Kousuke Baba

Disrupted-In-Schizophrenia 1, a candidate gene for schizophrenia, participates in neurite outgrowth

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation that segregated with schizophrenia in a Scottish family. Predicted DISC1 product has no significant homology to other known proteins. Here, we demonstrated the existence of DISC1 protein and identified fasciculation and elongation protein zeta-1 (FEZ1) as an interacting partner of DISC1 by a yeast two-hybrid study. FEZ1 and its nematode homolog are reported to represent a new protein family involved in axonal outgrowth and fasciculation. In cultured hippocampal neurons, DISC1 and FEZ1 colocalized in growth cones. Interactions of these proteins were associated with F-actin. In the course of neuronal differentiation of PC12 cells, upregulation of DISC1/FEZ1 interaction was observed as along with enhanced extension of neurites by overexpression of DISC1. The present study shows that DISC1 participates in neurite outgrowth through its interaction with FEZ1. Recent studies have provided reliable evidence that schizophrenia is a neurodevelopmental disorder. As there is a high level of DISC1 expression in developing rat brain, dysfunction of DISC1 may confer susceptibility to psychiatric illnesses through abnormal development of the nervous system.

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.

Association analysis of FEZ1 variants with schizophrenia in Japanese cohorts

BACKGROUND DISC1 has been suggested as a causative gene for psychoses in a large Scottish family. We recently identified FEZ1 as an interacting partner for DISC1. To investigate the role of FEZ1 in schizophrenia and bipolar disorder, case-control association analyses were conducted in Japanese cohorts. METHODS We performed a mutation screen of the FEZ1 gene and detected 15 polymorphisms. Additional data on informative polymorphisms were obtained from public databases. Eight single nucleotide polymorphisms (SNPs) were analyzed in 119 bipolar disorder and 360 schizophrenic patients and age- and gender-matched control subjects. All genotypes were determined with the TaqMan assay, and selected samples were confirmed by sequencing. RESULTS The two adjacent polymorphisms displayed a nominally significant association with schizophrenia (IVS2+ 1587G>A, p = .014; 396T<A or Asp123Glu, p = .024). Homozygotes with the Glu123 allele were observed in only a small portion (2%) of schizophrenia patients but not in control subjects or bipolar patients. Conversely, no SNPs displayed allelic, genotypic, or haplotypic associations with bipolar disorder. CONCLUSIONS A modest association between FEZ1 and schizophrenia suggests that this gene and the DISC1-mediated molecular pathway might play roles in the development of schizophrenia, with FEZ1 affecting only a small subset of Japanese schizophrenia patients.

The superficial layers of the superior colliculus are cytoarchitectually and myeloarchitectually disorganized in the reelin-deficient mouse, reeler

The causative gene for the reeler mouse is reelin which encodes Reelin protein, an extracellular molecule. In the present study, we have examined the cytoarchitecture, myeloarchitecture, and afferent/efferent systems of the superior colliculus (SC) of the reeler mouse. In the reeler, the laminar structures of the superficial three layers of the SC were disorganized and intermingled into a single layer, i.e., the superficial fused layer (SuF), as previously reported in the reelin-deficient SRK rat (Sakakibara et al., Develop. Brain Res. 141:1-13). Next, we have investigated the course and terminals of visual corticotectal and retinotectal projections with an injection of biocytin into the visual cortex or an injection of cholera toxin subunit B into the retina, respectively. In the reeler, anterogradely labeled visual corticotectal and retinotectal fibers took an aberrant course within the SuF, resulting in abnormal myeloarchitecture of the superficial SC of the reeler. Retrograde labeling of tectospinal tract neurons could not show any differences between the normal and reeler mice, suggesting that the deep layers of the reeler SC are cytoarchitectually normal. In situ hybridization and immunohistochemical studies have shown that reelin mRNA and Reelin protein were both recognized in the normal SC. These results suggest that Reelin protein plays some roles in histogenesis of the superficial layers of the SC.

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.

