Acute Polyglutamine Expression in Inducible Mouse Model Unravels Ubiquitin/Proteasome System Impairment and Permanent Recovery Attributable to Aggregate Formation

Animals.

Mice: previously described HD94 (Yamamoto et al., 2000), R6/1 (Mangiarini et al., 1996), and UbGFP-line1 (Lindsten et al., 2003) mice were bred at Centro de Biología Molecular “Severo Ochoa” animal facility. Mice were housed five per cage with food and water available ad libitum and maintained in a temperature-controlled environment on a 12/12 h light/dark cycle with light onset at 07:00 A.M. All experiments were performed in accordance with institutional guidelines approved by the ethical committee of Consejo Superior de Investigaciones Científicas (CSIC). UbGFP:HD94 mice result from the breeding of the double transgenic mice tTA+UbGFP, obtained from the breeding of CaMKIIα-tTA mice (Mayford et al., 1996) with UbGFP-line1 mice (Lindsten et al., 2003), with TetO mice (carrying the bidirectional tet-responsive promoter followed by a chimeric mouse/human exon 1 with a polyQ expansion of 94 repeats) (Yamamoto et al., 2000). UbGFP:R6/1 mice result from the breeding of R6/1 mice (Mangiarini et al., 1996) with UbGFP-line1 mice (Lindsten et al., 2003).

Doxycycline treatment.

UbGFP:HD94 mice and their control littermates were given doxycycline (Sigma-Aldrich) (0.5 g/L) in drinking water ad libitum for 8 or 12 weeks from embryonic day 16. This paradigm was chosen based on our previous observation that it results in full shut-down of transgene expression in the same conditional mouse model of Huntington disease (Diaz-Hernandez et al., 2005).

Riluzole treatment.

Riluzole (1.2 g/L or 2 g/L) (Sigma-Aldrich) was complexed with hydroxypropyl-β-cyclodextrin (Sigma-Aldrich) in water (4.5% w/v) and administered in light-protected drinking bottles. UbGFP:HD94 mice after 3.5 weeks of transgene expression or 3-week-old R6/1 mice were given riluzole for 3 d, either at 1.2 g/L for all 3 d or at 2 g/L for the first day and then at 1.2 g/L for the remaining 2 d (in view of the hypoactivity induced by 2 g/L riluzole when it was given for >1 d). Control mice were given 4.5% w/v hydroxypropyl-β-cyclodextrin.

Stereotaxic injection.

Five 18-month-old UbGFP male mice received bilateral intrastriatal injections with lactacystin in one hemisphere and with vehicle (water) in the other hemisphere. Mice were anesthetized with isoflurane (1-chloro-2,2,2-trifluoroethyl-difluoromethylether) (Inibsa) diluted in a 50% O₂/50% NO₂ mixture. Mice were kept under anesthesia during the surgery with the aid of a gas mask. The scalp was incised along the midline, and two holes were made at the appropriate stereotaxic coordinates (mediolateral, 1.5 mm; anteroposterior, 0.5 mm; dorsoventral, 1.2 mm) according to the atlas of Paxinos and Franklin (2001). Intrastriatal administration of 2 μl of lactacystin at 10 μg/μl (right side) and 2 μl of distilled H₂O (left side) was performed using a 10 μl Hamilton syringe. Lactacystin and H₂O were infused at a rate of ∼1 μl/min. After the injection, the needle was kept for additional 2 min and then slowly withdrawn.

Tissue preparation.

In all experiments, wild-type and transgenic animals were processed in parallel and have been treated identically. Briefly, mice were killed by CO₂, and brains were removed and halved sagittally immediately after decapitation. Hemispheres were fixed with 4% paraformaldehyde in Sorensen phosphate buffer (PFA) overnight at 4°C, and then were either embedded in 3.5% bacteriological agar in distilled H₂O [brains for green fluorescent protein (GFP)-fluorescence study], or immersed in 30% sucrose in PBS for 48 h for cryoprotection and then in OCT (Optimal Cutting Temperature, Tissue-Tek) (brains for immunofluorescence or immunohistochemistry). Hemispheres were serially cut into 30 μm slices, using either a vibratome (Vibratome 1500) (for GFP-fluorescence study) or a freezing microtome (Leica) (cryoprotected hemispheres for immunofluorescent and immunohistochemical analysis), and placed in a solution containing 30% glycerol, 30% ethylene-glycol, 30% dH₂O, and 10% phosphate buffer at pH 7.2.

