Yang Luo

Laurent Chatre

Shaden Melhem

Zayana M Al-Dahmani

Natalie Z M Homer

Anneke Miedema

Leo E Deelman

Matthew R Groves

Martin Feelisch

Nicholas M Morton

Amalia Dolga

Harry Van Goor

Thiosulfate sulfurtransferase deficiency promotes oxidative distress and aberrant NRF2 function in the brain

Keywords: Thiosulfate sulfurtransferase (TST), Reactive species, Antioxidant, Oxidative distress, Nrf2 signaling…

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1) was discovered as an enzyme that detoxifies cyanide by conversion to thiocyanate (rhodanide) using thiosulfate as substrate; this rhodanese activity was subsequently identified to be almost exclusively located in mitochondria. More recently, the emphasis regarding its function has shifted to hydrogen sulfide metabolism, antioxidant defense, and mitochondrial function in the context of protective biological processes against oxidative distress. While TST has been described to play an important role in liver and colon, its function in the brain remains obscure. In the present study, we therefore sought to address its potential involvement in maintaining cerebral redox balance in a murine model of global TST deficiency (Tst -/-mice), primarily focusing on characterizing the biochemical phenotype of TST loss in relation to neuronal activity and sensitivity to oxidative stress under basal conditions. Here, we show that TST deficiency is associated with a perturbation of the reactive species interactome in the brain cortex secondary to altered ROS and RSS (specifically, polysulfide) generation as well as mitochondrial OXPHOS remodeling. These changes were accompanied by aberrant Nrf2-Keap1 expression and thiol-dependent antioxidant function. Upon challenging mice with the redox-active herbicide paraquat (25mg/kg i.p. for 24hrs), Tst -/-mice displayed a lower antioxidant capacity compared to wildtype controls (C57BL/6J mice). These results provide a first glimpse into the molecular and metabolic changes of TST deficiency in the brain and suggest that pathophysiological conditions associated with aberrant TST expression and/or activity renders neurons more susceptible to oxidative stress-related malfunction.

