Ken Ichiro Kubo

A schizophrenia-associated mutation of DISC1 perturbs cerebral cortex development

Disrupted-In-Schizophrenia-1 (DISC1), originally identified at the breakpoint of a chromosomal translocation that is linked to a rare familial schizophrenia, has been genetically implicated in schizophrenia in other populations. Schizophrenia involves subtle cytoarchitectural abnormalities that arise during neurodevelopment, but the underlying molecular mechanisms are unclear. Here, we demonstrate that DISC1 is a component of the microtubule-associated dynein motor complex and is essential for maintaining the complex at the centrosome, hence contributing to normal microtubular dynamics. Carboxy-terminal-truncated mutant DISC1 (mutDISC1), which results from a chromosomal translocation, functions in a dominant-negative manner by redistributing wild-type DISC1 through self-association and by dissociating the DISC1–dynein complex from the centrosome. Consequently, either depletion of endogenous DISC1 or expression of mutDISC1 impairs neurite outgrowth in vitro and proper development of the cerebral cortex in vivo. These results indicate that DISC1 is involved in cerebral cortex development, and suggest that loss of DISC1 function may underlie neurodevelopmental dysfunction in schizophrenia.

Rab GTPases-Dependent Endocytic Pathways Regulate Neuronal Migration and Maturation through N-Cadherin Trafficking

Although membrane trafficking pathways are involved in basic cellular functions, the evolutionally expanded number of their related family proteins suggests additional roles for membrane trafficking in higher organisms. Here, we show that several Rab-dependent trafficking pathways differentially participate in neuronal migration, an essential step for the formation of the mammalian-specific six-layered brain structure. In vivo electroporation-mediated suppression of Rab5 or dynamin to block endocytosis caused a severe neuronal migration defect in mouse cerebral cortex. Among many downstream endocytic pathways, suppression of Rab11-dependent recycling pathways exhibited a similar migration disorder, whereas inhibition of Rab7-dependent lysosomal degradation pathways affected only the final phase of neuronal migration and dendrite morphology. Inhibition of Rab5 or Rab11 perturbed the trafficking of N-cadherin, whose suppression also disturbed neuronal migration. Taken together, our findings reveal physiological roles of endocytic pathways, each of which has specific functions in distinct steps of neuronal migration and maturation during mammalian brain formation.

DISC1-dependent switch from progenitor proliferation to migration in the developing cortex

Regulatory mechanisms governing the sequence from progenitor cell proliferation to neuronal migration during corticogenesis are poorly understood. Here we report that phosphorylation of DISC1, a major susceptibility factor for several mental disorders, acts as a molecular switch from maintaining proliferation of mitotic progenitor cells to activating migration of postmitotic neurons in mice. Unphosphorylated DISC1 regulates canonical Wnt signalling via an interaction with GSK3β, whereas specific phosphorylation at serine 710 (S710) triggers the recruitment of Bardet–Biedl syndrome (BBS) proteins to the centrosome. In support of this model, loss of BBS1 leads to defects in migration, but not proliferation, whereas DISC1 knockdown leads to deficits in both. A phospho-dead mutant can only rescue proliferation, whereas a phospho-mimic mutant rescues exclusively migration defects. These data highlight a dual role for DISC1 in corticogenesis and indicate that phosphorylation of this protein at S710 activates a key developmental switch.

Recruitment of PCM1 to the Centrosome by the Cooperative Action of DISC1 and BBS4 A Candidate for Psychiatric Illnesses

CONTEXT A role for the centrosome has been suggested in the pathology of major mental illnesses, especially schizophrenia (SZ). OBJECTIVES To show that pericentriolar material 1 protein (PCM1) forms a complex at the centrosome with disrupted-in-schizophrenia 1 (DISC1) and Bardet-Biedl syndrome 4 protein (BBS4), which provides a crucial pathway for cortical development associated with the pathology of SZ. To identify mutations in the PCM1 gene in an SZ population. DESIGN Interaction of DISC1, PCM1, and BBS proteins was assessed by immunofluorescent staining and coimmunoprecipitation. Effects of PCM1, DISC1, and BBS on centrosomal functions and corticogenesis in vivo were tested by RNA interference. The PCM1 gene was examined by sequencing 39 exons and flanking splice sites. SETTING Probands and controls were from the collection of one of us (A.E.P.). PATIENTS Thirty-two probands with SZ from families that had excess allele sharing among affected individuals at 8p22 and 219 white controls. MAIN OUTCOME MEASURES Protein interaction and recruitment at the centrosome in cells; neuronal migration in the cerebral cortex; and variant discovery in PCM1 in patients with SZ. RESULTS PCM1 forms a complex with DISC1 and BBS4 through discrete binding domains in each protein. DISC1 and BBS4 are required for targeting PCM1 and other cargo proteins, such as ninein, to the centrosome in a synergistic manner. In the developing cerebral cortex, suppression of PCM1 leads to neuronal migration defects, which are phenocopied by the suppression of either DISC1 or BBS4 and are exacerbated by the concomitant suppression of both. Furthermore, a nonsense mutation that segregates with SZ spectrum psychosis was found in 1 family. CONCLUSIONS Our data further support for the role of centrosomal proteins in cortical development and suggest that perturbation of centrosomal function contributes to the development of mental diseases, including SZ.