GFP-fluorescence, immunofluorescence, and immunohistochemistry.

For GFP-fluorescence detection, slices were transferred to slides, incubated with Hoechst 33342 (DAPI) (Biologics) (2.5 μg/ml) for 10 min, and then air dried in darkness and coverslipped with Fluorosave. Immunofluorescence staining was performed pretreating free-floating slices with 0.1% Triton X-100 for 15 min and glycine 1 m for 30 min at room temperature, followed by blocking solution (1% BSA and 0.1% Triton X-100) for 1 h. Next, slices were incubated for 2 h at room temperature with primary antibodies at the following dilutions: anti-GFP (rabbit, Invitrogen), anti-NeuN (mouse, Millipore Bioscience Research Reagents), anti-GFAP (rabbit, Promega), anti-CNPase (rabbit, Abcam), anti-iba1 (rabbit, Wako), anti-β-galactosidase (rabbit, ICN Biomed.-Cappel), and anti-N-terminal htt (mouse MAB5374, Millipore Bioscience Research Reagents). Then the sections were washed in PBS and incubated with secondary antibodies for 1 h (1:200) goat anti-rabbit TexasRed-conjugated (Invitrogen), goat anti-mouse TexasRed-conjugated (Invitrogen), goat anti-rabbit TexasRed-conjugated (Invitrogen), and goat anti-mouse OregonGreen-conjugated (Invitrogen). Finally, nuclei were stained by using DAPI (Biologics) (2.5 μg/ml) for 10 min, and then sections were air dried in darkness and coverslipped with Fluorosave. For immunohistochemistry, free-floating sections were pretreated for 30 min in 1% H₂O₂/PBS, followed by 1 h with 1% BSA, 5% FBS, and 0.2% Triton X-100; and then incubated overnight at 4°C with primary antibody at the following dilutions: anti-β-galactosidase (ICN Biomed.-Cappel; 1:5000); anti-GFP (Invitrogen; 1:5000); anti-N-terminal htt (MAB5374, Millipore Bioscience Research Reagents; 1:200); anti-cleaved caspase3 (Cell Signaling Technology; 1:50). Next, slices were incubated with avidin-biotin complex (Elite Vectastain kit, Vector Laboratories). Chromogen reactions were performed with diaminobenzidine (SIGMAFAST DAB, Sigma-Aldrich) and 0.003% H₂O₂ for 10 min. Finally, sections were coverslipped with Fluorosave.

Primary neuron culture and transfection.

Primary neuronal cultures were prepared according to modifications of established procedures (Huettner and Baughman, 1986; Dubinsky, 1989; Martin-Aparicio et al., 2001). Pups were killed at embryonic day 18. Embryo cortices and striata were dissected and dissociated with the Papain Dissociation System (Worthington). Cortical and striatal neurons were maintained in Neurobasal medium (Invitrogen) supplemented with 1% sodium pyruvate, 0.5 mm glutamine, 10% v/v horse serum, and 1% penicillin/streptomycin, and grown on 3 μg/ml laminin (Sigma) and 10 μg/ml poly-l-lysine-coated open μ-Slide with eight wells (chamber slide) for high quality microscopy (Ibidi). After 2 h of incubation, the culture medium was replaced with a Neurobasal medium supplemented with 1% sodium pyruvate, 0.5 mm glutamine, 2% B27. Cells were maintained in 95% air/5% CO₂ in a humidified incubator at 37°C. For inhibition of proteasome activity, neuronal cultures were adjusted to 50 μm MG-132 (Calbiochem). For inhibition of aggregation, neuronal cultures were adjusted to 400 μm PGL-135 or 40 μm riluzole. In both treatments, drugs were added 1 h after transfection, and primary cultures were incubated for an additional 16 to 24 h. Control cells were incubated with an equivalent amount of solvent, H₂O in the case of MG-132, DMSO in the case of PGL-135 (up to 0.3% final concentration), and cyclodextrin in the case of riluzole (6.75 × 10⁻³ w/v final concentration).

