Shinnosuke Yamada

Behavioral alterations associated with targeted disruption of exons 2 and 3 of the Disc1 gene in the mouse

Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration, axon/dendrite formation and synapse formation; during these processes, DISC1 acts as a scaffold protein by interacting with various partners. However, the lack of Disc1 knockout mice and a well-characterized antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we generated mice lacking exons 2 and 3 of the Disc1 gene and prepared specific antibodies to the N- and C-termini of DISC1. The Disc1 mutant mice are viable and fertile, and no gross phenotypes, such as disorganization of the brains cytoarchitecture, were observed. Western blot analysis revealed that the DISC1-specific antibodies recognize a protein with an apparent molecular mass of ~100 kDa in brain extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons and astrocytes. A deficiency of full-length Disc1 induced a threshold shift in the induction of long-term potentiation in the dentate gyrus. The Disc1 mutant mice displayed abnormal emotional behavior as assessed by the elevated plus-maze and cliff-avoidance tests, thereby suggesting that a deficiency of full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest that full-length Disc1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the pathophysiological functions of DISC1.

Nobiletin, a citrus flavonoid, ameliorates cognitive impairment, oxidative burden, and hyperphosphorylation of tau in senescence-accelerated mouse

Senescence-accelerated mouse prone 8 (SAMP8) is a model of aging characterized by the early onset of learning and memory impairment and various pathological features of Alzheimers disease (AD). Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from citrus peels, ameliorates learning and memory impairment in olfactory-bulbectomized mice, amyloid precursor protein transgenic mice, and NMDA receptor antagonist-treated mice. Here, we present evidence that this natural compound improves age-related cognitive impairment and reduces oxidative stress and tau phosphorylation in SAMP8 mice. Treatment with nobiletin (10 or 50mg/kg) reversed the impairment of recognition memory and context-dependent fear memory in SAMP8 mice. Treatment with nobiletin also restored the decrease in the GSH/GSSG ratio in the brain of SAMP8 mice. In addition, increases in glutathione peroxidase and manganese-superoxide dismutase activities, as well as a decrease in protein carbonyl level, were observed in the brain of nobiletin-treated SAMP8 mice. Furthermore, nobiletin reduced tau phosphorylation in the hippocampus of SAMP8 mice. Together, the markedly beneficial effects of nobiletin represent a potentially useful treatment for ameliorating the learning and memory deficits, oxidative stress, and hyperphosphorylation of tau in aging as well as age-related neurodegenerative diseases such as AD.

Girdin phosphorylation is crucial for synaptic plasticity and memory : a potential role in the interaction of BDNF/TrkB/Akt signaling with NMDA receptor

Synaptic plasticity in hippocampal neurons has been thought to represent a variety of memories. Although accumulating evidence indicates a crucial role of BDNF/TrkB/Akt signaling in the synaptic plasticity of the hippocampus, the mechanism by which Akt, a serine/threonine kinase, controls activity-dependent neuronal plasticity remains unclear. Girdin (also known as APE, GIV, and HkRP1), an actin-binding protein involved both in the remodeling of the actin cytoskeleton and in cell migration, has been identified as a substrate of Akt. Previous studies have demonstrated that deficit of neuronal migration in the hippocampus of Girdin-deficient (Girdin−/−) mice is independent on serine phosphorylation of Girdin at S1416 (Girdin S1416) by Akt. In the present study, we focused on the role of Girdin S1416 phosphorylation in BDNF/TrkB/Akt signaling associated with synaptic plasticity. We found that Girdin in the hippocampus was phosphorylated at S1416 in an activity-dependent manner. Phosphorylation-deficient knock-in mice (GirdinSA/SA mice), in which S1416 is replaced with alanine, exhibited shrinkage of spines, deficit of hippocampal long-term potentiation, and memory impairment. These phenotypes of GirdinSA/SA mice resembled those of Girdin+/− mice, which have 50% loss of Girdin expression. Furthermore, Girdin interacted with Src kinase and NR2B subunit of NMDA receptor, leading to phosphorylation of the NR2B subunit and NMDA receptor activation. Our findings suggest that Girdin has two different functions in the hippocampus: Akt-independent neuronal migration and Akt-dependent NR2B phosphorylation through the interaction with Src, which is associated with synaptic plasticity in the hippocampus underlying memory formation.

