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Proc Natl Acad Sci U S A. Jun 26, 2007; 104(26): 11062–11067.
Published online Jun 15, 2007. doi:  10.1073/pnas.0609621104
PMCID: PMC1904148
Neuroscience

Cellular prion protein regulates β-secretase cleavage of the Alzheimer's amyloid precursor protein

Abstract

Proteolytic processing of the amyloid precursor protein (APP) by β-secretase, β-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid β (Aβ) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrPC), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt–Jakob disease in humans, remains enigmatic. Because both APP and PrPC are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrPC in the proteolytic processing of APP. Cellular overexpression of PrPC inhibited the β-secretase cleavage of APP and reduced Aβ formation. Conversely, depletion of PrPC in mouse N2a cells by siRNA led to an increase in Aβ peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Aβ levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the β-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrPC on the β-secretase cleavage of APP required the localization of PrPC to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrPC via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Aβ is regulated by PrPC and may have implications for both Alzheimer's and prion diseases.

Keywords: lipid raft, proteolysis, scrapie, glycosaminoglycan

Alzheimer's disease (AD) is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles within the afflicted brain. The major constituents of senile plaques are the amyloid β (Aβ) peptides, which are derived from the proteolytic processing of the amyloid precursor protein (APP) (1). In the amyloidogenic pathway, β-secretase cleavage of APP yields a soluble N-terminal fragment sAPPβ, along with a short membrane-bound C-terminal fragment that is subsequently cleaved by γ-secretase to release the Aβ peptides. In the alternative, nonamyloidogenic pathway, α-secretase cleaves APP within the Aβ sequence, thus precluding the formation of Aβ, and releases a soluble N-terminal fragment sAPPα. The transmembrane aspartyl protease, β-site APP cleaving enzyme (BACE1), has been identified as β-secretase (2), members of the ADAM (a disintegrin and metalloprotease) family, particularly ADAM10 and ADAM17, are responsible for α-secretase cleavage (3), while a complex of at least four proteins, the presenilins, nicastrin, Aph-1, and Pen-2, constitutes the γ-secretase (2).

The prion protein (PrP) is the causative agent of the transmissible spongiform encephalopathies (TSEs) that include Creutzfeldt–Jakob disease (CJD), Gerstmann-Scheinker-Straussler (GSS) disease, kuru and fatal familial insomnia in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep (4). In these diseases, the normal cellular form of PrP (PrPC) undergoes a conformational change to the infectious form, PrPSc. The function of PrPC remains enigmatic, with roles in metal homeostasis, neuroprotective signaling, and cellular response to oxidative stress having been proposed (5, 6).

AD and CJD share a variety of neuropathological features (79), and the Val/Met-129 polymorphism in the gene encoding PrPC has been identified as a risk factor for early onset AD (10, 11). In addition, like APP, PrPC is shed from the cell surface by zinc metalloproteases and is subject to endoproteolysis by ADAM10 and ADAM17 (1215). As a result of these pathological, genetic, and mechanistic similarities, we were led to investigate whether PrPC alters the proteolytic processing of APP. We show that PrPC inhibits the β-secretase cleavage of APP and reduces Aβ formation. This effect is lost in scrapie-infected mouse brain or in cells expressing mutants of PrP associated with human prion disease. The regulation of β-secretase requires PrPC to be located in lipid rafts and is mediated by the N-terminal polybasic region of PrPC interacting with BACE1 via glycosaminoglycans (GAGs).

Results and Discussion

PrPC Inhibits the β-Secretase Cleavage of APP.

To investigate whether PrPC alters the proteolytic processing of APP, the cDNA encoding murine PrP was stably transfected into SH-SY5Y cells expressing APP695. In the transfected cells, PrPC appeared as a broad band of 32 to 40 kDa due to the differentially glycosylated forms (Fig. 1A). The presence of PrPC had no effect on the amount of APP695 holoprotein in the cell lysates (Fig. 1B) or on the amount of sAPPα in the cell medium (Fig. 1C). However, PrPC dramatically inhibited (97.5%) the shedding of sAPPβ (Fig. 1 D and E) and reduced the secretion into the conditioned medium of Aβ1–40 by 92% and of Aβ1–42 to an undetectable level (Fig. 1F). Similarly, expression of PrPC inhibited the amyloidogenic processing of endogenous APP in cells stably expressing BACE1 [supporting information (SI) Fig. 6]. In these cells, PrPC reduced the amount of sAPPβ by 95% and reduced Aβ1–40 and Aβ1–42 to undetectable levels.