Pax-6 is required for thalamocortical pathway formation in fetal rats.

Pax‐6, a transcription regulatory factor, has been demonstrated to play important roles in eye, nose, and brain development by analyzing mice, rats, and humans with a Pax‐6 gene mutation. We examined the role of Pax‐6 with special attention to the formation of efferent and afferent pathways of the cerebral cortex by using the rat Small eye (rSey2), which has a mutation in the Pax‐6 gene. In rSey2/rSey2 fetuses, cortical efferent axons develop with normal trajectory, at least within the cortical anlage, when examined with immunohistochemistry of the neuronal cell adhesion molecule TAG‐1 and 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine perchlorate (DiI) labeling from the cortical surface. A remarkable disorder was found in the trajectory of dorsal thalamic axons by immunostaining of the neurofilament and the neural cell adhesion molecule L1 and DiI labeling from the dorsal thalamus. In normal rat fetuses, dorsal thalamic axons curved laterally in the ventral thalamus without invading a Pax‐6‐immunoreactive cell cluster in the ventral part of the ventral thalamus. These axons then coursed up to the cortical anlage, passing just dorsal to another Pax‐6‐immunoreactive cell cluster in the amygdaloid region. In contrast, in rSey2/rSey2 fetuses, dorsal thalamic axons extended downward to converge in the ventrolateral corner of the ventral thalamus and fanned out in the amygdaloid region without reaching the cortical anlage. These results suggest that Pax‐6‐expressing cell clusters along the thalamocortical pathway (ventral part of the ventral thalamus and amygdala) are responsible for the determination of the axonal pathfinding of the thalamocortical pathway. J. Comp. Neurol. 408:147–160, 1999.

Characterization of the dipeptide repeat protein in the molecular pathogenesis of c9FTD/ALS

The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (c9FTD/ALS). Recently, it was reported that an unconventional mechanism of repeat-associated non-ATG (RAN) translation arises from C9orf72 expansion. Sense and anti-sense transcripts of the expanded C9orf72 repeat, i.e. the dipeptide repeat protein (DRP) of glycine-alanine (poly-GA), glycine-proline (poly-GP), glycine-arginine (poly-GR), proline-arginine (poly-PR) and proline-alanine (poly-PA), are deposited in the brains of patients with c9FTD/ALS. However, the pathological significance of RAN-translated peptides remains unknown. We generated synthetic cDNAs encoding 100 repeats of DRP without a GGGGCC repeat and evaluated the effects of these proteins on cultured cells and cortical neurons in vivo. Our results revealed that the poly-GA protein formed highly aggregated ubiquitin/p62-positive inclusion bodies in neuronal cells. In contrast, the highly basic proteins poly-GR and PR also formed unique ubiquitin/p62-negative cytoplasmic inclusions, which co-localized with the components of RNA granules. The evaluation of cytotoxicity revealed that overexpressed poly-GA, poly-GP and poly-GR increased the substrates of the ubiquitin-proteasome system (UPS), including TDP-43, and enhanced the sensitivity to a proteasome inhibitor, indicating that these DRPs are cytotoxic, possibly via UPS dysfunction. The present data indicate that a gain-of-function mechanism of toxic DRPs possibly contributes to pathogenesis in c9FTD/ALS and that DRPs may serve as novel therapeutic targets in c9FTD/ALS.

The outermost region of the developing cortical plate is crucial for both the switch of the radial migration mode and the Dab1-dependent "inside-out" lamination in the neocortex.

