BACE (β-secretase) modulates the processing of APLP2 in vivo

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 25 (2004) 642 – 649

BACE (h h-secretase) modulates the processing of APLP2 in vivo L. Pastorino, a A.F. Ikin, a S. Lamprianou, b N. Vacaresse, b J.P. Revelli, c K. Platt, c P. Paganetti, d P.M. Mathews, e S. Harroch, b and J.D. Buxbaum a,f,* a

Department of Psychiatry, Mount Sinai School of Medicine, New York, NY, 10029, USA Unite´ de Neurovirologie et Re´ge´ne´ration du Systeme Nerveux, Institut Pasteur, Paris, France c Lexicon Genetics, The Woodlands, TX, 77381, USA d Nervous System, Novartis Pharma AG, CH-4002, Basel, Switzerland e Nathan Kline Institute, Orangeburg, NY 10962, USA f Department of Neurobiology, Mount Sinai School of Medicine, New York, NY, 10029, USA b

Received 4 June 2003; revised 1 December 2003; accepted 3 December 2003

BACE is an aspartyl protease that cleaves the amyloid precursor protein (APP) at the h -secretase cleavage site and is involved in Alzheimer’s disease. The aim of our study was to determine whether BACE affects the processing of the APP homolog APLP2. To this end, we developed BACE knockout mice with a targeted insertion of the gene for h -galactosidase. BACE appeared to be exclusively expressed in neurons as determined by differential staining. BACE was expressed in specific areas in the cortex, hippocampus, cerebellum, pons, and spinal cord. APP processing was altered in the BACE knockouts with Ah h levels decreasing. The levels of APLP2 proteolytic products were decreased in BACE KO mice, but increased in BACE transgenic mice. Overexpression of BACE in cultured cells led to increased APLP2 processing. Our results strongly suggest that BACE is a neuronal protein that modulates the processing of both APP and APLP2. D 2004 Elsevier Inc. All rights reserved.

Introduction The core of the amyloid plaques, which are a characteristic feature of Alzheimer’s disease (AD), are composed of Ah peptides, generated by the processing of the larger amyloid precursor protein (APP) and aggregated as deposits in the brain parenchyma, causing neuronal death (Glenner and Wong, 1984; Glenner et al., 1984). For this reason, the enzymes responsible for the generation of these peptides (the so-called h- and g-secretases) are considered therapeutic targets important in the treatment of Alzheimer’s disease

Abbreviations: Ah, h-amyloid peptide; AD, Alzheimer’s disease; APP, amyloid precursor protein; APLP, amyloid precursor like protein; BACE, h-site APP cleaving enzyme; WT, wild-type; HET, heterozygous; KO, knockout; TG, transgenic; sAPLP2, soluble APLP2; AICD, APPintracellular domain; ER, endoplasmic reticulum; CNS, central nervous system. * Corresponding author. Mount Sinai School of Medicine, One Gustave L. Levy Place, box 1668, New York, NY, 10029. Fax: +1-212-828-4221. E-mail address: [email protected] (J.D. Buxbaum). Available online on ScienceDirect (www.sciencedirect.com.) 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2003.12.013

(AD) (Citron, 2002; Hardy and Selkoe, 2002; Wolfe et al., 2002). Proteins showing g-secretase (De Strooper et al., 1998; Kimberly et al., 2000; Selkoe and Wolfe, 2000; Wolfe et al., 1999) and hsecretase activity have been identified (Farzan et al., 2000; Hussain et al., 2000; Vassar and Citron, 2000); in particular, beta-site amyloid cleaving enzyme (BACE) has been characterized as the major h-secretase activity in vivo and in vitro, and is expressed both in CNS and peripheral tissues (Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999). Studies performed on synthetic substrates and in cultured cells show that BACE cleaves APP at the aspartate residue D1 that begins the sequence of the Ah peptides, generating the APP Cterminal fragment C99, which is associated with the membrane (Huse et al., 2002; Liu et al., 2002; Vassar et al., 1999; Yan et al., 1999). C99 is further cleaved by g-secretase, an activity that includes presenilin 1, at residues-40/42/43, generating the Ah peptides Ah1-40/42/43 (Duff et al., 1996; Scheuner et al., 1996). Recently, studies performed in vitro have demonstrated that BACE is able to cleave within the sequence of Ah at glutamic acid residue E11 generating C89, a shorter membrane-associated C-terminal fragment that can be further cleaved by g-secretase (Andrau et al., 2003; Huse et al., 2002; Lee et al., 2002; Liu et al., 2002). The resulting Ah11-40/42 peptides could be involved in the pathogenesis of Alzheimer’s disease, since they have been detected in brain extracts of AD patients (Huse et al., 2002), and they are known to exhibit fibrillogenic and neurotoxic characteristics in vitro (Pike et al., 1995). APP like protein 2 (APLP2) is a type I membrane-inserted protein belonging to the APP-superfamily (Sprecher et al., 1993; Wasco et al., 1992, 1993) that shares high homology with APP. Like in the case of APP, APLP2 is expressed both in CNS and peripheral tissues, and this anatomical distribution is conserved among species, from human to mouse (Bayer et al., 1999; Lorent et al., 1995; McNamara et al., 1998; Slunt et al., 1994). Also, it has been demonstrated that, like APP, APLP2 can be processed, generating a large extracellular soluble ectodomain (Paliga et al., 1997; Slunt et al., 1994). Recently, Scheinfeld et al. (2002) showed that APLP2 can be processed by g-secretase and that the generated intracellular C-terminal domain can translocate to the nucleus as

