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Antioxidant cytoprotection of peroxisomal peroxiredoxin-5 Geoffroy Walbrecq, Bo Wang, Sarah Becker, Amandine Hannotiau, Marc Fransen, Bernard Knoops

www.elsevier.com/locate/freeradbiomed

PII: S0891-5849(15)00107-0 DOI: http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.032 Reference: FRB12335

To appear in: Free Radical Biology and Medicine

Cite this article as: Geoffroy Walbrecq, Bo Wang, Sarah Becker, Amandine Hannotiau, Marc Fransen, Bernard Knoops, Antioxidant cytoprotection of peroxisomal peroxiredoxin-5, Free Radical Biology and Medicine, http://dx.doi.org/ 10.1016/j.freeradbiomed.2015.02.032

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Geoffroy Walbrecq a, Bo Wang b, Sarah Becker a, Amandine Hannotiau a, Marc Fransen b and Bernard Knoops a,* a Group of Animal Molecular and Cellular Biology, Institut des Sciences de la Vie,

Université catholique de Louvain, 1348 Louvain-la-Neuve, Belgium b Laboratory of Lipid Biochemistry and Protein Interactions, Department of Cellular and

Molecular Medicine, University of Leuven – KU Leuven, 3000 Leuven, Belgium

Running title: Peroxisomal PRDX5

*Corresponding author: Dr. Bernard Knoops, Group of Animal Molecular and Cellular

Biology, Institut des Sciences de la Vie, Université catholique de Louvain, 4-5 Place croix du sud, bte L7.07.02, 1348 Louvain-la-Neuve, Belgium. E-mail: [email protected]. Phone: +32 10 47 37 60. Fax: +32 10 47 35 15

Abstract

Peroxiredoxin-5 (PRDX5) is a thioredoxin peroxidase which reduces hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. This enzyme is present in the cytosol, mitochondria, peroxisomes and the nucleus in human cells. Antioxidant cytoprotective functions have been previously documented for cytosolic, mitochondrial and nuclear mammalian PRDX5.

However, the exact function of PRDX5 in peroxisomes is still not clear. The aim of this work was to determine the function of peroxisomal PRDX5 in mammalian cells, and more specifically in glial cells. In order to study the role of PRDX5 in peroxisomes, the endogenous expression of PRDX5 in murine oligodendrocyte 158N cells was silenced by

RNA interference. In addition, human PRDX5 was also overexpressed in peroxisomes using a vector coding for human PRDX5 whose unconventional peroxisomal targeting sequence 1

(PTS1; SQL) was replaced by the prototypical PTS1 SKL. Stable 158N clones were obtained. Antioxidant cytoprotective function of peroxisomal PRDX5 against peroxisomal and mitochondrial KillerRed-mediated ROS production as well as H 2O2 was examined using

MTT viability assays, roGFP2 and C11-BOBIPY probes. Altogether our results show that peroxisomal PRDX5 protects 158N oligodendrocytes against peroxisomal and mitochondrial

KillerRed- and H 2O2-induced oxidative stress.

Keywords

Peroxiredoxin-5; peroxisomes; mitochondria; oxidative stress; KillerRed; lipid peroxidation; roGFP2.

Abbreviations

3-AT, 3-amino-1,2,4-triazole; ATPB, mitochondrial ATP synthase; COX4, cytochrome C oxidase subunit 4; E, postnuclear supernatant; GSTK1, glutathione S-transferase Kappa 1;

Hsp90, heat shock protein 90; KR, KillerRed; L, light mitochondrial fraction; L-PRDX5, long form of peroxiredoxin-5; M, mitochondrial fraction; mt-KR, mitochondrial KillerRed;

N, nuclear fraction; NEM, N-ethyl-maleimide; po-KR, peroxisomal KillerRed; PRDX5,

2 peroxiredoxin-5; PTS1, peroxisomal targeting sequence 1; ROS, reactive oxygen species;

SOD1, superoxide dismutase 1; SP, remaining supernatant; S-PRDX5, short form of peroxiredoxin-5; TRX1, thioredoxin-1; TRX2, thioredoxin-2; X-ALD, X-linked adrenoleukodystrophy

Introduction

Peroxisomes are essential organelles which participate in multiple important metabolic processes, including the !-oxidation of fatty acids, plasmalogen synthesis and the metabolism of reactive oxygen species (ROS) [1]. Their essential roles are emphasized in various genetic diseases such as Zellweger syndrome and X-linked adrenoleukodystrophy

(X-ALD), in which the integrity of the organelle is compromised [2]. In addition, mice lacking peroxisomes specifically in oligodendrocytes develop physiopathological conditions similar to X-ALD, including demyelination and axonal damage [3]. Peroxisomes produce

ROS (hydrogen peroxide and superoxide radical) as part of their normal oxidative functions

(e.g the !-oxidation of a variety of fatty acids and fatty acid derivatives, the degradation of

D-amino acids) [4]. Peroxisomal ROS have been suggested to be implicated in aging [5-6], redox signaling [7-8], and the pathogenesis of several age-related and neurodegenerative disorders [9]. Peroxisomes also contain a variety of non-enzymatic (e.g. glutathione) and enzymatic antioxidants (e.g. catalase, superoxide dismutase 1 (SOD1), and glutathione S- transferase kappa 1 (GSTK1)) involved in the degradation of ROS. Several studies have demonstrated the role of catalase [10-13], SOD1 [8], and GSTK1 [8] in the antioxidant defense of peroxisomes. However, the role of mammalian peroxisomal PRDX5 as antioxidant cytoprotective enzyme remained to be established.

