Proteolytic Processing of Amyloid-β Precursor Protein by Secretases Does Not Require Cell Surface Transport
2004; Elsevier BV; Volume: 279; Issue: 45 Linguagem: Inglês
10.1074/jbc.m408474200
ISSN1083-351X
AutoresMikhail Khvotchev, Thomas C. Südhof,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoCleavage of amyloid-β precursor protein (APP) by α-,β-, and γ-secretases releases an extracellular fragment called APPS, small Aβ peptides, and a short APP intracellular domain that may provide a transcriptional signal analogous to the Notch intracellular domain. Notch cleavage is activated by extracellular ligands on the cell surface, but the cellular localization of APP cleavage remains unclear. We now show that in transfected cultured cells, the plasma membrane SNARE protein syntaxin 1A, when expressed as a full-length protein, disrupts the Golgi apparatus and blocks trans-Golgi traffic and exocytosis. In contrast, truncated syntaxin 1A1–243 selectively abolishes exocytosis but has no apparent effect on trans-Golgi traffic or Golgi structure, whereas further truncated syntaxins 1A1–236 and 1A1–230 have no effect on either exocytosis or Golgi traffic. Using these syntaxin 1A fragments, we demonstrated that blocking trans-Golgi traffic greatly impairs APP cleavage and AICD-dependent nuclear signaling, whereas blocking exocytosis alone does not affect either process, even though secretion of APPS and Aβ40 peptide is abolished. Our data suggest that, different from Notch, cleavage of APP is independent of cell surface regulation by extracellular ligands but may be controlled by intracellular signaling. Cleavage of amyloid-β precursor protein (APP) by α-,β-, and γ-secretases releases an extracellular fragment called APPS, small Aβ peptides, and a short APP intracellular domain that may provide a transcriptional signal analogous to the Notch intracellular domain. Notch cleavage is activated by extracellular ligands on the cell surface, but the cellular localization of APP cleavage remains unclear. We now show that in transfected cultured cells, the plasma membrane SNARE protein syntaxin 1A, when expressed as a full-length protein, disrupts the Golgi apparatus and blocks trans-Golgi traffic and exocytosis. In contrast, truncated syntaxin 1A1–243 selectively abolishes exocytosis but has no apparent effect on trans-Golgi traffic or Golgi structure, whereas further truncated syntaxins 1A1–236 and 1A1–230 have no effect on either exocytosis or Golgi traffic. Using these syntaxin 1A fragments, we demonstrated that blocking trans-Golgi traffic greatly impairs APP cleavage and AICD-dependent nuclear signaling, whereas blocking exocytosis alone does not affect either process, even though secretion of APPS and Aβ40 peptide is abolished. Our data suggest that, different from Notch, cleavage of APP is independent of cell surface regulation by extracellular ligands but may be controlled by intracellular signaling. Amyloid-β precursor protein (APP) 1The abbreviations used are: APP, amyloid-β precursor protein; AICD, APP intracellular domain; CTF, C-terminal fragment; DAPT, N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester; hGH, human growth hormone; NICD, Notch intracellular domain; SNARE, soluble N-ethylmaleimide-sensitive factor attachment receptor; PBS, phosphate-buffered saline; CMV, cytomegalovirus. of Alzheimer's disease is a ubiquitous membrane protein that is physiologically processed by site-specific proteolysis (1Bayer T.A. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Mol. Psychiatry. 1999; 4: 524-528Crossref PubMed Scopus (81) Google Scholar, 2Haass C. De Strooper B. Science. 1999; 286: 916-919Crossref PubMed Scopus (367) Google Scholar, 3Price D.L. Sisodia S.S. Annu. Rev. Neurosci. 1998; 21: 479-505Crossref PubMed Scopus (509) Google Scholar, 4Selkoe D.J. Trends Cell Biol. 1998; 8: 447-453Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar). First, cleavage of APP by α-or β-secretases releases a large fragment called APPS that contains most of the extracellular sequences of APP. A small extracellular stub, the transmembrane region, and the cytoplasmic tail of APP (referred to as "AICD" for APP intracellular domain) remain in the membrane after α/β-cleavage. These APP sequences are subsequently cleaved by γ-secretase at multiple sites in the transmembrane region (5Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Crossref PubMed Scopus (429) Google Scholar, 6Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. 