Galantamine ameliorates the impairment of recognition memory in mice repeatedly treated with methamphetamine: involvement of allosteric potentiation of nicotinic acetylcholine receptors and dopaminergic-ERK1/2 systems

Galantamine, a drug used to treat Alzheimers disease, inhibits acetylcholinesterase (AChE) and allosterically modulates nicotinic acetylcholine receptors (nAChRs) resulting in stimulation of catecholamine neurotransmission. In this study, we investigated whether galantamine exerts cognitive-improving effects through the allosteric modulation of nAChRs in an animal model of methamphetamine (Meth) psychosis. The mice treated with Meth (1 mg/kg.d) for 7 d showed memory impairment in a novel object recognition test. Galantamine (3 mg/kg) ameliorated the memory impairment, and it increased the extracellular dopamine release in the prefrontal cortex (PFC) of Meth-treated mice. Donepezil, an AChE inhibitor (1 mg/kg) increased the extracellular ACh release in the PFC, whereas it had no effect on the memory impairment in Meth-treated mice. The nAChR antagonist, mecamylamine, and dopamine D1 receptor antagonist, SCH 23390, blocked the ameliorating effect of galantamine on Meth-induced memory impairment, whereas the muscarinic AChR antagonist, scopolamine, had no effect. The effects of galantamine on extracellular dopamine release were also antagonized by mecamylamine. Galantamine attenuated the defect of the novelty-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). The ameliorating effect of galantamine on recognition memory in Meth-treated mice was negated by microinjection of an ERK inhibitor, PD98059, into the PFC. These results suggest that the ameliorating effect of galantamine on Meth-induced memory impairment is associated with indirect activation of dopamine D1 receptor-ERK1/2 following augmentation with dopaminergic neurotransmission in the PFC through the allosteric activation of nAChRs. Galantamine could be a useful therapeutic agent for treating cognitive deficits in schizophrenia/Meth psychosis, as well as Alzheimers disease.

Alterations of GABAergic and dopaminergic systems in mutant mice with disruption of exons 2 and 3 of the Disc1 gene.

Disrupted-in-schizophrenia-1 (DISC1) has been widely associated with several psychiatric disorders, including schizophrenia, mood disorders and autism. We previously reported that a deficiency of DISC1 may induce low anxiety and/or high impulsivity in mice with disruption of exons 2 and 3 of the Disc1 gene (Disc1(Δ2-3/Δ2-3)). It remains unclear, however, if deficiency of DISC1 leads to specific alterations in distinct neuronal systems. In the present study, to understand the role of DISC1 in γ-aminobutyric acid (GABA) interneurons and mesocorticolimbic dopaminergic (DAergic) neurons, we investigated the number of parvalbumin (PV)-positive interneurons, methamphetamine (METH)-induced DA release and the expression levels of GABAA, DA transporter (DAT) and DA receptors in wild-type (Disc1(+/+)) and Disc1(Δ2-3/Δ2-3) mice. Female Disc1(Δ2-3/Δ2-3) mice showed a significant reduction of PV-positive interneurons in the hippocampus, while no apparent changes were observed in mRNA expression levels of GABAA receptor subunits. METH-induced DA release was significantly potentiated in the nucleus accumbens (NAc) of female Disc1(Δ2-3/Δ2-3) mice, although there were no significant differences in the expression levels of DAT. Furthermore, the expression levels of DA receptor mRNA were upregulated in the NAc of female Disc1(Δ2-3/Δ2-3) mice. Male Disc1(Δ2-3/Δ2-3) mice showed no apparent differences in all experiments. DISC1 may play a critical role in gender-specific developmental alteration in GABAergic inhibitory interneurons and DAergic neurons.

Gene-wide association study between the methylenetetrahydrofolate reductase gene (MTHFR) and schizophrenia in the Japanese population, with an updated meta-analysis on currently available data

Methylenetetrahydrofolate reductase (MTHFR) is a critical molecule for single-carbon transfer reactions. Recent evidence suggests that polymorphisms of MTHFR are related to neural tube deficits and the pathogenesis of schizophrenia. While several studies have demonstrated associations between the gene encoding the MTHFR (MTHFR) polymorphisms and schizophrenia, these studies lack consistency. Therefore, we conducted a gene-wide association study (patients with schizophrenia = 696, control subjects = 747) and performed imputation analysis. Additionally, we performed meta-analysis on currently available data from 18 studies for two common functional polymorphisms (rs1801131 and rs1801133). There were no significant associations with schizophrenia in the single marker analysis for the seven tagging SNPs of MTHFR. In the haplotypic analysis, a nominally significant association was observed between the haplotypes, which included four SNPs (rs1801133, rs17421511, rs17037396, and rs9651118) and the schizophrenic patients. Additionally, the imputation analysis demonstrated there were several associated markers on the MTHFR chromosomal region. However, confirmatory analyses of three tagging SNPs (rs1801133, rs17037396, and rs9651118) and the top SNP (rs17421511) for the imputation results (patients with schizophrenia = 797, control subjects = 1025) failed to replicate the haplotypic analysis and the imputation results. These findings suggest that MTHFR polymorphisms are unlikely to be related to the development of schizophrenia in the Japanese population. However, since our meta-analysis results demonstrated strong support for association of rs1801133 with schizophrenia, further replication studies based on a gene-wide approach need to be considered.

Matrix metalloproteinase-3 is a possible mediator of neurodevelopmental impairment due to polyI:C-induced innate immune activation of astrocytes.

