-length APP to facilitate BACE1 cleavage in endocytic compartments (Figs 1a and 5a) followed by c-secretase cleavage (Fig. 6). In contrast, Ab generation from APPswe requires BACE1 cleavage to occur in the secretory pathway (Figs 1b, 2, 5a), followed by internalization of C99 into endocytic compartments for c-secretase cleavage (Fig. 5a, 6). Our results further support the recent data indicating that inhibition of the endocytosis in APPswe over-expressing mice decreased Ab secretion by 70% (Cirrito et al. 2008). In addition, our data agree with already published studies which report that the b-secretase cleavage of APPswe takes place in the secretory pathway (Haass et al. 1995). Similar to the observations in cells stably over-expressing APPwt (Perez et al. 1999), both Ab40 and Ab42 were decreased in APPswe mutants of the YENPTY-motif (Fig. 1e). This result further corroborates the fact that both Ab species are produced at the same site within a cell (Perez et al. 1999). The diminished Ab secretion indicates that C99DNPTYmyc is not efficiently transported from the secretory vesicles, in which they are generated by b-secretase cleavage of APPswe, to the location of c-secretase activity. Apart from the reduced Ab secretion, we observed a strong decrease in C99 levels derived from endocytosis deficient APPswe mutants. However, secretion of APPsbswe remained almost the same as in the endocytosis competent APPswe cells. Our investigations revealed that the decrease in C99 was not caused by enhanced c-secretase cleavage, or by increased lysosomal degradation (Fig. 4a and 4b). Instead, C99 generated from internalization deficient APPswe accumulated at the cell surface (Fig. 5b) similar to mature fulllength APPswe. This cell surface associated C99 became substrate of cell surface localized a-secretase activity similar to endocytosis deficient mutants of full-length APP (Fig. 5a). To demonstrate that a-secretase cleavage was responsible for the reduction in C99 levels, we introduced the F615P mutation into the APP695DNPTYmyc and APP695sweDNPTYmyc constructs. This inhibition prevented the C99 conversion to C83 (Fig. 5a). Interestingly,



this mutation not only restored Ab secretion in cells expressing endocytosis deficient APPswe mutants (APP695sweDNPTY-F615P-myc) through stabilization of C99, but it also increased Ab secretion in cells expressing internalization deficient mutants of APPwt (APP695DNPTYF615P-myc). As b-secretase cleavage of the latter mutants was strongly decreased, it is likely that the a-secretase cleavage inhibition stabilized those C99 stubs of APPwt generated in the secretory pathway similar to Swedish C99 fragments. This event would explain the increase in Ab generation through a-secretase inhibition in APP695DNPTYF615P-myc mutants (Fig. 5a). Most importantly, we additionally demonstrated that a-secretase shedding of internalization competent C99 diminishes Ab generation. Therefore, the inhibition of a-secretase cleavage of C99 elevated Ab secretion (Fig. 6c and d). These results indicated that a-secretase-mediated conversion of C99 to C83 is a crucial event that limits Ab production from APPwt as well as APPswe (Fig. 7).

The evidence presented in this study is in agreement with results on APP processing in mice over-expressing ADAM 10, a potential a-secretase candidate. ADAM10 overexpression reduced Ab formation and prevented the deposition of Ab plaques in a mouse model of AD. It was suggested that an increase in the a-secretase cleavage decreases the availability of the substrate for b-secretase and, thus, decreases Ab generation (Skovronsky et al. 2000; Postina et al. 2004). In addition, Ab levels were more strongly decreased than APPsb levels in ADAM10 over-expressing mice (Postina et al. 2004) and following PMA stimulation of the a-secretase activity in cell culture (Skovronsky et al. 2000). Therefore, our results indicate that increased a-secretase activity additionally prevents the Ab release through cleavage of the c-secretase substrate C99 (Fig. 5a and 6). Recently, several truncated APP/Ab peptides were identified in the cerebrospinal fluid, which were all ending one amino acid before the a-secretase cleavage site (Portelius