PRDX5 is a thioredoxin reductase which reduces H 2O2, alkyl hydroperoxides and peroxynitrite [14]. PRDX5 is an atypical 2-Cys PRDX. Its catalytic mechanism leads to the formation of an intramolecular disulfide bridge unlike other PRDXs that form an

3 intermolecular disulfide bridge between two monomers (for review see 14). Reduction of the oxidized PRDX5 is mediated by thioredoxin-1 (TRX1) in the cytosol and the nucleus, and thioredoxin-2 (TRX2) in mitochondria [14]. Among the six members of the mammalian

PRDX family, PRDX5 presents the widest subcellular distribution. Indeed, two alternative translation initiation sites lead to two forms of PRDX5: a long form (L-PRDX5) which is addressed to mitochondria, and a short form (S-PRDX5) which is located in the cytosol, the nucleus and peroxisomes [15-16]. The peroxisomal localization of S-PRDX5 is due to the presence of a C-terminal peroxisomal targeting sequence (PTS1; SQL) [17]. Our group and others have highlighted the specific roles of PRDX5 in different subcellular compartments in which the enzyme is present. Overexpressing S-PRDX5 and L-PRDX5 have been shown to protect Chinese hamster ovary cells against extracellular peroxides [18-19]. Overexpression of L-PRDX5 reduces hypoxia-mediated mitochondrial ROS [20], prevents MPP +-mediated apoptosis and could affect redox modulation of Ca 2+ transport by a possible ER- mitochondria crosstalk in SH-SY5Y neuroblastoma cells [21]. In addition, silencing of

PRDX5 in these cells resulted in an increase of MPP+-mediated apoptosis [22]. Interestingly, it must be noted that PMP20, the yeast ortholog of PRDX5 in Candida boidinii and

Hansenula polymorpha , is exclusively located in peroxisomes and has been found to be an essential antioxidant enzyme that exhibits a cytoprotective effect [23-25].

In this study, we selected 158N oligodendrocyte clones stably transfected to express different levels of PRDX5 in order to investigate the potential cytoprotective antioxidant role of peroxisomal PRDX5. Here, we report that the peroxisomal form of PRDX5 exerts a cytoprotective function against oxidative attacks induced by exogenous H 2O2 and photoactivated peroxisomal as well as mitochondrial KillerRed.

Materials and Methods

DNA manipulations and plasmids

The plasmids encoding po-KR and mt-roGFP2 have been described elsewhere [7]. The mammalian expression vectors pKillerRed-dmito (Bio-Connect), pcDNA3.1, pCR2.1 TOPO

(Invitrogen), pSILENCER3.1-H1 puro and pSILENCER3.1-H1 puro Negative control (here named p-sh-scrambledRNA) (Ambion) were commercially obtained. The pcDNA3.1 plasmid encoding po-Grx1-roGFP2 was constructed as follows: (i) the cDNA encoding po-

Grx1-roGFP2 was amplified by PCR (template: pLPCX-GRX1-roGFP2-SKL [26] kindly provided by Prof. Andreas Meyer, INRES, University of Bonn, Germany; primers: BamHI-

Grx1-roGFP2 5’-gacaggatccaccatggctcaagagtttgtg-3’ (forward) and Grx1roGFP2-Sal1 5’- attttgtcgacttaaagcttagacttg-3’(reverse)) and subcloned into PCR2.1-TOPO; (ii) the resulting plasmid was digested by SalI/BamHI and the insert was subcloned into pcDNA3.1 to generate the plasmid p-poGrx1-roGFP2. The plasmid encoding human po-PRDX5 was generated as follows: (i) the cDNA encoding human S-PRDX5 was amplified by PCR

(template: PCR2.1-PRDX5; primers: PRDX5-Hs-SKL-NotI 5’- ggcgcggccgctcagagctttgagatgatattgg-3’ (forward) and PRDX5-Cf-SpeI 5’- agcgactagtccgccatggccccaatcaaggtgggagatgc-3’ (reverse)) and subcloned into PCR2.1-

TOPO; (ii) the resulting plasmid was digested with NotI/SpeI to obtain the human po-

PRDX5 sequence, which was subsequently ligated into the pEF-BOS vector [18 , 27] to generate the plasmid p-poPRDX5. The plasmid p-poPRDX5-C47S was constructed the same way but pRE30-PRDX5-C47S was used as template. Mammalian vector pSILENCER

(Ambion) was used to silence murine PRDX5 expression. The shRNAs were designed according to ‘siRNA Target Finder’ at the Ambion website

(http://www.ambion.com/techlib/misc/siRNA_finder.html). The construction of the pSIl- siPRDX5 plasmid used for expressing siRNAs was performed as described previously [22].

The target sequence to silence murine PRDX5 expression was 5’-aaagaagcaggttgggagtgt-3’.

All the constructs were verified by sequencing.

