Title: Excess hydrogen sulfide and polysulfides production underlies a schizophrenia pathophysiology
Abstract: Article28 October 2019Open Access Source DataTransparent process Excess hydrogen sulfide and polysulfides production underlies a schizophrenia pathophysiology Masayuki Ide Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Department of Psychiatry, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Search for more papers by this author Tetsuo Ohnishi Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Manabu Toyoshima Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Shabeesh Balan orcid.org/0000-0002-1098-1290 Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Motoko Maekawa Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Chie Shimamoto-Mitsuyama Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Yoshimi Iwayama Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Support Unit for Bio-Material Analysis, Research Division, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Hisako Ohba Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Akiko Watanabe Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Takashi Ishii Research& Development Department, MCBI Inc, Tsukuba, Ibaraki, Japan Search for more papers by this author Norihiro Shibuya Department of Pharmacology, Sanyo-Onoda City University, Sanyo-Onoda, Yamaguchi, Japan Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan Search for more papers by this author Yuka Kimura Department of Pharmacology, Sanyo-Onoda City University, Sanyo-Onoda, Yamaguchi, Japan Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan Search for more papers by this author Yasuko Hisano Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Yui Murata Department of Molecular Brain Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Tomonori Hara Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Department of Organ Anatomy, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan Search for more papers by this author Momo Morikawa Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kenji Hashimoto Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan Search for more papers by this author Yayoi Nozaki Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Tomoko Toyota Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Yuina Wada Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan Search for more papers by this author Yosuke Tanaka Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Tadafumi Kato Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Akinori Nishi Department of Pharmacology, Kurume University School of Medicine, Kurume, Fukuoka, Japan Search for more papers by this author Shigeyoshi Fujisawa Laboratory for Systems Neurophysiology, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Hideyuki Okano Department of Physiology, Keio University School of Medicine, Tokyo, Japan Search for more papers by this author Masanari Itokawa Center for Medical Cooperation, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Nobutaka Hirokawa orcid.org/0000-0002-0081-5264 Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yasuto Kunii Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan Department of Psychiatry, Aizu Medical Center, Fukushima Medical University, Aizuwakamatsu, Fukushima, Japan Search for more papers by this author Akiyoshi Kakita Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan Search for more papers by this author Hirooki Yabe Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan Search for more papers by this author Kazuya Iwamoto Department of Molecular Brain Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Kohji Meno Research& Development Department, MCBI Inc, Tsukuba, Ibaraki, Japan Search for more papers by this author Takuya Katagiri Department of Pharmacy, Faculty of Pharmacy, Iryo Sosei University, Iwaki, Fukushima, Japan Search for more papers by this author Brian Dean The Florey Institute of Neuroscience and Mental Health, Howard Florey Laboratories, The University of Melbourne, Parkville, Vic., Australia The Centre for Mental Health, Swinburne University, Hawthorn, Vic., Australia Search for more papers by this author Kazuhiko Uchida Department of Molecular Oncology, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Search for more papers by this author Hideo Kimura Department of Pharmacology, Sanyo-Onoda City University, Sanyo-Onoda, Yamaguchi, Japan Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan Search for more papers by this author Takeo Yoshikawa Corresponding Author [email protected] orcid.org/0000-0003-2791-6679 Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Masayuki Ide Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Department of Psychiatry, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Search for more papers by this author Tetsuo Ohnishi Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Manabu