Calpain Inhibitor I Increases beta -Amyloid Peptide Production by Inhibiting the Degradation of the Substrate of gamma -Secretase
EVIDENCE THAT SUBSTRATE AVAILABILITY LIMITS beta -AMYLOID PEPTIDE PRODUCTION*

Lili ZhangDagger , Lixin Song, and Eric M. Parker

From the Department of Central Nervous System and Cardiovascular Research, Schering-Plough Research Institute, Kenilworth, New Jersey 07033

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) has been reported to have complex effects on the production of the beta -amyloid peptide (Abeta ). In this study, the effects of ALLN on the processing of the amyloid precursor protein (APP) to Abeta were examined in 293 cells expressing APP or the C-terminal 100 amino acids of APP (C100). In cells expressing APP or low levels of C100, ALLN increased Abeta 40 and Abeta 42 secretion at low concentrations, decreased Abeta 40 and Abeta 42 secretion at high concentrations, and increased cellular levels of C100 in a concentration-dependent manner by inhibiting C100 degradation. Low concentrations of ALLN increased Abeta 42 secretion more dramatically than Abeta 40 secretion. ALLN treatment of cells expressing high levels of C100 did not alter cellular C100 levels and inhibited Abeta 40 and Abeta 42 secretion with similar IC50 values. These results suggest that C100 can be processed both by gamma -secretase and by a degradation pathway that is inhibited by low concentrations of ALLN. The data are consistent with inhibition of gamma -secretase by high concentrations of ALLN but do not support previous assertions that ALLN is a selective inhibitor of the gamma -secretase producing Abeta 40. Rather, Abeta 42 secretion may be more dependent on C100 substrate concentration than Abeta 40 secretion.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The beta -amyloid peptide (Abeta )1 is the major protein component of the senile plaques found in the brain of Alzheimer's disease (AD) patients. Abeta is produced by proteolysis of a single transmembrane domain protein known as the amyloid precursor protein (APP) (reviewed in Refs. 1 and 2). The first step in Abeta production involves the cleavage of APP by an uncharacterized protease termed beta -secretase. Cleavage of APP by beta -secretase produces a large ectodomain protein known as APPsbeta , which is ultimately secreted, and a C-terminal 14-kDa membrane-bound fragment known as C100 (also termed C99 in some references). C100 is subsequently cleaved by gamma -secretase, another uncharacterized protease that cleaves within the transmembrane domain of C100 and produces the 39-43-amino acid Abeta peptides. Abeta 40 is the dominant species of Abeta secreted from cultured cells and is also more abundant in cerebrospinal fluid of normal and AD patients. Abeta 42, which comprises about 5-10% of total Abeta secreted from cultured cells, is more amyloidogenic and is the major species of Abeta that is deposited at the early stage of senile plaques formation.

Familial AD has thus far been associated with autosomal dominant mutations in the genes encoding APP, presenilin 1 (PS1), and presenilin 2 (2, 3). Multiple mutations in these three genes are associated with increased Abeta 42 production (4-6). Collectively, these data suggest that excessive Abeta 42 production is critical for the development of AD. Whereas the locations of mutations in the APP gene suggest that the mutations lead to increased Abeta 42 production by increasing cleavage of APP by beta - or gamma -secretase, the mechanism by which presenilin mutations increase Abeta 42 production remains unclear. Primary neuronal cultures derived from PS1 knock-out mice exhibit marked reduction of Abeta secretion (7), suggesting an essential role of PS1 in generating Abeta . Understanding the cellular mechanisms that regulate Abeta production will be a key step to unraveling the pathogenesis of AD.

Abeta production can also be modulated by peptide aldehyde protease inhibitors such as N-acetyl-leucyl-leucyl-norleucinal (ALLN, also known as calpain inhibitor I or LLnL) (5, 8-11). ALLN was first identified as a cysteine protease inhibitor (12), but at high concentrations it can also inhibit proteasome-associated activities (13). It has been reported that ALLN inhibits Abeta 40 production at concentrations that have little effect on or even increase Abeta 42 production (5, 9). These data are interpreted as evidence suggesting that Abeta 40 and Abeta 42 are produced by distinct gamma -secretases. In contrast, a recent study demonstrates that ALLN increased Abeta 40 and Abeta 42 production at low concentrations and decreased Abeta 40 and Abeta 42 production at higher concentrations (10). Thus, the reported effects of ALLN on Abeta production are conflicting, and the mechanism(s) by which ALLN modulates Abeta production are not clear. Nevertheless, ALLN may serve as an important tool to investigate the regulation of Abeta biosynthesis.

