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Notch1 Competes with the Amyloid Precursor Protein for γ-Secretase and Down-regulates Presenilin-1 Gene Expression

2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês

10.1074/jbc.m308480200

1083-351X

Alberto Lleó, Oksana Berezovska, Pavan P Ramdya, Hiroaki Fukumoto, Susan Raju, Tejal Shah, Bradley T. Hyman,

Prion Diseases and Protein Misfolding

Presenilin 1 (PS1) is a critical component of theγ-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and Notch1. Based on the fact that APP and Notch are processed by the same γ-secretase, we postulated that APP and Notch compete for the enzyme activity. In this report, we examined the interactions between APP, Notch, and PS1 using the direct γ-secretase substrates, Notch 1 Δextracellular domain (N1ΔEC) and APP carboxyl-terminal fragment of 99 amino acids, and measured the effects on amyloid-β protein production and Notch signaling, respectively. Additionally, we tested the hypothesis that downstream effects on PS1 expression may coexist with the competition phenomenon. We observed significant competition between Notch and APP for γ-secretase activity; transfection with either of two direct substrates of γ-secretase led to a reduction in the γ-cleaved products, Notch intracellular domain or amyloid-β protein. In addition, however, we found that activation of the Notch signaling pathway, by either N1ΔEC or Notch intracellular domain, induced down-regulation of PS1 gene expression. This finding suggests that Notch activation directly engages γ-secretase and subsequently leads to diminished PS1 expression, suggesting a complex set of feedback interactions following Notch activation. Presenilin 1 (PS1) is a critical component of theγ-secretase complex, which is involved in the cleavage of several substrates including the amyloid precursor protein (APP) and Notch1. Based on the fact that APP and Notch are processed by the same γ-secretase, we postulated that APP and Notch compete for the enzyme activity. In this report, we examined the interactions between APP, Notch, and PS1 using the direct γ-secretase substrates, Notch 1 Δextracellular domain (N1ΔEC) and APP carboxyl-terminal fragment of 99 amino acids, and measured the effects on amyloid-β protein production and Notch signaling, respectively. Additionally, we tested the hypothesis that downstream effects on PS1 expression may coexist with the competition phenomenon. We observed significant competition between Notch and APP for γ-secretase activity; transfection with either of two direct substrates of γ-secretase led to a reduction in the γ-cleaved products, Notch intracellular domain or amyloid-β protein. In addition, however, we found that activation of the Notch signaling pathway, by either N1ΔEC or Notch intracellular domain, induced down-regulation of PS1 gene expression. This finding suggests that Notch activation directly engages γ-secretase and subsequently leads to diminished PS1 expression, suggesting a complex set of feedback interactions following Notch activation. Amyloid-β protein (Aβ) 1The abbreviations used are: Aβamyloid-β proteinAPPβ-amyloid precursor proteinCTFcarboxyl-terminal fragmentC83CTF of 83 amino acidsC99CTF of 99 amino acidsPSpresenilinAICDAPP intracellular domainNICDNotch intracellular domainNΔECNotchΔECN1FLfull-length Notch1HEKhuman embryonic kidneyELISAenzyme-linked immunosorbent assayCBF1C-promoter binding factor 1. is a 40–42 residue peptide believed to play a central role in the pathogenesis of Alzheimer's disease (1Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11039-11041Crossref PubMed Scopus (200) Google Scholar). It arises from cleavage of a much larger precursor polypeptide, the β-amyloid precursor protein (APP), encoded by a gene on human chromosome 21. APP undergoes a proteolytic event by an α-secretase creating a large soluble fragment (α-APPs) and a membrane-retained carboxyl-terminal fragment (CTF) of 83 amino acids (C83). The resultant 10-kDa C83 can be cleaved by a presenilin (PS)-dependent γ-secretase to generate a small fragment