Sulindac Sulfide Is a Noncompetitive gamma -Secretase Inhibitor That Preferentially Reduces Abeta 42 Generation*

Yasuko TakahashiDagger , Ikuo HayashiDagger , Yusuke Tominari§, Kentaro Rikimaru§, Yuichi MorohashiDagger , Toshiyuki Kan§, Hideaki Natsugari, Tohru Fukuyama§, Taisuke TomitaDagger ||, and Takeshi IwatsuboDagger **

From the Dagger  Department of Neuropathology and Neuroscience, the § Department of Synthetic Natural Products Chemistry, and the  Department of Rational Medicinal Science, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, February 14, 2003, and in revised form, March 10, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Abeta 42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of gamma -secretase for Abeta 42 generation (gamma 42-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on gamma -secretase and preferentially inhibits the gamma 42-secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro gamma -secretase assay using recombinant amyloid beta  precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Abeta 40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for gamma 42-secretase in vitro. Our data suggest that SSide is a direct inhibitor of gamma -secretase that preferentially affects the gamma 42-secretase activity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease (AD)1 is a dementing neurodegenerative disorder of the elderly characterized pathologically by neuronal loss in the cerebral cortex accompanied by massive deposition of amyloid beta  peptides (Abeta ) as senile plaques (1). Abeta is produced by sequential proteolytic cleavages of the amyloid beta  precursor protein (beta APP) by a set of membrane-bound proteases termed beta - and gamma -secretases. The C-terminal length of Abeta generated by gamma -secretase is heterogeneous; Abeta 42 is a relatively minor molecular species of the Abeta secreted from cells, but it has a much higher propensity to aggregate and form amyloid compared with other Abeta species. These findings provide strong support for the hypothesis that the deposition of Abeta 42 is closely related to the pathogenesis of AD, implicating gamma -secretase as an important therapeutic target.

Mutations in PS1 or PS2 genes account for the majority of early onset familial AD, and these mutations cause an increase in the ratio or levels of production of Abeta 42 (1). It is known that PS is essential for the gamma -secretase-mediated intramembranous cleavage not only for beta APP but for other type I transmembrane proteins (e.g. Notch, ErbB4, E-cadherin, low density lipoprotein receptor-related protein, and CD44) (2). PS proteins undergo endoproteolysis to generate N- and C-terminal fragments and interact with other proteins (i.e. nicastrin, APH-1, and PEN-2) to form a high molecular weight (HMW) protein complex (3). The functional role of PS complex in gamma -secretase activity still remains unknown. However, aspartyl protease transition state analogue inhibitors of gamma -secretase, which harbors a hydroxyl ethylene isostere or a difluoro alcohol moiety, directly label PS fragments (4-6). In addition, a systematic analysis using a variety of PS mutants revealed that HMW complex formation of PS as well as conserved aspartyl residues within the transmembrane domain are essential for gamma -secretase activity (7-11). Finally, in vitro gamma -secretase activity is associated with PS HMW complex (12, 13). These data suggest that HMW PS complex corresponds to the gamma -secretase, an atypical membrane-embedded aspartyl protease, and that PS proteins harbor the catalytic center for gamma -secretase complex (3).

