Characterization of beta-secretase - Biochemical Society Transactions

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20 Macias, M. J., Hyvonen, M., Baraldi, E., Schultz, J., Sudol, M., Saraste, M. and Oschkinat, H. (1996) Nature (London) 382,646-649 21 Ermekova, K. S., Zambrano, N., Linn, H., Minopoli, G., Gertler, F., Russo, T. and Sudol, M. (1997) J. Biol. Chem. 272,32760-32768 22 Gertler, F. B., Neibuhr, K., Reinhard, M., Wehland, J. and Soriano, P. (1996) Cell 87, 227-239 23 Ferreira, A., Caceres, A. and Kosik, K. S. (1993) J. Neurosci. 13, 31 12-3123 24 Duclos, F., Boschert, U., Sirugo, G., Mandel, J.-L., Hen, R. and Koenig, M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 109-113

25 Blanco, G., Irving, N. G., Brown, S. D. M., Miller, C. C. J. and McLoughlin, D. M. (1998) Mamm.

Genome, in the press 26 Saras, J. and Heldin, C.-H. (1996) Trends Biochem. Sci. 21,455-458 27 Okamoto, M. and Sudhof, T. C. (1997) J. Biol. Chem. 272,31459-31464 28 Sudhof, T. C. (1995) Nature (London) 375, 645-653

Received 3 March 1998

Characterization of /?-secretase D. J. Stephens and B. M. Austen Department of Surgery, St George’s Hospital Medical School, Cranmer Terrace, London SW I 7 ORE, U.K.

Processing of amyloid precursor protein 1-Amyloid is a 40-43 residue neurotoxic peptide that is deposited in fibrillar form in senile plaques in the brains of Alzheimer’s disease patients. It is released by proteolysis of several transmembrane glycoproteins [amyloid precursor protein (APP) (molecular mass 100-140 kDa)] which are derived by differential splicing of the 19 exons of a single gene located on chromosome 21. During intracellular transport, APP is cleaved between Lys-687 and Leu-688 (numbered according to APP770; amino acids 16 and 17 of P-amyloid) by a-secretase to create fragments that are non-amyloidogenic. T h e N-terminus of P-amyloid is released by /j-secretase cleavage at Met-671 to generate the N-terminus of P-amyloid Asp-672. T h e importance of this cleavage is emphasized by the presence of a double APP mutation in a Swedish family at amino acids 670 and 671. Cleavage of the mutant Leu-Asp bond results in an 8-fold increase of /j-amyloid in transfected cells [ 13. Cleavage that releases the C-terminus of P-amyloid is termed y-secretase activity. In transgenic animals expressing mutant forms of APP or presenilins, the production of P-amyloid 1-42, which is more neurotoxic than P-amyloid 1-40, is up-regulated [2]. Prolyl endopeptidase [3] calpain [4] and a chymotryptic protease associated with the proteasome [S] have been suggested to act as Abbreviations used: APP, P-amyloid precursor protein; Man-6-PR, mannose-6-phosphate receptor.

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y-secretase. As proteasomes are cytoplasmic, exposure or release of APP from the lipid bilayer would be required for y-secretase to gain access to its cleavage point, predicted to lie within the transmembrane region. Alternatively y-secretase may be related to a recently reported novel metalloproteinase which cleaves the transmembrane region of sterol-regulatory element-binding proteins from within the membrane [6]. Production of P-amyloid 1-42 is not effected by brefeldin A, whereas monensin, which blocks transport through the Golgi apparatus, allows P-amyloid 1-42 to accumulate, indicating that y-secretase may be localized in the endoplasmic reticulum [7]. In contrast, the y-secretase that releases P-amyloid 1-40 is localized close to the cell surface [8]. Differential localization would be in keeping with different actions of the inhibitor MDL28170 on the production of [j-amyloid 1-40 and P-amyloid 1-42 [9].

