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NEUROPHARMACOLOGY

Targeted Antioxidative and Neuroprotective Properties of the Dopamine Agonist Pramipexole and Its Nondopaminergic Enantiomer SND919CL2x [(+)2-Amino-4,5,6,7-tetrahydro-6-Lpropylamino-benzathiazole Dihydrochloride]

Department of Neurology, University of Ulm, Ulm, Germany (R.D., B.S., A.C.L.); Department of Central Nervous System Research, Boehringer-Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany (F.G., E.B., B.H., C.D.-C., L.K.); Department of Medical Data Services, Boehringer-Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany (V.K.); Department of Drug Discovery Support, Boehringer-Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany (K.K.); and Institute of Clinical Chemistry and Pathological Biochemistry, Department of Pathological Biochemistry, Otto-von-Guericke-University, Magdeburg, Germany (L.S.)

Received July 8, 2005; accepted September 26, 2005.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Pramipexole has been shown to possess neuroprotective properties in vitro that are partly independent of its dopaminergic agonism. The site of neuroprotective action is still unknown. Using [³H]pramipexole, we show that the drug enters and accumulates in cells and mitochondria. Detoxification of reactive oxygen species (ROS) by pramipexole is shown in vitro and in vivo by evaluating mitochondrial ROS release and aconitase-2 activity, respectively. Pramipexole and its (+)-enantiomer SND919CL2X [low-affinity dopamine agonist; (+)2-amino-4,5,6,7-tetrahydro-6-L-propylamino-benzathiazole dihydrochloride] possess equipotent efficacy toward hydrogen peroxide and nitric oxide generated in vitro and inhibit cell death in glutathione-depleted neuroblastoma cells. IC₅₀ values ranged from 15 to 1000 µM, consistent with the reactivity of the respective radical and the compartmentalization of ROS generation and ROS detoxification. Finally, both compounds were tested in superoxide dismutase 1-G93A mice, a model of familial amyotrophic lateral sclerosis. SND919CL2X (100 mg/kg) prolongs survival time and preserves motor function in contrast to pramipexole (3 mg/kg), which shows an increase in running wheel activity before disease onset, presumably caused by the dopaminergic agonism. We conclude that both enantiomers, in addition to their dopaminergic activity, are able to confer neuroprotective effects by their ability to accumulate in brain, cells, and mitochondria where they detoxify ROS. However, a clinical use of pramipexole as a mitochondria-targeted antioxidant is unlikely, because the high doses needed for antioxidative action in vitro are not accessible in vivo due to dopaminergic side effects. In contrast, SND919CL2X may represent the prototype of a mitochondria-targeted neuroprotectant because it has the same antioxidative properties without causing adverse effects.

At higher concentrations (above 10 µM), PPX has been shown to be neuroprotective in vitro independent of the dopaminergic agonism (Le et al., 2000; Abramova et al., 2002; Gu et al., 2004 and references therein). In addition, SND, the (+)-enantiomer of PPX, has been shown to be neuroprotective as well (Abramova et al., 2002; Gu et al., 2004), although its affinity to dopamine receptors is approximately 100-fold less compared with PPX (Mierau, 1995).

Site and mechanism of the receptor-independent action have so far not been shown directly. Several studies described an antioxidative action of the drug and/or a preservation of mitochondrial function, resulting in inhibition of cell death (Cassarino et al., 1998; Kitamura et al., 1998; Kakimura et al., 2001; Abramova et al., 2002; Gu et al., 2004). Because both enantiomers are lipophilic cations, it has been hypothesized (Abramova et al., 2002) that they might accumulate in mitochondria, as predicted for other lipophilic cations (Trapp and Horobin, 2005). However, the concept that PPX and/or SND act intracellularly as mitochondrial-targeted antioxidants has not yet been proven.

Mitochondria-targeted antioxidants accumulate in mitochondria and show higher efficacy compared with untargeted antioxidants (Ross et al., 2005). They represent promising candidates to prevent or alleviate mitochondrial oxidative stress, which is involved in the pathogenesis of Alzheimer's diseases, Parkinson's disease, or amyotrophic lateral sclerosis (ALS) (Andersen, 2004). Because of the lack of an active uptake process, we propose that SND is able to enter neural cells and exert the same properties as PPX. Support for this proposal is given by its equal antioxidative efficacy toward hydrogen peroxide and nitric oxide when compared with PPX and by equipotent efficacy of SND and PPX to prevent cell death in glutathione-depleted neuroblastoma cells (Maher and Davis, 1996).

This study addresses those neuroprotective properties of PPX and SND, which are not mediated by stimulation of dopamine receptors. Initially, an intracellular site of action is demonstrated by evaluating the uptake properties of ³H-labeled PPX. We show for the first time that PPX enters neural cells and accumulates in mitochondria driven by mitochondrial membrane potential in a diffusion-limited process. In addition, PPX detoxifies ROS within the mitochondria as shown by inhibition of mitochondrial hydrogen peroxide (H₂O₂) release and by an increase in mitochondrial aconitase activity.

ALS is a devastating disease, progressing from mild motor symptoms to severe paralysis and premature death caused by the degeneration of motor neurons. Twenty percent of familial ALS cases are caused by mutations in superoxide dismutase 1 (SOD1), which expressed in mice result in a phenotype resembling the pathology in patients. Transgenic SOD1 mice represent the predominant model to study ALS pathogenesis and therapy. Neuronal cell death in ALS is at least in part associated with an increase in oxidative stress and mitochondrial alterations (Menzies et al., 2002). Mitochondrial dysfunction seems a likely candidate to explain many facets of ALS, because it is the earliest reported pathologic event in ALS mice (Bendotti et al., 2001). Mitochondrial pathology may contribute to generate a condition of oxidative stress, and indeed markers of oxidative damage (increased ROS flux, oxidatively modified proteins) have been found in cultured neuronal cells, in transgenic mice, and in patients as well (reviewed in Bendotti and Carri, 2004). Furthermore, PPX reduces oxidative stress in ALS patients (Pattee et al., 2003), pointing to the necessity to evaluate PPX and SND for their efficacy in an animal model for ALS.

To test for neuroprotection by PPX/SND in vivo, transgenic SOD1 mice were treated with both compounds. Treatment with PPX resulted in preonset motor hyperactivity; SND was able to prolong survival and improve motor performance without causing hyperactivity. This is the first report showing that PPX and SND are able to act as brain- and mitochondrial-targeted antioxidants, which most likely explains the receptor-independent neuroprotective effects observed in vitro and in vivo.

