From the
Neurobiology of Disease Laboratory and ||Genetics and Aging Research Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
Received for publication, January 15, 2003 , and in revised form, March 5, 2003.
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ABSTRACT |
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INTRODUCTION |
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The main pathogenic event that occurs in all forms of AD is the abnormal accumulation of amyloid -peptide (A
) into senile (or amyloid) plaques (2). A
is a 3943-amino acid peptide proteolytically derived from the amyloid precursor protein (APP). APP is first cleaved by
-site APP-cleaving enzyme 1 (BACE1) at the N terminus of A
(
-cleavage), producing a C-terminal fragment (
-APP-CTF) of
12 kDa, and subsequently in the transmembrane domain (
-cleavage) by a presenilin-harboring protease complex. The two major sites of
-cleavage are located at positions 40 and 42 of A
, generating A
40 and A
42, respectively.
The membrane lipid ceramide is the backbone of all complex sphingolipids and acts as a second messenger in many biological events. In addition, it regulates several biochemical and genetic events that occur during aging/senescence, including inhibition of phospholipase D and c-Fos-dependent signaling pathways, retinoblastoma protein dephosphorylation, arrest of the serum/growth factor-mediated activation of protein kinase C, and arrest of DNA synthesis (3, 4). Endogenous ceramide can be generated by either de novo synthesis or hydrolysis of sphingomyelin (SM) at the cell surface, the latter being the most important source of the active pool of ceramide (5, 6). The intracellular levels of ceramide increase progressively during aging in both cultured cells and the whole organ (7,8, 9, 10). In addition, brains from AD patients contain approximately three times more ceramide when compared with age-matched controls (11).
In this study, we investigated the role of the second messenger ceramide in the regulation of A generation through several biochemical approaches. We found that A
biogenesis is strictly regulated by the endogenous pool of ceramides, implicating the sphingomyelin, but not the glycosphingolipid, biosynthetic pathway in A
generation. We also found that the ceramide-dependent regulation of A
biogenesis is achieved via control of BACE1 steady-state levels, is specific for APP, and is not associated with cell death.
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EXPERIMENTAL PROCEDURES |
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Lipid Labeling and ExtractionLabeling of glycosphingolipids was performed using [9,10-3H]palmitic acid (60 Ci/mmol) (PerkinElmer Life Sciences). Cells were incubated in the presence of [3H]palmitic acid for at least 3 days (ad equilibrium) to allow steady-state labeling of palmitic-containing lipids. At the end of each treatment (see above), cells were washed twice in phosphate-buffered saline, scraped, and extracted in chloroform:methanol (2:1, v/v). After Folch extraction, the lipid phase was dried, resuspended in chloroform, and applied together with standards to a Silica Gel-G (EM Science, Gibbstown, NJ) thin layer chromatography (TLC) plate. Plates were developed in either chloroform, methanol, acetic acid, formic acid, water (70:30:12:4:2, v/v/v/v/v) (13) or chloroform, methanol, 9.8 mM CaCl2 (60:35:8, v/v/v) (14). Spots were scraped and counted in a liquid scintillation counter.
Antibodies and Western Blot AnalysisPolyclonal antibodies C7 and C8 against the C terminus of APP were a generous gift from Dr. Dennis J. Selkoe (Harvard Medical School, Boston, MA). The polyclonal antibodies against caveolin-1 came from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and those against BACE1 were from Abcam (Cambridge, UK). Monoclonal antibodies against poly(ADP-ribose) polymerase (PARP) (C-2-10) were obtained from Clontech Laboratories, Inc. (Palo Alto, CA), and those against tumor necrosis factor- (M32255
[GenBank]
a) were from Fitzgerald Industries International, Inc. (Concord, MA). The hybridoma antibody against Notch developed by S. Artavanis-Tsakonas was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA).
For Western blot analysis, total proteins (50100 µg/lane) were separated by NuPage 412% BisTris-polyacrylamide gel electrophoresis (Invitrogen) using MES running buffer (Invitrogen) and then blotted on Immun-BlotTM polyvinylidene difluoride membranes (Bio-Rad). Proteins were visualized using the LumiGLOTM protein detection kit (KPL, Gaithersburg, MD) as described by the manufacturer.
A Concentration DeterminationsFor A
determination, APP751 stably transfected CHO cell lines were grown in 6-well plates (BD Biosciences). When
8090% confluent, cells were washed in phosphate-buffered saline and incubated in 1 ml of fresh medium for 24 h (12). Secreted A
total and A
42 were quantitated by standard sandwich ELISA (A
ELISA Core Facility, Center for Neurological Diseases, Harvard Institutes of Medicine, Harvard Medical School).
