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• W2079333946 abstract "Alzheimer disease is associated with extracellular deposits of amyloid β-peptides in the brain. Amyloid β-peptides are generated by proteolytic processing of the β-amyloid precursor protein by β- and γ-secretases. The cleavage by secretases occurs predominantly in post-Golgi secretory and endocytic compartments and is influenced by cholesterol, indicating a role of the membrane lipid composition in proteolytic processing of the β-amyloid precursor protein. To analyze the role of glycosphingolipids in these processes we inhibited glycosyl ceramide synthase, which catalyzes the first step in glycosphingolipid biosynthesis. The depletion of glycosphingolipids markedly reduced the secretion of endogenous β-amyloid precursor protein in different cell types, including human neuroblastoma SH-SY5Y cells. Importantly, secretion of amyloid β-peptides was also strongly decreased by inhibition of glycosphingolipid biosynthesis. Conversely, the addition of exogenous brain gangliosides to cultured cells reversed these effects. Biochemical and cell biological experiments demonstrate that the pharmacological reduction of cellular glycosphingolipid levels inhibited maturation and cell surface transport of the β-amyloid precursor protein. In the glycosphingolipid-deficient cell line GM95, cellular levels and maturation of β-amyloid precursor protein were also significantly reduced as compared with normal B16 cells. Together, these data demonstrate that glycosphingolipids are implicated in the regulation of the subcellular transport of the β-amyloid precursor protein in the secretory pathway and its proteolytic processing. Thus, enzymes involved in glycosphingolipid metabolism might represent targets to inhibit the production of amyloid β-peptides. Alzheimer disease is associated with extracellular deposits of amyloid β-peptides in the brain. Amyloid β-peptides are generated by proteolytic processing of the β-amyloid precursor protein by β- and γ-secretases. The cleavage by secretases occurs predominantly in post-Golgi secretory and endocytic compartments and is influenced by cholesterol, indicating a role of the membrane lipid composition in proteolytic processing of the β-amyloid precursor protein. To analyze the role of glycosphingolipids in these processes we inhibited glycosyl ceramide synthase, which catalyzes the first step in glycosphingolipid biosynthesis. The depletion of glycosphingolipids markedly reduced the secretion of endogenous β-amyloid precursor protein in different cell types, including human neuroblastoma SH-SY5Y cells. Importantly, secretion of amyloid β-peptides was also strongly decreased by inhibition of glycosphingolipid biosynthesis. Conversely, the addition of exogenous brain gangliosides to cultured cells reversed these effects. Biochemical and cell biological experiments demonstrate that the pharmacological reduction of cellular glycosphingolipid levels inhibited maturation and cell surface transport of the β-amyloid precursor protein. In the glycosphingolipid-deficient cell line GM95, cellular levels and maturation of β-amyloid precursor protein were also significantly reduced as compared with normal B16 cells. Together, these data demonstrate that glycosphingolipids are implicated in the regulation of the subcellular transport of the β-amyloid precursor protein in the secretory pathway and its proteolytic processing. Thus, enzymes involved in glycosphingolipid metabolism might represent targets to inhibit the production of amyloid β-peptides. The deposition of amyloid β-peptides (Aβs) 1The abbreviations used are: Aβ, amyloid β-peptide; AD, Alzheimer disease; APP, β-amyloid precursor protein; APPS, soluble APP; CTF, C-terminal fragment; ECL, enhanced chemiluminescence; GSL, glycosphingolipid; HEK, human embryonic kidney; PDMP, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; TRITC, tetramethylrhodamine isothiocyanate; WGA, wheat germ agglutinin. in extracellular plaques is an invariant neuropathological feature of Alzheimer disease (AD) (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5170) Google Scholar, 2Aguzzi A. Haass C. Science. 