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. Author manuscript; available in PMC: 2009 Dec 26.
Published in final edited form as: Neuron. 2008 Dec 26;60(6):988–1009. doi: 10.1016/j.neuron.2008.10.047

Figure 5. Elevated BACE1 levels are correlated with increased eIF2α-P and amyloidogenesis in a chronic model of energy deprivation in vivo.

Figure 5

(A) Nine-month old Tg2576 mice were administered i.p. injections of saline (VEH), 1g/kg 2-deoxyglucose (2DG), or 80mg/kg 3-nitropropionic acid (3NP) once a week for 3 months (8-9 mice/group). One week after the final injection, hemibrains from each mouse were harvested and prepared for immunoblot and immunohistochemical analyses. Brain homogenates were prepared and 10μg of total protein per lane were used for immunoblot analysis of total BACE1 protein and β-actin. “+” lane: 5μg of BACE1-293 cell lysate as a BACE1 positive control.. “-“ lane: 10μg of BACE1-/- mouse brain homogenate as a BACE1 negative control. (B) 15μg of total protein per lane of brain homogenate from VEH, 2DG, 3NP treated Tg2576 mice were used for immunoblot analysis of eIF2α-P, eIF2α-T, and β-actin. “+” lane: 10μg of lysate from UV-treated 293 cells as a positive control for eIF2α-P. (C) Immunosignals in (A) and (B) were quantified by phosphorimager and expressed as percentage of vehicle (VEH). BACE1 levels and eIF2α-P/eIF2α-T ratios were significantly elevated in 2DG and 3NP treated Tg2576 mice compared to VEH (mean ± SEM; *, p<0.05; **, p<0.01; ***, p<0.001). (D) Guanidine-extracted brain homogenates from VEH, 2DG, and 3NP treated Tg2576 mice were analyzed using a human Aβ40 ELISA. Values were expressed as ng of Aβ40 per mg of total protein. A clear trend toward elevation of Aβ40 level was observed in 2DG and 3NP treated mice, although values did not reach statistical significance. Increases in Aβ40 levels tended to parallel the elevations of eIF2α-P and BACE1 for 2DG and 3NP treatments (compare C and D). (E) Fixed hemibrains of each mouse treated with VEH, 2DG, and 3NP were sectioned and stained with anti-Aβ antibody 4G8. The total number of 4G8-immunopositive plaques was counted in a set of eight evenly spaced parasagittal sections that spanned the entire medial-lateral dimension of each hemibrain. A clear trend toward an increase in plaque number was observed for both 2DG and 3NP treatments compared to VEH. Plaque numbers for the different treatments paralleled the relative levels of Aβ40, as expected (compare to 5D), as well as the levels of eIF2α-P and BACE1 (compare to 5C). (F) Representative parasagittal brain sections from VEH, 2DG, and 3NP treated Tg2576 mice were immunostained with 4G8 and micrographed at 4x. 4G8-positive plaques in the brains of 2DG and 3NP treated mice tended to be larger and more numerous than those in VEH treated mice.