called p3 precluding the formation of Aβ. In an analogous fashion, other APP holoproteins can be cleaved by another protease, β-secretase, generating a 12-kDa CTF (or C99) that is cleaved by the same γ-secretase to create Aβ. Although the generation of the different isoforms of Aβ and p3 has been intensively studied, the normal biological function of APP proteolysis remains unclear. Recent data suggest that APP could act as a cell surface receptor that mediates signaling through the release of the APP intracellular domain (AICD) (2Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1066) Google Scholar, 3Gao Y. Pimplikar S.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14979-14984Crossref PubMed Scopus (213) Google Scholar). amyloid-β protein β-amyloid precursor protein carboxyl-terminal fragment CTF of 83 amino acids CTF of 99 amino acids presenilin APP intracellular domain Notch intracellular domain NotchΔEC full-length Notch1 human embryonic kidney enzyme-linked immunosorbent assay C-promoter binding factor 1. Notch receptor is another type I transmembrane receptor that is critically required for a variety of signaling events and cell fate decisions during embryogenesis and in adulthood (4Artavanis-Tsanokas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (5024) Google Scholar, 5Fortini M.E. Nat. Rev. Neurosci. 2002; 3: 673-684Google Scholar). It has been shown that the Notch1 receptor continues to be expressed in the adult brain and inhibits neurite outgrowth in postmitotic neurons (6Berezovska O. Xia M.Q. Hyman B.T. J. Neuropathol. Exp. Neurol. 1998; 57: 738-745Crossref PubMed Scopus (128) Google Scholar, 7Berezovska O. McLean P. Knowles R. Frosh M. Lu F.M. Lux S.E. Hyman B.T. Neuroscience. 1999; 93: 433-439Crossref PubMed Scopus (194) Google Scholar, 8Sestan N. Artavanis-Tsanokas S. Rakic P. Science. 1999; 286: 741-746Crossref PubMed Scopus (500) Google Scholar). Full-length Notch is cleaved in the presence of its biological ligand Delta, and the generated Notch intracellular domain (NICD) rapidly translocates to the nucleus where it acts as a transcriptional coactivator (9Jack C. Berezovska O. Wolfe M.S. Hyman B.T. Brain Res. Mol. Brain Res. 2001; 87: 166-174Crossref PubMed Scopus (25) Google Scholar, 10Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1382) Google Scholar). Similar to APP proteolysis, Notch undergoes sequential cleavage by α- and PS-dependent γ-secretase-like activities, named S2 and S3 respectively (11De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1825) Google Scholar). These data suggest that the same PS-dependent proteolytic complex is involved in the processing of both APP and Notch. Thus, it seems conceivable that the different substrates compete for the same protease. We previously showed that endogenous Notch and APP are competitive substrates for γ-secretase activity; treatment of neurons with the native Notch ligand, Delta, induced a dose-dependent decrease in Aβ production, and overexpression of APP in neurons led to a decrease in Notch signaling (12Berezovska O. Jack C. Deng A. Gastineau N. Rebeck G.W. Hyman B.T. J. Biol. Chem. 2001; 276: 30018-30023Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, conflicting data were reported by Chen et al. (13Chen F. Yu G. Arawaka S. Nishimura M. Kawarai T. Yu H. Tandon A. Supala A. Song Y.Q. Rogaeva E. Milman P. Sato C. Yu C. Janus C. Lee J. Song L. Zhang L. Fraser P.E. St George-Hyslop P.H. Nat. Cell. Bio. 2001; 3: 751-754Crossref PubMed Scopus (112) Google Scholar) using human embryonic kidney (HEK)-293 cells overexpressing either C99 or NotchΔextracellular domain (NΔEC), in which no competition was observed for γ-secretase. In an effort to resolve these apparent conflicts in the present study, we examined the competition between Notch and APP under different circumstances. We postulated that if APP and Notch compete for γ-secretase activity, competition should only be observed with active Notch constructs that require a PS1-dependent cleavage, such as the truncated form NΔEC, but not with constructs that do not require this