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NATURE CELL BIOLOGY VOL 2 DECEMBER 2000 http://cellbio.nature.com 863

Upregulation of BiP and CHOP by the unfolded-protein response is independent of presenilin expression

Naoyuki Sato*, Fumihiko Urano†, Jae Yoon Leem*, Seong-Hun Kim*, Mingqing Li‡, Dorit Donoviel§, AlanBernstein§, Amy S. Lee‡, David Ron†, Margaret L. Veselits*, Sangram S. Sisodia* and Gopal Thinakaran*¶

*Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Knapp R212, 924 East 57th street, Chicago, Illinois, 60637, USA†Skirball Institute of Biomolecular Medicine, New York University Medical School, New York, New York 10016, USA

‡Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, LosAngeles, California, 90089, USA

§Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S-1A8,Canada.

¶e-mail: [email protected]

Presenilin 1 (PS1), a polytopic membrane protein, has a critical role in the trafficking and proteolysis of a selectedset of transmembrane proteins. The vast majority of individuals affected with early onset familial Alzheimer’s dis-ease (FAD) carry missense mutations in PS1. Two studies have suggested that loss of PS1 function, or expression ofFAD-linked PS1 variants, compromises the mammalian unfolded-protein response (UPR), and we sought to evaluatethe potential role of PS1 in the mammalian UPR. Here we show that that neither the endoplasmic reticulum (ER)stress-induced accumulation of BiP and CHOP messenger RNA, nor the activation of ER stress kinases IRE1α andPERK, is compromised in cells lacking both PS1 and PS2 or in cells expressing FAD-linked PS1 variants. We alsoshow that the levels of BiP are not significantly different in the brains of individuals with sporadic Alzheimer’s dis-ease or PS1-mediated FAD to levels in control brains. Our findings provide evidence that neither loss of PS1 andPS2 function, nor expression of PS1 variants, has a discernable impact on ER stress-mediated induction of the sev-eral established ‘readouts’ of the UPR pathway.

A lzheimer’s disease (AD), a progressive dementia in the elder-ly, is characterized pathologically by the presence of intracel-lular neurofibrillary tangles and the deposition of β-amyloid

(Aβ) peptides of 40–42 residues in the brains of effected individu-als1,2. A subset of AD with the age of onset in the fourth to sixth

decade is classified as familial early onset AD (FAD). FAD is inher-ited as an autosomal dominant disorder, and mutations in the genesencoding presenilin 1 (PS1) and presenilin 2 (PS2) co-segregatewith most pedigrees3–5. PS1, a polytopic membrane protein thataccumulates as endoproteolytic derivatives in vivo6, is required for

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Figure 1 UPR-mediated induction of BiP and CHOP expression in primaryPS1–/– and PS1+/– fibroblasts treated with tunicamycin. a, Primary mousefibroblasts derived from PS1–/– and PS1+/– embryos were incubated with tuni-camycin (2 µg ml−1) for 5 h. Detergent lysates of tunicamycin treated and controlcultures were analysed by immunoblotting with PS1NT, anti-PS1Loop, anti-KDEL,anti-CHOP and anti-β-tubulin antibodies. PS1-derived N- and C-terminal endoprote-olytic fragments are absent in PS1–/– fibroblasts. Anti-KDEL antibody reacted withBiP and GRP94 (another UPR-regulated protein). Note that treatment with tuni-camycin increased the levels of CHOP to levels comparable to those in PS1–/– and

PS1+/– fibroblasts; BiP polypeptide levels remained unchanged. Anti-β-tubulin anti-body indicated equivalent protein loading on all lanes. Asterisk indicates a PS1NT-reactive polypeptide that is unrelated to PS1 NTF. CTF, C-terminal fragment; NTF,N-terminal fragment. b, Subconfluent cultures of primary PS1–/– and PS1+/– fibrob-lasts were incubated with tunicamycin (Tun; 2 µg ml–1) for various periods (0, 2, 3,5 or 7 h). Total RNAs isolated from the fibroblasts were subjected to northern blotanalysis. Blots were sequentially hybridized with BiP, CHOP and GAPDH probes,and the signals quantified by Phosphorimaging. BiP and CHOP mRNA levels werenormalized to GAPDH mRNA.


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intramembraneous cleavage of β-amyloid precursor protein (APP),amyloid precursor-like protein 1, and Notch 1 (refs 7–9). Moreover,FAD-linked PS1 and PS2 variants elevate selectively the productionof longer, and highly fibrillogenic Aβ peptides (Aβ42)10–13. Recently,Niwa et al.14 reported that, under conditions that induce the UPR,PS1 regulates endoproteolysis and nuclear translocation of thetransmembrane protein kinases IRE1α and IRE1β. Moreover,Katayama et al.15 reported that expression of FAD-linked PS1 alsocompromises the mammalian UPR.

The UPR, which involves the coordinate transcriptional activa-tion of a set of genes that encode ER chaperones and certain cell-death signals, occurs as a result of the accumulation of misfoldedproteins in the ER16,17. The UPR is activated by agents that affect ERhomeostasis, such as tunicamycin, thapsigargin, Ca2+ ionophoresand reducing agents. In yeast, the most proximal reactions necessaryfor promoting the UPR are the dimerization and trans-autophospho-rylation of the ER-resident, transmembrane kinase Ire1p16,18. In mam-malian cells, two IRE1p homologues, IRE1α and IRE1β, have beenidentified. ER stress causes dissociation of BiP/GRP78 (animmunoglobulin-γ-binding protein) from the lumenal, stress-sensingdomain of IRE1α and IRE1β and leads to their oligomerization,which activates trans-autophosphorylation and downstream sig-nalling to the UPR18–21. The carboxy-terminal effector domain ofIREs may, under certain circumstances, be liberated from the full-length protein by proteolysis and translocated to the nucleus14.

The prototypical inducers of the UPR are tunicamycin, a nucle-oside antibiotic that inhibits N-glycosylation of target asparagine

residues in the lumenal domains of proteins expressed in the ER,and thapsigargin, an inhibitor of the ER Ca2+ ATPase. The cellularresponse to these ER stressors is to activate transcriptionally genesthat encode protein chaperones, such as BiP, GRP94, proteindisulphide isomerase and Erp72, and the transcription factorCHOP (CCAAT/enhancer-binding protein (C/EBP)-homologousprotein)16,22,23.