Fig. 7 A schematic diagram of APP processing. APP can be processed either via the non-amyloidogenic or the amyloidogenic pathway. In the non-amyloidogenic pathway, APP is first cleaved by asecretase in the Ab domain causing the secretion of the large, soluble ectodomain (APPsa) and the formation of a membrane bound C-terminal fragment of 83 amino acids (C83). Subsequently, the C83 is cleaved by c-secretase releasing the APP intracellular domain (AICD) and the non-amyloidogenic p3 fragment. In the amyloidogenic pathway APP is sequentially cleaved by the b-secretase and c-secretase.

APP processing by BACE1 is a prerequisite for Ab formation. b-secretase forms the N-terminus of the peptide resulting in the secretion of the slightly truncated ectodomain (APPsb) and the formation of membrane bound C-terminal fragment of 99 amino acids (C99). csecretase cleavage of C99 generates the C-terminus of Ab and liberates Ab together with AICD. Furthermore, Ab formation is limited by a-secretase processing of C99. a-Secretase cleaves C99 to generate C83, which is subsequently cleaved by c-secretase generating p3 and AICD.



et al. 2009a). Among other shorter peptides the authors reported an Ab1–14 and an Ab1–15 peptide, which could both be reduced by b-secretase and a-secretase inhibitor treatments (Portelius et al. 2009b). This in vivo existence of C-terminal truncated Ab-species is consistent with the consecutive cleavage of APP by b-secretase and a-secretase described here. These observations further strengthen previous proposals that the activation of a-secretases should be considered a valuable therapeutic target for the treatment of AD. They also support the suggestion that a decrease in the a-secretase activity promotes the development of AD (Postina et al. 2004). The findings presented here also indicate that the final c-secretase cleavage step in the generation of Ab from APPwt and APPswe occurs both at the cell surface and in endocytic, potentially, endosomal compartments. Therefore, retention of APP at the cell surface is beneficial in terms of reduced Ab production and subsequent Ab plaque burden. In addition, the production of Ab is partially interrupted by a-secretase mediated truncation of already generated C99. Our study strongly suggests that specific reduction in APP internalization or specific activation of a-secretase cleavage is a promising route towards slowing down the progression of Alzheimer’s disease.

Acknowledgments We thank Dr Dale Schenk for his generous gift of the APPsb specific antibodies, 192wt and 192swe. We also thank Barbara Klonus for providing the pLHCX-APP695sweDNPXY and the p12-C99DNPXY vectors and Dr Mirsada Causevic for critical reading of the manuscript. Sebastian Ja¨ger received a scholarship of the Boehringer-Ingelheim-Fonds and a grant of the MAIFOR – Forschungsfo¨rderungsprogrann der Universita¨t Mainz. This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) Grant PI379 3-3 (C.U.P.) and Bundesministerium fu¨r Bildung und Forschung (BMBF) Grant 01GI0719 to C.U.P.

Supporting information Additional Supporting Information may be found in the online version of this article: Fig. S1 (a) APPsa secretion was quantified in cell culture medium collected from CHOK1 cells over-expressing APP770swe and APP770sweDNPTY (as shown in Fig. 2, panel 3). (b) APPsbswe secretion in cell culture medium was determined (as shown in Fig. 2, panel 4). (c) C99 and C83 levels were also measured in cell lysates of APP770swe and in APP770sweDNPTY expressing cells (as shown in Fig. 2, panel 5). (d) Total Ab secretion was quantified by Western blotting of cell culture medium collected from the indicated cells (as shown in Fig. 2, panel 6). (e) Ab40 and Ab42 were measured in 24 h conditioned media, from cells expressing different mutations within the endocytosis domain GYENPTY, with a standard ELISA. Statistical analysis was performed using one-way ANOVA and Dunett’s post tests.

