A Connection between the Mitochondrial Permeability Transition Pore, Autophagy, and Cerebral Amyloidogenesis Vukic´ Sˇosˇkic´,† Martina Klemm,† Tassula Proikas-Cezanne,‡ Gerhard P. Schwall,† Slobodan Poznanovic´,† Werner Stegmann,† Karlfried Groebe,† Helmut Zengerling,† Rainer Schoepf,† Michael Burnet,§ and Andre´ Schrattenholz*,† ProteoSys AG, Carl Zeiss Strasse 51, D-55129 Mainz, Germany, Autophagy Laboratory, Department of Molecular Biology, University of Tu ¨ bingen, Auf der Morgenstelle 15, D-72076 Tu ¨ bingen, Germany, and Synovo GmbH, Paul Ehrlich Strasse 15, D-72076 Tu ¨ bingen, Germany Received October 23, 2007
In a drug reprofiling attempt, we explored novel neuroprotective properties of 4-azasteroids by synthesizing chemical affinity tags capturing adenine nucleotide translocator-1, as a potential target. Dutasteride inhibits the mitochondrial transition pore and induces an increase of autophagosomal structures in human cell lines. In vivo, a surprising reduction of the β-amyloid plaque load in a model for cerebral amyloidosis appears to connect release of neurotoxic peptides, mitochondrial apoptosis and autophagy. Keywords: Dutasteride • Finasteride • mitochondrial • cerebral amyloidosis • Alzheimer’s disease • excitotoxicity • autophagy • drug repositioning • chemical proteomics • systems biology
Introduction The optimization of drug specificities against one selected target and related screening for high affinities have not led to urgently needed treatments of central nervous system (CNS) disorders. On the contrary, successful drugs in the field rather have low selectivities and affinities and multiple targets.1 Given the complex and polyetiological molecular biological background, functional proteomic profiling of efficacy models for active compounds has the potential to contribute to a more systematic understanding of underlying neuronal mechanisms.2–4 The unexpected functional flexibility of many protein targets, and in particular the functional participation of redundant post-translational isoforms, requires a corresponding investigation of crosstalk, redundancy and multiple affinities of drugs that may exert combinatorial effects on cellular function.5 Using a functional in vitro model (previously characterized on the level of protein surrogate biomarker signatures for various types of neuronal stress),6–8 we found novel neuroprotective effects of the 4-azasteroid anticancer drugs Finasteride and Dutasteride. We then synthesized protein-reactive 4-azasteroid-derivatives as affinity tags for binding sites of interacting proteins and were able to enrich the mitochondrial adenine nucleotide translocators as potential novel targets from rat brain. Mitochondrial dysfunction contributes to the pathogenesis of various neurodegenerative diseases9 with pathophysiological consequences at the level of calcium-driven excitotoxicity and neuroinflammation.10–12 It is age-related and oxidative damage * Corresponding author: Dr. Andre´ Schrattenholz; phone, +49-61315019215; fax, +49-6131-5019211; e-mail,
[email protected]. † ProteoSys AG. ‡ University of Tu ¨ bingen. § Synovo GmbH.
2262 Journal of Proteome Research 2008, 7, 2262–2269 Published on Web 05/09/2008
leading to protein aggregation and dysfunction is thought to combine with individual injuries, lesions, or genetic risks to precipitate specific pathologies. Here, we present evidence that neuroprotective effects of 4-azasteroids go together with an inhibition of the mitochondrial transition pore and thus intrinsic apoptosis and also activate autophagy. Autophagy has recently attracted considerable attention as a key mechanism for the clearance of dysfunctional proteins or cells in the diseased brain, downstream of mitochondrial signals.13–17 The in vivo effect on cerebral amyloidosis is discussed in this context.