Abbreviations

TST

J o u r n a l P r e -p r o o f

Introduction

The enzyme thiosulfate sulfurtransferase (TST, EC 2.8.1.1) was first identified in 1933 as a cyanide (CN -) detoxification enzyme that utilizes thiosulfate (S2O3 2-) as sulfur donor to form thiocyanate (SCN -; also known as rhodanide). [START_REF] Lang | Die Rhodanbildung Im Tierkorper [Thiocyanogen in the Bodies of Animals[END_REF][START_REF] Hall | Which cyanide antidote?[END_REF] The use of thiosulfate as an antidote to cyanide poisoning relies on this enzymatic reaction. Another known rhodanese enzyme, 3mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2), shares a high sequence homology with TST, and both enzymes are thought to be evolutionarily closely related. [START_REF] Nagahara | Cytosolic Mercaptopyruvate Sulfurtransferase Is Evolutionarily Related to Mitochondrial Rhodanese: Striking similarity in active site amino acid sequence and the increase in the mercaptopyruvate sulfurtransferase activity of rhodanese by site-directed mutagenesis[END_REF][START_REF] Nagahara | Mercaptopyruvate sulfurtransferase role of amino acid residues in the active site of rat liver mercaptopyruvate sulfurtransferase: cDNA cloning, overexpression, and site-directed mutagenesis[END_REF] TST is abundantly expressed in various tissues and plays a vital role in three important biological processes: the metabolism of hydrogen sulfide (H2S; hereinafter referred to as sulfide), modulation of mitochondrial respiratory chain activity, and involvement in the thiol-dependent antioxidant system. [START_REF] Wang | Physiological implications of hydrogen sulfide: A whiff exploration that blossomed[END_REF][START_REF] Pagani | Interaction of rhodanese with mitochondrial NADH dehydrogenase[END_REF][START_REF] Bonomi | Rhodanese-mediated sulfur transfer to succinate dehydrogenase[END_REF][START_REF] Nandi | Rhodanese as a thioredoxin oxidase[END_REF] In the context of sulfide metabolism, TST oxidizes the endogenous gasotransmitter H2S in a similar fashion as sulfide quinone oxidoreductase (SQOR) and persulfide dioxygenase (ETHE1/PDO). [START_REF] Hildebrandt | Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria[END_REF] TST also has the capacity to form H2S from thiosulfate by utilizing dihydrolipoic acid (DHLA), the reduced form of α-lipoic acid. [START_REF] Villarejol | Rhodanese-catalyzed reduction of thiosulfate by reduced lipoic acid[END_REF][START_REF] Iciek | Ssulfhydration as a cellular redox regulation[END_REF] Balancing both functions, H2S oxidation and production, it seems that the primary function of TST is in the detoxification of H2S and less so in the production of H2S. [START_REF] Libiad | Polymorphic variants of human rhodanese exhibit differences in thermal stability and sulfur transfer kinetics[END_REF] TST is almost exclusively expressed in mitochondria, the organelle that synthesizes ATP by oxidative phosphorylation (OXPHOS) through stepwise reduction of oxygen in the Electron Transport Chain (ETC) and the generated proton gradient using five enzyme complexes (Complex I-V). [START_REF] Xu | Role of Mitochondria in Neurodegenerative Diseases: From an Epigenetic Perspective[END_REF][START_REF] Wang | Mitochondrial dysfunction in neurodegenerative diseases and the potential countermeasure[END_REF] TST activity appears to modulate reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) and other mitochondrial complexes including succinate dehydrogenase (Complex II) via interaction with their iron-sulfur clusters. [START_REF] Pagani | Interaction of rhodanese with mitochondrial NADH dehydrogenase[END_REF][START_REF] Bonomi | Rhodanese-mediated sulfur transfer to succinate dehydrogenase[END_REF] In addition to this interaction between mitochondrial activity and TST, phosphorylation of TST may modulate its activity and thereby affect the stability of sulfur in the iron-sulfur centers of Complexes III and IV, further impacting electron transport and ATP production. [START_REF] Kruithof | Unraveling the role of thiosulfate sulfurtransferase in metabolic diseases[END_REF] In mammalian cells, TST is linked to two thiol-dependent antioxidant systems, the thioredoxin system and the GSH system. [START_REF] Kruithof | Unraveling the role of thiosulfate sulfurtransferase in metabolic diseases[END_REF] TST exerts anti-oxidative properties via donating sulfur for these two systems and an increase of TST activity can (in)directly activate their reactive oxygen species (ROS) scavenging function. [START_REF] Libiad | Polymorphic variants of human rhodanese exhibit differences in thermal stability and sulfur transfer kinetics[END_REF][START_REF] Libiad | Thiosulfate sulfurtransferase-like domaincontaining 1 protein interacts with thioredoxin[END_REF] Oxidative stress has classically been defined as an imbalance in the generation of pro-oxidant and antioxidant species in favor of the former. [START_REF] Sies | Findings in redox biology: From H2O2 to oxidative stress[END_REF] A series of experimental observations promoted a redefinition of oxidative stress as a condition that is associated with alterations in redox signaling and control, and an updated interpretation of the original concept distinguishes physiological oxidative stress (or 'oxidative eustress') from excessive and deleterious oxidative stress (coined 'oxidative distress'). [START_REF] Jones | Redefining Oxidative Stress[END_REF] Besides ROS, some other types of short-lived molecules such as reactive nitrogen (RNS) and reactive sulfur species (RSS) contribute to cellular signaling processes and merit consideration specifically in the context of sensing and adaptation to changes in environmental and/or metabolic conditions. These considerations are part of the stress signaling paradigm and have recently been conceptualized in the form of the Reactive Species Interactome (RSI) framework. [START_REF] Koning | The reactive species interactome: evolutionary emergence, biological significance, and opportunities for redox metabolomics and personalized Medicine[END_REF][START_REF] Malard | The reactive species interactome in the brain[END_REF] The transcription factor Nrf2 (nuclear factor (erythroid-derived 2)-like 2) is a master regulator of cellular homeostasis that controls the expression of genes related to redox homeostasis. [START_REF] Suzuki | Molecular basis of the Keap1-Nrf2 system[END_REF] The J o u r n a l P r e -p r o o f primary mechanism of NRF2 regulation is at the protein level. The majority of research has focused on the involvement of the electrophile and redox sensor Kelch-like ECH-associated protein 1 (KEAP1) in regulating NRF2 protein levels in response to metabolic demands. [START_REF] Saito | Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response[END_REF] The association between KEAP1 and NRF2 is disrupted by electrophilic alteration or oxidation of cysteine thiols in KEAP1, enabling cells/organisms to respond to environmental stress. Following dissociation from KEAP1, NRF2 escapes destruction and targets antioxidant response element (ARE) genes, which leads to an enhancement of antioxidant capacity. [START_REF] Raghunath | Antioxidant response elements: Discovery, classes, regulation and potential applications[END_REF] Moreover, various studies linked the TST-related antioxidant system to nuclear factor erythroid 2-related factor 2 (Nrf2) signaling due to its transcriptional activation of GSH-related enzymes, one of the first lines of defense against oxidative stress. [START_REF] Xie | Hydrogen sulfide induces Keap1 S-sulfhydration and suppresses diabetes-accelerated atherosclerosis via Nrf2 activation[END_REF][START_REF] Cuadrado | Transcription factor NRF2 as a therapeutic target for chronic diseases: A systems medicine approach[END_REF][START_REF] Abdul-Aziz | Oxidative stress responses and NRF2 in human leukaemia[END_REF][START_REF] Yang | Hydrogen sulfide protects against cellular senescence via s-sulfhydration of keap1 and activation of Nrf2[END_REF] Here, H2S mediates direct persulfidation of KEAP1 (Kelch-like ECH-associated protein 1) and thereby contributes to sulfide-mediated NRF2 regulation. [START_REF] Zhao | Hydrogen Sulfide Alleviates Liver Injury Through the S-Sulfhydrated-Kelch-Like ECH-Associated Protein 1/Nuclear Erythroid 2-Related J o u r n a l P r e -p r o o f Factor 2/Low-Density Lipoprotein Receptor-Related Protein 1 Pathway[END_REF] Several studies have demonstrated that TST provides an essential protective function against disease. [START_REF] Kruithof | Unraveling the role of thiosulfate sulfurtransferase in metabolic diseases[END_REF] Studies investigating the mechanistic effects of TST, which were identified in polygenic lean mouse adipocytes, have indicated its potential as a predictor for diabetes. [START_REF] Abdul-Aziz | Oxidative stress responses and NRF2 in human leukaemia[END_REF] In an Icelandic cohort of almost 700 people, an inverse correlation was observed between TST mRNA levels in subcutaneous adipose tissue and body mass index (BMI). [START_REF] Morton | Genetic identification of thiosulfate sulfurtransferase as an adipocyte-expressed antidiabetic target in mice selected for leanness[END_REF] Upregulation of adipose TST was detected in lean mice challenged with a high-fat diet (HFD), further supporting its likely protective effects. [START_REF] Morton | Genetic identification of thiosulfate sulfurtransferase as an adipocyte-expressed antidiabetic target in mice selected for leanness[END_REF] Additionally, mice with transgenic Tst overexpression in mature adipocytes, showed resistance against HFD-induced obesity and exhibited higher protein expression of mitochondrial superoxide dismutase 2 (SOD2) and higher mRNA levels of peroxiredoxin 3 (Prdx3) compared to control mice, while cytosolic superoxide dismutase 1 (SOD1) remained unchanged. [START_REF] Morton | Genetic identification of thiosulfate sulfurtransferase as an adipocyte-expressed antidiabetic target in mice selected for leanness[END_REF] These findings provide evidence of an interaction between TST and mitochondrial ROS. Consistent with a function of TST for ROS removal, knockdown of Tst in the preadipocyte cell line 3T3-L1 generated higher levels of mitochondrial ROS in the presence of oxidative stress stimuli. [START_REF] Morton | Genetic identification of thiosulfate sulfurtransferase as an adipocyte-expressed antidiabetic target in mice selected for leanness[END_REF] Furthermore, ROS-related adiponectin release from 3T3-L1 adipocytes was decreased in cells treated with the TST inhibitor, 2-PTS (2-propenyl thiosulfate), while adiponectin was increased following addition of the TST substrate thiosulfate. [START_REF] Morton | Genetic identification of thiosulfate sulfurtransferase as an adipocyte-expressed antidiabetic target in mice selected for leanness[END_REF] TST enables H2S detoxification in colon tissue, and has reduced expression in colon mucosa of patients with ulcerative colitis and colon cancer. [START_REF] Levine | Fecal hydrogen sulfide production in ulcerative colitis[END_REF][START_REF] Picton | Mucosal protection against sulphide: Importance of the enzyme rhodanese[END_REF][START_REF] Kaczor-Kamińska | The expression and activity of rhodanese, 3-mercaptopyruvate sulfurtransferase, cystathionine γ-lyase in the most frequently chosen cellular research models[END_REF] During the development of dextran sulfate sodium (DSS) induced colitis in mice, the expression of Tst mRNA, protein and activity significantly decrease. [START_REF] Taniguchi | Rhodanese, but not cystathionine-γ-lyase, is associated with dextran sulfate sodium-evoked colitis in mice: A sign of impaired colonic sulfide detoxification?[END_REF] Taken together, these findings indicate that TST has an important function in maintaining the redox balance in colon and adipocytes, but there are no studies elucidating the importance of TST in redox signaling within the brain. A clinical report of rhodanese (i.e. TST) deficiency in a rare neurological and mitochondrial human disorder known as Leber's Hereditary Optic Atrophy (LHON) [START_REF] Poole | Deficiency of thiosulphate sulphurtransferase (rhodanese) in Leber's hereditary optic neuropathy[END_REF] prompted us to investigate the underlying molecular and metabolic changes of TST deficiency in the brain. From physiology to pathology, the brain consumes about 20% of the whole-body supply of oxygen and energy [START_REF] Abdul-Aziz | Oxidative stress responses and NRF2 in human leukaemia[END_REF] . The cerebral cortex, with its intense neuronal activity and high metabolic activity, has a particularly high susceptibility to perturbation by oxidative stress. [START_REF] Cobley | 13 reasons why the brain is susceptible to oxidative stress[END_REF][START_REF] Vandresen-Filho | Cerebral cortex, hippocampus, striatum and cerebellum show differential susceptibility to quinolinic acid-induced oxidative stress[END_REF][START_REF] Trombetta-Lima | Mitochondrial dysfunction in neurodegenerative diseases: A focus on iPSC-derived neuronal models[END_REF] In Alzheimer's Disease, increased oxidative stress is associated with the progression of the neurodegenerative pathology along with amyloid and hyperphosphorylated tau. [START_REF] Sbodio | Redox mechanisms in neurodegeneration: From disease outcomes to therapeutic opportunities[END_REF][START_REF] Marmolejo-Garza | Transcriptomic and epigenomic landscapes of Alzheimer's disease evidence mitochondrial-related pathways[END_REF] This is due to high mitochondrial generation of ROS, including superoxide anion (O2 • -) and hydrogen peroxide (H2O2). [START_REF] Koning | The reactive species interactome: evolutionary emergence, biological significance, and opportunities for redox metabolomics and personalized Medicine[END_REF][START_REF] Rink | Significance of brain tissue oxygenation and the arachidonic acid cascade in stroke[END_REF] Hence, we hypothesized that TST plays a vital role in enabling an appropriate redox balance in the brain, and that global gene silencing of TST in mice would provide novel insights into the central role of TST in neurological antioxidant signaling and tissue protection.

