Caspase-3 activation in the VaD brain.

<p><b>(A-F):</b> Representative immunofluorescence double labeling within the human VaD brain utilizing an antibody to active caspase-3 (green, Panels A and D) and TauC3 (red, Panels B and E), with the overlap images shown in Panels C and F. Labeling of active caspase-3 was evident within pretangles that co-localized with TauC3 (arrows, C). Co-localization of the two antibodies was also evident within plaques although TauC3 gave a much weaker fluorescence signal (F). In fibrillary NFTs only TauC3 was present (arrowhead, C and arrow, F). <b>(G-I)</b>: Representative immunofluorescence double labeling with active caspase-3 (green, G) and the nuclear stain, DAPI (blue, H) indicating labeling within blood vessels of the VaD brain (I). Note the appearance of cuboid, elongated nuclei that typically define endothelial cell nuclei (arrows, H). All scale bars represent 10 μm.</p

Caspase-cleaved tau in the human vascular dementia brain.

<p><b>(A)</b>: Representative labeling from a VaD case utilizing the TauC3 antibody illustrating staining in the hippocampus within apparent NFTs. <b>(B):</b> Representative staining in the hippocampus indicating labeling of TauC3 within neuritic plaques (arrowhead), as well as apparent NFTs (arrow). <b>(C):</b> Correlation of TauC3 labeling with Braak & Braak stage. For six VaD cases in which the Braak & Braak stage was known, the number of TauC3-positive NFTs was counted three separate times, averaged and then plotted versus Braak & Braak stage. A positive correlation (R² = .070) was observed between these two variables. <b>(D)</b>: Western blot analysis utilizing the TauC3 antibody was carried out utilizing brain extracts from the frontal cortex (FCTX) or cerebellum (CBL) of four VaD cases. Lanes 1 (Case 6, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132637#pone.0132637.t001" target="_blank">Table 1</a>) and 2 (Case 4, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132637#pone.0132637.t001" target="_blank">Table 1</a>) are VaD cases that had Stage A plaque load, whereas lanes 3 (Case 3, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132637#pone.0132637.t001" target="_blank">Table 1</a>) and 4 (Case 2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132637#pone.0132637.t001" target="_blank">Table 1</a>) were designated as having a plaque load of 0. A band at 50 kDa corresponding to caspase-cleaved tau truncated at Asp⁴²¹ was identified in the FCTX of all four VaD cases and two of four cases in the CBL. The bottom panel of D depicts an identical experiment except transferred proteins were probed with HT7 (1:1,000), an antibody that detects total, full-length (FL) tau. <b>(E and F):</b> Low (E) and high magnification (F) of representative labeling from a VaD case utilizing the TauC3 antibody illustrating staining in the dentate gyrus of the hippocampus within numerous, round translucent structures. All scale bars represent 10 μm, except for Panel E, which represents 50 μm.</p

Confirmation of caspase-cleaved tau within corpora amylacea.

<p><b>(A):</b> Representative bright field staining in a VaD hippocampal brain section utilizing PAS that specifically labels CA in brain tissue. Labeled CA (magenta color, arrows) were in the vicinity of neurons in the granule cell layer of the dentate gyrus that were counter-stained with hematoxylin. <b>(B):</b> Representative bright-field labeling of numerous CA in the hippocampus of a VaD case utilizing an anti-ubiquitin, a specific marker for CA. <b>(C and D):</b> Representative immunofluorescence double-labeling in a VaD case at high magnification (C) and low magnification (D) indicating the co-localization of ubiquitin (green) together with the TauC3 antibody (red) within CA. In Panel D, the nuclei were also stained with nuclear stain, DAPI. All scale bars are equivalent to 10 μm except for Panel D, which represents 50 μm.</p

PHF-1 labeling of corpora amylacea within the hippocampus of the VaD brain.

<p><b>(A)</b>: Representative bright-field DAB labeling from a VaD case utilizing the PHF-1 antibody illustrating staining in the hippocampus within NFTs. <b>(B)</b>: High magnification of a single PHF-labeled NFT indicated the presence of small circular structures (arrows) of the same size and shape as CA in close proximity to the labeled neuron. <b>(C)</b>: In this example, a PHF-labeled neuron appeared to exhibit intracellular inclusions that were of the same size and shape as identified CA (arrow). <b>(D)</b>: Numerous CA labeled with PHF-1 were documented in plaque-rich regions within the hippocampal region of VaD cases. All scale bars represent 10 μm.</p

Co-localization of caspase-cleaved tau with NFTs in the VaD brain.

