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• W2040373875 abstract "Processing of amyloid precursor protein (APP) is a well acknowledged central pathogenic mechanism in Alzheimer disease. However, influences of age-associated cellular alterations on the biochemistry of APP processing have not been studied in molecular detail so far. Here, we report that processing of endogenous APP is down-regulated during the aging of normal human fibroblasts (IMR-90). The generation of intracellular APP cleavage products C99, C83, and AICD gradually declines with increasing life span and is accompanied by a reduced secretion of soluble APP (sAPP) and sAPPα. Further, the maturation of APP was reduced in senescent cells, which has been shown to be directly mediated by age-associated increased cellular cholesterol levels. Of the APP processing secretases, protein levels of constituents of the γ-secretase complex, presenilin-1 (PS1) and nicastrin, were progressively reduced during aging, resulting in a progressive decrease in γ-secretase enzymatic activity. ADAM10 (a disintegrin and metalloprotease 10) and BACE (β-site APP-cleaving enzyme) protein levels exhibited no age-associated regulation, but interestingly, BACE enzymatic activity was increased in aged cells. PS1 and BACE are located in detergent-resistant membranes (DRMs), well structured membrane microdomains exhibiting high levels of cholesterol, and caveolin-1. Although total levels of both structural components of DRMs were up-regulated in aged cells, their particular DRM association was decreased. This age-dependent membrane modification was associated with an altered distribution of PS1 and BACE between DRM and non-DRM fractions, very likely affecting their APP processing potential. In conclusion, we have found a significant modulation of endogenous APP processing and maturation in human fibroblasts caused by age-associated alterations in cellular biochemistry. Processing of amyloid precursor protein (APP) is a well acknowledged central pathogenic mechanism in Alzheimer disease. However, influences of age-associated cellular alterations on the biochemistry of APP processing have not been studied in molecular detail so far. Here, we report that processing of endogenous APP is down-regulated during the aging of normal human fibroblasts (IMR-90). The generation of intracellular APP cleavage products C99, C83, and AICD gradually declines with increasing life span and is accompanied by a reduced secretion of soluble APP (sAPP) and sAPPα. Further, the maturation of APP was reduced in senescent cells, which has been shown to be directly mediated by age-associated increased cellular cholesterol levels. Of the APP processing secretases, protein levels of constituents of the γ-secretase complex, presenilin-1 (PS1) and nicastrin, were progressively reduced during aging, resulting in a progressive decrease in γ-secretase enzymatic activity. ADAM10 (a disintegrin and metalloprotease 10) and BACE (β-site APP-cleaving enzyme) protein levels exhibited no age-associated regulation, but interestingly, BACE enzymatic activity was increased in aged cells. PS1 and BACE are located in detergent-resistant membranes (DRMs), well structured membrane microdomains exhibiting high levels of cholesterol, and caveolin-1. Although total levels of both structural components of DRMs were up-regulated in aged cells, their particular DRM association was decreased. This age-dependent membrane modification was associated with an altered distribution of PS1 and BACE between DRM and non-DRM fractions, very likely affecting their APP processing potential. In conclusion, we have found a significant modulation of endogenous APP processing and maturation in human fibroblasts caused by age-associated alterations in cellular biochemistry. Down-regulation of endogenous amyloid precursor protein processing due to cellular aging. VOLUME 281 (2006) PAGES 2405-2413Journal of Biological ChemistryVol. 