L. Pastorino et al. / Mol. Cell. Neurosci. 25 (2004) 642–649

part of a transcriptionally active complex, like the APP-intracellular-domain (AICD) (Baek et al., 2002; Cao and Sudhof, 2001). Little is known about the function of APLP2. Although studies performed both in cultured cells (White et al., 1998) and in knockout animals (Heber et al., 2000) have shown that it might have functions similar and/or redundant to those of APP, APLP2 cannot contribute to the generation of Ah peptides since it does not contain the sequence for Ah. Thus, the processing of APLP2 is not amyloidogenic, although it may involve a similar set of secretase activities, such as g-secretase. In this study, we developed knockout animals to study BACE expression and function as a potential modulator of APLP2 processing.

643

introduces an IRES h-galactosidase (h-gal) cassette, replacing wild-type BACE with an allele containing the h-gal gene. Since expression of the h-gal gene is now driven by the BACE promoter, this strategy enables us to study the normal expression of BACE by following the expression of the h-gal reporter as described below. Animals were genotyped by PCR and Southern blot. The mutant allele was present only in the heterozygous and knockout animals. As expected, the wild-type allele was absent in the knockout animals (Fig. 1B), leading to lack of BACE protein expression in knockout brain extracts, whereas brain extracts from wild-type animals exhibited two bands corresponding to the mature and immature forms of BACE (Fig. 1C). Animals with disruption in BACE in one or both alleles did not show gross abnormalities, and the genotypes were transmitted as expected for Mendelian inheritance.

Results Localization of BACE expression in the central nervous system Characterization of BACE deficient mice BACE knockout mice were generated by deletion of exon 1, which contains the start codon (Fig. 1A). The disrupted allele

We used h-gal expression, assayed by h-gal activity, to study the regional distribution of BACE in situ (Fig. 2). h-Gal activity was detected in the following brain areas: cortex, hippocampus,

Fig. 1. Generation of a BACE knockout. (A) BACE knockout (KO) mice were generated by deleting a region of exon 1, which was replaced with a DNA sequence containing an expression cassette for the reporter gene h-galactosidase. (B) The presence of the wild-type (WT) or mutant (KO) allele was assessed both by PCR (left) and Southern blot (right) of genomic DNA in wild-type, heterozygous (HET), and knockout animals. (C) BACE was not detectable by immunoblot in the Triton X-100 extracts from cortex and hippocampus of BACE KO mice with a BACE-specific antibody.

644

L. Pastorino et al. / Mol. Cell. Neurosci. 25 (2004) 642–649

Fig. 2. BACE is expressed in neuronal cells in the CNS. Coronal sections (rostral to caudal) of P10 mouse brain (a – c) and sagittal sections of adult mouse brain (d – f) were stained for Gal4, NeuN, or GFAP. Squares in c, d, and e indicate hippocampal areas (RS, PV, DG, Den), whole hippocampus (dV, eV, e), and cortex (d), which were further magnified and depicted alongside. Cerebellum is depicted in f with magnified details in f V and f. PL, prelimbic area; Cl, claustrum; Pir, piriform cortex; Den, dorsal endopiriform cortex, Ven, ventral endopiriform cortex; DG, dentate gyrus; CA1, CA3, fields of hippocampus; mol, molecular layer of hippocampus; V, trigeminal nucleus, Ves, vestibular nucleus; PV, paraventricular thalamic nucleus; RS retrosplenial cortex.

hypothalamus, cerebellum, pons, and in the spinal cord. In the cortex, BACE was expressed in the claustrum, piriform, dorsal and ventral endopiriform, and retrosplenial from the most rostral to the caudal part of the structure, both in 10 days old (P10) and adult animals (Figs. 2a, b, c, d, d, e). The expression in the diencephalon was restricted to the paraventricular nuclei of the hypothalamus (containing corticotrophin releasing factor neurons) (Fig. 2c). In the pons, h-gal expression was observed in tegmental, parabrachial, vestibular, and trigeminal nuclei in young and adult animals (data not shown). These structures are involved in neurovegetative functions. The expression in the spinal cord was restricted to the dorsal horn, which receives inputs from sensory neurons of the dorsal root ganglia (data not shown). To determine which types of cells express BACE, we performed differential immunostaining to visualize neuronal (anti-NeuN) and glial (anti-GFAP) cell populations (Figs. 2e, e, e, f). BACE expression was observed in pyramidal cells and their distal dendrites extended into the CA field (lacunosum and molecular layers) of the hippocampus. The cell bodies of these cells were also immunostained with anti-NeuN antibodies, confirming their neuronal phenotype. Within the dentate gyrus of the hippocampus, h-gal activity was localized within NeuN-positive granular cells particularly in the proximal dendrites within the molecular layer. Both CA and DG molecular layers receive direct inputs from the entorhinal cortex via the perforant pathway,