In contrast to the previous studies that reported that the UPR isimpaired by loss of PS1 function or expression of mutant PS1, wehave failed to find defects in the induction of BiP and CHOP inprimary PS1–/– fibroblasts, immortalized PS1–/–/PS2–/– cell lines,mouse N2a neuroblastoma and human 293 cell lines expressing adominant-negative loss-of-function PS1 mutant or several FAD-linked PS1 variants. Furthermore, the steady-state levels of BiPprotein appear to be unaltered in brain tissue of individuals withPS1-mediated FAD or in brain tissue of transgenic mice express-ing mutant PS1. Collectively, our results show that neither theexpression of FAD-linked variants, nor the loss of PS1 functioncompromises the activation of BiP and CHOP by the UPR.

ResultsInduction of UPR does not require PS1 and PS2. Niwa et al.14 havereported that in a PS1–/– fibroblast line the activation of BiP mRNAwas impaired under conditions that induce the UPR14. To re-examine this issue, we assessed the coordinate induction of expres-sion of the ER-resident chaperone BiP and the nuclear protein

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Figure 2 ER stress signalling and the induction of BiP and CHOP mRNAs inPS1–/–/PS2–/– double-null cells. a, The genotype of PS1 and PS2 alleles in wild-type (pBD6 and pBD18) and PS1–/–/PS2–/– double-null cell lines (BD1 and BD8) wasanalysed as described34,35. PCR products amplified from the wild-type (WT) and tar-geted (MUT) alleles are marked. b, ER stress-activated phosphorylation of endoge-nous IRE1α and PERK were analysed by combined immunoprecipitation/ westernblot analysis of lysates from wild-type (pBD6) and PS1–/–/PS2–/– double-null cells(BD1) treated with tunicamycin (Tun) or thapsigargin (Tg) for the indicated time peri-ods. In lanes 7 and 14 immunoprecipitates were treated with alkaline phosphatase(AP) before being loaded on the gel. P-IRE1, phosphorylated IRE1α; P-PERK, phos-

phorylated PERK. C, untreated control. c, The levels of BiP and CHOP mRNAs inwild-type (pBD6) and PS1–/–/PS2–/– double-null cells (BD1) were examined by north-ern blot analysis. Cells were untreated or treated with 2.5 µg ml–1 tunicamycin forthe indicated time periods. Blots were hybridized with actin probes without remov-ing BiP probes to visualize both mRNA species. d, The levels of BiP and CHOPmRNAs in wild-type and PS1–/–/PS2–/– double-null cell lines were examined asdescribed above. Cells were treated as follows: C, untreated control; Tun,2.5 µg ml–1 tunicamycin for 7 h; Tg, 1 mM thapsigargin for 7 h. Blots werehybridized with actin probes as in c.


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CHOP in primary fibroblasts generated from PS1–/– and PS1+/–

mouse embryos. Western blot analyses did not show differences inthe steady-state levels of BiP and CHOP between PS1–/– and PS1+/–

fibroblasts under basal conditions, or after treatment with tuni-camycin for 5 h (Fig. 1a). As protein accumulation studies may notreflect accurately induction of gene expression, we examined thekinetics of UPR-induced accumulation of mRNAs encoding BiP orCHOP in PS1–/– and PS1+/– primary fibroblasts incubated for vari-ous periods of time in tunicamycin. BiP and CHOP mRNAhybridization signals from northern blots were quantified by phos-phorimaging and normalized to GAPDH mRNA levels. As expected,the levels of BiP and CHOP mRNA were induced, and accumulatedin a time-dependent fashion after treatment of PS1–/– or PS1+/–

fibroblasts with tunicamycin. Notably, the kinetics and levels ofaccumulated transcripts derived from either gene were no differentin primary PS1–/– or PS1+/– fibroblasts (Fig. 1b).

We next considered whether endogenous expression of the PS1homologue, PS2, might compensate for loss of PS1 function inPS1–/– cells, and so blunt potential changes in the UPR. To assesswhether UPR-related gene expression is affected by the lack of bothPS1 and PS2, we immortalized blastocyst-derived cells from wild-type mouse embryos (Fig. 2a; cell lines pBD6 and pBD18) orembryos with targeted disruptions of both PS1 and PS2 (Fig. 2a;cell lines BD1, BD8), and induced ER stress by incubating cellmonolayers with tunicamycin and thapsigargin. Northern blotanalyses showed a time-dependent accumulation of BiP and CHOPmRNA in wild-type pDB6 cells and PS1–/–/PS2–/– mutant BD1 cells(Fig. 2c). Analyses of additional clonal lines showed that the steady-state levels of BiP mRNA differed in the four lines of wild-type andPS1–/–/PS2–/– cells examined, but levels of BiP and CHOP mRNA

increased in all clones after treatment with the ER stressors tuni-camycin and thapsigargin (Fig. 2d). These data strongly suggestthat PS1 and PS2 are not required for ER stress-induced upregula-tion of BiP and CHOP mRNA.

As IRE1α and IRE1B sense the ER stress-induced accumulationof misfolded proteins and subsequently signal the activation of theUPR (reviewed in refs 16, 20), we determined whether PS1 or PS2is required for IRE1 activation. We examined the ER stress-inducedphosphorylation of IRE1α in wild-type and PS1–/–/PS2–/– cells aftertreatment with tunicamycin or thapsigargin. As expected24, a time-dependent increase in the levels of IRE1α polypeptides that exhib-ited retarded mobility on treatment with tunicamycin and thapsi-gargin was observed in wild-type cells (Fig. 2b). The shift in elec-trophoretic mobility of IRE1α is caused only by phosphorylation,as treatment of the immunoprecipitates with alkaline phosphataserestores these species to a molecular weight similar to that observedin untreated cells. Similarly, and as predicted from our northernblot studies of BiP and CHOP mRNA, phosphorylation of IRE1αwas unaffected in PS1–/–/PS2–/– cells exposed to either tunicamycinor thapsigargin (Fig. 2b).

In response to ER stress, the UPR-mediated transcriptional acti-vation of selected mRNAs is accompanied by the induction of aparallel pathway that signals the inhibition of de novo protein syn-thesis. This latter pathway is dependent on the activity of PERK, aprotein kinase that phosphorylates the translation initiation factoreIF2α, thereby inhibiting an early step in mRNA translation24–26.PERK and IRE1s are coordinately regulated by similar upstreamsignals, and PERK activation is also associated with trans-autophosphorylation, which retards the protein’s mobility onSDS–PAGE gels21,24. We therefore sought to examine the phospho-