Fig. 1.
PrPC inhibits the β-secretase cleavage of APP. SH-SY5Y cells expressing APP695 were stably transfected with cDNA-encoding murine PrP. (A) Detection of PrPC in cell lysates with 3F4. (B) Detection of the APP in cell lysates with Ab54. (C) Detection ...

Because PrPC decreased the production of both sAPPβ and Aβ, it can be concluded that the observed inhibitory effect is at the level of the β-secretase cleavage of APP, rather than an effect on γ-secretase. One possible explanation for this observation would be an alteration in the levels of expression of BACE1. However, the presence of PrPC had no significant effect on the level of expression of BACE1 (SI Fig. 7). Another possibility is that PrPC is competing with APP as a substrate for BACE1. However, neither the shedding nor the endoproteolytic processing of PrPC was increased in cells overexpressing BACE1 (SI Fig. 8), indicating that PrPC is not processed by BACE1.

Reduction of PrP by siRNA Increases Aβ Production, and Aβ Levels Are Increased in the Brains of PrP-Null Mice.

To confirm that PrPC regulates the production of Aβ in another cell system and using a different approach, we used siRNA duplexes to down-regulate the expression of endogenous PrPC in the mouse neuroblastoma N2a cell line. The specific siRNA reduced the level of PrPC expression by 60%, whereas the scrambled siRNA had no effect (Fig. 2 A and B). Cells treated with the specific siRNA had no difference in the amount of APP holoprotein (Fig. 2A). However, cells depleted of PrPC secreted increased amounts of Aβ1–40 and Aβ1–42 into the conditioned medium compared to untreated cells or those treated with the scrambled siRNA (Fig. 2C).

Fig. 2.
Depletion of PrPC increases Aβ peptide production. N2a cells were transfected with siRNA targeted against PrP or a scramble siRNA. (A) Detection in cell lysates of PrP with SAF-32, APP, and actin. (B) Quantification of multiple PrP immunoblots ...

To determine whether a reduced level of PrPC would lead to increased Aβ levels in the brain, we compared the amount of Aβ in brains from 129OlaPrP−/− mice to that in wild-type 129Ola controls. PrP was undetectable in the PrP−/− mice, whereas the level of APP holoprotein was similar to that in the wild-type mice (Fig. 2D). However, the levels of both Aβ1–40 and Aβ1–42 were significantly increased in the PrP−/− mice (Fig. 2E), providing direct evidence that PrPC regulates the production of Aβ in the brain. It should be noted that increased levels of murine Aβ do not result in amyloid plaque deposition (16). A recent study reported that bigenic mice carrying mutant human APP and wild-type Syrian hamster PrP had increased amyloid plaques but no significant alteration in Aβ1–40 or Aβ1–42 levels compared to control mice carrying just the mutant APP (17). Because these control mice have endogenous levels of murine PrPC, which may be maximally inhibiting the β-cleavage of APP (see Fig. 1), the overexpression of hamster PrPC may not then lead to further inhibition of APP processing and Aβ production, but instead may have a secondary affect on Aβ aggregation.

The Polybasic N Terminus of PrPC and Its Localization to Lipid Rafts Are Required for the Inhibition of β-Secretase.

To determine the mechanism by which PrPC inhibits the β-secretase cleavage of APP, we examined the effect of a number of PrP constructs (Fig. 3A). All of the anchored PrP constructs were expressed in the SH-SY5Y cells at a very similar level to wild-type PrP (Fig. 3B), and, as shown previously (1820), all were present at the cell surface. The amount of APP695 holoprotein in the lysates from the cells expressing the different PrP constructs was similar, and no significant difference was detected in the shedding of sAPPα from any of the cell lines (Fig. 3B). However, although sAPPβ shedding, which is a direct measure of β-secretase activity, was dramatically reduced from cells transfected with either wild-type PrP or PrPΔoct, none of the other constructs had any significant effect on sAPPβ shedding (Fig. 3C), excluding the possibility that an overexpression artifact might cause the observed reduction in β-cleavage of APP. Because PrPΔoct inhibited the β-cleavage of APP similarly to wild-type PrP, the copper binding octapeptide repeats are not required for this effect. The lack of inhibitory effect on sAPPβ production by PrPΔN, which is missing the four residues (KKRP) at the N terminus of the mature protein, and PrP-DA, in which the N terminus is tethered to the membrane, indicate that the N-terminal polybasic region is critically required for PrPC to inhibit the β-cleavage of APP. Neither PrPΔGPI, which is not membrane-attached, nor PrP-CTM, which is anchored by a transmembrane domain and is excluded from cholesterol-rich lipid rafts (19), reduced sAPPβ shedding. Thus, to inhibit the β-cleavage of APP, it would appear that PrPC has to be localized to cholesterol-rich lipid rafts. This conclusion would be consistent with rafts being the site where the processing of APP by BACE1 preferentially occurs (21, 22), although there is a report that BACE1 can cleave APP outside of rafts (23).