representing the only entrance from the cortex into the hippocampus. In the cerebellum, h-gal activity was detected in Purkinje cells (Figs. 2f, f V, f U). h-gal activity did not overlap with GFAP-immunostaining, indicating that BACE is not expressed in glial cells (Figs. 2 eV, e, f). We also examined h-gal activity in primary glial cultures prepared from neocortices of newborn animals. We could not detect any h-gal activity in astrocytes, microglia, or oligodendrocytes suggesting that BACE is primarily a neuronal molecule (data not shown). In addition to the brain, h-gal was expressed in peripheral tissues such as spleen and intestine (data not shown). All together, these results are consistent with previously published data (Hussain et al., 1999; Vassar et al., 1999). Alteration of APP processing in BACE deficient mice Since BACE is the major aspartyl protease to cleave APP, we were interested to know how lack of BACE will affect APP processing. To this end, we analyzed the levels of holo-APP and C-terminal fragments in membrane extracts of brains from BACE knockout animals (Fig. 3). The ratio of mature APP to immature APP increased significantly in BACE knockout compared to wildtype mice (Figs. 3A, B) indicating that the lack of expression of BACE leads to impaired processing of mature APP. In BACE transgenic mice (Bodendorf et al., 2002), the ratio of mature to

L. Pastorino et al. / Mol. Cell. Neurosci. 25 (2004) 642–649

645

Fig. 3. Altered APP processing in BACE knockout mice. (A) Extracts from brains of BACE KO mice, BACE transgenic (Tg) mice and their relative controls (WT) were immunoblotted with 369. Mature (APPmat), immature (APPimm), and the C-terminal fragments of APP are indicated. (B) Ratios of APP mature vs. APP immature were quantified in WT, BACE KO, and transgenic (Tg) mice. Data represent mean F SEM of three experiments run in duplicate. *P < 0.05; **P < 0.005 Fisher’s post hoc comparison test.

immature APP was significantly decreased compared to wild-type mice (Figs. 3A, B). The altered processing of holoAPP in the knockout animals was paralleled by an altered pattern of the APP C-terminal fragments (Fig. 3A). In wild-type animals, four bands with apparent molecular weights between 14.5 and 10.0 kDa were detected by immunoblotting with the C-terminal antibody 369, whereas only the two lower bands were detected in BACE knockout brain extracts. By means of immunoblot analysis from lysates of cells expressing APPswe or APP751 with or without BACE, we were able to identify the larger bands in the wild-type animals as C99 and C89 APP C-terminal fragments (data not shown). Our data show that the formation of C99 and C89 was decreased in BACE knockout mice, whereas C89 was elevated in transgenic animals Table 1 Ah40 and Ah42 measurements from wild-type (WT), heterozygote (HET) and knockout (KO) mice

WT HET KO

Ah40

Ah42

15 F 1 13 F 2 <3.1

5F1 5F1 <3.1

Measurements were obtained by ELISA of brain extracts. Values are expressed as fmol/ml DEA extract (Rozmahel et al., 2002) and are means F SEM from three brains each. Levels of Ah40 and Ah42 in the brain of knockout mice were below the detection level of the assay.

Fig. 4. Altered processing of APLP2 in BACE knockout mice. (A) Levels of soluble APLP2 and APLP2 C-terminal fragments were detected by Western blot in brain extracts from BACE KO mice, BACE transgenic (Tg) mice and their relative controls (WT). Asterisk marks the approximately 12kDa band that is absent from KO brains and enriched in Tg brains. (B) Levels of soluble APLP2 (sAPLP2) and C-terminal fragment were quantified in WT, BACE KO, and Tg mice. Data represent mean F SEM of three experiments run in duplicate; sAPLP2: *P < 0.05; **P < 0.005. Cterminal fragment: *P < 0.05 Fisher’s post hoc comparison test.

(Fig. 3A). Levels of endogenous murine Ah40 and Ah42 decreased to undetectable levels in the BACE knockouts (Table 1). Levels of these peptides in heterozygous animals were indistinguishable from those in wild type. BACE modulates the processing of APLP2 Like APP, APLP2 is processed generating a large soluble Nterminal fragment and a smaller membrane-associated C-terminal stub. In addition, APLP2 and BACE co-localize in the Golgi and ER (Bayer et al., 1999; Hussain et al., 1999; Paliga et al., 1997; Vassar et al., 1999) and exhibit similar brain regional distribution (McNamara et al., 1998; Vassar et al., 1999). This suggests that BACE could be involved in the processing of APLP2. To address this, we examined APLP2 cleavage fragments in BACE wild-type, knockout, and transgenic mice using a set of antibodies specific for either the N-terminal or the C-terminal domains of APLP2. The levels of soluble APLP2 (sAPLP2) were reduced in BACE knockout mice compared to wild-type mice, but increased in

646

L. Pastorino et al. / Mol. Cell. Neurosci. 25 (2004) 642–649

Fig. 5. Human BACE modulates the processing of human APLP2. (A) Immunoblot of COS-7 cell lysates transfected with APLP2-myc (Control) or APLP2myc and BACE ( + BACE), probed for myc with 9E10. (B) Immunoblot of media from cells probed for soluble APLP2 (sAPLP2) with an APLP2 N-terminal antibody.