Cell culture, C11-BODIPY, transfection and immunofluorescence microscopy

The murine oligodendrocyte 158N cell line [28], which was provided by Prof. Said

Ghandour (University of Strasbourg, France), was cultured in DMEM 4.5 g d-glucose/L with

GlutaMAX, supplemented with 10% FCS. The cell line was maintained at 37 °C in 5% CO 2 in the presence of 100 U/ml streptomycin and 100 U/ml penicillin. Cells were transfected using a Nucleofector instrument according to manufacturer’s instructions (Amaxa

Biosystems). Immunostaining was performed as described previously [15], using 1:200 mouse anti-mitochondrial ATP synthase (ATPB; Abcam), 1:100 sheep anti-catalase

(Biodesign) (Figs. 3 and 7C), 1:50 goat polyclonal anti-catalase (GeneTex) (Figs. 1A, 5A and 5B), 1:200 rabbit anti-PRDX5 [29], or 1:50 mouse monoclonal anti-human PRDX5 (286

6F7; Millipore). C11-BODIPY 581/591 (Molecular Probes) at 1 µM was incubated with growth medium without red phenol for 30 min at 37°C, then the free thiols were clamped with 10 mM of N-ethyl-maleimide (NEM) (Sigma) for 10 min at 37°C. Finally, the cells were fixed in 4% paraformaldehyde before mounting and imaging. Confocal images were acquired with a Zeiss 710 confocal microscope (Carl Zeiss) which was equipped with Plan-

Apochromat 63x/1.40 NA objective (oil immersion). RoGFP2 is a mutated enhanced green fluorescent protein with two cysteine residues exposed at the outer surface of the !-barrel which forms a disulfide bond in oxidizing conditions [30-31]. The formation of this disulfide bond changes the excitation wavelength of the roGFP2 [31]. Thus, the roGFP2 presents two excitation wavelengths (i) at Ϣ 490 nm when the roGFP2 is reduced and (ii) at Ϣ 400 nm when the roGFP2 is oxidized [31]. The ratio of the two roGFP2 emissions (at Ϣ 510nm) allows the monitoring of the redox status of the surrounding environment [30-31]. Measurements of roGFP2 redox status were performed on a motorized IX-81 microscope equipped with a temperature, humidity, and CO 2-controlled incubation chamber and analyzed as described elsewhere [7].

Subcellular fractionation

15x10 6 158N cells were homogenized in 250 µl of homogenization solution at pH 7.2 (250 mM sucrose, 5 mM Mops, 1 mM EDTA, 0,1% ethanol, 1 mM DTT) by three strokes of

Potter-Elvehjem homogenizer. Briefly, the homogenate was centrifuged at 500 g for 10 min to pellet the nuclear fraction (N). The postnuclear supernatant (E) was centrifuged at 2330 g for 10 min to pellet the heavy mitochondrial fraction (M). The resulting supernatant was centrifuged at 13000 g for 20 min to pellet the light mitochondrial fraction (L). The remaining supernatant (SP) and the M fraction were centrifuged at 20000 g for 60 min through a self-generating Percoll gradient (40% Percoll (w/v), 220 mM sucrose, 1 mM

Mops, 0.1% ethanol, 1 mM DTT, pH 7.2). Equal volume fractions were collected from the bottom of the tube with a peristaltic pump.

Generation of stable 158N clones expressing different levels of PRDX5

158N cells were transfected with combination of plasmids using a Nucleofector instrument according to manufacturer’s instructions (Amaxa Biosystems). Three days after transfection, cells were exposed to 4 µg/ml puromycin for selection. Subsequently, stably transfected cell clones were obtained by isolation of individual clones by dilution. PRDX5 expression was analyzed by immunofluorescence and Western blotting (see Figs. 2, 3 and 7A-C).

Incorporation of replication arrest sequence from pEF-BOS vector in the genome of control and shPRDX5 cells was verified by PCR on genomic DNA using the primers: terpef-f 5’- gcatccccttggctgtgaggcc-3’ (forward) and terpef-r 5’-caggagacgcgtcggtgatgttcggg-3’ (reverse)

(data not shown). Incorporation of sh scrambled-RNA sequence from p-scrambled-sh-RNA in the genome of control and po-PRDX5 high cells was verified by PCR on genomic DNA using the primers: f-pSIL 5’-gcgattaagttgggtaacgc-3’ (forward) and r-pSIL 5’- cacaggaaacagctatgacc-3’ (reverse) (data not shown). Genomic DNA was extracted from

5x10 6 cells using DNeasy Blood and Tissue kit (Qiagen) following manufacturer’s instructions.

Western blotting

Western blotting was performed as described previously [18] with the following diluted antibodies: 1:5000 mouse monoclonal anti-catalase (CAT-505; Sigma), 1:2000 mouse

7 monoclonal anti-cytochrome c oxidase subunit 4 (COX4; Molecular Probes), 1:1000 mouse monoclonal anti-heat shock protein 90 (Hsp90; Millipore), 1:5000 rabbit polyclonal anti-

PRDX3 [22], 1:5000 rabbit polyclonal anti-PRDX5 [29], 1:200 mouse monoclonal anti- human PRDX5 (286 6F7; Millipore), 1:2000 rabbit polyclonal anti-actin (Sigma), and chemiluminescence ECL Western blotting detection was performed as previously described

[18].

Light irradiation and cell viability assays

For the cell viability, lipid peroxidation, and Western blotting assays, the cells were seeded in 96- or 24-well cell culture plates or 35 mm tissue culture dishes (Falcon), respectively, and irradiated with green light (irradiance Ϣ 15 mW/cm 2) using a KL1500LCD cold light source with a green insert filter (P/N 158 304; Schott). Light dose (J/cm 2) was calculated by multiplying the irradiance (W/cm 2) by the exposure time (s). MTT cell viability assays were performed as described elsewhere [8].

