Homodimerization Protects the Amyloid Precursor Protein C99 Fragment from Cleavage by γ‑Secretase

The amyloid precursor protein (APP) is a single-span integral membrane protein whose C-terminal fragment C99 is cleaved within the transmembrane helix by γ-secretase. Cleavage produces various Aβ peptides that are linked to the etiology of Alzheimer’s disease. The transmembrane helix is known to homodimerize in a sequence-specific manner, and considerable controversy about whether the homodimeric form of C99 is cleaved by γ-secretase exists. Here, we generated various covalent C99 homodimers via cross-linking at engineered cysteine residues. None of the homodimers was cleaved <i>in vitro</i> by purified γ-secretase, strongly suggesting that homodimerization protects C99 from cleavage

Multi-modal analysis of the four AD mouse strains studies in this cross-sectional [¹⁸F]-florbetaben PET study.

<p>Upper images represent group averaged sagittal PET slices, normalised to the cerebellum and overlayed on an MRI mouse atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116678#pone.0116678.ref039" target="_blank">39</a>]. Dots indicate corresponding assessments of SUVR_CTX/CBL in individual mice. Dashed lines express the estimated time dependent progression in PS2APP (red; five months: N = 5; eight months: N = 7; 10 months: N = 6; 12 months: N = 2; 16 months: N = 6, 19 months: N = 6), G384A (green; five months: N = 2; 16 months: N = 1) and APP/PS1dE9 (purple; 12 months: N = 2; 24 months: N = 2) mice, fitted with a polynomial function (for the purposes of illustration). Longitudinal progression in APPswe mice is indicated by a continuous blue line. Lower images depict representative <i>ex vivo</i> autoradiography results; autoradiography of APP/PS1dE9 mice and young G384A mice was performed <i>in vitro</i>. WT level expresses the mean SUVR_CTX/CBL of pooled WT mice (N = 22).</p

Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM

<div><p>Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified <i>in silico</i> in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.</p></div

Cross-Sectional Comparison of Small Animal [¹⁸F]-Florbetaben Amyloid-PET between Transgenic AD Mouse Models

<div><p>We aimed to compare [¹⁸F]-florbetaben PET imaging in four transgenic mouse strains modelling Alzheimer’s disease (AD), with the main focus on APPswe/PS2 mice and C57Bl/6 mice serving as controls (WT). A consistent PET protocol (N = 82 PET scans) was used, with cortical standardized uptake value ratio (SUVR) relative to cerebellum as the endpoint. We correlated methoxy-X04 staining of β-amyloid with PET results, and undertook <i>ex vivo</i> autoradiography for further validation of a partial volume effect correction (PVEC) of PET data. The SUVR in APPswe/PS2 increased from 0.95±0.04 at five months (N = 5) and 1.04±0.03 (p<0.05) at eight months (N = 7) to 1.07±0.04 (p<0.005) at ten months (N = 6), 1.28±0.06 (p<0.001) at 16 months (N = 6) and 1.39±0.09 (p<0.001) at 19 months (N = 6). SUVR was 0.95±0.03 in WT mice of all ages (N = 22). In APPswe/PS1G384A mice, the SUVR was 0.93/0.98 at five months (N = 2) and 1.11 at 16 months (N = 1). In APPswe/PS1dE9 mice, the SUVR declined from 0.96/0.96 at 12 months (N = 2) to 0.91/0.92 at 24 months (N = 2), due to β-amyloid plaques in cerebellum. PVEC reduced the discrepancy between SUVR-PET and autoradiography from −22% to +2% and increased the differences between young and aged transgenic animals. SUVR and plaque load correlated highly between strains for uncorrected (R = 0.94, p<0.001) and PVE-corrected (R = 0.95, p<0.001) data. We find that APPswe/PS2 mice may be optimal for longitudinal amyloid-PET monitoring in planned interventions studies.</p></div

Determination of the sheddase cleavage amino acid sequence in mEpEX.

