Panel A: Aβ oligomers triggers the release of m-HKI from mitochondria. Western immunoblot analysis of HKI levels in mitochondrial fractions from cultured cortical neurons exposed to 500 nM Aβ oligomers (ADDLs) (D) for 24 hours. Cytochrome c oxidase was used as a loading control. Bars correspond to means ± SE from at least three independent experiments carried out in duplicate. Panel B: Time-dependent toxicity of ADDLs (white bars) or Aβ monomers (gray bars) to mature hippocampal neurons measured using the MTT reduction assay. Values represent the means ± SD of the relative absorbance measured using 6 wells for each experimental condition for three independent experiments. (*) indicates a statistically significant (p<0.005) difference relative to control cultures. Panel C: Aβ oligomers decrease ATP levels in mature cortical neurons. Cultures were exposed to vehicle or 500 nM ADDLs for 12 hours. Values represent means ± SD of the relative levels measured in five independent experiments.

Neuronal cortical cultures were maintained in the absence or in the presence of Aβ for 48 hours and cell viability was determined using the Live/Dead assay as described in “Materials and Methods”. Live cells are identified by green calcein fluorescence and dead cells are identified by red ethidium bromide fluorescence. Representative images from control (vehicle-treated) cultures (Panel A), cultures exposed to 20 µM Aβ (Panel B) or cultures treated with 20 µM Aβ+30 mM 2-DOG (Panel C). Panel D: Quantitative analysis of neuronal viability was carried out using Image J (described in “Methods”). A total of 3,000–4,000 cells were analyzed in each experimental condition. Bars show means ± SD from four different experiments (carried out in triplicate). (*) and (**) indicates a statistically significant (p<0.005 or p<0.01, respectively) difference relative to control cultures. Scale bar corresponds to 100 µm. Panel E: Effects of 2-DOG on neuronal HKI levels. Western immunoblot analysis of total HKI levels from cultured cortical neurons exposed to 30 mM 2-DOG for 48 hours. Cyclophilin B was used as a loading control. Bars correspond to means ± SD from four independent experiments.

Panel A: Western immunoblot analysis of HKI levels in mitochondrial fractions from cultured cortical neurons exposed to 5 µM Aβ for 24 hours. Cytochrome c oxidase was used as a loading control. Bars correspond to means ± SE from at least three independent experiments carried out in duplicate. Panel B: HK activity as measured in mitochondrial enriched fractions from cultured cortical neurons exposed to 5 µM Aβ for 24 hours. Bars correspond to means ± S.D. from three independent experiments carried out in duplicate. Panel C: HK1 activity as measured in cortical neurons exposed to 5 µM Aβ for 24 hours. Bars represent the means ± SD from three independent experiments carried out in duplicate. (*) indicates a statistically significant (p<0.005) difference relative to control (vehicle-treated) cultures. Panel D: HK activity in mitochondria isolated from adult rat brains was measured in the absence or in the presence of Aβ (5 µM or 20 µM; 1 hour). Values represent means ± SD of the activity measured in two independent experiments carried out in triplicate.

Representative DCF fluorescence images from control (vehicle-treated) cultures (Panel A), cultures exposed to 5 µM Aβ for 6 hours (Panel B) or cultures exposed to 5 µM Aβ+30 mM 2-DOG for 6 hours (Panel C). When present, 2-DOG was added 10 minutes before Aβ. In order to allow direct comparison between ROS levels, identical conditions and exposure times for image acquisition were employed for all experimental conditions. Panel D: Quantitative analysis of DCF fluorescence from five independent experiments (∼ 1,500 cells analyzed per experimental condition in each experiment) using ImageJ software (NIH Windows version). (*) indicates a statistically significant (p<0.005) difference relative to control cultures. (**) and (***) indicates a statistically significant (p<0.005) difference relative to Aβ-treated cultures. Scale bar corresponds to 100 µm. Panel E: 2-DOG decreases neuronal G6P levels in neurons exposed to Aβ. Glucose-6P levels were measured in control cortical neurons or neurons exposed to 5 µM Aβ+30 mM 2-DOG or 30 mM 2-DOG. Values represent means ± SD from three experiments (carried out in duplicate) using independent neuronal cultures. (*) indicates a statistically significant (p<0.001) difference relative to control cultures.