Statistical analysis

Statistics were performed using JMP 9.0 software (SAS). ANOVA2 with Tukey ‘s post-hoc test was chosen to determine statistical differences for MTT assays and roGFP2 measurements.

Results

Peroxisomal PRDX5 expression in murine oligodendrocyte 158N cell line

The murine oligodendrocyte 158N cell line was used as previous studies showed that peroxisomes are present in these cells and are involved in myelin metabolism [32]. We first examined the subcellular PRDX5 localization using immunofluorescence. PRDX5 was localized in peroxisomes by catalase codetection (Fig. 1A) and in mitochondria by colocalization with mitochondrial membrane ATP synthase (ATPB) (Fig. 1B). To confirm immunofluorescence localization of PRDX5 in peroxisomes and mitochondria, PRDX5

8 localozation was also examined using subcellular fractionation on Percoll gradient. Equal volume fractions were analyzed by Western blotting. Our data show that PRDX5 is present in fractions enriched with COX4 (mitochondrial marker), catalase (peroxisomal marker) and

Hsp90 (cytosolic marker) (Figs. 1C and 1D). These two experiments showed that the peroxisomal form of PRDX5 is indeed expressed in the 158N cell line.

Selection of 158N clones expressing PRDX5 in peroxisomes

Two mammalian expression vectors were used to modulate the expression of PRDX5 in peroxisomes. The first vector is a pSILENCER vector (p-shPRDX5) coding for a RNA hairpin targeted to mRNA coding for murine PRDX5. This vector was used to silence endogenous murine PRDX5 expression. The second vector is a pEF-BOS vector coding for a human S-PRDX5 variant in which the unconventional peroxisomal targeting sequence 1

(PTS1) SQL was replaced by the prototypical PTS1 SKL (p-po-PRDX5) to force the cell to address PRDX5 to peroxisomes. The RNA hairpin targeted to the murine PRDX5 mRNA did not recognize the human PRDX5 mRNA. Indeed, human neuroblastoma SH-SY5Y cells transfected with p-shPRDX5 did not show a decrease of PRDX5 expression after puromycin selection (data not shown). These two vectors and their respective control (p-sh-scrambled and pEF-BOS-empty) were transfected to select clones.

After transfection of 158N cells with various combinations of control vectors (p-sh- scrambled and pEF-BOS-empty), p-shPRDX5 and p-po-PRDX5, stable clones were selected. The stable clones were named control (p-sh-scrambled and pEF-BOS-empty), shPRDX5 (p-shPRDX5 and pEF-BOS-empty), po-PRDX5 high (p-sh-scrambled and p-po-

PRDX5), or shPRDX5/po-PRDX5 (p-shPRDX5 and p-po-PRDX5). Expression levels of

PRDX5 in these clones were analyzed by immunofluorescence (Fig. 2A) and immunoblotting (Fig. 2B) using a polyclonal antibody recognizing human and murine

PRDX5. In the shPRDX5 clone, lower PRDX5 expression was observed compared to control clone by immunofluorescence (Fig. 2A) and was 2-fold lower as measured by

Western blotting (Fig. 2B and 2C). The po-PRDX5 high clone presented a 3.2-fold overexpression compared to the control clone (Fig. 2B and 2C). PRDX5 expression in po-

PRDX5 high clone was mainly localized in peroxisomes but some PRDX5 was not properly targeted and remained in the cytosol (Fig. 2A). Immunofluorescence showed that in the shPRDX5/poPRDX5 clone, a punctuated distribution of PRDX5 was present, which is typical of a peroxisomal localization (Fig. 2A). Immunoblotting revealed a 0.3-fold lower expression of shPRDX5 clone compared to the control clone (Fig. 2B and 2C). Note that shPRDX5/poPRDX5 clones were selected in order to keep a PRDX5 expression level similar to control clones. Peroxisomal expression of human S-PRDX5 was assessed by immunofluorescence using a specific monoclonal antibody recognizing human PRDX5 but not murine PRDX5 and a specific antibody targeted against catalase (peroxisomal marker).

In po-PRDX5 high and shPRDX5/po-PRDX5 clones, human PRDX5 was largely located in peroxisomes (Fig. 3C and 3D, respectively).

Resistance and sensitivity of 158N clones to oxidative stress

To investigate the antioxidant protection of peroxisomal PRDX5, 158N clones with different expression levels and subcellular localizations of PRDX5 were exposed to several kinds of acute oxidative stress, including increasing concentrations of H 2O2 or tBHP. Cell death, assessed with MTT assay, was significantly higher in control and shPRDX5 cells compared to po-PRDX5 and shPRDX5/po-PRDX5 cells exposed to 100 µM H 2O2 (Fig. 4). At 250 µM

H2O2, no significant difference was observed. At the same concentrations of tBHP, no significant difference was observed and cell death was above 70% in each condition (data not shown).