	Materials and Methods

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Chemicals
All cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA). Pramipexole, SND919CL2x [(+)2-amino-4,5,6,7-tetrahydro-6-L-propylamino-benzathiazole dihydrochloride], and EUK-134 [manganese 3-methoxy-N,N'-bis(salicylidene)ethylenediamine chloride] were provided by Boehringer Ingelheim GmbH (Ingelheim, Germany). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) in the highest purity available, unless stated otherwise.

Cell Culture
Astroglial Cells. Brains of rat pups (1–3 days) were removed and dissociated with 1% trypsin/0.05% DNase in Hanks' balanced salt solution (HBSS) for 30 min at 37°C. After addition of fetal calf serum-containing culture medium, the cells were dissociated by triturating using a Pasteur pipette and centrifuged for 5 min at 300g. Afterward, the cells were resuspended in culture medium [Dulbecco's modified Eagle's medium; 10% (v/v) heat-inactivated fetal calf serum; 6 mM L-glutamine; 100 U/ml penicillin; 100 µg/ml streptomycin; and 2.5 mg/l amphotericin[and 2 x 10⁵ living cells/well were seeded in 24-well plates. The cells were incubated at 37°C, 5% CO₂ for 14 to 21 days before performing experiments. Culture medium was changed twice a week.

Cerebellar Granule Cells. These cells were isolated from 7-day-old rat pups and were purified by density sedimentation. In brief, cerebella were removed and dissociated with 1% trypsin/0.05% DNase in HBSS for 13 min at room temperature. After two washing steps with HBSS to dilute trypsin, 0.5% DNase in HBSS was added and the cerebella were dissociated by triturating using a Pasteur pipette. The cells were subsequently centrifuged for 15 min at 1300g through a 40% (v/v) Percoll solution to separate them from larger cell types. The cells were harvested from the pellet and washed in ice-cold Basal modified Eagle's medium supplemented with 2 mM glutamine followed by centrifugation for 5 min at 150g. The cells were plated on poly-L-lysine-coated 24-well plates in basal modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 25 mM KCl, 100 U/ml penicillin, and 100 µg/ml streptomycin. After 24 h in culture, the cells were treated with cytosine arabinoside in a final concentration of 5 µM. Cultures were maintained at 37°C, 5% CO₂ for 7 to 8 days without a change in medium before performing the experiments.

HT-22 Cells. Hippocampal neuroblastoma cells derived from mice (HT-22 cells) were cultured routinely in Dulbecco's modified Eagle's medium (10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin), 5% CO₂, and 100% humidity. 2 x 10⁴ cells were seeded into a 96-well plate and left for 16 h to grow to 80% confluence. This was followed by incubation in culture medium with 5 mM glutamate, with or without the additional presence of PPX, SND (both 0.008–1 mM), or EUK-134 (2–250 µM) for 24 h. Subsequently, cell survival was assessed by the capacity of cells to reduce Alamar Blue (Serotec, 10% in culture medium, incubation for 1 h) to resorufin using a Fluoroskan Ascent fluorescence plate reader [Thermo Electron Corporation (Waltham, MA)]. The wavelengths for excitation and emission were set at 530 and 590 nm, respectively. The obtained fluorescence values were normalized to those obtained from untreated cells (% control). The cells were a kind gift from P. Maher (The Scripps Research Institute, La Jolla, CA).

Preparation of Brain Mitochondria. Four to twelve male C57BL/6 mice (20–25 g in weight; 5–6 months) were used for the preparation of forebrain mitochondria according to a modified method of Sims (1990). The mice were fasted overnight and sacrificed by decapitation. All steps were performed at 4°C. After rapid isolation of the brain and dissection of the cerebella, the remaining forebrains were washed in isolation buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 5 mM HEPES, pH 7.4) to remove blood. The forebrains were sliced and homogenized in 10% (w/v) isolation buffer with a Teflon in-glass potter (eight strokes, 750 rpm). The homogenate was centrifuged for 4 min at 1400g. The supernatant was transferred to another tube, and the pellet was homogenized and centrifuged as above. The residual pellet was discarded, and the pooled supernatants were centrifuged for 5 min at 30,700g. The pellet was resuspended in 3 ml of 15% (v/v) Percoll in HES buffer (5 mM HEPES, 1 mM EDTA, and 0.32 M sucrose, pH 7.4) and layered on the top of a discontinuous 25 to 40% (v/v) Percoll/HES gradient (3.5 ml each). The Percoll gradient was centrifuged for 5 min at 30,700g, and the interphase (between 25 and 40% Percoll) diluted 1:4 with EDTA-free isolation buffer and centrifuged for 10 min at 16,700g. The supernatant was discarded, and the pellet was washed once more in EDTA-free isolation buffer and centrifuged for 10 min at 7300g. The resulting pellet was resuspended in 20 to 30 µl/mice forebrain in EDTA-free isolation buffer (20 mg of protein/ml). All subsequent experiments were performed within 4 h after isolation where the mitochondria were strictly coupled and possessed a respiratory control ratio of 6 if malate and pyruvate were used as substrates. Contamination of cytosol by determination of lactate dehydrogenase in the homogenate and the mitochondria was 8%.

Uptake Experiments (Cells). All steps were carried out using 37°C prewarmed solutions, unless stated otherwise. For analyzing the uptake of [³H]PPX (69 mCi/mmol, GE Healthcare, Little Chalfont, Buckinghamshire, UK), the astroglial cells were washed twice with 500 µl of incubation buffer (20 mM HEPES, 145 mM NaCl, 1.8 mM CaCl₂, 5.4 mM KCl, 1 mM MgCl₂, 0.8 mM Na₂HPO₄, and 5 mM glucose, pH 7.4). Afterward, the cells were preincubated for 10 min in 200 µl of incubation buffer containing glucose (10 mM) and/or inhibitors. Uptake of [³H]PPX was started by the addition of 200 µl of [³H]PPX (6 µCi/ml) in incubation buffer with or without unlabeled PPX (0–30 mM). The incubation was terminated by exhausting the incubation buffer following a washing step with 600 µl of ice-cold incubation buffer. The cells were lysed with 300 µl of NaOH (1 M) for 1 h at 22°C. The alkaline lysate was neutralized with 2 M HCl, and radioactivity was determined by liquid scintillation counting in a LS-3801 Beckman Coulter counter (Fullerton, CA). The cerebellar granule cells were incubated with the same protocol, with the exception of the incubation buffer (20 mM HEPES, 5.6 mM NaCl, 1.8 mM CaCl₂, 145 mM KCl, 1 mM MgCl₂, 0.8 mM Na₂HPO₄, and 5 mM Glc, pH 7.4). The uptake of [³H]PPX was calculated by subtracting the obtained amount of radioactivity in the absence of cells from the amount in the presence of cells. The concentration of [³H]PPX added to the incubation medium was 43.5 nM, calculated by taking the radioactive concentration as well as the specific radioactivity of the used [³H]PPX stock into account (1 mCi/ml, 69 Ci/mmol). The obtained values were normalized to the protein content and expressed in disintegrations per minute/milligram. The data represented correspond to the mean ± S.D. of three independent experiments.

