Drug
Trehalose was obtained from Pfanstiehl Inc (Illinois, USA) as trehalose dyhydrate, a white crystalline powder. In its solid form was stored at room temperature in a ventilated area. For in vitro and in vivo studies was prepared in water, as specified in the following sections.
Cell culture and treatments
The mouse neuroblastoma cell line (Neuro-2A cells) was obtained from the American Type Culture Collection cell biology bank (CCL-131) and maintained in Dulbecco’s modified Eagle’s medium (complete medium; Gibco) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin, at 37 °C in 5% CO2/air atmosphere. Neuro-2A transduction with lentiviral particles expressing mutant ataxin-3 72 CAG repeats (MutAtx-3) was performed as previously described [8]. Two weeks post-infection, cells were plated and treated with trehalose (1 mM, 10 mM or 100 mM) or control (vehicle) for 0.5 h, 1 h, 3 h, 6 h, 24 h, 48 h and 72 h. Treatments started 24 h after platting and medium was changed after 48 h. Trehalose incubations were repeated every 24 h and cells from all conditions were collected 72 h after platting.
Resazurin assay
Cells were incubated with 0.1 mg/mL resazurin solution, diluted 1:10 in DMEM culture medium, for 45 to 60 min at 37 °C. From each well, 100 µl triplicates were placed into a 96-well plate and the absorbance was read at wavelengths of 570 nm and 600 nm. The absorbance ratio 570/600 nm was calculated and expressed as percentage of control.
RNA extraction and RT-qPCR
Total RNA was extracted by using the NucleoSpin® RNA extraction kit, according to manufacturer’s instructions (Macherey–Nagel). RNA was eluted in 40 µl of nuclease-free water and total RNA was quantified by optical density (OD) using a Nanodrop 2000 Spectrophotometer (Thermo Scientific). RNA purity was evaluated by measuring the ratio of OD at 260 and 280 nm. 1000 ng of total RNA were treated with DNase (Qiagen) and then reverse transcribed into double stranded cDNA by using the iScript cDNA Synthesis Kit® (BioRad). cDNA samples were stored at − 20 °C until use.
Real-time PCR was performed using a standard SYBR-Green® PCR kit protocol on a StepOne® Detection System (Life Technologies). RT-PCR was carried out in 10 µL reaction, which included cDNA product (diluted 1:10), 1× SsoAdvanced SYBR Green Supermix and 0.5 µM of forward and reverse primers. Primers for SIRT1, LC3B, p62 and Beclin-1 were pre-designed and validated by QIAGEN (QuantiTect Primers, QIAGEN). Primers for mutant ataxin-3 and HPRT-1 were designed and validated by our group. Reverse-transcription and non-template controls were run in parallel. All reactions were performed in duplicate, according to the manufacturer’s recommendations: 95 °C for 30 s, followed by 45 cycles at 95 °C for 5 s and 60 °C for 30 s. HPRT-1 was used as reference gene. The mRNA fold increase or fold decrease with respect to control samples was determined by the delta-delta Ct method.
Animals and experimental groups
Transgenic mouse model
A colony of MJD transgenic mice (MJDTg; C57BL/6 background) expressing the N-terminal-truncated human ataxin-3 with 69 glutamine repeats and a N-terminal hemagglutinin (HA) epitope, driven specifically in cerebellar Purkinje cells by the L7 promoter [21], was maintained at CNC animal facility by backcrossing heterozygous males with C57BL/6 females. For this experiment, 14 MJD transgenic female mice were weaned and genotyped at 4 weeks of age. Females have a less aggressive phenotype than males (unpublished data) and were therefore used in this experiment to avoid the use of weakened animals that could reach the humane critical endpoints over the long course of the experiment. Animals were housed in groups (2–5 per cage, depending on cohort study) in plastic cages (365 × 207 × 140 mm) with food and water ad libitum, and maintained on a 12-h light/dark cycle at a room with constant temperature (22 ± 2 °C) and humidity (55 ± 15%). The animals were allowed 1 week of acclimation to the surroundings before the beginning of the behavioral tests. Physical state of animals was evaluated daily and weight measured every week.
MJD Tg mice from different progenitors were randomly distributed into control and treatment group (7 animals/group) and then tested for behavior background before beginning the treatment. Trehalose was orally administered diluted in drinking distilled water at a final concentration of 2% (w/v) to the treatment group, from week 5 to week 35 of age. The control group was treated with vehicle (distilled water). A fresh solution of 2% Trehalose in water was prepared and changed twice a week until euthanasia of the animals.