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Figure 3 Expression of the FAD-linked PS1 C410Y variant does not affect theUPR in stable N2a cell lines. a, PS1 expression was analysed in detergent lysatesprepared from parental N2a cell line Swe.10, or independent stable Swe.10 deriva-tives that express human wild-type PS1 (Wt.9 and Wt.15), or the FAD-linked PS1C410Y mutant (C410Y.6 and C410Y.11). Full-length PS1 (PS1 FL), and N- and C-ter-minal proteolytic derivatives (NTF and CTF, respectively) are indicated. Quantitativeanalysis of secreted Aβ40 and Aβ42 in the media conditioned by stable lines wasperformed using two-site ELISAs. The ratio Aβ42 / total Aβ was calculated fromthree independent experiments (nine samples) and the mean (± s.e.) is plotted. Cellsexpressing C410Y mutant PS1 secrete roughly twofold more Aβ42 than do Swe.10or wild-type PS1 lines; the total amount of Aβ40 was not significantly differentbetween control, wild-type PS1 and mutant PS1 cells. Asterisk, P < 0.0001. b, ERstress-activated phosphorylation of endogenous IRE1α and PERK was analysed incells treated with tunicamycin (Tun; 2 µg ml–1) for 5 h. Migration of both ER kinases

were retarded, indicative of phosphorylation, in tunicmycin treated wild-type andC410Y cells. c, The levels of BiP mRNA in wild-type PS1 and C410Y mutant PS1 celllines were analysed by northern blot. Cells were treated with tunicamycin (2 µg ml–1)for 5 h. Blots were sequentially hybridized with BiP and GAPDH probes, and the sig-nals quantified by Phosphorimaging. BiP mRNA levels were normalized to GAPDH lev-els and plotted. d, Stable N2a cell lines that express wild-type PS1 or C410Y PS1variant were co-transfected with the reporter plasmid BiP (–154CAT) and the controlSV40 β-galactosidase plasmid. Cells were treated with 2 µg ml–1 tunicamycin for 10or 20 h (three dishes each) before collection. Lysates were normalized for transfec-tion efficiency by β-galactosidase activity. BiP promoter activity was determined bythe percentage conversion of [14C]chloramphenicol to its acetylated forms and quan-tified by phosphorimaging. Fold-induction relative to untreated cultures for each lineis plotted. Treatment with tunicamycin induced transcription from the BiP (−154CAT)plasmid to similar levels in wild-type and PS1 mutant cell lines.


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rylation of PERK as a surrogate marker for ER stress signalling. Asshown in Fig. 2b, treatment with tunicamycin or thapsigarginresulted in a similar shift in PERK mobility in wild-type andPS1–/–/PS2–/– cells. Collectively, these results show that PS1 and PS2

are not required for the activation of ER stress kinases IRE1α andPERK, nor the induction of BiP and CHOP mRNA after ER stress.UPR induction in stable cell lines expressing FAD-linked PS1mutants. Katayama et al. 15 have shown that expression of FAD-linked


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equal protein loading (bottom). b, To quantify the levels of BiP and CHOP polypep-tides, blots containing primary antibodies were incubated with [125I]-labelled second-ary antibodies, and signal intensity was quantified by phosphorimaging and plotted.Despite the differences observed at time 0, the levels of BiP and CHOP uniformlyincreased after 8 and 16 h of tunicamycin treatment in wild-type PS1 and mutantPS1 cultures.

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pools expressing M146L and ∆E9 variants as compared with wild-type PS1 pool. C,untreated control. b, RNA was isolated from subconfluent wild-type PS1 and mutantPS1 cultures treated for 5 or 10 h with tunicamycin (2 µg ml–1) or thapsigargin (Tg;300 nM). The levels of BiP and GAPDH mRNA were analysed by northern blot.Hybridization signals were quantified by phosphorimaging and BiP signals were nor-malized to GAPDH mRNA levels and plotted. Note that no obvious difference in theinduction of BiP mRNA was observed between the different pools of cells analysed.


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PS1 impairs the UPR. We re-examined this issue in stable mouseN2a neuroblastoma cell lines that express a FAD-linked PS1 C410Yvariant. Western blot analysis using PS1 antibodies showed theexpression of human wild-type PS1 and mutant PS1 polypeptidesin these lines; and, as described previously6,27, overexpression of thehuman PS1 polypeptides resulted in the ‘replacement’ of mostmurine PS1 derivatives (Fig. 3a). Furthermore, as expected fromprevious results10,11, two-site enzyme-linked immunosorbant assays(ELISAs)28 confirmed that the levels of Aβ42 secreted by the PS1C410Y cell lines are twofold higher than those secreted by parentalSwe.10 or wild-type PS1 lines (Aβ42/total Aβ ratio Swe.10 andwild-type PS1 lines is 3.01 ± 0.09 versus C410Y lines, 6.37 ± 0.24;P < .0001) (Fig. 3a).

To determine whether the ER stress-mediated activation ofIRE1α or PERK is impaired by expression of mutant PS1, we treat-ed PS1 C410Y cell lines with tunicamycin for 5 h and examined thephosphorylation of IRE1α and PERK as described above. On expo-sure of the cells to tunicamycin, the migration of IRE1α and PERKwere retarded in all lines tested, indicating comparable activation ofboth kinases (Fig 3b). Quantification of BiP mRNA levels by north-ern blots, followed by normalization to GAPDH mRNA levels,showed that tunicamycin treatment results in increased accumula-tion of BiP mRNA comparable to levels in wild-type and PS1C410Y cell lines (Fig. 3c). As an independent measure of BiPmRNA induction, we also examined the effect of tunicamycintreatment on BiP promoter activity. For these studies, we transfect-ed wild-type and C410Y lines with BiP(–154CAT), a plasmid thatretains all the ER stress-inducible properties of the rat BiP promot-er29. As described previously29, treatment with tunicamycin causeda time-dependent increase in the promoter activity. Notably, thepromoter activity was induced to comparable levels in the wild-type PS1 and C410Y PS1 stable lines, indicating that the UPR-mediated increase in BiP transcription is not influenced by theexpression of FAD-linked PS1 variant (Fig. 3d).

To confirm the results of RNA analysis, we incubated the wild-type PS1 and C410Y mutant cell lines with tunicamycin for variousperiods of time, and performed western blots using anti-KDEL andanti-CHOP antibodies. We detected bound BiP and CHOP anti-bodies with [125I]-labelled secondary antibodies and quantifiedthem by phosphorimaging. These analyses showed that the basallevels of CHOP protein varied, but the levels of BiP polypeptidewere comparable in the four cell lines (Fig. 4a). After tunicamycin

treatment, CHOP protein accumulated to higher, but comparablelevels in wild-type PS1 and mutant PS1 cell lines. Quantification ofthe signals showed that the levels of BiP increased markedlybetween 8 and 16 h after tunicamycin treatment, whereas CHOPlevels increased after 4 h of treatment (Fig. 4b). Notably, we failedto find any defect in tunicamycin-induced accumulation of BiP orCHOP in the cell lines expressing the FAD-linked C410Y PS1mutant. Similar results were obtained in stable N2a cell linesexpressing additional FAD-linked mutants, including PS1 M146Lor PS1∆E9 (data not shown), and in stable human 293 cell lines inwhich wild-type PS1, PS1 A246E, or PS1∆E9 expression is regulat-ed by a muristerone-inducible promoter (data not shown).Induction of the UPR in stable pools of human 293 cells express-ing mutant PS1. Although we used over ten independent mouseN2a and human 293 cell lines expressing wild-type PS1 or mutantPS1 polypeptides, it was possible that the discordant results15 werecaused by clonal or cell-type variability. To obtain unambiguousconfirmation that the UPR is unimpaired by the expression of

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ionophore A23781 (7 µM) for 16 h. Detergent lysates were analysed by blottingwith PS1NT and anti-KDEL antibodies. Note that all three agents caused markedincreases in the levels of BiP and GRP94 polypeptides whether wild-type PS1 ormutant PS1 was expressed.