BACE transgenic mice compared to wild-type mice (Figs. 4A, B). These differences were paralleled by differences in the levels of a C-terminal fragment, as the fragment migrating at approximately 12 kDa was absent from the BACE knockout extracts and accumulated in the BACE transgenic (Figs. 4A, B). To ensure that the alteration in APLP2 processing in the knockout and transgenic mice was a result of altering BACE levels and that this effect is not limited to mouse, we examined APLP2 processing in cells co-transfected with human BACE and APLP2. This resulted in increased levels of the APLP2 C-terminal fragment as well as increased levels of secreted APLP2 (Fig. 5).

Discussion The aim of this study was to characterize BACE expression and function using BACE knockout mice as a model. We have shown here that BACE knockout mice provide a suitable model for the study of BACE activity in the brain, and that BACE regulates the processing of APLP2 in vivo and in vitro. BACE expression, visualized by h-gal activity, was restricted to defined neuronal populations in different brain areas, consistent with previous Northern and in situ studies (Bennett et al., 2000; Vassar et al., 1999). The pattern of BACE expression overlapped with structures that are known to accumulate h-amyloid peptides and to develop amyloid plaques. Consistent with previous reports (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001), our BACE knockout animals exhibited impaired processing of the mature form of APP that was reflected in the lack of formation of the h-secretase-dependent C99 and C89 C-terminal fragments. Furthermore, the fact that the production of the C89 C-terminal fragment is inhibited in our knockout animals

provides direct evidence that this fragment is generated specifically by BACE activity and not by other putative secretases. This is consistent with previous studies performed in vitro (Andrau et al., 2003; Liu et al., 2002). The opposite effect was seen in BACE transgenic mice, such that C99 and C89 fragments accumulated (Bodendorf et al., 2002; Fig. 3A). Ah levels decreased in BACE knockouts (as previously reported by Cai et al., 2001) but were unchanged in heterozygotes, suggesting that a 50% decrease in BACE levels may not affect Ah generation. The processing of APLP2 was also impaired in our BACE knockout mice. In particular, we observed reduced levels of secreted extracellular soluble APLP2, which was paralleled by the lack of formation of the APLP2 C-terminal fragment migrating at approximately 12 kDa in membrane extracts. Opposite effects were seen in BACE transgenic mice. Overexpression of BACE in cultured cells also caused increased APLP2 processing. It is important to note that the studies in cultured cells made use of human APLP2, indicating that the processing of both murine and human APLP2 is strongly modulated by BACE. Little is known about the physiological role of APLP2. Since it lacks the Ah sequence, direct or indirect proteolytic activity of hand g-secretase would not result in the generation of amyloidogenic products. It has been demonstrated recently that, like APP, APLP2 can be processed by g-secretase, generating intracellular Cterminal fragments with transcriptional activity (Scheinfeld et al., 2002). Since in APP g-secretase acts downstream of h-secretase, it is possible that the modulation of APLP2 cleavage by BACE can lead to g-cleavage and the formation of the APLP2 intracellular domain fragment, which can then translocate to the nucleus. However, more detailed studies are required to confirm this hypothesis and to better understand the role of BACE-mediated processing of APLP2.

L. Pastorino et al. / Mol. Cell. Neurosci. 25 (2004) 642–649

Experimental methods

647

specific for BACE (Pastorino et al., 2002; see below for sample preparation).