Uptake Experiments (Mitochondria). All steps were carried out at 22°C, unless stated otherwise. For measuring the uptake of [³H]PPX, 50 µl of mitochondria (0–1.6 mg/ml) were preincubated for 2.5 min in incubation buffer (100 mM KCl, 100 mM sucrose, 10 mM Tris, and 1 mM MgCl₂, pH 7.4) containing energy substrates and/or mitochondrial inhibitors. Uptake of [³H]PPX was started by the addition of 200 µl of [³H]PPX (6 µCi/ml) in incubation buffer with or without unlabeled PPX (0–30 mM). The incubation was terminated after 2 min, unless stated otherwise, by exhausting the incubation buffer with a cell harvester over a filter mate following a washing step with 200 µl of ice-cold incubation buffer. Afterward, the filter was dried and fused with a MeltiLex sheet (PerkinElmer Life and Analytical Sciences, Boston, MA) in the microwave. Radioactivity was determined by scintillation counting in a 1450 MicrobetaPlus liquid scintillation counter (PerkinElmer Wallac, Gaithersburg, MD). The specific uptake of [³H]PPX was determined as the amount of radioactivity in the presence of mitochondria from which the amount of radioactivity in vigorously sonicated mitochondria was subtracted. The data represent the mean ± S.D. of three independent experiments.

Hydrogen Peroxide. Hydrogen peroxide was measured using the Amplex Red (AR)-horseradish peroxidase (HRP) method (Invitrogen). HRP catalyzes the H₂O₂-dependent oxidation of nonfluorescent AR to fluorescent resorufin (Zhou et al., 1997). Because HRP does not cross membranes, the assay only detects H₂O₂, which has been released from the mitochondria. The reaction was started by the addition of mitochondria (0.1 mg/ml) to a mixture containing Amplex Red (10 µM), HRP (0.4 U/ml), and succinate (2.5 mM; control) in incubation buffer (see "Uptake Experiments"). In further conditions, PPX (300 µM), malonate (Malo, 10 mM), the SOD-catalase mimic EUK-134 (Melov et al., 2001), EUK (30 µM), or ATP (1 mM) was added. Fluorescence intensity was measured in the kinetic mode using the fluorescence plate reader as described above. The rate of resorufin generation was measured each 5 s for a total time of 5 min and normalized to the control incubation after linear regression of the obtained fluorescence intensities. The data represent the mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the control incubation.

In a further set of experiments, hydrogen peroxide (80%) and, to a smaller extent, superoxide (20%) were generated by xanthine oxidase (0.3 mU/ml) and hypoxanthine (0.1 mM) (Fridovich, 1970) in a solution containing PPX, SND, or EUK-134 at the indicated concentrations. After 20 min, ROS generation was stopped by the addition of allopurinol (1 mM) and the nondetoxified H₂O₂ was detected by the addition of AR and HRP (10 µM, 0.4 U/ml). Fluorescence of generated resorufin was determined in the endpoint mode. The obtained values were normalized to the amount of resorufin generated in the absence of detoxifying molecule. The specificity for hydrogen peroxide was verified with SOD (300 mU/ml), which did not affect the oxidation of Amplex Red. The data represent the mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the incubation in absence of antioxidants.

Nitric oxide was generated in vitro by the addition of 100 µl of the nitric oxide donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA; 0.1 and 0.3 mM) to 100 µl of a solution containing 4,5-diaminofluorescein (DAF; 5 µM) and PPX or SND. The generation of DAF-triazole fluorescence was measured at room temperature every 30 s during 30 min. The obtained slopes were plotted against the concentration of PPX. The data represent mean ± S.D. of a representative experiment, each done in triplicate.

Animals. All of the required procedures were approved by the local ethics committee for animal experimentation. The animals were handled according to the German laws for animal experimentation. The mice were housed in groups of four (C57BL/6 mice purchased from Janvier, Le Genest Saint Isle, France) or individually (SOD1-G93A mice) under standardized conditions (temperature 21°C, relative humidity 55%, 12-h light/12-h dark cycle, lights on at 7:00 AM) and maintained under ad libitum food and water throughout the experiments. Male Tg(SOD1-G93A)1Gur/J and female B6SJLF1/J mice were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Mice were cross-bred, and first generation offspring were screened for the presence of the transgene (tail biopsy) according to Jackson ImmunoResearch Laboratories (http://jaxmice.jax.org/).

Treatment of Animals
Schedule for Plasma and Brain Levels of SND and PPX. Male C57BL/6 mice (weighing 25–30 g) were treated twice daily (7:00 AM and 7:00 PM) with PPX or SND (four in each group; 200 mg/kg) via the p.o. route for 4 consecutive days. On day 5, the animals were sacrificed 12 h after the last treatment. Blood was collected, anticoagulated with EDTA, centrifuged to obtain plasma, and stored at –20°C. Brains were rapidly removed, blotted with paper to remove excess surface blood, and stored at –20°C.

Schedule for Aconitase Activity. Male C57BL/6 mice (weighing 25–30 g) received two intraperitoneal injections (9:00 AM and 5:00 PM) of PPX (30 mg/kg, n = 8) or vehicle (water, n = 6) for four consecutive days. On day 4, a further injection was given (9:00 AM) and, 30 min later, the animals were sacrificed and mitochondria were isolated as described above.