Experimental Procedures One-Dimensional Polyacrylamide Gel Electrophoresis (1D-PAGE) and Matrix-Assited Laser Disorpsion Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry. Electrophoresis, silver staining, in-gel digestion and MALDI-TOF mass spectrometry were essentially performed as described previously.18 Mass spectra were internally mass calibrated using trypsin auto digestion peptide signals as reference values. Mass measurement accuracies were typically e50 ppm. For identification of proteins, the peptide masses were searched against the NCBI nonredundant protein database (www.ncbi.nlm.nih.gov, version 09/2002) using MASCOT software version 1.8 (Matrix Science, London, U.K.).18 Cell Culture Experiments and Calcium Imaging. These experiments were performed as described previously.6–8 The significance of differences was evaluated using t tests. APPPS1 Experiments. APPPS1 transgenic mice, expressing both KM670/671NL mutated human APP and L166P mutated human PS1 under the Thy-1 promoter element,19,20 were treated with the compounds from the age of 126 days after birth (DAB) to 158 DAB. Mice were treated with either the vehicle 10.1021/pr700686x CCC: $40.75
2008 American Chemical Society
Connection between MPTP, Autophagy, and Cerebral Amyloidogenesis (0.5% methyl cellulose, 0.25% Lecithin, 0.1% microcrystalline cellulose) or a commercial formulation of Dutasteride (100 mg/ kg) suspended in 0.5% (w/v) methyl cellulose and 0.25% (w/v) Lecithin once daily by gavage at a time corresponding to the first third of the resting period after the dark cycle. On completion of the dosing period, animals were sacrificed by lethal narcosis followed by collection of blood by cardiac puncture and recovery of brain material for sectioning and extraction of drug and relevant peptides. Samples were snapfrozen using liquid nitrogen immediately after collection. All procedures were conducted in conformity with applicable German and EU laws on animal experimentation and the study was approved by a government appointed ethics committee. Brains were removed and postfixed at 4 °C in 4% paraformaldehyde (PFA), dehydrated in 30% sucrose, and frozen. Serial coronal 40 µm sections were cut with a microtome and collected in cryoprotectant (30% glycerol and 45% ethylene glycol in PBS) and stored at -20 °C until use. Free-floating sections were processed for immunohistochemistry as described elsewhere.21 Briefly, sections were washed in TBS and blocked with 3% goat or donkey serum (Vector Laboratories, Inc., Burlingame, CA) in 0.3% Triton-X-100 (Fisher, Fair Lawn, NJ). The sections were incubated overnight with primary antibodies at 4 °C in 2% serum and 0.3% TritonX-100, washed three times with TBS and incubated for 3 h with biotin-conjugated secondary antibodies. After repeated TBS washing, sections were stained by complexing with SG blue (Vectastain ABC elite kit; Vector Laboratories). Sections were mounted on precleaned glass microscope slides (Superfrost Plus; Langenbrinck, Teningen, Germany), dehydrated with an alcohol series, cleared in xylene and coverslipped in a xylene soluble mounting medium (Pertex; medite GmbH, Burgdorf, Germany). Amyloid load was estimated on every 12th section throughout the entire neocortex. Plaque load of β-amyloid