Fig. 6. N548 mutant cells show occasional aggregates by immunofluorescence. N548 mutant cells were seeded on 22-mm² glass coverslips and incubated with DMSO or rotenone for 12 h. Cells were fixed with ice-cold acetone:methanol (50:50) for 5 min and were washed three times with PBS. The cells were incubated with anti-htt primary antibody (MAB2166), followed by goat anti-mouse Alexa 488-conjugated (Molecular Probes) antibody for another 1 h. Cells were briefly washed in PBS after each antibody and were incubated with DNA-binding dye Hoechst 33342 (1 mM) for 10 min to visualize nuclei (blue staining). Cells were viewed under a fluorescence microscope (excitation, 485 nm; emission, 535 nm), and images were acquired and processed by using ImageJ (National Institutes of Health, Bethesda, MD) to visualize htt (grayscale or green) and DNA (blue) and to generate composite figures. (A) Cells that were incubated with secondary antibody alone (negative controls) and untransfected parental cells served as controls for endogenous rat htt. Most N548 mutant cells (N548 mut) showed diffuse, largely cytosolic staining. Occasional cells (<1%) showed clear aggregates, with some cells showing solitary aggregates (arrow) (A) and others showing multiple aggregates (arrow) (B). Phase-contrast images of cells showing aggregates (B, bottom). (C) Parental and N548 mutant cells were incubated with DMSO, rotenone (10 mM), or BOC-D-Fmk (50 mM) for 6 h; cell lysates were prepared in radioimmunoprecipitation assay buffer with 2% SDS, and then the lysates were filtered through a 0.2-mm cellulose acetate membrane. The SDS insoluble aggregates on the filter were assayed by Western blotting for htt by using the MAB2166 antibody.

Fig. 7. A schematic of the glucose metabolic pathway shows the sites of action of the various inhibitors used in our study. Glucose is metabolized by glycolysis in the cytoplasm and generates two molecules each of ATP and pyruvate per molecule of glucose used. Pyruvate is further metabolized by the tricarboxylic acid cycle (TCA) in the mitochondrial matrix and generates reduced NADH and FADH₂. These reduced products transfer their electrons to the electron transport chain (ETC) located in the inner mitochondrial membrane. The ETC consists of 4 multimeric protein complexes (complexes I to IV) that transfer electrons down an electrochemical gradient to complex IV, where the electrons are transferred to O₂. Electron transfer is coupled to generation of an electrochemical gradient by pumping protons into the mitochondrial intermembrane space. This voltage gradient is used to generate ATP from ADP and inorganic phosphate at complex V. The end product is 36 ATP molecules per molecule of glucose metabolized by oxidative phosphorylation. The site of action of various mitochondrial and glycolytic inhibitors is indicated (red bars), and the inhibitors are shown in red text. 2,4-Dinitrophenol (DNP) uncouples the mitochondrial membrane potential by "leaking" protons back across the intermembrane space without generation of ATP (based in part on ref. 1).

1. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1994) Molecular Biology of the Cell (Garland, NY), 3rd Ed, Chap 14.

Fig. 8. Progressive degeneration in the Drosophila HD model and alleviation of degeneration by rotenone. (A) The average number of rhabdomeres per ommatidium was counted at 1 and 9 days posteclosion by using the pseudopupil technique. Error bars represent one SE. *, Significant differences in the number of rhabdomeres between 1-day- and 9-day-old animals based on Student's t test (P < 0.05). (B) A dose-dependent rescue of rhabdomere degeneration by rotenone. The average number of total rhabdomeres in vehicle (DMSO), rotenone, and sodium butyrate (NaB)-treated flies at day 9 is shown. Error bars represent one SE. *, Significant differences in the number of rhabdomeres between DMSO and drug-treated animals based on Student's t test (P < 0.05).

Fig. 9. Oligomycin (Oligo) decreases ATP levels in the absence of glycolysis. N548 mutant cells were grown in media without (-Glu,) or with (+Glu) glucose and treated with oligomycin or DMSO (-) for 8 h and assayed for ATP levels by a luminescence-based assay. Pyruvate (1 mM), the end product of glycolysis that is used by mitochondria for oxidative phosphorylation, was included in the media to enable mitochondrial ATP production to continue in the absence of glycolysis.

Fig. 10. NADH and reactive oxygen species (ROS) levels do not affect N548 mutant cell viability. Dose-response of NAD⁺ (A) and NADH (B) on N548 mutant cell viability in serum-deprived media is shown. Viability was assessed by trypan blue dye exclusion. One thousand cells were counted per sample, and the data shown are from one representative experiment of at least two independent experiments. (C) ROS levels were measured after incubating N548 mutant cells with different concentrations of mitochondrial inhibitors or H₂O₂ (positive control) for 2 h, H₂DCFDA dye (2′,7′-dichlorodihydrofluorescein diacetate; 10 mM) was loaded for 30 min, and fluorescence (excitation, 490 nm; emission, 530 nm) was measured after an additional 30 min on a plate reader. The data represent the average of six replicate samples; error bars represent one SD. The horizontal bar shows the level of ROS in two sets of DMSO controls (C). *, Significant differences (P < 0.0001), as determined by Student's t test.

Fig. 11. Inhibitors of glycolysis and ATP synthetase activate survival signaling. N548 mutant cells were treated with the glycolysis inhibitor NaF (A) or the ATP synthetase inhibitor oligomycin (oligo) (B), and the levels of activated ERK and/or AKT were assayed by Western blotting at the indicated time points.

Fig. 12. Rotenone rescues cell death and activates ERK/AKT in mitochondrial DNA-deficient cells. (A) Expression of mitochondrial encoded complex IV (Cox I) and nuclear-encoded complex II subunits (70-kDa subunit) of mitochondrial complexes were determined by Western blotting in two independent (#1 and #2) mitochondrial-deficient (r⁰) and parental (P) N548 mutant cells. Tubulin served as a loading control. (B) Two r⁰ cell lines were treated with rotenone (Rot) (10 mM) or DMSO for 6 h and analyzed for ERK/AKT activation by Western blotting. (C) In a parallel experiment, r⁰ cells were subjected to a viability assay after 2 days in serum-deprived medium with DMSO or 10 mM rotenone treatment. At least 1,000 cells were counted per sample; the experiment is representative of three independent experiments.

Fig. 13. Inhibitors of AKT and ERK activation abrogate the rescue by rotenone. N548 mutant cells were treated with rotenone (10 mM) in the presence of increasing concentrations of inhibitors of AKT activation [AKT inhibitor VIII or the upstream inhibitor of phosphoinositol-3 kinase LY294002 (Top)] or with inhibitors of the MAPK pathway that is upstream of ERK (Bottom). U0126 inhibits both ERK activators MEK1 and MEK2, whereas the MEK1 inhibitor only targets MEK1. Cell viability was determined after 2 days in serum-deprived media by using a fluorescence-based assay (calcein acetoxymethyl ester). The results are the averages ± SD of an experiment performed in triplicate. Rescue with rotenone alone (first point on the left) is represented by a red horizontal line and was used as the baseline to assess the decrease in viability caused by the inhibitors of AKT and ERK activation.