An Increase in Peroxiredoxin 6 Expression Induces Neurotoxic A1 Astrocytes in the Lumbar Spinal Cord of Amyotrophic Lateral Sclerosis Mice Model

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease with selective degeneration of motor neurons. It has been reported that an increase in the levels of inflammatory cytokines and glial cells such as reactive astrocytes is closely involved in the pathological progression of ALS. Recently, the levels of neuropathic cytotoxic (A1) astrocytes among reactive astrocytes have reportedly increased in the central nervous system of ALS mice, which induce motor neuron degeneration through the production of inflammatory cytokines and secretion of neuropathic factors. Hence, elucidating the induction mechanism of A1 astrocytes in ALS is important to understand the mechanism of disease progression in ALS. In this study, we observed that the expression of peroxiredoxin 6 (PRDX6), a member of the peroxiredoxin family, was markedly upregulated in astrocytes of the lumbar spinal cord of SOD1G93A mice model for ALS. Additionally, when PRDX6 was transiently transfected into the mouse astrocyte cell line C8-D1A and human astrocytoma cell line U-251 MG, the mRNA expression of complement C3 (a marker for A1 astrocyte phenotype) and inflammatory cytokines was increased. Furthermore, the mRNA expression of C3 and inflammatory cytokine was increased in C8-D1A and U-251 MG cells stably expressing PRDX6, and the increased mRNA expression was significantly suppressed by MJ33 (lithium[1-hexadecoxy-3-(2,2,2-trifluoroethoxy) propan-2-yl] methyl phosphate), an inhibitor of the phospholipase A2 activity of PRDX6. Our results suggest that the expression of PRDX6 in astrocytes plays an important role in the induction of A1 astrocytes and expression of inflammatory cytokines in the ALS mice model.


Introduction
Amyotrophic lateral sclerosis (ALS) is a serious neurodegenerative disease that causes the degeneration of peripheral motor nerves and damage to central motor neurons, resulting in muscle weakness and atrophy [1].Approximately 90% of ALS is sporadic, and 10% is familial (hereditary).To date, ALS1 (Cu/Zn superoxide dismutase-1: SOD1) has been discovered as the causative gene of familial ALS [2].In patients with ALS, an increase in reactive astrocyte levels has been observed in lumbar spinal cord lesions, and an increase in inflammatory cytokine levels, such as those of tumour necrosis factor (TNF) and interleukin-1β (IL-1β), has been reported in the cerebrospinal fluid [3].These observations suggest that the increase in reactive astrocyte and inflammatory cytokine levels is deeply involved in the pathological progression of ALS [4].A study has reported that there are two forms of reactive astrocytes: neurotoxic astrocytes (A1) and neuroprotective astrocytes (A2).A1 astrocytes produce inflammatory cytokines and secrete neurotoxic factors [5].In addition, an increase in the number of A1 astrocytes has been reported in the lumbar spinal cord of the ALS mice model [6,7]; however, their induction mechanism is unclear.
Peroxiredoxins (PRDXs) are a ubiquitous and abundant family of proteins important for the antioxidant defence system and regulation of cell signalling pathways [8].In mammals, six different types of PRDXs (PRDX1-PRDX6) are expressed by different genes [9].PRDX6 is selectively highly expressed in astrocytes of the central nervous system [10] and has calcium-independent phospholipase A 2 activity [11].Therefore, PRDX6 is thought to play an important role in the progression of inflammation via the modulation of the arachidonic acid cascade in the central nervous system [12].A study has reported that chronic inflammation in the central nervous system plays an important role in the pathological progression of ALS [13].
In this study, we examined the expression of PRDX6 in the lumbar spinal cord of ALS mice and role of PRDX6 in the induction of A1 astrocytes.

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 SOD1 G93A [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.

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% CO 2 air as previously described [15].For transfection, U-251 MG and C8-D1A cells were seeded on a 24-well plate at 1 × 10 5 cells/well and 2.0 × 10 5 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 SOD1 G93A .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 SOD1 G93A plasmid.For SOD1 G93A 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].48 h 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 × 10 5 cells/well for real-time RT-PCR.