To gain insight into the antioxidant protection of peroxisomal PRDX5 against ROS production in peroxisomes and mitochondria, we triggered targeted oxidative stress using

KillerRed (KR) [7]. KR is a photosensitizer developed from a mutated red fluorescent protein which generates ROS upon green light illumination [33]. KR has been reported to

10 produce superoxide, singlet oxygen and hydrogen peroxide [33-34]. However, recent studies demonstrate that the major ROS formed by KR is superoxide [35-36]. We employed peroxisomal and mitochondrial targeted variants of KR [7] to generate oxidative stress in these compartments. We first verified peroxisomal and mitochondrial localizations of po-KR and mt-KR in 158N cells with specific markers (Figs. 5A and 5B, respectively). Po-KR and mt-KR showed colocalization with catalase and ATPB, respectively. Levels of cell death induced by the phototoxicity of peroxisomal KillerRed (po-KR) and mitochondrial KillerRed

(mt-KR) were similar for the control clone (Figs. 6A and 6B, respectively). Cell death of the shPRDX5 clone was statistically higher than that of control clone in po-KR-induced stress

(Fig. 6A). Moreover, the clones overexpressing peroxisomal PRDX5 showed a marked reduction of cell death compared to the control clone after KR-induced stress (Figs. 6A and

6B). Taken together, these results suggest that, in peroxisomes, PRDX5 exerts an antioxidant protection.

Is cell death decrease in overexpressing peroxisomal PRDX5 clones exposed to oxidative stress linked to PRDX5 peroxidatic activity?

Since addressing human PRDX5 to peroxisomes increased cell viability against KR-induced

ROS generation both in peroxisomes and mitochondria, we examined whether this cytoprotection was dependent on PRDX5 peroxidatic activity. We selected 158N clones overexpressing peroxisomal PRDX5-C47S, a PRDX5 mutant which lacks peroxidatic activity [37]. Po-PRDX5-C47S and shPRDX5/po-PRDX5-C47S clones were generated

(Figs. 7 A, B and C), both expressing the enzymatically inactive human peroxisomal S-

PRDX5 with the C47S and SKL mutations, and respectively expressing either shscrambled

RNA or shPRDX5 RNA. The relative expression of the mutant PRDX5 was assessed by immunofluorescence (Fig. 7A) and quantified by Western blotting (Fig. 7B). Immunoblots revealed a 3-fold and a 2.3-fold higher PRDX5 expression for respectively the po-PRDX5-

C47S and the shPRDX5/po-PRDX5-C47S clones (Fig. 7B). In po-PRDX5-C47S and

11 shPRDX5/po-PRDX5-C47S clones, human PRDX5 was found to colocalize with catalase as expected (Fig. 7C).

The PRDX5-C47S clones were exposed to oxidative stress induced by po-KR and mt-KR and found to be as vulnerable to KR-induced oxidative stress as the control clone (Fig. 7D and 7E, respectively). However, note that the po-PRDX5-C47S clone after po-KR activation showed no significant difference compared to non-illuminated cells (Fig. 7D). These results showed that the peroxidatic activity should be partly responsible for the cytoprotection of peroxisomal PRDX5. However, other factors not yet identified could be involved in this protection.

Overexpressing peroxisomal PRDX5 prevents lipid peroxidation induced by po-KR

As it was reported that po-KR and mt-KR induce oxidative stress and generate lipid peroxidation, and that this effect can be counteracted by overexpressing antioxidant enzymes

[8], we examined whether the peroxisomal PRDX5 could also protect against po-KR- and mt-KR-induced lipid peroxidation. Therefore, we used the lipid peroxidation sensor C11-

BODIPY 581/591. C11-BODIPY 581/591 is a fluorescent fatty acid analogue which has a comparable sensitivity to oxidation to that of endogenous fatty acyl moieties [38]. Upon peroxidation, the red fluorescence of this sensor shifts to green fluorescence. As this probe and KR display overlapping red fluorescence, the visualization of the lipid peroxidation was only detected in the green channel [8]. Control and shPRDX5 clones displayed an increase in green channel imaging after illumination for both po-KR and mt-KR (Figs. 8 and 9, respectively). The percentage of green positive cells ( Ϣ 10%) was comparable for both po-KR and mt-KR. However, the clones overexpressing peroxisomal PRDX5 (po-PRDX5 high and shPRDX5/po-PRDX5) did not show an increase in the green channel imaging (Figs. 8 and

9).

Effects of catalase inhibition on peroxisomal and mitochondrial redox balance

To highlight the role of peroxisomal PRDX5 in the balance of intracellular redox status, we used targeted variants of roGFP2 and we treated 158N clones, presenting different levels of

PRDX5 expression, with a catalase inhibitor (3-amino-1,2,4-triazole (3-AT)) [7].

Peroxisomal and mitochondrial redox status were assayed after transfection of the 158N clones with vectors expressing po-Grx1-roGFP2 and mt-roGFP2 [7 , 26]. The correct localization of po-Grx1-roGFP2 and mt-roGFP2 in 158N cells was verified by immunofluorescence with peroxisomal and mitochondrial markers (catalase and ATPB respectively) (Figs. 5A and 5B, respectively). Compared to the control clone in basal conditions, the peroxisomal redox status of the shPRDX5 and shPRDX5/poPRDX5 clone was slightly but significantly higher. Surprisingly, in non treated conditions, the peroxisomal redox status of the po-PRDX5 high was significantly higher (Fig. 10A). Catalase inhibition by 3-AT resulted in a slight but significant decrease of the peroxisomal redox status of the control, shPRDX5 and po-PRDX5 high clones (Fig. 10A). In basal conditions, the mitochondrial redox status of overexpressing peroxisomal PRDX5 clones (po-PRDX5 high and shPRDX5/po-PRDX5) were slightly but significantly higher than the control clone (Fig.

