Thy-1-GPI anchor redirects PrP^C to the apical site.

<p>(A) Cells stably expressing PrP^CWT and PrP^C-GPIThy-1 were grown in Transwells for 4 to 5 days, processed for immunocytochemistry, and analyzed with confocal microscopy. YZ sections (left) and view on the membrane (right) at the level of tight junctions stained for ZO-1 (red) confirm both polarization and confluency of cells and show increased apical signal for PrP^C-GPIThy-1 (green). (B) After staining with PrP 3F4 antibody under non-permeabilizing conditions, serial Z-stacks from the bottom to the top were taken. YZ sections show transversal cut through cells at the level of the dashed line in mid. PrP^C-GPIThy-1 was found at the apical membrane when compared to PrP^CWT. Scale bars are 10 µm. (C) Cells grown in Transwells labeled with EZ-Link Sulfo-NHS-SS-Biotin either apically (a) or basolaterally (b) were processed for Western blotting for PrP^C and E-Cadherin (as control of cell polarization) in parallel. The graph (three independent experiments) shows mean percentages ± SEM of apical (a) or basolateral (b) amount of protein when compared to the total amount which is set at 100%.</p

Schematic drawing of constructs used in this study.

<p>Shown are the maps of PrP^CWT, PrP^CG1, PrP^CG2, and PrP^CG3 with N-terminal signal sequence (ss) and C-terminal GPI-anchor signal (ss GPI-anchor) (dark boxes) and the mutations introduced to delete N-gylcosylation sites.</p

PrP^C-GPIThy-1 is glycosylated and transported to the plasma membrane.

<p>(A) Schematic presentation of GPI-anchored PrP^CWT and the PrP^C fusion protein with the GPI-anchor of Thy-1 (PrP^C-GPIThy-1). The substitution of the GPI-anchor signal sequence (ss) of the PrP for the one of Thy-1 is indicated. (B) Western blots of PrP^CWT and PrP^C-GPIThy-1 stably expressed in MDCK cells. A clone with a similar expression level as PrP^CWT was chosen. The glycotype of di-, mono-, and non-glycosylated PrP^C-GPIThy-1 is unchanged. (C) Assessment of non-permeabilized membrane localization of PrP^CWT and PrP^C-GPIThy-1 by confocal microscopy shows plasma membrane localization of both proteins (scale bar is 10 µm). (D) Sucrose density gradient centrifugation of 1% Triton-X100 extraction at 4°C of PrP^CWT and PrP^C-GPIThy-1 cells reveal localization of both in flotillin enriched DRMs. Fractions were taken from the top (fraction 1) to the bottom (fraction 12).</p

Cell surface biotinylation assay confirms a role of the N-glycans in polarized sorting of PrP^C.

<p>Cells were grown in Transwells for 4–5 days until fully polarized and labelled with EZ-Link Sulfo-NHS-SS-Biotin either on the apical (a) or the basolateral (b) side. Cells were processed for PrP^C (recognized with the 3F4 antibody) and E-cadherin Western blotting in parallel. The graph indicates densitometric evaluation of Western blots of at least 3 independent experiments, expressed as mean percentages ± SEM apical (a) or basolateral (b) of total protein found, which is set at 100%.</p

N-glycosylation of PrP^C affects polar sorting in MDCK cells.

<p>MDCK cells stably expressing PrP^CWT, PrP^CG1, PrP^CG2 or PrP^CG3 were grown in Transwells for 4 to 5 days until they were fully polarized. (A) Cells were separately stained with the 3F4 antibody (green) followed by permeabilisation and staining with an antibody against ZO-1 (red), a constituent of tight junctions, indicating the cell polarity. Confocal microscopy of a Z-stack of PrP^CWT (left) at the level of tight junctions stained with ZO-1, and YZ-sections (right) of all glycomutants indicate both the integrity of the polarized monolayer and a redistribution of PrP^CG1 and PrP^CG2 to the apical compartment when compared to PrP^CWT and PrP^CG3. Localization of the apical (a) and basolateral (b) compartment is indicated. (B) After immunocytochemistry under non-permeabilising conditions with the 3F4 antibody, serial Z-stacks from the bottom to the top were taken with confocal microscopy. YZ images shows transversal cut trough cells at the mid level, marked with a dashed line. PrP^CWT and PrP^CG3 were mainly found in the basolateral compartment whereas PrP^CG1 and PrP^CG2 were mainly found in both compartments. Scale bars represent 10 µm.</p

Physiological membrane localization of PrP^C glycomutants.