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Addressing this question is not only essential for understanding the function of APP but also has implications for the role of the AICD, which may be activated either by an extracellular ligand or by intracellular signals. To our knowledge, no manipulation is currently available that selectively inhibits exocytosis and would allow addressing this question directly. Tetanus toxin light chain effectively impairs regulated exocytosis of synaptic vesicles by inactivating synaptobrevin/vesicle-associated membrane protein (33Link E. Edelmann L. Chow J.H. Binz T. Yamasaki S. Eisel U. Baumert M. Südhof T.C. Niemann H. Jahn R. Biochem. Biophys. Res. Commun. 1992; 189: 1017-1023Crossref PubMed Scopus (256) Google Scholar, 34Schiavo G. Benfenati F. Poulain B. Rossetto O. Polverino de Laureto P. Das Gupta B.R. Montecucco C. Nature. 1992; 359: 832-835Crossref PubMed Scopus (1446) Google Scholar) and also cleaves a ubiquitous synaptobrevin homolog called cellubrevin (35McMahon H. Ushkaryov Y.A. Link E. Edelmann L. Binz T. Niemann H. Jahn R. Südhof T.C. Nature. 1993; 364: 346-349Crossref PubMed Scopus (401) Google Scholar). However, overexpression of tetanus toxin light chain does not impair constitutive exocytosis (36Khvotchev M. Ren M. Takamori S. Jahn R. Südhof T.C. J. Neurosci. 2003; 23: 10531-10539Crossref PubMed Google Scholar) and thus cannot be used to interfere with all exocytosis. In this paper, we exploit the unexpected observation that a fragment of the plasma membrane SNARE protein syntaxin 1A is a selective inhibitor of all exocytosis but does not alter trans-Golgi traffic, whereas full-length syntaxin 1A blocks traffic through the Golgi complex as described previously (37Rowe J. Corradi N. Malosio M.L. Taverna E. Halban P. Meldolesi J. Rosa P. J. Cell Sci. 1999; 112: 1865-1877Crossref PubMed Google Scholar). This allows for the first time testing of whether surface expression of APP is required for cleavage. We find that in cells deficient in constitutive exocytosis, α/β-cleavage and γ-cleavage of APP proceed normally in contrast to cells where Golgi traffic is disrupted, demonstrating that the biology of APP is fundamentally different from that of Notch. Plasmids and Antibodies—Expression vectors for human growth hormone (hGH) (pHGH-CMV5), full-length syntaxin 1A (pCMV-syntaxin 1A1–288), various APP derivatives, and the reporter plasmids pG5E1B-luc and pCMV-LacZ were described previously (9Cao X. Südhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1055) Google Scholar, 38Biederer T. Cao X. Südhof T.C. Liu X. J. Neurosci. 2002; 22: 7340-7351Crossref PubMed Google Scholar, 39McMahon H.T. Missler M. Li C. Südhof T.C. Cell. 1995; 83: 111-119Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 40Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Vectors encoding C-terminally truncated syntaxin 1A were generated by subcloning an EcoRI fragment from corresponding pGEX-KG vectors. pcDNA3.1-mycBACE1 encoding BACE1 was kindly provided by Dr. G. Yu (University of Texas Southwestern Medical Center). The polyclonal APP (U955) and syntaxin (I378 and I379) antibodies were described previously (9Cao X. Südhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1055) Google Scholar, 39McMahon H.T. Missler M. Li C. Südhof T.C. Cell. 1995; 83: 111-119Abstract Full Text PDF PubMed Scopus (389) Google Scholar). Monoclonal antibodies against the extracellular sequences of APP (1G7 and 5A3) were a kind gift of Dr. E. Koo (University of California–San Diego). Transfection and Transactivation Assays—For APP trafficking and glycosylation analyses, HEK293 cells were transfected in 6-well plates with 200 ng of APP expression vector and 1 μg of empty vector or various syntaxin 1A vectors/well using FuGENE 6 transfection reagent (Roche Applied Science). For BACE experiments, HEK293 cells were transfected with 0.5 μg of APP expression vector, 1.5 μg of empty vector or various syntaxin 1A vectors, and 50 ng of BACE expression vector or empty vector. Transfected cells and medium were collected 36–48 h post-transfection. The cells were washed in PBS and lysed in SDS-PAGE loading buffer for immunoblotting or processed for treatment with PNGFase (New England Biolabs), O-glycosidase, and neuraminidase (Sigma) according to the manufacturer's recommendations. To detect secreted APP, 1-ml aliquots of the cell culture medium were collected, and proteins were precipitated with trichloroacetic acid. Transactivation assays were performed in HEK293 and COS cells essentially as described in Refs. 9Cao X. Südhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1055) Google Scholar and 38Biederer T. Cao X. Südhof T.C. Liu X. J. Neurosci. 