Toyoshima Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Shabeesh Balan orcid.org/0000-0002-1098-1290 Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Motoko Maekawa Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Chie Shimamoto-Mitsuyama Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Yoshimi Iwayama Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Support Unit for Bio-Material Analysis, Research Division, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Hisako Ohba Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Akiko Watanabe Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Takashi Ishii Research& Development Department, MCBI Inc, Tsukuba, Ibaraki, Japan Search for more papers by this author Norihiro Shibuya Department of Pharmacology, Sanyo-Onoda City University, Sanyo-Onoda, Yamaguchi, Japan Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan Search for more papers by this author Yuka Kimura Department of Pharmacology, Sanyo-Onoda City University, Sanyo-Onoda, Yamaguchi, Japan Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan Search for more papers by this author Yasuko Hisano Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Yui Murata Department of Molecular Brain Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Tomonori Hara Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Department of Organ Anatomy, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan Search for more papers by this author Momo Morikawa Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kenji Hashimoto Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan Search for more papers by this author Yayoi Nozaki Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Tomoko Toyota Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Yuina Wada Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan Search for more papers by this author Yosuke Tanaka Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Tadafumi Kato Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Akinori Nishi Department of Pharmacology, Kurume University School of Medicine, Kurume, Fukuoka, Japan Search for more papers by this author Shigeyoshi Fujisawa Laboratory for Systems Neurophysiology, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Hideyuki Okano Department of Physiology, Keio University School of Medicine, Tokyo, Japan Search for more papers by this author Masanari Itokawa Center for Medical Cooperation, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Search for more papers by this author Nobutaka Hirokawa orcid.org/0000-0002-0081-5264 Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yasuto Kunii Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan Department of Psychiatry, Aizu Medical Center, Fukushima Medical University, Aizuwakamatsu, Fukushima, Japan Search for more papers by this author Akiyoshi Kakita Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan Search for more papers by this author Hirooki Yabe Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan Search for more papers by this author Kazuya Iwamoto Department of Molecular Brain Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Kohji Meno Research& Development Department, MCBI Inc, Tsukuba, Ibaraki, Japan Search for more papers by this author Takuya Katagiri Department of Pharmacy, Faculty of Pharmacy, Iryo Sosei University, Iwaki, Fukushima, Japan Search for more papers by this author Brian Dean The Florey Institute of Neuroscience and Mental Health, Howard Florey Laboratories, The University of Melbourne, Parkville, Vic., Australia The Centre for Mental Health, Swinburne University, Hawthorn, Vic., Australia Search for more papers by this author Kazuhiko Uchida Department of Molecular Oncology, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan Search for more papers by this author Hideo Kimura Department of Pharmacology, Sanyo-Onoda City University, Sanyo-Onoda, Yamaguchi, Japan Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan Search for more papers by this author Takeo Yoshikawa Corresponding Author [email protected] orcid.org/0000-0003-2791-6679 Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan Search for more papers by this author Author Information Masayuki Ide1,2,‡, Tetsuo Ohnishi1,‡, Manabu Toyoshima1, Shabeesh Balan1, Motoko Maekawa1, Chie Shimamoto-Mitsuyama1, Yoshimi Iwayama1,3, Hisako Ohba1, Akiko Watanabe1, Takashi Ishii4, Norihiro Shibuya5,6, Yuka Kimura5,6, Yasuko Hisano1, Yui Murata7, Tomonori Hara1,8, Momo Morikawa9, Kenji Hashimoto10, Yayoi Nozaki1, Tomoko Toyota1, Yuina Wada1,11, Yosuke Tanaka9, Tadafumi Kato12, Akinori Nishi13, Shigeyoshi Fujisawa14, Hideyuki Okano15, Masanari Itokawa16, Nobutaka Hirokawa9, Yasuto Kunii17,18, Akiyoshi Kakita19, Hirooki Yabe17, Kazuya Iwamoto7, Kohji Meno4, Takuya Katagiri20, Brian Dean21,22, Kazuhiko Uchida23, Hideo Kimura5,6 and Takeo Yoshikawa *,1 1Laboratory of Molecular Psychiatry, RIKEN Center for Brain