281Issue 18PreviewPAGES 2407-2408: Full-Text PDF Open Access Aging is the most prevailing risk factor of Alzheimer disease, even though the biochemical basis of this association is unknown. A significant pathological feature of Alzheimer disease is the appearance of senile plaques that are composed primarily of amyloid β (Aβ), 2The abbreviations used are: Aβ, amyloid β; APP, amyloid precursor protein; ADAM10, a disintegrin and metalloprotease 10; AICD, APP intracellular domain; APP, amyloid precursor protein; BACE, β-site APP cleaving enzyme; CTF, C-terminal fragment; DRM, detergent-resistant membranes; NHF, normal human fibroblast; PDL, population doubling level; PS1, presenilin-1; sAPP, soluble APP; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MβCD, methyl-β-cyclodextrin. a 38–42-amino-acid peptide derived from proteolytic processing of the ubiquitously expressed amyloid precursor protein (APP) (1Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (11025) Google Scholar). At least three APP processing secretases are identified. ADAM10 (α-secretase) is involved in non-amyloidogenic processing and cleaves APP within the Aβ domain, whereby release of Aβ is prevented and soluble sAPPα is secreted (2Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (985) Google Scholar). Amyloidogenic processing is driven by BACE (β-secretase), which cleaves APP at the N-terminal site of the Aβ domain (3Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3308) Google Scholar). Subsequently, a complex of presenilin, nicastrin, anterior pharynx defective-1 and presenilin enhancer-2 (γ-secretase complex) cleaves the generated C-terminal fragments (CTFs) C83 or C99 at the C-terminal site of the Aβ domain (4Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld J. 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 (713) Google Scholar, 5Lee S.F. Shah S. Li H. Yu C. Han W. Yu G. J. Biol. Chem. 2002; 277: 45013-45019Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 6Yu G. Nishimura M. Arawaka S. Levitan D. Zhang L. Tandon A. Song Y.Q. Rogaeva E. Chen F. Kawarai T. Supala A. Levesque L. Yu H. Yang D.S. Holmes E. Milman P. Liang Y. Zhang D.M. Xu D.H. Sato C. Rogaev E. Smith M. Janus C. Zhang Y. Aebersold R. Farrer L.S. Sorbi S. Bruni A. Fraser P. St. George-Hyslop P. Nature. 2000; 407: 48-54Crossref PubMed Scopus (824) Google Scholar). This results in the generation of the APP intracellular domain (AICD), and in the amyloidogenic pathway, Aβ is released (7Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5170) Google Scholar, 8Haass C. EMBO J. 2004; 23: 483-488Crossref PubMed Scopus (487) Google Scholar). BACE and the γ-secretase complex have been well described to be associated to detergent-resistant membranes (DRMs) or lipid rafts (9Riddell D.R. Christie G. Hussain I. Dingwall C. Curr. Biol. 2001; 11: 1288-1293Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 10Vetrivel K.S. Cheng H. Lin W. Sakurai T. Li T. Nukina N. Wong P.C. Xu H. Thinakaran G. J. Biol. Chem. 2004; 279: 44945-44954Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). These well structured membrane microdomains exhibit high levels of cholesterol, sphingolipids, and structural protein compounds, such as caveolin (11Simons K. Vaz W.L. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 269-295Crossref PubMed Scopus (1361) Google Scholar). They function as specialized membrane compartments for channeling and integrating external stimuli into downstream pathways and are also implicated in membrane trafficking (12Stuermer C.A. Plattner H. Gustafsson J.A. Biochem. Soc. Symp. 2005; : 109-118PubMed Google Scholar, 13Lucero H.A. Robbins P.W. Arch. Biochem. Biophys. 2004; 426: 208-224Crossref PubMed Scopus (153) Google Scholar, 14Verkade P. Simons K. Histochem. Cell Biol. 1997; 108: 211-220Crossref PubMed Scopus (64) Google Scholar). Modulation of cellular cholesterol levels has been shown to affect amyloidogenic APP processing by altering the constitution of DRMs and thereby the potential of BACE processing activity (15Simons M. Keller P. Dichgans J. Schulz J.B. Neurology. 