Epidemiological studies have shown that long term treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) prevents the development of AD (reviewed in Refs. 14 and 15). Recently, a prospective, population-based cohort study provided strong evidence that the long term use of NSAIDs significantly reduced the risk of AD (16). NSAIDs affect the inflammatory response by direct inhibition of cyclooxgenase (COX) enzymes. Moreover, recent studies indicate that NSAIDs are involved in transcriptional regulation by the modulation of Ikappa B kinase beta  or peroxisome proliferator-activated receptors (PPAR) (reviewed in Ref. 17). It has been believed that NSAIDs might influence the AD pathology by inhibiting the inflammation response (e.g. activation of microglia and astroglia) in brains (14). In fact, administration of NSAIDs affects the inflammatory profile and inhibits the progression of amyloid deposition in the brains of beta APP transgenic mice (18-20). Contrary to these traditional views, however, it has recently been shown that a subset of NSAIDs (ibuprofen, sulindac sulfide (SSide), indomethacin, and R-flurbiprofen) selectively decrease the secretion of Abeta 42 from cultured cells independently of COX activity and lowers the amount of soluble Abeta 42 in the brains of transgenic mice (21, 22). In cultured cells, the decrease in Abeta 42 secretion caused by SSide was accompanied by an increase in Abeta 38 generation, whereas the Notch site-3 cleavage activity to generate Notch intracellular domain (NICD) was not significantly affected. In contrast, other NSAIDs including sulindac sulfone (SSone), which is a metabolite of SSide and inactive for COX, had no significant effect on gamma -secretase activity. These data suggest that some of the NSAIDs may affect the pathogenetic process of AD by directly inhibiting the gamma -secretase activity, causing a shift in the cleavage site. Furthermore, in contrast to the previously developed gamma -secretase inhibitors, the treatment with NSAIDs may be free from side effects caused by inhibiting the intramembranous cleavage of other substrates. However, the molecular mechanism whereby NSAIDs inhibit Abeta 42 generation, as well as the target protein of NSAIDs to modulate gamma -secretase activity, still remains unclear. Here we analyzed the enzymatic property of gamma -secretase by an in vitro gamma -secretase assay using recombinant substrates and CHAPSO-solubilized membrane as an enzyme source, with special reference to the effect of NSAIDs on intramembranous cleavage of beta APP and Notch.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Compounds and Peptides-- Synthesis of DAPT (23) was performed by a standard solution phase peptide synthesis utilizing the Cbz protecting group. The detailed synthetic procedure will be reported elsewhere.

NSAIDs used in this study (SSide, Ssone, and naproxen) were purchased from Biomol (Plymouth Meeting, PA) and were dissolved in dimethyl sulfoxide (Me2SO). L-685,458 and standard Abeta peptides were purchased from Bachem AG (Bubendorf, Switzerland).

Cell Culture and Treatment by NSAIDs-- The expression vectors encoding beta APPNL in pCEP4 (Invitrogen) and NotchDelta E in pCS2 were provided by Drs. K. Maruyama (Saitama Medical School) and R. Kopan (Washington University), respectively. A stable Neuro2a (N2a) cell line doubly expressing beta APPNL and NotchDelta E (N2a NL/N) was generated as described previously (24). To analyze the effect of NSAIDs on gamma -secretase activity, N2a NL/N cells were cultured at confluency in Dulbecco's modified Eagle's medium containing 10 mM butyric acid to drive protein expression in the presence of various concentrations of NSAIDs for 48 h. Culture media were collected and subjected to BAN50/BA27 or BAN50/BC05 ELISAs (25). Immunoblot analysis using C4 (anti-beta APP C terminus, provided from Dr. Y. Ihara (University of Tokyo)) or anti-c-Myc (Roche Applied Science) was performed as described previously (8, 9).

Purification of Recombinant Substrates-- cDNAs encoding the C-terminal 99 amino acids of human beta APP or 101 amino acids of mouse Notch 1 fused to FLAG tag at the C terminus and harboring an additional Met at the N terminus was generated by PCR and subcloned into pTrcHis2A (Invitrogen) (C100-FmH and N102-FmH, respectively) (26). A cDNA encoding C100-FmH carrying I716F mutation (11, 27) was generated by the long PCR protocol using a cDNA encoding C100-FLAG in pTrcHis2A vector as a template. All constructs were sequenced using Thermo SequenaseTM (Amersham Biosciences) on an automated sequencer (Li-Cor). Recombinant proteins were expressed in Escherichia coli and purified by nickel-chelating affinity chromatography as described previously (26).

Preparation of Solubilized gamma -Secretase Fractions from Cultured Cells-- All cell lines were maintained as described previously (25) and grown at confluency. Membrane fractions were prepared as described previously (9, 28, 29). The membrane pellets were resuspended in HEPES buffer to yield a protein concentration of 5-10 mg/ml and were stored at -70 °C. The membranes were solubilized by 1% CHAPSO (Wako, Osaka, Japan) combined with 1 M NDSB-256 (Calbiochem) for 60 min at 4 °C and centrifuged at 100,000 × g for 60 min. We defined the supernatant (~5 mg of protein/ml) as the solubilized gamma -secretase fraction, which was stored at -80 °C until use. All procedures were performed at 4 °C.