<p>(A) Characterization of glycomutants (PrP^CG1, PrP^CG2, and PrP^CG3) and PrP^CWT for the study by Western blot analysis, using an antibody directed against the 3F4 epitope. Clones with similar amounts of overexpressed 3F4 tagged PrP^C as assessed by densitometric analysis of Western blots were used for these analyses (see graph). Relative expression of various PrP^C forms is shown in percentages of PrP^CWT that was set to 100%. (B) Assessment of plasma membrane (non-permeabilized) and intracellular (permeabilized) localization of PrP^C glycomutants by confocal microscopy shows presence of PrP^C at the plasma membrane and intracellularly (scale bar is 10 µm). (C) Assessment of DRMs localization of PrP^C glycomutants by Triton X-100 extraction at 4°C and sucrose density gradient centrifugation showing correct localization of PrP^C glycomutants with flotillin-positive DRM containing fractions.</p

Amyloid-Precursor-Protein-Lowering Small Molecules for Disease Modifying Therapy of Alzheimer's Disease

<div><p>Alzheimer's disease (AD) is the most common form of dementia in the elderly with progressive cognitive decline and memory loss. According to the amyloid-hypothesis, AD is caused by generation and subsequent cerebral deposition of β-amyloid (Aβ). Aβ is generated through sequential cleavage of the transmembrane Amyloid-Precursor-Protein (APP) by two endoproteinases termed beta- and gamma-secretase. Increased APP-expression caused by APP gene dosage effects is a risk factor for the development of AD. Here we carried out a large scale screen for novel compounds aimed at decreasing APP-expression. For this we developed a screening system employing a cell culture model of AD. A total of 10,000 substances selected for their ability of drug-likeness and chemical diversity were tested for their potential to decrease APP-expression resulting in reduced Aβ-levels. Positive compounds were further evaluated for their effect at lower concentrations, absence of cytotoxicity and specificity. The six most promising compounds were characterized and structure function relationships were established. The novel compounds presented here provide valuable information for the development of causal therapies for AD.</p></div

Characterization of the six best compounds.

<p>A) Flowchart of the screening of the 10,000 compound library DIVER Set. 10,000 compounds were screened and hits were analyzed by serial dilutions (100 µM, 50 µM, 10 µM, 1 µM). Compounds effective at lower concentrations were checked for cytotoxicity and the non-cytotoxic ones were further analyzed by western blot, ELISA and RT-PCR. B) Structures of the highly potent 5 compounds (A, C–F) in the DIVER Set library which have an specific effect on APP/Aβ-production at a concentration of a minimum of 10 µM and are not cytotoxic. The structure of compound B is added below.</p

Characterization of a new cell-based assay for screening of APP-lowering small molecules.

<p>A) Expression of APP/Aβ in APPsw transfected HEK 293 cells and in HEK 293 cells. APPsw cells and HEK cells were fixed, labelled with 6E10 antibody and stained with Cy3 anti-mouse IgG for detection of APP/Aβ (red). Nuclei were stained with DAPI (blue). Scale bar 25 µm. B) Supernatants of HEK and APPsw cells were characterized via dot blot with the 6E10 antibody. APPsw cells produce a higher level of sαAPP than control HEK cells. C) Representative example of a dot blots from the screening stage of the study. Supernatants of compound-treated APPsw cells and controls (solvent-treated APPsw, extreme left and right lane). With this approach 80 compounds could be assessed in parallel. Only compounds reducing the signal in four independent experiments were evaluated as “positive”.</p

Assessment of APP, Aβ₄₀ and Aβ₄₂ levels in compounds-treated APPsw cells.

<p>A) Western blot of the cell lysates and the supernatants of APPsw cells after 3 day incubation with compounds (10 µM). Arrows indicate fully glycosylated mature, incompletely glycosylated immature APP and sαAPP. β-actin serves as loading control. First Graph showing relative expression of mAPP and imAPP normalized to expression of actin, untreated APPsw controls were set to 1. Results are shown as mean±S.D., n = 5, ***p<0.001. Second Graph showing relative expression of sαAPP normalized to expression of actin, untreated APPsw controls were set to 1. Results are shown as mean±S.D., n = 5, ***p<0.001. B) Aβ₄₀ quantification by ELISA with the supernatant of APPsw cells after 3 days of compound incubation (10 µM). Aβ₄₀ and Aβ₄₂ levels were decreased significantly. Untreated APPsw cells and HEK cells are used as controls. Data are the mean ± S.D., n = 3, **p<0.01, ***p<0.001. C) Aβ₄₂ quantification by ELISA with the supernatant of APPsw cells after 3 days of compound incubation (10 µM). Aβ₄₀ and Aβ₄₂ levels were decreased significantly. Untreated APPsw cells and HEK cells are used as controls. Data are the mean ± S.D., n = 3, **p<0.01, ***p<0.001. D) mRNA levels of APP were measured by RT-PCR in treated APPsw cells and HEK cells, untreated APPsw cells were set to 1. Compound B reduces the amount of APP-mRNA. Data are the mean of two experiments.</p