FACS Analysis
To identify subpopulations of A1/A2 astrocytes, we used as a marker of C3 for A1 astrocyte and S100a10 for A2 astrocyte.48 h after the transfection of C8-D1A cells with the PRDX6 expression vector, these cells were fixed in 4% PFA/PBS for 15 min at 4 °C and then passed through a nylon mesh (40 μm).The cells were permeabilized with 0.1% tween-20 containing PBS for 6 h at 4 °C, and then treated with Alexa Fluor 647-conjugated anti-C3 (Novus biologicals, CO, USA) and Alexa Fluor 488-conjugated anti-S100a10 (R and D systems, MN, USA) antibodies in 1% bovine serum albumin containing PBS for 24 h.The number of C3 positive cells and S100a10 positive cells were determined by flow cytometry (BD FACSAriaTM Fusion Cell Sorter instrument) and analysed using FACSDIVA software.Obtained events were gated in a Forward scatter (FSC) intensity and Side scatter intensity dot plot to eliminate debris.Cells were gated on an FSC intensity and FSC peak dot plot to eliminate doublet.A minimum of 10,000 cells with in the gated region were analysed.

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.

PRDX6 Expression is Increased in Astrocytes of the Lumbar Spinal Cord of the ALS Mice Model
Male Tg mice showed significant weight loss from 15 weeks of age compared with age-matched WT mice (Fig. 1a, P < 0.05).In the rotarod test, male Tg mice showed a significant reduction in the time spent on the rod from 18 weeks of age compared with age-matched WT mice (Fig. 1b, P < 0.05).By the age of 23 weeks, most male Tg mice developed hindlimb paralysis and could not get up.
Thereafter, we examined the expression of PRDX6 mRNA in the lumbar spinal cord (L3-L5) of WT and male Tg mice.Real-time RT-PCR analysis revealed no significant difference in the level of PRDX6 mRNA in 5-and 10-week-old male Tg mice compared with age-matched WT mice; however, the level of PRDX6 mRNA in 15-and 20-week-old male Tg mice was significantly higher than that in age-matched WT mice (Fig. 2a, P < 0.01).Next, we examined the localisation of PRDX6 in the lumbar spinal cord of 20-week-old WT and male Tg mice using immunofluorescence.NeuN (a marker for neurons) immunoreactivity was observed in grey matter, where neuronal cell bodies were localised, in both WT and male Tg mice in a round shape.In contrast, PRDX6 immunoreactivity was observed in the white and grey matter in the lumbar spinal cord of WT and male Tg mice in a spindle shape (Fig. 2b).The localisation of PRDX6 was not consistent with that of the NeuN in the grey matter of the lumbar spinal cord of both WT and male Tg mice (Fig. 2b).In WT mice, GFAP (a marker for reactive astrocytes) immunoreactivity was observed in the white and grey matter of the lumbar spinal cord in a spindle shape (Fig. 2c).The numbers of GFAP-and PRDX6immunoreactive cells in the white and grey matter of the lumbar spinal cord were higher in male Tg mice than in WT mice.PRDX6 immunoreactivity was colocalised with GFAP-immunoreactive cells in the white and grey matter of the lumbar spinal cord, suggesting that PRDX6 expression is increased in the astrocytes of the lumbar spinal cord of the ALS mice model.

Neurotoxic Reactive Astrocytes and Inflammatory Cytokines are Increased in the Lumbar Spinal Cord of the ALS Mice Model
We investigated the mRNA expression of inflammatory cytokines and C3, a marker molecule for A1 astrocyte, in the lumbar spinal cord of 20-week-old WT and male Tg mice using real-time RT-PCR.mRNA levels of TNF, IL-1β, and IL-6 were higher in the lumbar spinal cord of male Tg mice than in that of WT mice (Fig. 3a−c, P < 0.05).In addition, the C3 mRNA level was higher in the lumbar spinal cord of male Tg mice than in that of WT mice (Fig. 3d, P < 0.05), suggesting that A1 astrocytes were induced in the lumbar spinal cord of male Tg mice and that the production of inflammatory cytokines was increased in A1 astrocytes.