Generation of exon 1 BACE knockout mice Histochemical staining The BACE mutant mice were generated in collaboration with Lexicon Genetics, Inc. (The Woodlands, TX). The BACE targeting vector was derived using the Lambda KOS system (Wattler et al., 1999). The Lambda KOS phage library, arrayed into 96 superpools, was screened by PCR using exon 1-specific primers (BACE-2:5V CACAAGGCCCGGGCTCAC) and (BACE-7: 5VCTGCCTACGGTCATCTCCACATAG). The PCR-positive phage superpools were plated and screened by filter hybridization using the 267-bp amplicon derived from primers BACE-2 and BACE-7 as a probe. Three pKOS genomic clones, pKOS-53, pKOS-58, and pKOS-90 were isolated from the library screen and confirmed by sequence and restriction analysis. Gene-specific arms (5 V-TCGTCGTCTCCTCTCGTGCGCTACGGATTT) and (5 VCCATGCATGAAGGAGGGTTGCTAGATCTGA) were appended by PCR to a yeast selection cassette containing the URA3 marker. The yeast selection cassette and pKOS-90 were co-transformed into yeast, and clones that had undergone homologous recombination to replace a 437-bp region containing exon 1 with the yeast selection cassette were isolated. The yeast cassette was subsequently replaced with a LacZ/Neo selection cassette to complete the targeting vector. The NotI linearized targeting vector was electroporated into 129/SvEvBrd (Lex-1) ES cells. G418/FIAU-resistant ES cell clones were isolated, and correctly targeted clones were identified and confirmed by Southern analysis using a 368-bp 5V external probe (39/38), g e n e r a t e d b y P C R u s i n g p r i m e r s ( B A C E - 3 9 : 5 VC T TA AT G G T T T C A AT T G A C A ) a n d ( B A C E - 3 8 : 5 VGATCTTGTGTCAGAAAAACAAAG), and a 537-bp 3V external probe (24/40), amplified by PCR using primers (BACE-24: 5VGGGAGTCTGCAGGTCTTAAGG) and (BACE-40: 5 VCAACCTCACGCCTTCTCCAC). Southern analysis using probe 39/38 detected a 11 Kb wild-type band and 7.5 kb mutant band in PvuII digested genomic DNA, while probe 24/40 detected a approximately 15-kb wild-type band and 5.8-kb mutant band in XhoI-digested genomic DNA. Two targeted ES cell clones were microinjected into C57BL/6 (albino) blastocysts. The resulting chimeras were mated to C57BL/6 (albino) females to generate mice that were heterozygous for the BACE mutation. Genotyping of BACE knockout mice To screen for heterozygous and knockout mice by PCR we used a 5V primer recognizing the Neo cassette and a 3V primer recognizing part of the genomic DNA downstream the Neo cassette. To screen for wild-type mice, we designed a 5V primer directed to part of exon 1 that was deleted in the mutant allele, and the 3V primer described above. For the identification of the genotype by Southern blot, we used primers BACE-24 and BACE-40 (described above) on XhoIdigested genomic DNA blotted onto nylon (Schleicher and Schuell Inc.). The probe was amplified by PCR from BACE cDNA, labeled with 32P-a-dCTP (ICN) using Rediprime IIk (Amersham Pharmacia Biotech Inc.) and purified on a ProbeQuantk G-50 micro column (Amersham Pharmacia Biotech Inc.). Hybridization was performed using QuickHyb (Stratagene), according to manufacturer’s instructions. For detection of BACE in the membrane fraction of cortex and hippocampus, we performed immunoblotting using an antibody

Brain sections were prepared essentially as described (Harroch et al., 2002). Briefly, brains were perfused transcardially with 2% paraformaldehyde, 0.2% glutaraldehyde, and incubated for 30 min in the same solution. The fixed brains were then cut into 100-Am sections with a vibratome. Sections were incubated in X-galcontaining solution in phosphate-buffered saline (PBS) and mounted onto slides. For the double staining experiment, the same sections were further fixed in 100% ethanol for 5 min, re-hydrated in PBS and incubated overnight in PBS containing 5% goat serum, 0.3% Triton X-100 and antibodies to Neu N (1/200) or GFAP (1/ 200). Sections were then washed and incubated with secondary anti-mouse FITC, or anti-rabbit rhodamine. Analysis of APP and APLP2 processing from mouse brain Whole brains from wild-type, knockout and transgenic mice were homogenized in solution A (Sol. A), a PBS buffer containing 10 mM EDTA, 2 mM EGTA, 0.01 mM PMSF, and a set of protease inhibitors (Complete, Roche). Samples were centrifuged at 1000  g for 10 min, to remove debris, and then spun at 100,000  g for 40 min. The supernatants, representing the soluble fraction, were separated from the pellet, representing the membrane fraction. The pellet was further treated in Sol. A containing 0.1% Triton X-100, to extract membrane-inserted proteins, and spun at 100,000  g for 40 min. The resulting supernatant represented the Triton X-100 membrane extracted fraction. Approximately 50 Ag of samples prepared in Laemmli buffer were loaded onto 6% SDSPAGE or 10 – 20% Tris-Tricine gels (Invitrogen). For detection of APP full-length forms and APP C-terminal fragments in the Triton X-100 membrane extracted fraction, we used 369 antibody, a polyclonal antibody raised against the APP intracellular domain. For detection of APLP2 full-length and/or Cterminal fragments in the Triton X-100 membrane extracted fraction, we used specific antibodies (Calbiochem), raised against the intracellular domain of APLP2. Secreted APLP2 was detected using an antibody raised against the full-length protein (Calbiochem). These antibodies do not cross-react with APP or the other APLP species (data not shown). After blotting, levels of mature and immature APP and levels of soluble APLP2 were evaluated using 125I-protein A, and membranes were exposed to a PhosphorImager screen for quantification. Levels of APLP2 full-length and C-terminal fragments, as well as APP C-terminal fragments, were evaluated using Super Signal West Pico Chemiluminescent Substrate (Pierce), and quantified using NIH Image1.63 (NIH). Analysis of APLP2 processing from cultured cells APLP2 with a C-terminal myc tag was produced by subcloning human APLP2 from pBlueScript (a gift from Dr. W. Wasco, Harvard Medical School, Charlestown, MA) into pcDNA3.1myc (Invitrogen). COS7 cells were transfected with either APLP2-myc or APLP2-myc and BACE-V5 (Pastorino et al., 2002). Forty-eight hours after transfection, media were collected and cells lysed. Equal amounts of protein were ran on a 10 – 20% Tricine gel, blotted, and probed for APLP2 C-terminal fragments with 9E10