cleavage, such as NICD, or with forms that require ligand binding for its activation, such as full-length Notch. In this report, we extend the previous findings and show that transfection with either of two direct substrates of γ-secretase, C99 or NΔEC, lead to a reduction in the γ-cleaved products (NICD or Aβ respectively). However, surprisingly, we found that NICD also decreased Aβ production from C99. We found that this decrease in Aβ was associated with a down-regulation in the expression of PS1 gene at both the transcriptional and protein levels. Plasmids—Full-length Notch1 (N1FL) and two truncated forms of Notch1 (NΔEC and NICD) cloned into pBos vectors (generous gift of Gerry Weinmaster, UCLA), were used in the transfection experiments. NΔEC contains the cytoplasmic portion and the membrane-spanning region of the molecule (5374–7836 bp), and NICD contains the active signaling portion of the Notch1 molecule (5476–7836 bp) (9Jack C. Berezovska O. Wolfe M.S. Hyman B.T. Brain Res. Mol. Brain Res. 2001; 87: 166-174Crossref PubMed Scopus (25) Google Scholar, 14Berezovska O. Frosch M. McLean P. Knowles R. Koo E. Kang D. Shen J. Lu F.M. Lux S.E. Tonegawa S. Hyman B.T. Brain Res. Mol. Brain Res. 1999; 69: 273-280Crossref PubMed Scopus (62) Google Scholar, 15Lu F.M. Lux S.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5663-5667Crossref PubMed Scopus (69) Google Scholar). Cells were transfected with human full-length APP or with a deletion mutant containing only the C99 cloned into a pSecTag2B vector (Invitrogen) (16Kinoshita A. Whelan C.M. Smith C.J. Mikhailenko I. Rebeck G.W. Strickland D.K. Hyman B.T. J. Neurosci. 2001; 21: 8354-8361Crossref PubMed Google Scholar) and with β-galactosidase as an internal control for transfection efficiency. Transfection with an empty pBos vector served as a negative control for each experiment. Cell Culture Conditions and Transient Transfection—N2a mouse neuroblastoma or HEK293 cells were cultured in OPTI-MEMI with 5% fetal bovine serum at 37 °C with 5% CO2 in a tissue culture incubator. Transient transfection was performed using Superfect reagents (Qiagen, Valencia, CA) according to the manufacturer's instructions. The cells were plated onto 12-well plates 1 day before transfection. The day of transfection, 1.5 μg of plasmid DNA was dissolved in 75 μl of Dulbecco's modified Eagle's medium, and 7.5 μl of Superfect reagent was added. The mixture was left for 5–10 min at room temperature, and 400 μl of growth medium was added to the mixture. Then, 475 μl of this mixture was added to each well. After 2 h, cells were washed with PBS, and growth medium was added to cells. Fe65-dependent APP Luciferase Transactivation Assay—HEK cells were cotransfected with the APP-Gal4 construct (a generous gift from Dr. Thomas Südhof, Univ. of Texas Southwestern Medical Center), which was cloned from APP 695 with the Gal4 sequence spliced into the cytoplasmic tail, in conjunction with pG5E1B-luc, a luciferase reporter plasmid with a Gal4 activation-dependent binding domain and Fe65 (2Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1066) Google Scholar). The generation of Fe65 with a C-terminal myc tag has been reported elsewhere (17Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (440) Google Scholar). CBF1 Luciferase Assay—The CBF1-luciferase assay was performed as described previously (18Berezovska O. Jack C. McLean P. Aster J.C. Hicks C. Xia W. Wolfe M.S. Kimberly W.T. Weinmaster G. Selkoe D.J. Hyman B.T. J. Neurochem. 2000; 75: 583-593Crossref PubMed Scopus (114) Google Scholar). Briefly, cells were transfected with a CBF1 luciferase reporter plasmid (19Hsieh J.J. Henkel T. Salmon P. Robey E. Peterson M.G. Hayward S.D. Mol. Cell. Biol. 1996; 16: 952-959Crossref PubMed Scopus (398) Google Scholar) and β-galactosidase as an internal control for transfection efficiency. The assay was performed 24 h after transfection using an LKB 1251 luminometer, and results were normalized to β-galactosidase expression levels. ELISA and Western Blots—For Aβ ELISA, we used 500 μl of conditioned medium collected 24 h after transfection. The capture antibody was BNT77 (directed against Aβ11–28), and the detection antibody was horseradish peroxidase-conjugated BA27 for Aβ40 (directed against Aβ1–40) and horseradish peroxidase-conjugated BC05 for Aβ42 (directed against Aβ35–43) (20Asami-Odaka A. Ishibashi Y. Kikuchi T. Kitada C. Suzuki N. Biochemistry. 