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• W2073539123 abstract "Familial Alzheimer's disease (FAD) presenilin 1 (PS1) mutations give enhanced calcium responses upon different stimuli, attenuated capacitative calcium entry, an increased sensitivity of cells to undergo apoptosis, and increased γ-secretase activity. We previously showed that the FAD mutation causing an exon 9 deletion in PS1 results in enhanced basal phospholipase C (PLC) activity (Cedazo-Minguez, A., Popescu, B. O., Ankarcrona, M., Nishimura, T., and Cowburn, R. F. (2002) J. Biol. Chem. 277, 36646-36655). To further elucidate the mechanisms by which PS1 interferes with PLC-calcium signaling, we studied the effect of two other FAD PS1 mutants (M146V and L250S) and two dominant negative PS1 mutants (D257A and D385N) on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular calcium concentrations ([Ca2+]i) in SH-SY5Y neuroblastoma cells. We found a significant increase in basal PI hydrolysis in PS1 M146V cells but not in PS1 L250S cells. Both PS1 M146V and PS1 L250S cells showed a significant increase in carbachol-induced [Ca2+]i as compared with nontransfected or wild type PS1 transfected cells. The elevated carbachol-induced [Ca2+]i signals were reversed by the PLC inhibitor neomycin, the ryanodine receptor antagonist dantrolene, the general aspartyl protease inhibitor pepstatin A, and the specific γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. The cells expressing either PS1 D257A or PS1 D385N had attenuated carbachol-stimulated PI hydrolysis and [Ca2+]i responses. In nontransfected or PS1 wild type transfected cells, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester and pepstatin A also attenuated both carbachol-stimulated PI hydrolysis and [Ca2+]i responses to levels found in PS1 D257A or PS1 D385N dominant negative cells. Our findings suggest that PS1 can regulate PLC activity and that this function is γ-secretase activity-dependent. Familial Alzheimer's disease (FAD) presenilin 1 (PS1) mutations give enhanced calcium responses upon different stimuli, attenuated capacitative calcium entry, an increased sensitivity of cells to undergo apoptosis, and increased γ-secretase activity. We previously showed that the FAD mutation causing an exon 9 deletion in PS1 results in enhanced basal phospholipase C (PLC) activity (Cedazo-Minguez, A., Popescu, B. O., Ankarcrona, M., Nishimura, T., and Cowburn, R. F. (2002) J. Biol. Chem. 277, 36646-36655). To further elucidate the mechanisms by which PS1 interferes with PLC-calcium signaling, we studied the effect of two other FAD PS1 mutants (M146V and L250S) and two dominant negative PS1 mutants (D257A and D385N) on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular calcium concentrations ([Ca2+]i) in SH-SY5Y neuroblastoma cells. We found a significant increase in basal PI hydrolysis in PS1 M146V cells but not in PS1 L250S cells. Both PS1 M146V and PS1 L250S cells showed a significant increase in carbachol-induced [Ca2+]i as compared with nontransfected or wild type PS1 transfected cells. The elevated carbachol-induced [Ca2+]i signals were reversed by the PLC inhibitor neomycin, the ryanodine receptor antagonist dantrolene, the general aspartyl protease inhibitor pepstatin A, and the specific γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. The cells expressing either PS1 D257A or PS1 D385N had attenuated carbachol-stimulated PI hydrolysis and [Ca2+]i responses. In nontransfected or PS1 wild type transfected cells, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester and pepstatin A also attenuated both carbachol-stimulated PI hydrolysis and [Ca2+]i responses to levels found in PS1 D257A