mRNA Expression Levels of PRDX6, C3, and Inflammatory Cytokines are Increased by ALS Causative Gene SOD1 G93A Transduction in Cultured Astrocytes
We examined the effect of mutant human G93A SOD1 (mSOD1) expression on the mRNA expression of PRDX6, inflammatory cytokines, and C3 in C8-D1A cells.48 h after the transfection of C8-D1A cells with the WT SOD1 or mSOD1 expression vectors, the protein expression of human SOD1 was confirmed by western blotting (Fig. 4a).The mRNA expression of PRDX6 was significantly increased by the transient expression of WT SOD1 (Fig. 4b, P < 0.05) or mSOD1 (Fig. 4b, P < 0.01).The mRNA expression of TNF, IL-6, and C3 was also significantly increased by the expression of WT SOD1 (Fig. 4c−e, P < 0.05) or mSOD1 (Fig. 4c−e, P < 0.01).mSOD1 expression induced higher mRNA expression levels of PRDX6, TNF, IL-6, and C3 than WT SOD1 expression (Fig. 4b−e, P < 0.05).

Transfection of Cultured Astrocytes with PRDX6 Expression Vector Causes an Increase in mRNA Expression Levels of Inflammatory Cytokines and C3
We investigated whether PRDX6 is involved in the induction of A1 astrocytes and expression of inflammatory cytokines in U-251 MG cells and C8-D1A cells.48 h after the transfection of U-251 MG cells with the human PRDX6 expression vector, we observed an approximately 15-fold Data represent the mean ± SEM.Statistical analysis was performed using the two-tailed unpaired Student's t test.b Motor coordination was assessed in mice using the rotarod test (n = 7-12).Data represent the mean ± SEM.Statistical analysis was performed using two-way analysis of variance followed by Dunnett's test (*P < 0.05, **P < 0.01 vs. age-matched WT mice) increase in the PRDX6 mRNA expression level compared with endogenous PRDX6 mRNA (Fig. 5a, P < 0.01).The transient expression of PRDX6 significantly increased the mRNA expression levels of IL-1β and TNF in U-251 MG cells (Fig. 5b and c, P < 0.01).C3 mRNA expression was significantly increased by the transient expression of PRDX6 in U-251 MG cells (Fig. 5d, P < 0.01), while the expression of S100a10 mRNA, a marker of A2 astrocytes, was unchanged (Fig. 5e).Transfection of C8-D1A cells with the human PRDX6 expression vector led to the expression of human PRDX6 mRNA in the cells (Fig. 5f, P < 0.01).The transient expression of PRDX6 significantly increased the mRNA levels of IL-6 and TNF in C8-D1A cells (Fig. 5g  and h, P < 0.01).C3 mRNA expression was also significantly increased by the transient expression of PRDX6 in C8-D1A cells (Fig. 5i, P < 0.01), while the S100a10 mRNA  5j), suggesting that the increased expression of PRDX6 mRNA in cultured astrocytes was related to the induction of A1 astrocytes and increase in the mRNA expression levels of inflammatory cytokines.
Furthermore, we examined the effect of the transfection of PRDX6 on the induction of A1/A2 astrocytes using FACS analysis in C8-D1A cells (Fig. 6a and e).The results showed that the percentage of C3 high /S100a10 low (Q4) cells was 44.0% in C8-D1A cells treated with a cultured medium prepared from microglia without LPS treatment (MCM; Fig. 6d), whereas the percentage of C3 high /S100a10 low (Q4) cells increased to 62.4% with a cultured medium prepared from LPS-treated microglia (LPS-MCM), which is known to induce A1 astrocytes (Fig. 6e) [5].Similarly, the percentage of C3 high / S100a10 low (Q4) cells was 42.0% in C8-D1A cells transfected with empty vector (Fig. 6b), whereas the percentage of C3 high / S100a10 low (Q4) cells increased to 71.1% in C8-D1A cells transfected with PRDX6 expression vector (Fig. 6c).However, the percentage of C3 high /S100a10 high (Q2) cells and the percentage of C3 low /S100a10 high (Q1) cells were not increased in LPS-MCM-treated C8-D1A cells (Fig. 6e) and in PRDX6 transfected C8-D1A cells (Fig. 6c), suggesting that PRDX6 does not affect the induction of A2 astrocytes in C8-D1A cells, but may only be involved in the induction of A1 astrocytes.