10B). As previously observed with wild-type MEFs [7], catalase inhibition in the control and the shPRDX5 clones resulted in a slight but significantly increase of the mitochondrial redox status. Remarkably, in clones overexpressing peroxisomal PRDX5, treatment with 3-AT did not result in an increase of the mitochondrial redox status.

Discussion

Recent studies demonstrated that peroxisomal ROS can lead to cell death and may be also involved in a complex interorganellar crosstalk [7-8 , 13]. Moreover, the relative importance of several antioxidant enzymes in the reduction of these peroxisomal ROS was underlined [8

, 13]. In this context, we examined the antioxidant role of PRDX5 in peroxisomes.

Here, we show that PRDX5 is indeed localized in peroxisomes of the mouse oligodendrocyte

158N cell line. Subsequently, using different constructs, we selected stable clones with

13 different levels of human PRDX5 expression. Our results show that overexpression of peroxisomal PRDX5 exerts a cytoprotective role against oxidants such as exogenously applied H 2O2.

We extended our observations on peroxisomal PRDX5 by using cell model assays based on the phototoxicity of KR [8]. This cell model allowed us to trigger localized oxidative stress in peroxisomes and mitochondria. Our results show that global silencing of PRDX5 sensitizes the cells to po-KR and mt-KR phototoxicity. Furthermore, overexpression of the peroxisomal PRDX5 protected against po-KR and mt-KR derived ROS. Moreover, overexpression of PRDX5 in peroxisomes preserved from lipid peroxidation induced by KR- derived ROS. PRDX5 could probably act in concert with GSTK1 in peroxisomes to detoxify lipid peroxides. GSTK1 has a peroxidase activity [39] and GSTK1 overexpression was shown also to protect against lipid peroxidation induced by po-KR- and mt-KR-induced stress [8]. Here, it must be pointed out that PRDX5 is more efficient to reduce organic

6 7 -1 -1 peroxides and peroxynitrite (rate constant in the 10 -10 M s range) compared to H 2O2

(rate constant of 3 ± 0.5x10 5 M -1 s -1 ) [40]. Interestingly, catalase overexpression did not act as a lipid peroxidation quencher following po-KR- and mt-KR-induced stress [8]. These data suggest that PRDX5 could act both by preventing lipid peroxidation by previous decomposition of peroxides (with a higher affinity for peroxynitrite) or by direct lipid peroxide reduction. Two other peroxidases which reduce lipid peroxides in the cytosol and the membranes are PRDX6 [41] [42] and GPx4 [43] [44] [45]. Notably, GPx4 was shown also to protect mitochondria from lipid peroxidation [46]. Thus, GSTK1, PRDX5, PRDX6 and GPx4 could act with their specific subcellular localization to control lipid peroxidation and/or subsequent redox cell signaling.

Here, we report also that a peroxisomal PRDX5 mutant that does not display peroxidatic activity did not protect from phototoxicity induced by po-KR and mt-KR for the shPRDX5/po-PRDX5-C47S clone. However, cells from the po-PRDX5-C47S clone were protected from mt-KR-induced phototoxicity. These results suggest that the cytoprotective

14 effect of the PRDX5 is partially linked to its peroxidatic activity. The cytoprotection observed in the po-PRDX5-C47S could be due to compensation by other peroxisomal antioxidant enzymes. In this context, we showed that liver cells from pig, in which the L-

PRDX5 targeting was lost during evolution, express more PRDX3 and GPX4 than rat liver cells [47].

Our observations also strengthen the concept of a redox cross-talk between peroxisomes and mitochondria [7]. Indeed, overexpression of peroxisomal PRDX5 protected from a mitochondrial oxidative stress induced by KR. In addition, we confirmed that inhibiting catalase by 3-AT disturbs the mitochondrial redox status but overexpressing peroxisomal

PRDX5 counteracts this effect.

Surprisingly, inhibiting catalase by 3-AT led to a decrease or maintained the peroxisomal redox status for all clones. Previous studies have shown that inhibition of catalase by 3-AT of mouse embryonic fibroblasts (MEF) or catalase deficient-MEF maintain their peroxisomal redox status while their mitochondrial redox status is increased [7]. In addition, inhibiting catalase of human fibroblats by 3-AT led to activity inhibition of mitochondrial aconitase

[48] and chronic treatment by 3-AT resulted in proliferation of peroxisomes [12]. Moreover, catalase deficient-yeast Hansenula polymorpha cells showed different chronological life spans (CLS) upon growth conditions [49]. Several hypotheses can be ruled out to explain those responses. First, absence or decrease of catalase could increase the activity or the import of other peroxisomal antioxidant enzymes, which in turn would induce the decrease or maintain of the peroxisomal redox status. Second, a crosstalk from peroxisomes to mitochondria in which H 2O2 or peroxisomal lipid metabolites are involved, could induce mitochondrial redox status increase. However, 3-AT could either lead to a yet unknown inhibition of a mitochondrial antioxidant enzyme or perturb the mitochondrial electron transport chain leading to an increase of mitochondrial redox status.

In conclusion, our results showed that peroxisomal PRDX5 exerts an antioxidant protection, and that PRDX5 protects from lipid peroxidation induced by KR-phototoxicity. However, the relative role of PRDX5 compared to other antioxidant enzymes in peroxisomes remains to be elucidated.