or PS1 D385N dominant negative cells. Our findings suggest that PS1 can regulate PLC activity and that this function is γ-secretase activity-dependent. Mutations in genes encoding presenilins are responsible for the majority of early onset familial Alzheimer's disease (FAD) 1The abbreviations used are: FAD, familial Alzheimer's disease; AD, Alzheimer's disease; PS, presenilin; PI, phosphoinositides; PLC, phospholipase C; APP, β-amyloid precursor protein; Aβ, β-amyloid; AICD, APP intracellular domain; IP3, inositol 1,4,5-trisphosphate; ER, endoplasmic reticulum; DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester; AM, acetoxymethyl; PBS, phosphate-buffered saline; NT, nontransfected; WT, wild type; ANOVA, analysis of variance; ΔE9, exon 9 deleted. 1The abbreviations used are: FAD, familial Alzheimer's disease; AD, Alzheimer's disease; PS, presenilin; PI, phosphoinositides; PLC, phospholipase C; APP, β-amyloid precursor protein; Aβ, β-amyloid; AICD, APP intracellular domain; IP3, inositol 1,4,5-trisphosphate; ER, endoplasmic reticulum; DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester; AM, acetoxymethyl; PBS, phosphate-buffered saline; NT, nontransfected; WT, wild type; ANOVA, analysis of variance; ΔE9, exon 9 deleted. cases. Presenilins are multipass membrane proteins that form a high molecular complex with other binding partners (Aph-1, Pen-2, and Nicastrin) so as to execute an intramembraneous proteolytic activity known as γ-secretase cleavage (1De Strooper B. Neuron. 2003; 38: 9-12Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar). The γ-secretase complex cleaves several integral membrane proteins; of particular interest for AD pathogenesis is the β-amyloid precursor protein (APP), which is processed to different peptide fragments (2Selkoe D.J. J. Clin. Invest. 2002; 110: 1375-1381Crossref PubMed Scopus (211) Google Scholar), including β-amyloid (Aβ) and the APP-intracellular domain (AICD). Although Aβ is secreted in the extracellular milieu, the AICD was found to translocate to the nucleus and form a transcriptively active complex with the nuclear adaptor protein Fe65, in a manner similar to the intracellular domain of Notch (3Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1044) Google Scholar). The releases of both Aβ and AICD from the C-terminal fragment of APP are dependent on γ-secretase activity (4Sastre 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 (423) Google Scholar, 5Yu 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 (198) Google Scholar).Different FAD mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes were shown to increase production of the longer and more fibrillogenic forms of Aβ (6Scheuner D. Eckman C. Jensen M. Song X.M. Citron M. Suzuki N. Bird T.D. Hardy J. Hutton M. Kukull W. Larson E. Levy-Lahad E. Viitanen M. Peskind E. Poorkaj P. Schellenberg G. Tanzi R. Wasco W. Lannfelt L. Selkoe D. Younkin S. Nat. Med. 1996; 2: 864-870Crossref PubMed Scopus (2247) Google Scholar). There is a wealth of data showing Aβ to be neurotoxic in various experimental paradigms (7Small D.H. Mok S.S. Bornstein J.C. Nat. Rev. Neurosci. 2001; 2: 595-598Crossref PubMed Scopus (351) Google Scholar). Another pathogenic pathway by which mutated PS1 triggers neurodegeneration is the sensitization of cells to undergo death by apoptosis (8Popescu B.O. Ankarcrona M. J. Cell. Mol. Med. 2000; 4: 249-261Crossref PubMed Scopus (14) Google Scholar), although there is as yet no proof to link the proapoptotic effects of mutant PS1 to increased Aβ secretion. Under stress conditions, the cells expressing mutant PS1 show increased caspase-3, caspase-12, and calpain activation (9Kovacs D.M. Mancini R. Henderson J. Na S.J. Schmidt S.D. Kim T.W. Tanzi R.E. J. Neurochem. 