Multiple Mechanisms Underlie Neurotoxicity by Different Types of Alzheimer's Disease Mutations of Amyloid Precursor Protein
2000; Elsevier BV; Volume: 275; Issue: 44 Linguagem: Inglês
10.1074/jbc.m005332200
ISSN1083-351X
AutoresYuichi Hashimoto, Takako Niikura, Yuko Ito, Ikuo Nishimoto,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoWe examined a neuronal cell system in which single-cell expression of either familial Alzheimer's disease (FAD) gene V642I-APP or K595N/M596L-APP (NL-APP) in an inducible plasmid was controlled without affecting transfection efficiency. This system revealed that (i) low expression of both mutants exerted toxicity sensitive to both Ac-DEVD-CHO (DEVD) and glutathione ethyl ester (GEE), whereas wild-type APP (wtAPP) only at higher expression levels caused GEE/DEVD-resistant death to lesser degrees; (ii) toxicity by the V642I mutation was entirely GEE/DEVD sensitive; and (iii) toxicity by higher expression of NL-APP was GEE/DEVD resistant. The GEE/DEVD-sensitive death was sensitive to pertussis toxin and was due to Go-interacting His657-Lys676 domain. The GEE/DEVD-resistant death was due to C-terminal Met677-Asn695. APP mutants lacking either domain unraveled elaborate intracellular cross-talk between these domains. E618Q-APP, responsible for non-AD type of a human disease, only exerted GEE/DEVD-resistant death at higher expression. Therefore, (i) different FAD mutations in APP cause neuronal cell death through different cytoplasmic domains via different sets of mechanisms; (ii) expression levels of FAD genes are critical in activating specific death mechanisms; and (iii) toxicity by low expression of both mutants most likely reflects the pathogenetic mechanism of FAD. We examined a neuronal cell system in which single-cell expression of either familial Alzheimer's disease (FAD) gene V642I-APP or K595N/M596L-APP (NL-APP) in an inducible plasmid was controlled without affecting transfection efficiency. This system revealed that (i) low expression of both mutants exerted toxicity sensitive to both Ac-DEVD-CHO (DEVD) and glutathione ethyl ester (GEE), whereas wild-type APP (wtAPP) only at higher expression levels caused GEE/DEVD-resistant death to lesser degrees; (ii) toxicity by the V642I mutation was entirely GEE/DEVD sensitive; and (iii) toxicity by higher expression of NL-APP was GEE/DEVD resistant. The GEE/DEVD-sensitive death was sensitive to pertussis toxin and was due to Go-interacting His657-Lys676 domain. The GEE/DEVD-resistant death was due to C-terminal Met677-Asn695. APP mutants lacking either domain unraveled elaborate intracellular cross-talk between these domains. E618Q-APP, responsible for non-AD type of a human disease, only exerted GEE/DEVD-resistant death at higher expression. Therefore, (i) different FAD mutations in APP cause neuronal cell death through different cytoplasmic domains via different sets of mechanisms; (ii) expression levels of FAD genes are critical in activating specific death mechanisms; and (iii) toxicity by low expression of both mutants most likely reflects the pathogenetic mechanism of FAD. Alzheimer's disease familial Alzheimer's disease amyloid β-protein pertussis toxin APP695 with K595N/M596L mutations wild-type APP glutathione ethyl ester acetyl-l-aspartyl-l-glutaminyl-l-valyl-l-aspart-1-al ecdysone retinoid X receptor F11 cells stably overexpressing both EcR and RXR fetal bovine serum the domain His657-Lys676 the domain Met677-Asn695 APP695 lacking His657-Lys676 APP695 lacking Met677-Asn695 hereditary cerebral hemorrhage with angiopathy Dutch type Alzheimer's disease (AD),1 the most prevalent neurodegenerative disease, is characterized by neuronal loss and extracellular senile plaques, whose major constituent is Aβ amyloid, cleaved