Science, Wako, Saitama, Japan 2Department of Psychiatry, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan 3Support Unit for Bio-Material Analysis, Research Division, RIKEN Center for Brain Science, Wako, Saitama, Japan 4Research& Development Department, MCBI Inc, Tsukuba, Ibaraki, Japan 5Department of Pharmacology, Sanyo-Onoda City University, Sanyo-Onoda, Yamaguchi, Japan 6Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan 7Department of Molecular Brain Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan 8Department of Organ Anatomy, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan 9Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 10Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan 11Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan 12Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Center for Brain Science, Wako, Saitama, Japan 13Department of Pharmacology, Kurume University School of Medicine, Kurume, Fukuoka, Japan 14Laboratory for Systems Neurophysiology, RIKEN Center for Brain Science, Wako, Saitama, Japan 15Department of Physiology, Keio University School of Medicine, Tokyo, Japan 16Center for Medical Cooperation, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 17Department of Neuropsychiatry, School of Medicine, Fukushima Medical University, Fukushima, Japan 18Department of Psychiatry, Aizu Medical Center, Fukushima Medical University, Aizuwakamatsu, Fukushima, Japan 19Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan 20Department of Pharmacy, Faculty of Pharmacy, Iryo Sosei University, Iwaki, Fukushima, Japan 21The Florey Institute of Neuroscience and Mental Health, Howard Florey Laboratories, The University of Melbourne, Parkville, Vic., Australia 22The Centre for Mental Health, Swinburne University, Hawthorn, Vic., Australia 23Department of Molecular Oncology, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +81 48 467 5968; Fax: +81 48 467 7462; E-mail: [email protected] EMBO Mol Med (2019)11:e10695https://doi.org/10.15252/emmm.201910695 See also: M Simonneau (December 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mice with the C3H background show greater behavioral propensity for schizophrenia, including lower prepulse inhibition (PPI), than C57BL/6 (B6) mice. To characterize as-yet-unknown pathophysiologies of schizophrenia, we undertook proteomics analysis of the brain in these strains, and detected elevated levels of Mpst, a hydrogen sulfide (H2S)/polysulfide-producing enzyme, and greater sulfide deposition in C3H than B6 mice. Mpst-deficient mice exhibited improved PPI with reduced storage sulfide levels, while Mpst-transgenic (Tg) mice showed deteriorated PPI, suggesting that "sulfide stress" may be linked to PPI impairment. Analysis of human samples demonstrated that the H2S/polysulfides production system is upregulated in schizophrenia. Mechanistically, the Mpst-Tg brain revealed dampened energy metabolism, while maternal immune activation model mice showed upregulation of genes for H2S/polysulfides production along with typical antioxidative genes, partly via epigenetic modifications. These results suggest that inflammatory/oxidative insults in early brain development result in upregulated H2S/polysulfides production as an antioxidative response, which in turn cause deficits in bioenergetic processes. Collectively, this study presents a novel aspect of the neurodevelopmental theory for schizophrenia, unraveling a role of excess H2S/polysulfides production. Synopsis This study proposes a novel concept that excess hydrogen sulfide production (sulfide stress) underlies a schizophrenia pathophysiology in the realm of neurodevelopmental hypothesis of the disease. Targeting the metabolic pathway of hydrogen sulfide provides a novel therapeutic approach. Mpst-deficient mice exhibited improved prepulse inhibition (PPI), a typical schizophrenia-relevant endophenotype, with reduced sulfide levels, while Mpst-transgenic mice showed deteriorated PPI. Postmortem brains and iPS-derived cells from a subset of schizophrenia patients displayed evidence for sulfide stress. Sulfide stress condition stemmed from insults in developing brain in mouse models and elicited dampened energy metabolism. MPST expression level in hair follicles has a potential to stratify schizophrenia patients with sulfide stress. Introduction Schizophrenia is a severe mental illness featuring three major symptomatic domains: positive symptoms (hallucinations, delusions, etc.), negative symptoms (affective flattening, avolition, etc.), and cognitive deficits (disorganized thought, etc.) (American Psychiatric Association, 2013). This illness exhibits a life-time prevalence of approximately one percent worldwide. Repeated relapses of psychotic symptoms often lead to a deterioration of brain function, and eventually to end-stage illness in some cases, characterized by persistent symptoms and profound functional