Schedule of SOD1-G93A Mice. Animals were treated by supplementing the diet with PPX or SND. This minimizes stress caused by a chronic treatment (e.g., i.p. injection) and, because of the continuous intake, lowers peak dose effects. Only transgenic animals (SOD1-G93A; 1Gur/JD) were included in the study. Group I (six males and five females) received control diet (altromin Granulat 8/15; Altromin GmbH, Lage, Germany) and water, group II (7 males, 8 females) received control diet and PPX in the drinking water (3 mg/kg/day), and group III (six males and nine females) received SND in the diet (100 mg/kg/day) and water. Consumption of drinking water and pellets was tested in regular intervals of 7 days. Drug administration started at day 45 and was continued until removal of the animal from the study. To assess the effects of the drug treatment, the lifetime of each animal was recorded (date of birth until removal from the study or death of the animal) and compared by Kaplan-Meier survival analysis followed by log rank Mantel-Cox test.

Motor Activity of SOD1-G93A Mice The motor activity of SOD1-G93A mice was assessed by monitoring the running wheel activity of each mouse. It correlates directly with the revolutions per minute generated by each animal in the running wheel. At 38 days of age, the animals were placed into cages equipped with a running wheel. Running wheel activity of each mouse was recorded for 12 h from 8:00 PM to 8:00 AM each day. Analysis of the data was accomplished using program Maus Vital supplied by Laserund Medizin-Technologie Berlin (LMTB, Berlin, Germany). Animals were removed from the study if they reached disease end-stage characterized by running wheel activity <10 rpm/12 h or disability to rise within 30 s.

For the evaluation of activity, performance values were averaged within the treatment groups for each day of age. The resulting time-performance values for each treatment group were fitted by the three parametric sigmoidal curves (f(x) = ax^c/(x^c + b^c), in which "a" represents the upper asymptote and therefore presymptomatic performance, "b" represents the day of half-maximal performance, and "c" represents the disease progression as the decline of running wheel activity per day. The fit parameters and 95% confidence intervals of the fit parameters were calculated by means of nonlinear regression. The treatment groups were compared by these fit parameters. A statistically significant difference between two treatment groups according to a fit parameter was achieved if the 95% confidence intervals of the fit parameters did not overlap. The statistical evaluation was performed using the software package SAS, version 8.2 (SAS Institute, Cary, NC).

Postmortem Analysis
Plasma and Brain Levels of PPX or SND. For quantification of PPX or SND in brain, the tissue was homogenized with 0.05 M phosphoric acid. An aliquot of the homogenate was spiked with standard solutions or water and internal standard and then extracted two times with tert-butyl-methylether at pH 10. Plasma samples were diluted with standard solutions or water and an internal standard and then extracted two times with tert-butyl-methylether at pH 10. The extracts were evaporated under dry nitrogen at 40°C and reconstituted with a mixture of methanol/water/formic acid (27/75/0.1). All extracts were diluted prior to analysis to fit into the calibrated concentration range. This solution was injected into the liquid chromatography/MS/MS system consisting of an Agilent 1100 high pressure liquid chromatography system (Agilent Technologies, Palo Alto, CA) and a API4000 mass spectrometer (Applied Biosystems, Foster City, CA). Chromatography took place on a Kromasil RP18 column (2.1 mm i.d. x 30 mm, 5-µm particle size) with a gradient from 0.1% formic acid in 10 mM ammonium acetate to 0.1% formic acid in acetonitrile/water [90/10 (v/v)] in 4 min. Samples and internal standard were detected by electrospray ionization-MS/MS in multiple reaction-monitoring mode. The limit of quantification was 2 nM. For both analytes, a linear calibration range from 2 to 4000 nM was observed. The shown results are the mean ± S.E.M. of four animals.

Mitochondrial Aconitase. Mitochondrial aconitase is reversibly inactivated by superoxide and has therefore been used as a marker for steady-state concentration of superoxide in vitro and in vivo (reviewed by Tarpey et al., 2004). Mitochondrial aconitase activity was measured by the method of Gardner et al. (1994), which was slightly modified. Mitochondria (10 mg/ml) were diluted in lysis buffer containing 10 mM Tris/HCl, 0.6 mM MnCl₂, 20 µM D,L-fluorocitrate, and 1% (v/v) Triton X-100, pH 7.4, and incubated for 30 min on ice. Twenty microliters of lysate was added to a well of a 96-well microtiter plate followed by the addition of a prewarmed solution (37°C) containing 0.4 mM NADP, 1.2 mM MnCl₂, and 2 U/ml isocitrate dehydrogenase in buffer [10 mM Tris/HCl, 0.6 mM MnCl₂, 20 µM D,L-fluorocitrate, and 1% (v/v) Triton X-100]. After a 5-min preincubation, the generation of NADPH was started by the addition of 10 mM citrate in buffer. The increase in absorbance was followed each 15 s for a period of 10 min in an iEMS plate reader (Thermo Electron Corporation). Aconitase activity was calculated by using an absorbance coefficient of 6.22 mM^–1x cm^–1 for NADPH and a thickness of 0.5 cm for 200 µl in the well, which was normalized to the protein content afterward.

Additionally, a monoclonal antibody raised against the 23 C-terminal amino acids of aconitase-2 (mitochondrial aconitase) from rat (nanoTools; Antikörpertechnik, Teningen, Germany) was used to study the expression of the protein in mitochondria. This antibody (Clone 29 A4) recognizes a single band with an apparent mass of 82 kDa in human A431 cells as well as mouse and rat forebrain mitochondria. For Western blotting, equal amounts of mitochondrial lysates (5 µg) were reduced with dithiothreitol and subjected to SDS/polyacrylamide gel electrophoresis on a 10% polyacrylamide gel. Afterward, aconitase-2 expression was analyzed by a standard Western blot protocol by probing the used nitrocellulose membranes with the anti-aconitase-2 antibody (2 µg/ml) using an enhanced chemiluminescence detection system (Applied Biosystems).

Presentation of Data and Statistical Analysis. All in vitro experiments were carried out on at least three independent cell cultures or mitochondrial preparations. For experiments performed in vivo, each individual animal was regarded as representing an independent experiment. Therefore, the results are presented as mean values ± S.D. of n experiments in vitro or as mean values ± S.E.M. of n animals when performed in vivo. In the figures, the bars were omitted if they were smaller than the symbols representing the mean values. Unless stated otherwise, multiple comparisons of data were analyzed by ANOVA followed by the Bonferroni post hoc test using SigmaStat, version 2.03 (SyStat Software, Inc., Richmond, CA). A p value of <0.05 was considered statistically significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
To test for an intracellular site of action, the uptake of [³H]PPX into neural cells, both cerebellar granule cells and astroglial cells, was examined. The uptake of PPX increases with incubation time and reaches constant levels after incubation for 20 min (Fig. 1A). After reaching plateau, the amount of [³H]PPX obtained in the cell lysates does not differ significantly between neurons (0.7 ± 0.2 pmol/mg) and astroglial cells (0.60 ± 0.09 pmol/mg). Additionally, the amount in astroglial cells converts to an intracellular concentration of 160 nM [³H]PPX by taking the specific volume for astroglial cells (4 µl/mg protein) into account (Dringen and Hamprecht, 1998).