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RESULTS |
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To confirm the above results in a different cell type and to assess whether the changes in the rate of A generation were determined by changes in only
- or in both
- and
-cleavage of APP, we used H4 (human neuroglioma) cells stably transfected with either full-length APP751 or the C-terminal 105 amino acids of APP (APPC-105), which mimic
-APP-CTF (12). C6-ceramide increased the steady-state levels of both
- and
-APP-CTFs in H4 cells expressing full-length APP751 without any evident effect on APP expression or maturation (Fig. 2a). Significantly, no effect was observed on the steady-state levels of APPC105 (Fig. 2a), indicating that the changes in A
secretion were due to changes in
- but not
-cleavage of APP. Again, we did not detect any effect on cell viability (data not shown) or caspase-mediated cleavage of full-length PARP (Fig. 2b). Finally, C6-ceramide did not stimulate the "
-like" cleavage of tumor necrosis factor-
by tumor necrosis factor-
-converting enzyme (TACE) or the furin-dependent cleavage of full-length Notch (Fig. 2b).
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Taken together, the above results suggest that the second messenger ceramide regulates the rate of A generation by affecting
-, but not
-cleavage of APP. Reduced
-cleavage of APP is unlikely to lower A
levels. Instead, most reports indicate that it would elevate A
under normal cellular conditions (15). Finally, low levels of C6-ceramide did not induce apoptotic cell death of CHO and H4 cell lines under our experimental conditions.
Endogenous Ceramide Levels Regulate A GenerationTo confirm that endogenous ceramide and not only cell-permeable analogues can modulate A
production, we used additional biochemical approaches known to regulate the endogenous pool of active ceramide. The different approaches used in these studies are schematically described in Fig. 3. nSMase increases the intracellular pool of ceramide by hydrolysis of cell surface SM. In contrast, FB1 inhibits ceramide-synthase, preventing the biosynthesis of ceramide and all the other glycosphingolipids beyond the ceramide moiety. In addition to nSMase and FB1, we also used NB-DGJ, a biochemical inhibitor of the ceramide-specific glycosyltransferase. NB-DGJ blocks the glycosphingolipid, but not the SM, biosynthetic pathway and does not affect the levels of the signaling-active ceramide (16, 17).
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Neither nSMase nor FB1 affected cell viability under the conditions used in our studies, as assessed by the uptake of trypan blue and by the release of the cytosolic enzyme lactate dehydrogenase into the media (data not shown). When used at 0.25 µM, nSMase produced a 60% decrease of SM levels (Fig. 4a). Similar to C6-ceramide, nSMase increased both ceramide levels (Fig. 4a) and A
secretion (Fig. 4b) by
2-fold. The increase in A
secretion was accompanied by increased steadystate levels of both
- and
-APP-CTFs in the absence of any evident effect on APP expression or maturation (Fig. 4c).
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As seen with nSMase, FB1 treatment reduced SM by 50% (Fig. 5a). However, in contrast with C6-ceramide and nSMase, FB1 reduced ceramide levels by
6070% (Fig. 5a). Decreased endogenous ceramide levels were paralleled by a corresponding reduction in A
total and A
42 secretion into the media by
50% (Fig. 5b). Steady-state levels of
- and
-APP-CTF also decreased, with no apparent effect on APP expression or maturation (Fig. 5c). Most importantly, exogenous C6-ceramide could recover A
levels reduced by FB1 (Fig. 5d), confirming that decreased ceramide levels were responsible for lowering A
in the first place. In contrast to nSMase and FB1, NB-DGJ, which did not affect the endogenous pool of the signaling active ceramide (Fig. 6a), was not able to produce any effect on A
secretion (Fig. 6b) or APP processing (Fig. 6c).
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Finally, we assessed the effect of nSMase, FB1, and NB-DGJ on - and
-secretase cleavage of APP by using H4 (human neuroglioma) cells stably transfected with either full-length APP751 or with APPC105. As seen in CHO cells, both nSMase and FB1 reduced SM levels while producing opposite effects on ceramide levels. nSMase increased, whereas FB1 reduced ceramide levels by
30 and
50%, respectively (data not shown). Again, the changes in ceramide production were paralleled by similar changes in the steady-state levels of both
- and
-APP-CTFs; nSMase increased, whereas FB1 reduced APP CTFs (Fig. 7, a and b). Significantly, no effect was observed on the steady-state levels of APPC105 (Fig. 7, a and b), indicating that the changes in A
secretion were only due to changes in
- and not
-cleavage of APP. As already observed in CHO cells, NB-DGJ did not affect ceramide levels or APP processing (Fig. 7c).
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Overall, our results indicate that both cell-permeable analogues and endogenous ceramides regulate A generation by affecting
-, but not
-cleavage of APP. Although ceramide levels also modulate
-cleavage of APP, this clip does not directly contribute to A
generation and could be regulated by separate cellular events. Moreover, our data argue for a direct effect of ceramide on BACE1 activity instead of APP trafficking, because
-cleavage of APP is not affected by ceramide in APPC105-expressing H4 cells. Finally, biotinylation of cell surface proteins, subcellular fractionation studies, and analysis of APP mature/immature ratios revealed that ceramide levels do not significantly affect APP trafficking or localization (data not shown).