Aknowledgements

This study was supported by the “Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture" (FRIA), the DIANE research program of the Walloon region (10/15-

026), the FWO (G.0754.09), and the “Bijzonder Onderzoeksfonds van de K.U.Leuven”

(DBOF/10/059 and OT/14/100). We would like to thank Prof. Andreas Meyer (University of

Bonn) for providing GRX1-roGFP and Prof. Said Ghandour (University of Strasbourg) for providing murine oligodendrocyte 158N cell line. We would like also to thank Marc Pirson

(Catholic University of Louvain) for critical reading of the manuscript.

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Figure legends

Figure 1. Subcellular localization of PRDX5 in 158N cell line. Immunostaining of PRDX5 and colocalization with ( A) catalase (peroxisomal marker) and ( B) ATPB (mitochondrial marker). Nuclei were stained with DAPI. Scale bars: 10 µm. ( C) Subcellular fractionation on

Percoll gradient. Nuclear (N), heavy mitochondrial (M) and ( D) cytosolic/microsomal fractions (SP) were analyzed for PRDX5 and for marker enzyme by Western blotting,

PRDX5 is present in fractions enriched in COX4 (mitochondrial marker), catalase

(peroxisomal marker) and Hsp90 (cytosolic marker).

Figure 2. Endogenous and peroxisomal human PRDX5 expression in transfected 158N clones. Silencing of endogenous PRDX5 expression and overexpression of human peroxisomal PRDX5 was examined by ( A) immunofluorescence and ( B) Western blotting and total expression levels were quantified ( C). Bands arise from the same blot but were reorganized to respect the disposition of clones on the figure. Scale bars: 10 µm.

Figure 3. Subcellular localization of human po-PRDX5 in 158N clones. Immunostaining of human po-PRDX5 and colocalization with catalase (peroxisomal marker). Scale bars: 10

µm.

Figure 4. Viability assays on 158N clones exposed to hydrogen peroxide. Cells were exposed to increasing concentrations of hydrogen peroxide (50, 100 and 250 µM) for 24 h, and MTT viability assay was performed. n=9 per group, mean values come from three independent experiments followed by ANOVA2 statistical analysis. Significance versus without treatment: a,b,c,d p<0.05, aa,bb,cc,dd p<0.01, aaa,bbb,ccc,ddd p<0.0001.

Figure 5. (A) Colocalization by immunofluorescence of po-Grx1-roGFP2 and po-KR with catalase (peroxisomal marker. ( B) Colocalization by immunofluorescence of mt-roGFP2 and mt-KR with ATPB (mitochondrial marker). Nuclei were stained with DAPI. Scale bars: 10

µm.

Figure 6. Viability assays on 158N clones. 158N clones were transiently transfected with a plasmid encoding peroxisomal ( A) or mitochondrial ( B) KillerRed, after three (peroxisomal) or two (mitochondrial) days, cells were illuminated for 1h (black) or not illuminated (no treatment, grey) to green light. 24h post-illumination, MTT assays were performed. n=9 per group. Mean values come from three independent experiments followed by ANOVA2 statistical analysis. Significance versus no treatment is indicated by * p<0.05 , ** p<0.01,

*** p<0.0001. The values from illuminated control clone condition were also compared to the other illuminated conditions ( p<0.05).

Figure 7. Subcellular localization and expression of mutant PRDX5-C47S in 158N clones and viability assays. ( A) Immunostaining of endogenous murine and exogenous human

PRDX5. ( B) Western blotting analysis of PRDX5 mutants and ( C) blot quantification. Bands arise from the same blot but were reorganized to respect the disposition of clones on the figure. Immunostaining of human PRDX5-C47S and colocalization with catalase. 158N clones were transiently transfected with a plasmid encoding peroxisomal ( D) or mitochondrial ( E) KillerRed, after three (peroxisomal) or two (mitochondrial) days, cells were illuminated for 1h (dark) or not illuminated (no treatment, grey) to green light. 24h post-illumination, MTT assays were performed. n=9 per group. Mean values from three independent experiments followed by ANOVA2 statistical analysis. Significance versus no treatment is indicated by * p<0.05 , ** p<0.01, *** p<0.0001. The values from illuminated control clone were also statistically compared to the other illuminated conditions ( p<0.05).

Figure 8. Sensing of peroxisomal KillerRed-induced lipid peroxidation in 158N clones.

158N clones were transiently transfected with a plasmid encoding peroxisomal KR (po-KR).

After three days, cells were illuminated (54 J/cm 2) for 1h or not illuminated (0 J/cm 2) to green light. After illumination, cells were incubated for 30 min at 37°C with BODIPY C11

(10 µM). Then cells were incubated with NEM (10 mM) for 10 min at 37°C. Finally, the cells were fixed in PFA 4% for 30 min at RT and then visualized in fluorescence microscopy as described in “Materials and Methods”. Oxidation of BODIPY-C11 results in a shift of the emission spectra from red to green. Scale bars: 10 µm.

Figure 9. Sensing of mitochondrial KillerRed-induced lipid peroxidation in 158N clones.

158N clones were transiently transfected with a plasmid coding for mitochondrial KR (mt-

KR). After two days, cells were illuminated (54 J/cm 2) for 1h or not illuminated (0 J/cm 2) to green light. After illumination, cells were incubated for 30 min at 37°C with BODIPY C11

(10 µM). Then cells were incubated with NEM (10 mM) for 10 min at 37°C. Finally, the

24 cells were fixed in PFA 4% for 30 min at RT and then visualized in fluorescence microscopy as described in “Materials and Methods”. Oxidation of BODIPY-C11 results in a shift of the emission spectra from red to green. Scale bars: 10 µm.

