Immunohistochemical analysis of the localization of the Reelin proteases in the hippocampus of 3xTg-AD mice and their <i>non-transgenic</i> controls.

<p>(<b>A–C</b>) Immunoperoxidase labeling using anti-tPA (<b>A</b>), anti-ADAMTS-4 (<b>B</b>), and anti-ADAMTS-5 antibodies (<b>C</b>). (<b>D</b>) Semi-quantitative analysis of the tPA, ADAMTS-4 and -5 immunoreactivity (IR) in striatum oriens (so), striatum radiatum (sr), striatum lacunosum moleculare (slm), and Dentate Gyrus molecular layer (DG ml). AU, arbitrary units, represent mean background-corrected pixel brightness measured on 4 sections per animal (n = 3 per genotype). *p<0.05, **p<0.01, statistics based on unpaired <i>t-test</i> with Welch's correction. (<b>E</b>) Optimized pepsin pre-treatment protocol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047793#pone.0047793-Doehner1" target="_blank">[50]</a> allowed the detection of ADAMTS-4 IR in extracellular protein depositions throughout the hippocampus of aged mice (lower and higher magnification). Scale bars: <b>A–C</b> = 500 µm; <b>E</b> = 200 µm.</p

ADAMTS-5 degrades Reelin.

<p>(<b>A–D</b>) Anti-Reelin (G10, N-terminal antibody) immunoblots (IB). (<b>A</b>) Recombinant ADAMTS-5 (40 ng/µl) cleaves Reelin at both, its N- and C-cleavage site and further degrades the N-terminal fragment (asterisks). ADAMTS-5 activity is abolished after addition of TIMP-3 (20 ng/µl). (<b>B</b>) Pull-down (PD, using the G10 anti-Reelin antibody, diluted at 1:200, 1:100, 1:50) and subsequent IB with the same antibody revealed the existence of smaller N-terminal fragments (asterisks) in <i>wild-type</i> hippocampus homogenates. (<b>C</b>) Full length Reelin after 52 h incubation at 37°C (lane 1). ADAMTS-5 (80 ng/µl) was added to the FL-Reelin medium (indicated by +), which was previously incubated for 48 h with ADAMTS-4 (20 ng/µl, lane 2) or tPA (50 ng/µl, lane 4).Cleaved Reelin was incubated with ADAMTS-5 for additional 4 hours at 37°C (lanes 3 and 5). (<b>D</b>) Recombinant ADAMTS-1 (10, 20, 40 ng/µl) does not cleave Reelin. (<b>E</b>) Reelin enriched-medium was transferred to HEK293 cells expressing the indicated protease. The samples were collected 24 h after the medium transfer. (<b>F</b>) Schematic summary of Reelin processing and the possible modulations of this process. Dashed lines symbolize indirect effects of serpins and MMP-9 on Reelin processing, which are not mediated by tPA and ADAMTS-4, respectively. X = unknown ECM mediator.</p

Biochemical analysis of Reelin preoteolytic processing and protein levels of the identified Reelin proteases in young and old wild-type mice.

<p>Immunoblots using (<b>A</b>) anti-tPA (H-90), (<b>B</b>) anti-ADAMTS-4 (PA1-1749A), (<b>C</b>) anti-ADAMTS-5 (ab41037), (<b>D</b>) anti-Reelin (G10), (<b>E</b>) anti-Reelin (142), (<b>F</b>) anti-Actin (MAB1522) antibody. Lanes represent different animals. Hippocampus lysates from young (4 weeks) and old (16 months) animals were processed on the same gel and membrane. No difference in Reelin cleavage or levels of Reelin proteases is observed between young and old animals. However, a prominent Reelin-positive band of ∼60 kDa was selectively observed in the aged animals.</p

MMP-9 activates Reelin-cleaving proteases.

<p>(<b>A–D</b>) Anti-Reelin (G10, N-terminal antibody) immunoblots (IB). (<b>A</b>) Recombinant MMP-9 (10 ng/µl) induces Reelin cleavage. (<b>B</b>) Action of MMP-9 (20 ng/µl) on Reelin cleavage could be suppressed with TIMP-3 (10, 15, 20 ng/µl), α2M (10, 20, 40 ng/µl), and also with trypsin inhibitors SBTI and Apro (500 and 150, 1000 and 300, 2000 and 600 ng/µl). The framed lane belongs to the same IB (asterisk on the right blot), but was overexposed for visualization of cleaved Reelin. (<b>C</b>) Incubation of FL-Reelin and recombinant ADAMTS-4 (10 ng/µl), ADAMTS-5 (20 ng/µl), or MMP-9 (10 ng/µl) after heating the FL-Reelin medium at 80°C for 10 min. (<b>D</b>) Expression of the MMP-9 cDNA did not increase Reelin cleavage in Reelin expressing HEK293 cells (left), although we could confirm the synthesis of MMP-9 in these cells using anti-MMP-9 antibody (right). Short vertical lines at the bottom of the blots denote that the last lanes, from the same blot, were joined for visual presentation.</p

tPA cleaves Reelin at its C-terminal cleavage site.

<p>(<b>A</b>) Schematic representation of the Reelin fragments detected with G10 anti-Reelin antibody (left) and immunoblot (IB) of hippocampus lysate from a 3 month-old wild-type mouse, showing full-length Reelin (F, 460 kDa), C-terminal cleaved (C, 380 kDa), and N-terminal cleaved (N, 160 kDa) Reelin fragments (right). (<b>B-F</b>) Anti-Reelin IB (G10, N-terminal antibody). (<b>B</b>) 100 mM Furin Inhibitor I (FI-I) inhibits Reelin cleavage in HeLa cells expressing Reelin. After 12 hours incubation of FI-I or DMSO alone, medium was collected (0 h) and further incubated at 37°C to check for potential Reelin degradation. (<b>C</b>) Recombinant tPA (25 ng/µl) cleaves Reelin at its C-terminal cleavage site. Recombinant serpin E1 (12.5, 37.5, 100 ng/µl) inhibits tPA action. (<b>D</b>) Neither the metalloproteinase inhibitors TIMP-3 (40 ng/µl) or α2M (40 ng/µl), nor the trypsin inhibitors SBTI/Aprotinin (1000 and 300 ng/µl) affect Reelin cleavage by tPA (25 ng/µl). (<b>E</b>) Co-expression of tPA and its inhibitors in Reelin expressing HEK293 cells. Sample (medium) for IB was collected after 12 h incubation. (<b>F</b>) Cross-linking Reelin with BS3 (30 minutes on ice) revealed that C-terminal cleavage does not affect Reelin dimerization (asterisk). The additional band observed with C-terminally cleaved Reelin (two asterisks) may represent higher-order oligomers. Note that the top of the gel is marked with the horizontal line. Indicated hours represent incubation times. All IB blots are representatives of three independent experiments.</p

Co-localization of ADMTS-4 and Reelin in the <i>striatum radiatum</i> of aged animals.

<p>(<b>A–D</b>) Double-immunofluorescence staining using anti-Reelin (red) and anti- ADAMTS-4 and anti-ADAMTS-5 (green) antibodies on brain sections of 15 month-old non-transgenic and 3xTg-AD mice. Co-localization analysis revealed a selective overlap of ADAMTS-4 IR (<b>A,B</b>), but not ADAMTS-5 IR (<b>C,D</b>), with Reelin in extracellular deposits in the striatum radiatum (sr). DAPI (blue) counterstaining was used for labeling the cell nuclei. Scale bars: 20 µm.</p