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Methods

Experimental animals

All the experiments were performed according to international guidelines set out by the ethical committees of the University of Edinburgh and within the framework of the Animals (Scientific Procedures) Act (1986) of the United Kingdom Home Office. Animals were maintained in standard housing conditions with a 12-hour light and 12-hour dark cycle (7 a.m. to 7 p.m.) and ad libitum access to rodent chow. All studies used female mice housed in cages of 3-6 individual littermates. The female mice used for the study originated from C57BL/6N Tst -/- mice [START_REF] Morton | Genetic identification of thiosulfate sulfurtransferase as an adipocyte-expressed antidiabetic target in mice selected for leanness[END_REF] backcrossed onto a C57BL/6J genetic background for more than 10 generations. Pieces of brain cortex were snap frozen and stored at -80 o C until use.

Induction of oxidative stress in vivo using paraquat

To test the efficacy of paraquat dichloride (PQ, 36541-SIGMA) as an inducer of oxidative stress in the brain cortex tissue in vivo, PQ (in physiological saline 0.9%) was administered to WT and Tst -/-mice by intraperitoneal injection at a concentration of (25 mg/kg body weight). 24 hours later, mice were culled by decapitation and organs were harvested for analysis.

Total ATP, GSH/GSSG and MPST activity measurements

Total ATP content was measured with the CellTiter-Glo 2.0 Luminescent assay (G9241, Promega). GSH/GSSG content and ratio were measured with the Glutathione GSH/GSSG Assay Kit (MAK440, Sigma-Aldrich) following the manufacturer's instructions. MPST enzyme activity was detected with MPST ELISA kit (MBS9912889, Mybiosource) according to manufacturer's instructions. All analyses were performed using freshly extracted total protein from brain cortex samples, and using microplate multimode reader Spark (Tecan). For MPST enzyme activity, absorbance reading were obtained by a Synergy H4 Hybrid Reader (Biotek).

Protein extraction and immunoblotting

Protein extraction from brain cortex samples was performed using 50 mM Tris⋅HCl pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100, 0.1% (w/v) SDS. Extracted proteins were centrifuged to remove sample debris. The protein content was determined with the Bicinchoninic acid (BCA) Protein Assay (23227, ThermoFisher Scientific/Pierce). 20 μg of protein was loaded for SDS/PAGE. After blotting, Trans-blot Turbo nitrocellulose membranes (Bio-rad) were stained with Ponceau S staining solution for protein loading reference pictures, then cut for different membrane immunostaining, and blocked with blocking buffer (5% (w/v) milk in 1× Trisbuffered saline (TBS), 0.1% (v/v) Tween) for 1 h. Blocked membranes were incubated with primary antibodies in blocking buffer overnight at 4°C, and finally with HRP-conjugated secondary antibodies for 1 h at room temperature. Detection was performed using Clarity Western ECL substrate (Bio-Rad) and Odyssey Fc Imager (LI-COR). Representative immunoblots are shown. Primary antibodies include TST (HPA003643, Atlas Antibodies), MPST (Ab224043, Abcam), GPX4 (SAB4300725, Sigma-Aldrich), OXPHOS complexes (total OXPHOS Rodent WB Antibody Cocktail, AB110413, ABCAM), SOD1 (HPA001401, Sigma-Aldrich), SOD2 (HPA001814, Sigma-Aldrich), KEAP1 (MABS5164, Millipore), NRF2 (AB62352, ABCAM).

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Mitochondrial isolation and high-resolution respirometry

Approximate 50 mg of brain cortex from C57BL/6J and Tst -/-mice were harvested and mitochondria were isolated utilizing a semi-automated pump-controlled tissue rupture system (0.7 mL/min) (PCC) and syringes (SGE, Trajan© Scientific, Australia) attached to a cell homogenizer (Isobiotech, EMBL Heidelberg, Germany) installed on a pump (ProSense, Oosterhout, NL). [START_REF] Honrath | SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake[END_REF] The isolated mitochondria were suspended in isolation buffer (250mM sucrose, 20mM HEPES, 3mM EDTA, pH 7.5). The mitochondrial concentration was quantified by Pierce® BCA Protein Assay Kit (Thermo Scientific). All the experiments were conducted on ice.

The mitochondrial respiration parameters were analyzed using an O2K oxygraphy (Oroboros systems, Innsbruck, Austria). 250 µg crude mitochondria from wildtype and Tst -/-mouse cortexes were monitored under continuous stirring at 750 rpm in 1 mL MiR05 (0.5 mM EGTA, 3 mM MgCl2, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH2PO4, 20mM HEPES, 110mM D-Sucrose, BSA, 1 g/l essentially fatty acid free, at pH7.4). Oxygen polarography was performed at 37 °C and the oxygen flux per tissue mass (pmol O2/s/mg) were recorded in realtime using the DatLab5 software. The mitochondrial respiration was assessed with the use of different substrates as follows: 5 mM pyruvate, 2 mM malate and 1.5 µM FCCP to uncouple all the ETC complexes and measure complex I respiration; subsequently, 0.5 µM rotenone was added to inhibit complex I, followed by 10 mM succinate to measure complex II respiration. In addition, 2.5 µM antimycin A was added to prevent both complex I and II mediated respiration due to inhibition of complex III, followed by 2 mM ascorbate combined with 0.5 mM TMPD to measure complex IV-mediated respiration. The measurement sequence was completed by injection of 10 mM sodium dithionite.