Increasing epidemiological evidence indicates that prenatal infection and childhood central nervous system infection with various viral pathogens enhance the risk for several neuropsychiatric disorders. Polyriboinosinic-polyribocytidilic acid (polyI:C) is known to induce strong innate immune responses that mimic immune activation by viral infections. Our previous findings suggested that activation of the innate immune system in astrocytes results in impairments of neurite outgrowth and spine formation, which lead to behavioral abnormalities in adulthood. To identify candidates of astrocyte-derived humoral factors that affect neuronal development, we analyzed astrocyte-conditioned medium (ACM) from murine astrocyte cultures treated with polyI:C (polyI:C-ACM) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Through a quantitative proteomic screen, we found that 13 protein spots were differentially expressed compared with ACM from vehicle-treated astrocytes (control-ACM), and characterized one of the candidates, matrix metalloproteinase-3 (Mmp3). PolyI:C treatment significantly increased the expression levels of Mmp3 mRNA and protein in astrocytes, but not microglia. PolyI:C-ACM was associated with significantly higher Mmp3 protein level and enzyme activity than control-ACM. The addition of recombinant Mmp3 into control-ACM impaired dendritic elongation of primary cultured hippocampal neurons, while the deleterious effect of polyI:C-ACM on neurite elongation was attenuated by knockdown of Mmp3 in astrocytes. These results suggest that Mmp3 is a possible mediator of polyI:C-ACM-induced neurodevelopmental impairment.

The expression of HMGA1a is increased in lymphoblastoid cell lines from schizophrenia patients

The high-mobility group A protein 1a (HMGA1a) is a well-documented DNA-binding protein acting as an architectural transcription regulator. Recently, HMGA1a protein has been identified as a hypoxia-inducible RNA-binding trans-acting factor for aberrant splicing of presenilin-2 (PS2) pre-mRNA observed in the brains of sporadic Alzheimers disease. Interestingly, this aberrant splicing of PS2 was also observed in the brains of bipolar disorder and schizophrenia. Many downstream genes under the control of HMGA1a could be associated with schizophrenia. On the other hand, many gene transcripts are aberrantly spliced in schizophrenia. Therefore, we examined the expression at the mRNA and protein levels of this DNA- and RNA-binding factor HMGA1a in the lymphoblastoid cell lines obtained from 16 schizophrenia patients with age-matched controls. We observed markedly higher HMGA1a mRNA and the increased HMGA1a protein in the nuclear fractions of schizophrenia patients. In contrast, there were no significant differences in the expression levels of HMGA1b, which is an alternatively spliced isoform of HMGA1a. The present study is the first to report a significant upregulation of HMGA1a in schizophrenia, suggesting its potential roles in both transcription and splicing of target genes linked with schizophrenia.

Induction of interferon-induced transmembrane protein 3 gene expression by lipopolysaccharide in astrocytes.

Astrocytes are important modulators of the immune and inflammatory reactions in the central nervous system. We have recently demonstrated the role of interferon-induced transmembrane protein 3 (IFITM3) in long-lasting neuronal impairments in mice following neonatal immune challenge by injections of the double-stranded RNA analog polyriboinosinic polyribocytidylic acid. Here, we show that IFITM3 is induced after lipopolysaccharide (LPS) treatment in cultured astrocytes. The induction of IFITM3 by LPS was completely suppressed by the addition of anti-interferon-β (IFN-β) antibody. In addition, neutralization of tumor necrosis factor-α (TNF-α) with its antibody partially inhibited the induction of IFITM3, suggesting that LPS induces IFITM3 through autocrine secretion of IFN-β and TNF-α. Furthermore, experiments using pharmacological inhibitors suggest that LPS induces IFITM3 through activation of TANK-binding kinase 1, p38 mitogen-activated protein kinase, and nuclear factor-κB pathways. Together, these findings may provide new insight into the role of IFITM3 in the pathogenesis of neurodevelopmental diseases associated with immune activation.

No association between the protein tyrosine phosphatase, receptor‐type, Z Polypeptide 1 (PTPRZ1) gene and schizophrenia in the Japanese population

NRG1‐ERBB signaling influences the risk for schizophrenia pathology. A recent study has reported that MAGI1, MAGI2, and protein tyrosine phosphatase, receptor‐type, Z polypeptide 1 (PTPRZ1; located on 7q31.3) gene products regulate the NRG1‐ERBB4 signaling pathway, and PTPRZ1 is associated with schizophrenia in a Caucasian population. By applying a gene‐based association concept, we analyzed any association between PTPRZ1 tagging SNPs and schizophrenia in the Japanese population (576 schizophrenics and 768 controls). After linkage disequilibrium analysis, 29 single nucleotide polymorphisms (SNPs) were genotyped using a 5′‐exonuclease allelic discrimination assay. We found a significant association of one tagging SNP in a genotype‐wise analysis (P = 0.007); however, this might be resulted from type I error due to multiple testing (P = 0.17 after SNPSpD correction). No association was observed between schizophrenic patients and controls in either allelic, genotypic, or haplotypic analyses. Our results therefore suggest that PTPRZ1 is unlikely to be related to the development of schizophrenia in the Japanese population.