2; in Braak stages V-VI, small numbers of UBL immunoreactive pyramidal cells remaining in the CA1 precluded optical density analyses). The ratio was slightly, but non-significantly, elevated in the CA2/3 field from Braak stage groups III-IV and V-VI when compared to Braak stage group 0-I-II, and a similar trend was observed in the CA4 field (Fig. 2). Optical density measurements in the nucleoplasm and cytoplasm

correlated directly across all Braak staged groups in CA2/3 as well as in CA4, but did not correlate in the CA1 field (data not shown). We detected statistically significant (Spearman r = 0.7, Romidepsin clinical trial P = 0.01) correlation between more advanced age and higher nucleoplasm/cytoplasm UBL immunoreactivity optical density ratio values in CA1, but not CA2/3 or CA4. The relationship between UBL protein and a marker of advanced stage NFT including extracellular “ghost NFT” (X-34) or an antibody that also recognizes pre/early NFT (AT8) was examined

using multiple-label fluorescence confocal microscopy (Figs 3, 4). The pattern of UBL immunofluorescence was consistent with our observations using the same antibody and chromogen-based immunohistochemistry with light microscopy (Fig. 3). In multiple-labeled (UBL, AT8, DAPI, X-34) sections from Braak stage 0-I-II cases, we observed pyramidal neurons with UBL immunofluorescence in the cytoplasm and nucleoplasm, the latter co-labeled with DAPI (Fig. 3A–D). Braak 0-I-II cases had no AT8- or X-34-positive NFT in the hippocampus, although sparse, scattered AT8 immunofluorescent neuritic elements were observed in the CA fields (Fig. 3E–H). In Braak stages III-IV and V-VI cases, we observed a complex pattern of UBL/AT8 or UBL/X-34 co-localization

check details in CA fields. Neurons with light cytoplasmic and prominent nucleoplasmic UBL immunofluorescence co-localized AT8, but had little or no X-34, (Fig. 3I–L,M–P,M′–P′). The majority of UBL-immunofluorescent Tyrosine-protein kinase BLK pyramidal neurons in the CA2/3 region were AT8- and X34-negative, yet surrounded by numerous AT8-immunofluorescent neurites (Fig. 3I–L). Pyramidal neurons in CA1 and subiculum of Braak stages V-VI cases had UBL immunofluorescence co-localized with X-34, and very little or no AT8 immunofluorescence and no DAPI labeling, indicative of extracellular “ghost” NFT (eNFT, Fig. 3M–P,M″–P″). UBL immunoreactive neuritic elements were also detected within X-34 labeled amyloid plaques in the CA1 and DG molecular layer (not shown). A small number of AT8-positive neurons lacking UBL immunofluorescence were observed in the CA1 region of Braak V-VI cases. The overall pattern of UBL/AT8/X-34 immunofluorescence in a representative Braak stage VI case is illustrated diagrammatically in Figure 4. The present study investigated UBL immunoreactivity in the hippocampus from non-AD and clinically diagnosed AD cases stratified by Braak stages, in relation to markers of primarily advanced stage NFT (the pan-amyloid marker X-34) and the antibody clone AT8 which also recognizes pre/early NFT.