In Vitro gamma -Secretase Assay-- In vitro gamma -secretase assay was performed as described previously (26) with some modifications. Each recombinant substrate at defined concentrations was incubated together with the solubilized gamma -secretase fraction (250 µg/ml) in 1× gamma  buffer (HEPES buffer containing 0.25% CHAPSO, 5 mM EDTA, 5 mM 1,10-phenanthroline, 10 mg/ml phosphoramidon, Complete protease inhibitor mixture (Roche Applied Science)) with or without gamma -secretase inhibitors (including NSAIDs) at 4 or 37 °C for 3 h. Control reactions were performed in the presence of 1% Me2SO. The reaction was stopped by boiling the reaction mixtures for 2 min. The samples were centrifuged, and the supernatants were analyzed by BAN50/BA27, BAN50/BC05, BNT77/BA27, or BNT77/BC05 ELISAs for de novo generation of Abeta (25). For the immunoblot analyses of de novo generated peptides, the total proteins in the supernatants were precipitated by trichloroacetic acid and analyzed by immunoblotting with BAN50 (anti-Abeta ), anti-c-Myc (Roche Applied Science), or anti-FLAG M2 (Sigma). Tris/Bicine/urea high resolution Abeta immunoblot analysis was performed as described previously (30).

    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
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Characterization of the gamma -Secretase Activity in Vitro-- We first characterized the biochemical and enzymatic properties of the gamma -secretase activity as detected by our in vitro gamma -secretase assay. For this purpose, we generated a recombinant protein substrate C100-FmH, based on the amino acid sequence of the C-terminal fragment of beta APP fused to FLAG-myc-His6 tag sequences at the C terminus (Fig. 1). De novo generation of Abeta peptides from recombinant C100-FmH incubated with membranes of HeLa cells as an enzyme source required the presence of 0.25% CHAPSO, whereas Triton X-100 or SDS abolished the gamma -secretase activity (data not shown), which was consistent with the previous observations (12). Incubation of HeLa cell membranes with wild-type (wt) C100-FmH predominantly generated Abeta 40, in addition to Abeta 42 as a minor species (Fig. 2A). However, the relatively low levels of de novo generation of Abeta 42 hampered detailed pharmacological and enzymatic analyses of Abeta 42-generating activity from wt C100-FmH. Thus, we introduced the I716F mutation into the recombinant substrate, which had been described to cause a dramatic increase in Abeta 42 generation in intact cell-based assays (27). De novo Abeta 42 generation from I716F mutant (mt) C100-FmH was significantly increased, whereas the production of Abeta 40 was almost totally abolished, suggesting that mt C100-FmH served as an optimal substrate for gamma 42-secretase (Fig. 2A). These proteolytic activities were recovered from the solubilized membrane fraction by 1% CHAPSO containing 1 M NDSB-256. We next examined the effects of the two well characterized gamma -secretase inhibitors, L-685,458 and DAPT, on Abeta generation in our in vitro assay (12, 23, 31). Both compounds inhibited Abeta -generating activities in a similar, concentration-dependent fashion (Fig. 2B). We then studied the Abeta -generating activities in membranes of various cell lines including embryonic fibroblasts derived from PS1/2 double knockout mice (9, 32). Consistent with the results of intact cell-based assays, de novo production of Abeta peptides was almost totally abolished in membrane fractions from PS1/2 double knockout cells, and these activities can be immunoprecipitated with antibodies against PS1 (Fig. 2C and data not shown). These data suggest that recombinant wt and mt C100-FmHs were processed by the endogenous, PS-dependent gamma 40- and gamma 42-secretase activities that enzymatically generate Abeta 40 and -42 polypeptides, respectively. We further analyzed the enzymatic properties of gamma -secretase activities of intact or solubilized membrane fractions from HeLa cells in vitro. The apparent Km value for the processing of either wt or mt substrate to generate Abeta 40 or Abeta 42, respectively, by gamma -secretase activities was ~0.5 µM (Fig. 2D). The progress curve was linear during the 6-h reaction time, and the pH dependence of gamma -secretase activity was broad, ranging from pH 5 to 9 (data not shown). Furthermore, we observed a gamma -secretase-dependent in vitro generation of the C-terminally tagged APP intracellular domain fragments (AICD-FmH) that is produced as the C-terminal counterpart of Abeta , by incubation of either wt or mt C100-FmH with solubilized HeLa membranes (Fig. 2E). These data are consistent with the previous reports (12, 33, 34) on the in vitro gamma -secretase assays using different types of recombinant C100 with or without C-terminal tags.