1995; 34: 10272-10278Crossref PubMed Scopus (153) Google Scholar). The antibodies were a generous gift of Takeda Pharmaceuticals, Japan. For the Western blot analysis, the cell lysates were adjusted to equal protein concentrations and electrophoresed in 10–20% SDS-polyacrylamide Tris-glycine gels for PS1 expression. The immunoblotting was performed with the PS1 loop antibody (1:300, Chemicon), mouse actin (1:500), and APP C8 antibody (1:500, a gift of D. J. Selkoe), followed by detection with a horseradish peroxidase-conjugated secondary antibody. Bands on films were quantitated using Odyssey software (LI-COR Biosciences, Lincoln, NE), and the values were normalized to actin expression. RNA Extraction and Real-time Quantitative PCR—RNA was extracted from cells 24 h after transfection using TRIzol reagent (Invitrogen), chloroform, and precipitated with isopropyl alcohol. RNA was purified using the RNeasy mini-kit (Qiagen), and 5 μg of total RNA was used to synthesize cDNA in a 20-μl reaction using the SuperScript First-Strand Synthesis System for reverse transcriptase-PCR (Invitrogen). Mouse PS1 gene was amplified by using the primers 5′-ATCTAAGGCACAGGCCCGA-3′ and 5′-TTGGTAAGGCCTCAGCAACC-3′. GAPDH was used as an internal control gene to normalize for the amount of RNA. Real-time PCR was performed in an iCycler iQ Thermal Cycler (Bio-Rad). 1 μl of cDNA was added to 25 μl of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Each sample was run in triplicate, and analysis of relative gene expression was done by using the 2—ΔΔCt method (21Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (133017) Google Scholar). Briefly, the relative change in gene expression is calculated by subtracting the threshold cycle (ΔCt) of the target gene (PS1) from the internal control gene (GAPDH). Based on the fact that the amount of cDNA doubles in each PCR cycle (assuming a PCR efficiency of 100%), the final fold-change in gene expression is calculated by using the following formula: relative change = 2——ΔΔCt (21Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (133017) Google Scholar). APP C99, a Direct Substrate for γ-Secretase, Decreases Notch Signaling—We postulated that if APP and Notch compete for γ-secretase, we should observe a reduction in Notch signaling after transfection with APP C99. HEK cells were cotransfected with NΔEC and APP C99 or an empty vector; CBF1 luciferase activity in the lysates was measured 24 h after transfection as a measure of Notch signaling. The results were normalized to β-galactosidase expression levels. We observed a marked reduction (63%, p = 0.01) in CBF1 luciferase activity in cells transfected with APP C99 compared with empty vector (Fig. 1). A statistically significant reduction (compared with an empty vector) in the endogenous Notch signaling was also seen in cells transfected with C99 without activating the Notch pathway with NΔEC (data not shown). Transfection with Activated Notch Constructs Leads to a Decrease in Aβ Production—To test the hypothesis that Notch1 and APP are competitive substrates, we transfected N2a cells with N1FL and the two truncated forms of Notch1 (NΔEC and NICD) and measured Aβ production using ELISA in the conditioned media 24 h after transfection. We reasoned that, in the absence of ligand, N1FL would have minimal effect, that NΔEC, the truncated membrane-bound form of Notch that requires PS1-dependent cleavage, would lead to a reduction in Aβ levels if it competed with APP for γ-secretase, and that NICD, which does not require γ-secretase, would not alter Aβ levels. N1FL had only a small effect on Aβ production, perhaps because of activation by endogenous ligands (Table I). However, both truncated forms of Notch (NΔEC and NICD) led to a decrease in Aβ production.Table