Figure 2

Minocycline effects on amyloid precursor protein metabolism. Cortical brain homogenates from Tg animals treated with vehicle (Tg Pl) or minocycline (Tg Mino) were subjected to western blotting and ELISA to determine the levels of Aβ species, full-length APP (flAPP) and CTF. (A) Representative western blot of cortical homogenates from Tg animals treated with vehicle or with minocycline, using 6E10 antibody. Note the strong down-regulation of the approximately 12-kDa ir band (indicated with an asterisk) which likely represents a mixture of β-CTF of APP and Aβ trimers. (B) Densitometric analysis of the 12-kDa band detected with 6E10. Values were normalized on neuronal specific β-tubulin. Minocycline-treated animals showed significantly lower levels compared with placebo (**P < 0.01, Student's t-test). (C) Correlation analysis of the levels of the 12-kDa band ir to 6E10 and the levels of iNOS per sample. The correlation was found to be highly significant (r = 0.81; P = 0.004, Spearman's correlation), where Tg Placebo animals (black dots) had the highest levels of 6E10-ir and iNOS, while Tg Mino (yellow dots) mice displayed reduced levels of ir with 6E10 and lower levels of iNOS. (D) Representative western blots of cortical (ctx) homogenates from placebo and minocycline-treated animals using the pab27576 antibody. This antibody recognizes an epitope on the C-terminus of APP and therefore detects two prominent bands in the blots: a high molecular weight band (about 100 kDa), corresponding to flAPP and a faster band (approximately 12 kDa) corresponded to the CTF fragments. A calibration curve using known amounts of recombinant C100 was run in the same gel for quantification purposes (the recombinant peptide ran slightly slower due to the presence of a C-terminal hexa-His tag). (E) Quantification of flAPP and CTF levels in cortical homogenates from Tg Placebo and Tg-treated animals. FlAPP relative optical density values were normalized on neuronal specific β-tubulin. Note the significant down-regulation of flAPP (**P < 0.01, Student's t-test) in Tg Mino compared with Tg Placebo. CTF absolute levels were extrapolated from the calibration curve of recombinant CTF. There was a non-significant trend towards reduction following minocycline treatment. Normalization of the relative optical density of CTF over the relative optical density of flAPP confirmed a non-significant trend towards a reduction (ratio CTF/flAPP). All data were analyzed with Student's t-test. (F) Quantification of human Aβ levels in cortical homogenates from Tg Placebo and Tg Mino using ELISA. The treatment reduced both Aβ40 and Aβ42, but the effect did not reach significance (P = 0.09 and 0.08, respectively, Student's t-test; see main text for exact values). (G) The ratios between Aβ (pg/mg total protein) and CTF (ng/mg total protein) were calculated per sample and compared across groups. We observed 24.29 ± 6.43 pg of Aβ42 and 15.14 ± 3.08 pg of Aβ40 per each nanogram of CTF. Minocycline treatment resulted in a reduction of the ratio (which did not reach significance), suggesting that less Aβ was produced per CTF molecule after the treatment with minocycline.