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Fig. 1.   Schematic representation of recombinant substrates used in this study. C100-FmH consists of an N-terminal Met, beta APP-(597-695) and the consecutive FLAG, c-Myc, and His tags. Similarly, N102-FmH is composed of Notch-(1699-1799) with an N-terminal Met and C-terminal FLAG/Myc/His tag. De novo generated Abeta peptides and intracellular domains (black box and white boxes, respectively) were detected by two-site ELISAs (BAN50/BNT77-BA27/BC05) or using sensitive immunoblotting methods. The location of I716F mutation in mt C100-FmH is indicated by star.


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Fig. 2.   Characterization of the in vitro gamma -secretase activity. A, de novo generation of Abeta 40 (open columns) or Abeta 42 (filled columns) peptides from recombinant substrates in vitro using HeLa cell membranes as enzyme source and quantitated by ELISAs. Names of recombinant proteins used as substrates are indicated below the columns. B, inhibitory potencies of solubilized gamma -secretase activity by DAPT (left panel) and L-685,458 (right panel). De novo generated levels of Abeta 40 from wt substrate (open circles) or Abeta 42 from mt substrate (filled triangles) were evaluated by ELISAs. The data are indicated as % of those observed in the absence of given inhibitors. C, generation of Abeta 40 from wt (open columns) or Abeta 42 from mt (filled columns) substrates by membrane fractions from various cell lines. CHO, Chinese hamster ovary; MDCK, Madin-Darby canine kidney. D, Michaelis-Menten plot for the generation of Abeta 40 from wt substrate (open circles) or Abeta 42 from mt substrate (filled triangles). E, AICD-FmH generation from recombinant substrates in vitro. Total proteins in the samples incubated overnight at the indicated temperature (4 or 37 °C) in the presence or absence of DAPT were precipitated by trichloroacetic acid and analyzed by immunoblotting. Recombinant C100-FmH and the cleavage product thereof (AICD-FmH) are indicated by the arrowhead and arrow, respectively. F, generation of NICD-FmH from recombinant substrates in vitro as in E. Recombinant N102-FmH and cleavage products (NICD-FmH) are indicated by the arrowhead and arrow, respectively.

Intramembranous cleavage of gamma -secretase substrates generates the intracellular domain fragments that are liberated from the membrane and mediate the signal pathways from plasma membrane to nucleus (2). Notably, a proteolytic generation of Notch intracellular domain (NICD) by PS-dependent gamma -secretase is the most well known example, and potential side effects caused by the blockade of Notch pathway by gamma -secretase inhibitors are emerging problems (35, 36). To analyze the gamma -secretase activity to generate NICD in vitro, we generated a recombinant N102-FmH substrate composed of a 101-residue fragment of murine Notch1 beginning close to the S2 cleavage site and containing transmembrane domain fused to a FLAG-myc-His C-terminal tag (Fig. 1). After incubation of N102-FmH with the membrane fraction, we observed the appearance of an NICD-like polypeptide migrating slightly faster than N102-FmH, which was diminished by treatment with DAPT in a dose-dependent manner (Fig. 2F and data not shown). Thus, the recombinant N102-FmH polypeptide is also cleaved by gamma -secretase to generate C-terminally tagged NICD (NICD-FmH) in vitro, which was consistent with the recent report by Wolfe and colleagues (37, 38).

