Inducible expression of mutant human DISC1 in mice is associated with brain and behavioral abnormalities reminiscent of schizophrenia

A strong candidate gene for schizophrenia and major mental disorders, disrupted-in-schizophrenia 1 (DISC1) was first described in a large Scottish family in which a balanced chromosomal translocation segregates with schizophrenia and other psychiatric illnesses. The translocation mutation may result in loss of DISC1 function via haploinsufficiency or dominant-negative effects of a predicted mutant DISC1 truncated protein product. DISC1 has been implicated in neurodevelopment, including maturation of the cerebral cortex. To evaluate the neuronal and behavioral effects of mutant DISC1, the Tet-off system under the regulation of the CAMKII promoter was used to generate transgenic mice with inducible expression of mutant human DISC1 (hDISC1) limited to forebrain regions, including cerebral cortex, hippocampus and striatum. Expression of mutant hDISC1 was not associated with gross neurodevelopmental abnormalities, but led to a mild enlargement of the lateral ventricles and attenuation of neurite outgrowth in primary cortical neurons. These morphological changes were associated with decreased protein levels of endogenous mouse DISC1, LIS1 and SNAP-25. Compared to their sex-matched littermate controls, mutant hDISC1 transgenic male mice exhibited spontaneous hyperactivity in the open field and alterations in social interaction, and transgenic female mice showed deficient spatial memory. The results show that the neuronal and behavioral effects of mutant hDISC1 are consistent with a dominant-negative mechanism, and are similar to some features of schizophrenia. The present mouse model may facilitate the study of aspects of the pathogenesis of schizophrenia.     

A review of Disrupted-In-Schizophrenia-1 (DISC1): neurodevelopment, cognition, and mental conditions.

Disrupted-In-Schizophrenia-1 (DISC1) is a promising candidate gene for schizophrenia (SZ) and bipolar disorder (BP), but its basic biology remains to be elucidated. Accumulating genetic evidence supports that DISC1 is associated with some aspects of cognitive functions relevant to SZ and BP. Here, we provide a summary of the current updates in biological studies of DISC1. Disrupted-In-Schizophrenia-1, preferentially expressed in the forebrain, has multiple isoforms with potential posttranslational modifications. Disrupted-In-Schizophrenia-1 protein occurs in multiple subcellular compartments, which include the centrosome, microtubule fractions, postsynaptic densities, actin cytoskeletal fractions, the mitochondria, and the nucleus. Recent studies have clarified that DISC1 mediates at least centrosome-dynein cascade and cyclic adenosine monophosphate (cAMP) signaling. Furthermore, both cytogenetic and cell biological studies consistently suggest that an overall loss of DISC1 function (either haploinsufficiency or dominant-negative, or both) may be associated with SZ and BP. On the basis of these findings, production of DISC1 genetically engineered mice is proposed as a promising animal model for SZ and BP. Several groups are currently generating DISC1 mice and starting to characterize them. In this review, the advantages and disadvantages of each animal model are discussed.     

Disrupted in schizophrenia 1 genotype and positive symptoms in schizophrenia.

BACKGROUND: Converging evidence has demonstrated an association between the Disrupted in Schizophrenia 1 (DISC1) gene and schizophrenia (SZ). Within the DISC1 gene, a single nucleotide polymorphism (SNP), Ser704Cys, has been associated with the structure and function of the hippocampus. Because positive symptoms in SZ have also been associated with hippocampal structure and function, we hypothesized that variation in a DISC1 haplotype containing Ser704Cys would be significantly associated with positive symptomatology in SZ. METHODS: We tested for an association between variation in a haplotype block within the DISC1 gene containing Ser704Cys and lifetime history of positive symptoms in 199 Caucasian patients with SZ. RESULTS: We detected significant associations between a DISC1 haplotype containing Ser704Cys and Ser704Cys genotype and lifetime severity of delusions in SZ. CONCLUSIONS: These data suggest that that the effect of DISC1 genetic variation might be associated with positive symptoms in patients with SZ.     