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Figure 7 Western blot analysis of BiP protein levels in brains of FAD patientsand mutant PS1 transgenic mice. a, Human cortical brain tissue of six age-matched controls (lanes 1–6), three FAD patients with a PS1 I143T mutation (lanes7–9), one FAD patient with a PS1 G384A mutation (lane 10) and six patients withsporadic AD (lanes 11–16) was homogenized in SDS extraction buffer. Aliquots oflysates were fractionated by SDS–PAGE, and immunoblotted with anti-KDEL (top),anti-calnexin (middle) and anti-β-tubulin (bottom) antibodies. The age at death of allindividuals is indicated. Note the marked differences in calnexin polypeptide levels,and the abnormal migration of β-tubulin in two FAD samples. b, The levels of BiPwere quantified from several western blots developed using [125I]-labelled secondaryantibodies, and normalized relative to β-tubulin levels. Statistical significance wasexamined using ANOVA followed by Scheffé’s test. BiP levels were not significantlydifferent between control and FAD samples (P = 0.17) or control and sporadic AD(SpAD) samples (P = 0.36). c, SDS homogenates were prepared from brain tissueof non-transgenic mice (NTg), or transgenic mice expressing wild-type human PS1(lines R8-1, S8-4), or FAD-linked A246E (lines B2-5 and N5) and M146L (lines I5 andE5) variants. Aliquots of lysates were fractionated by SDS–PAGE and immunoblot-ted with anti-PS1Loop and anti-KDEL antibodies. Note the lack of correlationbetween mutant PS1 C-terminal fragment expression (top) and BiP polypeptide lev-els (middle). Blots were re-probed with β-tubulin antibody (bottom) to visualize differ-ences in the amounts of proteins loaded on different lanes.


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mutant PS1, we generated stable pools of human 293 cells transfect-ed with bicistronic expression plasmids that encode PS1 and thehygromycin selection marker. Western blot analysis using PS1amino-terminal antibody showed transgene-derived expression offull-length and cleaved PS1 derivatives in cells transfected with wild-type, M146L and H163R PS1 complementary DNA, and uncleavedmutant PS1 polypeptide (owing to the lack of the site of endoprote-olysis) in cells transfected with PS1∆E9 cDNA. Consistent with ourresults from N2a cell lines, incubation in tunicamycin or thapsigar-gin led to comparable activation of IRE1α and PERK in stable 293pools expressing wild-type PS1, or M146L or ∆E9 variants (Fig. 5a).Furthermore, quantification of the BiP mRNA signals from north-ern blots showed that, regardless of the expression of wild-type ormutant PS1, BiP mRNA levels were induced to similar levels by theER stress agents tunicamycin and thapsigargin (Fig. 5b).

To confirm these results, we exposed subconfluent wild-type PS1and mutant PS1 cultures to tunicamycin, thapsigargin, or the Ca2+

ionophore, A23781, for 16 h and examined the levels of BiP andGRP94 polypeptides. Western blot analysis revealed that the levels offull-length PS1 or processed PS1 NTF were not affected by the treat-ments (Fig. 6, top panels). In contrast, all three ER stress agents test-ed increased markedly the levels of BiP and GRP94 polypeptides.Notably, we did not observe a defect in the induction of either BiP orGRP94 in cells that express FAD-linked M146L, H163R or ∆E9 vari-ants, as compared with vector control cells or cells that express wild-type PS1 (Fig. 6, bottom panels). Collectively, our results show thatexpression of FAD-linked mutant PS1 has no influence on UPR-related gene expression in cultured mammalian cell lines.BiP protein levels in human and transgenic mouse brains. Katayamaet al.15 have reported that the levels of BiP were reduced by ~75% inthe brains of individuals with FAD. We also examined the levels ofBiP in detergent homogenates prepared from the brains of sixpatients with sporadic AD, six age-matched controls, three patientswith a PS1 I143T mutation and one patient with a PS1 G384A muta-tion30 (Fig. 7a). Quantitative western blot analysis using anti-KDELantibody revealed subtle differences in the levels of BiP between indi-vidual samples in each group; however, quantification of BiP relativeto β-tubulin showed that the differences between groups were notstatistically significant (Fig. 7b). Re-probing the blot using anti-calnexin antibody showed marked differences in the levels of the twocalnexin-related polypeptides in three of the four FAD samples andtwo control samples. Notably, the expression of calnexin is not affect-ed by the UPR30. Similarly, probing with β-tubulin antibody alsorevealed differential migration of β-tubulin in two of the four FADsamples (Fig. 7a, lanes 8 and 9). Furthermore, Coomassie staining ofproteins also revealed distinct banding patterns for several polypep-tides in the samples examined (see Supplementary Information).

The differences in calnexin levels and β-tubulin mobility exem-plify the inherent limitations in analysing levels of specific polypep-tides in human brain, owing to the many variables associated withneurodegeneration, disease duration, pre- and postmortem hydroly-sis, and sample storage. We therefore examined BiP levels in freshlydissected brains of 2–3-month-old transgenic mice that expresshuman wild-type PS1 or FAD-linked M146L or A246E variants (Fig.7c); these mice have been characterized extensively with respect toPS1 expression and Aβ production6,31,32. Quantitative western blotanalyses of detergent homogenates prepared from the brains of acohort of animals of each genotype did not show differences in thelevels of BiP polypeptide between transgenic mice expressing wild-type or mutant PS1 (Fig. 7c). Collectively, our results show that thesteady-state levels of BiP polypeptide are not altered detectably in thebrains of humans or transgenic mice that express FAD-linked PS1variants.