1999; 73: 2278-2285Crossref PubMed Scopus (47) Google Scholar, 10Chan S.L. Culmsee C. Haughey N. Klapper W. Mattson M.P. Neurobiol. Dis. 2002; 11: 2-19Crossref PubMed Scopus (88) Google Scholar); high oxyradical production; and perturbed calcium homeostasis (11Mattson M.P. J. Neurovirol. 2002; 8: 539-550Crossref PubMed Scopus (120) Google Scholar). Moreover, FAD PS1 mutants were shown to disrupt calcium signaling not only in response to toxic stimuli but also to the physiological signals of inositol 1,4,5-trisphosphate (IP3), bradykinin, and glutamate (12Leissring M.A. Paul B.A. Parker I. Cotman C.W. LaFerla F.M. J. Neurochem. 1999; 72: 1061-1068Crossref PubMed Scopus (160) Google Scholar, 13Schneider I. Reverse D. Dewachter I. Ris L. Caluwaerts N. Kuiperi C. Gilis M. Geerts H. Kretzschmar H. Godaux E. Moechars D. Van Leuven F. Herms J. J. Biol. Chem. 2001; 276: 11539-11544Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The fact that all analyzed FAD PS1 mutations have been found to alter calcium signaling could be of relevance to AD pathogenesis, especially because deregulation of [Ca2+]i is also a constant finding in cells from AD patients and in AD experimental models (14LaFerla F.M. Nat. Rev. Neurosci. 2002; 3: 862-872Crossref PubMed Scopus (796) Google Scholar). Increased [Ca2+]i can also result in enhanced Aβ generation (15Querfurth H.W. Selkoe D.J. Biochemistry. 1994; 33: 4550-4561Crossref PubMed Scopus (214) Google Scholar), whereas in turn cells treated with Aβ show increased [Ca2+]i (16Mattson M.P. Cheng B. Davis D. Bryant K. Lieberburg I. Rydel R.E. J. Neurosci. 1992; 12: 376-389Crossref PubMed Google Scholar). In contrast secreted APP stabilizes calcium homeostasis (17Guo Q. Robinson N. Mattson M.P. J. Biol. Chem. 1998; 273: 12341-12351Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar).A large proportion of calcium mobilization and regulation in neurons can be attributable to cholinergic neurotransmission (18Berridge M.J. Neuron. 1998; 21: 13-26Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar). Phospholipase C (PLC), which predominantly couples to muscarinic receptors via G proteins, was shown to be essential for transduction of cholinergic signals in hippocampal and cortical neurons (19Kim D. Jun K.S. Lee S.B. Kang N.G. Min D.S. Kim Y.H. Ryu S.H. Suh P.G. Shin H.S. Nature. 1997; 389: 290-293Crossref PubMed Scopus (260) Google Scholar). Activated PLC hydrolyzes plasma membrane phosphoinositides (PI) to form the second messengers IP3 and diacylglycerol, which release calcium from the endoplasmic reticulum (ER) and activate protein kinase C, respectively (20Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (814) Google Scholar). Cholinergic signaling is impaired in AD at different levels (21Auld D.S. Kornecook T.J. Bastianetto S. Quirion R. Prog. Neurobiol. 2002; 68: 209-245Crossref PubMed Scopus (568) Google Scholar), and muscarinic receptor-stimulated PI hydrolysis is disturbed in vitro by both Aβ and apolipoprotein E4 (22Cedazo-Minguez A. Cowburn R.F. FEBS Lett. 2001; 504: 45-49Crossref PubMed Scopus (14) Google Scholar), two molecules involved in AD pathogenesis.Serial reports by Leissring and co-workers (12Leissring M.A. Paul B.A. Parker I. Cotman C.W. LaFerla F.M. J. Neurochem. 1999; 72: 1061-1068Crossref PubMed Scopus (160) Google Scholar, 23Leissring M.A. Yamasaki T.R. Wasco W. Buxbaum J.D. Parker I. LaFerla F.