In this study, the effects of ALLN on Abeta 40 and Abeta 42 secretion are examined in detail and the mechanism by which ALLN modulates Abeta secretion is further defined. The data provide the novel insights that substrate availability plays a major role in regulating Abeta 40 and Abeta 42 production by gamma -secretase.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Antibodies W02, G2-10, and G2-11 were obtained from Dr. Konrad Beyreuther (University of Heidelberg, Heidelberg, Germany). W02 recognizes an epitope at amino acids 5-8 of the Abeta peptide, and G2-10 and G2-11 specifically recognize the C terminus of Abeta 40 and Abeta 42, respectively (14). Antibody 54 was obtained from Dr. Barry Greenberg (Cephalon, Inc., West Chester, PA) and recognizes the secreted APP ectoprotein formed after beta -secretase cleavage (APPsbeta ) (15). Antibody 14, which recognizes an N-terminal domain of PS1(16), was obtained from Dr. Samuel Gandy (Cornell University, New York). N-Acetyl-leucyl-leucyl-norleucinal (ALLN) was purchased from Boehringer Mannheim. All tissue culture reagents used in this work were from Life Technologies, Inc.

cDNA Constructs, Cell Culture, and Transfection of Cultured Cells-- A human APP695 cDNA clone with the Swedish mutation (APPsw) and a cDNA encoding the the C-terminal 99 amino acids of APP plus an N-terminal methionine (hereafter referred to as C100) were obtained from Dr. Barry Greenberg. C100 was cloned into the expression vector pcDNA3.1 (Invitrogen, San Diego, CA). The SPC100 construct consists of the N-terminal 18 amino acids of APP appended to the N terminus of C99 as described by others (17). To prepare the SPC100 construct, residues 19-596 of the APP695 cDNA were deleted by using the Seamless Cloning Kit (Stratagene, La Jolla, CA). The resulting SPC100 construct was cloned into the pcDNA3.1 vector. The APP London mutation (18) was introduced into C100 and SPC100 constructs using the QuickChangeTM site-directed mutagenesis kit (Stratagene). The human cDNAs encoding wild type PS1 and mutant PS1 with the exon 9 deletion (PS1Delta E9) (19) were obtained from Dr. Peter St. George-Hyslop (University of Toronto, Toronto, Canada). PS1Delta E9 sometimes is referred to as exon 10 deletion (20), as an alternate 5'-untranslated region exon was missed in the initial characterization of PS1 genomic structure (19).

Human embryonic kidney 293 cells were purchased from American Type Culture Collection (Rockville, MD) and were grown in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin. For transient expression of C100 and SPC100, 293 cells were seeded into 6-well plates, and transfection was conducted 2 days later when the cells reached 60-70% confluence. Cells were transfected by means of LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer's instructions. To prepare 293 cells stably expressing APPsw, 293 cells were transfected as described above. About 24 h after transfection, cells were passed to media containing 0.4 mg/ml G418, and G418-resistant clones were analyzed for Abeta secretion by ELISA (see below). Clones secreting high levels of Abeta were expanded and maintained in media supplemented with 0.2 mg/ml G418.

ALLN Treatment-- APPsw cells and cells transiently expressing C100 or SPC100 were treated with various concentrations of ALLN for 16 h. The conditioned media were then collected, centrifuged at 10,000 × g for 5 min to remove cell debris, and stored at -20 °C prior to ELISA and Western blot analysis. The cell monolayers were washed with cold phosphate-buffered saline and stored frozen at -20 °C prior to Western blot analysis.

Western Blot-- APPsbeta was detected in conditioned media by Western blot analysis with antibody 54. C100 and SPC100 were identified in cell lysates with antibody W02. Visualization was performed with an ECL kit (Amersham Pharmacia Biotech) according to the manufacturer's procedure.