Transfection was performed 1 d after plating. Cells were transiently transfected with 1 μg DNA and 3 μl Lipofectamine (Invitrogen) in 100 μl Neurobasal medium. The mixture was carefully (drop by drop) directly added to the medium (without antibiotics) and incubated for 2 h in 95% air/5% CO₂ in a humidified incubator at 37°C. Next, cells were washed three times with PBS and added to Neurobasal medium supplemented with 1% sodium pyruvate, 0.5 mm glutamine, 2% B27, and 1% penicillin/streptomycin. Transfection efficiency was controlled by counting the number of transfected neurons corrected by the total number of neurons (a minimum of 300 cells were analyzed per experimental condition). Transfection efficiency was between 3% and 6% across all experiments.

Robotic microscope imaging system and image analysis.

For immunohistochemistry analysis, Axioskop2 plus microscope and a CCD camera (Coolsnap FX color) was used. For fluorescence detection, an inverted Axiovert200 (Zeiss) microscope was used. Plan-Apochromat 10× (numerical aperture 0.45), 20× (numerical aperture 0.6), and 40× objectives were used. Xenon lamp XBO (75W/2) illumination was supplied by a liquid light guide to reduce electrical noise. Images were detected and digitized with a Hamamatsu C9100-02 digital camera and Universal Imaging MetaMorph software.

Stage movements and focusing were executed with computer-controlled stepper motors. Fluorescence excitation and emission filters [GFP: 473-498, 552-577 86007bs; cyan fluorescent protein (CFP): 426-446/422-432, 455 dichroic long-pass, 460-500; yellow fluorescent protein (YFP): 490-510, 510-560, HQ520LP; and DAPI: 359-371, 450LP] were moved into or out of the optical path with each program loop by two filter wheels (Sutter Instruments) under computer control. The whole system is mounted on a vibration isolation table to reduce noise. Computer commands that perform and coordinate automated stage movements, filter wheel movements, and focusing were generated with software programs that combine custom-designed and commercially available algorithms.

For analysis of transfected primary neurons, wells were scanned and images were taken once every 2 h (from 16 h to 24 h after transfection) to avoid photobleaching. The exposure time (70 milliseconds) and the sensitivity (30) were the same for YFP and CFP fluorescence and among experiments. Neurons were maintained in 95% air/5% CO₂ in a humidified incubator at 37°C between images. Measurements of CFP and YFP fluorescence were extracted from files generated with automated imaging by automated analysis software (ImageJ). CFP and YFP fluorescence was estimated by measuring fluorescence intensity over a region of interest that corresponded to the cell soma. These CFP and YFP intensity values were background-subtracted by using an adjacent area of the image. We classify neurons in increase or decrease of YFP fluorescence when the final fluorescence intensity increases or decreases in >50% respect to the initial fluorescence intensity respectively.

For analysis of GFP-positive cells on brain sections, 30 μm sagittal sections spanning the whole striatum (taken 120 μm apart) were entirely scanned and photographed. GFP fluorescence intensity over a region of interest that corresponds to a cell soma (based on roundness and size) was extracted from files generated with automated imaging by automated analysis software (ImageJ) and background-subtracted by using an adjacent area of the image. We classify neurons as positive for increased GFP fluorescence when its fluorescence intensity is >50% over background.

TUNEL assay.

Mice were anesthetized and transcardially perfused with 4% PFA in PBS for 10 min. Brains were postfixed in 4% PFA for 2 h at 4°C and cryoprotected. Sections were treated following the protocol of the In Situ Cell Death Detection Kit, POD (Roche).

Filter trap assay.

Hemibrains from vehicle- or riluzole-treated mice Ub-GFP:R6/1 (n = 5 for each genotype) were used to perform filter trap assay. The isolation of nuclei from total brain and the preparation of aggregates were performed as previously described (Davies et al., 1999). The cellulose acetate filters were incubated with the MAB5374 anti-N-terminal htt antibody (mouse, Millipore Bioscience Research Reagents) at 4°C overnight in 5% nonfat dried milk. A secondary goat anti-mouse immunoglobulins/HRP (DAKO; 1:1000), followed by ECL detection reagents (PerkinElmer), was used for immunodetection.