Fig. 3.
The polybasic N terminus of PrPC and its localization to lipid rafts are required for the inhibition of β-secretase. (A) Schematic of the PrP constructs used. Murine wtPrP comprises a 22-aa N-terminal sequence (criss-cross box), a positively charged ...

The Inhibitory Action of PrPC on β-Secretase Involves GAGs.

Next we investigated whether PrPC directly interacts with BACE1. Coimmunoprecipitation experiments demonstrated that PrPC physically interacts with BACE1, but not with APP (Fig. 4A). However, PrPC did not inhibit the activity of BACE1 toward a quenched fluorescent peptide substrate (SI Fig. 9), indicating that PrPC does not regulate the processing of APP through direct inhibition of the enzymatic activity of BACE1. Because the N-terminal region of PrP, which is ablated in PrPΔN, is known to participate in GAG binding (24), we investigated whether GAGs are involved in the mechanism by which PrPC inhibits the β-secretase cleavage of APP. Although wild-type PrP bound to heparin-coated ELISA plates in a concentration-dependent manner, PrPΔN did not bind above background levels (Fig. 4B), indicating that the N-terminal KKRP sequence is necessary for the binding of GAGs to PrP. We next investigated whether incubating cells with heparin could reverse the effect of PrPC on the amyloidogenic processing of APP. Incubation of SH-SY5Y cells with heparin had no effect on the expression level of the APP holoprotein or on the shedding of sAPPα (Fig. 4C). In contrast, heparin increased the amount of sAPPβ shed from the cells in a concentration-dependent manner (Fig. 4 C and D) and reduced to 41.2 ± 7.3% (n = 3) the amount of BACE1 coimmunoprecipitated with PrP (Fig. 4A). Although heparin increased sAPPβ production in the absence of PrPC, the fold increase in sAPPβ production was higher in the cells expressing PrPC (2.16 ± 0.22-fold compared to 6.22 ± 1.22-fold, respectively) (Fig. 4E), thus showing that PrPC is required to obtain the maximal effect of heparin on sAPPβ shedding.

Fig. 4.
The effect of GAGs on APP metabolism correlates with their binding to PrPC. (A) Membranes from SH-SY5Y cells expressing PrP and BACE1 were solubilized with CHAPSO, immunoprecipitated with 3F4 in the absence or presence of 4 μM heparin, and the ...

Having established that heparin could restore sAPPβ shedding from cells expressing PrP and disrupt the physical interaction between PrPC and BACE1 (Fig. 4 A–E), we investigated whether other GAGs could restore sAPPβ production and, if so, whether the same GAGs were also capable of binding to PrPC. SH-SY5Y cells expressing wild-type PrP were incubated with hyaluronic acid, dextran sulfate, chondroitin sulfate A, low molecular weight (LMW) heparin, or polymerized heparin. None of the GAGs affected the level of APP holoprotein or the shedding of sAPPα, except for hyaluronic acid, which slightly reduced the amount of holoprotein and the shedding of sAPPα (Fig. 4F). In contrast, dextran sulfate and LMW heparin restored sAPPβ shedding to 55% and 47%, respectively, of that from untransfected cells, whereas heparin restored the level of sAPPβ shedding to 62% (Fig. 4 F and G). Hyaluronic acid and chondroitin sulfate A both failed to restore the shedding of sAPPβ (Fig. 4 F and G). We next examined the binding of the various GAGs to PrPC (Fig. 4H). Relative to heparin, dextran sulfate and LMW heparin bound to wild-type PrP with 58% and 43% efficiencies, respectively. In contrast, hyaluronic acid and chondroitin sulfate A did not bind to wild-type PrP. Thus, those GAGs that were capable of binding to PrPC were also able to restore the β-cleavage of APP, and the extent of binding to PrPC directly correlated with the effect on APP processing. Because heparin has been shown to interact directly with BACE1 (25), a possible mechanism by which PrPC regulates the β-cleavage of APP is through the N terminus of PrPC interacting via GAGs with one or more of the heparin binding sites on BACE1 within a subset of cholesterol-rich lipid rafts, thereby restricting access of BACE1 to APP.