DiscussionThe UPR, a pathway activated by agents that induce the accumula-tion of misfolded, secretory and membrane proteins in the ER,

involves the transcriptional induction of genes encoding proteinssuch as ER chaperones and selected transcription factors16. Recentstudies in PS1-deficient fibroblasts have suggested that PS1 mayhave a role in induction of the UPR14, and analysis of cells express-ing FAD-linked PS1 variants have indicated that these cells may berendered more susceptible to ER stress by impairing the UPR sig-nalling pathway15. Here we re-examined the involvement of PS1 inthe UPR using several experimental models. Contrary to expecta-tions, we now report that neither the activation of ER-stress kinas-es IRE1α and PERK, nor the coordinate induction of BiP andCHOP mRNA and protein is impaired either in cells lacking PS1function or in stable N2a neuroblastoma and human 293 cellsexpressing FAD-linked PS1 variants. In addition, and in contrast toprevious work that reported diminished steady-state levels of BiP inbrains of patients with PS1-linked FAD15, we failed to find signifi-cant decreases in the levels of BiP in the brains of individuals withsporadic AD, or patients with FAD carrying PS1 mutations I143Tand G384A, as compared with controls. These latter findings werefurther strengthened by our demonstration that the levels of BiPprotein are nearly indistinguishable in brains of transgenic miceexpressing wild-type human PS1 or PS1 M146L and A246E variants.

Our results suggest that neither the lack of PS1 function nor theexpression of FAD-linked PS1 variants has significant effect on theER stress-induced elevation in levels of BiP and CHOP mRNAs orprotein during the UPR. Although our findings differ from those ofNiwa et al.14 and Katayama et al.15, the discrepancies may have aris-en from variation in experimental design between the studies. Forexample, Niwa et al.14 derived their conclusions from a single analy-sis of BiP mRNA induction in PS1+/+ and PS1–/– fibroblasts. In con-trast, we used primary PS1–/– and PS1+/– fibroblasts at second orthird passage and performed several analyses of independentmarkers of the UPR. As constitutive expression of PS2 in PS1-deficient cells might have compensated for the loss of PS1 func-tion, we generated immortalized PS1–/–/PS2–/– cell lines andshowed that, in these compound homozygous lines, treatmentwith either tunicamycin or thapsigargin leads to the accumulationof BiP and CHOP mRNAs in a manner similar to that observed inwild-type cultures. Moreover, we showed that in PS1–/–/PS2–/– cellsUPR activation, as revealed by induction of IRE1α and PERKphosphorylation, is indistinguishable from that in control cells.

In the studies by Katayama et al.15, tunicamycin-mediatedincreases in the accumulation of BiP mRNA were quantified bydensitometric scanning of autoradiographic signals, and theseanalyses were performed either in a single line (each) of human SK-N-SH neuroblastoma cells stably transfected with vector, wild-typePS1, A246E or ∆E9 mutant PS1 cDNAs, or in transiently transfect-ed human 293 cells. To obviate potential artefacts resulting fromclonal variability, or transient overexpression of a polytopic proteinsuch as PS1, we used over ten independent mouse N2a and human293 cell lines expressing four different FAD-linked PS1 variants. Inaddition, we confirmed our findings using stable pools of N2a and293 cells expressing human wild-type PS1 or independent mutantPS1. To obtain unambiguous measures of changes in BiP andCHOP mRNA, we quantified signals by direct phosphorimaging ofnorthern blots or dried gels containing products from polymerasechain reaction with reverse transcription (RT–PCR). We also con-firmed the mRNA results by the analysis of BiP and CHOP proteinlevels using quantitative western blot with [125I]-labelled secondaryantibodies and phosphorimage analysis. Finally, Katayama et al.15

measured the steady-state levels of BiP protein in human brainextracts prepared in a mild non-ionic detergent, NP-40. To avoidthe potential problems associated with inefficientsolubilization/extraction of proteins from the lipid-rich, amyloid-laden brain tissue, we homogenized human tissue in a chaotropicbuffer containing 4% SDS. We further validated our in vivo resultsby western blot analysis of steady-state BiP protein levels in brainsof non-transgenic and PS1 transgenic mice, sources that are free ofthe potential problems associated with diseased brain tissue.



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The mechanism(s) by which mutations in PS1 predispose indi-viduals to FAD is not clearly defined. Analysis of FAD-linked PS1 andPS2 variants in a number of cell-culture models provides evidencethat expression of mutant PS1 perturbs calcium homeostasis andprobably causes ER stress33. UPR is well known to trigger both sur-vival responses (for example, inhibition of protein synthesis, inducedexpression of ER chaperones) and activated expression of certaingenes associated with cell death (for example, CHOP). As such, amodel in which PS1 is involved in the cellular response to ER stress,such that mutant PSI may cause impairment of the UPR pathwayseems highly attractive. Our results, however, provide strong evi-dence that induction of BiP (survival response) and CHOP (cell-death signalling) expression by ER stressors is not effected by the lackof PS1 function or the expression of FAD-linked PS1 variants.Alternatively, we suggest that, if FAD-linked PS variants alter ERstress signalling in vivo, these processes may be driven by mecha-nisms that are distinct from the classical UPR pathway.

MethodsGeneration of PS1/PS2 double-null cell lines and stable PS1 cell lines.Blastocysts were flushed from PS1+/–/PS2–/– intercrossed animals34,35 or wild-type control mice (E3.5) in

M2 medium and plated on feeders (as for embryonic stem (ES) cells)36. After 5 days, the inner cell

mass was picked off and disaggregated in trypsin and replated onto fresh feeders. After a period of

12 days, the cultures were passaged onto gelatin-coated plates and grown until confluent. The cultures

were then maintained in ES cell media (plus LIF) in uncoated dishes. Small refractory cells that grew

out of the cultures (resembling endoderm-derived cells) were immortalized with SV40 large tumour

antigen. Genotypes of immortalized wild-type and PS1–/–/PS2–/– cells were confirmed by PCR as

described34,35. To induce ER stress, cells were treated with tunicamycin (2.5 µg ml–1) or thapsigargin

(1 mM) for various periods of time as indicated in figure legends.

To generate stable PS1 lines, a mouse N2a neuroblastoma cell line (Swe.10; ref. 37) that constitu-

tively expresses human APP695 carrying the ‘Swedish’ double mutation was transfected with cDNAs

encoding wild-type PS1, PS1D385A mutant, or the FAD-linked C410Y variant, and independent

clones were isolated. Stable N2a Swe.10 and human 293 pools were generated by transfecting cells with

empty bicistronic vector (pIRE1hyg; Clontech), or pIRE1hyg containing wild-type PS1 or the FAD-

linked M146L, H163R or DE9 mutant cDNA, and stable transfectants were selected in medium con-

taining 0.4 mg ml–1 hygromycin38. About 200–300 colonies, visible after 3 weeks of growth in selection

medium, were pooled for further studies. Tunicamycin (2 µg ml–1), thapsigargin (300 nM) or A23781

(7 µM) was added to subconfluent cultures for various periods as indicated in the figure legends.