Discussion
Many researchers have adopted male Tg mice expressing the human SOD1 G93A [B6SJL-Tg (SOD1*G93A)1Gur/J] as an animal model of ALS because the morphological and cytological changes in the spinal cord in this model are similar to those observed in patients with ALS [17].It has been reported that weight loss and loss of motor function occur in the ALS mice model [18][19][20].Consistent with previous reports, weight loss in SOD1 G93A mice was observed from 15 weeks of age, and loss of motor function was observed from 18 weeks of age in this study.Reactive astrocyte proliferation has been observed in the autopsy spinal cord of patients with ALS [21].In this study, a significant increase in GFAP-positive reactive astrocytes in the lumbar spinal cord was observed in SOD1 G93A mice even before the disease onset (Supplement data).Furthermore, we newly observed that the mRNA expression of PRDX6 was significantly increased in the spinal cord from 15 weeks of age prior to the loss of motor function in SOD1 G93A mice.
As the levels of various inflammatory cytokines, such as TNF and IL-1β, are increased in the cerebrospinal fluid of patients with ALS [22,23], the involvement of inflammation in the pathological progression of ALS is now attracting attention.It has been reported that the levels of A1 astrocytes, which produce inflammatory cytokines, are increased in the lumbar spinal cord of the ALS mice model [24,25].In this study, we also observed a significant increase in the mRNA expression levels of inflammatory cytokines and C3 in the lumbar spinal cord of SOD1 G93A mice compared with age-matched WT mice.In addition, the expression of PRDX6 was increased in the lumbar spinal cord astrocytes of SOD1 G93A mice.Furthermore, the increase in the mRNA expression of C3 and inflammatory cytokines was observed in astrocyte cell lines transiently or stably expressing PRDX6, while no increase in the mRNA expression of S100a10 was observed.These results suggest that the expression of PRDX6 is important for the induction of A1 astrocytes and production of inflammatory cytokines in astrocytes.
The calcium-independent phospholipase A 2 /arachidonic acid/cyclooxygenase pathway has been shown to play an Fig. 4 Expression of PRDX6, inflammatory cytokines, and C3 in C8-D1A cells transiently transfected with human SOD1 G93A .C8-D1A cells were transiently transfected with empty vector (mock) or vector containing human wild-type SOD1 (WT SOD1) or SOD1 G93A (mSOD1) using Fugene HD transfection reagent.a 48 h after the transfection, the expression of SOD1 protein was examined by western blotting.Data shown are representative of three independent experiments.b−e 48 h after the transfection, the mRNA expression of PRDX6 (b), TNF (c), IL-6 (d), and C3 (e) was examined using real-time RT-PCR (n = 4).Data are expressed as arbitrary units normalised to β-actin and represent the mean ± SEM.Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey-Kramer test.*P < 0.05, **P < 0.01, significant deference between bracketed values important role in the production of inflammatory cytokines and development of pathological features in the ALS mice model.Cyclooxygenase inhibitors such as celecoxib suppress the production of inflammatory cytokines and reduce disease progression in the ALS mice model [26].In addition, the intracerebroventricular administration of antisense oligonucleotides of calcium-independent phospholipase A 2 suppresses disease progression in the ALS mice model [27].In this study, MJ33, an inhibitor of the calcium-independent phospholipase A 2 activity of PRDX6, suppressed the increase in the mRNA expression levels of C3 and inflammatory cytokines in astrocyte cell lines stably expressing PRDX6.Altogether, the calcium-independent phospholipase A 2 activity of PRDX6 and its downstream arachidonic U-251 MG (a−e) and C8-D1A (f−j) cells were transiently transfected with empty vector (Mock) or vector containing human PRDX6 (PRDX6) using Fugene HD transfection reagent.48 h after the transfection, the mRNA expression of PRDX6 (a, f), IL-1β (b), IL-6 (g), TNF (c, h), C3 (d, i), and S100a10 (e, j) was examined using real-time RT-PCR (n = 5).Data are expressed as arbitrary units normalised to β-actin and represent the mean ± SEM.Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey-Kramer test (**P < 0.01 vs. Mock) acid-cyclooxygenase pathway may be important for the induction to A1 astrocytes with damaging effects on neurons and production of inflammatory cytokines; consequently, this activity may be important for disease progression in the ALS mice model.
In this study, the increased expression of PRDX6 was observed in the lumbar spinal cord of the SOD1 G93A mice and cultured astrocyte cell lines transfected with the mSOD1 expression vector, suggesting that mSOD1 is involved in the induction of PRDX6.The exact mechanisms underlying the induction of PRDX6 are under investigation.It has been reported that transcriptional factors such as CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ are involved in the regulation of PRDX6 mRNA expression [11,28,29].These transcriptional factors are activated by aberrant protein aggregation [30], and mSOD1 causes aberrant protein aggregation and activates C/EBP signalling in human neuroglioma cells and embryonic kidney 293 cells [31][32][33][34].Therefore, the increased expression of PRDX6 observed in the present study may involve a C/EBP-mediated signalling pathway due to abnormal protein aggregation of mSOD1.In this study, an increase in the mRNA expression of PRDX6 was also observed in cultured astrocyte cell lines transfected with the WT SOD1 expression vector.In this context, studies have reported that WT SOD1 can form aggregates, albeit more weakly than mSOD1 [31,35,36].Although our results and previous reports [31,35,36] strongly suggest that the formation of intracellular aggregates such as mSOD1 in astrocytes increases the expression level of PRDX6, further studies are needed to elucidate the exact induction mechanism of PRDX6 expression.
In this study, we showed two major findings.First, PRDX6 is upregulated in the astrocytes of the lumbar spinal cord of the ALS mice model.Second, the expression of PRDX6 induces A1 astrocytes and inflammatory cytokines via calcium-independent phospholipase A 2 activity in cultured astrocyte cell lines.Therefore, our results suggest that the expression of PRDX6 plays a crucial role in the induction of A1 astrocytes in the ALS mice model.

