Excess hydrogen sulfide and polysulfides production underlies a schizophrenia pathophysiology.

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 (H2 S)/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 H2 S/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 H2 S/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 H2 S/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 H2 S/polysulfides production.

Global analysis of dynamic changes in lipid raft proteins during T-cell activation.

Lipid rafts are considered as specialized microdomains within the plasma membrane with unique lipid compositions different from surrounding membranes. Following T-cell receptor (TCR) stimulation, lipid rafts assemble in T-cell/antigen-presenting cell (APC) contact site known as the immunological synapse, inner leaflets of which serve as activation or docking sites for downstream signaling components. To understand the signaling events occurring in lipid rafts, we globally analyzed dynamic changes in lipid raft proteins during TCR/CD28 costimulation using 2-D fluorescence difference gel electrophoresis. We detected multiple spots whose intensities were enhanced after costimulation, and identified proteins in these spots by PMF. Identified proteins include Src family tyrosine kinases, tyrosine phosphatase, phosphatidylinositol 3-kinase (PI3-kinase), actin-binding proteins, and regulators for small GTPases. Of particular interest, a number of pleckstrin homology (PH) domain-containing proteins were identified. Biochemical and histochemical analyses confirmed the translocation of these proteins from cytosol to lipid rafts. We also demonstrated that these proteins assembled at the T-cell/APC interface. These results indicate the efficacy of our system to systematically analyze dynamics of lipid raft proteins during extracellular stimulation.

Alterations of DNA copy number and expression in genes involved in cell cycle regulation and apoptosis signal pathways in gamma-radiation-sensitive SX9 cells and -resistant SR-1 cells.

In the present study, genomic differences related to sensitivity to radiation were examined by comparative genomic hybridization and GeneChip 45K microarray in SX9 cells (radiation-sensitive) and their parental line, SR-1 (radiation-resistant). SX9 cells have defective DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity. DNA-PKcs is a DNA double-strand break repair protein that maintains chromosomal stability through nonhomologous end joining. However, the molecular basis of the radiation sensitivity of SX9 cells is unclear. Flow cytometry analysis showed that SR-1 and SX9 cells had a larger G2/M-phase population at 12 h after 4 Gy gamma irradiation, while only SR-1 cells progressed to G1/S at 24-36 h. SX9 and SR-1 cells had similar patterns of DNA copy number alteration, but the gains were observed on chromosome 9 (cent-E2), 11 (cent-A3), and 12 (C1-E) only in SX9 cells. Expression of genes located on those regions is higher in SX-9 cells than in SR1 cells, and the regions include genes associated with apoptosis and cell cycle regulation. Time-course data for gene expression at 0, 1, 3, 6 and 12 h after 4 Gy gamma irradiation revealed that the genes whose expression was altered in SX9 cells but not in SR-1 cells are in 16 clusters. Three of these clusters included genes for cell cycle regulation: JNK, PKC (PRKC) and ceramide cascade protein. These results suggest that amplification and altered expression of genes associated with cell cycle and apoptosis regulators in DNA-PK-deficient SX9 cells affect the differences in response to gamma radiation between SX9 and SR-1 cells.

A novel hematopoietic adaptor protein, Chat-H, positively regulates T cell receptor-mediated interleukin-2 production by Jurkat cells.

Chat (Cas/HEF1-associated signal transducer) is a novel adaptor protein with an N-terminal Src homology-2 domain and C-terminal Cas/HEF1 association domain. We report here the molecular cloning of Chat-H, the hematopoietic isoform of Chat. Chat-H has an extended N-terminal domain besides the known Chat domain structures, suggesting a unique function of Chat-H in hematopoietic cells. Jurkat transfectants overexpressing Chat-H show a marked increase in interleukin-2 production after costimulation of T cell receptor and CD28. The degree of JNK activation is enhanced substantially in the Chat-H transfectants upon costimulation. The Src homology-2 domain mutant of Chat-H loses this signal modulating activity. Expression of the Cas/HEF1 association domain mutant exhibits a dominant negative effect on both JNK activation and interleukin-2 production. We further found that Chat-H forms a complex with Pyk2H and enhances its tyrosine 402 phosphorylation, an up-regulator of the JNK pathway. These results suggest that Chat-H positively controls T cell function via integrating the costimulatory signals.

TCR engagement induces proline-rich tyrosine kinase-2 (Pyk2) translocation to the T cell-APC interface independently of Pyk2 activity and in an immunoreceptor tyrosine-based activation motif-mediated fashion.

The relocation of kinases in T lymphocytes during their cognate interaction with APCs is essential for lymphocyte activation. We found that the proline-rich tyrosine kinase-2 (Pyk2) is rapidly translocated to the T cell-APC contact area upon T cell-specific recognition of superantigen-pulsed APCs. Stimulation with anti-CD3-coated latex microspheres was sufficient for Pyk2 reorientation, and the coengagement of CD28 boosted Pyk2 redistribution. Nevertheless, Pyk2 translocation did not result in its recruitment to lipid rafts. Two results support that Pyk2 translocation was independent of its kinase activity. First, Lck activity was required for TCR-induced Pyk2 translocation, but not for TCR-induced Pyk2 activation. Second, a kinase-dead Pyk2 mutant was equally translocated upon TCR triggering. In addition, Lck activity alone was insufficient to induce Pyk2 reorientation and activation, requiring the presence of at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Despite the dependence on functional Lck and on phosphorylated ITAM for Pyk2 translocation, the ITAM-binding tyrosine kinase zeta-associated protein 70 (ZAP-70) was not essential. All these data suggest that, by translocating to the vicinity of the immune synapse, Pyk2 could play an essential role in T cell activation and polarized secretion of cytokines.