ELISA Analysis of Abeta Peptides-- Sandwich ELISA assays were developed to measure Abeta 40 and Abeta 42 using the combination of antibodies G2-10/W02 and G2-11/W02, respectively. Both antibody G2-10 and G2-11 are more than 100-fold selective for Abeta 40 and Abeta 42, respectively (14), and the sensitivity of these assays are about 50-100 pg/ml. Briefly, Nunc MaxiSorb immunoassay plates were coated overnight at 4 °C with 0.4 µg/well G2-10 in 100 mM NaHCO3 (pH 9.5) or with 1 µg/well G2-11 in 100 mM Tris-HCl (pH 7.4). Subsequently, the antibody solution was removed, and the wells were incubated overnight at 4 °C with 5% bovine serum albumin in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl (TBS). The wells were then washed with TBS plus 0.05% Tween 20 (TTBS) and were stored at 4 °C for up to 6 months. Conditioned media were diluted with 10% bovine serum albumin in TBS to yield a final concentration of 2% bovine serum albumin, and 100 µl of diluted media was added to each well along with 40 ng/well biotinylated W02. Biotinylation of W02 was performed with the EZ-LinkTM Sulfo-NHS-LC-Biotinylation kit (Pierce) according to the manufacturer's instructions. The plate was incubated at 4 °C with gentle shaking either overnight (for Abeta 40 measurement) or for at least 24 h (for Abeta 42 measurement). The plate was then washed five times with TTBS, and 100 µl of 0.5 µg/ml horseradish peroxidase-conjugated NeutrAvidin (Pierce) was added to each well and incubated at room temperature for 1 h. The color was developed with the TMB-H2O2 system (Kirkegaard & Perry Laboratories, Gaithersburg, MD) according to the manufacturer's instructions, and absorption at 450 nm was measured on a plate reader.

Metabolic Labeling of C100 with [35S]Methionine-- 293 cells were seeded in 60-mm dishes and transiently transfected with C100 as described above. About 40 h after transfection, cells were incubated in methionine- and cysteine-free DMEM medium for 1 h and were then labeled for 1 h with 150 µCi/ml [35S]methionine. The cells were subsequently washed and either kept frozen at -20 °C (pulse) or incubated with fresh complete DMEM (chase) in the absence and presence of 25 µM ALLN. The chase media and cell monolayers were collected at different time points and kept frozen until further analysis. C100 and Abeta peptide were immunoprecipitated with antibody W02 from radiolabeled cells and chased media, respectively. The cells were solubilized with 0.6 ml/dish of 1× RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS), and 0.5 ml of cell lysate was used for each immunoprecipitation assay with 3 µg of antibody W02. For immunoprecipitation of Abeta from chased media, 0.2 ml of 5× RIPA was added to 1 ml of media together with 3 µg of antibody W02. Forty microliters of Protein G plus Protein A-agarose (Calbiochem) was added to each immunoprecipitation reaction, and the mixtures were rocked overnight at 4 °C before being centrifuged at 10,000 × g for 2 min. The pellets were then washed twice with 1× RIPA buffer and once with 10 mM Tris-HCl (pH 7.5). The immunocomplexes were denatured in SDS-polyacrylamide gel electrophoresis sample buffer and boiled for 5 min before being processed by electrophoresis and autoradiography.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of C100 and SPC100-- In addition to full-length APP with the Swedish mutation, two truncated APP constructs were used in this study to examine the processing of APP by gamma -secretase (see Fig. 1A). The construct designated as C100 consists of the methionine initiation codon plus the C-terminal 99 amino acids of APP. The N terminus of C100 corresponds to the beta -secretase cleavage site, i.e. the N terminus of the Abeta peptide. The construct designated SPC100 consists of the methionine initiation codon, the 16 amino acid signal peptide of APP, and the first two amino acids of APP appended to the N terminus of C100. C100 and SPC100 are similar to the constructs previously reported by Dykes et al. (17) except that the expression vector pcDNA3.1 was used instead of pCEP4. Western blot analysis of extracts from 293 cells transiently expressing SPC100 detected a 14-kDa band that co-migrated with native C100 (Fig. 1B). These data confirm that the signal peptide of SPC100 was removed. As reported previously (21), the level of expression of C100 was much higher in cells transiently expressing SPC100 than in cells transiently expressing C100 (Fig. 1B).