Lentiviral-based mouse model
Twenty-five C57/Bl6 mice (Charles River, France), males, 8 weeks old, were housed in groups of 6 per cage, in plastic cages (365 × 207 × 140 mm) with food and water ad libitum, and maintained on a 12-h light/dark cycle at a room with constant temperature (22 ± 2 °C) and humidity (55 ± 15%). Males were used in this experiment to ensure comparability with previous studies using this model. The animals were stereotaxically-injected in the striatum with lentiviral vectors encoding mutant human ataxin-3 with 72 CAG repeats, as previously described [22]. After recovery from surgery, mice were randomly distributed into control (n = 13) and treatment groups (n = 12). The treatment group was orally-administered with trehalose diluted in drinking water at a final concentration of 2% (w/v) and the control group was treated with the vehicle (water). After 2 and 4 weeks of drug administration, mice were euthanized and brains collected for analysis (western bot and immunohistochemistry, respectively).
Behavioral testing
Mice were trained on a battery of motor tests starting at 4 weeks of age (P21–25) and tested for behavioural background at 5 weeks of age (before the beginning of trehalose treatment, t = 0). Behaviour was then evaluated at different time points (t = 2, t = 6, t = 11, t = 14, t = 17, t = 20, t = 24 and t = 28 weeks of treatment) by an experienced operator in a blind fashion way. All tests were performed in the same dark room after at least 60 min of acclimatization.
Rotarod performance test
Motor coordination and balance were evaluated in a rotarod apparatus (Letica Scientific Instruments, Panlab, Barcelona, Spain). Mice were placed on the rotarod at a constant speed (5 rpm) and at accelerated speed (4 to 40 rpm in 5 min) and the latency to fall was recorded for a maximum of 5 min. Mice were allowed to perform four trials for each test and time point, with at least 30 min rest between trials. For analysis, the mean latency to fall off the rotarod of 4 trials was used.
Grip strength test
A grip strength test was performed to assess mice neuromuscular function. The grip strength of forelimbs was determined using a device consisted of a 300-g metal grid with a scale on. The animal was hung with its forepaws on the grid. The strength was determined as the weight pulled (g) from the scale. The test was performed during 10–15 consecutive trials and the mean of four best performances was taken to analysis. Mice body weight was used as a normalization factor.
Footprint analysis
Footprint analysis was performed at 28 weeks post-initiation of treatment. To obtain footprint patterns, mice front and forefeet were coated with blue and red non-toxic paints, respectively. Mice were allowed to walk on a blank greenish paper along a 100 cm long, 10 cm wide runway (with 15 cm high walls). Stride length was measured as the average distance of forward movement between each stride. Frontbase width and hindbase width were measured as the average distance between left and right front and hind footprints, respectively. These values were determined by measuring the perpendicular distance of a given step to a line connecting its opposite preceding and proceeding steps. The distance from left or right front footprint/hind footprint overlap was measured to evaluate uniformity of step alternation. A sequence of five consecutive steps was chosen for evaluation, excluding footprints made at the beginning and at the end of the run. Measurements were all made by the same operator. The mean value of each set of five was considered for each animal.
Brain tissue collection
For histological analysis, the animals were given an avertin overdose (2.5 × 200 mg/g, i.p.) and were transcardially perfused with a phosphate solution (0.1 M) followed by fixation with 4% paraformaldehyde (PFA; Fluka, Sigma, Buchs, Switzerland). The brains were removed, post-fixed in 4% PFA for 24 h at 4 °C, and then cryoprotected by immersion in 25% sucrose/phosphate buffer for 48 h at 4 °C. The brains were frozen at − 80 °C and then the entire cerebellum (transgenic model) or brain (lentiviral-based model) was sliced into 30-μm midsagittal or 25-μm coronal sections, respectively, using a cryostat (LEICA CM3050S, Leica Microsystems) at − 21 °C. Sections were collected in anatomical series and stored at 4 °C as free-floating sections in phosphate buffered saline (PBS) supplemented with 0.05 mM sodium azide until processing.
For western blot analysis (lentiviral-based model), animals were euthanized by cervical dislocation. Tissue punches from striatum were collected and keep at -80 °C until use.
Histological analysis of brain tissue
Cresyl violet staining
Eight sagittal sections along the extent of the mice left hemicerebellum (inter-section distance of 240 µm, which corresponds to eight sections of 30 µm) were mounted in gelatin covered microscope slides and dried at room temperature. Sections were then stained with cresyl violet for 5 min, differentiated in 70% ethanol, dehydrated by passing through 95% ethanol, 100% ethanol and xylene solutions and mounted with Eukitt (Sigma-Aldrich).