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8590-8593Crossref PubMed Scopus (85) Google Scholar, 24Leissring M.A. LaFerla F.M. Callamaras N. Parker I. Neurobiol. Dis. 2001; 8: 469-478Crossref PubMed Scopus (47) Google Scholar) have shown that mutant PS1 enhances calcium signaling mediated by caged IP3 in Xenopus oocytes. By imaging calcium release with high resolution line scanning confocal microscopy, they found that mutant PS1 induced an increase of ER calcium stores without affecting the number or the sensitivity of IP3 receptors in the ER (24Leissring M.A. LaFerla F.M. Callamaras N. Parker I. Neurobiol. Dis. 2001; 8: 469-478Crossref PubMed Scopus (47) Google Scholar). A recent study revealed that phosphoinositide/calcium signaling is dependent on the γ-secretase activity of PS1, because PS1 knock-out cells or cells treated with γ-secretase inhibitors showed no calcium signals upon bradykinin stimulation (25Leissring M.A. Murphy M.P. Mead T.R. Akbari Y. Sugarman M.C. Jannatipour M. Anliker B. Muller U. Saftig P. De Strooper B. Wolfe M. Golde T. LaFerla F.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4697-4702Crossref PubMed Scopus (252) Google Scholar). In a previous work (26Cedazo-Minguez A. Popescu B.O. Ankarcrona M. Nishimura T. Cowburn R.F. J. Biol. Chem. 2002; 277: 36646-36655Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) we have shown that the FAD mutation-causing deletion of PS1 exon 9 (PS1 ΔE9) enhances basal [Ca2+]i, as a result of an increased PLC upstream of ER IP3 receptors. Currently it is still unclear whether this novel mechanism of calcium homeostasis alteration is common for all FAD PS1 mutants or is induced by only the PS1 ΔE9 mutation. To further elucidate the pathways by which PS1 interferes with cholinergic signal transduction, we transfected SH-SY5Y neuroblastoma cells to express wild type PS1 (PS1 WT), the FAD-causing PS1 M146V or PS1 L250S mutants, or the dominant negative PS1 D257A or PS1 D385N mutants lacking γ-secretase activity (27Kimberly W.T. Xia W. Rahmati T. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2000; 275: 3173-3178Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). These cell lines were used to measure basal and carbachol-stimulated PI hydrolysis and [Ca2+]i responses. We show that PS1 M146V but not PS1 L250S generates an increased basal PLC activity. However both FAD mutants gave increased basal and carbachol-stimulated [Ca2+]i. We also show that abolition of γ-secretase activity by pharmacological inhibition or by expression of dominant negative PS1 results in the attenuation of carbachol-stimulated PI hydrolysis and the following [Ca2+]i signals. Together, these data suggest that PS1 γ-secretase activity regulates PLC activity.EXPERIMENTAL PROCEDURESMaterials—Chemicals and isotopes were purchased from the following companies: myo-[2-3H]inositol (10 Ci/mmol) from PerkinElmer Life Sciences. Carbamylcholine chloride (carbachol), Dowex 1X8-200 (chloride form), dantrolene, pepstatin A, and probenecid were from Sigma-Aldrich. Neomycin (neomycin sulfate) and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) were from Calbiochem-Novabiochem (Darmstadt, Germany). Fluo-3 acetoxymethyl (AM) ester and Pluronic F-127 were purchased from Molecular Probes (Europe BV, Leiden, The Netherlands). All other chemicals were standard laboratory reagents.Cell Lines, Cell Culture, and Transfections—Human SH-SY5Y neuroblastoma cells were cultured at 37 °C, 5% CO2 in minimum essential medium with Earle's salts containing 10% fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin. The transfected cells were additionally supplemented with 200 μg/ml geneticin. All of the cell culture supplies were purchased from Invitrogen (European Division).Transfection of cDNA constructs for mutant PS1 into SH-SY5Y cells was performed using DOTAP liposomal transfection reagent (Roche Applied Science) in accordance with the manufacturer's protocol. Stable transfectants were first selected in 500 μg/ml geneticin for several days, and then the geneticin concentration was reduced to 200 μg/ml. The expression levels of the transfected PS1 were determined by Western blotting as described previously (28Tanii H. Ankarcrona M. Flood F. Nilsberth C. Mehta N.D. Perez-Tur J. Winblad B. Benedikz E. Cowburn R.F. Neuroscience. 