Increasing evidence has implicated the low density lipoprotein receptor-related protein (LRP) and the adaptor protein FE65 in Alzheimer's disease pathogenesis. We have shown previously that LRP mediates ␤-amyloid precursor protein (APP) processing and affects amyloid ␤-protein and APP secretion and APP-c-terminal fragment generation. Furthermore, LRP mediates APP processing through its intracellular domain. Here, we set out to examine whether this interaction is of direct or indirect nature. Specifically, we asked whether adaptor proteins such as FE65 influence the LRP-mediated effect on APP processing by forming a protein complex. In coimmunoprecipitation experiments, we confirmed the postulated APP-FE65 and the LRP-FE65 interaction. However, we also showed an LRP-FE65-APP trimeric complex using pull-down techniques. Because FE65 alters APP processing, we investigated whether this effect is LRP dependent. Indeed, FE65 was only able to increase APP secretion in the presence of LRP. In the absence of LRP, APP secretion was unchanged compared with the LRP knock-out phenotype. Using RNA short interference techniques against FE65, we demonstrated that a reduction in FE65 protein mimics the LRP knock-out phenotype on APP processing. These results clearly demonstrate that FE65 acts as a functional linker between APP and LRP.

Accumulation of the amyloid ␤ (A␤) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted A␤ generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and A␤ generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced ␤-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced A␤ generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease. The major defining pathological hallmark of Alzheimer disease (AD)2 is the accumulation of amyloid ␤ protein (A␤), a neurotoxic peptide derived from ␤-and ␥-secretase cleavages of the amyloid precursor protein (APP). The vast majority of APP is constitutively cleaved in the middle of the A␤ sequence by ␣-secretase (ADAM10/TACE/ADAM17) in the non-amyloidogenic pathway, thereby abrogating the generation of an intact A␤ peptide. Alternatively, a small proportion of APP is cleaved in the amyloidogenic pathway, leading to the secretion of A␤ peptides (37-42 amino acids) via two proteolytic enzymes, ␤-and ␥-secretase, known as BACE1 and presenilin, respectively (1).The proteolytic processing of APP to generate A␤ requires the trafficking of APP such that APP and BACE1 are brought together in close proximity for ␤-secretase cleavage to occur. We and others have shown that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytosis receptor (2), binds to APP and alters its trafficking to promote A␤ generation. The loss of LRP substantially reduces A␤ release, a phenotype that is reversed when full-length (LRP-FL) or truncated LRP is transfected in LRP-deficient cells (3, 4). Specifically, LRP-CT lacking the extracellular ligand binding regions but containing the transmembrane domain and the cytoplasmic tail is capable of rescuing amyloidogenic processing of APP and A␤ release in LRP deficient cells (3). Moreover, the LRP soluble tail (LRP-ST) lacking the transmembrane domain and only containing the cytoplasmic tail of LRP is sufficient to enhance A␤ secretion (5). This activity of LRP-ST is achieved by promoting APP/BACE1 intera...

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in A␤ production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/␤-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/ Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS ؊/؊ ) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS ؉/؉ cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS ؊/؊ cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.Mutations in genes encoding presenilin 1 (PS1) 2 and presenilin 2 (PS2) account for the vast majority of early onset familial Alzheimer disease (FAD) (1, 2). Presenilins are multipass transmembrane proteins that form the core enzymatic activity of the ␥-secretase complex together with nicastrin, pen-2, and aph-1. These four proteins are required for regulated intramembrane proteolysis of multiple type I transmembrane proteins, including APP and Notch, such that the loss of any one of these four proteins destabilizes the complex and abrogates ␥-secretase activity (3). Accordingly, null mice for any one of these components display embryonic lethality associated with severe malformations of the axial skeleton and cerebral hemorrhage, resembling that of Notch deficiency (3). As expected, this activity is conserved in Caenorhabditis elegans and Drosophila where the presenilin complex functions to facilitate Notch signaling (4, 5).FAD mutations in PS1 and PS2 alter the activity of the ␥-secretase complex, leading to the change in the ratio of A␤ to that favoring A␤42 generation and accelerated amyloid deposition in brain (6). Although the amyloid hypothesis is the leading model for AD pathogenesis, the aggressive early age of disease onset by PS1 FAD mutations have...

The major defining pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid beta protein (Abeta), a small peptide derived from beta- and gamma-secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that beta- and gamma-secretase activities of BACE1 and presenilin, respectively, are concentrated in intracellular lipid raft microdomains. However, the manner in which APP normally traffics to lipid rafts is unknown. In this study, using transient transfection and immuno-precipitation assays, we show that the cytoplasmic domain of low-density lipoprotein receptor-related protein (LRP) interacts with APP and increases Abeta secretion and APP beta-CTF (C-terminal fragment) generation by promoting BACE1-APP interaction. We also employed discontinuous sucrose density gradient ultracentrifugation to show that the LRP cytoplasmic domain-mediated effect was accompanied by greatly increased localization of APP and BACE1 to lipid raft membranes, where beta- and gamma-secretase activities are highly enriched. Moreover, we provide evidence that endogenous LRP is required for the normal delivery of APP to lipid rafts and Abeta generation primarily in the endocytic but not secretory pathway. These results may provide novel insights to block Abeta generation by targeting LRP-mediated delivery of APP to raft microdomains.

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