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Fig. 1.   Protein expression and Abeta secretion in cells expressing C100 and SPC100. A, schematic diagram of the C100 and SPC100 expression constructs. B, Western blot analysis of lysates prepared from cells expressing C100 and SPC100. The Western blot was probed with antibody W02 as described under "Experimental Procedures." C and D, concentration of Abeta 40 (C) and Abeta 42 (D) in the conditioned media from cells expressing C100 and SPC100. Abeta 40 and Abeta 42 were quantitated by ELISA assay as described under "Experimental Procedures." The data shown are from one transfection and are representative of more than five transfections.

293 cells transiently expressing SPC100 secreted 2-3-fold more Abeta 40 and Abeta 42 than cells transiently expressing C100 (Fig. 1, C and D), consistent with a previous report (21). The increased Abeta secretion from cells expressing SPC100 relative to cells expressing C100 is probably due to the higher level of C100 in these cells (Fig. 1B). Despite the quantitative differences in Abeta secretion from cells expressing C100 and SPC100, the relative amounts of Abeta 42 and Abeta 40 secreted by cells expressing the two constructs (i.e. the Abeta 42:Abeta 40 ratios) were similar (Fig. 2A). The effects of APP and PS1 FAD mutations on Abeta secretion from cells expressing C100 and SPC100 were also tested. As reported previously (16), extracts from 293 cells expressing wild type PS1 displayed a 48-kDa product corresponding to full-length PS1 as well as a 33-kDa N-terminal fragment of PS1 (Fig. 2B). Extracts from 293 cells expressing the PS1 mutant PS1Delta E9 displayed a full-length PS1 protein with slightly greater mobility than that of full-length wild type PS1 due to the deletion of exon 9 in this mutant (Fig. 2B). The 33-kDa N-terminal fragment of PS1, which is derived primarily from endogenous PS1, was not increased significantly by overexpression of PS1wt or PS1Delta E9. As previously reported for full-length APP (4), the secretion of Abeta 42 from cells expressing either C100 or SPC100 was selectively increased by co-expression of PS1Delta E9 or by introduction of the London mutation into C100 or SPC100 (Fig. 2A). Co-expression of PS1Delta E9 did not alter Abeta 40 secretion from cells expressing either C100 or SPC100, nor did co-expression of wild type PS1 affect Abeta secretion from cells expressing either construct (data not shown). Co-expression of wild type PS1 or PS1Delta E9 did not affect the levels of C100 protein in cells expressing either C100, SPC100, C100-London, or SPC100-London (Fig. 2C and data not shown). Thus, gamma -secretase processing of C100 derived from either the C100 or SPC100 constructs is qualitatively similar as evidenced by the similar relative secretion of Abeta 40 and Abeta 42 (i.e. the Abeta 42:Abeta 40 ratio) and the similar effect of APP and PS1 FAD mutations on Abeta secretion.


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Fig. 2.   Modulation of Abeta production by APP and PS1 FAD mutations in cells expressing C100 and SPC100. Expression vectors containing cDNAs encoding C100, 100-lon, SPC100, or SPC100-lon) were transiently transfected into 293 cells together with either an empty expression vector or an expression vector containing cDNAs encoding PS1wt or PS1Delta E9. About 24 h after transfection, the cells were fed with fresh media, and conditioned media were collected the next day. The concentrations of Abeta 40 and Abeta 42 in the conditioned media were determined by ELISA assay as described under "Experimental Procedures." The cell monolayers were washed with ice-cold PBS and stored at -20 °C before analysis of protein expression by Western blot as described under "Experimental Procedures." A, Abeta 42:Abeta 40 ratio in the conditioned media. B, Western blot analysis of PS1 expression with antibody 14. C, Western blot analysis of C100 expression in the cell lysates with antibody W02. The data shown here are from one experiment and are representative of three independent transfections.