Detection of the ROS O 2 •-and H 2 O 2 , RNS NO and ONOO -, and the RSS H 2 S and H 2 S n

Fluorescent molecules were used to measure ROS, specifically O2 -(DHE, D7008, Sigma-Aldrich) and H2O2 (DCF-DA, D6883, Sigma-Aldrich), the RNS NO (DAF-2DA AB145283, ABCAM) and ONOO -(DHR123, D1054, Sigma-Aldrich), and the RSS H2S (7-Azido-4-Methylcoumarin, 802409, Sigma-Aldrich) and H2Sn (Sulfane Sulfur Probe 4/SSP4, SB10-10, Tebubio) in freshly extracted total proteins from brain cortex samples. This was done by spectrofluorometry using a microplate multimode reader Spark (Tecan).

Total SOD activity, catalase activity, total nitric oxide synthase activity, and total peroxidase activity

Total SOD activity was measured with the Superoxide Dismutase Activity Assay Kit (AB65354, ABCAM), which assesses the inhibition activity of xanthine oxidase by SOD.

Catalase activity was assessed with the Catalase Activity Assay Kit (AB83464, ABCAM). Total Nitric Oxide Synthase activity was measured with the Nitric Oxide Synthase (NOS) Activity Assay Kit (MAK407, Sigma-Aldrich). Total peroxidase activity was assessed with the Peroxidase Activity Assay Kit (MAK092, Sigma-Aldrich). Analyses were performed following manufacturer's instructions, with freshly extracted total proteins from brain cortex samples, and using microplate multimode reader Spark (Tecan).

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Glutamate content

Glutamate concentration was measured with the Glutamate Assay Kit (AB138883, ABCAM). Analysis was performed following manufacturer's instructions, with freshly extracted total proteins from brain cortex samples, and using microplate multimode reader Spark (Tecan).

RNA isolation, cDNA synthesis and quantitative real-time PCR

RNA was extracted from tissue slices according to the instruction of miRNeasy ® Mini Kit (Qiagen, Germany). Total RNA concentration was quantified by Nanodrop® (ND-1000 Spectrophotometer). Equal amount of cDNA synthesis and amplification were performed using Superscript II kit and RANDOM primers (Thermo Fisher Scientific, USA), according to the manufacture's protocol. The cDNA synthesis mix are incubated as follows: 10 mins at 25

Thiosulfate measurements in the cortex using LC-MS/MS

Frozen cortex (~25 mg, exact weight recorded) was homogenised in 160 mM EPPS, 16 mM DTPA and monobromobimane, pH 8.0 buffer (300 µL) and acetonitrile (300 µL) with 46 mM monobromobimane (MBBr) (25 µL) for 30 min, room temperature in the dark. The reaction was stopped with ethyl acetate (1 mL), the mixture centrifuged (400 x g for 15 min at 4 °C), and the upper organic layer (~800 µL) passed through a 96-well Filter+ Plate (Biotage, Sweden), along with an MBBr-derivatised calibration curve of thiosulfate (0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 20, 90 and 100 µM), followed by PLD+ extraction (Biotage, Sweden), reduction to dryness and resuspension in water (100 µL). Samples were analyzed on an I-Class UPLC system (Waters, UK) on a 16 min chromatographic run using an HSS T3 (2.1 x 150 mm; 1.8 mm) column kept at 50°C. MBB-thiosulfate was eluted using water (0.1% formic acid) / methanol (0.1% formic acid) system at 0.4 mL/min from 0-100% methanol over 6 mins, reequilibrating to 0% methanol at 15 mins. Multiple reaction monitoring parameters for the thiosulfate MBB derivative was: m/z 303.0 → 205.1 on a QTrap 6500+ (AB Sciex) with the following compound settings: declustering potential -65 V, collision exit potential -15 V, collision energy -18 V. Source conditions were -4.5 kV ion spray voltage, 600 °C temperature, gases (GS1=40, GS2=60, Curtain gas= 40 units). Analyst ® v1.7 was used for instrument control and data acquisition. Data analysis was carried out using MultiQuant v3.0 (AB Sciex). Linear regression analysis of the peak areas of MBB-thiosulfate was used to calculate the quantity of thiosulfate in each sample, corrected for tissue mass.

Statistics

The data are presented as mean ± standard deviation (SD). Statistical differences between two conditions were calculated using an unpaired T-test. Statistical differences among more than two conditions were calculated using an one-way ANOVA test. Statistical analysis was performed in GraphPad Prism (version 9.1.0), and repeat numbers are specified in the figure legends.

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Results

Tst -/-cerebral cortex exhibits a steady H 2 S level and a dysregulated GSH pathway

To investigate how TST influences the GSH pathway in the brain, we performed studies in the cortical brain region of conventional knock-out mice in which Tst deletion (termed Tst -/-) has been successfully validated (Fig. 1A). Although the difference in thiosulfate concentrations in the cerebral cortex between wildtype and knockout mice did not reach statistical significance (likely due to small sample size), mean thiosulfate concentration was approx. 3-fold higher in Tst -/-mice compared to WT (Fig. 1B). Next, sulfide and polysulfide concentrations were assessed in the cortex. While tissue steady-state levels of both, H2S and H2Sn were found to be maintained at similar levels in Tst -/-and C57BL/6J mice, mean concentrations were lower for H2S and higher for polysulfides (Fig. 1B). To investigate whether MPST can compensate for the lack of TST in the brain of the Tst -/-mice, we analysed Mpst mRNA and protein levels.

MPST protein and Mpst mRNA was decreased in Tst -/-mice (Fig. S1A-D). Interestingly, the MPST activity was not changed, although it tended to be higher in the brain of Tst -/-mice compared to C57BL/6J mice (Fig. S1E). To verify whether the GSH pathway was altered in the brain of Tst -/-mice, we measured its GSH and GSSG content. GSH was 36% lower in Tst -/- mice, while its oxidized form, GSSG, increased 5 times, causing the GSH/GSSG ratio to decline 7.2-fold in Tst -/-mice compared to wildtype controls (Fig. 1C). This indicates a redox imbalance secondary to TST enzyme activity loss. Moreover, we found a significant decrease of GPX4 protein (Fig. 1D; Fig. S1F) and the transcriptional level of GR significantly decreased in Tst -/-brain cortex (Fig. 4J). Altogether, our results established that expression and activity of TST exert a major effect on both H2S metabolism and the GSH antioxidant pathway (Fig. 1E).

TST deletion increases ATP in the cerebral cortex and elevates ROS via modulation of OXPHOS activity

Since TST exists almost exclusively in mitochondria and is capable of modulating mitochondrial respiratory complexes, we examined the impact of Tst deletion on mitochondrial activity in the cortical brain areas. For that purpose, we performed total ATP measurements generated by OXPHOS via luminescence. Total ATP was increased by 22% in the Tst -/-mice cortex compared with C57BL/6J mice (Fig. 2A). Next, we measured the protein levels of OXPHOS complexes, and found no significant differences between the two mouse genotypes (Fig. 2B; Fig. S2A). In contrast, basal respiration and complex IV activity represented by oxygen consumption rate were markedly increased in the cortex of Tst -/-mice, while no significant differences in Complex I capacity were observed among either mouse genotypes (Fig. 2C). The increase of complex IV activity in the absence of TST indicates an impact of this enzyme on mitochondrial capacity. The function of TST as modifier of GSH to form glutathione persulfide (which is a more efficient ROS scavenger than GSH itself) has been well characterized. [START_REF] Libiad | Polymorphic variants of human rhodanese exhibit differences in thermal stability and sulfur transfer kinetics[END_REF][START_REF] Libiad | Thiosulfate sulfurtransferase-like domaincontaining 1 protein interacts with thioredoxin[END_REF] We next determined the extent of O2-derived reactive species formation from OXPHOS. The O2 .-level was 10% higher in the cortex of Tst -/-mice, while tissue levels of H2O2 were 57% higher in the cerebral region of Tst -/-mice compared to C57BL/6J control mice J o u r n a l P r e -p r o o f (Fig. 2D).