2003; 302: 814-818Crossref PubMed Scopus (200) Google Scholar). Aβ derives from the β-amyloid precursor protein (APP) by proteolytic processing, which involves sequential cleavages by proteases called β- and γ-secretases (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5170) Google Scholar, 3Annaert W. de Strooper B. Annu. Rev. Cell Dev. Biol. 2002; 18: 25-51Crossref PubMed Scopus (198) Google Scholar, 4Walter J. Kaether C. Steiner H. Haass C. Curr. Opin. Neurobiol. 2001; 11: 585-590Crossref PubMed Scopus (158) Google Scholar). APP is a type I membrane protein that is transported from the endoplasmic reticulum via the Golgi compartment to the cell surface and undergoes maturation by N′- and O′-glycosylation (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5170) Google Scholar, 3Annaert W. de Strooper B. Annu. Rev. Cell Dev. Biol. 2002; 18: 25-51Crossref PubMed Scopus (198) Google Scholar, 4Walter J. Kaether C. Steiner H. Haass C. Curr. Opin. Neurobiol. 2001; 11: 585-590Crossref PubMed Scopus (158) Google Scholar). Within the secretory pathway and at the cell surface APP is predominantly cleaved by α-secretase, resulting in the secretion of soluble APP (APPS) (5Sisodia S.S. Koo E.H. Beyreuther K. Unterbeck A. Price D.L. Science. 1990; 248: 492-495Crossref PubMed Scopus (746) Google Scholar). Because α-secretase cleaves APP within the Aβ domain, this cleavage precludes the generation of Aβ. Alternatively, APP can be cleaved by β-secretase. The cleavage of APP by β-secretase occurs predominantly in endosomal and lysosomal compartments after internalization from the cell surface (6Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (773) Google Scholar, 7Koo E.H. Squazzo S.L. Selkoe D.J. Koo C.H. J. Cell Sci. 1996; 109: 991-998Crossref PubMed Google Scholar). The C-terminal fragments (CTFs) of APP resulting from α- or β-secretase cleavage can be cleaved within the transmembrane domain by γ-secretase to release p3 and Aβ, respectively (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5170) Google Scholar, 3Annaert W. de Strooper B. Annu. Rev. Cell Dev. Biol. 2002; 18: 25-51Crossref PubMed Scopus (198) Google Scholar, 8Steiner H. Haass C. Nat. Rev. Mol. Cell Biol. 2000; 1: 217-224Crossref PubMed Scopus (144) Google Scholar). The proteolytic processing of APP is influenced by the lipid composition of cellular membranes, as demonstrated by the pharmacological modulation of cellular cholesterol levels (9Wolozin B. Neuron. 2004; 41: 7-10Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 10Hartmann T. Trends Neurosci. 2001; 24: S45-S48Abstract Full Text Full Text PDF PubMed Google Scholar, 11Simons K. Ehehalt R. J. Clin. Investig. 2002; 110: 597-603Crossref PubMed Scopus (920) Google Scholar, 12Kovacs D.M. Fausett H.J. Page K.J. Kim T.W. Moir R.D. Merriam D.E. Hollister R.D. Hallmark O.G. Mancini R. Felsenstein K.M. Hyman B.T. Tanzi R.E. Wasco W. Nat. Med. 1996; 2: 224-229Crossref PubMed Scopus (513) Google Scholar). In addition, the inhibition of acyl-coenzyme A:cholesterol acyltransferase (ACAT) also led to strong reduction of Aβ generation in cultured cells and transgenic mice, indicating that cholesterol esters also influence proteolytic processing of APP (13Puglielli L. Konopka G. Pack-Chung E. Ingano L.A. Berezovska O. Hyman B.T. Chang T.Y. Tanzi R.E. Kovacs D.M. Nat. Cell Biol. 2001; 3: 905-912Crossref PubMed Scopus (387) Google Scholar, 14Hutter-Paier B. Huttunen H.J. Puglielli L. Eckman C.B. Kim D.Y. Hofmeister A. Moir R.D. Domnitz S.B. Frosch M.P. Windisch M. Kovacs D.M. Neuron. 2004; 44: 227-238Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). These effects on APP processing might involve altered cleavage of APP by α-secretase and/or β-secretase, probably by redistribution of APP and secretases between distinct membrane microdomains (15Abad-Rodriguez J. Ledesma M.D. Craessaerts K. Perga S. Medina M. Delacourte A. Dingwall C. de Strooper B. Dotti C.G. J. Cell Biol. 2004; 167: 953-960Crossref PubMed Scopus (293) Google Scholar, 16Kojro E. Gimpl G. Lammich S. Marz W. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5815-5820Crossref PubMed Scopus (728) Google Scholar, 17Runz H. Rietdorf J. Tomic I. de Bernard M. Beyreuther K. Pepperkok R. Hartmann T. J. Neurosci. 