was estimated on every 12th section throughout the entire neocortex: Sections were digitized under constant brightness in all images. Image data were converted to gray, and for evaluations of the cerebral cortex, bands of 250 pixels width below the convexity of the brain were processed and further analyzed. Quantification of the plaque regions was performed by automated image analysis, using normalized gray values within the brain sections. Plaque areas were identified as all tissue regions with gray values exceeding 30% of the range of gray values encountered in the respective brain section. Accumulated area and volume () sum of gray values in the region of interest) of the entire brain section and of the plaque areas were computed. Results were specified in the form of relative plaque areas and volumes () ratios of plaque areas and volumes over total areas and volumes in the individual brain sections). For each experimental group, means and standard deviations of relative plaque areas and volumes were computed. Experimental and control groups were compared using two-sided unpaired t tests. Autophagy Assays. Human HEK293 and G361 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/10% fetal calf serum (FCS) at 37 °C, 5% CO2. HEK293 cells were incubated for 3 h with Finasteride (1 µm) or Rapamycin (300 nM), and LC3 protein was detected in triplicate experiments using an anti-LC3 antiserum (a gift from Tamotsu Yoshimori, National Institute of Genetics, Japan) and standard ECL detection procedures. In the presence or absence of Wortmannin (233 nM), G361 cells were treated with Finasteride (1 or 5 µm)
research articles
or Rapamycin (100 nM), and LC3 protein was detected by ECL. Nonspecific bands that appeared using the LC3 antiserum reflected equal loading; alternatively, using a monoclonal GAPDH Antibody (Roche), standard ECL detections were conducted. Synthesis of Affinity Tags, General. Unless otherwise noted, materials obtained from commercial suppliers were used without further purification. S-2-Pyridyl-3-oxo-4-aza-5R-androstane-17β-thiocarboxylate (1) was prepared by following the method of Rasmusson et al.22 Tetrahydrofuran (THF) was distilled from sodium/benzophenone immediately prior to use. Melting points were measured on a Bibby Sterlin Stuart melting point apparatus SMP10 and are uncorrected. 1H NMR spectra were determined on a Bruker DRX400 spectrometer at 400 MHz. Mass spectra were measured by Bruker Daltonics Ultraflex MALDI TOF/TOF mass spectrometer. N-Boc-Aminoethyl-3-oxo-4-aza-5r-androstane-17β-carboxamide (2). A suspension of S-2-pyridyl-3-oxo-4-aza-5Randrostane-17β-thiocarboxylate (1) (500 mg, 1.07 mmol) in a mixture of 500 mg (2.77 mmol) of N-Boc-1,2-diaminoethane and 16.0 mL of THF was stirred at 24 °C for 16 h. The solution was concentrated and the reaction mixture was placed on a column of silica gel and was eluted with 1:1 acetone-CH2Cl2. Obtained yield was 550 mg, mp 130-132 °C. NMR (DMSO-d6) δ 0.53 (s, 3 H, 18-CH3), 0.76 (s, 3 H, 19-CH3), 1.12-1.40 (m, 4 H), 1.35 (s, 9 H, t-Bu), 1.41-1.64 (m, 8 H), 1.66, 194 > 1.80 (m, 2 H), 1.95, 194 > 2.05 (m, 1 H), 1.91-1.99 (m, 2 H), 2.12-2.22 (m, 2 H), 2.93, 194 > 3.00 (m, 4 H), 3.16 (s, 1 H), 3.31 (s, 1 H), 6.31 (t, 1 H, J ) 4 Hz), 7.26 (s, 1 H), 7.43 (t, 