Gene therapy for neurological disorders

ABSTRACT Introduction: Many nervous system disorders are minimally responsive to existing treatments but they are potential candidates for gene therapy, an approach that can correct the genetic abnormalities contributing to its pathogenesis at molecular level. Gene therapy involves either the introduction of a replacement allele into cells to compensate for loss of gene function or the silencing of dominant mutant allele that is pathologic to cells. Areas covered: This review discusses the currently available gene therapy techniques, potential problems derived from gene therapy strategies and the recent development of gene therapy to treat neurological disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and strokes. Expert opinion: Gene therapy may revolutionize the treatment of neurological disorders in the coming decades but there are still great challenges ahead. The strength of gene therapy has been emphasized in the overexpression of therapeutic genes. However, in a number of dominantly inherited nervous system diseases, the ideal therapeutic goal would be to inhibit the expression of disease-causing allele. Gene silencing strategies by single-stranded antisense oligonucleotides and RNA interference represent a major breakthrough. Clinical trials using these approaches for dominant diseases are likely to be implemented in the near future.

A novel histone deacetylase 1 and 2 isoform-specific inhibitor alleviates experimental Parkinson's disease

With increased histone deacetylase (HDAC) activity and histone hypoacetylation being implicated in neurodegeneration, HDAC inhibitors have been reported to have considerable therapeutic potential. Yet, existing inhibitors lack specificity and may show substantial adverse effect. In this study, we identified a novel HDAC1/2 isoform-specific inhibitor, K560, with protective effects against 1-methyl-4-phenylpyridinium (MPP(+))- and/or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal death in both in vitro and in vivo Parkinsons disease model. K560 attenuated cell death induced by MPP(+) in differentiated SH-SY5Y cells through the sustained expression of an antiapoptotic protein, X-linked inhibitor of apoptosis (XIAP). Inhibition of XIAP expression by locked nucleic acid antisense oligonucleotides abolished the protective effect of K560. Inactivation of mitogen-activated protein kinase cascades, reduced p53 phosphorylation, and down-regulation of p53-upregulated modulator of apoptosis on K560 treatment were also observed. Furthermore, pre- and post-oral administration of K560 to mice prevented MPTP-induced loss of dopaminergic neurons in substantia nigra, suggesting that selective inhibition of HDAC1 and HDAC2 by K560 may pave the way to new strategies for Parkinsons disease treatment.

Role of ARF4L in Recycling Between Endosomes and the Plasma Membrane

The human ADP-ribosylation factor-like protein, ARF4L is a member of the ARF family, which are small GTP-binding proteins that play significant roles in vesicle transport and protein secretion. However, little is known about the physiological roles of ARF4L. In this study, to understand the biological functions of ARF4L, we carried out immunocytochemical analysis of ARF4L molecules with mutations in the functional domains. ARF4L was shown to be distributed to the plasma membrane following binding to GTP (Q80L), and into endosomes following binding to GDP (T35N). Moreover, the inactive-form of ARF4L (T35N) causes localization of transferrin receptors to the endosomal compartment, while the active form (Q80L) causes transport to the plasma membrane. These findings indicate that ARF4L drive the transport of cargo protein and subsequent fusion of recycling vesicles with the plasma membrane for maintenance of the cell surface.

Go-sha-jinki-Gan (GJG) ameliorates allodynia in chronic constriction injury model mice via suppression of TNF-α expression in the spinal cord

Background Alternative medicine is noted for its clinical effect and minimal invasiveness in the treatment of neuropathic pain. Go-sha-jinki-Gan, a traditional Japanese herbal medicine, has been used for meralgia and numbness in elderly patients. However, the exact mechanism of GJG is unclear. This study aimed to investigate the molecular mechanism of the analgesic effect of GJG in a chronic constriction injury model. Results GJG significantly reduced allodynia and hyperalgesia from the early phase (von Frey test, p < 0.0001; cold-plate test, p < 0.0001; hot-plate test p = 0.011; two-way repeated measures ANOVA). Immunohistochemistry and Western blot analysis revealed that GJG decreased the expression of Iba1 and tumor necrosis factor-α in the spinal cord. Double staining immunohistochemistry showed that most of the tumor necrosis factor-α was co-expressed in Iba1-positive cells at day 3 post-operation. GJG decreased the phosphorylation of p38 in the ipsilateral dorsal horn. Moreover, intrathecal injection of tumor necrosis factor-α opposed the anti-allodynic effect of GJG in the cold-plate test. Conclusions Our data suggest that GJG ameliorates allodynia in chronic constriction injury model mice via suppression of tumor necrosis factor-α expression derived from activated microglia. GJG is a promising drug for the treatment of neuropathic pain induced by neuro-inflammation.