2002; 22: 1679-1689Crossref PubMed Google Scholar, 18Ehehalt R. Keller P. Haass C. Thiele C. Simons K. J. Cell Biol. 2003; 160: 113-123Crossref PubMed Scopus (926) Google Scholar). Apart from cholesterol, glycosphingolipids (GSLs) have also been implicated in the pathogenesis of AD. It has been shown that the levels of several gangliosides are altered in AD brains (19Cutler R.G. Kelly J. Storie K. Pedersen W.A. Tammara A. Hatanpaa K. Troncoso J.C. Mattson M.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2070-2075Crossref PubMed Scopus (840) Google Scholar). In addition, the ganglioside GM1 binds to Aβ and might contribute to early deposition of the peptide in amyloid plaques (20Yanagisawa K. Odaka A. Suzuki N. Ihara Y. Nat. Med. 1995; 1: 1062-1066Crossref PubMed Scopus (481) Google Scholar, 21Yanagisawa K. Ihara Y. Neurobiol. Aging. 1998; 19: S65-S67Crossref PubMed Scopus (80) Google Scholar, 22Hayashi H. Kimura N. Yamaguchi H. Hasegawa K. Yokoseki T. Shibata M. Yamamoto N. Michikawa M. Yoshikawa Y. Terao K. Matsuzaki K. Lemere C.A. Selkoe D.J. Naiki H. Yanagisawa K. J. Neurosci. 2004; 24: 4894-4902Crossref PubMed Scopus (217) Google Scholar). The biosynthesis of GSLs starts with the generation of glucosylceramide from UDP-glucose and ceramide by glucosylceramide synthase (Fig. 1A). Glucosylceramide represents the precursor of a large variety of GSLs (23Kolter T. Proia R.L. Sandhoff K. J. Biol. Chem. 2002; 277: 25859-25862Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) that are transported in the secretory pathway from the Golgi to the cell surface (11Simons K. Ehehalt R. J. Clin. Investig. 2002; 110: 597-603Crossref PubMed Scopus (920) Google Scholar, 24Sprong H. van der Sluijs P. van Meer G. Nat. Rev. Mol. Cell Biol. 2001; 2: 504-513Crossref PubMed Scopus (480) Google Scholar). The physiological functions of GSLs include the regulation of cell adhesion, cell differentiation, and signal transduction (24Sprong H. van der Sluijs P. van Meer G. Nat. Rev. Mol. Cell Biol. 2001; 2: 504-513Crossref PubMed Scopus (480) Google Scholar, 25Kolter T. Sandhoff K. Brain Pathol. 1998; 8: 79-100Crossref PubMed Scopus (76) Google Scholar, 26Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2551) Google Scholar). Dysfunction of GSL degradation is associated with several inherited diseases that are characterized by the accumulation of GSLs in endosomal/lysosomal compartments (27Marks D.L. Pagano R.E. Trends Cell Biol. 2002; 12: 605-613Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 28Schuette C.G. Doering T. Kolter T. Sandhoff K. Biol. Chem. 1999; 380: 759-766Crossref PubMed Scopus (27) Google Scholar). Here we sought to analyze the role of GSLs in the proteolytic processing of APP and the generation of Aβ. By using different cell types that express endogenous APP, we demonstrate that depletion of cells from GSLs results in reduced secretion of soluble APP and Aβ. Our data indicate that GSLs are implicated in the transport of APP in the secretory pathway and its expression at the cell surface, thereby altering the proteolytic processing by secretases. Reagents and Antibodies—d- and l-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), C6-ceramide, and TRITC-labeled wheat germ agglutinin (WGA) were obtained from Sigma. Purified gangliosides from bovine brain were obtained from Fidia Research Laboratories (Abano Therme, Italy). Antibodies 5313 and 6687, recognizing the N- and C-terminal domains of APP, respectively, were described earlier (29Walter J. Capell A. Hung A.Y. Langen H. Schnolzer M. Thinakaran G. Sisodia S.S. Selkoe D.J. Haass C. J. Biol. Chem. 1997; 272: 1896-1903Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and generously provided by Dr. C. Haass. Polyclonal antibodies 2964 against Aβ were raised by inoculation of rabbits with synthetic Aβ40. Monoclonal antibody 6E10 was obtained from Signet Inc. Cell Culture and Treatment—B16 and GM95 cells were obtained from the RIKEN cell bank (Tokyo, Japan) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Human embryonic kidney (HEK) 293 and HeLa cells were also cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. SH-SY5Y cells were maintained in RPMI supplemented with 15% fetal calf serum. PDMP was dissolved in water at concentrations of 50 mm and applied to cells as indicated (Figs. 1, 2, 3, 4, 5, 6). C6-ceramide was dissolved at a concentration of 10 mm in ethanol. Cells were cultured in the absence or presence of PDMP, ceramide, or exogenous GSLs as indicated (Figs. 1, 2, 3, 4, 5, 6). Control cells were incubated with the carrier alone.Fig. 3GSL depletion selectively inhibits secretion of APPS in SH-SY5Y cells.A, human SH-SY5Y cells were cultured in the absence (-) or presence (+) of 25 μm PDMP for 48 h and then pulse-labeled with [35S]methionine for 15 min. One set of cells was immediately lysed after the pulse. Another set of cells was incubated for an additional 2 h in the absence or presence of PDMP. APP was immunoprecipitated from the cell lysates (bottom) and chase media (top) and separated by SDS-PAGE. Radiolabeled APP was detected by phosphorimaging. The migration of APPS and full-length APP (fl APP) for the different splice variants APP751/770 and APP695 is indicated by arrowheads. B, quantification of APP secretion was carried out by phosphorimaging. Values represent the means of three independent experiments ± S.D. C, total protein secretion was analyzed by trichloroacetic acid precipitation of proteins from the conditioned medium after the chase period. Radioactivity was determined by liquid scintillation counting. Values represent the means of three independent experiments ± S.D. D, cells were treated with 10 μm C6-ceramide for 48 h, and APP was immunoprecipitated from conditioned media and cell lysates and detected by Western blotting. The band marked by an asterisk likely represents mature APP695.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4GSLs regulate secretion of Aβ. A, human SH-SY5Y cells were cultured in the absence (-) or presence (+) of 25 μm PDMP for 48 h, and endogenously generated Aβ was immunoprecipitated from conditioned media and detected by Western immunoblotting. B, Aβ secretion was quantified by ECL imaging and normalized to cellular APP. C and D, SH-SY5Y cells were cultured in the absence (-) or presence (+) of exogenous GSLs, and the secretion of Aβ was analyzed. E, membranes were isolated from PDMP-treated (+) and untreated (-) cells. After separation by SDS-PAGE, CTFs were detected by Western immunoblotting. CTFs generated by β-secretase cleavage (CTFβ) and α-secretase cleavage (CTFα), respectively, are indicated by arrowheads. The CTF generated by alternative β-secretase cleavage is indicated by an asterisk. F, quantification of the relative amounts of CTFs was done by ECL imaging. Values represent the means of three independent experiments ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5GSL depletion decreases expression of APP at the cell surface.A and B, cell surface proteins of control (-) and PDMP-treated (+) HEK293 cells were labeled with sulfo-N-hydroxysuccinimide-biotin and isolated with streptavidin-conjugated agarose beads as described under “Materials and Methods.” Precipitates were separated by SDS-PAGE, and endogenously expressed APP (A) or Fas (B) was detected by Western immunoblotting (right sections). As a control, cellular levels of APP (panel A, left section) and Fas (panel B, left section) were also analyzed by Western immunoblotting of isolated cell membranes with the respective antibodies. IP, immunoprecipitation. C, control (left) or PDMP-treated cells (right) were stained with TRITC-labeled WGA as described under “Materials and Methods” to detect cell surface-located glycoproteins and analyzed by fluorescence microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Suppression of GSL biosynthesis affects maturation and stability of APP.A, after culturing in the absence or presence of 10 μm PDMP for 48 h, HeLa cells were labeled with [35S]methionine for 10 min and chased for the indicated time periods (see “Materials and Methods”). APP was immunoprecipitated from cell lysates, separated by SDS-PAGE, and detected by phosphorimaging. The migration of mature (m) and immature (im) APP is indicated by arrowheads. B