BDNF+/− mice exhibit deficits in oligodendrocyte lineage cells of the basal forebrain

Previous work indicated that brain‐derived neurotrophic factor (BDNF), through the trkB receptor, increases DNA synthesis in oligodendrocyte (OLG) progenitor cells (OPCs) and differentiation of postmitotic OLGs of the basal forebrain (BF). In the present studies, BDNF knockout animals were used to investigate BDNF's effects on OLG lineage cells (OLCs) in vivo. OLCs of the BF were found to express the trkB receptor, suggesting they are responsive to BDNF. Immunohistochemistry using NG2 and CC1 antibodies was utilized to examine the numbers of NG2+ OPCs and CC1+ postmitotic BF OLGs. At embryonic day 17 (E17), BDNF−/− animals display reduced NG2+ cells. This reduction was also observed in BDNF+/− mice at E17 and at postnatal day 1 (P1), P14, and adult stage, suggesting that BDNF plays a role in OPC development. BDNF+/− mice do not exhibit deficits in numbers of CC1+ OLGs. However, myelin basic protein, myelin associated glycoprotein, and proteolipid protein are reduced in BDNF+/− mice, suggesting that BDNF plays a role in differentiation. These data indicate that progenitor cells and myelin proteins may be affected in vivo by a decrease in BDNF. © 2010 Wiley‐Liss, Inc.     

Levels of BDNF impact oligodendrocyte lineage cells following a cuprizone lesion

Previous work in culture has shown that basal forebrain (BF) oligodendrocyte (OLG) lineage cells respond to BDNF by increasing DNA synthesis and differentiation. Further, in the BF in vivo, reduced levels of BDNF as seen in BDNF(+/-) mice result in reduced numbers of NG2+ cells and deficits in myelin proteins throughout development and in the adult, suggesting that BDNF impacts the proliferating population of OLGs as well as differentiation in vivo. In this study, to investigate the roles BDNF may play in the repair of a demyelinating lesion, the cuprizone model was used and the corpus callosum was examined. BDNF protein levels were reduced after cuprizone treatment, suggesting that the demyelinating lesion itself elicits a decrease in BDNF. To analyze the effects of a further reduction of BDNF on OLG lineage cells following cuprizone, BDNF(+/-) mice were evaluated. These mice exhibited a blunted increase in the NG2 response at 4 and 5 weeks of cuprizone treatment. In addition, BDNF(+/-) mice exhibited decreased levels of myelin proteins during the demyelination and remyelination processes with no change in the total number of OLGs. These effects appear to be relatively specific to OLG lineage cells as comparable changes in CD11b+ microglia, GFAP+ astrocytes, and SMI32+ injured axons were not observed. These data indicate that BDNF may play a role following a demyelinating lesion by regulating the numbers of progenitors and the abilities of demyelinating and differentiating cells to express myelin proteins.     

Characterization of striatal phenotypes in heterozygous Disc1 mutant mice,a model of haploinsufficiency

Disrupted-in-Schizophrenia 1 (DISC1) is a susceptibility gene for several psychiatric illnesses. To study the pathogenesis of these disorders, we generated Disc1 mutant mice by introducing the 129S6/SvEv 25-bp deletion Disc1 variants into the C57BL/6J strain. In this study, we used heterozygous Disc1 mutant (Het) mice to evaluate the DISC1 haploinsufficiency model of schizophrenia. No changes in locomotor behaviors were observed in Het mice; however, after amphetamine injection, greater locomotor activity was observed in Het mice compared with wild-type (WT) mice. Moreover, amphetamine-induced elevations of c-Fos expression and dopamine level in the striatum were greater in Het mice than in WT controls, suggesting an altered dopaminergic regulation in the striatum of Het mice. Compared with those in WTs, the striatal protein levels of dopamine transporter and D2 dopamine receptor were increased in Het mice, while D1 dopamine receptor level was decreased. DISC1 interacting proteins, GSK3α and GSK3β, were downregulated in Het mice, whereas the levels of PDE4B and CREB were not altered. Morphologically, the complexities of striatal median spiny neurons (MSNs), parvalbumin-positive interneurons and Iba1-positive microglia were all decreased in Het mice. The density and head diameter of dendritic spines in the MSNs of Het mice were also reduced. Our results indicate that mice lacking one WT Disc1 allele are more sensitive to psychostimulant amphetamine challenge, which might be attributed to the altered structure and function of the striatal dopaminergic system. Here, we demonstrated striatal phenotypes in heterozygous Disc1 mutant mice, which could be a promising model of DISC1 haploinsufficiency.     