Purification of IBs by magnetic sorting and analysis in flow cytometer.

Huntingtin-containing IBs from vehicle- or riluzole-treated UbGFP:R6/1 mice were obtained as previously described (Diaz-Hernandez et al., 2006). Briefly, brains were split along the midline, and one hemibrain of each animal was homogenized in a glass homogenizer with 6 ml of cold buffer 1 (10 mm Tris, 1 mm EGTA, 0.15 m NaCl, 10% sucrose, 0.1% Triton X-100; and protease inhibitors: 2 mm phenylmethylsulfonyl fluoride; 10 μg/ml aprotinin; 10 μg/ml leupeptin; and 10 μg/ml pepstatin). After centrifugation at 27,000 × g for 20 min at 4°C, the supernatant (S1) containing cytosol and organelles was discarded and the pellet (P1) containing IBs was again homogenized in buffer 2 (10 mm Tris, 1 mm EGTA, 0.15 m NaCl, 10% sucrose; and protease inhibitors: 2 mm phenylmethylsulfonyl fluoride; 10 μg/ml aprotinin; 10 μg/ml leupeptin; and 10 μg/ml pepstatin), before it was centrifuged again at 27,000 × g for 20 min. Pellet P2 was resuspended in 12 ml of buffer 2 and filtered through a 30-μm-diameter pore filter. The F1 filter fraction was then centrifuged at 27,000 × g for 20 min, and the resulting pellet P3 containing the huntingtin-containing IBs was resuspended in 2 ml of buffer 2. The IBs were then selected by magnetic sorting. For this, resuspended pellet P3 was incubated at 37°C for 1 h with a mouse monoclonal antibody against N-terminal htt (MAB5374; 1:200). After two rounds of centrifugation and resuspension in buffer 2, the IBs were incubated at 37°C for 1 h with rat anti-mouse IgG1 antibodies labeled with microbeads. The IBs labeled with microbeads were then retained on a column by a magnetic field, while the unlabeled material passed through. Finally, the magnetic field was removed from the column and the retained IBs were eluted with 750 μl of buffer 2 and stored at −70°C until their use in further analysis. Finally, 30 μl of each sample were taken to a final 100 μl volume with PBS and analyzed on a FACsCalibur flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences).

Western blot analysis.

Extracts for Western blot analysis from cultures were prepared by homogenizing cells in loading-buffer (50 mm Tris-HCl, pH 6.8, 100 mm dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and boiled for 5 min. Extracts for Western blot analysis from mouse brain were prepared by homogenizing the brain areas in ice-cold extraction buffer consisting of 20 mm HEPES pH 7.4, 100 mm NaCl, 20 mm NaF, 1% Triton X-100, 1 mm sodium orthovanadate, 5 mm EDTA, and protease inhibitors (2 mm PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin). The samples were homogenized and centrifuged at 15,000 × g for 20 min at 4°C. The resulting supernatant was collected, and protein content was determined by Bradford. Total protein (15 μg) was electrophoresed on 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Schleider and Schuell). The filters were incubated with the antibodies anti-N-terminal htt (mouse, Millipore Bioscience Research Reagents), anti-GFP (mouse, Santa Cruz Biotechnology) and anti-β-actin (mouse, Sigma-Aldrich), at 4°C overnight in 5% nonfat dried milk. A secondary goat anti-mouse immunoglobulins/HRP (DAKO; 1:1000), followed by ECL detection reagents (PerkinElmer), was used for immunodetection.

Proteasome activity assays.