and C, quantification of APP maturation (B) and stability (C). In PDMP-treated cells (open squares) the maturation of APP is significantly decreased as compared with untreated cells (closed circles) (B). In addition, the stability of cellular APP is reduced in PDMP-treated cells (C). Values represent means of three independent experiments ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cell Staining with TRITC-labeled WGA—To visualize cell surface glycoproteins, cells grown on coverslips were fixed in 4% paraformaldehyde and incubated with TRITC-labeled WGA (Sigma) for 30 min. Samples were analyzed by fluorescence microscopy on a Nikon Eclipse E800 fluorescence microscope. Metabolic Labeling, Immunoprecipitation, and Immunoblotting—For pulse-chase experiments, cells were starved at 37 °C in methionine-free, serum-free medium for 45 min and then labeled with [35S]methionine/[35S]cysteine (MP Biomedicals Inc.) at 37 °C for 10 min. Cells were then washed with phosphate-buffered saline and chased in medium supplemented with 10% fetal calf serum and excess amounts of unlabeled methionine for the indicated periods of time. Cells were lysed in STEN buffer (50 mm Tris, pH 7.6, 150 mm NaCl, and 2 mm EDTA) supplemented with 1% Nonidet P-40, 1% Triton X-100, and 2% bovine serum albumin on ice for 10 min. Lysates were clarified by centrifugation for 20 min at 14000 × g and immunoprecipitated for 3 h at 4 °C. After separation by SDS-PAGE, proteins were transferred to nitrocellulose membrane (Schleicher & Schuell Inc.) and analyzed by autoradiography or phosphorimaging. Alternatively, proteins were detected by immunoblotting using enhanced chemiluminescence reagent ECL (Amersham Biosciences). Analysis of Total Protein Secretion—Whatman 3MM paper was cut into squares (2 × 2 cm), soaked with 10% trichloroacetic acid (w/v), and dried. Conditioned chase media (25 μl) of radiolabeled cells was applied to the paper and left to dry. The filter papers were washed in 5% trichloroacetic acid (w/v), absolute ethanol, and acetone (twice each). Subsequent to drying, the filter papers were transferred to scintillation counter vials containing 5 ml of Optiphase Highsafe II scintillation mixture. The activities of 35S in the samples were counted on a scintillation counter using a 60-s time window. Isolation of Membranes and Detection of GM1—Cells were scraped from the culture dishes and incubated in hypotonic buffer (10 mm Tris, pH 7.3, 10 mm MgCl2, 1 mm EDTA, and 1 mm EGTA) for 10 min on ice. Cells were then homogenized by passing 15 times through a 21-gauge needle and centrifuged for 10 min at 1000 rpm to pellet nuclei. The resulting supernatant was centrifuged 30 min at 16000 × g. Pelleted membranes were separated by SDS-PAGE, and GM1 was detected by Western immunoblotting with horseradish peroxidase-conjugated cholera toxin (Sigma). Biotinylation of Cell Surface Proteins—To label cell surface proteins, cells were washed three times with ice-cold phosphate-buffered saline and incubated on ice with phosphate-buffered saline containing 0.5 mg/ml EZ-Link™ sulfo-N-hydroxysuccinimide-biotin (Perbio Inc.) for 30 min. Cells were then washed three times with ice-cold phosphate-buffered saline supplemented with 20 mm glycine and finally lysed with STEN buffer containing 1% Nonidet P-40 and 1% Triton X-100. Biotinylated proteins were precipitated from cleared lysates with streptavidin-conjugated agarose beads (Sigma) and separated by SDS-PAGE. The respective proteins were then detected by Western immunoblotting. Reverse Transcription PCR—Total RNA was isolated from B16 and GM95 cells using TRIzol, followed by reverse transcription to obtain cDNA. Semi-quantitaive PCR was then performed for APP and actin cDNA with 18 cycles. Primer pairs were 5′-GGTGGACTCTGTGCCAGC-3′ and 5′-TCCGTTCTGCTGCATCTTGG-3′ for APP and 5′-TGCGTGACATCAAAGAGAAG-3′ and 5′-GCTCATAGCTCTTCTCCAGG-3′ for actin. Data Analysis and Statistics—In metabolic labeling experiments, band intensities were analyzed with a phosphorimaging device (FLA2000; Fuji) and the Fuji Image Gauge 3.0 software. For enhanced chemiluminescence detection, signals were measured and analyzed using an ECL imager (ChemiDoc™ XRS; Bio-Rad) and the Quantity One software package (Bio-Rad). For quantitations, three independent experiments (n = 3) were carried out. Statistical analysis was carried out using Student's t test. Significance values are as follows: *, p < 0.05; **, p < 0.01 (Figs. 3 and 4). To analyze the role of GSLs in the proteolytic processing of APP and Aβ generation, GSL biosynthesis was inhibited with PDMP, a competitive inhibitor of glucosylceramide synthase that has been shown previously to efficiently decrease GSL biosynthesis in cultured cells (30Rosenwald A.G. Pagano R.E. J. Lipid Res. 1994; 35: 1232-1240Abstract Full Text PDF PubMed Google Scholar, 31Kok J.W. Babia T. Filipeanu C.M. Nelemans A. Egea G. Hoekstra D. J. Cell Biol. 1998; 142: 25-38Crossref PubMed Scopus (42) Google Scholar, 32Naslavsky N. Shmeeda H. Friedlander G. Yanai A. Futerman A.H. Barenholz Y. Taraboulos A. J. Biol. Chem. 1999; 274: 20763-20771Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 33Mutoh T. Tokuda A. Inokuchi J. Kuriyama M. J. Biol. Chem. 1998; 273: 26001-26007Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Treatment of HEK293 or HeLa cells with PDMP for 48 h led to a significant decrease in GSL biosynthesis as demonstrated by a strong reduction of GM1 levels (Fig. 1B). The treatment of cells with PDMP at the concentrations used did not affect cell viability (Supplemental Fig. 1, available in the on-line version of this article). We therefore used this approach to investigate the role of GSLs in APP processing. HEK293 or HeLa cells were treated with PDMP, and APP was immunoprecipitated from conditioned media and lysates. As demonstrated in Fig. 1, the secretion of APPS into conditioned media was markedly decreased in both HEK293 and HeLa cells that were treated with PDMP (Fig. 1, C-F). The decrease in APP secretion was observed for both variants APPS-α and APPS-β (Supplemental Fig. 2, available in the on-line version of this article). We also assessed the effect of PDMP on the precursor-product relationship of cellular and secreted APP in a pulse-chase experiment. In control cells, ∼8% of 35S-labeled cellular APP was secreted into the conditioned media after 2 h. In the same time period, only ∼ 2.5% of cellular APP was secreted in PDMP-treated cells (not shown). Together, these data demonstrate that PDMP treatment significantly decreases the secretion of APP. It should be noted that the effects of PDMP on APP secretion were selectively observed in non-transfected cells, but not in cells that stably overexpress APP (data not shown). This might be due to a tight regulation of the interaction of APP with membrane lipids (see “Discussion”). We therefore used exclusively non-transfected cells for the further experiments. To prove that the effects of PDMP are due to decreased levels of GSLs, we first analyzed the effect of short term treatment with PDMP. In contrast to long term treatment (48 h), incubation of cells with PDMP for only 2 h did not significantly reduce levels of GSLs (data not shown). Under these conditions, the secretion of APPS was not significantly changed (Fig. 2A). We also assessed the effect of the inactive enantiomer l-PDMP that does not inhibit glucosylceramide synthase. Long term treatment of cells for 48 h with l-PDMP did not reduce APPS secretion (Fig. 2B). Together, these control experiments indicate that the reduced secretion of APPS observed upon cell treatment with PDMP is due to decreased GSL levels in cellular membranes. We next tested the effect of exogenous GSLs on proteolytic processing of APP. Cells were cultured in the presence or absence of purified bovine brain gangliosides, and levels of cellular and secreted APP were analyzed. The addition of exogenous GSLs significantly increased the cellular levels of endogenous APP as well as the secretion of APPS (Fig. 2C). Together, these data demonstrate a regulatory role of GSLs in the cellular metabolism of APP. The levels of endogenous Aβ in the conditioned media of HEK293 were below the detection limits (data not shown), likely due to efficient cleavage of APP by α-secretase in this cell type (34Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1763) Google