Dominant negative DISC1 mutant mice display specific social behaviour deficits and aberration in BDNF and cannabinoid receptor expression

Objectives. Disrupted in schizophrenia 1 (DISC1) is considered the most prominent candidate gene for schizophrenia. In this study, we aimed to characterize behavioural and brain biochemical traits in a mouse expressing a dominant negative DISC1mutant (DN-DISC1). Methods. DN-DISC1 mice underwent behavioural tests to evaluate object recognition, social preference and social novelty seeking. ELISA was conducted on brain tissue to evaluate BDNF levels. Western blot was employed to measure BDNF receptor (TrkB) and cannabinoid receptor CB1. Results. The mutant DISC1 mice displayed deficits in preference to social novelty while both social preference and object recognition were intact. Biochemical analysis of prefrontal cortex and hippocampus revealed a modest reduction in cortical TrkB protein levels of male mice while no differences in BDNF levels were observed. We found sex dependent differences in the expression of cannabinoid-1 receptors. Conclusions. We describe novel behavioural and biochemical abnormalities in the DN-DISC1 mouse model of schizophrenia. The data shows for the first time a possible link between DISC1 mutation and the cannabinoid system.     

Disruption of exploratory and habituation behavior in mice with mutation of DISC1: an ethologically based analysis

Disrupted-in-schizophrenia-1 (DISC1) is a gene that has been functionally linked with neurodevelopmental processes and structural plasticity in the brain. Clinical genetic investigations have implicated DISC1 as a genetic risk factor for schizophrenia and related psychoses. Studies using mutant mouse models of DISC1 gene function have demonstrated schizophrenia-related anatomical and behavioral endophenotypes. In the present study, ethologically based assessment of exploratory and habituation behavior in the open field was conducted in DISC1 (L100P), wild-type (WT), heterozygous (HET), and homozygous (HOM) mutant mice of both sexes. Ethological assessment was conducted in an open-field environment to explore specific topographies of murine exploratory behavior across the extended course of interaction from initial exploration through subsequent habituation (the ethogram). During initial exploration, HET and HOM DISC1 mutants evidenced increased levels of locomotion and rearing to wall compared with WT. A HOM-specific increase in total rearing and a HET-specific increase in sifting behavior and reduction in rearing seated were also observed. Over subsequent habituation, locomotion, sniffing, total rearing, rearing to wall, rearing free, and rearing seated were increased in HET and HOM mutants vs. WT. Overall, grooming was increased in HOM relative to other genotypes. HET mice displayed a selective decrease in habituation of sifting behavior. These data demonstrate impairment in both initial exploratory and habituation of exploration in a novel environment in mice with mutation of DISC1. This is discussed in the context of the functional role of the gene vis à vis a schizophrenia phenotype as well as the value of ethologically based approaches to behavioral phenotyping.     

Neu differentiation factor regulates tau protein and mRNA in cultured neonatal oligodendrocytes.

Axonal signals activate myelinogenesis via regulation of the extent to which oligodendrocyte (OLG) processes wrap around the axon. The cytoskeleton in OLG processes is actively involved in myelination and is a putative target for axonal regulation of myelination. The axon-associated neuregulins may regulate the cytoskeleton extensions in OLG processes. Here, we report that the neuregulin neu differentiation factor (NDF) increases the expression of tau mRNA and tau protein in OLGs. Treatment of neonatal OLGs with alpha-NDF or beta-NDF resulted in dramatic increases in the length of OLG processes, which appeared either as singular unbranched extensions or as a network of extensively branched processes. By immunoblot analysis with tau-1 mAb, which recognizes the dephosphorylated form of the tau proteins, neonatal OLGs treated with alpha-NDF or beta-NDF, had an increase in tau protein levels. The increase of tau levels in beta-NDF-treated cells is much greater than the twofold increase present in alpha-NDF-treated cells. By immunoblot analysis with the phosphorylation-insensitive tau-5 mAb, beta-NDF-treated cells had a twofold increase in tau. Immunoblot analysis suggest that alpha-NDF and beta-NDF promote a twofold increase in the tau protein levels in OLG, with the beta-factor also promoting a tau dephosphorylation. Using promoters spanning the amino-terminal region of tau, we found that OLGs treated with alpha-NDF or beta-NDF contained approximately twofold more tau mRNA than untreated cells. However, there was no qualitative difference between control and NDF-treated cells in the pattern of tau mRNA isoforms expressed. A model is proposed in which the axonal NDF-induced regulation of tau expression in OLGs may be part of the mechanism by which the axon regulates myelination.     