2000; 95: 593-601Crossref PubMed Scopus (41) Google Scholar). We also confirmed expression of mutant PS1 mRNA in SH-SY5Y cells by sequencing following reverse transcription. Establishment of SH-SY5Y cells overexpressing wild type PS1 has been previously reported by our lab (28Tanii H. Ankarcrona M. Flood F. Nilsberth C. Mehta N.D. Perez-Tur J. Winblad B. Benedikz E. Cowburn R.F. Neuroscience. 2000; 95: 593-601Crossref PubMed Scopus (41) Google Scholar).cDNA Constructs—The two-step PCR method was employed to construct PS1 cDNA with an artificial mutation of either D257A or D385N, using wild type PS1 in pcDNA3 (Invitrogen) as the template. Two sets of PCR reactions were performed each using: 1) a forward primer for the N terminus of PS1 (5′-CATATGACAGAGTTACCTGCACCG-3′) and a reverse primer for the sequence containing a point mutation (D257A, 5′-CAGCCACTAAAGCATATACTG-3′ or D385N, 5′-GAAAATGAAATTTCCCAATCC-3′) and 2) a forward primer for the sequence containing a point mutation (D257A, 5′-CAGTATATGCTTTAGTGGCTG-3′, or D385N, 5′-GGATTGGGAAATTTCATTTTC-3′) and a reverse primer for the C terminus of PS1 (5′-CTAGATATAAAATTGATGG-3′). The resultant two PCR fragments were mixed and hybridized to obtain the second template, which was amplified by PCR using the primers for both terminals of PS1. The resultant full-length PS1 cDNA with point mutations was inserted into the pcDNA3.1 vector (Invitrogen). The vectors containing cDNA constructs of FAD-associated PS1 mutations (M146V and L250S) were a gift from Prof. John Hardy (NIA, National Institutes of Health, Bethesda, MD). All of the cDNA constructs were sequenced for verification.Immunoblot Analysis—To obtain cell extracts, the cells were washed twice with ice-cold Ca2+- and Mg2+-free phosphate-buffered saline (PBS), harvested with a cell scraper, and collected by 3,000 rpm centrifugation for 3 min. The pellets were resuspended in 100 μl of lysis buffer (20 mm Tris-HCl, 137 mm NaCl, 2 mm EDTA, 2% Nonidet P-40, 2% Triton X-100, and 400 μg/ml protease inhibitors Complete™ Mini; Roche Applied Science) and centrifuged at 11,000 rpm for 10 min at 4 °C. The samples were stored at -20 °C until use. The Pierce BCA protein assay kit was used to quantify the protein amounts in cell extracts. PS1 expression was analyzed by separating equivalent protein amounts by SDS-PAGE using 10% acrylamide gels. The proteins were transferred onto Hybond ECL nitrocellulose membranes (Amersham Biosciences) by trans-blot electrophoretic transfer for 4 h at a constant current of 200 mA. After blocking for 1 h using 5% (w/v) dried milk in Tris-buffered solution containing 0.1% Tween 20, the membranes were incubated overnight with an N-terminal PS1 antibody (28Tanii H. Ankarcrona M. Flood F. Nilsberth C. Mehta N.D. Perez-Tur J. Winblad B. Benedikz E. Cowburn R.F. Neuroscience. 