Synthetic peptide substrates Candidate proteases for p-secretase include metalloendopeptidase (3.4.24.14 [ lo]), gelatinase [ l l ] , cathepsin S [12] and cathepsin D [13]. As tools to identify P-secretase, we have produced an affinity-purified antibody directed to the cleaved N-terminus of /j-amyloid using a synthetic peptide of the N-terminal 10 residues of P-amyloid (DAEFRHDSGY) (NTP4) coupled via a C-terminal Cys residue to a protein carrier. T h e antibody has been shown not to cross-react with whole APP expressed in transfected 293

Suspect Proteins in Neurodegeneration

HEK cells [141. From 3sS-methionine-labelled cells, anti-NTp4 immunoprecipitates P-amyloid and a 13 kDa amyloidogenic fragment. T o measure and detect APP, we have raised affinitypurified antibodies to the C-terminal 15 residues of APP (CT15) coupled to an N-terminal Cys residue. Anti-CT15 immunoprecipitates and reacts in Western blots with the variously glycosylated forms of APP, and also immunoprecipitates the 13 kDa fragment, indicating that it contains the domain of APP from the p-secretase cleavage site to the C-terminus of the protein and that it is a metabolic precursor of p-amyloid. The processing of APP to the C-terminal fragment is reported to be inhibited by ammonium chloride and chloroquine, suggesting that it is produced in an acidic compartment in the endosomes or lysosomes [15]. Cathepsin D is located in lysosomes, endosomes and the transGolgi network, and also in senile plaques in Alzheimer’s brain tissue [ 161. We have synthesized a peptide P 1 (TTRPGSGLTNI KTEEISEVKMDAEFRHDSGY)which overlaps the P-secretase cleavage site in APP as a substrate for p-secretase (the P-amyloid sequence is shown in bold type). We found by HPLC and mass spectrometry that cathepsin D cleaved P1 into a mixture of four peptides, giving 80% cleavage between Lys-670 and Met-671 and 20% between Glu-668 and Val-669 [17]. Thus, cathepsin D is unlikely to be p-secretase, although the protease is known to enhance the production of 5 kDa and 12 kDa amyloidogeneic fragments from APP [ 181. T h e anti-NTP4 antibodies were used to identify p-secretase activity in cell extracts by monitoring cleavage of peptide P 1 overlapping the p-secretase cleavage site. T o identify p-secretase activity on SDS/PAGE gels, extracts of rat whole brain, human cortex and human white blood cells isolated from fresh donor blood were subjected to electrophoresis on 6% polyacrylamide SDS/PAGE. The separated proteins were transferred to nitrocellulose sheets, which were then incubated in 50 mM Tris/HCI, 5 mM CaC12, 5 mM zinc acetate, 2% Triton X-100 (pH 6.5) overnight to renature proteases. The nitrocellulose sheet was then incubated in a solution of peptide P1 (0.3 mglml) to allow the renatured secretase to cleave at the N-terminus of the P-amyloid sequence and the released N-terminal sequence to bind to the nitrocellulose at the location of the secretase. Excess peptide was removed by washing in phosphate-buffered salline containing 0.05% Tween-20 and 5% casein.

Western blots were developed after incubating in 1:200 dilutions of anti-NTP4. The results showed that the dominant band derived from human brain detergent extracts had a molecular mass of approximately 150 000 Da. A band of the same molecular mass but much lower intensity was present in extracts of rat brain and human white blood cell extracts. The presence of a strong band in human brain would be in keeping with previous evidence that neurons are the major cell type that produce P-amyloid [ 191, whereas rat p-amyloid has a dissimilar N-terminal sequence to human P-amyloid. A weak band of >200000 Da was also present in all extracts. A Western blot of the same extracts in which the nitrocellulose was not incubated with the P2 peptide did not show either of these bands. Thus, the 150 000 Da band may represent p-secretase activity. As reducing agents were not added to extracts prior to electrophoresis, it is uncertain whether the 150000 Da band represents a subunit, or an oligomeric form of the enzyme, or a complex of p-secretase activity with other proteins.

