Fig 2: ADAMTS-5 degrades Reelin.(A–D) Anti-Reelin (G10, N-terminal antibody) immunoblots (IB). (A) 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) 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 wild-type hippocampus homogenates. (C) 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). (D) Recombinant ADAMTS-1 (10, 20, 40 ng/µl) does not cleave Reelin. (E) Reelin enriched-medium was transferred to HEK293 cells expressing the indicated protease. The samples were collected 24 h after the medium transfer. (F) 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.

Fig 3: Co-localization of ADMTS-4 and Reelin in the striatum radiatum of aged animals.(A–D) 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 (A,B), but not ADAMTS-5 IR (C,D), with Reelin in extracellular deposits in the striatum radiatum (sr). DAPI (blue) counterstaining was used for labeling the cell nuclei. Scale bars: 20 µm.