Immunofluorescent staining
Immunofluorescent staining was performed in eight sagittal sections over the extent of the mice left hemicerebellum (inter-section distance of 240 µm, which corresponds to eight sections of 30 µm). Free-floating sections were washed with PBS 0.1 M and blocked for 1 h at room temperature in 0.3% Triton X-100 in PBS 0.1 M supplemented with 10% normal goat serum (NGS; Gibco). Sections were then incubated with the following primary antibodies: mouse monoclonal anti-HA antibody (1:1000; InvivoGen, San Diego, CA, USA) and rabbit polyclonal anti-calbindin-28 K antibody (1:1000; Merck Millipore) overnight at 4 °C. After incubation, sections were washed three times with PBS and then incubated with the corresponding secondary antibody goat anti-mouse conjugated to the fluorophore 488 (1:200; Molecular Probes, Oregon, USA), diluted in blocking solution, for 2 h, at room temperature. Finally, sections were washed three times in PBS, counterstained with 4′,6-diamidino-2-phenylindole, washed again and mounted with Fluorsave (Calbiochem, Germany).
Immunohistochemistry
Immunohistochemistry was performed in twelve coronal sections covering the extent of the mice striata (25 μm-thick sections at 200 μm intervals). After the blockage of endogenous peroxidases with phenylhidrazyne/phosphate solution, free-floating sections were washed with PBS 0.1 M and blocked for 1 h at room temperature in blocking solution (0.1% Triton X-100 in PBS 0.1 M supplemented with 10% normal goat serum). Sections were processed overnight at 4 °C in blocking solution with the following primary antibodies: a polyclonal rabbit anti-ubiquitin antibody (1:300; Enzo Life Sciences) and a polyclonal rabbit anti–DARPP-32 antibody (1:1000; Merck Millipore), followed by 2-h incubation at room temperature with the respective biotinylated goat anti-mouse or anti-rabbit antibodies (1:200; Vector Laboratoires). Bound antibodies were visualized using the Vectastain ABC kit, with 3,30-diaminobenzidine tetrahydrochloride (DAB metal concentrate, Pierce) as substrate. Dry sections were mounted in gelatin-coated slides, dehydrated with ethanol solutions and xylene and mounted in Eukit (Sigma-Aldrich).
Quantitative analysis of histological sections
Quantification of cerebellum volume (transgenic model)
Quantification was made over eight cresyl violet staining sagittal sections over the extent of the mice left hemicerebella in a blind fashion. Mosaic pictures of these sections were taken using a PALM Laser microdissection microscope (Carl Zeiss, Germany) with a 20× objective. Volume was assessed by measuring the area of the cerebellum in each section using Fiji software. Hemicerebellum final volume was extrapolated using the following formula: volume = (area × section thickness) × number of sections.
Quantification of cerebellum layers thickness (transgenic model)
Quantification was made over four cresyl violet-stained sagittal sections. Mosaic pictures were taken using a PALM Laser microdissection microscope (Carl Zeiss, Germany) with a 20× objective. For each section, layers length was blindly determined using Fiji software. Layers thickness was assessed by measuring the mean width of cerebellum layers (molecular + granular + Purkinje cell layers) at interlobular regions.
Quantification of Purkinje cells and mutant ataxin-3 aggregates (transgenic model)
Quantitative analysis of number of Purkinje cells (calbindin-positive cells) and mutant ataxin-3 aggregates (HA aggregates) was performed over eight sections. To calculate the number of calbindin-positive cells, mosaic pictures were taken using a PALM Laser microdissection microscope (Carl Zeiss, Germany) with a 20× objective. Images were visualized with ImageJ software and cells were then manually counted by an operator in a blind fashion. Total number (no) of purkinje cells per hemicerebellum was calculated by extrapolation, using the formula:
$${\text{no}}\;{\text{of}}\;{\text{purkinje}}\;{\text{cells}}\, = \,\left( {{\text{no}}\;{\text{of}}\;{\text{cells in section 1}}\, + \,{\text{no of cells in}}\;{\text{section}}\;2\, + \, \cdots \, + \,{\text{no of cells}}\;{\text{in}}\;{\text{section}} \; 8} \right) \times 8 {\text{ sections}}$$
Total number of HA aggregates was manually counted by an operator in a blind fashion by visualizing immunostained sections using a Axioskop 2 plus microscope (Carl Zeiss) and calculated by extrapolation, using the formula:
$${\text{no of mutant ataxin{-}3 aggregates}}\, = \,\left( {{\text{no of aggregates in section 1}}\, + \,{\text{no of aggregates in}}\;{\text{section 2}} + \cdots + {\text{no of aggregates in}}\;{\text{section 8}}} \right) \times 8 {\text{ sections}}$$
Quantification of mutant ataxin-3 aggregates size (transgenic model)
Quantitative analysis of mutant ataxin-3 aggregates size was performed over four sections. Three representative pictures of lobule IX were taken using a Cell Observer Spinning Disk (Carl Zeiss, Germany) with a 100× objective. Z projections were visualized and the diameter was manually determined by an operator in a blind fashion using Fiji software.
