Intracellular Retention of Caveolin 1 in Presenilin-deficient Cells (2004)

Mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early onset familial Alzheimer's disease. PS, a critical component of γ-secretase, is responsible for the intramembranous cleavage of amyloid precursor protein and Notch. Other physiological functions have been assigned to PS without any clear identification of the mechanisms underlying these multiple biological roles. The early embryonic lethality of PS1 and PS2 double knock-out (PS1/2 null) mice prevents the evaluation of physiological roles of PS. To investigate new functions for presenilins, we performed a proteomic approach by using cells derived from PS1/2 null blastocysts and wild type controls. We identified a presenilin-dependent cell-surface binding of albumin. Binding of albumin depends on intact caveolae on the cellular surface. Abnormal caveolin 1 localization in PS1/2 null cells was associated with a loss of caveolae and an absence of caveolin 1 expression within lipid rafts. Expressing PS1 or PS2 but not the intracellular form of Notch1 in PS1/2 null cells restored normal caveolin 1 localization, demonstrating that presenilins are required for the subcellular trafficking of caveolin 1 independently from Notch activity. Despite an expression of both caveolin 1 and PS1 within lipid raft-enriched fractions after sucrose density centrifugation in wild type cells, no direct interaction between these two proteins was detected, implying that presenilins affect caveolin 1 trafficking in an indirect manner. We conclude that presenilins are required for caveolae formation by controlling transport of intracellular caveolin 1 to the plasma membrane. Mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early onset familial Alzheimer's disease. PS, a critical component of γ-secretase, is responsible for the intramembranous cleavage of amyloid precursor protein and Notch. Other physiological functions have been assigned to PS without any clear identification of the mechanisms underlying these multiple biological roles. The early embryonic lethality of PS1 and PS2 double knock-out (PS1/2 null) mice prevents the evaluation of physiological roles of PS. To investigate new functions for presenilins, we performed a proteomic approach by using cells derived from PS1/2 null blastocysts and wild type controls. We identified a presenilin-dependent cell-surface binding of albumin. Binding of albumin depends on intact caveolae on the cellular surface. Abnormal caveolin 1 localization in PS1/2 null cells was associated with a loss of caveolae and an absence of caveolin 1 expression within lipid rafts. Expressing PS1 or PS2 but not the intracellular form of Notch1 in PS1/2 null cells restored normal caveolin 1 localization, demonstrating that presenilins are required for the subcellular trafficking of caveolin 1 independently from Notch activity. Despite an expression of both caveolin 1 and PS1 within lipid raft-enriched fractions after sucrose density centrifugation in wild type cells, no direct interaction between these two proteins was detected, implying that presenilins affect caveolin 1 trafficking in an indirect manner. We conclude that presenilins are required for caveolae formation by controlling transport of intracellular caveolin 1 to the plasma membrane. γ-Secretase activity is responsible for the cleavage of the transmembrane domain of the amyloid precursor protein (APP), 1The abbreviations used are: APP, amyloid precursor protein; AD, Alzheimer's disease; PS, presenilin; PS1/2 null, cells derived from PS1 and PS2 double null mice; BSA, bovine serum albumin; Cav, caveolin; NICD, Notch 1 intracellular domain; CEM, caveolin-enriched membrane; WT, wild type; PBS, phosphate-buffered saline; GFP, green fluorescent protein; mAb, monoclonal antibody; pAb, polyclonal antibody; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MES, 4-morpholineethanesulfonic acid; ER, endoplasmic reticulum; Cav1, caveolin 1. releasing the amyloid peptide Aβ and the APP intracellular domain. Aβ is a major component of amyloid plaques characteristic of Alzheimer's disease (AD). Presenilins (PSs) are the active core of γ-secretase (1Herreman A. Sermeels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (452) Google Scholar, 2Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat. Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (361) Google Scholar). Different molecular components of the γ-secretase complex were identified and include, in addition to PSs, Nicastrin, Aph1, and Pen-2 (3Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld M. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurney M. Myers R.L. Himes C.S. Hiebsch R. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar). The PS-dependent proteolytic process is involved in the release of the cytoplasmic domains of an increasing range of type I integral membrane receptors such as Notch (4De 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; 388: 518-522Crossref Scopus (1808) Google Scholar, 5Struhl G. Greenwald L. Nature. 1999; 398: 522-525Crossref PubMed Scopus (706) Google Scholar, 6Ye Y. Lukinova N. Fortini M.E. Nature. 1999; 398: 525-529Crossref PubMed Scopus (462) Google Scholar), ErbB-4 (7Ni C.Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar), E-cadherin (8Marambaud P. Shioi J. Serban G. Georgakopoulos A. Sarner S. Nagy V. Baki L. Wen P. Efthimipoulos S. Shao Z. Wisniewski T. Robakis N.K. EMBO J. 2002; 21: 48-56Crossref Scopus (626) Google Scholar), and others. PSs, in addition to their role as γ-secretase catalytic subunits, have an important function in subcellular trafficking of a selected group of proteins such as APP, Trk receptor (9Naruse 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 (333) Google Scholar), Nicastrin (10Herreman A. Van Gassen A.G. Bentahir M. Nyabi O. Craessaerts K. Mueller U. Annaert W. De Strooper B. J. Cell Sci. 2003; 116: 1127-1136Crossref PubMed Scopus (166) Google Scholar), and Pen-2 (11Wang H. Luo W.J. Zhang Y.W. Li Y.M. Thinakaran G. Greengard P. Xu H. J. Biol. Chem. 2004; 279: 40560-40566Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). PSs mediate additional physiological functions, including roles in calcium homeostasis, neurite outgrowth, apoptosis, and synaptic plasticity (12Sisodia S.S. Kim S.H. Thinakaran G. Am. J. Hum. Genet. 1999; 65: 7-12Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Because deleting ps1 and ps2 genes in mice leads to an early embryonic lethality (13Donoviel D.B. Hadjantonakis A.K. Ikeda M. Zheng H. Hyslop P.S. Bernstein A. Genes Dev. 1999; 13: 2801-2810Crossref PubMed Scopus (410) Google Scholar), the role of presenilins cannot be fully examined in double knock-out mice. Therefore, we used cells derived from PS1 and PS2 double null (termed PS1/2 null) blastocysts and their WT controls (14Berechid B.E. Kitzmann M. Foltz D.R. Roach A.H. Seiffert D. Thompson L.A. Olson R.E. Bernstein A. Donoviel D.B. Nye J.S. J. Biol. Chem. 2002; 277: 8154-8165Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) to perform a differential proteomic approach. We identified a presenilin-dependent defect in albumin binding reflecting an abnormal caveolae-dependent pathway. We showed that caveolin 1 was mislocalized in PS1/2 null cells leading to abnormal caveolae formation and an absence of caveolin1 in lipid rafts. Overexpression of PSs in PS1/2 null cells was sufficient to restore normal caveolin localization and function. We could not detect any physical interaction between presenilins and caveolin 1. These results imply that presenilins are responsible, in an indirect manner, for caveolin 1 transport to the cell surface to form caveolae. Materials—On embryonic day E3.5, blastocysts were flushed from wild type or PS1+/–, PS2+/–-intercrossed mice (13Donoviel D.B. Hadjantonakis A.K. Ikeda M. Zheng H. Hyslop P.S. Bernstein A. Genes Dev. 1999; 13: 2801-2810Crossref PubMed Scopus (410) Google Scholar) into M2 medium and plated on mitotically inhibited feeder cells. Five days later, the inner cell mass was removed, trypsinized, and plated onto new feeder cells. After 12 days, the cells were replated onto gelatin-coated dishes, grown until confluent, and then maintained in embryonic stem cell media (high glucose Dulbecco's modified Eagle's medium, 15% fetal bovine serum, 2 mml-glutamine, 1% penicillin/streptomycin, 1× non-essential amino acids, sodium pyruvate, recombinant leukemia-inhibitory factor) on uncoated plates. For each cell line, PCR geno-typing was performed as described previously (13Donoviel D.B. Hadjantonakis A.K. Ikeda M. Zheng H. Hyslop P.S. Bernstein A. Genes Dev. 1999; 13: 2801-2810Crossref PubMed Scopus (410) Google Scholar). These cells were named blastocyst-derived cells. SV40 transformation was used to generate rapidly growing cell lines as described previously (33Palacino 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). These cells were named SV40-transformed blastocyst-derived cells. NICD-myc6, PS1, and PS2 cDNA constructs were described previously (14Berechid B.E. Kitzmann M. Foltz D.R. Roach A.H. Seiffert D. Thompson L.A. Olson R.E. Bernstein A. Donoviel D.B. Nye J.S. J. Biol. Chem. 2002; 277: 8154-8165Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). GFP-Cav1α cDNA construct was a generous gift from the laboratory of Dr. Van Deurs (Sweden). Two-dimensional Gel Electrophoresis—Total protein extracts from WT, PS1/2 null, and PS1/2 null cells transiently transfected with the presenilin 1 cDNA (blastocyst-derived and SV40-transformed blastocyst-derived cells) were analyzed by two-dimensional electrophoresis by the method of O'Farrell. The ampholine mixture used for the first dimension electrofocusing gel was composed of 60% (w/v) of ampholine, pH 5–7, and 40% of ampholine, pH 3–10. The second dimension was performed on SDS-12% polyacrylamide gels. Gels were then stained by Coomassie Blue and BSA-sequenced with a MALDI-TOF mass spectrometer (Carol Beach, University of Kentucky). Endocytosis Assays—Blastocyst-derived PS1/2 null cells and WT controls were grown in complete medium on 25-mm glass coverslips. When cells were 75–80% confluent, the medium was replaced with 2 ml of complete medium containing FITC-conjugated albumin (Sigma) at a final concentration of 10 μg/ml. Cells were incubated at 37 °C for 5 and 30 min, washed in PBS, and fixed in 3.7% formaldehyde for 10 min. The cells were washed for 20 min in PBS and mounted on a slide. Virtually identical experiments were carried out with FITC-conjugated transferrin (10 μg/ml, sigma). PS1/2 null cells were grown on 60-mm tissue culture dishes and transiently transfected with the presenilin 1 or presenilin 2 cDNA using Lipofectamine Plus (Invitrogen) according to the manufacturer's instructions. The transfected cells were plated on 25-mm coverslips in 6-well plates after 24 h. FITC-albumin uptake was then examined in transfected cells 36 h after the initial transfection. Cells transfected with presenilin 1 or presenilin 2 were detected by immunostaining with anti-presenilin 1 pAb (oncogene) and anti-presenilin 2 pAb (oncogene), respectively, and a Texas Red-conjugated secondary antibody (Jackson ImmunoResearch). Immunofluorescence Microscopy—Transfected or untransfected blastocyst-derived PS1/2 null cells and WT controls were plated on 18-mm glass coverslips (Fisher), fixed for 10 min in PBS containing 3.7% formaldehyde, rinsed with PBS, and incubated for 2 min in permeabilization buffer (PBS, 0.1% Triton X-100). Cells were then washed with PBS and incubated for 10 min in blocking buffer (PBS, 0.5% BSA) and labeled with corresponding antibodies: anti-FLAG mAb (M2, Sigma); anti-caveolin 1 pAb (Transduction Laboratories); anti-PS1 pAb (H70, Sc Biotechnology); and anti-Myc mAb (9E10). After rinsing with PBS, secondary antibody Alexa 488 or Alexa 555 donkey anti-mouse and Alexa-conjugated donkey anti-rabbit (Molecular Probes) were added for a period of 30 min. A confocal fluorescence microscope (Olympus, North-western University, Molecular Pharmacology and Biological Chemistry, Prof. R. J. Miller's laboratory) was used for visualization of FITC-BSA and FITC-transferrin (Fig. 2 and Fig. 3B). A fluorescence microscope (Leica, DRMA2, Institut de Génétique Humaine) was used for visualization of secondary for Fig. experiments were performed as described previously Kitzmann M. M. A. J. Cell Sci. PubMed Google expression albumin uptake in PS1/2 null cellular proteins were from PS1/2 null cells transiently transfected with the presenilin 1 cDNA blastocyst-derived and blastocyst-derived cells and by two-dimensional of the first dimension between pH 5 and pH is by The localization of on the two-dimensional gel in PS1/2 null cells PS1 is by blastocyst-derived PS1/2 null cells were transiently transfected with the cDNA encoding for PS1 and PS2 cells were to FITC-albumin for 30 min. Cells were then rinsed in PBS formaldehyde and immunostaining with anti-PS1 and localization of caveolin blastocyst-derived WT and PS1/2 null cells were and localization of and was by indirect 5 WT and PS1/2 null cells were transiently transfected with the cDNA encoding cells were by fluorescence after formaldehyde and 10 blastocyst-derived PS1/2 null cells were transiently transfected with the cDNA encoding and cells were and with anti-FLAG and anti-caveolin 1 5 PS1/2 null cells were transiently transfected with the cDNA encoding 24 cDNA encoding PS1 and or and was into PS1/2 null cells. cells were and with anti-PS1 and anti-Myc blastocyst-derived PS1/2 null cells and WT controls were and by