Tst deficiency reprograms the antioxidant defense against endogenous ROS. To determine whether antioxidant expression/activity changed in the cortex of Tst -/-mice, we checked both protein and total enzyme activity levels of superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2). Both, SOD1 and SOD2 are vital endogenous antioxidant enzymes. The expression of SOD1 and SOD2 proteins was unchanged between the two groups, whereas total SOD enzyme activity showed a 34% decrease in the Tst -/-mice (Fig. 3A; Fig. S3A). When H2O2 is excessively produced, catalase participates in H2O2 breakdown. The catalase activity in Tst - /-was 7.6% higher relative to that observed in C57BL/6J mice (Fig. 3B). In addition, the total peroxidase from Tst -/-mice exhibited unaltered activity compared with C57BL/6J mice (Fig. 3C).

In the context of the RSI framework, we also measured nitric oxide synthase (NOS) activity and RNS (NO and ONOO -) production. NOS activity and NO content were similar in the brain cortex of either genotype (Fig. S3B andD), while ONOO -amount in cortexes of Tst -/-mice was 10% lower than in C57BL/6J mice (Fig. S3C). In summary, we found decreased SOD enzyme activity, increased catalase activity and unchanged peroxidase activity, when ROS is elevated due to the global loss of TST in murine brain tissue as schematically depicted in Fig. 3D. Sulfide is an endogenously produced gaseous signaling molecule via CBS, CSE and MPST. Polysulfides (H2Sn) became novel reactive sulfur species, which is derived from H2S with MPST. TST converts thiosulfate produced via non-enzymatic reactions to sulphite. Then oxidation of sulphite to sulphate is dependent on the glutathione system. GPX4 and GR is the cofactor of conversion between GSH and GSSG.

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Tst absence results in decrease of Nrf2-Keap1 pathway expression level and increased glutamate content

Activation of the Nrf2-Keap1 system results in an antioxidant defense response which is, at least in part, regulated by H2S and KEAP1 protein level. [START_REF] Suzuki | Molecular basis of the Keap1-Nrf2 system[END_REF][START_REF] Saito | Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response[END_REF][START_REF] Zhao | Hydrogen Sulfide Alleviates Liver Injury Through the S-Sulfhydrated-Kelch-Like ECH-Associated Protein 1/Nuclear Erythroid 2-Related J o u r n a l P r e -p r o o f Factor 2/Low-Density Lipoprotein Receptor-Related Protein 1 Pathway[END_REF] To investigate whether Tst deficiency causes a dysregulation of Nrf2 signaling, qRT-PCR was conducted for genes regulated by antioxidant response elements (AREs) under control of NRF2. Nrf2 and Keap1 mRNA were significantly decreased in Tst -/-cortical brain samples compared with C57BL/6J mice (Fig. 4A andB). NRF2 protein showed significant reduction and KEAP1 protein had an increased expression in Tst -/-mice brain cortexes when compared to those of control mice (Fig. 4C-E). At the same time, different ARE genes including Hmox1 and Txn2 mRNA showed relative reduction in the case of Tst deficiency, while Gstm1, Txn1, Noq1and Cat revealed no significant difference between the two types of mice (Fig. 4G).

GCLC, GCLM and GR are important ARE genes for proteins protecting against oxidative stress. All these genes were found to be significantly reduced in the Tst -/-brain (Fig. 4G). Since both GCLC and GCLM are critical for de novo GSH biosynthesis we also measured glutamate content and found a 1.8-fold increase in Tst -/-compared to wildtype mice, corroborating the effect of Tst deletion on this important antioxidant pathway (Fig. 4F).

Tst -/-mouse brain displayed deteriorated antioxidant capacity when facing paraquatinduced oxidative stress

In order to show the effects of TST loss when facing oxidative stress, we analyzed several oxidative stress markers including ROS, RSS, GSH/GSSG contents and GPX4 protein abundance. Under basal TST deficiency condition, ROS showed a significant increase in the Tst -/-mouse brains. Upon challenging both genotypes of mice with 25mg/kg paraquat (PQ) for 24hrs, both genotypes of mouse brain cerebral cortices showed significantly increased ROS compared to their basal levels. Cerebral cortex of Tst -/-mice showed 1.5-fold increase of H2O2 compared to those of C57BL/6J mice (Fig. 5A). As for reactive sulfur species, brain cortexes of Tst -/-mice showed similar amounts of H2S in the cortical brain area compared to C57BL/6J mice. Upon PQ challenging, there was no difference in both H2S and H2Sn intensity between the two genotypes (Fig. 5B).

The parameters relevant to the GSH content were also assessed. Following paraquat stimulation, both mouse genotypes had significantly higher GSH content compared to their basic levels in brain tissue. However, GSH amount generated in the brain cortex of Tst -/-mice was less than that of C57BL/6J mice. In contrast to the accumulation of GSSG in basal cerebral cortical area of Tst -/-mice, PQ-treated Tst -/-mice also had relatively lower GSSG compared to C57BL/6J mice. Therefore, in the cerebral cortex of PQ-treated mice the ratio of GSH/GSSG showed a significant increase when compared with C57BL/6J mice (Fig. 5C). Furthermore, we tested GPX4 protein expression and found that GPX4 had higher levels in PQ injected animals in response to acute redox damage in both mouse genotypes. In the mouse brain of Tst -/-mice, J o u r n a l P r e -p r o o f GPX4 protein level was slightly lower than in C57BL/6J mice (Fig. 5D). In summary, this additional challenge experiment showed an overall deteriorated antioxidant capacity in murine cerebral cortices in conditions of global TST deficiency.

J o u r n a l P r e -p r o o f -and H2O2 of mice brain cortexes in the presence (hatched pattern) or absence (no pattern) of 25mg/kg 24hrs from C57BL/6J (black) (n=4-5) and Tst -/-mice (n=4). B. Relative H2S and H2Sn concentrations of mice brain cortexes in the presence (hatched pattern) or absence (no pattern) of 25mg/kg 24hrs from C57BL/6J (black) (n=4-5) and Tst -/-mice (n=4). C. GSH and GSSG contents in mice cortexes in the presence (hatched pattern) or absence (no pattern) of 25mg/kg 24hrs from C57BL/6J (black) (n=4-5) and Tst -/-mice (n=4). D. Quantification of immunoblots of cerebral cortical area for GPX4 protein level in mice cortexes in the presence (hatched pattern) or absence (no pattern) of 25mg/kg 24hrs from C57BL/6J (black) (n=3) and Tst -/-mice (n=3). The immunoblots are shown in Fig S4.