Effect of Sulindac Sulfide on gamma -Secretase Activity in Vitro-- It has been reported recently (21, 22) that a subset of NSAIDs lower Abeta 42 without affecting Notch processing in cultured cells. To gain more insights into the effect of NSAIDs on APP and Notch processing, we have chosen three NSAIDs, i.e. sulindac sulfide (SSide), sulindac sulfone (SSone), and naproxen, to treat N2a NL/N cells stably coexpressing beta APPNL and NotchDelta E (24). We confirmed a specific decrease in Abeta 42 secretion by treatment with 10-30 µM SSide, whereas the secretion of Abeta 40 as well as Notch processing was not affected (Fig. 3). Treatment with 100 µM of SSide caused cell death presumably by inducing apoptosis, resulting in marked decrease in Abeta generation as well as in total protein expression (17). The IC50 value for Abeta 42 secretion of SSide was 30.6 ± 2.8 µM. SSone and naproxen had no effect either on Abeta 40 or Abeta 42 secretion as well as on Notch cleavage up to 100 µM.


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Fig. 3.   Inhibitory effects of NSAIDs on Abeta secretion and Notch processing in N2a NL/N cells. A, ELISA quantitation of Abeta 40 (open circles) and Abeta 42 (filled triangles) secretion from N2a NL/N cells in the presence of NSAIDs at the indicated concentrations. The data are indicated as % of those observed in the absence of inhibitors. B, immunoblot analysis of the expression and processing of beta APP and Notch in N2a cell lysates treated by NSAIDs at the indicated concentrations. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-beta APP antibody C4 (upper and middle panel) or anti-c-Myc (lower panel), respectively. Protein bands corresponding to beta APPNL or NotchDelta E and their cleavage products (C83 or NICD, respectively) are indicated by arrows and arrowheads, respectively.

To examine whether SSide modulates gamma -secretase activity by direct or indirect mechanisms (e.g. altering the trafficking of substrates or enzymes, affecting the secretion or degradation of Abeta 42 peptides, or modifying the transcription of gamma -secretase-related genes), we analyzed the in vitro gamma -secretase activity in solubilized membrane fraction in the presence of NSAIDs. We observed an inhibition of gamma 42-secretase activity by SSide in a dose-dependent manner. The IC50 value of SSide for inhibiting gamma 42-secretase activity in vitro was 20.2 ± 2.6 µM (Fig. 4A). We found a decrease in slope by the increase of the concentration of SSide in the plot of rate against the enzyme concentration, suggesting that SSide is not an irreversible or pseudo-irreversible inhibitor (Fig. 4B). Moreover, when we dialyzed the solubilized gamma -secretase fraction pretreated with SSide against CHAPSO buffer without SSide, gamma -secretase activity was almost totally recovered (Fig. 4C). From these data, it was strongly suggested that the genuine molecular target of SSide is the gamma -secretase complex and that SSide works as a reversible gamma -secretase inhibitor.


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Fig. 4.   Inhibitory effects of NSAIDs on in vitro gamma -secretase activity. A, ELISA quantitation of de novo generated Abeta 40 (open circles) and Abeta 42 (filled triangles) from recombinant substrates in the presence of NSAIDs at the indicated concentrations. The data are indicated as % of those generated in the absence of inhibitors. Note that SSide, but not SSone or naproxen, inhibits both gamma 40- and gamma 42-secretase activity at high concentrations. B, plot of activity rate against the enzyme concentration under coincubation with 0 (open circles), 25 (filled circles), 50 (open squares), and 75 µM (filled squares) SSide. C, de novo Abeta 42 generation by dialyzed membrane fraction preincubated with 100 µM SSide. Note that the dialysis of SSide-preincubated gamma 42-secretase resulted in the almost total recovery of the enzymatic activity.

In contrast to the results in cultured cells, the application of SSide at low concentrations (1-25 µM) caused a transient, but significant, increase in Abeta 40 generation in vitro (Fig. 4A). Moreover, SSide diminished the de novo generation of Abeta 40, in addition to that of Abeta 42, at high concentrations (50-100 µM). Thus, SSide has an inhibition potency against gamma 40-secretase activity at high concentrations, whereas it elevates the gamma 40-secretase activity at sub-inhibitory doses. It was reported that the decrease in Abeta 42 secretion by SSide was accompanied by a dose-dependent increase in Abeta 38 secretion (21). To determine whether the decrease in Abeta 42 (plus Abeta 40) production caused by high concentrations of SSide affects that of Abeta 38 in vitro, we analyzed the de novo generated Abeta species by high resolution immunoblotting (30). We observed a dose-dependent increase in Abeta 38 generation in the low concentration ranges (1-25 µM) accompanied by a decrease in Abeta 42 production, although the in vitro generation of Abeta peptides including Abeta 38 was entirely inhibited by high concentrations of SSide (data not shown). These results suggest that SSide is a bona fide gamma -secretase inhibitor directly affecting the membrane-embedded protease complex, exhibiting distinct inhibitory potencies against Abeta 38-, Abeta 40-, and Abeta 42-generating activities of gamma -secretase.