Modulation of Abeta Production by ALLN-- ALLN and related peptide aldehyde protease inhibitors have multiple effects on APP processing. In addition to modulating Abeta production, it has been observed that these inhibitors can also potentiate the alpha -secretase pathway and enhance the secretion of APPsalpha (5, 8). The altered alpha -secretase processing may indirectly affect the beta -secretase pathway as the two proteases compete for a common substrate. Consequently, the effect of ALLN on Abeta production in cells expressing APP is complex, and it is very difficult to distinguish the specific action of ALLN on the gamma -secretase cleavage step. Since C100 is the product of beta -secretase cleavage and is the native substrate for gamma -secretase (1), Abeta production by cells expressing C100 or SPC100 reflects only gamma -secretase processing. We therefore utilized cells expressing C100 and SPC100 to study the effect of ALLN on the gamma -secretase cleavage reaction without the influence of this inhibitor on alpha - and beta -secretase processing. In cells expressing C100 or APPsw, both Abeta 40 and Abeta 42 secretions were increased by treatment with low concentrations of ALLN and decreased by treatment with high concentrations of ALLN (Fig. 3, A and C). Abeta 42 secretion from cells expressing C100 or APPsw was increased much more dramatically by low concentrations of ALLN than was Abeta 40 secretion (Fig. 3, A and C). In contrast, ALLN had only concentration-dependent inhibitory effects on Abeta 40 and Abeta 42 secretion from cells expressing SPC100 (Fig. 3B).


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Fig. 3.   Effects of ALLN treatment of Abeta production. About 24 h after transfection, 293 cells transiently expressing C100 (A), SPC100 (B), or APPsw (C) were treated overnight with various concentrations of ALLN. Conditioned media were collected and analyzed for Abeta 40 (open circle ) and Abeta 42 () concentration by ELISA as described under "Experimental Procedures." The data shown are the average of duplicate transfections in a single experiment and are representative of three independent experiments.

Examination of cell lysates revealed that ALLN increased the cellular level of C100 in a concentration-dependent manner in cells expressing C100 and APPsw, whereas it did not affect C100 protein levels in cells expressing SPC100 (Figs. 4 and 5A). The ALLN-induced increase in C100 in APPsw cells was not due to increased beta -secretase activity since ALLN did not potentiate the secretion of APPsbeta from these cells (Fig. 5B). The increase in C100 protein level induced by ALLN in cells expressing C100 or APPsw was not a result of inhibition of gamma -secretase since concentrations of ALLN that increased cellular C100 protein levels also increased both Abeta 40 and Abeta 42 production. In addition, pulse-chase experiments demonstrated that at concentrations of ALLN that increased Abeta production (data not shown), this compound decreased the rate of C100 turnover in cells expressing C100 (Fig. 6), suggesting that ALLN increases C100 protein levels by inhibiting C100 degradation.


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Fig. 4.   Effect of ALLN on cellular C100 levels in cells expressing C100 and SPC100. 293 cells transiently expressing C100 or SPC100 were incubated with various concentrations of ALLN as described in Fig. 3. After conditioned media were collected, cell monolayers were washed with cold PBS and lysed with SDS sample buffer. C100 levels in the cell lysates were determined by Western blot analysis with antibody W02 as described under "Experimental Procedures." Lanes 1-6, cell lysates from cells expressing C100 treated with the indicated concentrations of ALLN; lanes 7-12, cell lysates from cells expressing SPC100 treated with the indicated concentrations of ALLN.


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Fig. 5.   Regulation of APP processing by ALLN in cells expressing APPsw. 293 cells expressing APPsw were incubated overnight with ALLN at the indicated concentrations. Conditioned media were then collected, and cell monolayers were washed with cold PBS and lysed in SDS sample buffer. A, Western blot analysis of APP and C100 in lysates of cells expressing APPsw following ALLN treatment. The Western blot was probed with antibody W02. B, Western blot analysis of APPsbeta in conditioned media collected from cells expressing APPswe following ALLN treatment. APPsbeta was detected with antibody 54.


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Fig. 6.   Pulse-chase labeling study of C100 metabolism. About 36 h after transient transfection, cells expressing C100 were labeled with [35S]Met for 1 h. The cell monolayers were then washed with PBS and chased for 1 and 3 h in the presence and absence of 25 µM ALLN. Antibody W02 was used to precipitate C100 from cell lysates. Immunoprecipitates were separated on 14% SDS-polyacrylamide gel electrophoresis, and the gel was further processed for fluorography. Lane 1, cells pulse-labeled for 1 h; lanes 2 and 3, cells chased for 1 and 3 h without ALLN; lanes 4 and 5, cells chased for 1 and 3 h in the presence of ALLN.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In cells expressing APP, the protease inhibitor ALLN has recently been shown to inhibit selectively the production of Abeta 40 at concentrations that have little effect on the production of Abeta 42 (5, 9). These data were interpreted as indicating that distinct gamma -secretases are responsible for Abeta 40 and Abeta 42 production. Although these data are intriguing, a recent study suggests that the effects of ALLN on Abeta secretion from cells expressing APP are complex (10). To elucidate the mechanisms by which ALLN modulates Abeta production and, by inference, gamma -secretase activity, this study examined the effects of ALLN on Abeta production in more detail.