Fig. S2 (a) APPsbwt secretion was measured in wild-type APP695myc cells, APP695DNPTYmyc cells and APP695DNPTYmyc cells containing an a-secretase cleavage mutation, F615P (as shown in Fig. 5a, panel 5). (b) APPsbswe secretion was determined in the above mentioned cells (as shown in Fig. 5a, panel 4). (c) Total Ab levels were measured in the same cells (as shown in Fig. 5a, panels 7 and 8). (d) CHOKI cells stably over-expressing C99myc or C99DNPTYmyc were treated with 10 mM NH4Cl to neutralize intracellular acidic organelles. C99, C83 and Ab levels were examined with the indicated antibodies. (e) Ab levels were quantified and normalized to the levels of C99. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

References Bueler H., Fischer M., Lang Y., Bluethmann H., Lipp H. P., DeArmond S. J., Prusiner S. B., Aguet M. and Weissmann C. (1992) Normal development and behaviour of mice lacking the neuronal cellsurface PrP protein. Nature 356, 577–582. Buxbaum J. D., Liu K. N., Luo Y., Slack J. L., Stocking K. L., Peschon J. J., Johnson R. S., Castner B. J., Cerretti D. P. and Black R. A. (1998) Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J. Biol. Chem. 273, 27765–27767. Caporaso G. L., Gandy S. E., Buxbaum J. D. and Greengard P. (1992) Chloroquine inhibits intracellular degradation but not secretion of Alzheimer beta/A4 amyloid precursor protein. Proc. Natl. Acad. Sci. USA 89, 2252–2256. Chen W. J., Goldstein J. L. and Brown M. S. (1990) NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J. Biol. Chem. 265, 3116–3123. Chyung J. H. and Selkoe D. J. (2003) Inhibition of receptor-mediated endocytosis demonstrates generation of amyloid beta-protein at the cell surface. J. Biol. Chem. 278, 51035–51043. Chyung J. H., Raper D. M. and Selkoe D. J. (2005) Gamma-secretase exists on the plasma membrane as an intact complex that accepts substrates and effects intramembrane cleavage. J. Biol. Chem. 280, 4383–4392. Cirrito J. R., Kang J. E., Lee J., Stewart F. R., Verges D. K., Silverio L. M., Bu G., Mennerick S. and Holtzman D. M. (2008) Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron 58, 42–51. Citron M., Oltersdorf T., Haass C., McConlogue L., Hung A. Y., Seubert P., Vigo-Pelfrey C., Lieberburg I. and Selkoe D. J. (1992) Mutation of the beta-amyloid precursor protein in familial Alzheimer’s disease increases beta-protein production. Nature 360, 672–674. Citron M., Teplow D. B. and Selkoe D. J. (1995) Generation of amyloid beta protein from its precursor is sequence specific. Neuron 14, 661–670. Cook D. G., Forman M. S., Sung J. C., Leight S., Kolson D. L., Iwatsubo T., Lee V. M. and Doms R. W. (1997) Alzheimer’s A beta(1-42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nat. Med. 3, 1021–1023. De Strooper B., Umans L., Van Leuven F. and Van Den Berghe H. (1993) Study of the synthesis and secretion of normal and artificial



