Materials
Dulbecco’s modified Eagle’s medium, penicillin/streptomycin solution, and fungizone were purchased from Sigma (St Louis, MI, USA). Foetal calf serum was obtained from Nichirei (Tokyo, Japan). The RNA Isolation Kit was obtained from Promega (Madison, WI, USA). Primers for real-time RT-PCR and genotyping were purchased from Invitrogen (Waltham, MA, USA). Reagents for real-time RT-PCR, such as THUNDERBIRD SYBR qPCR Mix, were obtained from Toyobo (Osaka, Japan). Rabbit polyclonal anti-PRDX6 antibody was purchased from Abcam (ab73350; Cambridge, UK). Horseradish peroxidase-conjugated anti-rabbit IgG antibody was obtained from Cell Signaling Technology (7074S; Danvers, MA, USA). Mouse monoclonal anti-neuronal nuclei (NeuN) antibody was obtained from Millipore (MAB377; Burlington, MA, USA). Mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody was purchased from Progen (61011, Heidelberg, Germany). Rabbit polyclonal anti-SOD1 and horse-radish peroxidase-conjugated goat polyclonal anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (sc-11407 and sc-1615HRP, respectively; Dallas, TX, USA). SOD1 human untagged pCMV6-XL5 plasmid and PRDX6 human untagged pCMV6-XL5 plasmid were purchased from Origene (Rockville, MD, USA). MJ33 (lithium[1-hexadecoxy-3-(2,2,2-trifluoroethoxy) propan-2-yl] methyl phosphate), a selective inhibitor of the calcium-independent phospholipase A2 activity of PRDX6, was acquired from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals were obtained from Nacalai Tesque (Kyoto, Japan).
Animals
All mice experiments were performed in accordance with the protocols approved by the Committee for the Ethical Use of Experimental Animals and Safety Committee for Recombinant DNA Experiments at Setsunan University (approval ID: K08-13/08.04.14.2.S.017) and Animal Care and Use Committee at Nihon University (approval no: AP13P001 and AP19PHA026). All efforts were made to minimise animal suffering, reduce the number of animals used, and utilise alternatives to in vivo techniques. Male transgenic (Tg) mice expressing the human SOD1G93A [B6SJL-Tg (SOD1*G93A)1Gur/J; Jackson Laboratories, Bar Harbor, ME, USA] were used as the ALS mice model. The mice were bred with background-matched B6SJL wild-type (WT) female mice in the animal facility. The progeny was genotyped and used for subsequent studies. Genotyping analysis was performed in accordance with the Jackson Laboratory protocols for male Tg mice as previously described [14].
Male mice were used for the experiments throughout this study. All animals were housed in standard metallic mouse cages (19.5 × 29.5 × 15 cm) with a corncob under a 12/12-h light/dark cycle. The humidity was 55%, the temperature was 23 ± 1°C, and food and water were available ad libitum. The rotarod test was performed during the light period. We euthanised the animals when they could no longer right themselves 30 s after being placed on their sides.
Behavioural Test
In this study, we analysed the motor function and coordination of the ALS mice model.
Beginning at 7 weeks old, all animals (n = 19/genotype) were weighed and evaluated for signs of a motor deficit using the accelerated rotarod test. For this test, the time for which an animal could remain on the rotating cylinder of a rotarod apparatus (Muromachi Kikai, Tokyo, Japan) was measured. Each animal was given three tries, and the longest latency to fall was recorded. The apparatus had an initial speed of 6 rpm and gradually accelerated at a rate of 0.11 rpm/s.