The cell was fixed in buffer containing at Cells were then rinsed in in a for 1 h at and onto were at of each and with and visualization and on a was performed at the of of and were by of cellular proteins and centrifugation sucrose as described J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PS1/2 null cells and WT controls were grown in dishes, and 2 cells were used to the Cells were washed with 10 ml of phosphate-buffered and into 2 ml of buffer MES, 1% Triton mixture and with a The was to 40% sucrose by the addition of 2 ml of sucrose in Triton and in the of an A sucrose was by with ml of sucrose and ml of sucrose in The were at in an for h at and 12 fractions of 1 ml were from the of the To the of proteins within the of each were with in 20 of 1× and to on by The used are as anti-PS1 pAb (H70, Sc pAb Sc Biotechnology); mAb (Transduction Laboratories); mAb (Transduction Laboratories); and 1 mAb (Transduction anti-mouse or anti-rabbit secondary antibody was used to with to new functions for presenilins, we used a differential proteomic approach (Fig. We the two-dimensional of extracts from WT and PS1/2 null cells. of were in WT cells and in PS1/2 null cells. We performed the differential proteomic approach with SV40-transformed blastocyst-derived cells (Fig. and blastocyst-derived cells (Fig. proteins to the and a protein was to blastocyst-derived cells and their SV40 protein was with a MALDI-TOF mass spectrometer and identified as bovine serum albumin that is a defect in the binding of albumin from the fetal bovine serum in the absence of cells in the uptake and transport of albumin in W. B. M.P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). 1 null embryonic in the of albumin but not transferrin B. W. Zhang Li M. D. H. B. W. M.P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). in cells is by the binding of albumin to a protein in caveolae L. A. M. N. J. Cell Biol. PubMed Scopus Google Scholar). Therefore, the defect of binding of albumin in PS1/2 null cells (Fig. could be to the abnormal caveolae-dependent pathway. We examined the of WT and PS1/2 null cells to the fluorescent Fig. 2 that PS1/2 null cells to FITC-albumin after 30 min of in to WT cells cell surface with FITC-albumin within 5 min of To that the of expression from the presenilins not affect we examined the intracellular uptake of a second fluorescent to be in Fig. 2 FITC-transferrin was rapidly in both WT and PS1/2 null cells. PS1/2 null cells a defect in binding of that in caveolin cells B. W. Zhang Li M. D. H. B. W. M.P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). To that the defect in caveolae-dependent in PS1/2 null cells was of a of PS we examined the expression of the presenilin cDNA was sufficient to restore albumin binding in PS1/2 null cells. in Fig. the two-dimensional gel of proteins in PS1/2 null cells PS1 was with that of the corresponding WT cells Fig. The of was detected on two-dimensional of SV40-transformed blastocyst-derived PS1/2 null cells or blastocyst-derived PS1/2 null cells We by FITC-albumin uptake in blastocyst-derived PS1/2 null cells PS1 (Fig. or PS2 (Fig. These results that caveolae is PS1 or PS2 Because the in PS1/2 null cells is to the in cells B. W. Zhang Li M. D. H. B. W. M.P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), we caveolin 1 was in PS1/2 null cells. we were to detect caveolin 1 expression in PS1/2 null cells. R. J. S. N. J. C. A. and M. We to investigate caveolin1 was in PS1/2 null cells in to WT cells. We the to We first caveolin1 localization by We then caveolae formation by we at the of caveolin 1 with lipid by sucrose density to caveolin 1 between WT and PS1/2 null cells, we performed indirect experiments (Fig. of WT cells, caveolin 1 was at the plasma membrane with (Fig. in PS1/2 null cells, caveolin 1 showed localization (Fig. localization of caveolin 1 in PS1/2 null cells was after expression of (Fig. and with caveolin R. J. S. N. J. C. A. and M. that is an intracellular of caveolin 1 in PS1/2 null cells. To caveolin 1 membrane localization was we examined expression of presenilin 2 cDNA was sufficient to a in caveolin 1 in Fig. expression of 2 in PS1/2 null cells of caveolin 1 from localization of PS1 cDNA in PS1/2 null cells (Fig. to be as in WT cells no in localization was after the of the Notch (Fig. construct in PS1/2 null cells (Fig. These results that caveolin 1 is mislocalized in PS1/2 null cells and that of the PS1/2 null mice are to a loss of Notch 1 function (13Donoviel D.B. Hadjantonakis A.K. Ikeda M. Zheng H. Hyslop P.S. Bernstein A. Genes Dev. 1999; 13: 2801-2810Crossref PubMed Scopus (410) Google Scholar), presenilins are involved in the subcellular localization of caveolin 1 independently from Notch activity. of caveolin 1 in cells caveolae de formation of or of caveolin 1 expression a loss of caveolae K. Sci. U. S. A. PubMed Scopus Google Scholar, H. B. M.P. Am. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. L. Y. N. K. J. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). We PS1/2 null cells mislocalized caveolin 1 were to form caveolae. are at the plasma membrane by The of caveolae in PS1/2 null cells was by We a of caveolae at the plasma membrane of cells (Fig. in to WT cells (Fig. of PS1/2 null cells showed caveolae at the plasma membrane with of WT cells. R. J. S. N. J. C. A. and M. These that presenilins are involved in caveolae are a of to as lipid rafts. are by their in Triton and their in density fractions sucrose J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We performed of of WT and PS1/2 null cell proteins been previously that fractions in for caveolae such as the and the endoplasmic M.P. J. Z. A. M. J. Cell Biol. PubMed Scopus Google Scholar, Z. Li S. M.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of and proteins in the and the respectively, was examined to the of with and (Fig. have been described as a new of proteins associated with caveolae P. M.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). (Fig. 1 expression was to fractions and 5 implying that with Triton of lipid from and not detected in 1 expression was not to and was in fractions in WT cells. previously U. W. Nat. 1998; PubMed Scopus Google Scholar, M. Y. 1998; PubMed Scopus Google Scholar, D.R. G. C. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), PS1 was in 1 expression was detected in 5 in WT cells (Fig. but not in PS1/2 null cells (Fig. PS1/2 null cells, caveolin 1 was in and in density fractions with Triton that in the absence of presenilins, caveolin 1 was not detected in the density fractions in caveolae and the that caveolae not 1 expression was no associated with lipid in PS1/2 null cells, implying that not caveolae but lipid be by a in presenilins to the molecular responsible for caveolin 1 in PS1/2 null cells, we presenilin 1 and caveolin 1 were to with Despite a of caveolin 1 and PS1 in lipid (Fig. we were not to detect any interaction between presenilin 1 and caveolin 1 by in WT from or from these results that PS1 or PS2 expression is required for a localization of caveolin 1 in caveolae at the plasma membrane but that the absence of presenilins affect in caveolin 1 we performed a differential proteomic approach using blastocyst-derived WT and PS1/2 null cells in to new roles of presenilins Alzheimer's disease. using we identified a presenilin-dependent caveolin 1 localization leading to an absence of caveolae presenilins are 1 be added to the of proteins with a presenilin-dependent We an intracellular of caveolin 1 in PS1/2 null cells (Fig. The localization of caveolin 1 in PS1/2 null cells with by these caveolin 1 was The localization of caveolin 1 in the absence of presenilins The by presenilins, such as the caveolin 1 localization and caveolae formation not a complex between caveolin 1 and presenilin and is from Notch 1 (Fig. 1 to the lipid fractions of the of caveolin B. W. M. H. M.P. Am. 2004; Scholar). the absence of 1 from lipid fractions in PS1/2 null cells is not of caveolin 1 implying that the by presenilins transport proteins to lipid is and affect a protein involved in the of proteins to lipid rafts. are important roles in a of cellular and in we to an important new role for not form caveolae but form lipid with APP was in membrane fractions with C. A. G. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that be important in the of to lipid was to of APP C. Sci. U. S. A. 2003; PubMed Scopus Google Scholar), and presenilins were in caveolae in plaques K. T. Tomita T. Iwatsubo T. M.P. T. J. 1999; PubMed Google Scholar). γ-secretase complex was to be in lipid of and H. W. T. Li T. N. Wong P.C. Xu H. Thinakaran G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). We that in presenilins be in the trafficking of proteins such as is responsible for lipid These lipid form the for γ-secretase activity. A of γ-secretase complex is and to the cell membrane to the of new to the cleavage of APP and the of We for and the laboratory of Dr. Van Deurs for GFP-Cav1α We and from the of for their in and of for the in protein