Aβ Levels Are Increased in Cells Expressing Disease-Associated Mutants of PrP and in Scrapie-Infected Brain.

Two mutants of PrP, PG14 and A116V, which are associated with familial CJD and GSS, respectively (26, 27), did not inhibit the β-cleavage of APP when expressed in the SH-SY5Y cells (Fig. 3), suggesting that in certain forms of prion disease due to mutations in PrP there may be an increase in the production of Aβ. During prion disease, the proteinase-sensitive PrPC undergoes a conformational conversion to the proteinase-resistant PrPSc and may lead to an alteration in the β-cleavage of APP. In the brains of two strains (79A and 87V) of scrapie-infected mice, there was a significant increase in the amount of proteinase K-resistant PrPSc (Fig. 5A). Although the level of APP holoprotein was unchanged between uninfected control mice and the scrapie-infected mice (Fig. 5B), the amount of Aβ was increased significantly in the scrapie-infected mice (Fig. 5C). Interestingly the amounts of Aβ1–40 and Aβ1–42 were higher in the mice with the shorter prion disease incubation time (79A, 146 days; 87V, 350 days). These results suggest that during prion disease, when PrPC undergoes a conformational conversion to PrPSc, the inhibition of β-cleavage of APP may be lost, resulting in an increase in the amount of Aβ.

Fig. 5.
Scrapie infection increases Aβ peptide production. Mice infected with scrapie strains 79A or 87V were killed at 146 and 350 days, respectively. The right cerebral hemisphere from each mouse was used to prepare soluble and membrane fractions, and ...

To investigate whether the Val/Met-129 polymorphism in human PrPC would alter the production of Aβ, brains from mice whose endogenous PrP gene had been replaced with the human PrP gene with MM or VV 129 genotypes (28) were analyzed. Although there was no difference in the amount of Aβ1–42 (0.188 ± 0.015 vs. 0.184 ± 0.015 pmol/g; P = 0.348) between the MM and VV homozygous mice, respectively, there was a significant increase in the amount of Aβ1–40 (0.359 ± 0.026 vs. 0.324 ± 0.015 pmol/g; P = 0.014) in the MM mice compared to the VV mice.

Conclusions

We have identified a new role for PrPC in inhibiting the β-secretase cleavage of APP, thereby regulating the production of the neurotoxic Aβ peptide. Our data would predict that the lack of functional PrPC would lead to an increase in Aβ levels and potentially AD in humans. In this respect, in the two cases where a mutation (Y145stop or Q160stop) gives rise to truncated forms of PrP that fail to traffic to the cell surface, a diagnosis of AD was made, with the onset of clinical disease occurring in the fourth decade of life (29, 30). It is conceivable that small changes in PrPC levels in individuals may affect the proteolytic processing of APP in a subtle way over decades to affect long-term Aβ production that, in turn, could either accelerate or decelerate the deposition of amyloid in the brain. Our observations that the level of Aβ increases in scrapie-infected mice brains when PrPC is converted to PrPSc and that mutations in PrP that give rise to human prion diseases ablate the inhibitory effect of PrPC on the β-cleavage of APP suggest that the inhibition of β-secretase by PrPC is released in both TSEs and inherited prion diseases. Whether the increase in Aβ is, in part, responsible for the neurodegeneration observed in prion diseases and whether the increase in Aβ seen in humanized MM mice is linked to the Met/Val-129 polymorphism being a risk factor for early onset AD (11) awaits further study. In addition, these observations raise significant questions over whether depletion of PrPC (31) is a sound therapeutic approach for TSEs, but suggest that pharmacological interventions that mimic the effect of PrPC in inhibiting the β-secretase cleavage of APP may represent a therapeutic approach for AD.