Discussion

TST plays an important role in the oxidation of the endogenous gasotransmitter hydrogen sulfide, complementing the action of sulfide quinone oxidoreductase (SQOR) and persulfide dioxygenase (ETHE1/PDO). [START_REF] Hildebrandt | Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria[END_REF][START_REF] Libiad | Organization of the human mitochondrial hydrogen sulfide oxidation pathway[END_REF] Whether or not TST also contributes to antioxidant protection in the brain has not been investigated before. In Tst -/-mice, circulating thiosulfate and sulfide level were markedly elevated, while cerebral cortex displayed similar steady-state levels of sulfide and thiosulfate, as observed in the liver. [START_REF] Carter | The hepatic compensatory response to elevated systemic sulfide promotes diabetes[END_REF] This indicates that the response to Tst knockdown is organ specific in this chronic but viable sulfide elevation model. Starting from organ specificity, the free thiol antioxidant system of brain and liver revealed opposite responses in terms of GSH content. In Tst -/-mice, GSH was decreased in the cerebral cortex, while there was no difference in GSH content between the two genotypes in the liver. [START_REF] Carter | The hepatic compensatory response to elevated systemic sulfide promotes diabetes[END_REF] Since NADPH/H + dependent glutathione reductase (GR) is important for the recycling of GSSG to GSH [START_REF] Capelletti | Ferroptosis in liver diseases: An overview[END_REF] , its down-regulation in the brain of Tst -/-mice explains the alterations in GSSG levels observed. In support of an activation role of TST on ROS scavengers such as GSH, [START_REF] Kruithof | Unraveling the role of thiosulfate sulfurtransferase in metabolic diseases[END_REF] Tst -/-mice showed a lower GSH/GSSG ratio and higher ROS levels, indicating a redox signaling imbalance along with lower GPX4 and GR expression. Meanwhile, the Mpst transcription level of brain and liver in Tst -/-mice were reduced. In contrast, the liver functions as a major detoxification organ, and despite lower mRNA for Mpst protein levels were upregulated, perhaps for compensatorily enhanced sulfide removal, [START_REF] Carter | The hepatic compensatory response to elevated systemic sulfide promotes diabetes[END_REF] while MPST protein significantly decreased in the brain cortex and in mitochondria in Tst -/-mice.

TST interacts with iron-sulfur cluster proteins including Complex I (NADH dehydrogenase), Complex II (succinate dehydrogenase) and Complex III (Cytochrome bc1 ) of the mitochondrial respiratory chain) in oxidative pathway. [START_REF] Rydz | Sulfur administration in fe-s cluster homeostasis[END_REF] TST directly interacts with Complex I via sulfide transfer. [START_REF] Pagani | Interaction of rhodanese with mitochondrial NADH dehydrogenase[END_REF] In addition, TST transfers sulfane sulfur to Complex II and modifies its iron-sulfur structure via alteration of its phosphorylation status. Those functions led us to explore the metabolic phenotypes of the brain tissues of C57BL/6J and Tst -/-mice. The unexpected finding of mitochondrial activity remodeling was supported by several observations, including unchanged OXPHOS protein expression, increased basal respiration capacity and Complex IV activity in the presence of ascorbate and TMPD. However, the cerebral cortex of Tst -/-mice produced more ATP, and higher amounts of ROS compared to C57BL/6J control mice. ATP, the universal energy currency, is mainly synthesized in mitochondria by OXPHOS. The representative role of Complex IV as regulatory center of OXPHOS limits the rate of respiratory chain reaction. [START_REF] Kadenbach | Complex IV -The regulatory center of mitochondrial oxidative phosphorylation[END_REF] Hence, our finding of enhanced Complex IV activity is in line with the observation of enhanced ATP levels in Tst -/-mice.

J o u r n a l P r e -p r o o f ROS are generated during the incomplete reduction of O2 [START_REF] Bourgonje | Oxidative stress and redox-modulating therapeutics in Inflammatory bowel disease[END_REF][START_REF] Sies | Reactive oxygen species (ROS) as pleiotropic physiological signalling agents[END_REF] . In mitochondria, the major sources of O2 • -are the OXPHOS Complexes I and III. 20,49 O2 • -will be further processed quickly to H2O2 via SOD1 and SOD2. [START_REF] Zhao | Mitochondrial electron transport chain, ROS generation and uncoupling (Review)[END_REF] In the absence of TST, the significant inhibition of total SOD activity can be explained by excessive H2O2 in the brain cortex, which could possibly slow down H2O2 production as oxidative distress response. The metabolism of H2O2 to H2O is catalyzed by catalase, glutathione peroxidase, and peroxiredoxin 1. [START_REF] Pannala | Mechanistic characterization of the thioredoxin system in the removal of hydrogen peroxide[END_REF] Compared to O2 • -, the steadier feature of H2O2 allows for mitochondrial modulation of the cellular antioxidant system. [START_REF] Nolfi-Donegan | Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production, and methods of measurement[END_REF] The observed alterations in ROS metabolizing enzymes indicate the importance of TST in overall antioxidant capacity to protect the brain.

Our study of the Nrf2 pathway in Tst -/-mice highlights a potent interaction between Nrf2 signaling and the GSH antioxidant system [START_REF] Corsello | Role of hydrogen sulfide in nrf2-and sirtuindependent maintenance of cellular redox balance[END_REF][START_REF] Ryoo I Geun | Regulatory crosstalk between the oxidative stress-related transcription factor Nfe2l2/Nrf2 and mitochondria[END_REF][START_REF] Song | Nrf2 and ferroptosis: A new research direction for neurodegenerative diseases[END_REF][START_REF] Dodson | NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis[END_REF] . Importantly, Nrf2 is a major transcriptional factor regulating multiple processes related to GSH synthesis and regeneration. [START_REF] Yang | Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse: Novel model system for a severely compromised oxidative stress response[END_REF][START_REF] Chen | Glutamate cysteine ligase catalysis: Dependence on ATP and modifier subunit for regulation of tissue glutathione levels[END_REF] Beyond GSH content change, the transcriptional and protein levels of Nrf2 appear to be lower and its intracellular inhibitor Keap1 had higher protein expression in the brains of Tst -/-mice. A possible reason for the fact that TST deletion causes disrupted Nrf2-Keap1 signaling, might be found in the abnormal cysteine metabolism within the redox sensor protein KEAP1. TST's main function is sulfide oxidation as cysteine metabolism downstream, and KEAP1 protein contains 25 cysteines within mouse homologue, thus accumulated KEAP1 were detected in the brain of Tst -/-mice, consequently lower amount of Nrf2 protein was released from KEAP1 ubiquitin E3 ligase adapter, which further influenced the ARE gene transcription process. [START_REF] Saito | Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response[END_REF][START_REF] Dinkova-Kostova | Keap1, the cysteine-based mammalian intracellular sensor for electrophiles and oxidants[END_REF][START_REF] Naidu | C151 in KEAP1 is the main cysteine sensor for the cyanoenone class of NRF2 activators, irrespective of molecular size or shape[END_REF] In light with reduced transcription of Keap1, we found an increased KEAP1 protein level, and we suggest a post-transcriptional cellular redox-sensing mechanism as operative.

