BRI2 interacts with BACE1 and regulates its cellular levels by promoting its degradation and reducing its mRNA levels.

BRI2, a protein mutated in Familial British and Familial Danish Dementias, interacts with Amyloid Precursor Protein (APP) and reduces the levels of secreted APPß (sAPPß), which derives from APP cleavage by ß-secretase (BACE1). Exploring the mechanisms of this effect, we obtained data that BRI2 decreases the cellular levels of BACE1 thus reducing the ß-cleavage of APP. Deletion of N-terminal cytoplasmic or C-terminal extracellular sequences of BRI2 neither affected its interaction with BACE1 or APP (Fotinopoulou et al., 2005) nor the reduction in the levels of BACE1 and sAPPß. These results suggest that BRI2 may prevent access of BACE1 to APP and the BRI2/BACE1 interaction may mediate the reduction in BACE1 levels. In support, BRI2 expression induced lysosomal but not proteasomal degradation of BACE1. In parallel, BRI2 expression was also found to reduce BACE1 mRNA levels by 50%. This study adds novel information regarding the mechanism by which BRI2 affects APP processing and BACE1 levels.

Downregulation of AßPP enhances both calcium content of endoplasmic reticulum and acidic stores and the dynamics of store operated calcium channel activity.

The amyloid-ß protein precursor (AßPP) is a type-1 transmembrane protein involved in Alzheimer's disease (AD). It has become increasingly evident that AßPP, its protein-protein interactions, and its proteolytical fragments may affect calcium homeostasis and vice versa. In addition, there is evidence that calcium dysregulation contributes to AD. To study the role of AßPP in calcium homeostasis, we downregulated its expression in SH-SY5Y cells using shRNA (SH-SY5Y/AßPP-) or increased expression of AßPP695 by transfection (SH-SY5Y/AßPP+). The levels of cytosolic Ca2+ after treatment with thapsigargin, monensin, activation of capacitative calcium entry (CCE), and treatment with SKF, a store operated channel (SOCs) inhibitor, were measured by fura-2AM fluorimetry. SH-SY5Y/AßPP+ cells show reduced response to thapsigargin and reduced CCE, although this reduction is not statistically significant. On the other hand, we found that, relative to SH-SY5Y, SH-SY5Y/AßPP- cells show a significant increase in the response to thapsigargin but not in CCE and their SOCs were more susceptible to SKF inhibition. Additionally, downregulation of AßPP resulted in increased response to monensin that induces calcium release from acidic stores. The increase of calcium release from the endoplasmic reticulum and the acidic stores, when AßPP is downregulated, could be attributed to elevated Ca2+ content or to a dysregulation of Ca2+ transfer through their membranes. These data, along with already existing evidence regarding the role of AßPP in calcium homeostasis and the early occurring structural and functional abnormalities of endosomes, further substantiate the role of AßPP in calcium homeostasis and in AD.

BRI2 interacts with amyloid precursor protein (APP) and regulates amyloid beta (Abeta) production.

Transmembrane proteins BRI2 and amyloid precursor protein (APP) co-localize with amyloid beta (Abeta) lesions in sporadic Alzheimer disease and mutations in both precursor proteins are linked to early-onset familial cases of cerebral amyloidosis associated with dementia and/or cerebral hemorrhage. A specific interaction between BRI2 and APP was unveiled by immunoprecipitation experiments using transfected and non-transfected cells. The use of deletion mutants further revealed that stretches 648-719 of APP751 and 46-106 of BRI2, both inclusive of the full transmembrane domains, are sufficient for the interaction. Removal of most of the APP and BRI2 extracellular domains without affecting the interaction implies that both proteins interact when are expressed on the same cell membrane (cis) rather than on adjacent cells (trans). The presence of BRI2 had a modulatory effect on APP processing, specifically increasing the levels of cellular APP as well as beta-secretase-generated COOH-terminal fragments while decreasing the levels of alpha-secretase-generated COOH-terminal fragments as well as the secretion of total APP and Abeta peptides. Determining the precise molecular pathways affected by the specific binding between APP and BRI2 could result in the identification of common therapeutic targets for these sporadic and familial neurodegenerative disorders.

Presenilin 1 and cadherins: stabilization of cell-cell adhesion and proteolysis-dependent regulation of transcription.

Presenilin-1 (PS1) has gained intensive attention in relation to Alzheimer's disease, since it has been shown that PS1 mutations are linked to familial Alzheimer's disease (FAD), and that PS1 is a member of the high molecular weight complex of gamma-secretase, which generates the carboxyl end of beta-amyloid peptide (gamma-cleavage). A parallel line of evidence suggests that upon formation of cell-cell contacts, presenilin colocalizes with cadherins at the cell surface and stabilizes the cadherin-based adhesion complex. Under conditions stimulating cell-cell dissociation, cadherins are processed by a PS1/gamma-secretase activity, promoting disassembly of adherens junctions, and resulting in the increase of cytosolic beta-catenin, which is an important regulator of the Wnt/Wingless signaling pathway. PS1 also controls the cleavage of a number of transmembrane proteins at the interface of their transmembrane and cytosolic domains (epsilon-cleavage), producing intracellular fragments with a putative transcriptional role. Remarkably, cleavage of N-cadherin by PS1 produces an intracellular fragment that downregulates CREB-mediated transcription, indicating a role of PS1 in gene expression. PS1 mutations associated with FAD abolish production of the N-cadherin intracellular fragment and thus fail to suppress CREB-dependent transcription. These findings suggest an alternative explanation for FAD that is separate from the widely accepted 'amyloid hypothesis': dysfunction in transcription regulatory mechanisms.

Screening for glycosylation changes on recombinant human IgG using lectin methods.

Two lectin-binding methods were investigated as possible ways of monitoring the glycosylation of human monoclonal antibodies during their development and production. Carbohydrate composition was assessed in various preparations that were produced in different host cell types, cell sublines or batches of the same cells. The lectin binding was measured with ELISA and surface plasmon resonance (SPR). For comparative purposes, the monosaccharide content of many of the preparations was also measured by high-pressure anion exchange chromatography (HPAEC)/pulsed amperometric detection (PAD). Both lectin methods detected modifications in glycosylation when antibodies were produced in different ways; SPR was more sensitive than ELISA for some lectins and vice versa. Generally, the lectin results agreed with those obtained by the monosaccharide analysis; however, the former were much better for assessing N -acetylneuraminic acid changes. The latter were impossible to assess by HPAEC/PAD because of their low levels. The lectin-based methods also had the advantages that they were quicker to perform and required less expertise and could quickly identify structures that monosaccharide analysis might miss. It is suggested that, in the development of therapeutic proteins, monosaccharide analysis and/or oligosaccharide profiling is initially performed but later routine batches of the glycoprotein are screened with a lectin method. Of the two lectin methods used, SPR is much quicker when performing a screen, whereas ELISA is particularly useful for comparing a particular carbohydrate feature on different samples of the same glycoprotein.