648

L. Pastorino et al. / Mol. Cell. Neurosci. 25 (2004) 642–649

antibody. For soluble APLP2, equal volumes of media were ran on a gel blotted and probed for APLP2 using the N-terminal APLP2 antibody (Calbiochem). Quantification of Ab40 and Ab42 Peptide Levels Complete hemibrains from wild type heterozygote and BACE knockout littermates were homogenized in DEA buffer (0.2% Diethylamine/50 mM NaCl, 1:10 weight: volume) as previously described (Rozmahel et al., 2002) and endogenous murine Ah was quantified by sandwich ELISA. All ELISA measurements were done in two or more replica wells. Acknowledgments Authors are grateful to Stephen Schmidt, for helping with ELISA. This work was supported by The National Institutes of Aging grants AG10491 (to JDB), AG14996 (to JDB) and AG002219 (to JDB). References Andrau, D., Dumanchin-Njock, C., Ayral, E., Vizzavona, J., Farzan, M., Boisbrun, M., Fulcrand, P., Hernandez, J.F., Martinez, J., LefrancJullien, S., Checler, F., 2003. BACE1- and BACE2-expressing human cells: characterization of beta-amyloid precursor protein-derived catabolites, design of a novel fluorimetric assay, and identification of new in vitro inhibitors. J. Biol. Chem. 278, 25859 – 25866. Baek, S.H., Ohgi, K.A., Rose, D.W., Koo, E.H., Glass, C.K., Rosenfeld, M.G., 2002. Exchange of N-CoR corepressor and Tip60 coactivator complexes links gene expression by NF-kappaB and beta-amyloid precursor protein. Cell 110, 55 – 67. Bayer, T.A., Cappai, R., Masters, C.L., Beyreuther, K., Multhaup, G., 1999. It all sticks together—The APP-related family of proteins and Alzheimer’s disease. Mol. Psychiatry 4, 524 – 528. Bennett, B.D., Babu-Khan, S., Loeloff, R., Louis, J.C., Curran, E., Citron, M., Vassar, R., 2000. Expression analysis of BACE2 in brain and peripheral tissues. J. Biol. Chem. 275, 20647 – 20651. Bodendorf, U., Danner, S., Fischer, F., Stefani, M., Sturchler-Pierrat, C., Wiederhold, K.H., Staufenbiel, M., Paganetti, P., 2002. Expression of human beta-secretase in the mouse brain increases the steady-state level of beta-amyloid. J. Neurochem. 80, 799 – 806. Cai, H., Wang, Y., McCarthy, D., Wen, H., Borchelt, D.R., Price, D.L., Wong, P.C., 2001. BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat. Neurosci. 4, 233 – 234. Cao, X., Sudhof, T.C., 2001. A transcriptionally [correction of transcriptively] active complex of APP with Fe65 and histone acetyltransferase Tip60. Science 293, 115 – 120. Citron, M., 2002. Emerging Alzheimer’s disease therapies: inhibition of beta-secretase. Neurobiol. Aging 23, 1017 – 1022. De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhde, G., Annaert, W., Von Figura, K., Van Leuven, F., 1998. Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391, 387 – 390. Duff, K., Eckman, C., Zehr, C., Yu, X., Prada, C.M., Perez-tur, J., Hutton, M., Buee, L., Harigaya, Y., Yager, D., Morgan, D., Gordon, M.N., Holcomb, L., Refolo, L., Zenk, B., Hardy, J., Younkin, S., 1996. Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1. Nature 383, 710 – 713. Farzan, M., Schnitzler, C.E., Vasilieva, N., Leung, D., Choe, H., 2000. BACE2, a beta -secretase homolog, cleaves at the beta site and within the amyloid-beta region of the amyloid-beta precursor protein. Proc. Natl. Acad. Sci. U. S. A. 97, 9712 – 9717.