Figure 10. Intracellular redox status in 158N clones and under catalase inhibition. 158N clones were transiently transfected with plasmids encoding for mitochondrial roGFP2 (mt- roGFP2) or peroxisomal Grx1-roGFP2 (po-Grx1-roGFP2). After three days, the cells were exposed to 10 mM of 3-aminotriazole (catalase inhibitor) for 4 hours, then redox status was measured in mitochondria ( A) or peroxisomes ( B). 400/480 nm ratios were analyzed as described in [7]. Data were normalized to the control clone non-treated, followed by

ANOVA2 statistical analysis. Significance versus control is indicated by * p<0.05 , ** p<0.01,

*** p<0.0001. The values from the non-treated control clone were also statistically compared to the other non treated conditions ( p<0.05).

Walbrecq et al., Antioxidant cytoprotection of peroxisomal peroxiredoxin-5, MS #: FRBM-D-14-01029

Highlights

[ Peroxisomal PRDX5 exerts a cytoprotective function against exogenous H 2O2

[ Peroxisomal PRDX5 protects from peroxisomal and mitochondrial KillerRed- mediated ROS

[ Peroxisomal PRDX5 acts towards lipid peroxidation

Graphical Abstract (for review) Figure 1

Figure 1

A merged B merged

DAPI PRDX5 CAT DAPI PRDX5 ATPB

C Mitochondria MW (kDa)

55 COX4

17 PRDX5

D Peroxisomes Cytosol MW (kDa)

95 Hsp90

CAT 55

55 COX4

17 PRDX5 Figure 2

Figure 2

A control shPRDX5 po-PRDX5 high shPRDX5/po-PRDX5 PRDX5 PRDX5 PRDX5 PRDX5

B C 500 400 300 MW 200 (kDa) 100 0 17 PRDX5

42 β-Actin

CL 5 CL 2

CL 1 Figure 3

Figure 3

A B

C D Figure 4

Figure 4

Without treatment H2O2H2O2 50µM H2O2H2O2 100µM H2O2H2O2 250µM 140 120 c 100 d dd aaa 80 bbb ccc 60 aaa bbb 40 ddd Relative cell viability cell viability Relative

(avg control=100%;n=9)(avg 20 0 control shPRDX5 po-PRDX5 high shPRDX5/po-PRDX5 Figure 5

Figure 5 A B merged merged

po-GroGFP2 CAT po-KR mt-roGFP2 ATPB mt-KR Figure 6

Figure 6

A po-KR (>80%) 0 J/cm 2 54 J/cm 2

120 *** *** NS NS α α 100

80 α 60

40 Relative cell viability viability cell Relative

(avg control=100%;n=9)(avg 20

0 control shPRDX5 po-PRDX5 high shPRDX5/po-PRDX5

B mt-KR (>80%) 0 J/cm 2 54 J/cm 2

120 *** *** NS NS α 100 α

40 Relative cell viability viability cell Relative

(avg control=100%;n=9)(avg 20

0 control shPRDX5 po-PRDX5 high shPRDX5/po-PRDX5 Figure 7

Figure 7

A po-PRDX5-C47S shPRDX5/po-PRDX5-C47S B

MW (kDa) 17 PRDX5

C po-PRDX5-C47S shPRDX5/po-PRDX5-C47S 42 β-Actin merged merged 400 300 200 100 0

h-PRDX5 CAT h-PRDX5 CAT

po-KR (>80%) 0 J/cm 2 54 J/cm 2 NS 120 *** ***

100

80 viability 60

40 Relative cell Relative

(avg control=100%;n=8)(avg 20

0 control po-PRDX5-C47S shPRDX5/ po-PRDX5-C47S E mt-KR (>80%) 0 J/cm 2 54 J/cm 2 120 *** *** ***

100

Relative cell viability viability cell Relative 20 (avg control=100%;n=8)(avg 0 control po-PRDX5-C47S shPRDX5/ po-PRDX5-C47S Figure 8

Figure 8

0 J/cm 2 54 J/cm 2 merged green red merged green red A control B control ̴ 10%

C shPRDX5 D shPRDX5 ̴ 10%

E po-PRDX5 high F po-PRDX5 high

G shPRDX5/po-PRDX5 H shPRDX5/po-PRDX5 Figure 9

Figure 9

0 J/cm 2 54 J/cm 2 merged green red merged green red A control B control ̴ 10%

C shPRDX5 D shPRDX5 ̴ 10%

E po-PRDX5 high F po-PRDX5 high

G shPRDX5/po-PRDX5 H shPRDX5/po-PRDX5 Figure 10

Figure 10

A 200 po-Grx1-roGFP2 NT +3AT *** 180 α 160 140 * NS α 120 *** 100 80 60 40

400/480 nm (avg nm (avg 400/480 20 0 400/480nm (avg control=100%) (avg 400/480nm control shPRDX5 po-PRDX5 high shPRDX5/po-PRDX5

B 200 mt-roGFP2 NT +3AT 180 *** 160

140 *** *** ** α 120 α α 100 80 60 400/480 nm 400/480 40 20 0 400/480nm (avg control=100%) (avg 400/480nm control shPRDX5 po-PRDX5 high shPRDX5/po-PRDX5