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

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Abstract

BACE is an aspartyl protease that cleaves the amyloid precursor protein (APP) at the β-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 β-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 Aβ 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.

Introduction

The core of the amyloid plaques, which are a characteristic feature of Alzheimer's disease (AD), are composed of Aβ 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 β- and γ-secretases) are considered therapeutic targets important in the treatment of Alzheimer's disease (AD) Citron, 2002, Hardy and Selkoe, 2002, Wolfe et al., 2002. Proteins showing γ-secretase De Strooper et al., 1998, Kimberly et al., 2000, Selkoe and Wolfe, 2000, Wolfe et al., 1999 and β-secretase 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 β-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 Aβ peptides, generating the APP C-terminal 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 γ-secretase, an activity that includes presenilin 1, at residues-40/42/43, generating the Aβ peptides Aβ1-40/42/43 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 Aβ at glutamic acid residue E11 generating C89, a shorter membrane-associated C-terminal fragment that can be further cleaved by γ-secretase Andrau et al., 2003, Huse et al., 2002, Lee et al., 2002, Liu et al., 2002. The resulting Aβ11-40/42 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, Wasco et al., 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 γ-secretase and that the generated intracellular C-terminal domain can translocate to the nucleus as 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 Aβ peptides since it does not contain the sequence for Aβ. Thus, the processing of APLP2 is not amyloidogenic, although it may involve a similar set of secretase activities, such as γ-secretase.

In this study, we developed knockout animals to study BACE expression and function as a potential modulator of APLP2 processing.

Section snippets

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 introduces an IRES β-galactosidase (β-gal) cassette, replacing wild-type BACE with an allele containing the β-gal gene. Since expression of the β-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 β-gal reporter as described below. Animals were genotyped by PCR and Southern blot. The

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 β-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

Generation of exon 1 BACE knockout mice

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-2:5′ CACAAGGCCCGGGCTCAC) and (BACE-7: 5′-CTGCCTACGGTCATCTCCACATAG). The PCR-positive phage superpools were plated and screened by filter hybridization using the 267-bp amplicon derived from

Acknowledgements

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).

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