off from the transmembrane precursor APP (1Kang J. Lemaire H-G. Unterback A. Salbaum J.M. Masters C.L. Grezeschik K.H. Multhaup G. Beyreuther K. Måller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3957) Google Scholar). Genetic studies of early-onset FAD have demonstrated that structural alterations in APP cause AD. There are at least two different types of mutations reported in APP as established causes for FAD: Ile/Phe/Gly mutations at Val642 or the Asn/Leu mutation at Lys595/Met596 in APP695 (the numbering follows Kang et al. (1Kang J. Lemaire H-G. Unterback A. Salbaum J.M. Masters C.L. Grezeschik K.H. Multhaup G. Beyreuther K. Måller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3957) Google Scholar)). Despite the fact that neuronal death is a central abnormality in AD, exactly how these FAD-linked mutants of APP cause neuronal death has been little understood. Multiple groups (2Yamatsuji T. Okamoto T. Takeda S. Fukumoto H. Iwatsubo T. Suzuki N. Asami-Odaka A. Ireland S. Kinane T.B. Nishimoto I. Science. 1996; 272: 1349-1352Crossref PubMed Scopus (217) Google Scholar, 3Yamatsuji T. Okamoto T. Takeda S. Murayama Y. Tanaka N. Nishimoto I. EMBO J. 1996; 15: 498-509Crossref PubMed Scopus (109) Google Scholar, 4Wolozin B. Iwasaki K. Vito P. Ganjei J.K. Lacaná E. Sunderland T. Zhao B. Kusiak J.W. Wasco W. D'Adamio L. Science. 1996; 274: 1710-1713Crossref PubMed Scopus (392) Google Scholar, 5Zhao B. Chrest F.J. Horton Jr., W.E. Sisodia S.S. Kusiak J.W. J. Neurosci. Res. 1997; 47: 253-263Crossref PubMed Scopus (64) Google Scholar, 6Luo J.J. Wallace W. Riccioni T. Ingram D.K. Roth G.S. Kusiak J.W. J. Neurosci. Res. 1999; 55: 629-642Crossref PubMed Scopus (41) Google Scholar) have so far found that FAD-associated Val642 mutants of APP induce death through intracellular signaling cascades in neuronal cells. We (2Yamatsuji T. Okamoto T. Takeda S. Fukumoto H. Iwatsubo T. Suzuki N. Asami-Odaka A. Ireland S. Kinane T.B. Nishimoto I. Science. 1996; 272: 1349-1352Crossref PubMed Scopus (217) Google Scholar, 7Giambarella U. Yamatsuji T. Okamoto T. Matsui T. Ikezu T. Murayama Y. Levine M.A. Katz A. Gautam N. Nishimoto I. EMBO J. 1997; 16: 4897-4907Crossref PubMed Scopus (82) Google Scholar) also found that neuronal cell death by Val642-type FAD mutants of APP may be a potentially controllable process mediated by PTX-sensitive G protein Go and its βγ subunit. Wolozinet al. (4Wolozin B. Iwasaki K. Vito P. Ganjei J.K. Lacaná E. Sunderland T. Zhao B. Kusiak J.W. Wasco W. D'Adamio L. Science. 1996; 274: 1710-1713Crossref PubMed Scopus (392) Google Scholar) verified that the Val642-type mutant of APP induces death in PC12 cells in a PTX-sensitive manner, with the discovery that FAD-associated N141I presenilin-2 also causes PTX-sensitive death. Presenilin-2 is a member gene of the PS family, whose mutation is responsible for certain forms of FAD. One potential mechanism underlying the neurotoxicity of these FAD mutants is that neuronal death occurs by deposition of Aβ, particularly Aβ1–42/43, a longer version of Aβ polypeptides. Indeed, the deposition of AβX−42/43 is the earliest abnormality observed in AD brains (8Iwatsubo T. Odaka A. Suzuki N. Mizusawa H. Nukina N. Ihara Y. Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1568) Google Scholar); Aβ polypeptides, including Aβ1–42, kill neuronal cells in vitro (9Loo D.T. Copani A. Pike C.J. Whittemore E.R. Walencewicz A.J. Cotman C.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7951-7955Crossref PubMed Scopus (1045) Google Scholar, 10Gschwind M. Huber G. J. Neurochem. 1995; 65: 292-300Crossref PubMed Scopus (198) Google Scholar); and cellular secretion of AβX−42/43 increases by expression of FAD mutants of APP (2Yamatsuji T. Okamoto T. Takeda S. Fukumoto H. Iwatsubo T. Suzuki N. Asami-Odaka A. Ireland S. Kinane T.B. Nishimoto I. Science. 