C6-ceramide Reduces the Turnover Rate of BACE1To assess the effect of altered ceramide levels on BACE1, we asked whether C6-ceramide regulates either the subcellular/membrane distribution or the steady-state levels of this enzyme. Endogenous BACE1 was detected in H4 cells as a double band at around 6570-kDa (Fig. 8a). H4 cells were grown in the presence or absence of C-6 ceramide for up to 6 days and then analyzed for subcellular/membrane distribution or steady-state levels of BACE1. C6-ceramide did not affect the overall distribution of BACE1 among intracellular membranes or membrane microdomains (data not shown). However, C6-ceramide progressively increased BACE1 levels, reaching a plateau after approximately 4 days of treatment (Fig. 8a). No effect was observed on the steady-state levels of BACE2, a BACE1 homologue, or TACE (tumor necrosis factor--converting enzyme), a regulated form of
-secretase (data not shown).
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To assess whether post-transcriptional events lead to increased BACE1 protein levels, we used a CHO cell line stably expressing C-terminally Myc-tagged human BACE1 under the control of a cytomegalovirus promoter. When these cells were treated with C6-ceramide, the steady-state levels of both native and epitope-tagged BACE1 increased progressively, reaching a plateau after 4 days, at which point they were 3-fold higher than control (Fig. 8b). Most importantly, as observed for the rate of A
secretion (see Figs. 4 and 5), steady-state levels of BACE1 also paralleled changes in endogenous ceramide induced by FB1 and nSMase. Fig. 8c shows that FB1 reduced, whereas nSMase increased, the steady-state levels of BACE1, strongly suggesting that ceramide levels regulate A
generation by modulating the amount of enzyme available for
-secretase cleavage of APP.
Finally, we performed pulse-chase analysis and cycloheximide degradation assays to directly study changes in the turnover rate of BACE1. CHO cells stably expressing BACE1 were treated with C-6 ceramide followed by pulse-chase with radiolabeled methionine/cysteine to calculate the half-life of BACE1. Ceramide treatment increased the half-life of BACE1 from 1620 h to
30 h (Fig. 8d). It is worth noting that the levels of newly synthesized BACE1 found in ceramide-treated cells after 56 h of chase were very similar to those found in control cells after only 24 h of chase (Fig. 8d). Very similar results were obtained when the same cells were treated with cycloheximide to inhibit protein synthesis. Ceramide treatment significantly reduced the turnover rate of BACE1 and increased the half-life of preformed BACE1 from
20 to
56 h (Fig. 8e).
Taken together, the above data indicate that ceramide regulates A generation by affecting the steady-state levels of BACE1, the rate-limiting enzyme in the biogenesis of A
. They also suggest that the increase in BACE1 levels is, at least in part, the result of post-transcriptional stabilization of BACE1.
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DISCUSSION |
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Ceramide is a lipid second messenger involved in many biological events that regulate terminal differentiation of neurons, cellular senescence, proliferation, and death (7, 14, 18, 19). Depending on the cell type and the doses used, exogenously added ceramide has been shown to either activate or inhibit apoptosis (3, 18). Intracellular levels of ceramide increase during aging in both cultured cells and the whole organ (7, 8, 9, 10). In addition, senescent-like doses (10 µM) of ceramide are able to induce a senescent phenotype in young cultured cells (7, 9, 10). Under those conditions, ceramide has been shown to promote outgrowth and survival of cultured neurons (14, 18, 20).
In apparent contrast to the above studies, a chronic increase in intracellular ceramide can inhibit axonal elongation and receptor-mediated internalization of nerve growth factor and activate cell death (13, 21). In addition, it also reduces receptor-mediated internalization of lipoprotein-associated cholesterol (13), which is involved in the regulation of synaptogenesis (22). These effects may be part of a delicate set of events that occur during aging. Very recently, Han et al. (11) also reported that ceramide levels are increased more than 3-fold in the brains of AD patients when compared with age-matched controls.
In this study, we showed that ceramide levels control A biosynthesis, the first pathogenic event in the generation of senile (or amyloid) plaques. It is worth noting that our results with exogenous C6-ceramide (Figs. 1, 2, and 8) were observed at 10 µM concentration, which is known to produce a cellular concentration of active ceramide very close to that observed in senescent cells (7). Additionally, the signaling function of ceramide is likely to be required for its effect on A
generation, because the signaling inactive analog dehydroceramide did not produce any effect on A
production in our CHO cells (data not shown). Dehydroceramide is a naturally occurring ceramide that lacks the 45 trans double bond, which is required for the signaling activity but retains the stereochemical configuration of ceramides and is metabolized very similarly to ceramides (13, 18).
