226 Vingtdeux et al

Abstract ref 226

The amyloid precursor protein (APP) metabolism is central to pathogenesis of Alzheimer’s disease (AD). Parenchymal amyloid deposits, a neuropathological hallmark of AD, are composed of amyloid-beta peptides (Abeta). Abeta derives from the amyloid precursor protein (APP) by sequential cleavages by beta- and gamma-secretases. Gamma-secretase cleavage releases the APP intracellular domain (AICD), suggested to mediate a nuclear signaling. Physiologically, AICD is seldom detected and thus supposed to be rapidly degraded. The mechanisms responsible of its degradation remain unknown. We used a pharmacological approach and showed that several alkalizing drugs induce the accumulation of AICD in neuroblastoma SY5Y cell lines stably expressing APP constructs. Moreover, alkalizing drugs induce AICD accumulation in naive SY5Y, HEK and COS cells. This accumulation is not mediated by the proteasome or metallopeptidases and is not the result of an increased gamma-secretase activity since the gamma-secretase cleavage of Notch1 and N-Cadherin is not affected by alkalizing drug treatments. Altogether, our data demonstrate for the first time that alkalizing drugs induce the accumulation of AICD, a mechanism likely mediated by the endosome/lysosome pathway.

Introduction

The amyloid precursor protein (APP) metabolism is central to the pathogenesis of Alzheimer’s disease (AD). Extracellular amyloid deposits, a neuropathological hallmark of AD, are composed of the aggregation of amyloid-beta peptides (Abeta). Abeta is released from a type I transmembrane amyloid protein precursor (APP) by sequential cleavages. Carboxy-terminal fragments (CTFs) are first generated by alpha- or beta-secretase (for a review, see Suh and Checler, 2002). The alpha-secretase cleaves APP inside Abeta sequence (Buxbaum et al., 1998, Allinson et al., 2003). BACE1, the beta-secretase protease, releases the amino-terminus of Abeta (Vassar et al., 1999). The gamma-secretase further processes APP-CTFs (Sastre et al., 2001, Kim et al., 2003), releasing Abeta from beta-CTF and the APP intracellular domain (AICD or epsilon-CTF) from all APP-CTFs (Gu et al., 2001, Sastre et al., 2001, Weidemann et al., 2002, Andrau et al., 2003, Baulac et al., 2003, Vingtdeux et al., 2005).

Cytosolic domains are released by the gamma-secretase cleavage of several type I transmembrane proteins, such as Notch intracellular domain (NICD) or Cadherins intracellular domains (De Strooper et al., 1999, Marambaud et al., 2003). By similarities to Notch I signaling, AICD associates with nuclear factors Fe65 and Tip60 to form a trans-regulatory complex, regulating the expression of KAI1, Neprelysin or its own expression (Cao and Sudhof, 2001, Baek et al., 2002, Cao and Sudhof, 2004, von Rotz et al., 2004, Pardossi-Piquard et al., 2005). AICD produced physiologically is hardly detected in vivo and is rapidly degraded (Cupers et al., 2001). The mechanism responsible for its degradation remains uncertain. AICD is likely degraded by insulin-degrading enzyme (Edbauer et al., 2002, Farris et al., 2003) but not by the proteasome (Cupers et al., 2001). In contrast, the intracellular domain of Notch I (Gupta-Rossi et al., 2001), that of lipoprotein receptor protein (May et al., 2002) or that of p75NTR (Jung et al., 2003) is processed by the proteasome. The generation of AICD takes place at the plasma membrane and/or in early endosomes (Kaether et al., 2006). In addition, gamma-secretase activity has been localized at the endosomal and lysosomal membrane (Lah and Levey, 2000, Pasternak et al., 2003, Vetrivel et al., 2004). Together, those data suggest that the endosomal/lysosomal pathway is of importance for regulating AICD production and/or degradation.

In the present study, we investigated the role of the endosome/lysosome pathway for the regulation of AICD production using a pharmacological approach. Our results show that treatments of neuroblastoma SY5Y stably expressing APP constructs with several alkalizing agents result in an accumulation of AICD. We demonstrate that this accumulation did not result from an increased gamma-secretase proteolytic activity. Altogether, our results suggest that AICD accumulation is pH-sensitive and regulated via the endosome/lysosome pathway.

Section snippets

Antibodies

The APPCter-C17 is a well-characterized rabbit antibody raised against the last 17 amino acids of the human APP sequence (Sergeant et al., 2002, Casas et al., 2004, Santiard-Baron et al., 2005, Vingtdeux et al., 2005). The mouse monoclonal antibody, 8E5, was a generous gift from Dr. Peter Seubert (Elan Pharmaceuticals). 8E5 is raised against the 444–592 residues of human APP770. The mouse monoclonal antibody, 6E10 (Signet Labs), recognizes an epitope in the first 17 amino acids of the Abeta

AICD accumulates after long-term intracellular pH modulation

Intracellular pH was raised using ammonium chloride (NH4Cl) or chloroquine that are weak bases and bafilomycin A1 (BafA1) and concanamycin A (ConA) that are specific inhibitors of vacuolar H+-ATPase (Bowman et al., 1988). NH4Cl has been shown to alkalize endosomes (Gekle et al., 1995); chloroquine accumulates in lysosomes where it raises the pH (de Duve et al., 1974). BafA1 and ConA were shown to inhibit the acidification of endosomes and lysosomes (Umata et al., 1990, Yoshimori et al., 1991).

Discussion

The discovery of gamma-secretase has pioneered the mechanism of regulated intramembrane proteolysis. Intracellular domains released from several type I transmembrane proteins are essential for signaling pathways such as gene transactivation. For instance, release of NICD is essential for cell fate decisions, N-Cadherin ICD promotes the proteasomal degradation of transcription factor CBP and AICD regulates several gene expression (Jarriault et al., 1995, Marambaud et al., 2003, Cao and Sudhof,