1996; 272: 1349-1352Crossref PubMed Scopus (217) Google Scholar, 11Citron M. Oltersdorf T. Haass C. McConlogue L. Hung A.Y. Seubert P. Vigo-Pelfrey C. Lieberburg I. Selkoe D.J. Nature. 1992; 360: 672-674Crossref PubMed Scopus (1537) Google Scholar, 12Suzuki N. Cheung T.T. Cai X-D. Odaka A. Otvos L. Eckman C. Golde T.E. Younkin S.G. Science. 1994; 264: 1336-1340Crossref PubMed Scopus (1358) Google Scholar, 13Cai X-D. Golde T.E. Younkin S.G. Science. 1993; 259: 514-516Crossref PubMed Scopus (835) Google Scholar). It has been reported by multiple research groups that intracellular signaling mechanisms, including oxidative stress-relevant pathways (14Mark R.J. Keller J.N. Kruman I. Mattson M.P. Brain Res. 1997; 756: 205-214Crossref PubMed Scopus (149) Google Scholar, 15Pike C.J. Ramezan-Arab N. Cotman C.W. J. Neurochem. 1997; 69: 1601-1611Crossref PubMed Scopus (124) Google Scholar, 16Miranda S. Opazo C. Larrondo L.F. Munoz F.J. Ruiz F. Leighton F. Inestrosa N.C. Prog. Neurobiol. 2000; 62: 633-648Crossref PubMed Scopus (345) Google Scholar), calpain-activated cdk5 pathways (17Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Nature. 2000; 405: 360-364Crossref PubMed Scopus (911) Google Scholar), and caspase-dependent pathways (18Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2966) Google Scholar), mediate Aβ amyloid-induced neurotoxicity. Taken together, these observations suggest that countermeasures against neuronal cell death in these types of FAD and even sporadic AD might be feasible even after Aβ deposition if the countermeasures could suppress intracellular toxicity signals inside the neurons expressing the FAD genes or in the neurons exposed to Aβ-related insults. Therefore, it is important to understand fully the entire body of intracellular death signals generated by the expression of AD-causative genes. The aforementioned studies also suggest that FAD mutants of both APP and presenilin-2 may cause neuronal cell death through a common mechanism. On the other hand, it has not been determined whether the function of K595N/M596L-APP (NL-APP), another established cause of FAD, is relevant to neuronal cell death. In contrast, virus-mediated overexpression of wtAPP causes significant death in neuronal cells (19Nishimura I. Uetsuki T. Dani S.U. Ohsawa Y. Saito I. Okamura H. Uchiyama Y. Yoshikawa K. J. Neurosci. 1998; 18: 2387-2398Crossref PubMed Google Scholar, 20Bursztajn S. DeSouza R. McPhie D.L. Berman S.A. Shioi J. Robakis N.K. Neve R.L. J. Neurosci. 1998; 18: 9790-9799Crossref PubMed Google Scholar). The present study was thus conducted to investigate whether expression of NL-APP causes death in neuronal cells, like V642I-APP, and if so, whether both FAD mutants kill neuronal cells through the same mechanism, and in what relationship between the mutation-specific mechanisms and the toxicity of wtAPP. Here we report unexpectedly complicated potential of FAD mutants to cause neurotoxicity through different cytoplasmic domains via different sets of distinct mechanisms. wtAPP gene, the full-length APP695 cDNA (2Yamatsuji T. Okamoto T. Takeda S. Fukumoto H. Iwatsubo T. Suzuki N. Asami-Odaka A. Ireland S. Kinane T.B. Nishimoto I. Science. 1996; 272: 1349-1352Crossref PubMed Scopus (217) Google Scholar), was subcloned into pIND plasmid (Invitrogen). V642I-APP cDNA was described previously (2Yamatsuji T. Okamoto T. Takeda S. Fukumoto H. Iwatsubo T. Suzuki N. Asami-Odaka A. Ireland S. Kinane T.B. Nishimoto I. Science. 