mutants of murine amyloid precursor protein (APP): cleavage of APP occurs in a late compartment of the default secretion pathway. J. Cell Biol. 121, 295–304. Edbauer D., Winkler E., Regula J. T., Pesold B., Steiner H. and Haass C. (2003) Reconstitution of gamma-secretase activity. Nat. Cell Biol. 5, 486–488. Esch F. S., Keim P. S., Beattie E. C., Blacher R. W., Culwell A. R., Oltersdorf T., McClure D. and Ward P. J. (1990) Cleavage of amyloid beta peptide during constitutive processing of its precursor. Science 248, 1122–1124. Felsenstein K. M., Hunihan L. W. and Roberts S. B. (1994) Altered cleavage and secretion of a recombinant beta-APP bearing the Swedish familial Alzheimer’s disease mutation. Nat. Genet. 6, 251–255. Haass C. (2004) Take five–BACE and the gamma-secretase quartet conduct Alzheimer’s amyloid beta-peptide generation. EMBO J. 23, 483–488. Haass C., Koo E. H., Mellon A., Hung A. Y. and Selkoe D. J. (1992) Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments. Nature 357, 500–503. Haass C., Hung A. Y., Schlossmacher M. G., Teplow D. B. and Selkoe D. J. (1993) Beta-amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms. J. Biol. Chem. 268, 3021– 3024. Haass C., Hung A. Y., Selkoe D. J. and Teplow D. B. (1994) Mutations associated with a locus for familial Alzheimer’s disease result in alternative processing of amyloid beta-protein precursor. J. Biol. Chem. 269, 17741–17748. Haass C., Lemere C. A., Capell A., Citron M., Seubert P., Schenk D., Lannfelt L. and Selkoe D. J. (1995) The Swedish mutation causes early-onset Alzheimer’s disease by beta-secretase cleavage within the secretory pathway. Nat. Med. 1, 1291–1296. Hartmann T., Bieger S. C., Bruhl B. et al. (1997) Distinct sites of intracellular production for Alzheimer’s disease A beta40/42 amyloid peptides. Nat. Med. 3, 1016–1020. Hsiao K., Chapman P., Nilsen S., Eckman C., Harigaya Y., Younkin S., Yang F. and Cole G. (1996) Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 274, 99–102. Huse J. T., Pijak D. S., Leslie G. J., Lee V. M. and Doms R. W. (2000) Maturation and endosomal targeting of beta-site amyloid precursor protein-cleaving enzyme. The Alzheimer’s disease beta-secretase. J. Biol. Chem. 275, 33729–33737. Hussain I., Powell D., Howlett D. R. et al. (1999) Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol. Cell. Neurosci. 14, 419–427. Kaether C., Schmitt S., Willem M. and Haass C. (2006) Amyloid precursor protein and Notch intracellular domains are generated after transport of their precursors to the cell surface. Traffic 7, 408–415. King G. D., Perez R. G., Steinhilb M. L., Gaut J. R. and Turner R. S. (2003) X11alpha modulates secretory and endocytic trafficking and metabolism of amyloid precursor protein: mutational analysis of the YENPTY sequence. Neuroscience 120, 143–154. Koike H., Tomioka S., Sorimachi H., Saido T. C., Maruyama K., Okuyama A., Fujisawa-Sehara A., Ohno S., Suzuki K. and Ishiura S. (1999) Membrane-anchored metalloprotease MDC9 has an alphasecretase activity responsible for processing the amyloid precursor protein. Biochem. J. 343(Pt 2), 371–375. Koo E. H. and Squazzo S. L. (1994) Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J. Biol. Chem. 269, 17386–17389.