The gamma -secretase reaction was studied in 293 cells expressing either APPsw or various amounts of C100, the C-terminal fragment of APP that represents the immediate substrate of this enzyme. C100 was produced in 293 cells by expression of two constructs designated as C100 and SPC100 (Fig. 1). As reported previously, much higher levels of C100 were produced in cells expressing SPC100 than in cells producing C100 (21), presumably because the signal peptide present in SPC100 permits more efficient processing or sorting of the protein. The characteristics of Abeta production in cells expressing APPsw, C100 or SPC100 cells were similar. The relative amounts of Abeta 40 and Abeta 42 secreted by cells expressing C100 or SPC100 were similar as reflected by the similar Abeta 42:Abeta 40 ratios, and like the APP expression cells (4, 18), the secretion of Abeta 42 was specifically increased by co-expression of mutant PS1 or by introduction of the London mutation into the constructs (Fig. 2). These observations argue that the same gamma -secretase(s) is(are) responsible for Abeta production in cells expressing all three constructs.

Treatment of cells expressing C100 or APPsw with low concentrations of ALLN resulted in increased secretion of both Abeta 40 and Abeta 42 (Fig. 3, A and D), which is consistent with results reported by others (10). Interestingly, treatment of cells expressing either C100 or APPsw with low concentrations of ALLN also increased cellular C100 protein accumulation (Figs. 4 and 5A). Pulse-chase experiments in cells expressing C100 demonstrated that a low concentration of ALLN decreased the rate of C100 turnover (Fig. 6), whereas Abeta secretion was increased during the same period (data not shown). Taken together, these data suggest that low concentrations of ALLN increase Abeta production by inhibiting C100 turnover and, hence, increasing the amount of C100 substrate available for gamma -secretase cleavage (Fig. 7). This suggestion is supported by the observation that ALLN did not affect cellular C100 levels in cells expressing SPC100 and correspondingly did not increase Abeta secretion from these cells at any concentration. One implication of these results is that in addition to cleavage by gamma -secretase, C100 is normally degraded by a distinct ALLN-sensitive pathway (Fig. 7). Channeling of C100 into this alternative, ALLN-sensitive degradation pathway would prevent Abeta production (Fig. 7). The fact that ALLN did not increase cellular C100 levels in cells expressing SPC100 may be due to the fact that the ALLN-sensitive degradation pathways is overwhelmed by the much higher levels of cellular levels of C100 present in these cells. Low concentrations of ALLN also increase Abeta secretion from primary hippocampal cultures where only endogenous APP is expressed,2 suggesting that the ALLN-sensitive degradation of this APP intermediate represents a normal metabolic process and is not merely an artifact of overexpressing C100 in cultured cells. The presence of an ALLN-sensitive catabolic pathway for C100 may provide a mechanism by which cells regulate substrate availability for gamma -secretase and thus regulate cellular Abeta production. In this regard, the regulation of C100 metabolism may play an important role in AD pathogenesis.


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Fig. 7.   Model for metabolism of C100 by gamma -secretase and and ALLN-sensitive degradation pathway.

An alternative mechanism has been proposed to explain the ability of ALLN to increase Abeta secretion (22). Several protease inhibitors, including ALLN, are known to prevent the proteasome-mediated degradation of PS1 (22, 23). Based on this observation and the fact that presenilin mutations are associated with increases in Abeta 42 secretion, it has been suggested that the stabilization of presenilins by protease inhibitors like ALLN may directly potentiate Abeta 42 production in cells (22). Our data do not support this model for two reasons. First, the effects of ALLN and PS1 mutations are additive, i.e. low concentrations ALLN further increase the Abeta 42:Abeta 40 ratio in cells expressing mutant PS1 (22).3 Second, the fact that Abeta 42 production in cells expressing SPC100 can be increased by co-expression of a mutant PS1 (Fig. 2A), but not by ALLN (Fig. 3B), provides additional evidence that PS1 mutations and ALLN regulate Abeta 42 production by independent mechanisms.