2001; 57: 1089-1093Crossref PubMed Scopus (237) Google Scholar, 16Ehehalt R. Keller P. Haass C. Thiele C. Simons K. J. Cell Biol. 2003; 160: 113-123Crossref PubMed Scopus (926) Google Scholar, 17Abad-Rodriguez J. Ledesma M.D. Craessaerts K. Perga S. Medina M. Delacourte A. Dingwall C. De Strooper B. Dotti C.G. J. Cell Biol. 2004; 167: 953-960Crossref PubMed Scopus (293) Google Scholar). Although APP processing has been extensively studied in cellular models, the influence of cellular aging on the biochemistry of APP processing has not been investigated in molecular detail so far. Normal human fibroblasts (NHFs) exhibit a well established cellular aging model. In culture, they undergo a limited number of population doubling levels (PDLs) before entering a state of irreversible growth arrest (18Hayflick L. Moorhead P.S. Exp. Cell Res. 1961; 25: 585-621Crossref PubMed Scopus (5527) Google Scholar). At this end-stage of their in vitro mitotic life span, fibroblasts can be maintained and remain metabolically active but cannot be driven into further cell cycling (19Cristofalo V.J. Aging (Milano). 1999; 11: 1-3PubMed Google Scholar). Although the exact molecular mechanisms of this replicative senescence have not been fully understood, multiple age-dependent cellular alterations are described. These include morphological changes with enlarged cell size, dysregulation of protein degradation, as well as post-translational modification of proteins and unresponsiveness to external stimuli (20Campisi J. Dimri G.P. Nehlin J.O. Testori A. Yoshimoto K. Exp. Gerontol. 1996; 31: 7-12Crossref PubMed Scopus (35) Google Scholar, 21Cristofalo V.J. Pignolo R.J. Exp. Gerontol. 1996; 31: 111-123Crossref PubMed Scopus (57) Google Scholar). In this study, we examined age-associated alterations in the processing of endogenous APP by analyzing intracellular and secreted APP cleavage products throughout the life span of NHFs by immunoblotting as well as pulse-chase analysis. The impact of cholesterol on APP maturation was determined by depletion of intracellular cholesterol levels. We further focused on age-dependent changes in secretase levels, enzymatic activity of BACE, and the γ-secretase complex, as well as the impact of age-related alterations in DRM integrity on proteins involved in APP processing. Cell Line and Culture Conditions—IMR-90 NHFs (Coriell Institute for Medical Research, Camden, NJ) were grown in phenol red-free Dulbecco's modified Eagle's medium (Invitrogen) supplemented with antibiotics (Invitrogen), 1 mm sodium pyruvate (Invitrogen), and 10% charcoal-dextran-treated fetal calf serum (HyClone). These deficient culture conditions accelerate aging without influencing the resulting general aged phenotype, as described previously (22Atamna H. Robinson C. Ingersoll R. Elliott H. Ames B.N. FASEB J. 2001; 15: 2196-2204Crossref PubMed Scopus (39) Google Scholar). At subconfluency, cells were passaged by trypsinization. PDLs were calculated as (log Ch – log Cs)/log (2Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (985) Google Scholar), where Ch and Cs are defined as the cell number harvested and seeded, respectively. The aged phenotype was identified by use of a senescence-associated β-galactosidase staining kit (Cell Signaling), following the manufacturer's instructions. Immunoblotting—Adherent fibroblasts were scraped off the plates in lysis buffer (60 mm Tris-HCl, 2% SDS, 10% sucrose) sonicated briefly, and boiled for 5 min. Equal amounts of protein were separated on a SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane using a wet blot chamber (Bio-Rad). Commercial 4–12% NuPage BisTris gels (Invitrogen) were used to simultaneously separate high and low molecular weight proteins. For analysis of secreted APP, the medium was collected after 72 h, and the cells were scraped off the plates, as described above. According to the cell lysate protein concentration, the medium was loaded on an 8% SDS-polyacrylamide gel, separated, and transferred onto a nitrocellulose membrane. The blots were incubated overnight with the primary antibody at 4 °C. Proteins were detected using the ECL kit (Amersham Biosciences) after incubation for 2 h at room temperature with the appropriate secondary antibody. For detection of BACE, a biotinylated secondary antibody was used. Primary antibodies used were APP C-terminal antibody A8717 (Sigma), APP cytoplasmic domain polyclonal antibody CT-15 (kind gift of Dr. C. U. Pietrzik), APP mid-region polyclonal antiserum 863 (kind