1996; 272: 1349-1352Crossref PubMed Scopus (217) Google Scholar), and E618Q-APP was constructed by a site-directed mutagenesis, using a QuikChange Site-directed Mutagenesis Kit (Stratagene), with confirmation of generated mutations by sequencing. The sense and antisense primers used for E618Q construction were 5′-CTGGTGTTCTTTGCTCAAGATGTGGGTTCGAACAAAGGC-3′ and 5′-GCCTTTGTTCGAACCCACATCTTGAGCAAAGAACACCAG-3′, respectively. NL-APP cDNA was provided by Dr. T. Okamoto (RIKEN, Wako, Japan). These mutant APP cDNAs were subcloned to pIND with sequence confirmation. The pIND-encoded wtAPP, V642I-APP, or NL-APP was named as pIND-wtAPP, pIND-V642I-APP, or pIND-NL-APP, respectively. EGFP cDNA was purchased from CLONTECH (pEGFP-N1), and was also subcloned to pIND (pIND-EGFP). Glutathione ethyl ester (GEE) and PTX were from Sigma and Calbiochem-Novabiochem, respectively, and Ac-DEVD-CHO was from Peptide Institute Inc. Ponasterone (Invitrogen) was employed as EcD. Vitamin E and Aβ1–43 were from Wako Pure Chemicals and BACHEM, respectively. F11 cells were grown in Ham's F-12 plus 18% FBS and antibiotics. F11 cells are the hybrid of a rat embryonic day 13 primary cultured neuron with a mouse neuroblastoma NTG18. These cells are one of the best models for primary cultured neurons, exhibiting without differentiation factor treatment, a number of characteristics for primary neurons, including generation of action potentials (21Platika D. Boulos M.H. Baizer L. Fishman M.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3499-3503Crossref PubMed Scopus (179) Google Scholar). F11 cells (F11/EcR cells) overexpressing both EcR and RXR were established using the co-expression vector pVgRXR and Zeocin selection (Invitrogen). For transient transfection of the pIND plasmids, F11/EcR cells were seeded at 7 × 104 cells/well in a 6-well plate and cultured in Ham's F-12 plus 18% FBS for 12–16 h and transfected with EcD-inducible pIND plasmids (1 μg of pIND plasmids, 2 μl of LipofectAMINE, and 4 μl of plus reagent) in the absence of serum for 3 h. After subsequent incubation with Ham's F-12 plus 18% FBS for 12–16 h, cells were cultured with or without inhibitors in Ham's F-12 plus 10% FBS for 2 h, and EcD was then added to the media. Cell mortality was measured by trypan blue exclusion assay at 72 h after the onset of EcD treatment. Transfection efficiency was assessed with pEGFP-N1 by fluorescence microscopy. F11/EcR cells were transfected with this plasmid (1 μg of plasmid, 2 μl of LipofectAMINE, and 4 μl of plus reagent) in the absence of serum for 3 h. After subsequent incubation with Ham's F-12 plus 10% FBS for 48 h, transfection efficiency was assessed by: (i) dividing the number of green fluorescent cells by the total cell number in the same randomly chosen fields in each transfection; and (ii) calculating the mean ± S. D. of these ratios for each transfection. The mean ± S.E. of independent transfections was then calculated. For the Aβ experiment, F11/EcR cells were seeded at 7 × 104 cells/well in a 6-well plate or a 35 mm-dish and cultured in Ham's F-12 plus 18% FBS for 12–16 h. Cells were transfected with EcD-inducible pIND plasmids (1 μg of pIND plasmids, 2 μl of LipofectAMINE, and 4 μl of plus reagent) in the absence of serum for 3 h, then reseeded at 1.4 × 104cells/well in a 24-well plate, and cultured in Ham's F-12 plus 18% FBS for 18 h. Cells were then cultured with or without 25 μm Aβ1–43 in Ham's F-12 plus 10% FBS. Two hours after the onset of Aβ treatment, various concentrations of EcD or equivalent volumes of EtOH were added to the culture media and cells were cultured for an additional 72 h. Cell mortality was then measured by trypan blue exclusion assay. Trypan blue exclusion assay was performed as follows. At the termination of experiments, cells were suspended by pipetting gently, and 50 μl of 0.4% trypan blue solution (Sigma) was mixed with 200 μl of the cell suspension (final concentration 0.08%) at room