Scholar) (see Supplemental Fig. 2). Because neuronal cells secrete higher levels of Aβ, we used human neuroblastoma SH-SY5Y cells to prove a role of GSLs in the processing of APP by pulse-chase experiments. Two variants of endogenously expressed APP were detected after pulse labeling in cell lysates that represent distinct APP splice forms, including the neuron-specific APP695 form (Fig. 3A). The presence of distinct splice variants in SH-SY5Y cells was also confirmed by reverse transcription PCR using isoform-specific primers (Supplemental Fig. 3, available in the on-line version of this article). PDMP treatment did not affect the expression of the distinct APP variants, as demonstrated by the similar levels of cellular APP after pulse labeling (Fig. 3A). Also, no effect on cell viability was detected (Supplemental Fig. 1C). As observed in HEK293 and HeLa cells, PDMP resulted in a significant reduction of APPS secretion into the conditioned media of SH-SY5Y cells (Fig. 3, A and B). The reduction was observed for the APP751/770 as well as for the neuron-specific APP695 splice variants. In contrast to HEK293 cells, SH-SY5Y cells predominantly secrete APPS-β (not shown). Together, these data indicate that PDMP treatment inhibits secretion of both APPS-α and APPS-β. However, total protein secretion was not reduced upon PDMP treatment, indicating a selective role of GSLs in the secretion of APP (Fig. 3C). Because the inhibition of glucosylceramide synthase might lead to accumulation of its substrate ceramide, which was shown to alter the proteolytic processing of APP by stabilization of the β-site APP-cleaving enzyme BACE1 (35Puglielli L. Ellis B.C. Saunders A.J. Kovacs D.M. J. Biol. Chem. 2003; 278: 19777-19783Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), we tested the effect of C6-ceramide on the secretion of APPS. The treatment of cells with ceramide at concentrations of 10 μm, which was shown to stabilize BACE1 (35Puglielli L. Ellis B.C. Saunders A.J. Kovacs D.M. J. Biol. Chem. 2003; 278: 19777-19783Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), did not inhibit the secretion of APPS, indicating that the inhibition of APP secretion observed after PDMP was due to decreased levels of GSLs (Fig. 3D). To investigate the role of GSLs in the generation of Aβ, SH-SY5Y cells were incubated in the presence or absence of PDMP for 48 h, and Aβ was immunoprecipitated from conditioned media. The secretion of Aβ was significantly reduced upon the inhibition of GSL biosynthesis (Fig. 4, A and B). On the other hand, the addition of exogenous GSLs increased the secretion of Aβ (Fig. 4, C and D), indicating that GSLs participate in the generation of Aβ. We also analyzed the APP CTFs that derive from proteolytic processing of APP by β-secretase or α-secretase. Two major species of APP CTFs were detected that represent CTF-β and CTF-α resulting from β- and α-secretase cleavage, respectively (Fig. 4E). In addition, an intermediate band was detected that likely represents CTF-β′, a variant generated by an alternative cleavage of APP by β-secretase at Glu-11 within the Aβ domain (36Fluhrer R. Multhaup G. Schlicksupp A. Okochi M. Takeda M. Lammich S. Willem M. Westmeyer G. Bode W. Walter J. Haass C. J. Biol. Chem. 2003; 278: 5531-5538Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 37Lee E.B. Skovronsky D.M. Abtahian F. Doms R.W. Lee V.M. J. Biol. Chem. 2003; 278: 4458-4466Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Consistent with predominant secretion of APPS-β in this cell type, CTF-β was the predominant species in" @default.
• W2079333946 created "2016-06-24" @default.
• W2079333946 creator A5007309669 @default.
• W2079333946 creator A5015700940 @default.
• W2079333946 creator A5020194269 @default.
• W2079333946 creator A5051254091 @default.
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• W2079333946 date "2005-07-01" @default.
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• W2079333946 title "Inhibition of Glycosphingolipid Biosynthesis Reduces Secretion of the β-Amyloid Precursor Protein and Amyloid β-Peptide*[boxs]" @default.
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