Human and transgenic brain tissue.Human brain was obtained at autopsy, 1–10 h postmortem, in the Brain Resource Center, The Johns

Hopkins University School of Medicine, or in the Born-Bunge Foundation39. Cortical tissue from six

controls (age 40, 59 (two individuals), 67, 75 and 83 years), six cases of sporadic AD (age 55, 56, 59, 61,

76 and 80 years), three patients with FAD carrying a PS1 I143T mutation (36, 38 and 40 years) and

from one FAD patient carrying a PS1 G384A mutation (40 years) were used in this study. The controls

were free of any neurological diseases, and the cases of sporadic AD were pathologically confirmed

using standard CERAD criteria. Transgenic mice expressing wild-type human PS1 or the FAD-linked

A246E and M146L variants have been described6,11. Tissues were homogenized in a buffer containing

50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 4% SDS, and a protease inhibitor cocktail

(50 µg ml–1 leupeptin, 50 µg ml–1 pepstatin, 10 µg ml–1 aprotinin, and 0.25 mM phenylmethylsulphonyl

fluoride), briefly sonicated to reduce viscosity, and stored at –70 °C.

Protein expression studies.Cultured cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM

EDTA, 0.5% Nonidet P-40, 0.5% deoxycholate, 0.25% SDS and a protease inhibitor mixture (see

above). Lysates were briefly sonicated and fractionated by SDS–PAGE. Polyclonal PS1 antibodies, PS1NT

and anti−PS1Loop, and APP antibody, CT15, have been described6,40. Monoclonal antibodies raised

against CHOP, BiP (anti-KEDL) and β-tubulin were purchased from Santa Cruz Biotechnology,

StressGen Biotechnologies and Amersham Pharmacia, respectively. Immunoprecipitation/western blot

analysis of IRE1α and PERK was done as described described38. For quantitative immunoblotting

studies, blots were incubated with [125I]-labelled anti-mouse IgG (DuPont NEN), and signals quanti-

fied by phosphorimaging (Typhoon system; Molecular Dynamics). Secreted Aβ species ending at 40 or

42/43 were quantified using the well-characterized BNT77/BA27 or BC05 two-site ELISAs12,28, using

media conditioned by stable cell lines for 36 h. Statistical significance was examined using analysis of

variance (ANOVA) followed by Scheffé’s test.

Analysis of mRNA levels and BiP promoter activity.Total RNA was prepared by guanidinium thiocyanate lysis and CsCl ultracentrifugation, or by using

Trizol reagent (Life Technologies). For RNA blot analysis, total RNA (20 µg per lane) was fractionated

in a 1.2% formaldehyde agarose gel, transferred to a nylon membrane and hybridized with murine

CHOP, BiP, β-actin and GAPDH cDNA probes. Signal intensities were quantified from the blots by

phosphorimage analysis. For BiP promoter activity studies, N2a cells were co-transfected in 60-mm

dishes with 1.5 µg BiP promoter/CAT fusion gene –154 CAT29 and 0.75 µg pSV40 β-galactosidase plas-

mid. Cells were treated with 2 µg ml–1 tunicamycin for 10 or 20 h (three dishes for each time point)

before collection. The level of CAT activity was determined as described41.

RECEIVED 2 MAY; REVISED 11 JULY; ACCEPTED 22 SEPTEMBER;PUBLISHED 10 NOVEMBER 2000.

1. Glenner, G. G. & Wong, C. W. Alzheimer’s disease: initial report of the purification and characteri-

zation of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Comm. 120, 885–890

(1984).

2. Masters, C. L. et al. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc.

Natl Acad. Sci. USA 82, 4245–4249 (1985).

3. Sherrington, R. et al. Cloning of a gene bearing missense mutations in early-onset familial

Alzheimer’s disease. Nature 375, 754–760 (1995).

4. Levy-Lahad, E. et al. Candidate gene for the chromosome 1 familial Alzheimer’s disease locus.

Science 269, 973–977 (1995).

5. Rogaev, E. I. et al. Familial Alzheimer’s disease in kindreds with missense mutations in a gene on

chromosome 1 related to the Alzheimer’s disease type 3 gene. Nature 376, 775–778 (1995).

6. Thinakaran, G. et al. Endoproteolysis of presenilin 1 and accumulation of processed derivatives in

vivo. Neuron 17, 181–190 (1996).

7. De Strooper, B. et al. Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor

protein. Nature 391, 387–390 (1998).

8. Naruse, S. et al. Effects of PS1 deficiency on membrane protein trafficking in neurons. Neuron 21,

1213–1221 (1998).

9. De Strooper, B. et al. A presenilin-1-dependent gamma-secretase-like protease mediates release of

Notch intracellular domain. Nature 398, 518–522 (1999).

10. Scheuner, D. et al. Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer’s

disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial

Alzheimer’s disease. Nature Med. 2, 864–870 (1996).

11. Borchelt, D.R. et al. Familial Alzheimer’s disease-linked presenilin 1 variants elevate Abeta1-42/1-40

ratio in vitro and in vivo. Neuron 17, 1005–1013 (1996).

12. Tomita, T. et al. The presenilin 2 mutation (N141I) linked to familial Alzheimer disease (Volga

German families) increases the secretion of amyloid beta protein ending at the 42nd (or 43rd)

residue. Proc. Natl Acad. Sci. USA 94, 2025–2030 (1997).

13. Thinakaran, G. The role of presenilins in Alzheimer’s disease. J. Clin. Invest. 104, 1321–1327 (1999).

14. Niwa, M., Sidrauski, C., Kaufman, R. J. & Walter, P. A role for presenilin-1 in nuclear accumulation of

Ire1 fragments and induction of the mammalian unfolded protein response. Cell 99, 691–702 (1999).

15. Katayama, T. et al. Presenilin-1 mutations downregulate the signalling pathway of the unfolded-

protein response. Nature Cell Biol. 1, 479–485 (1999).

16. Kaufman, R. J. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene

transcriptional and translational controls. Genes Dev. 13, 1211–1233 (1999).

17. Pahl, H. L. Signal transduction from the endoplasmic reticulum to the cell nucleus. Physiol. Rev. 79,

683–701 (1999).

18. Shamu, C. E. & Walter, P. Oligomerization and phosphorylation of the Ire1p kinase during intracel-

lular signaling from the endoplasmic reticulum to the nucleus. EMBO J. 15, 3028–3039 (1996).

19. Sidrauski, C. & Walter, P. The transmembrane kinase Ire1p is a site-specific endonuclease that initi-

ates mRNA splicing in the unfolded protein response. Cell 90, 1031–1039 (1997).