<p><b>(A-C)</b>: Representative images from confocal immunofluorescence analysis in VaD utilizing TauC3 (green, A) and PHF-1 (red, B) with the overlap image shown in Panel C. Notice the filamentous nature of staining of PHF-1 as compared to TauC3. <b>(D and E)</b>: Representative immunofluorescence double labeling (arrows, D) and quantification of NFTs (E) double-labeled with TauC3 and PHF-1. Data show the number of NFTs labeled with TauC3 alone (blue bar), PHF-1 alone (green bar) or NFTs that were labeled with both antibodies (red bar). NFTs were identified in a 20X field within hippocampi sections by immunofluorescence overlap microscopy (n = 3 fields for 4 different VaD cases) ±S.E.M. *p = 5.46 x 10^-7 between PHF-1 alone and TauC3 + PHF-1 and #p = 4.49 x 10^-7 between TauC3 alone and TauC3 + PHF-1. Data indicated that roughly 90% of all labeled NFTS co-localized with both antibodies. <b>(F and G)</b>: Low- (F) and High-field (arrows, G) double immunofluorescence overlap images of corpora amylacea within the dentate gyrus of a representative VaD case showing co-localization of TauC3 (green) and PHF-1 (red). <b>(H-J)</b>: High magnification confocal images representing labeling of corpora amylacea with TauC3 (H), PHF-1 (I), and the merged image (J). Scale bars represent 10 μm in Panels D and G and 50 μm for Panel F.</p

Caspase-cleaved tau within neuritic plaques in the VaD brain.

<p><b>(A and B)</b>: Representative bright-field microscopy showing TauC3 labeling of neuritic plaques at low-field (arrows, A) and at high magnification (arrow, B). <b>(C-E)</b>: Representative immunofluorescence double labeling utilizing an anti-Aβ (clone 6E10) antibody (green, C), TauC3 (red, B), and the merged image (C) indicating co-localization of two markers (yellow, arrowheads). The arrow in Panel E reflects the labeling of a single NFT by TauC3 that is not labeled with the 6E10 antibody. <b>(F-G)</b>: Identical to Panels C-E depicting labeling in an additional VaD case. All scale bars represent 10 μm except Panel A, which represents 50 μm.</p

Case Demographics.

<p>PMI, postmortem interval in hours; NPD, neuropathological diagnosis.</p

Labeling of nApoECF antibody within the hippocampus co-localizes with PHF-1 and is regionally defined.

<p>(<b>A–C</b>): Representative immunofluorescence double-labeling in Case #3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080180#pone-0080180-t001" target="_blank">Table 1</a>) utilizing the nApoECF antibody (green, Panel A) and PHF-1 (red, Panel B) revealed strong co-localization of the two antibodies within Pick bodies of area CA1 (Panel C). (<b>D–F</b>): In contrast to area CA1, strong immunofluorescence was detected using PHF-1 (red, Panel E), but only weak labeling was observed within granule cells of the dentate gyrus following application of the nApoECF antibody (green, Panel D). Panel F shows the overlap image with yellow indicating Pick bodies double-labeled with both antibodies. (<b>G</b>): Quantification of Pick bodies double-labeled by PHF-1 and nApoECF. Data show the number of Pick bodies labeled with nApoECF alone (blue bar), PHF-1 alone (green bar) or those Pick bodies that were labeled with both antibodies (red bar) in area CA1. Pick bodies were identified in a 20× field within area CA1 by immunofluorescence overlap microscopy (n = 3 fields for 4 different Pick cases) ±S.D., *p<0.05. Data indicated that roughly 86% of all identified Pick bodies within area CA1 were labeled with both antibodies. (<b>H–J</b>): Double-labeled confocal immunofluorescence images with the nApoECF antibody (green, H), PHF-1 (red, I), and the two images overlapped (J). All scale bars represent 10 µm, except for confocal images in Panel H–J, which represent 50 µm. (<b>K</b>): High magnification confocal overlapped image representing nApoECF (green channel, left), PHF-1 (red channel, middle), and overlapped image (yellow/orange, right); notice filamentous nature of staining within the two Pick bodies. Scale bars for Panel K represent 5 µm.</p

Co-localization of the nApoECF antibody with caspase-cleaved tau.

<p>(<b>A–C</b>): Representative immunofluorescence double-labeling in Case #4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080180#pone-0080180-t001" target="_blank">Table 1</a>) within the CA1 region of the hippocampus in Pick's disease utilizing the nApoECF antibody (green, Panel A), caspase-cleaved tau (red, Panel B) and with the overlap image shown in Panel C. All scale bars represent 10 µm. (<b>D</b>): Quantification of Pick bodies double-labeled by TauC3 (caspase-cleaved tau) and nApoECF in area CA1 of the hippocampus. Data show the number of Pick bodies labeled with nApoECF alone (blue bar), Tauc3 alone (green bar) or those Pick bodies that were labeled with both antibodies (red bar). Pick bodies were identified in a 20× field within area CA1 by immunofluorescence overlap microscopy (n = 3 fields for 4 different Pick cases) ±S.D., *p<0.05. Data indicated that roughly 75% of all labeled Pick bodies within area CA1 were co-localized with both antibodies. (<b>E–G</b>): Double-labeled confocal immunofluorescence images with the nApoECF antibody (green, E), PHF-1 (red, F), and the two images overlapped (G). (<b>H</b>): High magnification confocal overlapped image representing nApoECF (green channel, left), TauC3 (red channel, middle), and overlapped image (yellow/orange, right). Scale bars in Panel H represent 5 µm.</p