temperature. Stained cells were counted within 3 min after the mixture with trypan blue solution. The mortality of cells was then determined as a percentage of trypan blue-stained cells in total cells. The cell mortality assessed by this method thus represents the population of dead cells in total cells, including both adhesive and floating cells at the termination of experiments. The basal death rates with or without pIND vector transfection with or without EcD treatment indicated the actual fraction of dead cells, but not artificial cell death occurred after detaching cells, as in situ staining of trypan blue-positive cells indicated the presence of similar fractions of dead cells. In all experiments shown in each figure presented in this study, we performed the experiments examining cell mortality (i) in the presence or absence of 40 μm EcD without transfection and (ii) in the presence or absence of 40 μmEcD with empty pIND transfection, both of which were constantly as low as the basal cell mortality in the absence of EcD with pIND-APP construct transfection (data not shown). Immunoblot analysis of expressed APP constructs and endogenous tubulin was performed as follows. Cell lysates (20 μg/lane) were submitted to SDS-polyacrylamide gel electrophoresis and separated proteins were transferred onto polyvinylidene difluoride sheets. After blocking, the blots were probed with the primary antibody (2.5 μg/ml anti-APP monoclonal antibody 22C11 (Roche Diagnostics) or 1/3000 dilution of anti-αtubulin monoclonal antibody TU-02 (Santa Cruz Biotechnology)) and 1/5000 dilution of the secondary antibody horseradish peroxidase-conjugated anti-mouse IgG antibody (Bio-Rad), followed by visualization of the immunoreactive bands by ECL (Amersham Pharmacia Biotech). The densities of the 120-kDa APP immunoreactive band and the 50-kDa tubulin band were, respectively, measured by densitometrical analysis. All of the experiments described in this study were repeated at least three times with independent transfections and treatments, each of which yielded essentially the same result. Statistical analysis was performed with Student's t test. We transfected F11/EcR cells with pIND-encoded mutant APP cDNA driven by an integrated EcD-responsive promoter (22No D. Yao T.P. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3346-3351Crossref PubMed Scopus (755) Google Scholar); about 1 day after transfection, we treated cells with EcD. In this context, the cDNA-encoding protein should express in a single cell in an EcD dose-dependent manner, uninfluenced by the variation in transfection efficiency. In addition, the transfection efficiency in F11/EcR cells with the present lipofection method was appreciably high and stable. We performed three independent transfections of F11/EcR cells with (constitutively active promoter-driven) pEGFP-N1 plasmid, which revealed the transfection efficiency to be 68.0 ± 3.1% as the mean ± S.E. (Fig.1 A). As expected, when F11/EcR cells were transfected with pIND-EGFP and treated with increasing concentrations of EcD, the expression of EGFP in a single cell was unidirectionally augmented, as the concentration of EcD became higher (Fig. 1 B), suggesting that a single-cell expression of pIND-encoded cDNA can be controlled by altering the concentrations of EcD in this system. We also confirmed that the expression of EGFP by ≤40 μm EcD was not affected by 100 μmAc-DEVD-CHO, 1 mm GEE, or 1 μg/ml PTX in cells transfected with pIND-EGFP (data not shown). We next examined whether wtAPP, V642I-APP, or NL-APP was expressed in proportion to the concentration of treated EcD (Fig. 1 C). Cells were transfected with pIND-encoded APP genes and treated with EcD. The result revealed that expression of either V642I-APP or NL-APP was not linearly augmented by EcD, and reached saturation by >10 μm EcD. In contrast, wtAPP was expressed proportionally to the EcD concentration under the same condition. From the literature (2Yamatsuji T. Okamoto T. Takeda S. Fukumoto H. Iwatsubo T. Suzuki N. Asami-Odaka A. Ireland S. Kinane T.B. Nishimoto I. Science. 