2002; 22: 7340-7351Crossref PubMed Google Scholar. The cells were co-transfected in 6-well plates with the following 4 plasmids: (a) pG5E1B-luc, 50 ng; (b) pCMV-LacZ, 100 ng; (c) pMst-APP-GV, pMST-APPC99-GV, or pMST-APPAICD-GV, 200 ng; (d) empty vector or various syntaxin vectors, 200 ng. For transactivation experiments in PC12 cells, the following different DNA ratios were employed: (a) 400 ng; (b) 200 ng; (c) 500 ng; (d) 500 ng. Transfections were performed using LipofectAMINE 2000 reagent (Invitrogen). Where indicated, cells were treated with 2 μm DAPT (Calbiochem) for 8 h. The cells were washed with PBS and harvested 48 h post-transfection in 0.2 ml of reporter lysis buffer (Promega) per well, and the luciferase and β-galactosidase activities were determined with the luciferase assay kit (Promega) using a microplate luminometer (Orion, Berthold Detection Systems), and the standard O-nitrophenyl-d-galactopyranoside (Sigma) method, respectively. The luciferase activity was standardized by the β-galactosidase activity as a control for transfection efficiency and general effects on transcription. Values shown are averages of transactivation assays carried out in duplicate, and repeated at least three times. SDS-PAGE and Immunoblotting—Tris-glycine and tricine SDS-PAGE and immunoblotting were performed as described (41Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar, 42Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar, 43Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44922) Google Scholar). For standard immunodetection on Western blots, enhanced chemiluminescence (ECL, Amersham Biosciences) was applied. Quantitative immunoblotting was performed using radiolabeled 125I secondary antibodies (Amersham Biosciences), and the signals were quantitated on a phosphorimaging device (Molecular Dynamics) using Image-Quant software (44Rosahl T.W. Spillane D. Missler M. Herz J. Selig D.K. Wolff J.R. Hammer R.E. Malenka R.C. Südhof T.C. Nature. 1995; 375: 488-493Crossref PubMed Scopus (623) Google Scholar). Measurement of Aβ40 Peptide Levels—HEK293 cells and medium were collected 48 h after transfections. The cells were washed with PBS, lysed in extraction buffer (5 m GnCl, PBS, pH 8.0) at 50 μl/well, and sonicated. The cell extracts were diluted 20-fold with sample buffer (PBS, pH 8.0, containing 5% bovine serum albumin, 5 mm EDTA, and complete protease inhibitor mixture (Roche Applied Science)), and cleared by centrifugation. Total proteins in 1-ml aliquots of cleared cell culture medium were trichloroacetic acid-precipitated, dissolved in 50 μl of extraction buffer, and diluted 20-fold with sample buffer. Aβ40 standards were prepared by dispersing the synthetic peptide in sample and extraction buffer mixture (20:1). Aβ40 levels in samples and standards were measured in duplicates by colorimetric enzyme-linked immunosorbent assay (BIOSOURCE) according to the manufacturer's recommendations. Miscellaneous Procedures—For measurements of constitutive exocytosis, HEK293 cells were co-transfected in 6-well plates using FuGENE 6 reagent with pHGHCMV5 and a control vector or various syntaxin 1A constructs at 1:20 ratio (typically 0.05 μg of pHGHCMV5 and 1 μg of a test plasmid/well). Constitutive hGH secretion was measured as described previously (36Khvotchev M. Ren M. Takamori S. Jahn R. Südhof T.C. J. Neurosci. 