View larger version (28K): [in this window] [in a new window]   Fig. 1. Uptake of [3H]PPX in neural cells. Uptake in astroglial cells (opened circles/bars) or cerebellar granule neurons (filled circles/bars) was started by the addition of [3H]PPX (3 µCi/ml) in incubation buffer containing 5 mM glucose and terminated at the indicated time (A) or after 10 min (B–D). B, uptake was modulated at 37°C by using an incubating buffer pH 7.4 (Ctrl) containing digitonin (Digi, 100 µg/ml) or adjusting the pH to 6 or 8, respectively. C and D, uptake was started by the addition of [3H]PPX (3 µCi/ml) and a variable concentration of unlabeled PPX (0–30 mM). C, uptake was normalized on time and expressed as the rate of [3H]PPX uptake. D, the specific rate of total PPX uptake was calculated by taking the excess of unlabeled PPX into account and is shown as a function of total PPX concentration in the incubation buffer. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with B, the control incubation for a given cell type, or with C, the incubation in the absence of unlabeled PPX, respectively.

Dilution of [³H]PPX with unlabeled PPX results in a dose-dependent inhibition of [³H]PPX uptake (Fig. 1C), which is caused by isotope dilution as shown in Fig. 1D. Multiplication of [³H]PPX uptake (Fig. 1C) by the molar excess of unlabeled PPX yields the rate of total PPX uptake (labeled plus unlabeled; Fig. 1D), which is proportional to the total PPX concentration (labeled plus unlabeled) over a range of seven orders of magnitude (Fig. 1D).

In a second set of experiments, entry of PPX into isolated mitochondria was measured and characterized. As observed in cells, the uptake of [³H]PPX in mitochondria strongly depends on mitochondrial integrity (Fig. 2A), as disruption of mitochondria by vigorous sonication or Triton X-100 results in a significant inhibition to 15% (Fig. 2A). In intact mitochondria, uptake reaches maximal values after 5 min in the presence of energy substrates malate plus pyruvate. By subtraction, the amount of [³H]PPX for unspecific binding (sonicated mitochondria) from the amount obtained in intact energized mitochondria, a specific uptake of 0.15 ± 0.01 pmol [³H]PPX/mg protein was obtained, which corresponds to an intramitochondrial concentration of 150 nM by accounting for a mitochondrial volume of 1 µl/mg (Halestrap and Quinlan, 1983).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Uptake of [3H]PPX in mitochondria. Mitochondria (control; open circles, bars) were lysed [1% (v/v) Triton X-100; open triangles], sonicated vigorously (SMP; filled triangles), or uncoupled with FCCP (1 µM; filled circles) and preincubated for 2.5 min in malate/pyruvate containing (2.5/5 mM) incubation buffer (all 0.2 mg/ml). Uptake was started by the addition of [3H]PPX (3 µCi/ml) and terminated at the indicated time (A). ***, p < 0.001 compared with incubation for 0 min in control mitochondria or FCCP-treated mitochondria; ###, p < 0.001 compared with incubation with sonicated mitochondria; , p < 0.001 compared with incubation with FCCP-treated mitochondria. B to E, uptake was terminated after 2 min of incubation. B, increasing amounts of mitochondria (0.1–0.4 mg/ml) were used. C, mitochondria (0.2 mg/ml) were preincubated in buffer with increasing osmolarity (160–960 mOsm/l adjusted with sucrose). ***, p < 0.001 compared with incubation in 320 mOsm/l incubation buffer. D, uptake was started by [3H]PPX (3 µCi/ml), which was diluted with unlabeled PPX (0–10 mM), normalized on time and taking the excess of unlabeled PPX into account. The specific rate of total PPX uptake is shown as a function of the total PPX concentration. E, uptake was measured in the presence of different energy substrates (buffer only, 5 mM succinate, 1 mM ATP, or 2.5 mM malate + 5 mM pyruvate). Depolarization of mitochondrial membrane potential was induced by the addition of ADP plus phosphate (both 1 mM), 1 µM valinomycin (Val), 100 nmol/mg CaCl2 + 1 mM phosphate, 5 µM rotenone (Rot), or 1 µM FCCP. ***, p < 0.001 compared with malate/pyruvate; , p < 0.05; , p < 0.01; , p < 0.001 compared with incubation in absence of energy substrates.

To evaluate the membrane potential as a driving force for mitochondrial [³H]PPX uptake, we used different energy substrates to polarize the mitochondrial membrane as well as different inhibitors to depolarize it (Fig. 2E). Any of the used energy substrates (malate/pyruvate, succinate, or ATP) results in a comparable [³H]PPX uptake. Withdrawal of these substrates in turn reduces uptake to 20% (p < 0.001). Furthermore, uptake in malate/pyruvate-energized mitochondria is significantly inhibited if mitochondrial membrane potential is reduced by the addition of ADP + P_i (57%), valinomycin (47%), calcium + P_i (44%), rotenone (41%), or FCCP (27%), respectively. After demonstrating entry of PPX into brain cells and mitochondria, we reevaluated the antioxidative properties of PPX and SND and compared the obtained efficacy with EUK-134, a potent SOD-catalase mimetic (Melov et al., 2001).

First, H₂O₂ and superoxide were generated by xanthine oxidase (Fridovich, 1970) in a cell-free assay where detoxifying enzymes and membranes are absent. This allows the evaluation of the dose response for the detoxification of in situ generated ROS by an antioxidant, independent of its uptake properties. Additionally, allopurinol was used to measure the efficacy of the antioxidant without the competition between antioxidant and the detection system.

As expected, EUK-134 was the most potent compound, which at a concentration of 300 µM was able to detoxify >95% ROS generated by xanthine oxidase. Both PPX and SND were less potent at this concentration (75% residual H₂O₂); however, a dose-dependent effect for detoxifying hydrogen peroxide by both enantiomers was observed (Fig. 3A). Comparable ROS detoxification was achieved at 3 µM EUK-134 and 1 mM for either PPX or SND. Additionally, both enantiomers showed equipotent efficacy in detoxification of xanthine oxidase-generated ROS (Fig. 3A).