Cell surface SM is mostly found in cholesterol-rich domains, which are specialized membrane microdomains highly enriched in SM, cholesterol, and the glycosphingolipid GM1 (23). Hydrolysis of SM has been reported to reduce the clustering of cholesterol into cholesterol-rich domains and to induce retro-transport of cholesterol from the plasma membrane to the endoplasmic reticulum (23). In the endoplasmic reticulum excess cholesterol activates the enzyme acyl-coenzyme A:cholesterol acyltransferase, which we have recently implicated with A generation (12). However, acyl-coenzyme A:cholesterol acyltransferase activation follows retro-transport of cholesterol, which occurs only in the presence substantial SM hydrolysis and massive sterol mobilization from cholesterol-rich domains (23). In our study, both nSMase and FB1 reduced the clustering of cholesterol into cholesterol-rich domains (data not shown), but they only induced a modest mobilization of sterols, which was not accompanied by acyl-coenzyme A:cholesterol acyltransferase activation (data not shown). In addition, although FB1 and nSMase had similar effects on SM levels and on cholesterol distribution between membrane microdomains, they produced opposite effects on A
generation. Those effects paralleled the changes in ceramide levels and could be reproduced by C6-ceramide. Finally, the reduction in A
biosynthesis produced by FB1 treatment was reversed by C6-ceramide, confirming that indeed ceramide was responsible for the changes observed in A
generation.
BACE1 is a type I integral membrane protein with an aspartyl protease motif in its lumenal domain that fulfills most of the requirements expected for a candidate -secretase (24). It is highly expressed in brain and neurons and colocalizes with Golgi and endosomal markers. BACE1 is the primary brain
-secretase and is highly increased in both protein levels and enzymatic activity in the neocortex of AD patients (25, 26). Disruption of BACE1 in AD transgenic mice almost completely abolished
-cleavage of APP together with the ability to generate A
(27), further confirming that BACE1 is indeed the long-searched APP
-secretase. Even if much is known about intracellular trafficking and post-translational modifications of BACE1, very little or nothing is known about regulation of BACE1 expression/activity. Very recently Tamagno et al. (28) have shown that oxidative stress is able to increase the expression of BACE1 together with
-cleavage of APP. In this previous work, BACE1 activation involved cell damage and most likely required transcriptional activation of BACE1. In contrast, our results indicate that the ceramide-dependent regulation of BACE1 expression occurs, at least in part, at the level of protein degradation. Whether ceramide also affects transcription and/or translation of BACE1 remains to be further analyzed. However, it is worth mentioning that for the stable transfection of CHOBACE1 cells we only used the coding region of BACE1, eliminating potential 5' and 3' regulatory elements.
In conclusion, our study implicates for the first time the lipid second messenger ceramide in the generation of A and proposes ceramide as a potential novel link between AD and aging. It also shows that ceramide regulates
-secretase activity at the level of BACE1 stabilization. Identification of the downstream molecules that mediate the ceramide-dependent regulation of BACE1 turnover may provide novel targets for the therapeutic treatment of AD patients.
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FOOTNOTES |
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These authors contributed equally to the execution of the experiments described in this study.
** Present address: Dept. of Bioscience and Biotechnology, Drexel University, Philadelphia, PA 19104.
¶ To whom correspondence may be addressed: Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, CAGN, Massachusetts General Hospital, Harvard Medical School, 114 16th St., Charlestown, MA 02129. Tel.: 617-724-1505; Fax: 617-724-1823; E-mail: pugliell{at}helix.mgh.harvard.edu. To whom correspondence may also be addressed: Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, CAGN, Massachusetts General Hospital, Harvard Medical School, 114 16th St., Charlestown, MA 02129. Tel.: 617-726-3668; Fax: 617-724-1823; E-mail: kovacs{at}helix.mgh.harvard.edu.
1 The abbreviations used are: AD, Alzheimer's disease; APP, amyloid precursor protein; -APP-CTF,
-APP-C terminal fragment;
-APP-CTF,
-APP-C terminal fragment; A
, amyloid
-peptide; BACE,
-site APP-cleaving enzyme; SM, sphingomyelin; C6-cer, C6-ceramide; FB1, fumonisin B1; nSMase, neutral sphingomyelinase; NB-DGJ, N-butyldeoxygalactonojirimycin; PARP, poly(ADP-ribose) polymerase; f. l., full-length; m-, mature; im-, immature; CHO, Chinese hamster ovary; MES, 4-morpholineethanesulfonic acid; ELISA, enzyme-linked immunosorbent assay; ZVAD, benzyloxycarbonyl-Val-Ala-Asp.
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ACKNOWLEDGMENTS |
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REFERENCES |
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