Susumu Tomita

Cleavage of Alzheimer’s Amyloid Precursor Protein (APP) by Secretases Occurs after O-Glycosylation of APP in the Protein Secretory Pathway IDENTIFICATION OF INTRACELLULAR COMPARTMENTS IN WHICH APP CLEAVAGE OCCURS WITHOUT USING TOXIC AGENTS THAT INTERFERE WITH PROTEIN METABOLISM

β-Amyloid peptide (Aβ) is a principal component of parenchymal amyloid deposits in Alzheimer’s disease. Aβ is derived from amyloid precursor protein (APP) by proteolytic cleavage. APP is subject to N- andO-glycosylation and potential tyrosine sulfation, following protein synthesis, and is then thought to be cleaved in an intracellular secretory pathway after or during these post-translational modifications. Studies utilizing agents that affect a series of steps in the protein secretory pathway have identified the possible intracellular sites of APP cleavage and Aβ generation within the protein secretory pathway. In the present study, using cells with normal protein metabolism, but expressing mutant APP with defectiveO-glycosylation, we demonstrated that the majority of APP cleavage by α-, β-, and γ-secretases occurs afterO-glycosylation. Cells expressing the mutant APP noticeably decreased the generation of the intracellular APP carboxyl-terminal fragment (αAPPCOOH), a product of α-secretase, and both Aβ40 and Aβ42 in medium, a product of β- and γ-secretases. Furthermore, we found that the cells accumulate the mutant APP in intracellular reticular compartments such as the endoplasmic reticulum. Agents that could ambiguously affect the function of specific intracellular organelles and that may be toxic were not used. The present results indicate that APP is cleaved by α-, β-, and γ-secretases in step(s) during the transport of APP through Golgi complex, where O-glycosylation occurs, or in compartments subsequent to trans-Golgi of the APP secretory pathway.

SREC-II, a new member of the scavenger receptor type F family, trans-interacts with SREC-I through its extracellular domain

The scavenger receptor expressed by endothelial cells (SREC) with an extremely large cytoplasmic domain, was originally identified in a human endothelial cell line. In this study, we have cloned a second isoform named SREC-II and shown that there is a heterophilic interaction between SREC-I and -II at their extracellular domains. The cDNA for murine SREC-II encodes an 834-amino acid protein with 35% homology to SREC-I. Similar to SREC-I, SREC-II contains multiple epidermal growth factor-like repeats in its extracellular domain. However, in contrast to SREC-I, SREC-II had little activity to internalize modified low density lipoproteins (LDL). A Northern blot analysis revealed a tissue expression pattern of SREC-II similar to that of SREC-I with predominant expression in human heart, lung, ovary, and placenta. Mouse fibroblast L cells with no tendency to associate showed noticeable aggregation when SREC-I was overexpressed in these cells, whereas overexpression of SREC-II caused only slight aggregation. Remarkably, intense aggregation was observed when SREC-I-expressing cells were mixed with those expressing SREC-II. Deletion of almost all of the cytoplasmic receptor domain had no effect on the receptor expression and cell aggregation, indicating that solely the extracellular domain is involved in cell aggregation. The association of SREC-I and -II was effectively suppressed by the presence of scavenger receptor ligands such as acetylated LDL and oxidized LDL. These findings suggest that SREC-I and -II show weak cell-cell interaction by their extracellular domains (termed homophilic trans-interaction) but display strong heterophilic trans-interaction through the extracellular epidermal growth factor-like repeat domains.

A BASIC AMINO ACID IN THE CYTOPLASMIC DOMAIN OF ALZHEIMER'S BETA -AMYLOID PRECURSOR PROTEIN (APP) IS ESSENTIAL FOR CLEAVAGE OF APP AT THE ALPHA -SITE

In Alzheimer’s disease (AD), the β-amyloid peptide (Aβ) is thought to be produced as a result of the aberrant metabolism of β-amyloid precursor protein (APP). We report that the APP cytoplasmic domain contains a novel and important signal for APP metabolism. A single amino acid mutation that changed arginine at amino acid 747 of APP770 (corresponding to position 672 of APP695) to a non-basic amino acid greatly increased the production of intracellular APP carboxyl-terminal fragment(s) cleaved at β-site(s) (CTFβ), but did not result in increased secretion of Aβ40 and Aβ42. This was not due to a simple intracellular accumulation of CTFβ resulting from a lack of γ-secretase. CTFβ derived from this mutant APP was generated and degraded as efficiently as CTFβ derived from wild-type APP. This result indicates that the increase in the quantity of CTFβ does not always give rise to more Aβ production, as was previously suggested by studies of a familial AD mutation of APP. These findings suggest that APP carrying the substitution mutation at this basic amino acid may be metabolized by another protein secretory pathway. Although these results have not completely elucidated why CTFβ derived from the mutant APP escapes from subsequent cleavage by γ-secretase, analysis of the processing pathway of this mutant APP should provide insights into the pathogenesis of the sporadic type of AD.

Regulation of X11L-dependent Amyloid Precursor Protein Metabolism by XB51, a Novel X11L-binding Protein

We isolated a cDNA encoding a novel protein, XB51, that interacts with the amino-terminal domain of the neuron-specific X11-like protein (X11L). The protein XB51 inhibited the association of X11L with amyloid precursor protein through a non-competitive mechanism and abolished the suppression of β-amyloid production by X11L. The majority of XB51 is localized around the nucleus and recovered in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) buffer-insoluble fraction when XB51 is expressed in cells. Association of XB51 with X11L changed the intracellular distribution of XB51 and resulted in redistribution of XB51 into the CHAPS buffer-soluble fraction. These observations suggest that XB51, together with X11L, plays an important role in the regulatory system of amyloid precursor protein metabolism and β-amyloid generation.

Expression and characterization of the Drosophila X11‐like/Mint protein during neural development

The X11‐like (X11L) protein was originally isolated as a protein bound to the cytoplasmic domain of the β‐amyloid precursor protein (APP), which is associated with Alzheimers disease. In mammals, X11L is believed to play an important role in the regulation of APP metabolism. Here we isolated and characterized the Drosophila X11L (dX11L) protein, also may be referred to this protein as Drosophila Mint (dMint), Lin 10 (dLin10) or X11 (dX11), is thought to be expressed in neuronal tissues from late embryonic through to the adult stages of the fly. The phosphotyrosine interaction domain of dX11L interacts with the cytoplasmic domain of the Drosophila amyloid precursor protein‐like (APPL) similar to the way human X11L (hX11L) interacts with APP. Overexpression of dX11L on post‐mitotic neurons had a lethal effect on flies and, when it was localized to the eye imaginal disc, disruption of compound eye morphology due to enhanced apoptosis of neuronal cells was observed. Overexpression of hX11L and the PDZ domain of dX11L resulted in identical eye phenotypes. The PDZ domain is highly conserved between Drosophila and human, and appears to be responsible for this phenotype. Our findings suggest that the X11L family may be involved with the regulation of apoptosis during neural cell development and that aberrant X11L function could be contribute in this way to the neuronal degeneration observed in Alzheimers disease.