View larger version (32K): [in this window] [in a new window]   Fig. 3. Antioxidative effects of PPX and SND related to hydrogen peroxide. A, ROS were generated by xanthine oxidase in a solution containing EUK-134 (black bars), PPX (light gray bars), or SND (dark gray bars) at the indicated concentrations. To avoid competition of the compounds with the detection system, ROS generation was stopped after 20 min by the addition of allopurinol (1 mM) and the nondetoxified H2O2 was detected by the addition of AR and HRP and normalized to the amount in the absence of an antioxidant (white bar). B, mitochondrial H2O2 release was started by the addition of mitochondria (0.1 mg/ml) to a mixture of 10 µM AR; 0.4 units/ml HRP, and 2.5 mM succinate (Ctrl) and compared with the rate of AR oxidation in the presence of PPX (300 µM) and EUK-134 (30 µM). Fluorescence of generated resorufin was detected every 30 s over a period of 5 min. After linear regression, the slopes were normalized to the control incubation and are given as mean ± S.D. from three independent experiments. C, oxidative stress was induced in HT-22 cells by 5 mM glutamate (Glu). Cells were treated with PPX (1 mM), SND (1 mM), and EUK-134 (250 µM) with or without simultaneous addition of glutamate. After 24-h incubation, cell viability was measured by the reduction of Alamar Blue. Measurement from vehicle-treated cells is shown as a control. Data shown are mean ± S.D. from eight independent experiments.

To show that PPX and EUK-134 are able to inhibit mitochondrial hydrogen peroxide release, we measured the release of hydrogen peroxide in succinate-energized mitochondria in the presence of PPX (300 µM) or EUK-134 (30 µM). Both compounds significantly inhibited the release of hydrogen peroxide to 73 and 58% respectively, compared with the values obtained in the absence of an antioxidant (Ctrl, Fig. 3B).

To evaluate whether these antioxidative properties of PPX and SND are sufficient to confer neuroprotection in a cellular model of oxidative stress qualitatively, HT-22 cells were treated with glutamate to induce cell death (Fig. 3C). HT-22 cells have been shown to be particularly sensitive to glutamate toxicity, which involves a glutamate/cysteine anti-porter; high extracellular levels of glutamate deplete the cells of cysteine, causing a decrease in cellular glutathione (and not the activation of glutamate receptors). This has been described to result in an early increase of ROS (5–10-fold) followed by a later but massive increase in ROS (200–400-fold) derived from mitochondria and paralleled by the time of cell death (Tan et al., 1998).

In our hands, the treatment of HT-22 cells with glutamate resulted in oxidative stress after 8 h of incubation, as observed by an increased oxidation of dihydro-2,7-dichloro-fluorescein-diacetate to the fluorescent 2,7-dichloro-fluorescein (data not shown). After a 24-h exposure to glutamate, cell viability was reduced to 13 ± 8% (###, p < 0.001 compared with untreated cells; Fig. 3C). In the absence of glutamate, incubation with PPX or SND (both at 1 mM) does not significantly affect cell viability, whereas EUK-134 (at 250 µM) reduces cell viability to 64 ± 8% (#, p < 0.05 compared with untreated cells). PPX, SND, and EUK were able to prevent glutamate-induced cell death (***, p < 0.001 for all three compounds). Protection of up to 82% remaining viability occurred at 1 mM (PPX and SND) and 250 µM (EUK-134). PPX, SND, and EUK-134 were able to protect cells in a dose-dependent manner (data not shown). EC₅₀ concentrations were determined by nonlinear regression as 370 ± 50, 190 ± 80, and 20 ± 1 µM, respectively (data given as mean ± S.D. of eight independent experiments). Multiple comparisons of the obtained EC₅₀ concentrations by ANOVA followed by Bonferroni's post hoc test resulted in no significant difference between PPX and EUK.

Because H₂O₂ decomposes within hours (Halliwell, 1999), we focused on the more reactive radical nitric oxide generated by the nitric oxide donor DETA, which in the presence of DAF yields the fluorescent DAF-triazole. Both PPX and SND (Fig. 4) equally inhibit the generation of the DAF-triazole. The IC₅₀ values determined by nonlinear regression for both enantiomers are 15 ± 2 µM (mean ± S.D. of three independent experiments).

View larger version (11K): [in this window] [in a new window]   Fig. 4. Antioxidative effects of PPX and SND related to nitric oxide generated by DETA. Generation of nitric oxide was initiated by the addition of DETA to a solution containing DAF and PPX (filled circles) or SND (open circles, both 0–10,000 µM). The generation of DAF-triazole fluorescence was measured at room temperature every 30 s during 30 min. The obtained slopes were plotted against the concentration of PPX. The data represent mean ± S.D. of a representative experiment, each done in triplicate.

This was followed by an experiment where mitochondria were isolated after PPX treatment of mice to determine mitochondrial aconitase activity, an indicator for steady-state levels of superoxide (Tarpey et al., 2004). PPX treatment for 4 days (2 x 30 mg/kg) increased mitochondrial aconitase activity by 42% to 67 ± 16 mU/mg compared with vehicle-treated animals (47 ± 9 mU/mg; Fig. 5A). This was not due to altered expression of mitochondrial aconitase, because protein levels were not affected by the PPX treatment (Fig. 5B). Consequently, we tested both compounds in mice expressing the G93A-mutant SOD1, a common and well described model of ALS, where neuronal cell death is associated with an increase in oxidative stress and mitochondrial alterations (Menzies et al., 2002; Bendotti and Carri, 2004).

View larger version (32K): [in this window] [in a new window]   Fig. 5. Antioxidative effect of PPX in vivo. Activity (A) and expression (B) of mitochondrial aconitase after treatment of C57BL/6 mice with PPX (2 x 30 mg/kg per day, n = 8) or vehicle (Ctrl, n = 6) have been determined as described under Materials and Methods. The data are shown as mean ± S.D. of n animals. **, p < 0.01 by unpaired Student's t test.

Since we found equipotent antioxidative properties of PPX and SND and no evidence for a transporter, which is involved in the uptake of PPX, we tested SND at a high dose of 100 mg/kg to achieve antioxidative action. PPX was given at a lower dose of 3 mg/kg [shown to be neuroprotective in models of dopaminergic neurodegeneration (Zou et al., 2000; Anderson et al., 2001)] to test for putative neuroprotection in the ALS model, which might be mediated via activation of dopamine receptors.