High concentrations of ALLN inhibited Abeta 40 and Abeta 42 production by cells expressing either APPsw or C100. High concentrations of ALLN may directly inhibit gamma -secretase and, hence, decrease Abeta production, but other effects of ALLN on cellular metabolism could also be responsible for this effect. In cells expressing SPC100, which gives rise to much higher cellular C100 levels than in cells expressing APPsw or C100, ALLN inhibited Abeta 40 and Abeta 42 production at all concentrations and the IC50 values of ALLN for inhibition of Abeta 40 and Abeta 42 production were very similar. This observation does not support the conclusion that ALLN selectively inhibits Abeta 40 production (5, 9), a finding that was interpreted as implying the existence of distinct gamma -secretases responsible for Abeta 40 and Abeta 42 production. Since ALLN modulates Abeta production by multiple mechanisms, as documented in this study and Ref. 10, it is difficult to use this compound to pharmacologically distinguish multiple gamma -secretases.

Whereas low concentrations of ALLN and other calpain/proteasome inhibitors increase Abeta 40 and Abeta 42 production, the increase in Abeta 42 production induced by these protease inhibitors is much more pronounced (Ref. 10, Fig. 3, A and D). Although a definitive explanation for this phenomenon cannot be discerned from the existing data, a reasonable model can be proposed. As discussed above and illustrated in the model diagrammed in Fig. 7, ALLN increases Abeta production by increasing the availability of the gamma -secretase substrate C100. Since ALLN increases Abeta 42 secretion more dramatically than Abeta 40 secretion, this model implies that the gamma -secretase cleavage reaction producing Abeta 42 is more dependent on substrate concentration than the reaction producing Abeta 40. In other words, the gamma -secretase that cleaves C100 to produce Abeta 42 has a higher Km for the substrate C100 than the gamma -secretase that cleaves C100 to produce Abeta 40. Thus, the ALLN-induced increase in the cellular concentration of the gamma -secretase substrate C100 will increase Abeta 42 secretion more than Abeta 40 secretion. The fact that Abeta 40 secretion was only slightly increased by ALLN would suggest that the gamma -secretase that produces Abeta 40 is nearly saturated with C100 under the conditions used in these experiments. This model would explain why Abeta 40 is the dominant Abeta species produced during normal physiologic processing of APP. It should be pointed out that this model and the experimental data that support it do not necessarily require the existence of two distinct gamma -secretase enzymes that independently produce Abeta 40 and Abeta 42. The major weakness of this model is the observation that the higher cellular levels of C100 seen in cells expressing SPC100 relative to cells expressing C100 is associated with similar increases in Abeta 40 and Abeta 42 secretion rather than a more selective increase in Abeta 42 secretion as would be predicted by the model. It is possible that the model is fundamentally correct but that increasing cellular C100 levels by expressing SPC100 rather than C100 is somehow biochemically or mechanistically different from increasing cellular C100 levels with ALLN. In any case, the hypothesis that substrate concentration is a more important determinant of Abeta 42 production than Abeta 40 production provides a novel framework for further experiments aimed at understanding the mechanisms regulating Abeta production.

    ACKNOWLEDGEMENTS

We thank Dr. Sam Gandy for providing antibody 14 and Drs. Peter St. George-Hyslop and Georges Levesque for presenilin cDNA constructs.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed: Schering-Plough Research Institute, K15-3-3600, 2015 Galloping Hill Rd., Kenilworth, NJ 07033. Tel.: 908-740-6559; Fax: 908-740-2383; E-mail: lili.zhang{at}spcorp.com.

2 P. Fraser and L. Zhang, unpublished data.

3 L. Zhang and L. Song, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Abeta , beta -amyloid peptide; AD, Alzheimer's disease; APP, beta -amyloid beta  precursor protein; APPsw, APP carrying Swedish mutations; ALLN, N-acetyl-leucyl-leucyl-norleucinal, also known as calpain inhibitor I or LLnL; C100, the C-terminal 100-amino acid fragment of APP; SPC100, C100 with an N-terminal signal peptide derived from the N-terminal 18 amino acids of APP; C100-lon and SPC100-lon, C100 and SPC100 carrying london mutation; DMEM, Dulbecco's modified Eagle's medium; FAD, familial Alzheimer's disease; PBS, phosphate-buffered saline; PS1, presenilin 1; ELISA, enzyme-linked immunosorbent assay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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