1996; 272: 1349-1352Crossref PubMed Scopus (217) Google Scholar, 3Yamatsuji T. Okamoto T. Takeda S. Murayama Y. Tanaka N. Nishimoto I. EMBO J. 1996; 15: 498-509Crossref PubMed Scopus (109) Google Scholar, 4Wolozin B. Iwasaki K. Vito P. Ganjei J.K. Lacaná E. Sunderland T. Zhao B. Kusiak J.W. Wasco W. D'Adamio L. Science. 1996; 274: 1710-1713Crossref PubMed Scopus (392) Google Scholar, 5Zhao B. Chrest F.J. Horton Jr., W.E. Sisodia S.S. Kusiak J.W. J. Neurosci. Res. 1997; 47: 253-263Crossref PubMed Scopus (64) Google Scholar, 6Luo J.J. Wallace W. Riccioni T. Ingram D.K. Roth G.S. Kusiak J.W. J. Neurosci. Res. 1999; 55: 629-642Crossref PubMed Scopus (41) Google Scholar, 23Gervais F.G. Xu D. Robertson G.S. Vaillancourt J.P. Zhu Y. Huang J. LeBlanc A. Smith D. Rigby M. Shearman M.S. Clarke E.E. Zheng H. Van Der Ploeg L.H. Ruffolo S.C. Thornberry N.A. Xanthoudakis S. Zamboni R.J. Roy S. Nicholson D.W. Cell. 1999; 97: 395-406Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar, 24Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2812) Google Scholar, 25Okamoto T. Takeda S. Giambarella U. Matsuura Y. Katada T. Nishimoto I. EMBO J. 1996; 15: 3769-3777Crossref PubMed Scopus (59) Google Scholar), we reasoned that in this system, V642I-APP and NL-APP may be induced by EcD similarly to the induction of wtAPP, but degraded through mechanisms involving caspase activation by the FAD mutants themselves, resulting in certain balanced expression. In accord with this idea, in the presence of 100 μm Ac-DEVD-CHO, an established cell-permeable inhibitor of caspases, expression of either FAD mutant became linear in relation to EcD concentrations (Fig. 1 C, right). Under the same conditions, expression of wtAPP was not affected by Ac-DEVD-CHO. These data suggest that the two FAD genes in pIND, like wtAPP in the same vector, were induced in proportion to the EcD concentration, and that both FAD mutants, but not wtAPP, were degraded by activating DEVD-sensitive mechanisms. The transfection of either pIND-encoded FAD gene in the presence of Ac-DEVD-CHO or in the transfection of pIND-wtAPP in its presence or absence, treatment with 10, 20, and 40 μm EcD resulted in the expression of APP immunoreactivity ∼2.5-, ∼4-, and ∼7-fold, respectively, of the basal expression, indicating that 10, 20, and 40 μm EcD caused ∼1.5-, ∼3-, and ∼6-fold expression of the transfected APP constructs, respectively. In F11/EcR cells, we examined whether and how robustly FAD mutants in pIND (pIND-V642I-APP and pIND-NL-APP) cause cell death by various concentrations of EcD. Fig.2 A indicates that in cells transfected with either pIND-V642I-APP or pIND-NL-APP, EcD augmented cell mortality dose dependently. In contrast, the vehicle ethanol caused no increase in cell mortality in either case. Treatment of non-transfected F11/EcR cells or vector-transfected F11/EcR cells with or without EcD resulted in low cell mortality around 10% for 72 h, the basal death rate of these cells (Fig. 2 A, upper panel). Considering that transfection efficiency was 60–70%, it followed that induction of either V642I-APP or NL-APP by ≥10 μm EcD caused death in most of the transfected cells after 72 h. Expression of wtAPP resulted in a different dose-response curve for death (Fig. 2 B, red closed circles). EcD caused little death in pIND-wtAPP-transfected cells at <20 μm, and stimulated death dose-dependently only at ≥20 μm, and to lesser degrees than the