Mice were killed by CO₂, and brains were removed. Brains were quickly dissected on an ice-cold plate. Striatal tissue was homogenized in extraction buffer (10 mm Tris-HCl, pH 7.8, 0.5 mm dithiothreitol, 5 mm ATP, 0.03% Triton X-100, and 5 mm MgCl₂). Lysates were centrifuged at 13,000 × g at 4°C for 20 min, and the resulting supernatants were placed on ice and assayed for protein concentrations by the Bradford method (Bio-Rad). For determination of proteasome activity, extracts were adjusted to 0.5 mg/ml total protein by dilution with extraction buffer. All assays were done in triplicate. Chymotrypsin-like activity was determined using the substrate Suc-LLVY-aminomethylcoumarin (AMC) (Sigma; 50 μm), and caspase-like activity was determined using the substrate Z-LLE-β-2-naphthylamine (Nap) (Sigma; 0.2 mm). Assay mixtures containing 10 μg of protein, substrate, and 50 mm HEPES–KOH, pH 7.5, are made up in a final volume of 400 μl. Incubations were performed at 37°C for 30 and 60 min. The cleavage products AMC and Nap were analyzed, after stopping the reaction with 1 ml of 10% SDS, in a fluorimeter (excitation/emission: 335/410 nm for Nap and 380/460 nm for AMC). Product formation was linear with time (at least for 60 min). Background activity (caused by nonproteasomal degradation) was determined by addition of the proteasome inhibitor lactacystin at a final concentration of 50 μm (Sigma) or MG-132 at a final concentration of 10 μm (Calbiochem).

Quantitative reverse transcription-PCR.

Mouse tissue RNA samples (n = 5 for UbGFP mice and n = 7 for UbGFP:HD94 mice in the experiment of acute N-mutHtt expression, and n = 7 for UbGFP mice and n = 6 for R6/1 mice in the experiment with riluzole treated mice) were analyzed for expression of the corresponding transgenes and uniformity across replicates. For each replicate, the striatum was dissected and RNA extracted with Qiagen RNeasy Protect kit (Qiagen). RNA was quantified in a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). All samples showed 260/280 ratio values around 2, which corresponds to pure RNA. Yield range was between 70 and 300 ng/μl. RNA Integrity was checked with the Agilent 2100 Bioanalyzer. RIN values were between 7.10 and 8.30 showing good integrity. Reverse transcription (RT) reactions were performed with High Capacity RNA-to-cDNA Master Mix with No-RT Control (Applied Biosystems PN 4390712). One microgram of total RNA from each sample was combined with 4 μl of master mix (includes a mixture of random primers and oligo-dT for priming). Reaction volume was taken to 20 μl with DNase/RNase free distilled water (Invitrogen). Thermal steps: 5′ × 25°C, 30′ × 42°C, and 5′ × 85°C. RT- amplifications were either negative or delayed >5 cycles compared to the corresponding RT+ reactions. Intron-spanning (β-actin) and non-intron-spanning (18S and GAPDH) assays were designed using Probe Finder software (Roche Applied Science). Primers for UbGFP transgene: 5′-GGGCACAAGCTGGAGTACAAC-3′ and 5′-GATGCCGTTCTTCTGCTTGTC-3′. Primers for N-mutHtt: 5′-CGGCTGAGGCAGCAGCGGCTGT-3′ and 5′-GCAGCAGCAGCAGCAACAGCCGCCACCGCC-3′. qPCR reactions were performed in triplicates in 10 μl final volume with the cDNA amount equivalent to 5 ng of total RNA, 250 nm of each primer, and 5 μl of Power Sybr Green PCR Master Mix (Applied Biosystems PN 4367659), which includes AmpliTaq Gold DNA Polymerase. For 18S, GAPDH, β-actin, and UbGFP genes we performed an initial denaturation of 10′ × 95°C followed by 40 two-step cycles (15′ × 95°C + 1′ × 60°C). We also included a melting curve from 60 to 95°C (2% ramp) at the end of the program to verify the specificity of the PCR. Fluorescence was acquired during the 60°C and melting steps. For N-mutHtt, the second step consisted of 34 four-step cycles (30′ × 94°C + 30′ × 68°C + 1′30′ × 72°C + 15′ × 82.6°C). Fluorescence was acquired during the 82.6°C step to avoid coquantification of a secondary peak with a smaller Tm that was detected in the melting curve. SDS 2.2.1 (Applied Biosystems) was used for data analysis. To normalize the data respect to the three control genes (18S, β-actin and GAPDH) two algorithms included in the program Genex (NormFinder and GeneNorm) were used.

Data analysis.

In all in vivo experiments, at least six animals per group were analyzed and data are presented as mean ± SEM. Significant differences between the groups were evaluated using Student t test in the case of parametric population, and Mann–Whitney test in the case of nonparametric populations. In all experiments performed in primary cultures, at least 300 transfected neurons were analyzed. Significant differences between groups in culture experiments were evaluated using two-proportion comparison test.