Materials and Methods

Cell Culture and Plasmids.

The SH-SY5Y cell line was cultured and cell lysates and conditioned medium were collected as described previously (32). Insertion of the coding sequence of murine PrP containing a 3F4 epitope tag into pIRESneo (BD Biosciences Clontech, Palo Alto, CA) and the generation of the PrP constructs have been reported previously (18, 20, 32). The coding sequence of human APP695 was inserted into the BstX I site of pIREShyg (BD Biosciences Clontech). The coding sequence of human BACE1 was inserted into the BamH I and BstX I sites of pIREShyg. For stable transfections, 30 μg of DNA was introduced to cells by electroporation and selection was performed in normal growth medium containing 500 μg/ml neomycin or 100 μg/ml hygromycin B (Gibco BRL, Paisley, U.K.). The cells were preincubated for 24 h in OptiMEM containing the stated GAG concentrations, washed in situ with OptiMEM, and incubated for a further 7 h in fresh OptiMEM containing the GAGs.

siRNA Transfection.

siRNAs corresponding to the murine Prnp gene from codon 392 to 410 (33) were synthesized by Dharmacon RNA Technologies (Lafayette, CO) and were supplied preduplexed. The sequences of the siRNAs are detailed in http://www.pnas.org/cgi/content/full/0609621104/DC1SI Methods. N2a cells were seeded at 10–15% confluency in a 12-well plate 24 h before transfection. siRNA (10 μl of the stock solution) was mixed with the corresponding half-volume of Oligofectamine reagent (Invitrogen, Paisley, U.K.) for 20 min and applied to the cells in a final volume made up to 0.5 ml with Opti-MEM. After incubation for 4 h at 37°C, 0.25 ml of Opti-MEM supplemented with 30% FBS and a penicillin/streptomycin mixture was added. Cells were cultured for 3 days at 37°C until confluent, after which medium was conditioned for 24 h.

Animals.

Inbred PrP knockout mice (129OlaPrP−/−) and 129Ola wild-type mice (34), and mice whose endogenous PrP gene had been replaced with the human PrP gene with MM or VV 129 genotypes (28), were analyzed at 4 weeks of age. Mice infected with scrapie strains 79A and 87V along with their respective age-matched controls were killed by cervical dislocation at 146 and 350 days, respectively, and the brains were immediately removed, rinsed in PBS, frozen in liquid nitrogen, and stored at −80°C before Aβ analysis. Animal care was in accordance with institutional guidelines.

Mouse Forebrain Fractionation and Proteinase K Treatment.

Cerebral hemispheres from killed mice were homogenized in 10 volumes of buffer A [5 mM Tris·HCl, 250 mM sucrose, 1 mM EGTA, and 5 mM EDTA (pH 7.4) containing a protease inhibitor mixture] by using 30 passes of a Dounce homogenizer. The homogenates were centrifuged at 5,000 × g for 10 min, and the resultant supernatant was centrifuged at 100,000 × g for 1 h. The supernatant (soluble fraction) was removed and the membrane pellet resuspended in 200 μl of buffer A lacking sucrose. For proteinase K digestion, aliquots of the resuspended membrane pellet were detergent-solubilized by the addition of an equal volume of buffer A lacking sucrose but containing 1% (wt/vol) sodium deoxycholate and 1% (vol/vol) Nonidet P-40. The samples were incubated at 4°C for 1 h and then centrifuged for 10 min at 11,600 × g. The detergent-soluble supernatant was incubated in the absence or presence of 20 μg/ml proteinase K for 1 h at 37°C.

Immunoprecipitation, SDS/PAGE, and Immunoelectrophoretic Blot Analysis.

