Tsuyoshi Hattori

SIMULTANEOUS ESTABLISHMENT OF MONOCLONAL ANTIBODIES SPECIFIC FOR EITHER CYCLOBUTANE PYRIMIDINE DIMER OR (6‐4)PHOTOPRODUCT FROM THE SAME MOUSE IMMUNIZED WITH ULTRAVIOLET‐IRRADIATED DNA

Six new monoclonal antibodies (TDM‐2, TDM‐3, 64M‐2, 64M‐3, 64M‐4 and 64M‐5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose‐dependent binding to UV‐irradiated DNA (UV‐DNA), decrease of binding to UV‐DNA after cyclobutane pyrimidine dimer photo‐reactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV‐DNA after photoisomerization of (6‐4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose‐dependent binding to UV‐DNA, unchanged binding to UV‐DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV‐DNA after photoisomerization of (6‐4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6‐4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6‐4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.

A novel DISC1-interacting partner DISC1-Binding Zinc-finger protein: implication in the modulation of DISC1-dependent neurite outgrowth

Disrupted-in-schizophrenia 1 (DISC1) is a gene disrupted by a (1;11) (q42.1;q14.3) translocation that segregates with major psychiatric disorders in a Scottish family. To investigate how DISC1 confers susceptibility to psychiatric disorders, we previously identified fasciculation and elongation protein zeta-1 and Kendrin as DISC1-interacting molecules in a yeast two-hybrid screen of a human brain complementary DNA library. Here, we have further identified a novel DISC1-interacting protein, termed DISC1-Binding Zinc-finger protein (DBZ), which has a predicted C2H2-type zinc-finger motif and coiled-coil domains. DBZ was co-immunoprecipitated with DISC1 in lysates of PC12 cells and rat brain tissue. The domain of DISC1 interacting with DBZ was close to the translocation breakpoint in the DISC1 gene. DBZ messenger RNA (mRNA) was expressed in human brains, but not in peripheral tissues. In situ hybridization revealed high expression of DBZ mRNA in the hippocampus, olfactory tubercle, cerebral cortex and striatum in rats. Because this pattern of localization was similar to that of the pituitary adenylate cyclase (PAC1) receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), which has recently been implicated in neuropsychological functions, we examined whether DISC1/DBZ interaction was involved in the PACAP signaling pathway. PACAP upregulated DISC1 expression and markedly reduced the association between DISC1 and DBZ in PC12 cells. A DISC1-binding domain of DBZ reduced the neurite length in PC12 cells after PACAP stimulation and in primary cultured hippocampal neurons. The present results provide some new molecular insights into the mechanisms of neuronal development and neuropsychiatric disorders.

Increased stathmin1 expression in the dentate gyrus of mice causes abnormal axonal arborizations.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in multiple brain functions. To clarify the cause of abnormal behavior in PACAP deficient-mice, we attempted the identification of genes whose expression was altered in the dentate gyrus of PACAP-deficient mice using the differential display method. Expression of stathmin1 was up-regulated in the dentate gyrus at both the mRNA and protein levels. PACAP stimulation inhibited stathmin1 expression in PC12 cells, while increased stathmin1expression in neurons of the subgranular zone and in primary cultured hippocampal neurons induced abnormal arborization of axons. We also investigated the pathways involved in PACAP deficiency. Ascl1 binds to E10 box of the stathmin1 promoter and increases stathmin1 expression. Inhibitory bHLH proteins (Hes1 and Id3) were rapidly up-regulated by PACAP stimulation, and Hes1 could suppress Ascl1 expression and Id3 could inhibit Ascl1 signaling. We also detected an increase of stathmin1 expression in the brains of schizophrenic patients. These results suggest that up-regulation of stathmin1 in the dentate gyrus, secondary to PACAP deficiency, may create abnormal neuronal circuits that cause abnormal behavior.

Dysbindin engages in c-Jun N-terminal kinase activity and cytoskeletal organization.

A number of reports have provided genetic evidence for an association between the DTNBP1 gene (coding dysbindin) and schizophrenia. In addition, sandy mice, which harbor a deletion in the DTNBP1 gene and lack dysbindin, display behavioral abnormalities suggestive of an association with schizophrenia. However, the mechanism by which the loss of dysbindin induces schizophrenia-like behaviors remains unclear. Here, we report that small interfering RNA-mediated knockdown of dysbindin resulted in the aberrant organization of actin cytoskeleton in SH-SY5Y cells. Furthermore, we show that morphological abnormalities of the actin cytoskeleton were similarly observed in growth cones of cultured hippocampal neurons derived from sandy mice. Moreover, we report a significant correlation between dysbindin expression level and the phosphorylation level of c-Jun N-terminal kinase (JNK), which is implicated in the regulation of cytoskeletal organization. These findings suggest that dysbindin plays a key role in coordinating JNK signaling and actin cytoskeleton required for neural development.