2003; 23: 10531-10539Crossref PubMed Google Scholar, 45Schlüter O.M. Khvotchev M. Jahn R. Südhof T.C. J. Biol. Chem. 2002; 277: 40919-40929Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) and is expressed as fold-over cellular hGH. Immunocytochemistry of transfected HeLa cells was performed essentially as described by Cao and Südhof (9Cao X. Südhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1055) Google Scholar) using polyclonal syntaxin antibodies and monoclonal antibodies to GM130 (Transduction Laboratories) and goat anti-rabbit or goat anti-mouse secondary antibodies coupled with Alexa Fluor 488 and Alexa Fluor 546 (Molecular Probes). The labeled cells were imaged on a Leica TCS SP2 confocal microscope. Stage-specific Inhibitors of Secretory Membrane Traffic Based on Syntaxin 1A—syntaxin 1A is a SNARE protein of the plasma membrane that mediates Ca2+-induced synaptic vesicle exocytosis and possibly other forms of exocytosis (46Chen Y.A. Scheller R.H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 98-106Crossref PubMed Scopus (870) Google Scholar, 47Jahn R. Lang T. Südhof T.C. Cell. 2003; 112: 519-533Abstract Full Text Full Text PDF PubMed Scopus (1228) Google Scholar). syntaxin 1A is composed of an N-terminal three-helical Habc domain, a SNARE motif that participates in core complex formation with other SNARE proteins, and a C-terminal transmembrane region (Fig. 1A). To test whether expression of different fragments of syntaxin 1A interferes with secretory membrane traffic, we employed four syntaxin 1A proteins, full-length syntaxin 1A1–288, and truncated syntaxin 1A1–243, 1A1–236, and 1A1–230. These proteins were co-expressed with hGH as a reporter protein that is constitutively secreted in non-neuronal cells (48Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (750) Google Scholar). We found that full-length syntaxin 1A1–288 or C-terminally truncated syntaxin 1A1–243 severely inhibited constitutive hGH secretion in HEK293 and HeLa cells, whereas truncated syntaxin 1A1–236 and syntaxin 1A1–230, which contain only 7 or 13 fewer residues, respectively, than syntaxin 1A1–243, had no significant effect (Fig. 1B and data not shown). The seven residues that are present in the inhibitory syntaxin 1A1–243 protein, but absent from the innocuous syntaxin 1A1–236 protein, are located in the middle in the SNARE motif. This suggests that the inhibitory effect of syntaxin 1A1–243 may be due to the formation of inappropriate SNARE complexes, which requires a minimal length of the SNARE motif for SNARE complex formation. However, it is surprising that full-length syntaxin 1A is also inhibitory because it should behave "normally." To explore whether the two inhibitory syntaxin 1A constructs act by distinct mechanisms, we examined the effect of transfected syntaxin 1A constructs on intracellular organelles using immunofluorescence staining for syntaxin 1A and organelle-specific markers. In agreement with previous studies (37Rowe J. Corradi N. Malosio M.L. Taverna E. Halban P. Meldolesi J. Rosa P. J. Cell Sci. 1999; 112: 1865-1877Crossref PubMed Google Scholar), full-length syntaxin 1A1–288 caused a disorganization of the Golgi apparatus in HeLa cells as visualized with an antibody to the Golgi protein GM130 (49Nakamura N. Rabouille C. Watson R. Nilsson T. Hui N. Slusarewicz P. Kreis T.E. Warren G. J. Cell Biol. 1995; 131: 1715-1726Crossref PubMed Scopus (675) Google Scholar) (Fig. 1C). Truncated syntaxin 1A1–243, however, had no effect on the morphology of the Golgi complex or any other organelle examined, despite the nearly equal inhibition of hGH secretion caused by this syntaxin construct (Fig. 1, B and C, and data not shown). Furthermore, other truncated syntaxins also induced no change (data not shown). Transfection of different syntaxins into HEK293 and Vero cells had identical effects on Golgi morphology, suggesting that these results are not dependent on the cell type used (data not shown). This observation indicates that syntaxin 1A1–243 may inhibit hGH secretion by selectively interfering with exocytosis, whereas full-length syntaxin 1A1–288 disrupts trans-Golgi traffic, and shorter truncated syntaxins, such as syntaxin 1A1–236, have no effect. The selective effect of syntaxin 1A1–243 appears to operate in all cells tested by a mechanism that involves Munc18 proteins. 