To characterize further the inhibitory mechanism of gamma 42-secretase activity by SSide, we performed the double-reciprocal plot analysis (Fig. 5A). We found that the Km value remained at a constant level, but the Vmax value was decreased under increasing concentrations of SSide, suggesting that SSide displayed a noncompetitive inhibition for gamma 42-secretase activity. Because transition state analogue gamma -secretase inhibitors (i.e. pepstatin or L-685,458) displayed linear noncompetitive inhibition profiles, a two-binding site model for intramembranous cleavage by gamma -secretase has been proposed (34). In this model, gamma -secretase complex is predicted to harbor a docking/anchoring site of substrates as well as a separate catalytic site. To determine whether SSide affects gamma 42-secretase activity by interacting with the catalytic site of gamma 42-secretase, we analyzed the inhibition profile of SSide by coincubation with L-685,458, a transition state analogue inhibitor that is expected to occupy the active site of gamma 42-secretase. If SSide binds to the docking/anchoring site, coincubation with SSide and L-685,458 would result in a synergistic inhibition of gamma 42-secretase and cause an increase in the inhibition slope. Unexpectedly, however, an addition of L-685,458 had no effect on the slope, raising the possibility that SSide may inhibit the gamma 42-secretase activity in a similar mechanism to that of a transition state analogue inhibitor, L-685,458 (Fig. 5B). Thus, SSide would compete for a similar binding site (i.e. catalytic site) with L-685,458 in the gamma 42-secretase complex or, alternatively, the binding of either of the inhibitors allosterically affects the interaction of the other.


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Fig. 5.   Characterization of the inhibitory effect of SSide on gamma 42-secretase. A, double-reciprocal plots for inhibition of gamma 42-secretase by 0 (open circles), 25 (filled circles), 50 (open squares), 75 (filled squares), and 100 µM (open triangles) SSide. The data are indicated as % of those observed in the absence of SSide. B, intercept replot of inhibition of gamma 42-secretase by SSide in the presence of 0 (open circles), 0.5 (filled circles), 1 (open squares), and 2 nM (filled squares) L-685,458. The data are indicated as % of those generated in the absence of inhibitors.

We next analyzed the effect of SSide on intracellular domain generation from recombinant substrates of beta APP and Notch in vitro (Fig. 6). We observed that the inhibition kinetics of AICD-FmH generation from C100-FmH by SSide was approximately similar to that of the Abeta generation; the proteolytic activity to release AICD-FmH from wt C100-FmH was increased by treatment with 10-25 µM SSide, whereas it was completely inhibited at 100 µM. In contrast, AICD-FmH production from mt C100-FmH was inhibited by SSide in a dose-dependent fashion at 10-100 µM. We then analyzed the effect of SSide on NICD-FmH generation from recombinant substrate in vitro. The endoproteolytic cleavage of N102-FmH was inhibited by high concentrations of SSide (250-500 µM), whereas it was not affected by SSide at concentrations up to 100 µM, which was consistent with the results obtained in cultured cells (Fig. 3B). These results demonstrate that SSide can inhibit the gamma -secretase activity for Notch as well as for beta APP, although its inhibition potency for Notch is much weaker than that for beta APP, especially for gamma 42-cleavage.