disabilities (Lewis & Lieberman, 2000). Though causal mechanisms are elusive, accumulating lines of evidence have shown abnormalities in early neurodevelopment processes, stemmed from genetic aberrations and environmental factors, such as maternal immune activation, for the etiopathogenesis of schizophrenia (neurodevelopmental hypothesis) (Knuesel et al, 2014; Estes & McAllister, 2016; Birnbaum & Weinberger, 2017). And for symptomatic treatments, drugs targeting dopaminergic systems are predominantly used (Murray et al, 2017). However, because currently available therapeutics has limitations in terms of efficacies and adverse effects, a new paradigm is needed for the development of novel drugs. Here, we hypothesize that examination of the traits and associated molecular underpinnings in inbred mouse strains could potentially identify as-yet-unknown pathophysiologies of schizophrenia. Pursuing this hypothesis, we have already reported that across 4 strains of mice, C57BL/6N (B6) mice exhibited the highest prepulse inhibition (PPI) scores while C3H/HeN (C3H) the lowest (Watanabe et al, 2007). PPI is the normal suppression of a startle response when a low-intensity stimulus immediately precedes an unexpected stronger startling stimulus. As a reproducible phenotypic marker, impaired PPI reflects sensorimotor gating deficits, and is typically regarded as an endophenotype for schizophrenia (Braff et al, 2001; Roussos et al, 2016). To explore the molecular signature underlying the behavioral differences between B6 and C3H, we performed proteomic analyses, using 2D-DIGE (two-dimensional difference gel electrophoresis) and MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). This screening step revealed an increase in the levels of a hydrogen sulfide (H2S)- and polysulfides- (H2S/polysulfides)-producing enzyme, Mpst (3-mercaptopyruvate sulfurtransferase; also known as 3MST) (Appendix Fig S1), in C3H mice compared to B6 animals. Then, we comprehensively assessed the biological possibility of the novel theory of "excessive H2S/polysulfides production" in schizophrenia, and pursued the mechanism underlying the functional consequence and causative origin of this phenomenon. Results Proteomic analyses of brain and splenic lymphocytes from B6 and C3H mice identified Mpst We performed proteomic analyses using brain and lymphocyte preparations from B6 and C3H mice to detect a homologous biomarker in the peripheral blood of schizophrenia patients. As shown in Fig 1A and B, and Appendix Tables S1 and S2, of the 1,093 spots identified from the brain using 2D-DIGE, 43 showed significant differences in expression between B6 and C3H animals, with our criteria of a fold change > 1.2 and P < 0.05. Of the 1,400 spots detected from the lymphocyte preparations, 131 showed significant differences in expression between B6 and C3H mice. Sixteen of these differentially expressed protein spots showed consistent change trends between the tissues from the two mouse strains (Appendix Table S2). Figure 1. Differential proteomic analysis of brain and lymphocyte tissues from B6 and C3H mice using 2D-DIGE A, B. Results of 2D-DIGE on frontal cortex tissue (A) and lymphocytes (B). Green and red rings denote spots showing significantly increased expression in B6 and C3H mice, respectively. C–G. Merged images of Cy3 (green) and Cy5 (red): green and red spots represent significantly increased expression of the corresponding protein isoforms in B6 and C3H mice, respectively. Nine (spot nos. 1, 2, 3, 4, 5, 10, 11, 12 and 13) out of the 16 spots were successfully identified as mortalin (Hspa9) (C), nucleophosmin (Npm1) (D), mercaptopyruvate sulfurtransferase (Mpst) (E), peroxiredoxin 6 (Prdx6) (F) and nucleoside diphosphate kinase B (Nme2) (G). Data information: Significance for differential expression between B6 and C3H was defined as P value of < 0.05 (unpaired two-tailed t-test) and fold change > 1.2. Yellow numbers indicate the 16 spots that showed consistent alterations between brain and lymphocyte samples from the two mouse strains. Source data are available online for this figure. Source Data for Figure 1 [emmm201910695-sup-0003-SDataFig1AB.pdf] Download figure Download PowerPoint We successfully identified the molecular entities of nine of these protein spots, by peptide mass fingerprinting (PMF), and found five different proteins: heat shock 70 kDa protein 9 (mortalin, Hspa9), nucleophosmin/nucleoplasmin (Npm1), Mpst, peroxiredoxin 6 (Prdx6), and nucleoside diphosphate kinase B (Nme2) (Appendix Tables S3 and S4). The proteins Hspa9, Npm1, Prdx6, and Nme2 appeared as paired spots at different isoelectric points (pI) (Fig 1C, D, F and G). Sequencing of the genes encoding these proteins revealed that each gene harbored single-nucleotide polymorphisms (SNPs) or insertion/deletion polymorphisms, which altered the amino acid sequences between the two mouse strains (Appendix Table