Glenner, G.G., Wong, C.W., 1984. Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 120, 885 – 890. Glenner, G.G., Wong, C.W., Quaranta, V., Eanes, E.D., 1984. The amyloid deposits in Alzheimer’s disease: their nature and pathogenesis. Appl. Pathol. 2, 357 – 369. Hardy, J., Selkoe, D.J., 2002. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353 – 356. Harroch, S., Furtado, G.C., Brueck, W., Rosenbluth, J., Lafaille, J., Chao, M., Buxbaum, J.D., Schlessinger, J., 2002. A critical role for the protein tyrosine phosphatase receptor type Z in functional recovery from demyelinating lesions. Nat. Genet. 32, 411 – 414. Heber, S., Herms, J., Gajic, V., Hainfellner, J., Aguzzi, A., Rulicke, T., von Kretzschmar, H., von Koch, C., Sisodia, S., Tremml, P., Lipp, H.P., Wolfer, D.P., Muller, U., 2000. Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members. J. Neurosci. 20, 7951 – 7963. Huse, J.T., Liu, K., Pijak, D.S., Carlin, D., Lee, V.M., Doms, R.W., 2002. Beta-secretase processing in the trans-Golgi network preferentially generates truncated amyloid species that accumulate in Alzheimer’s disease brain. J. Biol. Chem. 277, 16278 – 16284. Hussain, I., Powell, D., Howlett, D.R., Tew, D.G., Meek, T.D., Chapman, C., Gloger, I.S., Murphy, K.E., Southan, C.D., Ryan, D.M., Smith, T.S., Simmons, D.L., Walsh, F.S., Dingwall, C., Christie, G., 1999. Identification of a novel aspartic protease (Asp 2) as beta-secretase. Mol. Cell. Neurosci. 14, 419 – 427. Hussain, I., Powell, D.J., Howlett, D.R., Chapman, G.A., Gilmour, L., Murdock, P.R., Tew, D.G., Meek, T.D., Chapman, C., Schneider, K., Ratcliffe, S.J., Tattersall, D., Testa, T.T., Southan, C., Ryan, D.M., Simmons, D.L., Walsh, F.S., Dingwall, C., Christie, G., 2000. ASP1 (BACE2) cleaves the amyloid precursor protein at the beta-secretase site. Mol. Cell. Neurosci. 16, 609 – 619. Kimberly, W.T., Xia, W., Rahmati, T., Wolfe, M.S., Selkoe, D.J., 2000. The transmembrane aspartates in presenilin 1 and 2 are obligatory for gamma-secretase activity and amyloid beta-protein generation. J. Biol. Chem. 275, 3173 – 3178. Lee, E.B., Skovronsky, D.M., Abtahian, F., Doms, R.W., Lee, V.M., 2002. Secretion and intracellular generation of truncated Abeta in BACE expressing human neurons. J. Biol. Chem. 278, 4458 – 4466. Liu, K., Doms, R.W., Lee, V.M., 2002. Glu11 site cleavage and N-terminally truncated A beta production upon BACE overexpression. Biochemistry 41, 3128 – 3136. Lorent, K., Overbergh, L., Moechars, D., De Strooper, B., Van Leuven, F., Van den Berghe, H., 1995. Expression in mouse embryos and in adult mouse brain of three members of the amyloid precursor protein family, of the alpha-2-macroglobulin receptor/low density lipoprotein receptorrelated protein and of its ligands apolipoprotein E, lipoprotein lipase, alpha-2-macroglobulin and the 40,000 molecular weight receptor-associated protein. Neuroscience 65, 1009 – 1025. Luo, Y., Bolon, B., Kahn, S., Bennett, B.D., Babu-Khan, S., Denis, P., Fan, W., Kha, H., Zhang, J., Gong, Y., Martin, L., Louis, J.C., Yan, Q., Richards, W.G., Citron, M., Vassar, R., 2001. Mice deficient in BACE1, the Alzheimer’s beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat. Neurosci. 4, 231 – 232. McNamara, M.J., Ruff, C.T., Wasco, W., Tanzi, R.E., Thinakaran, G., Hyman, B.T., 1998. Immunohistochemical and in situ analysis of amyloid precursor-like protein-1 and amyloid precursor-like protein-2 expression in Alzheimer disease and aged control brains. Brain Res. 804, 45 – 51. Paliga, K., Peraus, G., Kreger, S., Durrwang, U., Hesse, L., Multhaup, G., Masters, C.L., Beyreuther, K., Weidemann, A., 1997. Human amyloid precursor-like protein 1-cDNA cloning, ectopic expression in COS-7 cells and identification of soluble forms in the cerebrospinal fluid. Eur. J. Biochem. 250, 354 – 363. Pastorino, L., Ikin, A.F., Nairn, A.C., Pursnani, A., Buxbaum, J.D., 2002. The carboxyl-terminus of BACE contains a sorting signal that regulates