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Fig. 6.   Characterization of the inhibitory effect of SSide on intracellular domain generation of beta APP and Notch in vitro. A-C, immunoblot analysis of de novo generation of AICD-FmH (arrow in A or B) or NICD-FmH (arrow in C) in the presence of SSide or SSone. The names of substrates used in the experiments are indicated below the lanes. D, densitometric analysis of the levels of AICD-FmH from wt substrate (open circles), AICD-FmH from mt substrate (filled triangles), and NICD-FmH (shaded squares). The averages of intensities of each band in two independent experiments are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It has been shown that a subset of NSAIDs selectively lowers the secretion of Abeta 42, although the molecular mechanism whereby NSAIDs affect the gamma -secretase activity remained unclear (21, 22). In this study, we established an in vitro gamma -secretase assay using recombinant wild type as well as mutant C100 as substrates and analyzed the effect of NSAIDs. We found that SSide, but not its metabolite SSone nor naproxen, directly inhibits the gamma -secretase activity derived from membrane fractions of HeLa cells in a dose-dependent manner. Moreover, we showed that SSide is a bona fide gamma -secretase inhibitor that has the highest inhibition potency against Abeta 42-cleaving activity compared with those for Abeta 38, Abeta 40, or Notch, with noncompetitive inhibition kinetics.

It has been extensively documented that NSAIDs exhibit various molecular targets, of which the primary target is COX, that converts arachidonic acid to prostaglandins (17). In addition, SSide has been shown to inhibit Ikappa B kinase beta  activity, activate PPARgamma , inactivate PPARdelta , inhibit Ras signaling, and reduce the proliferation and induce apoptosis of cancer cells (17). It has been shown that the Abeta 42-lowering effect of NSAIDs is independent of COX-inhibiting activity in cultured cells (21). We confirmed the Abeta 42-specific inhibition of Abeta secretion in culture cells by SSide, although the analysis of the inhibition profile at high concentrations was difficult because of the cell toxicity. To examine whether SSide directly inhibits gamma -secretase, we employed an in vitro assay system using solubilized membranes as enzyme sources and recombinant C100 polypeptides as substrates, and we demonstrated that SSide has the capacity to inhibit the total gamma -secretase activity, although cleavage at Abeta 42 site was most effectively inhibited. In addition, in vitro Abeta generation took place in the absence of any NTPs, suggesting that kinase activities (e.g. Ikappa B kinase beta ) are not involved in the regulation of gamma -secretase by SSide; we also observed that addition of ATP does not change the level of de novo Abeta generation, suggesting that gamma -secretase activity does not require energy.2 Finally, we showed that SSide displayed noncompetitive inhibition kinetics in vitro, which is a common characteristic of a number of gamma -secretase inhibitors (34). From these data, we postulate that SSide works as a gamma -secretase inhibitor that directly affects its activity by binding to the membrane-embedded protease complex.

To date, several gamma -secretase inhibitors have been documented to inhibit secretion of Abeta 40 and Abeta 42 in two different patterns. The peptide aldehydes and peptidomimetic inhibitors containing a difluoroketone or alcohol group increase Abeta 42 secretion at sub-inhibitory doses and diminish it at a high concentration, whereas they inhibit Abeta 40 generation in an absolutely dose-dependent manner (39-41). However, the rank order of inhibitory potencies of several peptide aldehydes against Abeta 40 and Abeta 42 are at similar levels, suggesting that a single gamma -secretase complex would generate Abeta 40 and Abeta 42 (40). In addition, the transition state analogue inhibitors of aspartyl proteases containing a hydroxyethylene isostere also inhibited the secretion of Abeta 40 and Abeta 42 to similar extents (12, 41). The compounds display similar inhibition kinetics between a cell-free system (i.e. incubation of the membrane fraction that harbors both enzyme and substrate) and an in vitro assay using recombinant substrates, suggesting that these compounds act directly on gamma -secretase and that the differences in the inhibition kinetics might depend on their binding sites or target molecule(s) within the gamma -secretase complex.