1 H, J ) 3 Hz). Molecular mass calculated for (M+): C26H44N3O4 462.325, found 462.327. Aminoethyl-3-oxo-4-aza-5-r-androstane-17-β-carboxamide hydrochloride (3). N-Boc-aminoethyl-3-oxo-4-aza-5R-androstane-17β-carboxamide (500 mg) was stirred with a mixture of 15 mL dioxane and 1.5 mL conc. HCl at room temperature for 60 min following evaporation of the solvent and acid in vacuum. To remove traces of HCl, the residue was dissolved in 5.0 mL of iPrOH and 10 mL of toluene was added and evaporation was repeated once again. Obtained aminoethy-3oxo-4-aza-5-R-androstane-17-β-carboxamide hydrochloride, mp 295, 194 > 296 °C; was used without further purification. NMR (DMSO-d6) δ 0.54 (s, 3 H, 18-CH3), 0.76 (s, 3 H, 19-CH3), 1.01, 194 > 1.45 (m, 8 H), 1.45-1.60 (m, 7 H), 1.60-180 (m, 3 H), 1.90-2.10 (m, 1 H), 2.75-2.85 (quartet, 2 H, J ) 2 Hz), 2.90-2.95 (d, 1 H, J ) 4 Hz), 3.20-3.40 (m, 2 H), 6.23 (s, 3 H, NH3+), 7.48 (s, 1 H), 7.69 (t, 1 H, J ) 3 Hz), 8.17 (s, 2 H). Molecular mass calculated for (M+): C21H35O2N3 361.273, found 361.276. Synthesis of N-[2-(N-Boc-biocytinyl)aminoethyl]-3-oxo-4aza-5-r-androstane-17-β-carboxamide (4). Aminoethyl-3-oxo4-aza-5-R-androstan-17-β-carboxamid hydrochloride (205 mg) was dissolved in 10 mL of methanol, and 235 mg of tert-butybiocytin, 250 µL of NMM and 165 mg of 4-(4,6-dimethoxy-1,3,5triazin-2-yl)-4-methylmorpholinium chloride were added. Reaction mixture was stirred at room temperature for 6 h. Solvent was then removed in vacuum and the products were purified by column chromatography on SiO2 using stepwise gradient of MeOH in dichloromethane (0-20%). Fractions containing products were pooled and solvent was evaporated in vacuum. Obtained yield of N-[(N-Boc-biocytinyl)aminoethyl]-3-oxo-4aza-5-R-androstane-17-β-carboxamide was 400 mg as solid material that upon heating shoves phase transition at 68-70 °C and completely melts at 98-100 °C. NMR (DMSO-d6) δ 0.53 (s, 3 H, 18-CH3), 0.75 (s, 3 H, 19-CH3), 0.76, 194 > 1.10 (m, 2 Journal of Proteome Research • Vol. 7, No. 6, 2008 2263
research articles H), 1.12-1.40 (m, 8 H), 1.35 (s, 9 H, t-Bu), 1.45-1.60 (m, 6 H), 1.60-1.80 (m, 2 H), 1.90-10 (m, 1 H), 2.75-2.85 (quartet, 2 H, J ) 2 Hz), 2.8-3.2 (m 10 H), 3.20-3.40 (m, 2 H), 3.60-4.00 (m, 12 H), 4.06-4.14 (m, 1 H), 4.23-4.32 (m, 1 H), 6.38 (s, 1 H), 6.43 (s, 1 H), 7.21 (t, 1 H, J ) 2 Hz), 7.82 (t, 1 H, J ) 4 Hz), 7.93 (t, 1 H, J ) 3 Hz). Molecular mass calculated for C42H70O7N7S (M+): 816.498, found 816.487. Synthesis of N-[2-(Biocytinyl)aminoethyl]-3-oxo-4-aza-5r-androstane-17-β-carboxamide Trifluoroacetate (5). A total of 230 mg (0.32 mmol) of N-[2-(N-Boc-biocytinyl)aminoethyl]3-oxo-4-aza-5-R-androstane-17-β-carboxamide was dissolved in a mixture of 2 mL of CH2Cl2 and 1 mL of TFA at room temperature. After 60 min, the mixture of CH2Cl2 and TFA was evaporated in vacuum. To remove traces of TFA, the residue was dissolved in 5.0 mL of iPrOH and 10 mL of toluene was added and evaporation was repeated once again. N-[2-(Biocytinyl)aminoethyl]-3-oxo-4-aza-5-R-androstane17-β-carboxamide trifluoroacetate was obtained as oily material that solidified upon standing used without further purification. NMR (DMSO-d6) δ 0.54 (s, 3 H, 18-CH3), 0.76 (s, 3 H, 19-CH3), 0.77-1.11 (m, 2 H), 1.14-1.42 (m, 8 H), 1.46-1.62 (m, 6 H), 1.7-1.90 (m, 2 H), 1.92-2.11 (m, 1 H), 2.75-2.89 (quartet, 2 H, J ) 2 Hz), 2.9-3.3 (m 10 H), 3.22-3.43 (m, 2 H), 3.60-4.00 (m, 12 H), 4.16-4.44 (m, 1 H), 4.43-4.43 (m, 1 H), 6.42 (s, 1 H), 6.45 (s, 1 H), 6.32 (s, 3 H, NH3+), 7.23 (t, 1 H, J ) 2 Hz), 7.82 (t, 1 H, J ) 4 Hz), 7.91 (t, 1 H, J ) 3 Hz). Molecular mass calculated for (M+): C37H62O5N7S 716.445, found 716.430. Synthesis of N-[2-(Isothiocyanate-biocytinyl)aminoethyl]3-oxo-4-aza-5-r-androstane-17-β-carboxamide (6). N-[2-(Biocytinyl)aminoethyl]-3-oxo-4-aza-5-R-androstane-17-β-carboxamide (0.40 mmol), 0.3 mL of NMM and 93 mg (97 mg, 0.42 mmol) of di-(2-pyridyl)thionocarbonate were dissolved in 1 mL of dry DMF. Reaction mixture was stirred at room temperature for 1 h. Solvent was then removed in vacuum and the products were purified by column chromatography on SiO2 using a stepwise gradient of iPrOH in chloroform (0-30%). Fractions containing products were pooled together and solvent was concentrated to small volume in vacuum. Fishhooks were precipitated with diethyl ether and dried in vacuum. They were kept at -18 °C until used. NMR (DMSO-d6) δ 0.54 (s, 3 H, 18CH3), 0.75 (s, 3 H, 19-CH3), 0.76-1.10 (m, 2 H), 1.12-1.40 (m, 6 H), 1.45-1.60 (m, 6 H), 1.60-180 (m, 2 H), 1.90-2.10 (m, 1 H), 2.75-2.85 (quartet, 2 H, J ) 2 Hz), 2.90-3.10 (m, 10 H), 3.20-3.30 (m, 2 H), 3.60-4.00 (m, 9 H), 4.06-4.14 (m, 2 H), 4.32-4.27 (m, 1 H), 6.12-6.16 (t, 1 H, J ) 4 Hz), 6.28-6.31 (d, 1 H, J ) 6 Hz), 6.38 (s, 1 H), 6.44 (s, 1 H), 7.27 (s, 1 H), 7. 36 (s, 1 H), 7.39 (t, 1 H, J ) 2 Hz), 7.62 (t, 1 H, J ) 4 Hz), 7.60 (t, 1 H, J ) 3 Hz), 7.94 (s, 1 H). Molecular mass calculated for (M+): C38H60O5N7S2 758.402, found 758.419. A summary of these synthetic procedures is given in Supplementary Figure S1.
Results In Figure 1, structures of the 4-azasteroids Dutasteride and Finasteride are shown (Figure 1A and B), 5-R-reductase inhibitors and anticancer drugs that originally have been approved to treat benign prostatic hyperplasia and hair loss,2,23 respectively. For affinity capture of proteins interacting with these compounds, we synthesized a chemically related bidentate affinity tag (Figure 1C), which is biotinylated for enrichment of the tag on avidin columns after the reactive thiocyanategroup has covalently reacted with vicinal amino acid residues 2264
Journal of Proteome Research • Vol. 7, No. 6, 2008
Šosˇkic´ et al.
Figure 1. Chemical structures of 4-azasteroids and related derivatives; (A) the chemical structures of Finasteride and Dutasteride, the two approved drugs which have been used in this neuroprotective reprofiling approach. (B) The corresponding active derivatives AEAA ) 4R, 6R-dimethyl-2-oxo-hexadecahydro-indeno [5,4-f] quinoline-7-carboxylic-(2-amino-ethyl)-amide hydrochloride and AACA ) 4R, 6R-dimethyl-2-oxo-hexadecahydroindeno [5,4-f] quinoline-7-carboxylic-acid are shown. (C) The biotinylated affinity tag is shown, as synthesized starting from AACA, and used for the subsequent capture and mass spectrometry-based identification of novel neuronal targets or proteins interacting with the drugs. The biotin moiety is used to capture the tag on an avidin column, the reactive SCN-group is meant to react covalently with binding pockets of target proteins in close proximity.