ITransfection with activated Notch constructs leads to a decrease in Aβ productionConditionnaTotal number of wells for each condition.Aβ40 meanp Compared to empty vectorAβ42 meanp Compared to empty vectorEmpty46100-100-N1FL1792.9nsbns, not significant.104.6nsNΔEC5181.4p < 0.00177.5p < 0.001NICD5186.6p < 0.00181.6p < 0.001a Total number of wells for each condition.b ns, not significant. Open table in a new tab We also examined the effects of cotransfecting the direct substrate NΔEC on the ability of APP-Gal4 to be cleaved by γ-secretase, releasing the Gal4-AICD fragment which activates a Gal4-dependent, artificially expressed luciferase plasmid (2Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1066) Google Scholar). It has been shown that the presence of the Fe65 adaptor protein is a requisite for the stabilization of the AICD fragment and its subsequent nuclear translocation for luciferase activation (2Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1066) Google Scholar, 22Kinoshita A. Whelan C.M. Smith C.J. Berezovska O. Hyman B.T. J. Neurochem. 2002; 82: 839-847Crossref PubMed Scopus (83) Google Scholar). To readily test any competitive effects of NΔEC on downstream activation by APP-Gal4, we cotransfected HEK cells with APP-Gal4, pG5E1B-luc, Fe65, and NΔEC or an empty vector. We observed an expected increase in the ability of APP-Gal4 to stimulate transcription when Fe65 is cotransfected, as compared with an empty vector control (Fig. 2). In addition, we found that NΔEC attenuated this phenomenon, decreasing the downstream signaling effects of the Fe65-stabilized AICD generated from the artificially expressed Gal4-dependent luciferase reporter plasmid. This finding is consistent with the reduction in Aβ40 and Aβ42 levels observed after transfection with NΔEC. To confirm that Notch1 signaling was indeed activated by the transfection with the truncated forms of Notch, we measured activation of a Notch downstream transcription factor, C-promoter binding factor 1 (CBF1), in the cell lysates. We cotransfected N2a cells with a CBF1 luciferase reporter construct (18Berezovska O. Jack C. McLean P. Aster J.C. Hicks C. Xia W. Wolfe M.S. Kimberly W.T. Weinmaster G. Selkoe D.J. Hyman B.T. J. Neurochem. 2000; 75: 583-593Crossref PubMed Scopus (114) Google Scholar, 19Hsieh J.J. Henkel T. Salmon P. Robey E. Peterson M.G. Hayward S.D. Mol. Cell. Biol. 1996; 16: 952-959Crossref PubMed Scopus (398) Google Scholar) and β-galactosidase as an internal control for transfection efficiency, and we measured luminescence 24 h after transfection. As expected, we found that NICD and NΔEC led to a robust increase in CBF1 luciferase activity in comparison to an empty vector (Fig. 3A). Next, we examined whether the decrease in Aβ secretion after transfection with truncated Notch1 constructs was due to any effects of the transfection on APP expression or processing. We performed Western blot analysis of cell lysates transfected with empty vector, NΔEC, or NICD for full-length APP expression and APP CTFs generation. Although after quantification we did not detect any significant change in the expression of any of these fragments, we observed a small non-significant increase in the expression of the APP CTFs after transfection with NΔEC (110.3% compared with an empty vector, Fig. 3B). Truncated Notch1 Constructs Induced Down-regulation of the Presenilin 1 Gene—We tested the hypothesis that the unexpected effects on Aβ secretion produced by NICD, which does not require PS1-dependent cleavage, were associated with changes in PS1 expression. RNA was extracted from N2a cells transfected with NΔEC, NICD, or an empty vector, and the expression of endogenous PS1 gene and GAPDH as a control gene were analyzed by using quantitative real-time PCR. We found that NICD and, to a lesser extent, NΔEC down-regulated the expression of the endogenous PS1 gene (Table II). Transfection with NICD and NΔEC was associated with an ∼40 and ∼20% reduction in PS1 expression, respectively. To investigate whether other γ-secretase substrates also have an effect on gene expression, we