Regarding basal running wheel activity (or presymptomatic performance), there is no significant difference between SND-treated animals (14,700 rpm/12 h [4200–15,200]) and the control group (15,400 rpm/12 h [14,600–16,200]) (Fig. 6A, 95% confidence interval). In contrast, PPX-treated animals show a significant increase by 55% to 23,900 rpm/12 h [23,200–24,600] in presymptomatic running wheel activity compared with the SND-treated animals and the control group (Fig. 6A).

View larger version (26K): [in this window] [in a new window]   Fig. 6. Treatment of SOD1(G93A) transgenic mice with PPX and SND. A, motor activity of mice treated with vehicle (Ctrl; open circles), SND (100 mg/kg p.o.; black circles), or PPX (3 mg/kg p.o.; gray circles) was measured by recording revolutions of each running wheel in the dark period (8:00 PM to 8:00 AM). Measurements were taken from day 80 to death. The resulting time performance values (insert given as mean ± S.E.M.) and the curve resulting from the nonlinear regression are shown for each group. B, survival times are represented in a Kaplan-Meier Plot. Statistical analysis was performed by a log rank Mantel-Cox test using the software package SAS, version 8.2.

No significant differences were obtained for the slopes in the inflection point of the motor performance curve (see parameter c under Materials and Methods), which ranged from –18 to –24 rpm/day among the different groups. In contrast, the time point of half-maximal motor performance occurs at day 103 [102–104] in vehicle-treated animals, which occurs significantly delayed at day 106 [105–106] or 109 [108–110] in PPX- or SND-treated animals, respectively.

Additionally, survival time in SND-treated SOD1 (G93A) mice (132 ± 2 days, mean ± S.E.M., n = 15) was significantly prolonged compared with the control group (125 ± 2 days, log rank test p = 0.011, mean ± S.E.M., n = 11) and in comparison to the PPX-treated group (122 ± 2 days, log rank test p = 0.002; mean ± S.E.M., n = 15). The survival times of the PPX-treated animals and the control group do not differ significantly (log rank Mantel-Cox test, p = 0.464; Fig. 6B).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
PPX has been proposed to accumulate in mitochondria (Abramova et al., 2002). Because cellular uptake is a prerequisite for penetration into mitochondria, we show that [³H]PPX enters astrocytes and neurons beyond nonspecific binding, indicated by reduced uptake in digitonin-permeabilized cells. Furthermore, [³H]PPX accumulates in astroglial cells as shown by the 3-fold intracellular concentration compared with incubation buffer. The mechanism of uptake is not transporter-mediated and therefore diffusion-limited because of the nonsaturable rate of total PPX uptake (labeled plus unlabeled), which is proportional to the total PPX concentration.

The increase in cellular uptake by alkalinization and inhibition by acidification might be explained by the pK values of PPX (5 and 11 for the aminothiazole and propylamino group, respectively). At pH 7.4, >98% PPX is protonated to the less permeable univalent cation; a pH increase raises the concentration of the neutral molecule, which penetrates the cells more easily than the protonated species.

Mitochondrial [³H]PPX uptake is proportional to mitochondrial amount and correlates with the intramitochondrial volume, because mitochondrial swelling or shrinkage results in an increase or a decrease in uptake, respectively. The ability of mitochondria to retain [³H]PPX is greatly affected by rupture of the mitochondrial membranes, showing that nonspecific binding of PPX to mitochondrial proteins is low. We conclude that PPX permeates the inner mitochondrial membrane and enters the matrix.

As in cells, mitochondrial uptake is not transporter-mediated but depends on mitochondrial membrane potential. We show that, in energized mitochondria, [³H]PPX influx is stimulated by a factor of two to six, irrespective of the energy substrate used, compared with all conditions where the generation of a membrane potential was prevented. The obtained intramitochondrial concentration of 150 nM in energized mitochondria accounts for a 3-fold accumulation of PPX within mitochondria compared with incubation buffer. The obtained values in FCCP-depolarized mitochondria (0.06 pmol/mg) most likely reflect the uniform distribution of PPX between incubation buffer and mitochondrial matrix (diffusion only), whereas, in energized mitochondria (0.15 pmol/mg), uptake of the cation is driven additionally by the membrane potential (negative inside).

The results are consistent with the accumulation of lipophilic cations in mitochondria driven by the mitochondrial membrane potential (reviewed by Ross et al., 2005). In addition, the model of Trapp and Horobin (2005) predicts the accumulation of strong bases (nonpermanent cations like PPX; pK₂ 11) in energized mitochondria, if the compound is sufficiently hydrophobic. Compound hydrophobicity is indicated by an equal octanol/water partitioning (log P = 0, PPX approximately –0.2, data not shown).

We conclude that apart from its binding to dopamine receptors, PPX can accumulate within cells and mitochondria. We assume that SND enters cells and mitochondria by the same mechanism, because no transporter is involved and the chiral configuration of the aminopropyl group does not affect the physicochemical determinants for uptake (pK values and hydrophobicity).

Although several studies addressed the antioxidative properties of PPX (e.g., Cassarino et al., 1998; Ferger et al., 2000), its efficacy might have been underestimated because of the efficient and competing detection systems (Floyd et al., 1984). Therefore, we reevaluated the antioxidative properties of PPX and SND targeted toward different reactive species and compartments; obtained efficacies were compared with EUK-134, a potent SOD-catalase-mimetic (Jung et al., 2001; Melov et al., 2001).

We show that PPX and (the equipotent) SND are weak H₂O₂ scavengers if generation and detoxification are measured in the same compartment in absence of a detection system (xanthine oxidase). In contrast, EUK-134 detoxifies the generated ROS in the 5 µM range, indicating a 100-fold higher efficacy compared with PPX or SND. However, the efficacy of EUK-134 decreased by a factor of five in the presence of a detection system (mitochondrial H₂O₂ release) or in HT-22 cells where cell death was induced by glutathione depletion (Murphy et al., 1989; Tan et al., 1998). Importantly, the loss of efficacy was not observed if PPX or SND was tested in these systems.