Role of internalization signal in APP We have explored the role of the endocytic pathway in production of /?-amyloid. PCR-directed site-specific mutagenesis was used to investigate the role of the consensus internalization signal, Tyr-Glu-Asn-Pro-Thr-Tyr,in the cytoplasmic tail of APP in the generation of P-amyloid. Using a cassette approach, and nested PCR, the essential tyrosine residues within this motif were mutated to alanine or phenylalanine. Constructs were then expressed in cultured cells, and endocytosis of mutant APPs measured by cell-surface biotinylation with a cleavable biotin, followed by immunoprecipitation with avidin and anti-CT15 after cleavage. The rates of internalization of cell surface mutant APPs at 37°C were measured after labelling the extracellular domain, by FACS scanning. APP processing was measured by immunoprecipitation of APP metabolites from 35 S-metabolically labelled cells with anti-CT15 and anti-NTp4. Biotinylation and FACS (Table 1) analyses showed that there were reduced rates of endocytosis of cell-surface APP containing the mutant Ala-Glu-Asn-Pro-Thr-Ala sequence in its cytoplasmic tail compared to the wild-type APP. In contrast, there was no change in the rates of endocytosis of single mutations in Tyr-Glu-AsnPro-Thr-Tyr. However, there was no change in

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processing of APP (Table Z), even in the double mutant.

Table I

SO2

FACS analysis of internalization of APP targeting mutants Duplicate aliquots of I x I O6 293 HEK cells in a volume of 50 pI of complete medium were incubated at 4°C for 90 min with the primary antibody mixture (5A3/1G7; from Dr. E. Koo, Brigham and Women's Hospital, Harvard, Boston, U.S.A.) at a dilution of I :20 (final concentration 50 pg/ml). Cells were washed twice in ice-cold PBS containing 2% foetal calf serum (FCS) before incubating at either 4°C in ice-cold complete medium or at 37°C in prewarmed complete medium for 30 min. Cells were chilled on ice and incubated in ice-cold complete medium containing fluorescein-isothiocyanate-conjugated anti-mouse F(ab')> fragments (Dako) at a dilution of I :20. After washing in ice-cold PBS containing 2% FCS and fixing in I % paraformaldehyde (freshly made) fluorescence measurements were performed on a BectonDickinson FACScan running LYSYS II software. Samples held at 4°C represented total cell-surface APP while those at 37°C represented APP remaining on the cell surface after endocytic internalization.

Mutant Tyr, Tyr Ala, Tyr Tyr, Phe Ala, Phe Tyr, Ala Ala, Ala

Labelling at 4°C (%) 34 34 40 7 24 72

Labelling at 37°C (%) 9

Internalized (%)

I1 2

3 51

lntracellular trafficking The production of P-amyloid is not effected by mutagenesis of internalization signals in the cytoplasmic domain of APP. Immunolocalization and measurements of processing performed in the presence of trafficking inhibitors suggests that P-processing of APP to P-amyloidogenic products occurs late in the secretary pathway, in

78 85 71 71 87 29

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lmmunofluorescent localization We have also used immunofluorescent labelling with anti-NTP4 and anti-CT15 and confocal microscopy to determine the subcellular localization of P-amyloid and the amyloidogenic 13 kDa product [14]. The staining for anti-NTP4 co-localized partly with staining of the mannose6-phosphate receptor (Man-6-PR) in the secretory pathway, in the trans Golgi network and late-endosomes (confirming the results of a number of other workers), but not with lysosoma1 or cell surface markers. Staining was more widespread than that of Man-6-PR. Incubation of the cells with brefeldin A increased separation of anti-NTP4 and anti-Man-6-PR, consistent with the localization of some of the 13 kDa fragment and/or P-amyloid in the endoplasmic reticulum.

Table 2 APP processing in 293 HEK cells transfected with internalization mutants Subconfluent transfected 293 HEK cells on 6 cm dishes were radiolabelled overnight with I00 pCi/ml ["S]rnethionine in I .5 rnl of methionine-free Dulbecco's modified Eagle's medium containing 10% foetal calf serum. After labelling, medium was collected and cells were lysed in I YO NP40 and I YO Triton X- 100. After immunoprecipitation with anti-CT I5 or anti-NTj4 and protein A-Sepharose, samples were boiled in SDS sample buffer and subjected to SDS/PAGE on 6% acrylamide gels or SDS/PAGE/Tricine gels ( 16.5% acrylamide). Bands were visualized by autoradiography. and quantified by densitometry on a Joyce Loebl Chromoscan 3 densitometer. Peak integrals corresponding to APP, the nonamyloidogenic 8 kDa C-terminal fragment and 8-amyloid were measured. Expression was normalized for each mutant by dividing the peak integral for each fragment by that for APP.