analyzed PS1 expression in N2a cells transfected with another direct substrate, C99. However, we did not observe a significant change compared with an empty vector.Table IITransfection with NICD downregulates endogenous PS1 gene expressionConditionChange in expression (mean ± SD)p valueEmpty vector (N = 5)100-NΔEC (n = 5)83 ± 18nsans, not significant.NICD (n = 5)58 ± 13<0.01C99 (n = 3)131 ± 35nsa ns, not significant. Open table in a new tab Finally, we examined the expression of endogenous PS1 at the protein level after transfection with Notch1 constructs. Western blot analysis of the cell lysates for endogenous PS1 expression showed that both NΔEC and NICD were associated with a reduction in PS1 CTF expression compared with transfection with an empty vector (Fig. 4, A and B). In accordance with the quantitative PCR results, we failed to observe a change in PS1 protein expression in cells transfected with C99. We showed previously that activation of endogenous Notch in primary neurons with Delta ligand caused a dose-dependent decrease in total Aβ secretion (12Berezovska O. Jack C. Deng A. Gastineau N. Rebeck G.W. Hyman B.T. J. Biol. Chem. 2001; 276: 30018-30023Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Furthermore, the opposite experiment confirmed that overexpression of APP in neuronal cultures derived from APP695Sw-overexpressing mice led to a reduction in Notch signaling assessed by a CBF1 luciferase transcription assay and by analysis of Notch C-terminal nuclear translocation in vitro and in vivo (12Berezovska O. Jack C. Deng A. Gastineau N. Rebeck G.W. Hyman B.T. J. Biol. Chem. 2001; 276: 30018-30023Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, there are apparent differences in the cleavage site and sequence specificity between APP and Notch, which suggests the possibility of different cleaving enzymes (23Sastre 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 (436) Google Scholar, 24Yu C. Kim S.H. Ikeuchi T. Xu H. Gasparini L. Wang R. Sisodia S.S. J. Biol. Chem. 2001; 276: 43756-43760Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Kimberly et al. (25Kimberly W.T. Esler W.P. Ye W. Ostaszewski B.L. Gao J. Diehl T. Selkoe D.J. Wolfe M.S. Biochemistry. 2003; 42: 137-144Crossref PubMed Scopus (107) Google Scholar), using an in vitro γ-secretase activity assay that cleaves both Notch- and APP-based substrates, showed that these substrates prevented each other's cleavage, which strongly suggests that the same γ-secretase complex is responsible for the processing of both substrates. The existence of multiple substrates that are cleaved by the same γ-secretase raises the possibility of competition among them for the enzymatic activity. However, there are conflicting reports regarding this issue. For instance, Chen et al. (13Chen F. Yu G. Arawaka S. Nishimura M. Kawarai T. Yu H. Tandon A. Supala A. Song Y.Q. Rogaeva E. Milman P. Sato C. Yu C. Janus C. Lee J. Song L. Zhang L. Fraser P.E. St George-Hyslop P.H. Nat. Cell. Bio. 2001; 3: 751-754Crossref PubMed Scopus (112) Google Scholar) did not observe competition between Notch and APP for γ-secretase in HEK cells transfected with NΔEC. To examine this issue in greater depth and to extend our previous studies, we analyzed the effect of the immediate γ-secretase substrates (NΔEC and C99) on Aβ production and Notch signaling. We found that transfection of cells with direct γ-secretase substrates led to a decrease in the formation of the γ-cleaved products from the alternative substrate. Transfection with C99, the direct substrate of γ-secretase, induced a marked reduction in Notch signaling, assessed by a CBF1 luciferase transcription assay. On the other hand, transfection of cells with NΔEC, a truncated form of Notch that requires a γ-secretase cleavage, induced a significant decrease in Aβ production measured with a sensitive and specific sandwich ELISA and in APP-Gal4 transcriptional activation. The effects on Aβ were not due to a change in APP expression, although a small nonsignificant increase in APP CTFs was seen after transfection with NΔEC. These