Quantitative Real-time Rt-pcr
For the quantitative measurement of PRDX6, cytokines, and C3 mRNA, total RNA was isolated from the lumbar spinal cord (L3-L5) tissues or cultured C8-D1A astrocyte cell line and U-251 MG astrocytoma using the SV Total RNA Isolation System (Promega). One microgram of total RNA was reverse-transcribed with M-MLV Reverse Transcriptase (Invitrogen) and random primers. One-twentieth of the total cDNA (50 ng of equivalent RNA) was used in each amplification reaction. Real-time RT-PCR was performed using Thermal Cycler Dice Real-time System (Takara, Shiga, Japan) and THUNDERBIRD SYBR qPCR Mix. For RT-PCR amplification of PRDX6, IL-1β, IL-6, and C3, an initial amplification was performed with a denaturation step at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s, primer annealing, and primer extension at 60°C for 30 s. For RT-PCR amplification of TNF, an initial amplification was performed with a denaturation step at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s, primer annealing at 55°C for 10 s, and primer extension at 72°C for 20 s. The desired amplification was confirmed by the melting curve analysis. The geometric mean of housekeeping gene β-actin was used as an internal control to normalise the variability in expression levels. Sequences of the primer pairs against mouse were: 5ʹ-GTC GAG AAG GAC GCT AAC AAC-3ʹ (PRDX6 forward) and 5ʹ-GGG TAG AGG ATA GAC AGC TTC AG-3ʹ (PRDX6 reverse); 5ʹ-GGT GCC TAT GTC TCA GCC TCT T-3ʹ (TNF forward) and 5ʹ-GCC ATA GAA CTG ATG AGA GGG AG-3ʹ (TNF reverse); 5ʹ-CCT CTC CAG CCA AGC TTC CT-3ʹ (IL-1β forward) and 5ʹ-TTT GGA AGC CCT TCA TC-3ʹ (IL-1β reverse); 5ʹ-TCC TAC CCC AAT TTC CAA TGC-3ʹ (IL-6 forward) and 5ʹ-CAT AAC GCA CTA GGT TTG CCG-3ʹ (IL-6 reverse); 5ʹ-CAC CGC CAA GAA TCG CTA C-3ʹ (C3 forward) and 5ʹ-GAT CAG GTG TTT CAG CCG C-3ʹ (C3 reverse); and 5ʹ-AGT GTG ACG TTG ACA TCC GTA-3ʹ (β-actin forward) and 5ʹ-GCC AGA GCA GTA ATC TCC TTC T-3ʹ (β-actin reverse). Sequences of the primer pairs against human were: 5ʹ-CGT GTG TTT GTT TTT GG-3ʹ (PRDX6 forward) and 5ʹ-TCT TCT TCA GGG ATG GTT GG-3ʹ (PRDX6 reverse); 5ʹ-CCC AGG CAG TCA GAT C-3ʹ (TNF forward) and 5ʹ-AGC TGC CCC TCA GCT T-3ʹ (TNF reverse); 5ʹ-AAT TTG AGT CTG CCC A-3ʹ (IL-1β forward) and 5'-AGT CAG TTA TAT CCT GGC CGC C-3ʹ (IL-1β reverse); 5ʹ-GTG GAA ATC CGA GCC GTT CTC T-3ʹ (C3 forward) and 5ʹ-GAT GGT TAC GGT CTG GTG A-3ʹ (C3 reverse); and 5ʹ-ACC GAG CGC GGC TAC AG-3ʹ (β-actin forward) and 5'-CTT AAT GTC ACG CAC GAT TTC C-3ʹ (β-actin reverse). The results of real-time RT-PCR were analysed using the second derivative maximum method.
Immunofluorescence
Mice were anaesthetised by the intraperitoneal administration of a single dose of MMB mixture (0.3 mg/kg medetomidine, 4 mg/kg midazolam, and 5 mg/kg butorphanol) and perfused by cardiac puncture with 4% paraformaldehyde/phosphate-buffered saline. We removed the lumbar spinal cords (L3-L5) and embedded them in paraffin for sectioning (at 5 µm thickness). After deparaffinising the sections with xylene, they were subjected to antigen retrieval by microwave irradiation (500 W, 10 min) in citric acid buffer (10 mM citric acid and 0.01% Tween-20) and hydrolysed with ethanol and water. After cooling, the sections were washed with Tris-buffered saline (TBS) containing 0.1% Triton X-100 and blocked with a blocking buffer at 20 ± 5°C for 30 min. The sections were incubated with antibodies against PRDX6 (1:200, rabbit, ab73350) and mouse monoclonal NeuN (1:200, mouse, MAB377) or mouse monoclonal anti-GFAP (1:100, mouse, 61011) at 4°C overnight. After washing with TBS containing 0.1% Tween-20, the sections were incubated with appropriate secondary antibodies at 4°C overnight. Secondary antibodies used were Alexa568-conjugated anti-rabbit IgG [1:200; Invitrogen (A11036)], biotin-labelled anti-mouse IgG [1:200; Dako (E0433), Santa Clara, CA, USA], and FITC-conjugated streptavidin [1:200; BD Bioscience (554060), Wyckoff, NJ, USA]. Immunostained sections were photographed using a fluorescence microscope (AZ-100M; Nikon, Tokyo, Japan), and preimmunised rabbit immunoglobulins were used as a negative control to confirm specific staining.