Neuropeptide Y neuronal network dysfunction in the frontal lobe of a genetic mouse model of schizophrenia

Neuropeptide Y (NPY) has been found to play a critical role in various mental functions as a neurotransmitter and is involved in the development of schizophrenia, a particularly intractable psychiatric disease whose precise etiology remains unknown. Recent molecular biological investigations have identified several candidate genes which may be associated with this disease, including disrupted-in-schizophrenia 1 (DISC1). The role of DISC1 would involve neurogenesis and neuronal migration. However, the functional consequences of this gene defect have not yet been fully clarified in neuronal systems. In the present study, to clarify the neuropathological changes associated with the function of DISC1, we explored how DISC1 dysfunction can induce abnormalities in the NPY neuronal network in the central nervous system. We performed immunohistochemical analyses (including the observation of the distribution and density) of prefrontal cortex specimens from DISC1-knockout (KO) mice, which are considered to be a novel animal model of schizophrenia. We then evaluated the number and size of NPY-immunoreactive (NPY-IR) neurons and the length of NPY-IR fibers. The number of NPY-IR neurons and the length of the fibers were decreased in the prefrontal cortex of DISC1-KO mice. The decrease was particularly prominent in the superficial regions, and the distribution of NPY-IR neurons differed between wild-type and DISC1-KO mice. However, the size of the neurons in the cortices of the DISC1-KO and wild-type mice did not differ markedly. Our findings suggest that dysfunction of DISC1 may lead to the alteration of NPY neurons and neurotransmission issues in NPY-containing neuron systems, which seem to play important roles in both the mental function and neuronal development. DISC1 dysfunction may be involved in the pathogenesis of schizophrenia through the impairment of the NPY neuronal network.     

Retroviral lineage analysis of fibroblast growth factor receptor signaling in FGF2 inhibition of oligodendrocyte progenitor differentiation

Fibroblast growth factor 2 (FGF2) inhibits oligodendrocyte progenitor cell (OPC) differentiation during development and limits remyelination following chronic demyelination. The current study examines the mechanism underlying this effect of FGF2 expression on OPC differentiation. Retroviral lineage tracing demonstrates a direct in vivo effect of FGF receptor (FGFR) signaling on OPC differentiation. Retrovirus expressing a dominant negative FGFR construct (FGFRdn) and green fluorescent protein (GFP) was injected into the dorsal columns of postnatal day 7 (P7) mice followed by perfusion at P28. Among the GFP-labeled cells, FGFRdn retrovirus generated a higher proportion of oligodendrocytes than did control infections. This result from FGFRdn expression in OPCs was similar to the result obtained in our previous study using control retrovirus in FGF2 null mice. Further, in vitro retroviral siRNA expression distinguishes the function of specific FGFR isoforms in OPC responses to FGF2. FGF2 inhibition of OPC differentiation was effectively blocked by siRNA targeted to FGFR1, but not FGFR2 or FGFR3. We propose a model of direct FGF2 activation of FGFR1 leading to inhibition of OPC differentiation. This signaling pathway may be an important regulator of oligodendrocyte generation during myelination in development and may perturb OPC generation of remyelinating oligodendrocytes in demyelinating disease.     

PAK1 Positively Regulates Oligodendrocyte Morphology and Myelination

The actin cytoskeleton is crucial for oligodendrocyte differentiation and myelination. Here we show that p21-activated kinase 1 (PAK1), a well-known actin regulator, promotes oligodendrocyte morphologic change and myelin production in the CNS. A combination of in vitro and in vivo models demonstrated that PAK1 is expressed throughout the oligodendrocyte lineage with highest expression in differentiated oligodendrocytes. Inhibiting PAK1 early in oligodendrocyte development decreased oligodendrocyte morphologic complexity and altered F-actin spreading at the tips of oligodendrocyte progenitor cell processes. Constitutively activating AKT in oligodendrocytes in male and female mice, which leads to excessive myelin wrapping, increased PAK1 expression, suggesting an impact of PAK1 during active myelin wrapping. Furthermore, constitutively activating PAK1 in oligodendrocytes in zebrafish led to an increase in myelin internode length while inhibiting PAK1 during active myelination decreased internode length. As myelin parameters influence conduction velocity, these data suggest that PAK1 may influence communication within the CNS. These data support a model in which PAK1 is a positive regulator of CNS myelination.SIGNIFICANCE STATEMENT Myelin is a critical component of the CNS that provides metabolic support to neurons and also facilitates communication between cells in the CNS. Recent data demonstrate that actin dynamics drives myelin wrapping, but how actin is regulated during myelin wrapping is unknown. The authors investigate the role of the cytoskeletal modulator PAK1 during differentiation and myelination by oligodendrocytes, the myelinating cells of the CNS. They demonstrate that PAK1 promotes oligodendrocyte differentiation and myelination by modulating the cytoskeleton and thereby internode length, thus playing a critical role in the function of the CNS.     

