Chemicals and reagents
R,S-Sulforaphane was purchased from LKT Laboratories, Inc (St. Paul, MN). Mannitol, sucrose, EGTA, HEPES, Tris, potassium phosphate dibasic (K2HPO4), magnesium chloride (MgCl2), malate, glutamate, oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), adenosine 5′-diphosphate (ADP), bovine serum albumin (BSA), percoll, succinate, rotenone, calcium, dimethylsulfoxide (DMSO) and tert-butyl hydrogen peroxide were obtained from Sigma–Aldrich (St. Louis, MO, USA). Ultra-pure potassium chloride was obtained from EM Sciences (Fort Washington, PA, USA).
Treatment of rats with sulforaphane
All experimental procedures were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals. Male 300 g Fischer 344 rats (Charles River) were injected intraperitoneally with sulforaphane at 10 mg/kg in 200 μL solution containing 40% DMSO and 60% isotonic saline. Animals received DMSO plus saline as the vehicle control.
Isolation of non-synaptic brain mitochondria
Rats were euthanized by decapitation and their forebrains rapidly removed, chopped and homogenized in ice-cold isolation buffer (225 mM mannitol, 25 mM sucrose, 10 mM Hepes, 1 mM EGTA, pH 7.4, at 4°C). The homogenate was centrifuged at 1,330g for 3 min. The pellets were re-suspended and re-centrifuged at 1,330 g for 3 min. The combined supernatants were centrifuged at 21,200g for 10 min. The pellets were re-suspended in 15% Percoll and layered on a 40%/23% Percoll gradient. The tubes containing the Percoll gradients were then centrifuged at 31,700g for 10 min. The mitochondrial fraction located at the interface between the 40% and 23% layers was removed, diluted with isolation buffer and centrifuged at 16,700g for 10 min. The purified mitochondrial pellet was re-suspended in isolation buffer and de-fatted bovine serum albumin was added to the suspension at 10 mg/mL. After a final centrifugation at 9,000 g for 10 min, the mitochondria were re-suspended in approximately 20 μL of isolation medium not containing EGTA or BSA. Mitochondrial protein concentrations were measured using a Lowry DC kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin used as concentration standards.
Mitochondrial oxygen consumption
The respiratory activities of isolated brain mitochondria were measured polarimetrically with a Clark-type oxygen electrode apparatus (Hansatech Instruments, Norfolk, England). Mitochondria were suspended at a protein concentration of 0.5 mg/ml in buffer containing 125 mM KCl, 20 mM HEPES, 2 mM K2HPO4, 0.01 mM EGTA, 1 mM MgCl2 (pH 7.0) at 37 °C, plus glutamate (5 mM) and malate (0.1 mM) or succinate (5 mM) plus the electron transport chain complex 1 inhibitor, rotenone (4 μM). Addition of ADP (0.5 mM) was used to initiate State 3 (phosphorylating) respiration. Oligomycin (2.5 μg/ml), an inhibitor of the mitochondrial ATP synthase, was used to induce State 4 (resting) respiration. The use of oligomycin to obtain the rate of respiration limited by the inner membrane proton permeability is necessary when magnesium is present as even small contamination of isolated mitochondria with Mg-dependent ATPases, e.g., the plasmalemmal sodium pump, can result in turnover of mitochondrially-generated ATP, resulting in an artificially high rate of State 4 respiration in the absence of oligomycin. Maximal respiration was initiated with the addition of the protonophore uncoupler, FCCP (54 nM). Rates of oxygen consumption are expressed as nmol O2/mg mitochondrial protein/min). The respiratory control ratio (RCR) is defined as the rate of ADP-stimulated oxygen consumption (State 3) divided by the rate of respiration determined in the presence of oligomycin (State 4).
Measurements of mitochondrial Ca2+-uptake and release and pyridine nucleotide redox state
Mitochondria were suspended in a cuvette at a protein concentration of 0.5 mg/ml in 2 ml of 125 mM KCl, 2 mM K2HPO4, 1 mM MgCl2, 20 mM Tris-HCl, 5 mM succinate, and 4 μM rotenone, pH 7.4 at 30°C. The use of the electron transport chain complex II substrate succinate in the presence of the complex I inhibitor rotenone allows for the mitochondrial pyridine nucleotide redox state (NAD(P) H/NAD(P)+) to be varied between totally reduced (no added pro-oxidant) to oxidized (after addition of pro-oxidants), independent of mitochondrial respiration and membrane potential. The medium free Ca2+ was measured fluorimetrically in the presence of the Ca2+-sensitive fluorescent dye Calcium Green 5N (0.1 μM), using excitation and emission wavelengths of 506 nm and 532 nm. Autofluorescence of reduced mitochondrial pyridine nucleotides (NADH plus NADPH) was measured simultaneously using excitation and emission wavelengths of 350 nm and 460 nm. Fluorescence measurements were performed on a Hitachi 2500 spectrofluorimeter, equipped with magnetic stirring and cuvette temperature control. Fluorescent wavelength settings cycled every 2 s for medium Ca2+ and NAD(P)H measurements.
Mitochondria were added to the cuvette and allowed to equilibrate, bioenergetically, for 2 min, as reflected by steady-state Calcium Green 5N and NAD(P)H fluorescence. CaCl2 was then added to the medium at a level that the mitochondria could completely accumulate with only a very slow rate of subsequent spontaneous release. At 200 s after the addition of Ca2+, either tert-butyl hydroperoxide or phenylarsine oxide were added as pro-oxidant inducers of PTP opening, as reflected by the release of accumulated Ca2+. In some experiments, the PTP inhibitor, cyclosporin A (1 μM), was present in the medium in which mitochondria were suspended. PTP opening was quantified by measuring the rate of increase in Calcium Green 5N fluorescence after the addition of pro-oxidants. The effect of these pro-oxidants on the pyridine nucleotide redox state was quantified by measuring the extent to which NAD(P)H autofluorescence was lost after their addition.
Immunoblot analysis of cyclophilin D
Isolated mitochondria were lysed in RIPA buffer containing a cocktail of protease and phosphatase inhibitors (Calbiochem). Equal amounts of mitochondrial protein (5 μg) from each sample were separated by SDS-PAGE (4–12% Bis-Tris gels) (Invitrogen) and transferred to PVDF membranes (Invitrogen), and then incubated with primary antibodies (overnight at 4 °C) with the mouse monoclonal antibodies anti-cyclophilin D antibody 1:500,000 (Mitosciences, Cat. #MSA04) anti-VDAC antibody 1:500,000 (Mitosciences, Cat. #MSA03). The membranes were then washed with PBST and incubated for 1 h at room T in HRP-conjugated anti-mouse antibodies (Millipore Cat. # 12–349) at 1:2000 dilution for 1 h at room T. The washed blots were then treated with enhanced chemiluminiscence detection reagent (Amersham Bioscience, UK). Densitometric analysis of the protein bands was performed using the Image J software.
Data are expressed as means ± S.E.M. of n different experiments. Differences between data obtained from mitochondria obtained from rats treated with sulforaphane or drug vehicle were assessed by students t-test. For data that are not normally distributed, the Mann-Whitney rank sum test was used. P<0.05 was considered to be statistically significant.