toxicity by the same concentrations of EcD in cells transfected with either pIND-FAD mutant. These data indicate that expression of V642I-APP or NL-APP as low as endogenous APP effectively killed neuronal cells and that wtAPP was toxic at only higher levels of expression. We next investigated whether Ac-DEVD-CHO affects cell death by either FAD gene. EcD-induced death of pIND-V642I-APP-transfected cells was greatly suppressed by 100 μm Ac-DEVD-CHO (Fig.2 B, blue closed circles). Surprisingly, the dose-response curve of V642I-APP-induced death in the presence of 100 μm Ac-DEVD-CHO was virtually identical to the dose-response curve of wtAPP-induced death. This was also the case with GEE, a cell-permeable antioxidant (Fig. 2 C, green closed circles). The dose-response curve of V642I-APP-induced death in the presence of 1 mm GEE was again virtually identical to its dose-response curve in the presence of 100 μmAc-DEVD-CHO and the curve of wtAPP-induced death. The EcD dependence of pIND-V642I-APP expression in the presence of 100 μmAc-DEVD-CHO was linear, equivalent to that in the presence of 1 mm GEE (data not shown), and virtually identical to that of pIND-wtAPP expression (Fig. 1 C, right). These data indicated that V642I turned on a specific death mechanism sensitive to both Ac-DEVD-CHO and GEE, and also suggested that death by wtAPP was resistant to both. In fact, toxicity by wtAPP was totally resistant to 100 μm Ac-DEVD-CHO (Fig. 2 D, blue closed circles) and 1 mm GEE (Fig. 2 D, green closed circles). Neither Ac-DEVD-CHO (Fig. 1 C) nor GEE (data not shown) affected the EcD-dependent expression of wtAPP. These data indicate that toxicity by wtAPP was through a mechanism completely different from the toxicity stimulated by the V642I mutation. Therefore, a novel possibility was raised that K595N/M596L might induce neuronal cell death through further different mechanisms. Both Ac-DEVD-CHO and GEE inhibited NL-APP-induced death, but not in the same curves as V642I-APP-induced death in the presence of either reagent (Fig.2 E). In the presence of 100 μm Ac-DEVD-CHO or 1 mm GEE, death by NL-APP occurred in mutually equivalent dose-response curves. The dose-response curve of NL-APP-induced death revealed that death by low expression of NL-APP was completely suppressed by Ac-DEVD-CHO and GEE, whereas death by higher induction of NL-APP was resistant to both reagents. EcD dependence of pIND-NL-APP expression was linear and equivalent to that of pIND-wtAPP in the presence of 100 μm Ac-DEVD-CHO (Fig. 1 C, right) or 1 mm GEE (data not shown). These data indicate that (i) low expression of both FAD mutants caused neuronal cell death GEE/DEVD sensitively, whereas wtAPP only at higher expression caused GEE/DEVD resistant death to lesser degrees; (ii) toxicity given by V642I was entirely GEE/DEVD-sensitive; and (iii) toxicity by higher induction of NL-APP was GEE/DEVD-resistant. To confirm that GEE acts on reactive oxygen species to block cell death by low induction of both FAD genes, we examined the effects of vitamin E and Aβ. Vitamin E acts as an antioxidant at 10–100 μm, and Aβ induces or enhances oxidative stress in neuronal cells (26Harris M.E. Hensley K. Butterfield D.A. Leedle R.A. Carney J.M. Exp. Neurol. 