Gene expression in the jimpy mutant: evidence for fewer oligodendrocytes expressing myelin protein genes and impaired translocation of myelin basic protein mRNA.

Myelin basic protein (MBP) and proteolipid protein (PLP) gene expression was investigated in the murine dysmyelinating mutant, jimpy and age-matched normal mice. MBP and PLP mRNA expression was examined in several brain regions by in situ hybridization histochemistry between 10 and 20 days postpartum. The results showed a general reduction in both PLP and MBP mRNA expression throughout in jimpy brain due primarily to fewer numbers of jimpy oligodendrocytes expressing these genes. A less severe, but significant, reduction in the level of PLP expression per oligodendrocyte was also observed in the mutant brain. Because of the diffuse pattern of MBP mRNA distribution in the controls it was not possible to determine whether MBP expression was reduced on a per cell basis in the jimpy mutant. Silver grains representing MBP mRNAs were diffusely distributed over myelinating regions in normal mice but, in contrast, the grains were primarily clustered over oligodendrocyte cell bodies in the jimpy brains. No significant differences in jimpy oligodendrocyte morphology were evident by galactocerebroside immunohistochemistry. These results suggested that the apparent MBP mRNA clustering was not due to truncated or reduced numbers of oligodendrocyte processes. Rather, they suggest that the inability of jimpy mice to incorporate MBP into myelin may be due, in part, to a disruption in the translocation of MBP mRNAs within the oligodendrocyte.     

Association Study Between the Pericentrin (PCNT) Gene and Schizophrenia

Disrupted-in-schizophrenia 1 (DISC1), a known genetic risk factor for schizophrenia (SZ) and major depressive disorder (MDD), interacts with several proteins and some of them are reported to be genetically associated with SZ. Pericentrin (PCNT) also interacts with DISC1 and recently single-nucleotide polymorphisms (SNPs) within the PCNT gene have been found to show significant associations with SZ and MDD. In this study, case-controlled association analysis was performed to determine if the PCNT gene is implicated in SZ. Nine SNPs were analyzed in 1,477 individuals (726 patients with SZ and 751 healthy controls). No significant difference was observed between the controls and the patients in allelic frequencies or genotypic distributions of eight SNPs. Although allelic distribution of rs11702684 was different between the two groups (P = 0.042), the difference did not reach statistical significance after permutation correction for multiple comparisons. In the haplotypic analysis, we could not find any significant association in our subjects, either. This gene may not play a major role independently in the etiology of SZ in the Japanese population.     

Mutant disrupted‐in‐schizophrenia 1 in astrocytes: Focus on glutamate metabolism

Disrupted‐in‐schizophrenia 1 (DISC1) is a genetic risk factor that has been implicated in major mental disorders. DISC1 binds to and stabilizes serine racemase to regulate production of D‐serine by astrocytes, contributing to glutamate (GLU) neurotransmission. However, the possible involvement of astrocytic DISC1 in synthesis, metabolism, reuptake, or secretion of GLU remains unexplored. Therefore, we studied the effects of dominant‐negative mutant DISC1 on various aspects of GLU metabolism by using primary astrocyte cultures and hippocampal tissue from transgenic mice with astrocyte‐restricted expression of mutant DISC1. Although mutant DISC1 had no significant effects on astrocyte proliferation, GLU reuptake, glutaminase, or glutamate carboxypeptidase II activity, expression of mutant DISC1 was associated with increased levels of alanine‐serine‐cysteine transporter 2, vesicular glutamate transporters 1 and 3 in primary astrocytes and in the hippocampus, and elevated expression of the NR1 subunit and diminished expression of the NR2A subunit of N‐methyl‐D‐aspartate (NMDA) receptors in the hippocampus, at postnatal day 21. Our findings indicate that decreased D‐serine production by astrocytic mutant DISC1 might lead to compensatory changes in levels of the amino acid transporters and NMDA receptors in the context of tripartite synapse. © 2014 Wiley Periodicals, Inc.