Quantification of DARPP-32 depleted volume, ubiquitin inclusions number and size (lentiviral model)
Quantification was made over twelve coronal sections per animal (25 μm-thick sections at 200 μm intervals) on the extent of the mouse striatum, in a blind fashion. Pictures of DARPP-32 stained sections were taken using a 5× objective using an Axioskop 2 Plus microscope (Carl Zeiss, Germany). Volume was assessed by measuring the area of the lesion in each section using Fiji software. Final volume was extrapolated as described above. Pictures of ubiquitin-stained sections were taken using a 20× objective, with the same microscope. The analyzed areas of the striatum encompassed the entire region containing ubiquitin aggregates. All inclusions were blindly automatically counted using Fiji software. Total number of ubiquitin aggregates was extrapolated as described for ataxin-3 aggregates. Data were normalized to the integrated lentivirus copy number, quantified as described below.
Protein extraction and preparation
Cellular extracts were obtained by scrapping cells with radioimmunoprecipitation assay (RIPA) buffer solution [50 mM Tris·HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate (SDS)] supplemented with protease inhibitors (Roche), 200 μM phenylmethylsulphonylfluoride, 1 mM dithiothreitol (DTT), 1 mM Na3VO4 and 10 mM NaF.
Tissue extracts were lysed in RIPA buffer, supplemented as described above, by 2 series of 4 s ultra-sound pulse (1 pulse/s). Total protein lysates were stored at − 20 °C and protein concentration was quantified with BCA protein assay (Pierce Biotechnology, Thermo Scientific, USA).
Western blotting
Protein samples were denaturated in SDS sample buffer (0.5 M Tris, 30% glycerol, 10% SDS, 0.6 M DTT, 0.012% bromophenol blue) for 5 min at 95 °C. Samples were then separated in a 4–12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Ponceau S staining was performed after transference and the membranes were blocked with 5% non-fat milk in TBS-T (137 mM NaCl, 20 mM Tris, 0.1% Tween 20, pH 7.6) following by incubation overnight at 4 °C with mouse monoclonal anti-β-actin antibody (clone AC74;1:5000; Sigma-Aldrich), mouse monoclonal anti-β-tubulin antibody (clone SAP.4G5;1:10,000; Sigma-Aldrich), mouse anti-GAPDH (1:500; Merck Millipore), mouse monoclonal anti-HA antibody (1:1500; InvivoGen, San Diego, CA, USA), rabbit monoclonal anti-p62 antibody (1:1500; Cell Signaling Technology) and rabbit monoclonal anti-LC3B antibody (1:1000; Cell Signaling Technology) diluted in blocking solution or in 3% bovine serum albumin (for cell signaling antibodies). After three washes with TBS-T, the membranes were incubated for 1 h, at room temperature, with an alkaline phosphatase-linked secondary antibody, specific to rabbit or mouse immunoglobulin G (1:20,000, Amersam Biosciences, GE Healthcare, UK). Immunoreactive bands were visualized using enhanced chemifluorescence (ECF) substrate in the Versa-Doc 3000 imaging system (Bio-Rad, USA) and densitometry of the bands was quantified using ImageJ software. The specific optical density was normalized to the total protein, measured by ponceau S, or to the amount of β-actin [23].
Integrated lentivirus copy number quantification
Twelve mounted histological slides were immersed in xylene for 2 days and then hydrated with ethanol solutions and water. Lentiviral-injected striata were removed with a scalpel and DNA extraction was performed using the GeneRead DNA FFPE Kit (Qiagen), starting from step 6 of manufacturer’s protocol. Purified cellular genomic DNA was then quantified by optical density (OD) using a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and the purity was evaluated by measuring the ratio of OD at 260 and 280 nm. Copy number of integrated lentiviruses (proviruses) present in the transduced cells of striatum was detected by qPCR using the Lenti-X Provirus Quantitation Kit (Takara) and according to the manufacturer’s instructions. Briefly, serial dilutions of the cellular gDNA were subjected to qPCR amplification alongside dilutions of a calibrated Provirus control template. The final result was expressed in terms of provirus copies (vg)/cell.
Statistical analysis
Raw data analysis was conducted using Prism GraphPad software. Outliers were excluded from analysis using Grubb’s test. For behavior results, mean values for each animal were calculated and a two-tailed Student’s t test (footprint analysis) or a trend analysis to compare linear regression slopes using the two-tailed t test hypothesis (other behavioral tests) was performed. For other analysis, statistics was performed using the two-tailed Student’s t test. Data were represented as mean ± SEM.