results confirm and extend the idea that, under several different experimental conditions in intact cells, substrates compete with each other for γ-secretase. However, we also unexpectedly observed that transfection with NICD, the active signaling portion of the Notch1 molecule that does not require γ-cleavage, also down-regulated Aβ production. This decrease was consistent and associated with an ∼40% down-regulation in endogenous PS1 gene expression detectable at both the transcriptional and the protein level. However, we cannot readily demonstrate whether the knock-down of the PS1 gene is responsible for the decrease in Aβ seen in our experiments. The effects of knocking-down presenilin 1 have been evaluated by obtaining lines heterozygous for PS1 (±) (26De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1575) Google Scholar, 27Palacino J.J. Berechid B.E. Alexander P. Eckman C. Younkin S. Nye J.S. Wolozin B. J. Biol. Chem. 2000; 275: 215-222Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Naruse S. Thinakaran G. Luo J.J. Kusiak J.W. Tomita T. Iwatsubo T. Qian X. Ginty D.D. Price D.L. Borchelt D.R. Wong P.C. Sisodia S.S. Neuron. 1998; 21: 1213-1221Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), by chronic pharmacological treatment with γ-secretase inhibitors (29Beher D. Wrigley J.D. Nadin A. Evin G. Masters C.L. Harrison T. Castro J.L. Shearman M.S. J. Biol. Chem. 2001; 276: 45394-45402Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), or by RNA interference techniques (30Refolo L.M. Eckman C. Prada C.M. Yager D. Sambamurti K. Mehta N. Hardy J. Younkin S.G. J. Neurochem. 1999; 73: 2383-2388Crossref PubMed Scopus (47) Google Scholar, 31Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (797) Google Scholar). Mice with total absence of PS1 (PS1—/—) die late in embryogenesis and exhibit defects in axial skeleton and cerebral hemorrhages (32Shen J. Bronson R.T. Chen D.F. Xia W. Selkoe D.J. Tonegawa S. Cell. 1997; 89: 629-639Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar). The complete absence of PS1 in neuronal cultures derived from PS1—/— mouse embryos inhibits the normal cleavage of APP, compromises the maturation of a tyrosine kinase receptor, and produces a dramatic drop in Aβ production (26De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1575) Google Scholar, 27Palacino J.J. Berechid B.E. Alexander P. Eckman C. Younkin S. Nye J.S. Wolozin B. J. Biol. Chem. 2000; 275: 215-222Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Naruse S. Thinakaran G. Luo J.J. Kusiak J.W. Tomita T. Iwatsubo T. Qian X. Ginty D.D. Price D.L. Borchelt D.R. Wong P.C. Sisodia S.S. Neuron. 1998; 21: 1213-1221Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). On the other hand, mice heterozygous for PS1 present a normal phenotype (32Shen J. Bronson R.T. Chen D.F. Xia W. Selkoe D.J. Tonegawa S. Cell. 1997; 89: 629-639Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar, 33Wong P.C. Zheng H. Chen H. Becher M.W. Sirinathsinghji D.J. Trumbauer M.E. Chen H.Y. Price D.L. van der Ploeg L.H. Sisodia S.S. Nature. 1997; 387: 288-292Crossref PubMed Scopus (648) Google Scholar), and the effects on APP processing in cell lines derived from these heterozygous mice embryos seem to be weak (26De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1575) Google Scholar). However, in an in vitro Aβ generation assay, knock-down of PS1 using RNA interference led to an almost complete abolition of Aβ production (31Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (797) Google Scholar). Although the effects of knocking down PS1 remain uncertain, our results suggest that the Notch pathway exerts some control on PS1 gene regulation that may ultimately affect APP processing. It will be interesting to learn if NICD also regulates other components of the γ-secretase complex. These data suggest that two types of phenomenon coexist in the regulation of γ-secretase, competition between substrates and downstream effects on gene regulation. These results depict a novel and more complex feedback relationship between γ-secretase and its substrates.