Similarly, icv injections of anti-p75-saporin and sham-lesions into 3- and 12-month-old WT mice (again with a subsequent observation period of 4 months) resulted in staining patterns indistinguishable from those in the related animal groups displayed in Figure 2
(data not shown). After ChAT immunolabelling in CPN of immunotoxin-treated animals, the success of immunolesion had been checked microscopically, while only hippocampi from mice with verified immunolesion of the MS/DB (n = 21) were applied to subsequent biochemical analysis. Western blotting of TBS-solubilized hippocampal tissues from 7-month-old mice did not show significant https://www.selleckchem.com/products/DAPT-GSI-IX.html differences in APP protein levels between control (n = 4) and immunolesioned
3xTg mice (n = 3; Figure 3a), whereas levels of its APP metabolites C99 and Aβ could not be detected by direct Western blotting at this time. Interestingly, protein levels of the astrocytic marker GFAP were significantly increased in immunolesioned 3xTg mice at 7 months of age (P < 0.05), when no obvious extracellular Aβ deposition is present in the hippocampal formation. Immunolesioned 3xTg mice at 16 months (n = 4) showed significantly increased levels of APP (P < 0.05) and C99 (P < 0.01) as well as monomeric Aβ (P < 0.05) for which only a faint band could be detected in the hippocampal lysates from untreated control 3xTg mice (n = 3). No differences in GFAP EPZ-6438 solubility dmso levels could be detected at this time (not shown). Next, we quantified
the levels of total tau protein in SDS-soluble fractions using anti-human tau as well as antibodies directed against a variety of phospho-tau epitopes, including MC-1, pS199, CP13, AT8 and pS422. No differences in any of these tau variants could be detected in 7-month-old immunolesioned 3xTg mice compared to their age-matched PD184352 (CI-1040) controls (Figure 4a). In contrast, 16-month-old immunolesioned 3xTg mice showed a significant increase in total tau levels (P < 0.05), as well as significantly increased protein levels using the phospho-tau specific antibodies MC-1, pS199 and CP13 (all P < 0.05). Using the antibodies AT8 and pS422, increased protein levels were detected in these mice, but failed to reach statistical significance (Figure 4b). In 16-month-old animals, immunofluorescence labelling of phospho-tau with AT8 and detection of total Aβ with a rabbit antiserum revealed strong hippocampal tau hyperphosphorylation and considerable β-amyloidosis even in tissue from naive mouse (Figure 5a), which was apparently enhanced in immunolesioned animals as exemplarily shown in Figure 5b. Additional co-staining elucidated phospho-tau-immunoreactivity for CP13 in close vicinity to hippocampal Aβ deposits in an immunolesioned animal (Figure 5c) and a naive mouse (not shown).
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