in binding pockets of interacting proteins. The precursor compound for the affinity tag is 4R,6R-dimethyl-2-oxo-hexadecahydro-indeno[5,4-f]quinoline-7-carboxylic-(2-amino-ethyl)-amide hydrochloride (AEAA), still has some residual activity as can be seen in Figure B. The neuroprotective activities of AEAA and one other intermediate, 4R,6R-dimethyl-2-oxohexadecahydro-indeno[5,4-f]quinoline-7-carboxylic-acid (AACA), Finasteride (Figure 2B), are reduced, but the compounds may serve as novel chemical lead structures. Dutasteride and Finasteride (Figure 1A and B) are both neuroprotective in vitro using a model based on neural differentiation of murine embryonic stem cells6–8 as shown in Figure 2A. Neurons which have previously been characterized by markers like synaptophysin respond to a brief pulse of the excitatory neurotransmitter glutamate with moderate calcium transients which are quantified by Fura-2 calcium imaging. In Figure 2A, representative images of neurons and their response to a low dose of glutamate are shown. This type of experiment can be repeated several times and serves as a control of functional integrity of the in vitro model. The consequences of subsequent exposures to neurotoxic insults can be quantified by counting surviving functional neurons.
Connection between MPTP, Autophagy, and Cerebral Amyloidogenesis
research articles
Figure 2. Finasteride and Dutasteride are neuroprotective in vitro and protect neural cells from cell death induced by transient intracellular calcium concentration surges. (A) Representative neurons are shown in phase contrast and Fura-2 calcium images, at the onset of a vitality control by a pulse application of 5 µm glutamate (middle) during which the neurons respond with a small but nontoxic calcium response (right), which can be repeated several times over the course of up to 2 h.6–8,51 Insults by pulse applications of 100 µm NMDA, 10 µm A-β1-40 or chemical ischemia (KCN, no glucose for 45 min) produce much more pronounced calcium transients, resulting in cell death or malfunction, quantified by vitality controls before and after respective experiments. (B) Neuroprotective effects of the compounds shown in Figure 1, each at 1 µm, are compared to negative (green bar: KCN, no glucose) and positive controls (10 nM EPO); testosterone has no significant effect. Whereas on average only about 8% of unprotected neurons survive the insult after 45 min, under neuroprotective conditions, between 20 and 78% of neurons are still functional after setting the insult. In each case, several hundred single cell Fura-2 images were quantified (n ) 300-500). (C) A table summarizes the EC50-values in each of the insults. Finasteride and Dutasteride protect neurons from calcium-related toxicities at concentrations in the low micromolar range.
The underlying paradigm in all insults is calcium toxicity: each of 10 µm β-amyloid1-40, 100 µm NMDA or chemical ischemia (3 mM KCN, glucose deprivation) induces massive surges of intracellular calcium, which does not return to steady state levels and subsequently results in neuronal death or dysfunction (see Experimental Procedures). In Figure 2B, in one example, it is shown that, after ischemic insult, less than 10% of functional neurons survive as compared to 100% in controls (not shown). Neuroprotective conditions like the positive control (10 nM EPO) can rescue 60-80% of neurons, as in the case of coapplication of each 1 µmM Finasteride or Dutasteride during the insult. Testosterone and the hormonally active products of 5-R-reductase activity in the brain, dihydrotestosterone, are shown to be nearly inactive in our model (Figure 2B). However, a small additive effect upon coapplication of testosterone and Dutasteride was observed. The ES cell line used is of male genotype, but no hormone production can be observed in the model, and the time course of experiments
(30-45 min) is too fast for more long-term steroidal effects on gene transcription. Both parent compounds are neuroprotective in all three insult conditions with EC50-values between 0.1 and 2 µM (Figure 2C). The application of the Dutasteride-related biotinylated affinity tag (Figure 1C) to raw rat brain resulted in the enrichment of a distinct group of mitochondrial proteins analyzed by 1DPAGE a shown in Figure 3. Protein identification from every band visible in the raw membrane extract or in the affinityenriched material was performed by MALDI-TOF mass spectrometry. The major proteins specifically retained by the affinity tag were ANT-1/2 (adenine nucleotide translocator 1/2), supposed integral components of the mitochondrial permeability transition pore (MPTP). To further investigate a potential modulation of the MPTP by 4-azasteroids as neuroprotective mode of action, we next assayed mitochondrial pore function of the MPTP, which can be quantified by Rhodamine-123, a fluorescent dye, which is Journal of Proteome Research • Vol. 7, No. 6, 2008 2265
research articles
Figure 3. Affinity capture of interacting proteins using a Dutasteride-related chemical affinity tag; the tag shown in Figure 1C was attached to beads, which were used to enrich proteins with the tag as described in the text. Corresponding fractions have been analyzed by silver-stained 1D gels:, and the lanes of 1D gels have been loaded with 10 µg, respectively, of raw membrane extract from rat brains (RME) or material enriched by the affinity tag (FinDut-AT). All visible bands were excised form gels, in-gel digested by trypsin and identified by MALDI-TOF as described.52 The proteins identified at positions marked by arrows were found exclusively in Dutasteride-related fractions but could not be identified in raw membranes or controls. These proteins were interacting with and enriched by the affinity tag, and considered potential novel binding sites of the parent compounds, Finasteride and Dutasteride. ANT stands for adenine nucleotide translocator. Details of mass spectrometry are included; MW is molecular weight, PMF is peptide mass fingerprint, pI is the isoelectric point.