Fig. 1
Fig. 1 Time course of disease progression in SOD1 G93A (Tg) mice.a Body weight was monitored twice a week and averaged (n = 7-12).Data represent the mean ± SEM.Statistical analysis was performed using the two-tailed unpaired Student's t test.b Motor coordination was assessed in mice using the rotarod test (n = 7-12).Data represent the mean ± SEM.Statistical analysis was performed using two-way analysis of variance followed by Dunnett's test (*P < 0.05, **P < 0.01 vs. age-matched WT mice)

Fig. 2
Fig. 2 Expression of PRDX6 mRNA in the lumbar spinal cord of WT and Tg mice.a The expression of PRDX6 mRNA at the age of 5, 10, 15, and 20 weeks in the lumbar spinal cord (L3-L5) of WT and Tg mice was examined using real-time RT-PCR (n = 5-8).Data are expressed as arbitrary units normalised to β-actin and represent the mean ± SEM.Statistical analysis was performed using a one-sided Mann-Whitney U test (**P < 0.01 vs. age-matched WT mice).b Photographs of the ventral horn show double immunostaining for NeuN and PRDX6 in the lumbar spinal cord of WT (20 weeks; upper panel) and Tg (20 weeks; lower panel) mice.Representative images from three independent experiments are shown.Scale bar: 50 μm.c Photographs of ventral horn show double immunostaining for GFAP and PRDX6 in the lumbar spinal cord of WT (20 weeks; upper panel) and Tg (20 weeks; lower panel) mice.Representative images from three independent experiments are shown.Scale bar: 50 μm

Fig. 3
Fig. 3 Expression of inflammatory cytokines, and C3 in the lumbar spinal cord of WT and Tg mice.The mRNA expression of TNF (a), IL-1β (b), IL-6 (c), and C3 (d) in the lumbar spinal cord (L3-L5) of WT (20 weeks) and Tg (20 weeks) mice was examined by real-time RT-PCR (n = 5).Data are expressed as arbitrary units normalised to β-actin and represent the mean ± SEM.Statistical analysis was performed using a one-sided Mann-Whitney U test (*P < 0.05 vs. WT mice)

Fig. 6 Fig. 7
Fig. 6 Effect of PRDX6 expression on induction of A1 and A2 astrocytes in C8-D1A cells.The expression of C3 and S100a10 in C8-D1A cells was analysed using FACS analysis.a Dot plots obtained from unstained C8-D1A cells.b C8-D1A cells were transfected with empty vector and stained with alexa fluor 647 conjugated