DISC1 regulates cell–cell adhesion, cell–matrix adhesion and neurite outgrowth

Disrupted-in-schizophrenia 1 (DISC1) is a promising susceptibility gene for major mental illness. Recent studies have implicated DISC1 in key neurodevelopmental processes, including neurite outgrowth, neuronal migration and proliferation. Here, we report that DISC1 regulates cell–cell and cell–matrix adhesion and neurite outgrowth. DISC1 overexpression increased expression of the adherence junction protein N-cadherin and enhanced cell–cell adhesion. The increased N-cadherin accumulated in the areas of cell–cell contact. DISC1 overexpression also enhanced cell–matrix adhesion by inducing expression of β1-integrin protein. In the presence of nerve growth factor (NGF), DISC1 overexpression increased β1-integrin expression at the cell membrane and growth cone. NGF-induced neurite extension was enhanced by DISC1, and anti-β1-integrin antibody reduced the neurite outgrowth of DISC1-overexpressing cells to the control level. Furthermore, DISC1 also regulated N-cadherin and β1-integrin expression at the cell membrane in primary neurons. We conclude that DISC1 regulates cell–cell adhesion and cell–matrix adhesion by regulating the expression of adhesion molecules.

DISC1–kendrin interaction is involved in centrosomal microtubule network formation

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation segregating with schizophrenia, bipolar disorder and other major mental illnesses in a Scottish family. We previously identified 446-533 amino acids of DISC1 as the kendrin-binding region by means of a directed yeast two-hybrid interaction assay and showed that the DISC1-kendrin interaction is indispensable for the centrosomal localization of DISC1. In this study, to confirm the DISC1-kendrin interaction, we examined the interaction between deletion mutants of DISC1 and kendrin. Then, we demonstrated that the carboxy-terminus of DISC1 is indispensable for the interaction with kendrin. Furthermore, the immunocytochemistry revealed that the carboxy-terminus of DISC1 is also required for the centrosomal targeting of DISC1. Overexpression of the DISC1-binding region of kendrin or the DISC1 deletion mutant lacking the kendrin-binding region impairs the microtubule organization. These findings suggest that the DISC1-kendrin interaction plays a key role in the microtubule dynamics.

Deletion of Atf6α impairs astroglial activation and enhances neuronal death following brain ischemia in mice.

To dissect the role of endoplasmic reticulum (ER) stress and unfolded protein response in brain ischemia, we investigated the relevance of activating transcription factor 6α (ATF6α), a master transcriptional factor in the unfolded protein response, after permanent middle cerebral artery occlusion (MCAO) in mice. Enhanced expression of glucose‐regulated protein78, a downstream molecular chaperone of ATF6α, was observed in both neurons and glia in the peri‐infarct region of wild‐type mice after MCAO. Analysis using wild‐type and Atf6α−/− mice revealed a larger infarct volume and increased cell death in the peri‐ischemic region of Atf6α−/− mice 5 days after MCAO. These phenotypes in Atf6α−/− mice were associated with reduced levels of astroglial activation/glial scar formation, and a spread of tissue damage into the non‐infarct area. Further analysis in mice and cultured astrocytes revealed that signal transducer and activator of transcription 3 (STAT3)‐glial fibrillary acidic protein signaling were diminished in Atf6α−/− astrocytes. A chemical chaperone, 4‐phenylbutyrate, restored STAT3‐glial fibrillary acidic protein signaling, while ER stressors, such as tunicamycin and thapsigargin, almost completely abolished signaling in cultured astrocytes. Furthermore, ER stress‐induced deactivation of STAT3 was mediated, at least in part, by the ER stress‐responsive tyrosine phosphatase, TC‐PTP/PTPN2. These results suggest that ER stress plays critical roles in determining the level of astroglial activation and neuronal survival after brain ischemia.