sequestered inside mitochondria at autoquenching concentrations. Upon activation of the MPTP by ischemia, the dye is released into the cytosol generating a strong fluorescent signal,24 as shown in Figure 4A and B for a representative neuron from the ESC model. We observe a dose-dependent inhibition of this type of signal by Dutasteride (and related compounds, not shown). The kinetics and EC50-values are very similar and in the same micromolar range as observed in the neuroprotection assays (Figure 4C). Elevated autophagic activity is marked by an increase of autophagosomal structures, reflected by an increase of autophagosomal membrane-bound LC3 (LC3 II) and a decrease of cytosolic, nonautophagosomal LC3 (LC3 I).25 Using this experimental setup in human G361 (Figure 5) and HEK293 (data not shown) cells, we found that Finasteride administration correlates with an increase of autophagosomal structures. Specifically, we detected an increase of membrane-bound LC3 II when cells were treated either with Finasteride or with Rapamycin (300 nM), used here as a positive control (Figure 5A). Critically, this effect was inhibited by co-treatment with Wortmannin (233 nM), a widely used inhibitor of autophagy. As dysfunctional autophagy has recently been correlated to the onset of neurodegeneration such as Alzheimer disease, we proceeded to 4-azasteroid administration in vivo using an established transgenic Alzheimer mouse model19,20 for further studies. Here, we used C57BL/6J transgenic mice (APPPS1-21) that coexpress the mutant human amyloid precursor protein KN670/ 2266
Journal of Proteome Research • Vol. 7, No. 6, 2008
Šosˇkic´ et al.
Figure 4. Dutasteride inhibits the opening of the mitochondrial permeability transition pore. In an assay employing Rhodamine123 for the measurement of mitochondrial potential, cells loaded with the dye were excited at 480 nm and emission was monitored at 565 nm. Rhodamine-123, which is trapped inside mitochondria at autoquenching concentrations (A), is released into the cytosol under ischemic conditions described, resulting in a strong increase of the fluorescence signal (B). The Rhodamine-123 signal (thought to be associated with MPTP opening24 is consistent with dose-response-relationships, kinetics and amplitudes of calcium transients shown in Figure 2, and can be inhibited by the presence of Dutasteride during the ischemic insult. (C) Each of the 5 experiments was performed 3 times, and in each case, the fluorescence signals from 150-200 cells were quantified.
671NL and the mutant presenilin 1 L166P under the control of a brain and neuron specific Thy-1 promoter element.20 Upon Dutasteride treatment, we observed a male-specific decrease of β-amyloid plaque load (Figure 6C). Quantifying plaque densities in each 5 individual female and male animals, we found a consistent overall decrease of plaque density of 11.5% in Dutasteride-treated males only, which amounts to reduction of 49% with regard to the normal increase in this models between months 4 and 5 (according to Radde et al.,20); Dutasteride-treated male mice in the current experiment exhibited an amyloid load of based on the plaque loads of individual sections (13-18 sections per animal in 5 male animals per group); the differences between the males in the two groups were significant with p-values