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Mutant

Mean peak integral (fl-amyloid)

Standard deviation (fl-amyloid)

Tyr. Tyr Ala. Tyr Tyr, Phe Ala, Phe Tyr, Ala Ala, Ala

0.037 0.032 0.038 0.032 0.030 0.033

0.01 I 0.01 I 0.0 I 4

0.009 0.0 I 2 0.01 I

Mean peak integral (8 kDa C-terminal fragment) 0.395 0.342 0.395 0.424 0.382 0.458

Standard deviation (8 kDa C-terminal fragment) 0.121 0. I05

0.092 0. ! 37 0.1 I I 0. I30

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Figure I Putative intracellular targeting and P-processing of APP

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I

I

I

I

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the trans Golgi network or late endosomes. A 13 kDa C-terminal fragment of APP would seem to be the main biosynthetic precursor of p-amyloid. T o account for these observations, and the report that P-amyloid 1-42 is generated in the endoplasmic reticulum, the 13 kDa fragment may be transported back from the trans Golgi network by a brefeldin-insensitive microtubulemediated retrograde pathway for further processing to 1-42, or to the cell surface for processing to 1-40. A schematic description of likely targeting events is shown in Figure 1. y-Processing of the 13 kDa fragment to /I-amyloid 1-42 in the endoplasmic reticulum may localize in the same subcellular compartment as the presenilins, and may be affected by the presence of familial Alzheimer's disease mutations in presenilins [Z]. Presenilin knockout mutants are known to be defective in production of P-amyloid 1-42. A recently described protein, endoplasmic reticulum amyloid binding protein (ERAB), related in sequence to hydroxysteroid dehydrogenase, is known to translocate from the endoplasmic reticulum to the cell surface in the presence of P-amyloid [21]; this protein could provide a mechanism of export for P-amyloid 1-42 (Figure 1). We are grateful to Research into Ageing and Immunogenetics for their support.

1 Citron, M., Oltersdorf, T., Haass, C., McConlogue,

1

L., Hung, A. Y., Seubert, P., Vigo-Pelfrey, C., Lieberburg, I. and Selkoe, D. J. (1992) Nature (London), 360, 672-674 2 Borchelt, D. R., Ratovitski, T., van Lare, J., Lee, M. K., Gonzales, V., Jenkins, M. A., Copeland, N. G., Price, D. L. and Sisodia, S. S. (1997) Neuron 19, 939-945 3 Shinoda, M., Toide, K., Ohsawa, I. and Kohsaka, S. (1997) Biochem. Biophys. Res. Commun. 235, 641-645 4 Yamazaki, T., Haass, C., Saido, T. C., Omura, S. and Ihara, Y. (1997) Biochemistry 36,8377-8383 5 Mundy, D. I. (1994) Biochem. Biophys. Res. Commun. 204, 333-341 6 Sakai, J., Duncan, E. A., Rawson, R. B., Hua, X., Brown, M. S. arid Goldstein, J. L. (1996) Cell 85, 1037-1046 7 Wild-Bode, C., Yamazaki, T., Capell, A., Leimer, U., Steiner, H., Ihara, Y. and Haass, C. (1997) J. Biol. Chem. 272, 16085-16088 8 Koo, E. H. and Squazzo, S. L. (1994) J, Biol. Chem. 269, 17386- 17389 9 Citron, M., Diehl, T. S., Gordon, G., Biere, A. L., Siebert, P. and Selkoe, D., J. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 13170-13175 to McDermott, J. R., Biggins, J. A. and Gibson, A. M. (1992) Biochem. Biophys. Res. Commun. 185, 746-752 11 LePage, R. N., Fosang, A. J., Fuller, S. J., Murphy, G., Evin, G., Beyreuther, K., Masters, C. L. and Small, D. H. (1995) FEBS Lett. 377, 267-270 12 Munger, J. S., Haass, C. A., Lemere, C. A., Shi, G. P., Wong, W. S., Teplow, D. B., Selkoe, D. J. and Chapman, H. A. (1995) Biochem. J. 311,299-305