gift of Dr. S. Weggen), human APP monoclonal antibody (clone 6E10) (Biocat), actin (Santa Cruz Biotechnology), ADAM10 (Chemicon), BACE (Ab-2) (Oncogene), caveolin-1 (Transduction Laboratories), nicastrin (Sigma), Notch (Santa Cruz Biotechnology), PS1 N-terminal (3110), which was raised against a fusion protein of the maltose-binding protein and amino acids 2–80 of human PS1. Quantitative Real-time Reverse Transcription-PCR—Total RNA from cells grown to subconfluency was prepared using the Absolutely RNA reverse transcription-PCR miniprep Kit (Stratagene) according to the manufacturer's instructions. Reverse transcription was performed on 500 ng of total RNA with the Omniscript reverse transcriptase kit (Qiagen) according to the supplier's instructions. The quantitative real-time PCR was carried out in triplicates containing 1 μl of cDNA, 100 pmol sense and antisense primer (sense, 5′-GGAGCTCCTTCCCGTGAATGG-3′; antisense, 5′-CGTAGCCGTTCTGCTGCATC-3′) and 12.5 μl of 2× SYBR Green Supermix (Bio-Rad) in a final volume of 25 μl. PCR was performed using the iCycler (Bio-Rad). PCR conditions were as follows: initial denaturation at 95 °C for 3 min, 35 cycles of 95 °C for 30 s, 59 °C for 20 s, 72 °C for 30 s, and final elongation at 72 °C for 5 min. The generation of specific PCR products was confirmed by melting curve analysis. The PCR cycle number that generated the first fluorescence signal above threshold (threshold cycle, Ct) was determined. Glyceraldehyde-3-phosphate dehydrogenase served for normalizations and calculation of ΔCt. Metabolic Labeling—IMR-90 cultures were incubated with methionine-free Dulbecco's modified Eagle's medium supplemented with 150 μCi/ml [35S]methionine/cystein for 15 min (pulse). Cells were lysed immediately or chased for the indicated time periods. APP and APP-CTFs were immunoprecipitated using the APP-CT antibody 140. This antibody was raised against the C-terminal 20 amino acids of human APP, which were conjugated to keyhole limpet hemocyanin before inoculation in rabbits. Immunoprecipitates were separated by SDS-PAGE. Radiolabeled proteins were detected and quantified by phosphorimaging. Determination of Free Cholesterol—Free unesterified cholesterol species were analyzed using the Amplex Red cholesterol assay kit (Molecular Probes) following the manufacturer's instructions. For determination of the cholesterol content in fractions of sucrose gradients for DRM isolation, 50 μl of each fraction were analyzed. Isolation of Detergent-resistant Membranes—Cells were scraped off the plates in phosphate-buffered saline and lysed in 300 μl of lysis buffer (20 mm CHAPS, 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride). The cell lysate was mixed with the same volume, 80% sucrose (in lysis buffer without CHAPS). The resulting 40% sucrose solution was overlaid by a step gradient of 1.3 ml of 30% and 0.25 ml of 5% sucrose. By centrifugation at 200,000 × g for 2 h at 4 °C, DRMs floated at the border of 5 and 30% sucrose. Fractions were collected, and 30 μl of each were analyzed by immunoblotting. Determination of β- and γ-Secretase Activity—Enzymatic activities of β- and γ-secretase were analyzed using the β-Secretase Activity assay kit (BioVision) or the γ-Secretase Activity assay kit (R & D Systems), respectively, following the manufacturer's instructions. For analysis of total cellular activity, the same amounts of cellular protein were used. Activity measurements in fractions of sucrose gradients for DRM isolation were done with 50 μl of each fraction. Statistical Analysis—Optical band densities from immunoblotting were measured with Aida Image Analyzer version 3.28 software (Ray-test). Mean values of data from at least three independent experiments were subsequently calculated and plotted as percentage change compared with young cells (PDL 20/23), which have been set to 100%. Standard deviations were calculated by analysis of variance using Sigma Stat software (SPSS Science). Significance was analyzed using the post hoc Tukey test. Levels of Mature APP and APP Cleavage Products Decrease during Cellular Aging—IMR-90 NHFs were sequentially passaged until growth arrest of the cells at PDL46. The aged