<p>(<b>A</b>) Schematic representation of mEpCAM-TF containing a TEV protease recognition site and a Flag-Tag in the mEpEX domain 42 amino acids before the predicted transmembrane domain. After cleavage by sheddases, the largest part of mEpEX can be removed through digestion with TEV protease and the resulting small fragment immunoprecipitated using Flag-specific antibodies. (<b>B</b>) Representative mass spectrometry spectrum of HEK293, NIH3T3, and mF9 cells stably expressing mEpCAM-TF and of vector control HEK293 cells as a control. Four major peak species are indicated. (<b>C</b>) Tabular overview of sheddase cleavage sites within mEpEX as determined upon mass spectrometric analysis and alignment to potential molecular weights. Calculated and determined masses are given in Dalton including error and charge of each peptide. (<b>D</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after treatment with DMSO, the metalloprotease protease inhibitor TAPI-1, and the phorbol ester PMA. (<b>E</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after treatment with DMSO, the BACE1 protease inhibitor C3, and after transient transfection of BACE1 expression plasmid. (<b>F</b>) Sequence alignment of murine and human EpCAM (top), and murine EpCAM and murine Trop-2 (bottom). metalloprotease protease cleavage (a-secretase) and BACE1 cleavage sites (b-secretase) are indicated.</p

Amyloid-PET and <i>ex vivo</i> autoradiography in PS2APP mice before and after PVEC.

<p><b>(A)</b> Comparison of uncorrected [¹⁸F]-florbetaben PET images (upper row), corresponding <i>ex vivo</i> autoradiography (mid row) and PVE-corrected PET (lower row) of representative PS2APP mice at 8, 12 and 19 months of age. Sagittal PET images captured 1.6 mm left of the midline were scaled to cerebellum and overlain on a 3T MRI mouse template [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116678#pone.0116678.ref013" target="_blank">13</a>]. PVEC was performed with a 10 region mask (four cerebral and six extracerebral VOIs). <b>(B)</b> Error-(%) (±SD) of uncorrected (black bar) and PVE-corrected (blue bar) data versus <i>ex vivo</i> autoradiography are shown for the whole group of PS2APP mice.</p

Schematic representation of EpCAM presenilin-dependent regulated intramembrane proteolysis (PS-RIP) and endocytosis.

<p>Murine EpCAM (mEpCAM) is cleaved at the plasma membrane to release soluble EpEX (smEpEX). The resulting C-terminal fragment (mCTF) is a substrate for γ-secretase, which cleaves mCTF to generate soluble, extracellular mEp-β fragments (γ-cleavage) and intracellular mEpICD fragments, which are prone to proteasomal degradation. Additionally, mEpICD can be endocytosed and processed either by BACE1 in acidic intracellular compartments (endosome) and/or by acidic hydrolases in lysosomes.</p

Comparison of uncorrected (A) and PVE-corrected (B) SUVR_CTX/CBL of the entire dataset.

<p>Dots indicate corresponding assessments of SUVR_CTX/CBL in individual mice. Dashed lines express the estimated time dependent progression in PS2APP (red; five months: N = 5; eight months: N = 7; 10 months: N = 6; 12 months: N = 2; 16 months: N = 6, 19 months: N = 6), G384A (green; five months: N = 2; 16 months: N = 1) and APP/PS1dE9 (purple; 12 months: N = 2; 24 months: N = 2) mice, fitted with a polynomial function (for the purposes of illustration). Longitudinal progression in APPswe mice is indicated by a continuous blue line. P-values for one-way ANOVA (incl. post hoc Tukey) testing of PS2APP and APPswe mice versus youngest littermates were as indicated: * p < 0.05; ** p < 0.005; *** p < 0.001.</p

Sequence conservation of cleavage sites in orthologs of EpCAM found in fishes, amphibians, birds, to placental mammals.

<p>Amino acid sequences of 52 orthologs of human EpCAM were aligned using ClustallW and sequence conservation of each amino acid was calculated (maximum score 11. Shown are the mean conservation score throughout all orthologs (mean) and conservation scores of single amino acids ranging positions p⁻³ to p⁺³ around determined cleavage sites of metalloproteases (<b>A</b>), BACE1 (<b>B</b>), γ-cleavage of γ-secretase (<b>C</b>), and ε-cleavage of γ-secretase (<b>D</b>).</p

Cleavage of endogeneous mEpCAM.

<p>Proteolytic cleavage of mEpCAM was addressed in mF9 (<b>A</b>) and E14TG2a (<b>B</b>) cells using membrane assays at pH 7 and pH 4. Membranes of mF9 and E14TG2a cells were incubated for 0 h and 24 h at 37°C and EpCAM fragments were detected in immunoblots using a mEpICD-specific antibody in combination with an HRP-conjugated secondary antibody. Inhibition of the γ-secretase complex was achieved upon treatment with DAPT where indicated. Shown are the representative results of three independent experiments.</p