1995; 131: 193-202Crossref PubMed Scopus (330) Google Scholar, 27Manelli A.M. Puttfarcken P.S. Brain Res. Bull. 1995; 38: 569-576Crossref PubMed Scopus (65) Google Scholar, 28Fu W. Luo H. Parthasarathy S. Mattson M.P. Neurobiol. Dis. 1998; 5: 229-243Crossref PubMed Scopus (157) Google Scholar). As shown in Fig.3 A, 100 μmvitamin E suppressed cell death by V642I-APP and NL-APP in dose-response curves virtually identical to those of V642I-APP-and NL-APP-induced death in the presence of 1 mm GEE, respectively. These data indicate that vitamin E precisely mimics the inhibitory effect of GEE. In contrast, Aβ potentiated the toxic actions of both V642I-APP and NL-APP. In cells with no transfection or with empty-pIND transfection, 25 μm Aβ1–43 had no effect on cell death (Table I). However, in the presence of 25 μm Aβ1–43, both V642I-APP and NL-APP exerted cytotoxicity at lower expression than that in the absence of Aβ, and caused death at 5–10% higher rates (Fig.3 B). These data suggest that Aβ enhances oxidative stress induced by both APP mutants, consistent with the study of Lockhartet al. (29Lockhart B.P. Benicourt C. Junien J.L. Privat A. J. Neurosci. Res. 1994; 39: 494-505Crossref PubMed Scopus (98) Google Scholar) reporting that Aβ peptide renders hippocampal neurons more sensitive to free radical attack without direct action on free radical generation.Table IToxic effect of Aβ amyloid on the basal death ratesNo transfectionpIND transfectionAbeta (−)Abeta (+)Abeta (−)Abeta (+)EcD (−)7.1 ± 1.3%9.2 ± 0.3%9.2 ± 0.7%8.0 ± 0.7%EcD (+)8.4 ± 1.4%8.1 ± 1.3%8.5 ± 0.6%8.9 ± 1.6%F11/EcR cells were transfected with (pIND transfection) or without (no transfection) empty pIND and treated with (Abeta (+)) or without (Abeta (−)) 25 μm Aβ1–43 with 40 μm EcD (EcD (+)) or an equivalent volume of EtOH (EcD (−)). Cell mortality was measured by trypan blue exclusion assay 72 h after the onset of EcD treatment. The values indicate mean ± S.D. of three independent experiments (in % dead cells of total cells). Open table in a new tab F11/EcR cells were transfected with (pIND transfection) or without (no transfection) empty pIND and treated with (Abeta (+)) or without (Abeta (−)) 25 μm Aβ1–43 with 40 μm EcD (EcD (+)) or an equivalent volume of EtOH (EcD (−)). Cell mortality was measured by trypan blue exclusion assay 72 h after the onset of EcD treatment. The values indicate mean ± S.D. of three independent experiments (in % dead cells of total cells). We next investigated the domains in APP responsible for each mechanism for death by V642I-APP, NL-APP, or wtAPP. A clue was that the His657-Lys676 domain (Domain 20) in APP interacts directly and specifically with the PTX-sensitive G protein Go in vitro (30Nishimoto I. Okamoto T. Matsuura Y. Okamoto T. Murayama Y. Ogata E. Nature. 1993; 362: 75-79Crossref PubMed Scopus (370) Google Scholar, 31Okamoto T. Takeda S. Murayama Y. Ogata E. Nishimoto I. J. Biol. Chem. 1995; 270: 4205-4208Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 32Brouillet E. Trembleau A. Galanaud D. Volovitch M. Bouillot C. Valenza C. Prochiantz A. Allinquant B. J. Neurosci. 1999; 19: 1717-1727Crossref PubMed Google Scholar). As shown in Fig.4 A, 1 μg/ml PTX completely suppressed death stimulated by the V642I mutation (but not completely by V642I-APP), indicating that the GEE/DEVD-sensitive mechanism for death by V642I-APP is entirely PTX-sensitive. This was also the case with NL-APP (Fig. 4 B). The dose-response curve of NL-APP-induced death indicated that (i) the GEE/DEVD-sensitive mechanism for death by low expression of NL-APP was totally sensitive to PTX; and (ii) the GEE/DEVD-resistant death mechanism by higher induction of NL-APP was resistant to PTX. In contrast, PTX did not affect the dose-response curve of wtAPP-induced death (Fig.2 C), indicating that (i) the observed PTX effects on death by FAD mutants were not artifacts; and (ii) wtAPP-induced death was PTX/GEE/DEVD-resistant. In the presence of 1 μg/ml PTX, EcD dependence of the expression of pIND-V642I-APP or pIND-NL-APP was similar to that of pIND-wtAPP (data not shown). We next examined the function of APP constructs lacking Domain 20: V642I-APPΔ20, NL-APPΔ20, or wtAPPΔ20 (V642I-APPΔ20 is V642
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