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13 Ladror, U. S., Snyder, S. W., Holzrnann T. F., Krafft, G. A. and Wang, G. T. (1994) J. Biol. Chern. 269, 18422-18428 14 Stephens, D. J. and Austen, B. M. (1996) J. Neurosci. Res. 46, 21 1-225 15 Estus, S., Golde, T. E., Kunishita, T., Blades, D., Lowery, D., Eisen, M., Usiak, M., Qu, X. M., Tabira, T., Greenberg, B. D. et al. (1990) Science 255, 726-728 16 Cataldo, A. M. and Nixon, R. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3861-3865 17 Austen, B. M. and Stephens, D. J. (1995) Biomedical Peptides, Proteins and Nucleic Acids 1, 243-246 18 Dreyer, R. N., Bausch, K. M., Fracasso, P.,

Harnrnond, P. J., Wunderlich, D., Wirak, D. D., Davis, G., Brini, C. M.,Buckolz, J. M., Konig, G. et al. (1994) Eur. J. Biochern. 224,265-271 19 Sirnons, M., de Strooper, B., Multhaup, G., Tienari, P. J., Dotti, C. G. and Beyreuther, K. (1996) J. Neurosci. 16, 899-908 20 Harter, C. and Mellrnan, I. (1992) J. Cell Biol. 117, 311-325 21 Yan, S. D., Ku, J., Soto, C., Chen, X., Zhu, H., Al-Mohanna, F., Collison, K., Zhu, A., Stern, E., Saido, T., Tohyama, M. et al. (1997) Nature (London) 389,689-696 Received 1 April 1998

Transgenic mouse models of Alzheimer’s disease D. J. S. Sirinathsinghji Merck Sharp and Dohrne Research Laboratories, Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, U.K.

Introduction Progress in the development of transgenic mouse models of Alzheimer’s Disease (AD) has come largely from the recent advances in the molecular genetics of the disease. Such studies have identified mutations in four different genes which are involved in early onset familial AD (FAD) [l-31. They are the /I-amyloid precursor protein (APP) gene on chromosome 21, the presenilin (PS)l gene on chromosome 14, the PS2 gene on chromosome 1 and the apolipoprotein E (APOE) gene on chromosome 19. At least five mis-sense mutations in the APP gene within or near the amyloid P-protein (AP) sequence have been identified leading to early-onset FAD and cerebral haemorrhage in several unrelated families. Three single-point mutations in exon 17 of APP at amino acid 717 (C terminus of AD) have been identified, with the valine residue substituted by either isoleucine, glycine or phenylalanine. In addition, a double-point mutation in the APP gene has been found in a large Swedish family with early onset (at about 55 years) FAD. T h e Abbreviations used: AP, arnyloid P-protein; AD, Alzheimer’s disease; APP, P-arnyloid precursor protein; APOE, apolipoprotein E; CADASIL, cerebral autosornal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; FAD, familial Alzheimer’s disease; LTP, long-term potentiation; MAP-2, rnicrotubule associated protein-2; PHF, paired helical filament; PS, presenilin.

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mutation consists of a lysine to asparagine and a methionine to leucine amino acid change at codons 670 and 671 respectively (Lys-670Asn, Met-671Leu) at the N-terminus of AS. Mutations in the PS1 gene accounts for the majority (approximately 20-50%) of early onset (at about 28-50 years) FAD cases and about 2-4% (or less) of all AD cases. At least 42 missense mutations and a splice site mutation have been identified so far. Two mutations have been found in the PS2 gene, Asn-141Ile and Met239Va1, in Volgan German families with onset ages of about 40-75 years. T h e APOEc4 allele is a susceptibility marker with incomplete penetrance, conferring a higher risk of developing AD at an earlier age. T h e plasma, fibroblasts and platelets from patients with APP or PS1 mutations all show increased levels of API-42(3), which is deposited much earlier than AP1-40 in the brains of AD and Down’s syndrome patients and is potentially more amyloidogenic [4]. T h i s evidence gives support to the current concept that the AP peptide is toxic to neurons and its increased generation from misprocessing of APP may be a major pathogenic mechanism. However, much evidence indicates that synapse loss, neuronal loss and paired helical filaments (PHFs) correlate better with clinical dementia than AP plaque load and distribution. Whether the early deposition of AP1-42(3),which may act as a nidus for AP1-40 accumulation, is the primary cause of AD