20. Sidrauski, C., Chapman, R. & Walter, P. The unfolded protein response: an intracellular signalling

pathway with many surprising features. Trends Cell Biol. 8, 245–249 (1998).

21. Bertolotti, A., Zhang, Y., Hendershot, L. M., Harding, H. P. & Ron, D. Dynamic interaction of BiP

and ER stress transducers in the unfolded- protein response. Nature Cell Biol. 2, 326–332 (2000).

22. Price, B. D. & Calderwood, S. K. Gadd45 and Gadd153 messenger RNA levels are increased during

hypoxia and after exposure of cells to agents which elevate the levels of the glucose-regulated pro-

teins. Cancer Res. 52, 3814–3817 (1992).

23. Wang, X. Z. et al. Signals from the stressed endoplasmic reticulum induce C/EBP-homologous pro-

tein (CHOP/GADD153). Mol. Cell. Biol. 16, 4273–4280 (1996).

24. Harding, H. P., Zhang, Y., Bertolotti, A., Zeng, H. & Ron, D. Perk is essential for translational regu-

lation and cell survival during the unfolded protein response. Mol. Cell 5, 897–904 (2000).

25. Shi, Y. et al. Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-

subunit kinase, PEK, involved in translational control. Mol. Cell. Biol. 18, 7499–7509 (1998).

26. Harding, H. P., Zhang, Y. & Ron, D. Protein translation and folding are coupled by an endoplasmic-

reticulum-resident kinase. Nature 397, 271–274 (1999).

27. Thinakaran, G. et al. Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by

competition for limiting cellular factors. J. Biol. Chem. 272, 28415–28422 (1997).

28. Suzuki, N. et al. An increased percentage of long amyloid beta protein secreted by familial amyloid

beta protein precursor (beta APP717) mutants. Science 264, 1336–1340 (1994).

29. Li, W.W., Alexandre, S., Cao, X. & Lee, A.S. Transactivation of the grp78 promoter by Ca2+ deple-

tion. A comparative analysis with A23187 and the endoplasmic reticulum Ca(2+)-ATPase inhibitor

thapsigargin. J. Biol. Chem. 268, 12003–12009 (1993).

30. Hendriks, L. et al. Processing of presenilin 1 in brains of patients with Alzheimer’s disease and con-

trols. NeuroReport 8, 1717–1721 (1997).

31. Borchelt, D. R. et al. A vector for expressing foreign genes in the brains and hearts of transgenic

mice. Genet. Anal. 13, 159–163 (1996).

32. Borchelt, D. R. et al. Accelerated amyloid deposition in the brains of transgenic mice coexpressing

mutant presenilin 1 and amyloid precursor proteins. Neuron 19, 939–945 (1997).

33. Mattson, M. P., Guo, Q., Furukawa, K. & Pedersen, W. A. Presenilins, the endoplasmic reticulum,

and neuronal apoptosis in Alzheimer’s disease. J. Neurochem. 70, 1–14 (1998).

34. Wong, P. C. et al. Presenilin 1 is required for Notch1 and DII1 expression in the paraxial meso-

derm. Nature 387, 288–292 (1997).

35. Donoviel, D. B. et al. Mice lacking both presenilin genes exhibit early embryonic patterning defects.

Genes Dev. 13, 2801–2810 (1999).

36. Hogan, B., Beddington, R., Costantini, F. & Lacey, E. Manipulating the Mouse Embryo: a Laboratory

Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1994).

37. Thinakaran, G., Teplow, D. B., Siman, R., Greenberg, B. & Sisodia, S. S. Metabolism of the

“Swedish” amyloid precursor protein variant in neuro2a (N2a) cells. Evidence that cleavage at the

“β-secretase” site occurs in the golgi apparatus. J. Biol. Chem. 271, 9390–9397 (1996).

38. Saura, C. A. et al. The non-conserved hydrophilic loop domain of presenilin (PS) is neither

required for PS endoproteolysis nor enhanced Aβ42 production mediated by familial Alzheimer’s


articles

disease-linked PS variants. J. Biol. Chem. 275, 17136–17142 (2000).

39. Cruts, M. et al. Molecular genetic analysis of familial early-onset Alzheimer’s disease linked to chro-

mosome 14q24.3. Hum. Mol. Genet. 4, 2363–2371 (1995).

40. Thinakaran, G. et al. Stable association of presenilin derivatives and absence of presenilin interac-

tions with APP. Neurobiol. Dis. 4, 438–453 (1998).

41. Li, M. et al. ATF6 as a transcription activator of the endoplasmic reticulum stress element: thapsi-

gargin stress-induced changes and synergistic interactions with NF-Y and YY1. Mol. Cell. Biol. 20,

5096–5106 (2000).

ACKNOWLEDGEMENTS

This work was supported by NIH 1PO1 grant AG14248 (S.S.S.), NIEHS grant ES08681 (D.R), NIH,

NCI grant CA27607 (A.S.L), the Alzheimer’s Association (G.T), the Adler Foundation (G.T. and J.Y.L),

and by an award to the University of Chicago’s Division of Biological Sciences under the Research

Resources Program for Medical Schools of the Howard Hughes Medical Institute (G.T.). N.S. and F.U

are supported by a research fellowship from the Japan Society for the Promotion of Science and

grants-in-aid from the Ministry of Education, Science, Culture and Sports, Japan. We thank C. A.

Saura, T. Tomita and T. Iwatsubo for Aβ analysis; D. R. Borchelt for transgenic mouse tissue; J.

Troncoso for control and sporadic AD brain tissue; and L. Hendriks and C. V. Broeckhoven for FAD

brain tissue.

Correspondence and requests for materials should be addressed to G.T. Supplementary information is

available on our World-Wide Web site (http://cellbio.nature.com) or as paper copy from the London

editorial office of Nature Cell Biology.