Proteins were immunoprecipitated and resolved by SDS/PAGE by using 7–17% polyacrylamide gradient gels and transferred to Immobilon P poly(vinylidene difluoride) membranes as previously described (32, 35). Antibody 3F4 recognizes an epitope tag at residues 108–111 of the murine prion protein, and antibody 6E10 recognizes amino acid residues 1–17 of the human Aβ sequence (both Signet Laboratories, Dedham, MA). Antibody 6H4 (Prionics AG, Zurich, Switzerland) recognizes the sequence DYEDRYYRE (human PrP: amino acids 144–152). Ab54 recognizes the C-terminal region of APP, and antibody 1A9 recognizes a neoepitope on sAPPβ formed after β-secretase cleavage of APP (36). Antibody 9B21 was raised to the catalytic domain of BACE1 by using BACE1-Fc fusion protein as immunogen. Antibody 22C11 (Chemicon International, Temecula, CA) recognizes amino acid residues 66–81 in the N terminus of APP. Antibody SAF-32 (Cayman Chemical, Ann Arbor, MI) recognizes the octapeptide repeat region located in the N-terminal region of PrP. Bound antibody was detected by using peroxidase-conjugated secondary antibodies in conjunction with the enhanced-chemiluminescence (ECL) detection method (Amersham Life Sciences, Buckinghamshire, U.K.).

ELISA Quantification of Aβ Peptides.

Mouse forebrains were homogenized in 10 volumes of 0.2% (vol/vol) diethylamine in 50 mM NaCl by 35 passes of a Dounce homogenizer. The homogenate was then centrifuged at 100,000 × g for 1 h, and the supernatants were neutralized by the addition of 1/10 volume of 0.5 M Tris·HCl (pH 6.8). The brain homogenates or conditioned medium from N2a cells (100 μl) was added to assay plates containing 50 μl of 0.02 M sodium phosphate (pH 7.0), 2 mM EDTA, 0.4 M NaCl, 0.2% BSA, 0.05% CHAPS, 0.4% Block Ace, 0.05% NaN3 and analyzed by using the BNT77/BA27 and BNT77/BC05 sandwich ELISA systems to detect Aβ1–40 and Aβ1–42, respectively (37). Human Aβ1–40 and Aβ1–42 in conditioned medium from SH-SY5Y cells were captured by using biotinylated 6E10. BioVeris-tagged Aβ C-terminal specific antibodies were then used to detect Aβ1–40 and Aβ1–42. Antibody–Aβ complexes were captured with streptavidin-coated dynabeads and assayed in a BioVeris M8 analyzer. Standard curves were constructed by using Aβ1–40 and Aβ1–42 dissolved in DMSO.

ELISA Quantification of GAG Binding to PrPC.

ELISAs were performed as described previously (38). Plates were coated with the desired GAG before blocking the wells with 3% BSA in PBS. After washing with PBS-Tween (0.05%), cell lysate protein was added over a concentration range of 1 to 100 μg/ml. After a 2-h incubation at room temperature, the plate was washed three times with PBS-Tween (0.05%) and incubated with a 1:3,000 dilution of 3F4 overnight at 4°C. The plate was washed three times with PBS-Tween (0.05%), and peroxidase-conjugated rabbit anti-mouse IgG was added to the wells. After a 1-h incubation at room temperature, the plate was washed by using PBS-Tween (0.05%) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (Roche Diagnostics Ltd., East Sussex, U.K.), and the absorbance was measured at 405 nm.

Statistical Analyses.

Results are given as mean ± SD. Statistical analyses were performed by using Student's t test (two-tailed), and the null hypothesis was rejected at the 0.05 level.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Takeda Chemical Industries for the BNT77, BA27, and BC05 antibodies. This work was supported by Medical Research Council of Great Britain Grant G9824728 (to A.J.T. and N.M.H.), European Union Grant QLG3-CT-2001–02353 (to N.M.H.), National Institutes of Health Grants NS042192 and NS048554–01 (to C.B.E.), the Alzheimer's Association (E.A.E.), the Robert H. and Clarice Smith and Abigail Van Buren Alzheimer's Disease Research Program, and the Mayo Foundation for Medical Education and Research (C.B.E. and E.A.E.). A.J.T. and N.M.H. are members of the Yorkshire Centre in the Alzheimer's Research Trust's Alzheimer's Disease Research Centre Network.

Abbreviations

amyloid β
AD
Alzheimer's disease
APP
amyloid precursor protein
BACE1
β-site APP cleaving enzyme
CJD
Creutzfeldt–Jakob disease
GAG
glycosaminoglycan
GSS
Gerstmann–Scheinker–Straussler
LMW
low molecular weight
PrP
prion protein
PrPC
cellular form of PrP
PrPSc
infectious form of PrP
sAPPα
soluble ectodomain of APP after α-cleavage
sAPPβ
soluble ectodomain of APP after β-cleavage
TSE
transmissible spongiform encephalopathy.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0609621104/DC1.

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