phenotype was determined using a senescence-associated β-galactosidase staining, which identified almost 100% of stained cells at PDL 46 (data not shown). Processing of endogenous APP throughout the in vitro life span of the NHF culture was analyzed by monitoring the protein levels of full-length APP, the intracellular cleavage products C99, C83, and AICD, as well as the secreted cleavage products sAPP and sAPPα at increasing PDLs by immunoblotting (Fig. 1). We observed ∼50% reduction in the ratio of mature to immature APP in senescent cells (Fig. 1A). Real-time PCR analysis did not show any decrease in APP gene expression (data not shown), and additionally, no significant alterations in immature APP protein levels were detected. Levels of the intracellular APP cleavage products C99 and C83, deriving from either β-or α-secretory cleavage, respectively, significantly decreased from young to old PDLs, resulting in reduced ratios of these C-terminal fragments (CTFs) to total or mature APP. C99 and C83 are further processed by the γ-secretase complex, leading to the generation of AICD. This ∼6-kDa APP-CTF is known to be rapidly degraded and has not been shown at endogenous cellular levels previously. To verify the AICD identity, we treated young fibroblasts (PDL 25) with the γ-secretase inhibitor H-5106 (Sigma), after which the ∼6-kDa band was not detectable anymore (Fig. 1B). In addition, a further APP C-terminal antibody was used, which is well described to be specific for AICD (23Goldgaber D. Lerman M.I. McBride O.W. Saffiotti U. Gajdusek D.C. Science. 1987; 235: 877-880Crossref PubMed Scopus (1026) Google Scholar). This antibody also recognized the 6-kDa band, emphasizing the identity of the fragment (supplemental Fig. 1). Thus, we suggest that this APP-derived fragment indeed resembles endogenous AICD. During aging, detectable levels of AICD decreased rapidly, reflecting the progressive decline in C99 as well as C83 generation. Therefore, we conclude that APP processing is gradually reduced throughout the life span of NHFs, resulting in lowered intracellular APP-CTF levels. To further confirm the age-related decline in endogenous APP processing, we analyzed the secreted cleavage products of APP (Fig. 1C). Although secretion of total sAPP and sAPPα decreased, no significant age-associated alteration in the ratio of sAPPα to sAPP was detected. Maturation of APP Is Reduced during Cellular Aging—Interestingly, we observed an age-associated reduction in levels of mature APP as well as APP-CTFs. We next examined the kinetics of APP metabolism by pulse-chase experiments. Analysis of the time course of APP maturation demonstrated a significant delay in generation of mature APP in senescent NHFs (Fig. 2A). Maturation of 50% APP (based on immature APP levels at t = 0 min) was reached after ∼15 min in young cells (PDL 23), whereas ∼24 min were needed in cells at PDL 46. As shown by immunoblotting (Fig. 1A), total levels of mature APP were significantly reduced in aged cells. The half-lives of mature as well as immature APP (∼60 min) did not show an age-associated alteration, suggesting that degradation rates of APP were unchanged during aging. Thus, the reduced levels of mature APP observed in cells at PDL 46, were caused by an age-associated down-regulation in the efficiency of APP maturation, rather than by an enhanced degradation or processing. We also found that APP-CTFs were generated earlier and reached higher levels in young cells as compared with aged cells (Fig. 2B). Together, these data demonstrate that maturation and proteolytic processing of APP is down-regulated during cellular aging. Age-associated Increased Cholesterol Levels Inhibit APP Maturation—We observed a significant reduction in APP maturation efficiency in cells at PDL 46, which resulted in decreased mature APP protein levels. Previously, it was shown that APP maturation is affected by increased cholesterol levels (24Galbete J.L. Martin T.R. Peressini E. Modena P. Bianchi R. Forloni G. Biochem. J. 2000; 348: 307-313Crossref PubMed Scopus (103) Google Scholar, 25Borroni B. Colciaghi F. Lenzi G.L. Caimi L. Cattabeni F. Di Luca M. Padovani A. Neurobiol. Aging. 