DISC1 (Disrupted-in-Schizophrenia-1) Regulates Differentiation of Oligodendrocytes

Disrupted-in-schizophrenia 1 (DISC1) is a gene disrupted by a translocation, t(1;11) (q42.1;q14.3), that segregates with major psychiatric disorders, including schizophrenia, recurrent major depression and bipolar affective disorder, in a Scottish family. Here we report that mammalian DISC1 endogenously expressed in oligodendroglial lineage cells negatively regulates differentiation of oligodendrocyte precursor cells into oligodendrocytes. DISC1 expression was detected in oligodendrocytes of the mouse corpus callosum at P14 and P70. DISC1 mRNA was expressed in primary cultured rat cortical oligodendrocyte precursor cells and decreased when oligodendrocyte precursor cells were induced to differentiate by PDGF deprivation. Immunocytochemical analysis showed that overexpressed DISC1 was localized in the cell bodies and processes of oligodendrocyte precursor cells and oligodendrocytes. We show that expression of the myelin related markers, CNPase and MBP, as well as the number of cells with a matured oligodendrocyte morphology, were decreased following full length DISC1 overexpression. Conversely, both expression of CNPase and the number of oligodendrocytes with a mature morphology were increased following knockdown of endogenous DISC1 by RNA interference. Overexpression of a truncated form of DISC1 also resulted in an increase in expression of myelin related proteins and the number of mature oligodendrocytes, potentially acting via a dominant negative mechanism. We also identified involvement of Sox10 and Nkx2.2 in the DISC1 regulatory pathway of oligodendrocyte differentiation, both well-known transcription factors involved in the regulation of myelin genes.

Gene and Expression Analyses Reveal Enhanced Expression of Pericentrin 2 (PCNT2) in Bipolar Disorder

BACKGROUND DISC1 has been suggested as a causative gene for psychoses in a large Scottish kindred. PCNT2 has recently been identified as an interacting partner of DISC1. In this study, we investigated the role of PCNT2 in bipolar disorder, by gene expression analysis and genetic association study. METHODS By TaqMan real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we examined the messenger RNA (mRNA) levels of PCNT2 in the postmortem prefrontal cortex of bipolar disorder (n = 34), schizophrenia (n = 31), and control subjects (n = 32), obtained from Stanley Array Collection. We also compared the mRNA levels of PCNT2 in the peripheral blood lymphocytes of bipolar disorder (n = 21), schizophrenia (n = 21), depression (n = 33), and control subjects (n = 57). For the association study, 23 single nucleotide polymorphisms (SNPs) were analyzed in 285 bipolar disorder patients and 287 age-and gender-matched control subjects, all of Japanese origin. The genotypes were determined by TaqMan assay. RESULTS Significantly higher expression of PCNT2 was observed in the brain samples of bipolar group, compared with the control (p = .001) and schizophrenia (p = .018) groups. In the peripheral blood lymphocytes also, a significantly higher expression of PCNT2 was observed in the bipolar group, compared with the control subjects (p = .043). However, none of the SNPs analyzed in our study showed a significant association with bipolar disorder; a weak tendency toward association was observed for two intronic SNPs. CONCLUSIONS Our findings suggest that elevated levels of PCNT2 might be implicated in the pathophysiology of bipolar disorder.

Association studies and gene expression analyses of the DISC1-interacting molecules, pericentrin 2 (PCNT2) and DISC1-binding zinc finger protein (DBZ), with schizophrenia and with bipolar disorder.

Disrupted‐in‐Schizophrenia 1 (DISC1) and its molecular cascade have been implicated in the pathophysiology of major psychoses. Previously, we identified pericentrin 2 (PCNT2) and DISC1‐binding zinc finger protein (DBZ) as binding partners of DISC1; further, we observed elevated expression of PCNT2 in the postmortem brains and in the lymphocytes of bipolar disorder patients, compared to controls. Here, we examined the association of PCNT2 with schizophrenia in a case–control study of Japanese cohorts. We also examined the association of DBZ with schizophrenia and with bipolar disorder, and compared the mRNA levels of DBZ in the postmortem brains of schizophrenia, bipolar and control samples. DNA from 180 schizophrenia patients 201 controls were used for the association study of PCNT2 and DBZ with schizophrenia. Association of DBZ with bipolar disorder was examined in DNA from 238 bipolar patients and 240 age‐ and gender‐matched controls. We observed significant allelic and genotypic associations of the PCNT2 SNPs, rs2249057, rs2268524, and rs2073380 (Ser/Arg) with schizophrenia; the association of rs2249057 (P = 0.002) withstand multiple testing correction. Several two SNP‐ and three SNP‐haplotypes showed significant associations; the associations of haplotypes involving rs2249057 withstand multiple testing correction. No associations were observed for DBZ with schizophrenia or with bipolar disorder; further, there was no significant difference between the DBZ mRNA levels of control, schizophrenia and bipolar postmortem brains. We suggest a possible role of PCNT2 in the pathogenesis of schizophrenia. Abnormalities of PCNT2, the centrosomal protein essential for microtubule organization, may be suggested to lead to neurodevelopmental abnormalities.