Fig. 3
ALDH2*2/*2 deficiency and daily treatment with ethanol for 11 weeks modulate brain metabolic state inducing upregulation of Alzheimer’s Disease-associated proteins and evidence of neuronal damage in mice. a) Protocol for alcohol dosing: WT and ALDH2*2/*2 mice were dosed daily with 1 g/kg/day ethanol for 11 weeks (weeks 9–20). Alzet pump releasing vehicle or Alda-1 (10 mg/kg/day) was implanted 1 week prior to onset and throughout ethanol treatment. b) ATP levels (using ATP colorimetric kit) were determined in freshly isolated brain mitochondria; Veh – Vehicle; A – Alda-1. c) Rates of hydrogen peroxide production were determined in state-4 respiration by Amplex-Red hydrogen peroxide assay using freshly isolated brain mitochondria; Veh – Vehicle; A – Alda-1. d) 4-HNE levels were measured in total brain homogenate using ELISA; Veh – Vehicle; A – Alda-1. e) Malondialdehyde content was determined using MDA lipid peroxidation assay in total brain homogenate; Veh – Vehicle; A – Alda-1. f) Aβ42 levels were determined using ELISA in brain mitochondrial fraction; Veh – Vehicle; A – Alda-1. g) Levels of phosphorylated tau, synaptophysin and cleaved caspase 3 were determined in total lysates by immunoblotting. β-actin was used as loading control; Veh – Vehicle; A – Alda-1. h) Levels of phosphorylated tau (Ser 416) were determined in total brain lysates by immunoblotting. β-actin was used as loading control. Protein levels were quantified, and data represented as fold change of WT Veh; Veh – Vehicle; A – Alda-1. i) Levels of synaptophysin were determined in total brain lysates by immunoblotting, as in h; Veh – Vehicle; A – Alda-1. j) Levels of cleaved caspase-3 were determined in total brain lysates by immunoblotting, as in h; Veh – Vehicle; A – Alda-1. k) Caspase-3 activity was determined in total brain homogenate using fluorometric assay based on the cleavage of substrate DEVD-AFC; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented fold of control for b, c, h-k and as absolute values for c, e, f. n = 6 per each group; probability by one-way ANOVA (with Holm-Sidak post hoc test)