2M. Khvotchev and T. C. Südhof, manuscript in preparation. Differential Effects of Syntaxin 1A Constructs on APP Cleavage and Glycosylation—We co-transfected APP with control vectors or the various syntaxin 1A constructs and examined the effect of the syntaxin fragments on the glycosylation and cleavage of APP in HEK293 cells. We used immunoblotting to estimate the levels of full-length APP and of the C-terminal cleavage products of APP in the cells and measured some of the cleavage products using quantitative immunoblotting in which protein amounts are determined by 125I-labeled secondary antibodies (44Rosahl T.W. Spillane D. Missler M. Herz J. Selig D.K. Wolff J.R. Hammer R.E. Malenka R.C. Südhof T.C. Nature. 1995; 375: 488-493Crossref PubMed Scopus (623) Google Scholar). Furthermore, to assess intramembranous γ-cleavage of APP, we compared untreated transfected cells with cells that had been treated with DAPT, an inhibitor of presenilin-dependent γ-secretase (50Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Alstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (797) Google Scholar). Finally, we employed two gel systems, a standard gel system (41Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar) to resolve larger proteins and a tricine gel system (42Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar) to separate small peptides such as the C-terminal fragments. In untreated transfected cells, APP was present in multiple bands that likely correspond to partially glycosylated immature and fully glycosylated mature species (6Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Crossref PubMed Scopus (318) Google Scholar, 51Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1038) Google Scholar). In addition, a small amount of the C-terminal APP fragments was detected (Fig. 2A). As expected, treatment with the γ-secretase inhibitor DAPT increased the abundance of the C-terminal fragments but had no effect on the banding pattern of full-length APP. When we co-expressed full-length syntaxin 1A1–288 with APP, no C-terminal fragment of APP was detectable, even after the addition of DAPT (Fig. 2A). Furthermore, glycosylation of APP was altered, as evidenced by the loss of the mature forms of APP on the immunoblots. In contrast, when we co-transfected truncated syntaxin 1A1–243 (which inhibits exocytosis) or syntaxin 1A1–236 (which has no effect on exocytosis), we observed no change in the production of the C-terminal fragment or the apparent glycosylation of APP (Fig. 2A). To ensure that the truncated syntaxin 1A1–243 did not cause a kinetic impairment in APP cleavage, we quantified the amount of accumulated C-terminal fragment of APP in cells after 20 min, 60 min, and 180 min of DAPT treatment (Fig. 2B). Quantitations were performed with 125I-labeled secondary antibodies and phosphorimaging detection. We observed no difference in the rate of APP cleavage between cells in which APP had been transfected alone or co-transfected with syntaxin 1A1–243 or syntaxin 1A1–236, whereas no C-terminal fragments were detectable after transfection with full-length syntaxin 1A (Fig. 2B and data not shown). APP is glycosylated by both N- and O-linked sugars (51Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1038) Google Scholar). To obtain a more detailed characterization of the glycosylation of APP after either trans-Golgi traffic or exocytosis were inhibited by co-expression of full-length syntaxin 1A1–288 or truncated syntaxin 1A1–243, respectively, we examined the effect of deglycosylating enzymes on the mobility of APP. Proteins from cells co-transfected with control vector or the various syntaxin expression vectors were treated with PNGFase (which removes N-linked sugars), with O-glycosidase (which digests O-linked sugars), and with neuraminidase (which cleaves sialic acid) (Fig. 2C). We found that the changes in electrophoretic mobility of APP induced by such treatments were identical between control-transfected and syntaxin 1A1–243-expressing cells, suggesting similar glycosylation patterns. In contrast, APP from cells expressing full-length syntaxin 1A1–288 exhibited only a small PNGase F-dependent change in electrophoretic mobility (Fig. 2C). This result indicates that similar to other treatments affecting the integrity of the Golgi apparatus or the exit of APP from the endoplasmic reticulum (52LeBlanc A.C. Goodyer C.G. J. Neurochem. 1999; 72: 1832-1842Crossref PubMed Scopus (35) Google Scholar, 53Piccini A. Fassio A. Pasqualetto E. Vitali A. Borghi R. Palmieri D. Nacmias B. Sorbi S. Sitia R. 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