2003; 24: 631-636Crossref PubMed Scopus (25) Google Scholar) and analysis of intracellular cholesterol in the NHF aging model detected an age-associated increase by 37% (±4, p = 0.006, n = 3) (data not shown), consistent with a previous study (26Nakamura M. Kondo H. Shimada Y. Waheed A.A. Ohno-Iwashita Y. Exp. Cell Res. 2003; 290: 381-390Crossref PubMed Scopus (18) Google Scholar). To determine whether age-related increased levels of cellular cholesterol affect APP maturation, we depleted cholesterol by treatment with methyl-β-cyclodextrin (MβCD), exactly as shown previously (27Kojro E. Gimpl G. Lammich S. Marz W. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5815-5820Crossref PubMed Scopus (728) Google Scholar). After cholesterol depletion, levels of mature APP were selectively increased in aged cells as compared with untreated cells, without any effect on immature APP levels (Fig. 3). This resulted in an increased ratio of mature to immature APP, almost to the level of young cells. Thus, APP maturation was recovered after depletion of cellular cholesterol, demonstrating that the age-associated decreased maturation efficiency of APP was directly affected by increased levels of intracellular cholesterol. Notably, MβCD treatment of young cells did not significantly affect APP maturation. Further, depletion of cholesterol is well described to affect the cleavage of APP, resulting in enhanced non-amyloidogenic processing (27Kojro E. Gimpl G. Lammich S. Marz W. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5815-5820Crossref PubMed Scopus (728) Google Scholar). However, we observed no significant influence on C83 levels after MβCD treatment. The rather short time of treatment, necessary to avoid the toxic potential of MβCD as well as the slow turnover of APP-CTFs, very likely did not permit resolution of these changes at levels of endogenous APP processing. In addition, analysis of secreted APP cleavage products did not detect differences in APP processing in comparison to untreated cells (data not shown). Age-associated Changes for Constituents of the γ-Secretase Complex—To analyze age-associated alterations in levels of enzymes involved in APP processing, we examined protein levels of ADAM10, BACE, PS1, and nicastrin throughout the life span of NHFs. Immunoblotting detected a selective down-regulation of PS1 and nicastrin, whereas ADAM10 and BACE protein levels were not altered (Fig. 4). Presenilin undergoes endoproteolysis, which results in a C- and N-terminal subunit (28Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (940) Google Scholar). Protein levels of the PS1 N-terminal subunit as well as of the full-length precursor protein were significantly reduced with increasing PDLs, without any change in the ratio of the PS1 N-terminal subunit to full-length protein. Therefore, cellular aging was not affecting PS1 maturation. Additionally, protein levels of mature nicastrin, another constituent of the γ-secretase complex, were down-regulated throughout the life span of the NHF culture, confirming that total γ-secretase complex levels were reduced during aging. Protein levels of ADAM10 exhibited no age-associated decrease. The inactive proform is cleaved by secretory proprotein convertases generating the active protease (29Anders A. Gilbert S. Garten W. Postina R. Fahrenholz F. FASEB J. 2001; 15: 1837-1839Crossref PubMed Scopus (187) Google Scholar). This maturation process was not exhibiting any changes with increasing PDLs. Also for BACE, no age-dependent alterations in total protein levels and maturation efficiency, mediated by a furin-like convertase (30Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.C. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), were detected (see supplemental Fig. 2 for graphical representations of maturation efficiencies). Age-associated decrease in DRM integrity affects the distribution of proteins involved in APP processing—To analyze age-associated alterations in membrane constitution and its influence on proteins involved in APP processing, we isolated DRMs, indicating lipid rafts, by sucrose gradient ultracentrifugation, as previously described (16Ehehalt R. Keller P. Haass C. Thiele C. Simons K. J. Cell Biol. 2003; 160: 113-123Crossref PubMed Scopus (926) Google Scholar, 17Abad-Rodriguez J. Ledesma M.D. Craessaerts K. Perga S. Medina M. Delacourte A. Dingwall C. De Strooper B. Dotti C.G. J. Cell Biol. 2004; 167: 953-960Crossref PubMed Scopus (293) Google Scholar). Thereby, due to their buoyant density, DRMs accumulate between 5 and 30% sucrose (Fig. 5, A and C, fraction 2). Analyzing cholesterol levels within fractions of DRM isolations, we found decreased levels within DRM fractions of aged cells (Fig. 5A), even though total intracellular cholesterol levels were increased, consistent with a previous report (26Nakamura M. Kondo H. Shimada Y. Waheed A.A. Ohno-Iwashita Y. Exp. Cell Res. 2003; 290: 381-390Crossref PubMed Scopus (18) Google Scholar). Furthermore, total caveolin-1 protein levels were progressively up-regulated during cellular aging and, interestingly, caveolin-1 was also observed to significantly migrate into the non-DRM fraction of senescent cells (Fig. 5, B and C, upper panels). To investigate whether this age-dependent disintegration of DRMs or reduction in total DRM levels influenced the localization of APP and its processing secretases, we analyzed their distribution between DRM and non-DRM fractions by immunoblotting (Fig. 5C). In agreement with previous reports, significant levels of BACE, PS1, and also APP were DRM-associated (9Riddell D.R. Christie G. Hussain I. Dingwall C. Curr. Biol. 2001; 11: 1288-1293Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 10Vetrivel K.S. Cheng H. Lin W. Sakurai T. Li T. Nukina N. Wong P.C. Xu H. Thinakaran G. J. Biol. Chem. 2004; 279: 44945-44954Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 16Ehehalt R. Keller P. Haass C. Thiele C. Simons K. J. Cell Biol. 2003; 160: 113-123Crossref PubMed Scopus (926) Google Scholar), and as expected, their immature precursors were almost completely excluded from DRMs of young cells. Senescent fibroblasts exhibited an altered pattern of distribution. Increased levels of immature APP accumulated within DRMs and significant levels of PS1 migrated into the non-DRM fraction, reflecting the age-associated reduction in DRM integrity or total levels. DRM-associated levels of total BACE were not decreased in senescent cells, but interestingly, mature BACE was almost completely replaced by the immature form, resulting in only marginal levels of DRM-associated mature protease. β-Secretase Enzymatic Activity Is Increased in Aged Cells and Migrates into the Non-DRM Fraction, whereas γ-Secretase Activity Progressively Decreases—Because we observed an age-related down-regulation in the generation of C99 and AICD, we analyzed alterations in enzymatic activity of β- and γ-secretase during cellular aging. β-secretase enzymatic activity was analyzed in total cell lysates and in fractions of DRM isolations. Interestingly, senescent fibroblasts exhibited a significant increase in total BACE activity by 44% in comparison to young and middle-aged cells (Fig. 6A). Within fractions of DRM isolations, β-secretase enzymatic activity was prominently present within DRMs of young cells (PDL 20) but migrated into the non-DRM fraction of cells at PDL 46 (Fig. 6B). Thus, we observed a permanent co-localization of enzymatic activity with the mature form of the enzyme. Analyzing changes in γ-secretase enzymatic activity during aging detected a progressive down-regulation in total enzymatic activity, which reflected the decrease in PS1 N-terminal subunit and nicastrin protein levels and, thus, total γ-secretase levels (Fig. 6C). To analyze whether the age-related reduction in γ-secretase activity exclusively affected APP processing or als" @default.
• W2040373875 created "2016-06-24" @default.
• W2040373875 creator A5019893954 @default.
• W2040373875 creator A5020194269 @default.
• W2040373875 creator A5046807483 @default.
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• W2040373875 date "2006-02-01" @default.
• W2040373875 modified "2023-09-30" @default.
• W2040373875 title "Down-regulation of Endogenous Amyloid Precursor Protein Processing due to Cellular Aging" @default.
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