L. Pastorino et al. / Mol. Cell. Neurosci. 25 (2004) 642–649 BACE trafficking but not the formation of total A(beta). Mol. Cell. Neurosci. 19, 175 – 185. Pike, C.J., Overman, M.J., Cotman, C.W., 1995. Amino-terminal deletions enhance aggregation of beta-amyloid peptides in vitro. J. Biol. Chem. 270, 23895 – 23898. Roberds, S.L., Anderson, J., Basi, G., Bienkowski, M.J., Branstetter, D.G., Chen, K.S., Freedman, S.B., Frigon, N.L., Games, D., Hu, K., JohnsonWood, K., Kappenman, K.E., Kawabe, T.T., Kola, I., Kuehn, R., Lee, M., Liu, W., Motter, R., Nichols, N.F., Power, M., Robertson, D.W., Schenk, D., Schoor, M., Shopp, G.M., Shuck, M.E., Sinha, S., Svensson, K.A., Tatsuno, G., Tintrup, H., Wijsman, J., Wright, S., McConlogue, L., 2001. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer’s disease therapeutics. Hum. Mol. Genet. 10, 1317 – 1324. Rozmahel, R., Huang, J., Chen, F., Liang, Y., Nguyen, V., Ikeda, M., Levesque, G., Yu, G., Nishimura, M., Mathews, P., Schmidt, S.D., Mercken, M., Bergeron, C., Westaway, D., St George-Hyslop, P., 2002. Normal brain development in PS1 hypomorphic mice with markedly reduced gamma-secretase cleavage of betaAPP. Neurobiol. Aging 23, 187 – 194. Scheinfeld, M.H., Ghersi, E., Laky, K., Fowlkes, B.J., D’Adamio, L., 2002. Processing of h-amyloid precursor-like protein-1 and -2 by g-secretase regulates transcription. J. Biol. Chem. 277, 44195 – 44201. Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T.D., Hardy, J., Hutton, M., Kukull, W., Larson, E., Levy-Lahad, E., Viitanen, M., Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco, W., Lannfelt, L., Selkoe, D., Younkin, S., 1996. 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. Nat. Med. 2, 864 – 870. Selkoe, D.J., Wolfe, M.S., 2000. In search of gamma-secretase: presenilin at the cutting edge. Proc. Natl. Acad. Sci. U. S. A. 97, 5690 – 5692. Sinha, S., Anderson, J.P., Barbour, R., Basi, G.S., Caccavello, R., Davis, D., Doan, M., Dovey, H.F., Frigon, N., Hong, J., Jacobson-Croak, K., Jewett, N., Keim, P., Knops, J., Lieberburg, I., Power, M., Tan, H., Tatsuno, G., Tung, J., Schenk, D., Seubert, P., Suomensaari, S.M., Wang, S., Walker, D., John, V., et al., 1999. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 402, 537 – 540. Slunt, H.H., Thinakaran, G., Von Koch, C., Lo, A.C., Tanzi, R.E., Sisodia, S.S., 1994. Expression of a ubiquitous, cross-reactive homologue of the

649

mouse beta-amyloid precursor protein (APP). J. Biol. Chem. 269, 2637 – 2644. Sprecher, C.A., Grant, F.J., Grimm, G., O’Hara, P.J., Norris, F., Norris, K., Foster, D.C., 1993. Molecular cloning of the cDNA for a human amyloid precursor protein homolog: evidence for a multigene family. Biochemistry 32, 4481 – 4486. Vassar, R., Citron, M., 2000. Abeta-generating enzymes: recent advances in beta- and gamma-secretase research. Neuron 27, 419 – 422. Vassar, R., Bennett, B.D., Babu-Khan, S., Kahn, S., Mendiaz, E.A., Denis, P., Teplow, D.B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M.A., Biere, A.L., Curran, E., Burgess, T., Louis, J.C., Collins, F., Treanor, J., Rogers, G., Citron, M., 1999. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286, 735 – 741. Wasco, W., Bupp, K., Magendantz, M., Gusella, J.F., Tanzi, R.E., Solomon, F., 1992. Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer disease-associated amyloid beta protein precursor. Proc. Natl. Acad. Sci. U. S. A. 89, 10758 – 10762. Wasco, W., Gurubhagavatula, S., Paradis, M.D., Romano, D.M., Sisodia, S.S., Hyman, B.T., Neve, R.L., Tanzi, R.E., 1993. Isolation and characterization of APLP2 encoding a homologue of the Alzheimer’s associated amyloid beta protein precursor. Nat. Genet. 5, 95 – 100. Wattler, S., Kelly, M., Nehls, M., 1999. Construction of gene targeting vectors from lambda KOS genomic libraries. Biotechniques 26 1150 – 1156, 1158, 1160. White, A.R., Zheng, H., Galatis, D., Maher, F., Hesse, L., Multhaup, G., Beyreuther, K., Masters, C.L., Cappai, R., 1998. Survival of cultured neurons from amyloid precursor protein knock-out mice against Alzheimer’s amyloid-beta toxicity and oxidative stress. J. Neurosci. 18, 6207 – 6217. Wolfe, M.S., Xia, W., Ostaszewski, B.L., Diehl, T.S., Kimberly, W.T., Selkoe, D.J., 1999. Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398, 513 – 517. Wolfe, M.S., Esler, W.P., Das, C., 2002. Continuing strategies for inhibiting Alzheimer’s gamma-secretase. J. Mol. Neurosci. 19, 83 – 87. Yan, R., Bienkowski, M.J., Shuck, M.E., Miao, H., Tory, M.C., Pauley, A.M., Brashier, J.R., Stratman, N.C., Mathews, W.R., Buhl, A.E., Carter, D.B., Tomasselli, A.G., Parodi, L.A., Heinrikson, R.L., Gurney, M.E., 1999. Membrane-anchored aspartyl protease with Alzheimer’s disease beta-secretase activity. Nature 402, 533 – 537.