Similar reduction in Nrf2 activation in Tst -/-mice using transcription factor binding site (TFBS) enrichment analysis were found earlier. [START_REF] Carter | The hepatic compensatory response to elevated systemic sulfide promotes diabetes[END_REF] Additionally, glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits together catalyze the rate-limiting step in GSH biosynthesis from glutamate under conditions of oxidative distress. [START_REF] Abdul-Aziz | Oxidative stress responses and NRF2 in human leukaemia[END_REF] Nrf2 -/-mice exhibited a decrease in GSH content and GCL levels, [START_REF] Hoang | Decreased ovarian glutathione and glutamate cysteine ligase subunit protein levels in Nrf2 knockout mice[END_REF] which supported our results on the brains of Tst -/- mice. The decrease in Nrf2 mRNA and protein translate into a reduced function of the GSH antioxidant system. Moreover, the decrease in Nrf2-mediated free thiol antioxidants influences various cellular processes, ranging from synthesis, oxidation and reduction of GSH. Among the heterogeneity of brain cell types, the expression of Tst mRNA is known to be highest in astrocytes (https://www.proteinatlas.org/ENSG00000128311-TST/single+cell+type/brain). Astrocytes comprise the majority of glial cells in the mammalian central nervous system (CNS) and serve as major provider of GSH for neurons. [START_REF] Sjöstedt | An atlas of the protein-coding genes in the human, pig, and mouse brain[END_REF][START_REF]Human Protein Atlas proteinatlas[END_REF] Astrocytes contribute to neuronal activity, and their interaction play distinct, cooperative roles in neuronal redox homeostasis. Astrocytes could initiate and promote an increase in gene expression of Nrf2-mediated antioxidant pathway in response to mild oxidative stress, which results in the synthesis of GSH using glutamate. [START_REF] Baxter | Adaptive regulation of the brain's antioxidant defences by neurons and astrocytes[END_REF] When astrocytes function abnormally, glutamate, which is an excitatory neurotransmitter, will accumulate to an excitotoxic level and contribute to harmful effects on neurons, eventually causing impaired brain function. [START_REF] Maher | The role of Ca2+ in cell death caused by oxidative glutamate toxicity and ferroptosis[END_REF] Thus, any deficiency in astrocyte capacity to produce GSH (or reduce GSSG) will render neurons particularly vulnerable to conditions or insults known to be associated with enhanced oxidative stress. [START_REF] Schousboe | Glutamate Metabolism in the Brain Focusing on Astrocytes[END_REF][START_REF] Mcbean | Cysteine, glutathione, and thiol redox balance in astrocytes[END_REF][START_REF] Raps | Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons[END_REF][START_REF] Makar | Ascorbate, Glutathione, Glutathicne Disulfide, and Enzymes of Glutathione Metabolism in Cultures of Chick Astrocytes and Neurons: Evidence that Astrocytes Play an Important Role in Antioxidative Processes in the Brain[END_REF][START_REF] Yudkoff | Glutathione turnover in cultured astrocytes: studies with [15N]glutamate[END_REF] Both oxidative stress and elevated glutamate contribute and are associated with brain pathologies. [START_REF] Maher | The role of Ca2+ in cell death caused by oxidative glutamate toxicity and ferroptosis[END_REF] The decreased Nrf2 expression, reduced J o u r n a l P r e -p r o o f GSH and increased glutamate content, together with an altered ROS landscape in the CNS, secondary to a loss of TST are therefore key observations of the present study.

Relevant to Nrf2 signaling, another important thiol-dependent antioxidant system is the thioredoxin (TXN) system. [START_REF] Masutani | The thioredoxin system in retroviral infection and apoptosis[END_REF] TXN2 is a mitochondria-specific protein, whereas TXN1 is found in the cytosol, both of them operate using NADPH as cofactor. [START_REF] Tang | Overexpression of thioredoxin-2 attenuates age-related muscle loss by suppressing mitochondrial oxidative stress and apoptosis[END_REF] Down-regulated Txn2 mRNA level observed in the cerebral cortex of Tst -/-mice, could be explained by loss of TST's function to interact with mitochondrial NADH dehydrogenase (Complex I) and other iron-cluster proteins. [START_REF] Pagani | Interaction of rhodanese with mitochondrial NADH dehydrogenase[END_REF] Our findings provide a better understanding of the role of TST in linking Nrf2-keap1 signaling and thiol antioxidant pathways.

When challenging mice with paraquat-induced oxidative stress, the ROS production in Tst -/- mouse brain was dramatically increased due to the hampered antioxidant capacity, indicating the importance of the interaction of TST with the antioxidant pathway. The nature of GSH is to provide a protective effect on free radical scavenging and H2O2 detoxification 73 therefore, after stimulation, increased GSH content was detected in both genotypes, although Tst -/-mouse brain had lower GSH levels than control mice. Unlike the basal condition, PQ-treated mice biosynthesized less GSSG when the brain tissue of Tst -/-mice was compared to the WT animals. PQ injection also induced an increase in GPX4 protein expression in response to excessive oxidative distress. GPX4 protein in Tst -/-mice was slightly lower than in C67BL/6J mice. In summary, this additional challenge experiment showed an overall deteriorated antioxidant capacity in murine cerebral cortex under the model of global TST deficiency.

In the context of brain function, both mitochondria and NRF2 functions are particularly important because the brain is highly susceptible to oxidative distress due to its high metabolic rate and relatively low levels of antioxidants compared to other tissues [START_REF] Bellissimo | Superoxide dismutase, glutathione peroxidase activities and the hydroperoxide concentration are modified in the hippocampus of epileptic rats[END_REF] . During oxidative stress, structural and functional damage occurs to neurons and disrupts the normal actions of neurotransmitters, leading to altered signaling from neurons. This can lead to impaired cognitive function, affect behavior, and contribute to the development and progression of neurological and neurodegeneration diseases [START_REF] Singh | Oxidative Stress: A Key Modulator in Neurodegenerative Diseases[END_REF] . Taken together, our data unmask several tissuespecific mechanisms linked to TST function that are related to mitochondrial and Nrf2-Keap1 signaled antioxidant functions, and the role of TST in maintaining the health and function of brain tissues.

Conclusion

Our study on mouse cerebral cortex revealed that TST deficiency promotes a dysregulation of the reactive species interactome through both generation of ROS and RSS, coupled with mitochondrial OXPHOS remodeling. In addition, TST deficiency elicits the dysregulation of thiol-dependent antioxidant systems with altered GSH levels and aberrant Nrf2 pathway expression. Moreover, the absence of TST is characterized by a deteriorated antioxidant buffering ability in the murine brain cortex when challenged with a redox cycler as demonstrated by paraquat in vivo.

Fig. 1 .

