The effects of chronic administration of Q-VD-OPh on Aβ deposition, caspase-7 activation, and the caspase-cleavage of tau in TgCRND8 mice. A) Cell-free inhibition of caspase-7 activity by Q-VD-OPh. Data display a concentration-dependent inhibition of caspase-7 cleavage of the substrate AMC-DEVD-pNA in a cell-free system consisting of active human recombinant caspase-7. The calculated IC₅₀ of Q-VD-OPh was determined to be approximately 48 nM. B-D) Three month-old TgCRND8 mice were injected 3 times a week for 3 months with 10 mg/kg of Q-VD-OPh (B) or vehicle (C). Data are representative of 3 independent experiments utilizing the monoclonal antibody to Aβ (clone 6E10, 1:400) and indicate the presence of extracellular Aβ deposits in the cortex and hippocampus of both animals (arrows). Panel D depicts semi-quantitative analysis of the number of Aβ plaques for vehicle or following Q-VD-OPh-treatment. E-J) Double-immunofluorescence images with active caspase-7 in green and 6E10 in red. (E-G) represent animal treated with Q-VD-OPh indicating that while caspase-7 activation was prevented (E), there was no apparent effect on plaque formation (F). Panel G represents the overlap image for Panels E and F. (H-J) Identical to above except panels represent a control animal treated with vehicle. In this case both active caspase-7 (H) and plaque labeling (I) are evident. (K-M) Prevention of the caspase-cleavage of tau following treatment with Q-VD-OPh. (K) Represents an animal treated with Q-VD-OPh, whereas Panels L and M represent vehicle controls following staining with the TauC3 antibody. No apparent staining was observed in the treated animal, while the presence of caspase-cleaved tau was evident in astrocytes (arrows, L) and extracellular plaques (M) in the vehicle controls. Scale bars represent 10 μm.

Caspase activation in 12 month-old TgCRND8 mice. A) Representative bright-field micrograph illustrating Aβ deposition in TgCRND8 mice utilizing the 6E10 mAB (1:400). B) Double-label immunofluorescence image with mAb 6E10 to detect extracellular Aβ deposits (red) and a polyclonal TauCCP antibody to detect caspase-cleaved tau (arrows, green). C) Representative image using TauC3 indicated the presence of caspase-cleaved tau within plaque-rich regions. D) Semi-quantitative analysis of the number of extracellular plaques labeled with 6E10 (blue bar), the TauCCP antibody (red bar) and TauC3 (green bar). N=6 different animals ± S.D. E) Western blot analysis of brain extracts probed with the mAb TauC3 (1:500) indicating the presence of caspase-cleaved tau in all four TgCRND8 mice with weak labeling in only one of four age-matched NonTg control mice. Bottom panel represents loading control blot following stripping and reprobing using a rabbit antibody to beta-actin (1:400). Data are representative of three independent experiments. F) Representative IH staining utilizing an antibody (APPccp) that detects caspase-cleaved APP. Staining with APPccp was localized within plaques of the neocortex. G) Representative IH analysis in TgCRND8 mice depicting staining with an antibody to active caspase-7. Caspase-7 labeling was evident in plaques and within neurons in the vicinity of plaques (G, arrows). H) Double-label immunofluorescence overlap image from a representative TgCRND8 mouse showing colocalization of active caspase-7 (green) together with the apoptotic label, propidium iodide (red). Propidium iodide staining was only evident in neuronal cell bodies that were also labeled with active caspase-7 (arrows, H). All scare bars represent 10 μm except for Panel C, which represents 20 μm.

Prevention of MC-1 labeling within astrocytes of TgCRND8 mice following chronic treatment with the caspase inhibitor, Q-VD-OPh. (A) Representative bright-field micrograph following labeling with MC-1 (1:500) in vehicle controls. (C and D) indicates the absence of MC-1 labeling in an animal following treatment with Q-VD-OPh. To confirm the presence of MC-1 labeling of astrocytes in vehicle controls, double-label immunofluorescence experiments were carried out utilizing MC-1 (red) and an antibody to GFAP (green). Co-localization of both antibodies was evident within astrocytes (arrow, B). All scale bars represent 10 μm.