S5 and Appendix Fig S2). The theoretical pI and molecular mass values calculated based on DNA sequences and possible posttranslational modifications were consistent with the observed spot profiles of these proteins in 2D gels, and were confirmed by 2D Western blotting (Fig 2A–J). By using 2D-DIGE, Mpst appeared as a single spot that was expressed in only C3H mice (Fig 1E). Two-dimensional Western blotting confirmed this spot for C3H animals (Fig 2F) and detected a faint spot for B6 mice at a different pI from the spot observed for C3H mice (Fig 2E). Sequencing of the Mpst gene revealed a Asp102Gly polymorphism (Appendix Table S5 and Appendix Fig S2), which can explain the differential mobility in the 2D gel between the two mouse strains. The Mpst protein catalyzes the transfer of a sulfur ion from 3-mercaptopyruvate to cyanide or other thiol compounds (Szabo, 2007; Kimura, 2015; Wallace & Wang, 2015), and this reaction produces H2S/polysulfides and detoxifies cyanide (Appendix Fig S1). The Asp102Gly polymorphism is predicted to have little effect ("benign") by the PolyPhen-2 algorithm (http://genetics.bwh.harvard.edu/pph2/). Indeed, the functional assay conducted by preparing Asp102 and Gly102 Mpst constructs showed no significant differences in enzymatic activity between the variants (Appendix Fig S3 and Appendix Table S6). Figure 2. Identified proteins visualized by 2D Western blotting A–J. Whole protein extracts from brain tissue of B6 (A, E, G, I). Whole protein extracts from brain tissue of C3H (B, F, H, J). Whole protein extracts from lymphocytes of B6 (C) and C3H (D). Npm3 expression levels were low in the brain (C, D). Hspa9 (mortalin) (A, B), Npm1 (nucleophosmin) (C, D), Mpst (mercaptopyruvate sulfurtransferase) (Mpst) (E, F), Prdx6 (peroxiredoxin 6) (G, H) and Nme2 (nucleoside diphosphate kinase B) (I, J) were detected by 2D Western blotting using the corresponding antibodies and chemiluminescence (red). The chemiluminescent signal of Nme2 was visualized by the LAS 3000 chemiluminescence image analyzer and the other signals were visualized by a Typhoon 9400. Data information: White crosses (+) indicate landmark spots. Spot numbers (indicated by arrows) correspond to the spot numbers in Fig 1. Yellow arrowheads (G, H) indicate the overoxidized form of Prdx6. Download figure Download PowerPoint The Mpst spot was the only protein to show differential expression, exhibiting lower expression in B6 mice than in C3H mice. The protein expression levels for Mpst were confirmed by standard Western blot analyses of B6 and C3H mice using both brains and splenic lymphocytes: significantly higher expression of Mpst was observed in the frontal cortex of the C3H mouse brain than in that of the B6 brain using both anti-Mpst N-terminus (Mpst-N, P = 0.03, Fig EV1A) and anti-Mpst C-terminus (Mpst-C, P = 0.02, Fig 3A) antibodies, though only a marginally increased expression of Mpst was observed in C3H mice in splenic lymphocytes (Mpst-N, P = 0.25; Mpst-C, P = 0.11) (Figs 3B and EV1B). Because of the nonsignificant differences in protein expression levels in lymphocytes between the two strains, we hereafter focused on mainly brain tissues. Click here to expand this figure. Figure EV1. Expression of Mpst examined using anti-N-terminal Mpst antibodies A, B. Mpst protein levels in the brain and lymphocytes from B6 (n = 4) and C3H (n = 4) mice were quantified by standard Western blotting with anti-N-terminal Mpst antibodies. The expression levels of Mpst were normalized using α-tubulin. Bar graphs show the mean expression levels of Mpst in brain (A) and lymphocyte (B) tissues. Data information: P values were calculated by using unpaired two-tailed t-test. The values represent the mean ± SD. Download figure Download PowerPoint Figure 3. Expression of Mpst/Mpst and genes encoding the other H2S-producing enzymes and profile of H2S metabolic states in mice A, B. Mpst protein levels in the brain and lymphocytes from B6 and C3H mice were quantified by standard Western blotting with anti-C-terminal Mpst antibodies (for anti-N-terminal Mpst antibodies, see Fig EV1). The expression levels of Mpst were normalized using α-tubulin. Bar graphs show the mean expression levels of Mpst in brain (A) and lymphocyte (B) tissues. C. Transcript expression levels of genes encoding three H2S-producing enzymes in the frontal cortex of B6 (n = 15) and C3H mice (n = 14). The values represent the mean ± SEM. D. H2S content in the frontal cortex of B6 (n = 8) and C3H mice (n = 8). The values are relative to those of B6 and represent mean ± SEM. E. Levels of labile sulfur in the frontal cortex of B6 (n = 10) and C3H mice (n = 10). The values are relative to those of B6 and represent mean ± SEM. F. Levels of bound sulfane sulfur in the frontal cortex of B6 (n = 10) and C3H mice (n = 10). The values are relative to those of B6 and represent mean ± SEM. Data information: P values wer