2000; 95: 593-601Crossref PubMed Scopus (41) Google Scholar) diluted 1:2000. The membranes were washed in Tris-buffered solution containing 0.1% Tween 20 for 15 min and then washed three times for 5 min. The secondary antibody (anti-rabbit horseradish peroxidase-linked; Amersham Biosciences) diluted in blocking solution (1:2000) was added to the membranes for 1 h at room temperature. The membranes were subsequently washed as described above, plus an additional wash of 1 h. Bound antibody was detected by the ECL method (Amersham Biosciences) after exposure to Hyperfilm MP (Amersham Biosciences).PI Hydrolysis Assay—The cells were cultured to 75-80% confluence in 75-cm2 flasks. One day prior to the experiment, the cells were changed to serum-free medium containing 5 μCi/ml myo-[2-3H]inositol and incubated for 24 h. PI hydrolysis was measured as described previously (22Cedazo-Minguez A. Cowburn R.F. FEBS Lett. 2001; 504: 45-49Crossref PubMed Scopus (14) Google Scholar). The cells were harvested by scraping with a rubber policeman in 4 ml of PBS. The contents were centrifuged at 1,500 rpm for 15 min. The pellets were washed twice with 37 °C PBS and resuspended in 3mlof37 °C Krebs-Henseleit bicarbonate buffer containing 10 mm LiCl (KHB/Li), gassed with 5% CO2, 95% O2, and centrifuged again (15,000 rpm, 15 min). The cell pellets were resuspended in 210 μl of KHB/Li and regassed, and 50 μl was added to glass centrifuge tubes containing 250 μl of KHB/Li buffer with or without 100 μl of carbachol. The tubes were incubated at 37 °C under an atmosphere of 5% CO2, 95% O2 with gentle agitation for 25 min. The incubations were stopped by adding 940 μl of chloroform:methanol (1:2). The tubes were incubated on ice for 30 min, and the phases were separated by adding 310 μl of chloroform and 310 μl of water followed by vortexing and centrifugation. 750 μl of the aqueous phase were removed, and labeled inositol phosphates were separated from myo-[3H]inositol by Dowex chromatography, as described by Berridge (29Berridge M.J. Cell Calcium. 1982; 3: 385-397Crossref PubMed Scopus (10) Google Scholar). The chloroform phase was removed, placed into scintillation vials, and allowed to evaporate before determination of “dpm lipid” by scintillation spectroscopy. The results were expressed as dpm inositol phosphates/(dpm inositol phosphates + dpm lipid). This unit is independent of the number of cells aliquoted in each tube and upon the degree of labeling of inositol phospholipids (30Fowler C.J. Court J.A. Tiger G. Bjorklund P.E. Candy J.M. Pharmacol. Toxicol. 1987; 60: 274-279Crossref PubMed Scopus (51) Google Scholar).For the treatment with DAPT, 1 day prior to experiment, the cells were changed to serum-free medium containing 5 μCi/ml myo-[2-3H]-inositol and incubated for 21 h. The medium was then replaced with that containing DAPT (1 μm) plus 5 μCi/ml myo-[2-3H]inositol for 3 h prior to PI hydrolysis measures.Intracellular Calcium Measurements—Intracellular calcium apparent concentrations were determined as described previously (31Popescu B.O. Cedazo-Minguez A. Popescu L.M. Winblad B. Cowburn R.F. Ankarcrona M. J. Neurosci. Res. 2001; 66: 122-134Crossref PubMed Scopus (20) Google Scholar). In brief, the cells cultured in 96-well plates were loaded with minimum essential medium without phenol red containing 5 μm Fluo-3 AM ester, 0.5% (v/v) Pluronic F-127, and 1 mm probenecid (90 min in the dark at room temperature). After loading, the cells were incubated for 120 min in minimum essential medium without phenol red with 1 mm probenecid in the dark at room temperature to allow intracellular esterases to decompose the Fluo-3 AM ester. For the measurements of basal apparent [Ca2+]i, the medium was removed, and Ca2+/Mg2+-free PBS was added. Basal apparent [Ca2+]i was measured repeatedly for 10 min at 37 °C to certify that a steady state of apparent [Ca2+]i was obtained. We found that basal apparent [Ca2+]i in the absence of treatments was steady during the entire incubation time courses used (26Cedazo-Minguez A. Popescu B.O. Ankarcrona M. Nishimura T. Cowburn R.F. J. Biol. Chem. 2002; 277: 36646-36655Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Neomycin