The molecular mechanism underlying the reciprocal regulation in Abeta 40 and Abeta 42 generation at low concentrations of peptide aldehyde inhibitors and SSide still remains unknown. One possible explanation is that a partial loss of gamma 40-secretase function by the low concentrations of peptide aldehydes or difluoroketone peptide mimetics would shift the substrate supply to gamma 42-secretase that is still active, thereby leading to overproduction of Abeta 42. In sharp contrast, SSide exhibited entirely novel inhibition profiles to preferentially inhibit Abeta 42 generation, which was accompanied by an increase in the production of Abeta 38 as well as Abeta 40 at sub-inhibitory concentration ranges in vitro. We speculate that SSide may act on a component that is distinct from those affected by peptide aldehydes or difluoroketone protease inhibitors. Alternatively, SSide and other inhibitors may exert opposite effects on a component that is involved in the determination of the position of a scissile bond to be cleaved in gamma -secretase complex. Unexpectedly, a coincubation study with the transition state analogue inhibitor, L-685,458, showed a direct competition with SSide, raising the possibility that SSide might directly act on the catalytic site, although the structure of SSide is not similar to any known substrates. An alternative possibility would be that SSide binds to the noncatalytic site of gamma -secretase complex and allosterically regulates the catalytic site in a way to dissociate substrates and active site-specific inhibitors, showing an apparent direct competition. Such reciprocal regulation of different proteolytic activities by protease inhibitors or substrates has been observed in proteasome that harbors three distinct proteolytic activities (i.e. chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide-hydrolyzing activities) (42-44). Ritonavir, an inhibitor of human immunodeficiency virus-1 protease, competitively inhibits the chymotrypsin-like activity, whereas trypsin-like activity is enhanced (43). Extensive studies using active site-specific inhibitors suggested that proteasome effectors/substrates (e.g. ritonavir) that cause reciprocal regulation might act on noncatalytic sites, rather than through binding to an active site. Further studies using derivatives of SSide that contain affinity moiety (e.g. photoreactive groups) are needed to obtain definitive proof that SSide acts directly on gamma -secretase.

It has been documented that almost all gamma -secretase inhibitors abolish the site-3 cleavage of Notch in cultured cells, with the exception of SSide and a nonpeptidic isocoumarin derivative, JLK6 (21, 45). However, it has been shown that JLK6 fails to inhibit gamma -secretase activity in vitro, suggesting that JLK6 is not a direct inhibitor of gamma -secretase (46). We found that SSide has the capacity to inhibit the endoproteolysis of Notch at the S3 site at much higher concentrations compared with those for Abeta inhibition in vitro, whereas other peptidic inhibitors (e.g. DAPT) abolished Notch cleavage with similar potencies to those for Abeta generation. These results suggest that gamma - and Notch secretases are pharmacologically distinct but related. Thus, it may be possible to avoid the envisaged side effects of gamma -secretase inhibitors caused by inhibition of Notch signaling by developing derivatives of SSide. Further attempts to define the molecular mechanisms of inhibition on gamma -secretase activity by SSide and to screen its derivatives specifically relevant to beta APP cleavage will facilitate not only the development of a novel therapeutic drug for AD but also our understanding of the unusual intramembranous proteolytic activity of gamma -secretase that cleaves membrane-spanning proteins at multiple positions.

    ACKNOWLEDGEMENTS

We thank Drs. Y. Ihara, B. De Strooper, and R. Kopan for providing antibody, mouse embryonic fibroblasts lacking PS1/2 and NDelta E cDNA, respectively, and Takeda Chemical Industries for continuous support for our studies.

    FOOTNOTES

* This work was supported by grants-in-aid from the Ministry of Health and Welfare, and the Ministry of Education, Science, Culture and Sports for the 21st Century Center of Excellence Program, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence may be addressed: Dept. of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4868; Fax: 81-3-5841-4708; E-mail: taisuke@ mol.f.u-tokyo.ac.jp.

** To whom correspondence may be addressed: Dept. of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4877; Fax: 81-3-5841-4708; E-mail: iwatsubo@mol. f.u-tokyo.ac.jp.

Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.M301619200

2 T. Tomita, Y. Takahashi, and T. Iwatsubo, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta  peptide; beta APP, amyloid-beta precursor protein; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; COX, cyclooxygenase; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester; ELISA, enzyme-linked immunosorbent assay; NICD, Notch intracellular domain; NSAIDs, nonsteroidal anti-inflammatory drugs; mt, mutant; PS, presenilin; SSide, sulindac sulfide; SSone, sulindac sulfone; wt, wild-type; Bicine, N,N-bis(2-hydroxyethyl)glycine; HMW, high molecular weight; AICD, APP intracellular domain; PPAR, peroxisome proliferator-activated receptors.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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