This is consistent with the uptake properties of PPX yielding cellular or mitochondrial accumulation, thus bypassing competition with the detection system (mitochondrial H₂O₂ release) and exerting inhibition of cell death at even lower concentrations (IC₅₀ at 300 µM). The uptake properties and the efficacy of PPX to scavenge H₂O₂ (accumulates 3-fold, IC₅₀ for H₂O₂ 1 mM) are sufficient to explain its neuroprotection in glutathione-depleted HT-22 cells (EC₅₀ 300 µM). Additionally, the equipotent efficacy of SND to protect HT-22 cells indicates a dopamine receptor-independent mechanism and is consistent with the assumption that SND enters cells and mitochondria. Mitochondrial ROS attenuation in HT22 cells is sufficient but not a prerequisite to protect HT-22 cells from cell death. Therefore, pathways by PPX/SND other than antioxidative action located to mitochondria cannot be excluded. However, alternative pathways were not examined in this paper.

The low efficacy of PPX and SND toward H₂O₂ might be the result of its low reactivity (Halliwell, 1999). Therefore, we evaluated the efficacy for more reactive species: nitric oxide or superoxide (both decompose within seconds). The inhibition of DAF-triazole generation in the presence of a nitric oxide donor and antioxidant (Nagata et al., 1999) was used to show that PPX and SND detoxify nitric oxide equipotently at the 15 µM level. We conclude that the antioxidative efficacy of PPX and SND, in addition to the spatial issues discussed above, depends on the reactivity of the respective radical because the IC₅₀ values for nitric oxide and H₂O₂ vary at least by a factor of 50.

We show that PPX treatment (2 x 30 mg/kg) results in an increase in mitochondrial aconitase activity in vivo, indicating a reduction of the superoxide steady-state level within mitochondria (Tarpey et al., 2004). Because treatment of these mice with a PPX dose of 2 x 200 mg/kg results in micromolar brain levels, even 12 h after the last treatment, the increase in mitochondrial aconitase might result from total brain concentrations within the same order of magnitude.

Aconitase measurements were performed by Gu et al. (2004), showing equipotent protection of mitochondria in dopaminergic and nondopaminergic cells in the presence of 10 µM PPX or SND, if added 48 h prior to complex I inhibitors. PPX causes a slight but not significant increase in total aconitase activity in 1-methyl-4-phenylpyridinium-treated SHSY-5Y cells. We hypothesize that either we achieved higher intramitochondrial concentrations or the protective effects observed by Gu et al. (2004) are related to the preincubation, which may alter gene expression and mediate protection by an alternative mechanism (e.g., Ling et al., 1998; Presgraves et al., 2004; Pan et al., 2005).

The present results support the concept of PPX and SND as mitochondria-targeted antioxidants, evidenced by equal antioxidative efficacy toward H₂O₂ and nitric oxide, and equipotent efficacy in preventing cell death in glutathione-depleted cells. Targeting of PPX to mitochondria is supported by the membrane potential-dependent uptake process in which no transporter is involved by higher efficacy for cellular protection compared with "direct scavenging" and finally by the ability of PPX to lower mitochondrial superoxide levels in vivo.

We conclude that the in vitro neuroprotective properties of PPX are independent of the chiral 6-propylaminogroup in the molecule. Therefore, the (+)-enantiomer SND, rather than PPX, might permit a sufficiently high dosage regimen to exert antioxidative and neuroprotective efficacy in vivo without causing side effects by overstimulation of dopamine receptors.

Therefore, we tested SND (at a high dose) and PPX (at a low dose) in an animal model for familial ALS, the SOD1-G93A mice. The dose of 100 mg/kg/day for SND was tested to achieve antioxidative action based on equipotent efficacy compared with PPX and the micromolar brain levels achieved in C57BL/6 mice. The used PPX dose of 3 mg/kg/day was chosen based on the expected dopaminergic stimulation to demonstrate that protection of dopaminergic neurons does not confer beneficial effects in the SOD1-G93A mouse. Indeed, we show that treatment with PPX does not increase survival time of the SOD1-G93A mice and conclude that the use of a dopamine agonist does not result in meaningful neuroprotection, although a loss of dopaminergic neurons has been described in these animals (e.g., Kostic et al., 1997) and in patients (e.g., Przedborski et al., 1996). PPX-treated animals display an increase in presymptomatic running wheel activity, probably a result of dopaminergic stimulation. Early after-disease onset, these animals have a decreased probability of survival (from day 115 to 133). This could be indicating that the demand on muscle cells is detrimental to survival, which has been observed in other studies as well (Mahoney et al., 2004).

The high-dose treatment with SND significantly prolongs survival time and improves motor performance (indicated by a delay of half-maximal performance) by 7 and 6 days, respectively. These effects are comparable with the treatment with 30 mg/kg riluzole (the only drug launched for ALS), which prolongs survival time by 8 days in our laboratory (B. Schwalenstoecker and A. C. Ludolph, unpublished observation). The absence of preonset hyperactivity in SND-treated animals suggests that the dose given does not confer dopaminergic stimulation.

Because disease progression was not altered, we conclude that SND acts as a neuroprotectant in the SOD1-G93A mice, resulting in a later onset of symptoms as well as an extended survival time. Based on the high dose, the equipotent antioxidative properties of PPX and SND and the lack of evidence for different distribution in vivo or in vitro, we propose that the beneficial effects of SND in the animals are mediated by SND brain- and mitochondria-targeted antioxidative property. However, mitochondrial action of SND in the SOD1-G93A mouse needs to be confirmed by direct measurement of mitochondrial ROS attenuation in this model, using specific endpoints such as modification of mitochondrial proteins or mitochondrial aconitase activity. In summary, SND might be a structural prototype, suitable for further chemical modification, to obtain a mitochondrial-targeted antioxidant with higher efficacy.

	Acknowledgements

 
We thank M. Boehringer, S. Brezina, K. Eltges, and S. Fleissner for excellent technical assistance.

	Footnotes

 
doi:10.1124/jpet.105.092312.

ABBREVIATIONS: PPX, pramipexole; ALS, amyotrophic lateral sclerosis; DETA, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; EUK-134, [manganese 3-methoxy-N,N'-bis(salicylidene)ethylenediamine chloride]; EUK, EUK-134; SND919CL2x, (+)2-amino-4,5,6,7-tetrahydro-6-L-propylamino-benzathiazole dihydrochloride; SND, SND919CL2x; DAF, 4,5-diaminofluorescein; HBSS, Hanks' balanced salt solution; AR, Amplex Red; MS, mass spectrometry; HRP, horseradish peroxidase; ROS, reactive oxygen species; ANOVA, analysis of variance; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

Address correspondence to: Lothar Kussmaul, Department of Central Nervous System Research, Boehringer-Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strae 65, 88397 Biberach an der Riss, Germany. E-mail: lothar.kussmaul{at}bc.boehringer-ingelheim.com

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