and dantrolene were added at indicated concentrations in minimum essential medium without phenol red during the 120 min of incubation time and in the PBS for the 10 min when basal apparent [Ca2+]i was measured. DAPT and pepstatin A were added to cells 24 h prior to experiments, in growth and incubation media and in the PBS for the 10 min when basal apparent [Ca2+]i was measured. After measuring basal apparent [Ca2+]i, the PBS was removed, and 100 μm carbachol in PBS solution at 37 °C was added (100 μl to each well). Carbachol was present for all subsequent apparent [Ca2+]i measures. A CytoFluor Series 4000 multiwell plate reader (PerSeptive Biosystems GmbH, Wiesbaden, Germany) was used to measure fluorescence, with the excitation and emission wavelengths being 485 and 530 nm, respectively. Apparent [Ca2+]i corresponding to fluorescent value F was calculated with the formula, [Ca2+]i = Kd(F - Fmin)/(Fmax - F), where Kd for Fluo-3-Ca2+ was taken as 390 nm, as indicated by the protocol from the manufacturer (Molecular Probes Europe BV). Fmin was determined by measuring the signal of the unloaded cells, and Fmax was determined by the addition to the cells of a 100 mm CaCl2 buffer containing 1% (v/v) Triton X-100.Statistical Analyses—Analyses of differences were carried out by ANOVA followed by Fisher's protected least significant distance post hoc test. A value of p < 0.05 was considered statistically significant.RESULTSPS1 Expression in Transfected Cell Lines—We stably transfected SH-SY5Y neuroblastoma cells to overexpress PS1 WT or FAD mutant PS1 (PS1 M146V or PS1 L250S) or the nonfunctional dominant negative PS1 mutants (PS1 D257A or PS1 D385N). The transfection of these PS1 variants in SH-SY5Y cells was confirmed by sequencing and by immunoblotting with an N-terminal PS1 antiserum (Fig. 1). Different clones of PS1 D257A (clones 2, 2A and 2C) and of PS1 D385N (clones 1A and 1B) were checked for levels of PS1 expression (Fig. 1). The pattern of PS1 protein expression in nontransfected (NT) cells and PS1 WT or FAD mutant PS1 transfected cells was similar to our previous reports, with most of the PS1 being identified as the ∼28-kDa N-terminal fragment resulting from its constitutive endoproteolytic cleavage (26Cedazo-Minguez A. Popescu B.O. Ankarcrona M. Nishimura T. Cowburn R.F. J. Biol. Chem. 2002; 277: 36646-36655Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 28Tanii H. Ankarcrona M. Flood F. Nilsberth C. Mehta N.D. Perez-Tur J. Winblad B. Benedikz E. Cowburn R.F. Neuroscience. 2000; 95: 593-601Crossref PubMed Scopus (41) Google Scholar, 31Popescu B.O. Cedazo-Minguez A. Popescu L.M. Winblad B. Cowburn R.F. Ankarcrona M. J. Neurosci. Res. 2001; 66: 122-134Crossref PubMed Scopus (20) Google Scholar). In the PS1 WT and FAD mutant PS1 transfectants, we observed a moderate accumulation of an ∼48-kDa full-length protein, probably caused by limited PS1 proteolytic processing (32Thinakaran G. Harris C.L. Ratovitski T. Davenport F. Slunt H.H. Price D.L. Borchelt D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: 28415-28422Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). In agreement with previous findings (33Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1674) Google Scholar, 34Yu G. Chen F. Nishimura M. Steiner H. Tandon A. Kawarai T. Arawaka S. Supala A. Song Y.Q. Rogaeva E. Holmes E. Zhang D.M. Milman P. Fraser P.E. Haass C. St George-Hyslop P. J. Biol. Chem. 2000; 275: 27348-27353Abstract Full Text Full Text PDF PubMed Google Scholar), cells expressing PS1 D257A or PS1 D385N showed an accumulation of full-length PS1, because of a lack of endoproteolysis in these PS1 variants. This