ErratumIn the fifth line of the abstract, ‘that’ should be deleted to read “Here we show that neither…”.In the penultimate line of the legend to Fig. 2, ‘mM’ should read ‘µM’.On p865, column 2, line 5, ‘IRE1B’ should read ‘IRE1β’.On p869, column 1, line 10, ‘PSI’ should read ‘PS1’.In the Methods, “Generation of PS1/PS2 double-null cells and stable PS1 cell lines”, line 9, ‘mM’ should read ‘µM’.For corrected version please see the print version of Nature Cell Biology vol. 2, no. 12, December 2000.


supplementary information


2

3

4

5

6

16 18 20 22

y = 0.241x-1.270 r2 = 0.966

APLP2

Log

Pho

spho

rimag

er u

nits

PCR cycle number

2

3

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16 18 20 22

y = 0.283x-1.202 r2 = 0.967

CHOP

Log

Pho

spho

rimag

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PCR cycle number

2

3

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16 18 20 22

y = 0.269x-0.871 r2 = 0.962

BiP

Log

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spho

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BiP

CHOP

APLP2

0 2 3 5 7 0 2 3 5 7

PS1 + / – PS1 – / –Genotype

Tunicamycin (h)

BiP

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0 2 4 6 8Hours of treatment

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6 ) CHOP

Hours of treatment

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+ / –

– / –

+ / –

– / –

b

a

Supplement Figure 1 Semi-quantitative RT-PCR analysis of BiP and CHOP mRNAlevels in PS1-/- and PS1+/- fibroblasts. a, mRNAs encoding BiP, CHOP, and APLP2were amplified from reverse-transcribed total RNA by 16, 18, 20, or 22 cycles ofPCR using 32P-5’ end-labeled sense primers, and quantified byg Phosphorimaging.The PCR cycle number was plotted versus the log of the Phosphorimager units cor-responding to the radiolabeled PCR product, and regression coefficients weredetermined. The inserts represent amplified PCR products visualized by

Phosphorimaging. b, Subconfluent cultures of primary PS1-/- and PS1+/- fibroblastswere incubated with tunicamycin (2 µg ml-1) for various periods (0, 2, 3, 5, or 7 h).Total RNAs isolated from the fibroblasts were subjected to RT-PCR (20 cycles ofPCR), and radiolabeled PCR products (left panels) were quantified byPhosphorimaging. The levels of BiP and CHOP mRNA (Phosphorimager units corre-sponding to radioactive products) were normalized to APLP2 mRNA.




CHOPsynthesis

Asp. 24Wild-type 9

+– +–

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68

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cAsp. 24Wild-type 9

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Tubulin

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d

Supplement Figure 2 Activation of the UPR is unimpaired by the expression of aloss of function PS1 mutant. a, Subconfluent cultures of stable N2a cell lines thatexpress human Wt PS1 (line Wt.9) or a dominant negative mutant, PS1 D385A (lineAsp.24), were incubated with tunicamycin (2 µg ml-1) for 5 h. In Wt.9 cells, PS1accumulates as full-length polypeptide (FL) and stable N- and C-terminal fragments(NTF and CTF) derived from endoproteolytic processing. In contrast, PS1 D385Amutant accumulates as full-length polypeptide. Due to the loss of PS1 function, α-and β-secretase-generated APP C-terminal fragments (APP CTFs) accumulate tohigh levels in Asp.24 cells. Western blot analyses revealed that tunicamycin treat-ment resulted in increased steady-state accumulation of CHOP protein in both Wt.9and Asp.24 cells; BiP levels remain unchanged. Reprobing with β-tubulin antibodiesconfirmed equal protein loading. b, Wt.9 and Asp.24 cells were metabolicallylabeled with [35S]methionine for 1 h, and the levels of APP β-CTFs in detergentlysate and secreted Aβ in conditioned medium were examined by immunoprecipita-tion analysis. As expected, α- and β-secretase-generated APP C-terminal fragmentsaccumulate to high levels with a concomitant decrease in the secretion of Aβ pep-tides in the Asp.24 cell line. c, The levels of BiP mRNA in Wt.9 and Asp.24 cellswere examined by Northern blot analysis. Cells were treated with tunicamycin (2 µgml-1) or thapsigargin (300 nM) for 5 h. Blots were sequentially hybridized with BiP

and GAPDH probes, and the signals quantified by Phosphorimaging. BiP mRNA lev-els were normalized to GAPDH levels and plotted. Northern blot analyses furtherconfirmed that treatment with tunicamycin or thapsigargin caused marked increas-es in the levels of BiP mRNA in Wt.9 and Asp.24 cells. Note that expression of thedominant negative PS1 mutant had no detectable effect on the ER stress-mediatedinduction of BiP mRNA compared to cells expressing Wt PS1. d, For biosyntheticlabeling studies, Wt.9 and Asp.24 were incubated with tunicamycin (2 µg ml-1) for 7h, and newly-synthesized proteins were pulse-labeled with [35S]-methionine for 15min. Aliquots of lysates (equivalent TCA-precipitable radioactive proteins) were frac-tionated by SDS-PAGE and visualized by Phosphorimaging. Arrowheads indicate twopolypeptides (that are remarkably similar to the reported molecular weights of theER chaperones GRP94 and BiP, respectively) whose synthesis is increased by tuni-camycin treatment; open circles indicate polypeptides whose synthesis isdecreased by tunicamycin treatment. Immunoprecipitation analysis with CHOP anti-bodies revealed induced, but indistinguishable, levels of synthesis of CHOP proteinin both Wt.9 and Asp.24 cells treated with tunicamycin




CHOP

APLP2

BiP

PS1

Wild-type 9 Asp.24

Tunicamycin (h) 0 2 3 5 7 0 2 3 5 7

0

0.5

1

1.5

2

2.5

3

0 2 4 6 8

Wild-type 9Asp.24

0

0.5

1

1.5

2

2.5

3

0 2 4 6 8

BiP CHOP

Wild-type 9Asp.24

Hours of treatment

Pho

spho

rimag

er u

nits

(10

6 )

Hours of treatment

Supplement Figure 3 Kinetic analysis of BiP and CHOP mRNA in N2a cell linesexpressing Wt PS1 or PS1 D385A mutant. a, Subconfluent cultures of Wt.9 Asp.24cells were incubated with tunicamycin (2 mg ml-1) for various periods of time (0, 2,3, 5 or 7 h). Total RNA was isolated and subjected to RT-PCR as described.Radiolabeled PCR products were fractionated on agarose gels (upper panels) andquantified by Phosphorimaging. The levels of BiP and CHOP mRNA were normalizedto APLP2 mRNA for each time point and plotted. Note that the levels of BiP andCHOP mRNA were induced in a time-dependent fashion, and neither the magnitudenor the kinetics of accumulation of BiP or CHOP mRNA differed between Wt.9 andAsp.24 cells

Controls

75 67 40 83 59 59 40 36 38 40 55 76 56 80 61 56Age

Familial AD Sporadic AD

20097

68

43

29

18

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Supplement Fig 4 Coomassie blue staining of brain homogenates of controls,FAD patients and sporadic AD patients. Human cortical brain homogenates of sixage-matched controls (lanes 1-6), three FAD patients with a PS1 I143T mutation(lanes 7-9), one FAD patient with a PS1 G384A mutation (lane 10), and six patientswith sporadic AD (lanes 11-16), were fractionated by SDS-PAGE, and stained withCoomassie brilliant blue R-250. The age at death of all individuals is indicated atthe top of each lane. Note the marked difference in the levels of few polypeptidesin some samples (marked by arrowheads) and diffused migration of severalpolypeptides in two FAD samples (lanes 8 and 9).

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