Figure 3
Minocycline inhibits β-site APP cleaving enzyme 1 in young, pre-plaque Tg mice. (A) BACE-1 activity was quantified from hippocampal homogenates using a well-characterized fluorometric assay. Fluorescent units (FU) data were normalized on Non Tg Placebo values. Note the significant up-regulation of BACE-1 activity in Tg Placebo compared to Non Tg Placebo (***P < 0.001), which was corrected following minocycline treatment (*P < 0.05 compared to Tg Placebo). (B) Specificity of the BACE-1 fluorometric assay: note how the fluorescent units detected from brain homogenates (of a Non Tg Placebo animal) are seven times higher than the background (bg). The signal was completely abolished by co-incubation with a specific BACE inhibitor (BACEi), and was in the range of activity of a recombinant human BACE protein (rhBACE, 2.5 μg). All the data were analyzed with one-way ANOVA followed by Tukey post-hoc test. (C) Specificity of the polyclonal BACE-1 antibody used for western blotting: the specific band (close to 75 kDa) appeared in brain extracts from post natal day 7 (pnd 7) and from adult cortex (ctx), while it was hardly detectable in cerebellum (cereb) sample and was not detectable in a glial preparation from pups. (D) Representative western blots of BACE-1, NFkB and IkB from the same hippocampal samples used for the enzymatic assay (ns = non-specific band recognized by the polyclonal antibody directed against NFkB). (E) Quantification data of BACE-1, NFkB and IkB from hippocampal homogenates. Band intensity was quantified via densitometry and the values were normalized on the neuronal specific-β-tubulin content. Note the significant up-regulation of BACE-1 levels in Tg Placebo, which was corrected by the minocycline treatment (P < 0.05). The levels of NFkB and BACE-1 in each sample strongly correlated (P < 0.001, Spearman's correlation analysis). Given the not-normal distribution of the data, the values were analyzed with Kruskal-Wallis test and Spearman's correlation analysis. BACEi: BACE inhibitor; bg; background; cereb: cerebellum; ctx: cortex; pnd: postnatal day; rhBACE: recombinant human BACE.