was more pronounced in D257A clones 2 and 2A and in D385N clone 1A (Fig. 1). D257A clones 2 and 2A also showed the presence of other PS1 fragments that presumably reflect caspase cleavage of overexpressed protein (Fig. 1).Effects of FAD Mutant PS1 and Dominant Negative Mutant PS1 on Basal and Carbachol-stimulated PI Hydrolysis—Basal PI hydrolysis values did not differ significantly in NT, PS1 WT, PS1 L250S, and PS1 D257A-2C cells (Fig. 2). In contrast, we found significantly higher PI hydrolysis in PS1 M146V cells (p < 0.05; ANOVA, Fisher's post hoc test) and significantly lower PI hydrolysis in PS1 D257A-2, PS1 D257A-2A, PS1 D385N-1A, and PS1 D385N-1B cells (p < 0.001; ANOVA, Fisher's post hoc test), as shown in Fig. 2. Stimulation with 100 μm carbachol of either the NT or PS1 WT cells gave an ∼2-fold increase in PI hydrolysis, comparable with that reported before (26Cedazo-Minguez A. Popescu B.O. Ankarcrona M. Nishimura T. Cowburn R.F. J. Biol. Chem. 2002; 277: 36646-36655Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). As compared with NT and PS1 WT cells, carbachol-stimulated PI hydrolysis was not significantly different either in PS1 M146V or PS1 L250S cells. Conversely, both PS1 D257A (p < 0.01 for all clones; ANOVA, Fisher's post hoc test) and PS1 D385N (p < 0.05 for clone 1A and p < 0.001 for clone 1B; ANOVA, Fisher's post hoc test) cells showed significantly lower PI hydrolysis upon stimulation with carbachol, as compared with NT and PS1 WT cells (Fig. 2). For further experiments, we selected PS1 D257A-2C and PS1 D385N-1B cells. These were the clones with the lowest level of PS1 expression among these cell lines. This was done to rule out potential artifacts caused by ER stress that could in theory occur because of overexpression of nonendoproteolyzed protein.Fig. 2Effects of FAD mutant presenilin 1 and dominant negative presenilin 1 on basal and carbachol-stimulated PI hydrolysis. PI hydrolysis was measured in basal and carbachol (100 μm) stimulation conditions. The histograms show the means ± S.E. (4 ≤ n ≤7). Statistical analysis of the results was carried out using ANOVA followed by Fisher's post hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001, as compared with values for both NT and PS1 WT cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of FAD Mutant PS1 and Dominant Negative Mutant PS1 on Basal and Carbachol-stimulated Intracellular Calcium Concentrations—We did not find any significant differences in the basal apparent [Ca2+]i values (means ± S.E., values given for time 0, before the addition of carbachol) in NT (39.4 ± 0.4 nm), PS1 WT (39.9 ± 1.0 nm), and PS1 D257A (38.6 ± 1.0 nm) cells (Fig. 3). Both the PS1 M146V and the PS1 L250S cells showed a significantly higher basal apparent [Ca2+]i value (45.7 ± 1.9 and 43.5 ± 1.2 nm, respectively, p < 0.05; ANOVA, Fisher's post hoc test) as compared with NT and PS1 WT cells (Fig. 3). In accordance with the basal PI hydrolysis data, PS1 D385N cells showed a significantly lower basal apparent [Ca2+]i (35.6 ± 0.5 nm, p < 0.05; ANOVA, Fisher's post hoc test) as compared with NT and PS1 WT cells (Fig. 3).Fig. 3Effects of FAD mutant presenilin 1 and dominant negative presenilin 1 on basal and carbachol-stimulated intracellular calcium concentrations. Basal apparent [Ca2+]i was repeatedly measured for 10 min to c" @default.
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• W2073539123 date "2004-02-01" @default.
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• W2073539123 title "γ-Secretase Activity of Presenilin 1 Regulates Acetylcholine Muscarinic Receptor-mediated Signal Transduction" @default.
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