Koo E. H., Squazzo S. L., Selkoe D. J. and Koo C. H. (1996) Trafficking of cell-surface amyloid beta-protein precursor. I. Secretion, endocytosis and recycling as detected by labeled monoclonal antibody. J. Cell Sci. 109(Pt 5), 991–998. Lai A., Sisodia S. S. and Trowbridge I. S. (1995) Characterization of sorting signals in the beta-amyloid precursor protein cytoplasmic domain. J. Biol. Chem. 270, 3565–3573. Lammich S., Kojro E., Postina R., Gilbert S., Pfeiffer R., Jasionowski M., Haass C. and Fahrenholz F. (1999) Constitutive and regulated alpha-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease. Proc. Natl. Acad. Sci. USA 96, 3922–3927. Leuchtenberger S., Maler J., Czirr E., Ness J., Lichtenthaler S. F., Esselmann H., Pietrzik C. U., Wiltfang J. and Weggen S. (2008) Nonsteroidal Anti-Inflammatory Drugs and Ectodomain Shedding of the Amyloid Precursor Protein. Neurodegener. Dis. 6(1–2), 1–6. Lichtenthaler S. F., Multhaup G., Masters C. L. and Beyreuther K. (1999) A novel substrate for analyzing Alzheimer’s disease gamma-secretase. FEBS Lett. 453, 288–292. Perez R. G., Squazzo S. L. and Koo E. H. (1996) Enhanced release of amyloid beta-protein from codon 670/671 ‘‘Swedish’’ mutant betaamyloid precursor protein occurs in both secretory and endocytic pathways. J. Biol. Chem. 271, 9100–9107. Perez R. G., Soriano S., Hayes J. D., Ostaszewski B., Xia W., Selkoe D. J., Chen X., Stokin G. B. and Koo E. H. (1999) Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42. J. Biol. Chem. 274, 18851–18856. Pietrzik C. U., Busse T., Merriam D. E., Weggen S. and Koo E. H. (2002) The cytoplasmic domain of the LDL receptor-related protein regulates multiple steps in APP processing. EMBO J. 21, 5691–5700. Portelius E., Brinkmalm G., Tran A. J., Zetterberg H., Westman-Brinkmalm A. and Blennow K. (2009a) Identification of novel APP/ Abeta isoforms in human cerebrospinal fluid. Neurodegener Dis 6, 87–94. Portelius E., Price E., Brinkmalm G., Stiteler M., Olsson M., Persson R., Westman-Brinkmalm A., Zetterberg H., Simon A. J. and Blennow K. (2009b) A novel pathway for amyloid precursor protein processing. Neurobiol. Aging [Epub ahead of print] doi:10.1016/ j.neurobiolaging.2009.06.002. Postina R., Schroeder A., Dewachter I. et al. (2004) A disintegrinmetalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model. J. Clin. Invest. 113, 1456–1464. Sambamurti K., Shioi J., Anderson J. P., Pappolla M. A. and Robakis N. K. (1992) Evidence for intracellular cleavage of the Alzheimer’s amyloid precursor in PC12 cells. J. Neurosci. Res. 33, 319–329. Sastre M., Steiner H., Fuchs K., Capell A., Multhaup G., Condron M. M., Teplow D. B. and Haass C. (2001) Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch. EMBO Rep 2, 835–841. Selkoe D. J. (2001) Alzheimer’s disease: genes, proteins, and therapy. Physiol. Rev. 81, 741–766. Seubert P., Oltersdorf T., Lee M. G. et al. (1993) Secretion of betaamyloid precursor protein cleaved at the amino terminus of the beta-amyloid peptide. Nature 361, 260–263. Sinha S., Anderson J. P., Barbour R. et al. (1999) Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402, 537–540.



Sisodia S. S. (1992) Beta-amyloid precursor protein cleavage by a membrane-bound protease. Proc. Natl. Acad. Sci. USA 89, 6075–6079. Skovronsky D. M., Moore D. B., Milla M. E., Doms R. W. and Lee V. M. (2000) Protein kinase C-dependent alpha-secretase competes with beta-secretase for cleavage of amyloid-beta precursor protein in the trans-golgi network. J. Biol. Chem. 275, 2568–2575. Thinakaran G., Teplow D. B., Siman R., Greenberg B. and Sisodia S. S. (1996) Metabolism of the ‘‘Swedish’’ amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the ‘‘betasecretase’’ site occurs in the golgi apparatus. J. Biol. Chem. 271, 9390–9397.

Vassar R., Bennett B. D., Babu-Khan S. et al. (1999) Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286, 735–741. Weidemann A., Eggert S., Reinhard F. B., Vogel M., Paliga K., Baier G., Masters C. L., Beyreuther K. and Evin G. (2002) A novel epsiloncleavage within the transmembrane domain of the Alzheimer amyloid precursor protein demonstrates homology with Notch processing. Biochemistry 41, 2825–2835. Yan R., Bienkowski M. J., Shuck M. E. et al. (1999) Membraneanchored aspartyl protease with Alzheimer’s disease beta-secretase activity. Nature 402, 533–537.