 1 Fig. 1. Tst -/-mice brain cortexes display a dysregulated GSH pathway. A-D: Data are expressed as mean ± SD, student Ttest. A. Tst mRNA and Tst protein levels of Tst -/-(red) and C57BL/6J (black) mice (n=4). B. Thiosulfate content in C57BL/6J mice (Black, n=3) and Tst -/-mice brain cortexes (Red, n=4) measured by HPLC-MS/MS. H2S and H2Sn concentrations were measured using fluorescent probes, and show that H2S decreased 5% and H2Sn increased 5% in the Tst -/-(red) compared with C57BL/6J mice (black) (n=4). C. GSH and GSSG contents in mice cortexes in the presence (black) or absence (red) of TST (n=4). D. Quantification of immunoblots of cerebral cortical area for GPX4 protein level in Tst -/-(red) compared with C57BL/6J mice (black) (n=3). The original blotting picture is shown in Fig S1F. E. Brief schematic showing H2S enzymatic pathway.Sulfide is an endogenously produced gaseous signaling molecule via CBS, CSE and MPST. Polysulfides (H2Sn) became novel reactive sulfur species, which is derived from H2S with MPST. TST converts thiosulfate produced via non-enzymatic reactions to sulphite. Then oxidation of sulphite to sulphate is dependent on the glutathione system. GPX4 and GR is the cofactor of conversion between GSH and GSSG.

Fig. 2 .

 2 Fig. 2. Tst -/-mice brain cortexes display metabolism reprogramming. A-D: Data expressed as mean ± SD, student T-test. A. Total ATP level of C57BL/6J (black) and Tst -/-(red) mice cortical area (n=4). B. OXPHOS protein levels of two genotypes detected by OXPHOS cocktail antibody (n=3). Quantification is normalized to ponceau stain. Immunoblot is displayed in Fig. S2. C. Corrected oxygen consumption rate of 250ug crude isolated mitochondria from mice cerebral cortices with various substrates. Respiratory capacity differences of basal respiration, Complex I, Complex II and Complex IV from C57BL/6J (black) and Tst -/-(red) were analyzed. (n=3 biological samples, independent experiments were repeated at least 3 times). D. Relative ROS contents as O2 .-and H2O2 of C57BL/6J (black) and Tst -/-(red) (n=4).

Fig. 3 .

 3 Fig. 3. Tst -/-mice cerebral cortical regions display reprogrammed antioxidant defense. A-D: Data expressed as mean ± SD, student T-test. A. SOD protein expression (n=3) and enzyme activity (n=4) levels of C57BL/6J (black) and Tst -/-(red) mice cortical area. Original immunoblots are presented in Fig.S3A. 2. B. Catalase enzyme activity measurements in C57BL/6J (black) and Tst -/-(red) mice (n=4). C. Peroxidase activity in C57BL/6J (black) and Tst -/-(red) mice (n=4). D. Created with Biorender.com. Brief scheme of response to excessive ROS with or without TST presence. SOD1 and SOD2 are vital endogenous antioxidant elements for H2O2 catalyzation from O2 -.

Fig. 4 .

 4 Fig. 4. Tst -/-mice brain cortexes display dysregulated Nrf2-keap1 signaling expression and increased glutamate content. A-B, D-G: Data expressed as mean ± SD, student T-test. A. Expression of Keap1 mRNA in C57BL/6J (black) (n=4) and Tst -/- (red) (n=4) mice levels of cortical area. B. Expression of Nrf2 mRNA in C57BL/6J (black) (n=4) and Tst -/-(red) (n=4) mice levels of cortical area. C. WB analysis of the two proteins. D. Quantification of immunoblots of cerebral cortical area for NRF2 protein level in mice cortexes in the presence from C57BL/6J (black) (n=3) and Tst -/-mice (n=3). E. Quantification of immunoblots of cerebral cortical area for KEAP1 protein level in mice cortexes in the presence from C57BL/6J (black) (n=3) and Tst -/-mice (n=3). F. Glutamate content measurements in both genotypes (n=4). G. Tst -/-mice brain cortexes display

Fig. 5 PQ

 5 Fig.5PQ challenge exacerbated Tst -/-mice brain cortexes disrupted antioxidant capacity. A-D: Data are expressed as mean ± SD, one-way ANOVA test. A. Relative ROS contents as O2 .-and H2O2 of mice brain cortexes in the presence (hatched pattern) or absence (no pattern) of 25mg/kg 24hrs from C57BL/6J (black) (n=4-5) and Tst -/-mice (n=4). B. Relative H2S and H2Sn concentrations of mice brain cortexes in the presence (hatched pattern) or absence (no pattern) of 25mg/kg 24hrs from C57BL/6J (black) (n=4-5) and Tst -/-mice (n=4). C. GSH and GSSG contents in mice cortexes in the presence (hatched pattern) or absence (no pattern) of 25mg/kg 24hrs from C57BL/6J (black) (n=4-5) and Tst -/-mice (n=4). D. Quantification of immunoblots of cerebral cortical area for GPX4 protein level in mice cortexes in the presence (hatched pattern) or absence (no pattern) of 25mg/kg 24hrs from C57BL/6J (black) (n=3) and Tst -/-mice (n=3). The immunoblots are shown in Fig S4.

  °C, 50mins at 42 °C, 15 mins under 70 °C via a Veriti 96 Well Thermal Cycler (Thermo Fisher Scientific). For qRT-PCR, 500 ng of cDNA samples were mixed with SYBR green, forward primers, and reverse primers (Primers used for quantification are shown in Supplementary Table.1). The qPCR was run on LightCycler® 480 II (Roche) and the protocol was as follows: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15s 95 °C and 60s 60 °C, and the dissociation curve is run under 15s 95 °C, 15s 60 °C, 15s 95 °C. RT-qPCR data was analyzed on LightCycler 480 software. Expression levels were normalized to GAPDH and displayed as 2-delta Ct.

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Acknowledgements

All authors greatly appreciate the technical assistance from Wierd Kooistra and Albert Gerding on experimental training. LC-MS/MS data were obtained at the University of Edinburgh, Edinburgh Clinical Research Facility, Mass Spectrometry Core, RRID:SCR_021833 using the AB SCIEX QTRAP 6500 plus system, RRID:SCR_021831. We acknowledge Joanna Simpson for expertise and Scott Denham for operation of the LC-MS/MS instrumentation. YL and HvG are members of COST (European Cooperation in Science and Technology) Action CA20121 (Bench to bedside transition for pharmacological regulation of NRF2 in noncommunicable diseases, BenBedPhar).

Funding Y.L. was supported by China Scholarship Council (Grant No.: 202008520033). Z. M. Al-Dahmani was supported by the Islamic Development Bank. A.M.D. was supported by a Rosalind Franklin Fellowship co-funded by the European Union and the University of Groningen. S.M. was supported by a British Heart Foundation 4Y PhD Scholarship (FS/4yPhD/F/20/34126). L.C was supported by the CNRS. Contribution Statement YL, AM and HvG designed and conceptualized the study, wrote and revised the manuscript. YL conducted parts of research, LC designed and performed parts of study, edited the manuscript. YL and LC analyzed the data. ZMM conducted parts of experiments. SM, NZMH and NMM performed experiments on the animal model, provided samples, and edited the manuscript. MG and MF revised the manuscript. AM and HvG supervised this study.

Data availability

Data will be made available on request.

Declaration of competing interest

The authors hereby declare that there is no competing financial interests or personal relationships that could have influenced the work in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online.

Reference:

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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