Cell Culture
The C8-D1A astrocyte cell line (astrocytic type I clone, mouse monoclonal anti-GFAP positive) cloned from the mouse cerebellum was obtained from the American Type Culture Collection (Manassas, VA, USA). U-251 MG cells, a human astrocytoma cell line, were obtained from the Japanese Cancer Research Resources Bank (Osaka, Japan). These cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated foetal calf serum, 100 µg/mL streptomycin, 100 IU/mL penicillin, and 1 µg/mL fungizone. Cells were maintained as monolayers at 37°C with 5% CO2 air as previously described [15]. For transfection, U-251 MG and C8-D1A cells were seeded on a 24-well plate at 1 × 105 cells/well and 2.0 × 105 cells/well, respectively.
Generation Of Sod1 Mutation
We introduced the SOD1 mutation (G93A) by site-directed mutagenesis using the PrimeSTAR Mutagenesis Basal Kit (Takara) according to the manufacturer’s instructions. We designed a primer in which the 281st guanine in the CDS region was replaced with cytosine to generate SOD1G93A. We used mutagenesis primers (Forward: 5ʹ-AAA GAT GCT GTG GCC GAT GTG TCT ATT GAA GAT-3ʹ, Reverse: 5ʹ-GGC CAC AGC ATG TTT GTC AGC AGT CAC-3ʹ), PrimeSTAR Mix, and a Thermal Cycler System (Bio-Rad, Hercules, CA, USA) to generate the SOD1G93A plasmid. For SOD1G93A generation, an initial amplification was performed with a denaturation step at 98°C for 10 s, followed by 35 cycles of denaturation at 98°C for 10 s, primer annealing at 55°C for 15 s, and primer extension at 72°C for 30 s. We transformed a competent cell (DH5α) by PCR product introduction and prepared a plasmid. Analysis of the sequence of the prepared plasmid was performed by Invitrogen, and we confirmed that the mutation was introduced into the SOD1 sequence.
Cell Transfection And Stable Cell Line Screening
Transfection was performed using Fugene HD transfection reagent (Promega). Twenty-four hours after seeding U-251 MG and C8-D1A cells, the cells were supplemented with 0.5 µg of plasmid per well [plasmid (µg): Fugene HD (µL) = 2:7]. Forty-eight hours after transfection, the cells were digested with 0.25% trypsin, and the cultures were transferred to plates for culture with Dulbecco’s modified Eagle’s medium containing 500 µg/mL G418 and 10% foetal calf serum for 30 days. The culture medium was replaced every 3 days. When resistant cell clones were observed, several individual cell clones were picked by the cloning cylinder, digested with 0.25% trypsin, and transferred to a new culture flask using an aseptic pipette for further culture. Established stable cell lines (clones) were seeded on a 24-well plate at 2.0 × 105 cells/well for real-time RT-PCR.
Western Blotting
Western blotting was performed as previously described [15, 16]. Cells were lysed in lysis buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulphate]. Equal amounts of protein (10 µg) were subjected to SDS-PAGE. After protein separation by electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane (Millipore). Nonspecific sites were blocked with 1% non-fat milk, and blots were incubated overnight at 4°C with rabbit polyclonal anti-SOD1 (1:1000) or anti-PRDX6 (1:1000) antibodies. After washing with TBS containing 0.5% Tween-20, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000) for 2 h at room temperature. Bands were detected using the enhanced chemiluminescence system, stripped with western blot stripping solution for 15 min at 20 ± 5°C and reproved with horse-radish peroxidase-conjugated anti-β-actin antibody (loading control).
Statistical analysis
Data are expressed as the mean ± standard error of the mean of 3–12 independent experiments. Statistical significance was evaluated using the two-tailed unpaired Student’s t-test for comparisons between groups for animal experiments or the one-way analysis of variance followed by the Tukey-Kramer test for multiple comparisons for real-time RT-PCR. For behavioural assessments, statistical significance was evaluated using the two-way analysis of variance followed by Dunnett’s test. All statistical analyses were performed using SPSS software PASW Statistics (18.0.0). P < 0.05 indicated a statistically significant difference.