Mammalian neocortex has a laminated structure that develops in a birth-date-dependent “inside-out” pattern. This layered structure is established by neuronal migration with sequential changes of the migratory mode regulated by several signaling cascades, including the Reelin–Disabled homolog 1 (Dab1) pathway. Although the importance of “locomotion,” the major migratory mode, has been well established, the physiological significance of the mode change from locomotion to “terminal translocation,” the final migratory mode, is unknown. In this study, we found that the outermost region of the mouse cortical plate has several histologically distinct features and named this region the primitive cortical zone (PCZ). Time-lapse analyses revealed that “locomoting” neurons paused transiently just beneath the PCZ before migrating into it by “terminal translocation.” Furthermore, whereas Dab1–knockdown (KD) neurons could reach beneath the PCZ, they failed to enter the PCZ, suggesting that the Dab1-dependent terminal translocation is necessary for entry of the neurons into the PCZ. Importantly, sequential in utero electroporation experiments directly revealed that failure of the Dab1-dependent terminal translocation resulted in disruption of the inside-out alignment within the PCZ and that this disrupted pattern was still preserved in the mature cortex. Conversely, Dab1–KD locomoting neurons could pass by both wild-type and Dab1–KD predecessors beneath the PCZ. Our data indicate that the PCZ is a unique environment, passage of neurons through which involves molecularly and behaviorally different migratory mechanisms, and that the migratory mode change from locomotion to terminal translocation just beneath the PCZ is critical for the Dab1-dependent inside-out lamination in the mature cortex.

Ectopic Reelin Induces Neuronal Aggregation with a Normal Birthdate-Dependent “Inside-Out” Alignment in the Developing Neocortex

Neurons in the developing mammalian neocortex form the cortical plate (CP) in an “inside-out” manner; that is, earlier-born neurons form the deeper layers, whereas later-born neurons migrate past the existing layers and form the more superficial layers. Reelin, a glycoprotein secreted by Cajal–Retzius neurons in the marginal zone (MZ), is crucial for this “inside-out” layering, because the layers are inverted in the Reelin-deficient mouse, reeler (Relnrl ). Even though more than a decade has passed since the discovery of reelin, the biological effect of Reelin on individual migrating neurons remains unclear. In addition, although the MZ is missing in the reeler cortex, it is unknown whether Reelin directly regulates the development of the cell-body-sparse MZ. To address these issues, we expressed Reelin ectopically in the developing mouse cortex, and the results showed that Reelin caused the leading processes of migrating neurons to assemble in the Reelin-rich region, which in turn induced their cell bodies to form cellular aggregates around Reelin. Interestingly, the ectopic Reelin-rich region became cell-body-sparse and dendrite-rich, resembling the MZ, and the late-born neurons migrated past their predecessors toward the central Reelin-rich region within the aggregates, resulting in a birthdate-dependent “inside-out” alignment even ectopically. Reelin receptors and intracellular adaptor protein Dab1 were found to be necessary for formation of the aggregates. The above findings indicate that Reelin signaling is capable of inducing the formation of the dendrite-rich, cell-body-sparse MZ and a birthdate-dependent “inside-out” alignment of neurons independently of other factors/structures near the MZ.

GABAergic Precursor Transplantation into the Prefrontal Cortex Prevents Phencyclidine-Induced Cognitive Deficits

Phencyclidine (PCP) is a noncompetitive NMDA receptor antagonist, and it induces schizophreniform cognitive deficits in healthy humans and similar cognitive deficits in rodents. Although the PCP-induced cognitive deficits appear to be accompanied and possibly caused by dysfunction of GABAergic inhibitory interneurons in the prefrontal cortex (PFC), the potential benefit(s) of GABAergic interneuron manipulations on PCP-induced cognitive deficits remains unexplored. In this study we show that when embryonic medial ganglionic eminence (MGE) cells, many of which differentiate into cortical GABAergic interneurons in situ, were grafted into the medial PFC (mPFC) of neonatal mice, they differentiated into a specific class of GABAergic interneurons and became functionally integrated into the host neuronal circuitry in adults. Prior MGE cell transplantation into the mPFC significantly prevented the induction of cognitive and sensory-motor gating deficits by PCP. The preventive effects were not reproduced by either transplantation of cortical projection neuron precursors into the mPFC or transplantation of MGE cells into the occipital cortex. The preventive effects of MGE cell transplantation into the mPFC were accompanied by activation of callosal projection neurons in the mPFC. These findings suggest that increasing GABAergic interneuron precursors in the PFC may contribute to the development of a cell-based approach as a novel means of modulating the PFC neuronal circuitry and preventing schizophreniform cognitive deficits.

Disrupted-in-Schizophrenia-1 (Disc1) is necessary for migration of the pyramidal neurons during mouse hippocampal development

The hippocampus has a highly ordered structure and is composed of distinct layers. Neuronal migration is an essential part of the process of the layer formation because neurons are primarily generated near the ventricle and must migrate to arrive at their final locations during brain development. Impairment of brain development is thought to underlie the etiology of psychiatric disorders. Consistent with this idea, many genetic risk factors for psychiatric disorders play critical roles during brain development. As one example, Disrupted-in-Schizophrenia-1 (DISC1) is a genetic risk factor for major psychiatric disorders and plays various roles during neurodevelopment. To examine the role of Disc1 in the hippocampal development, we suppressed expression of Disc1 in the CA1 region of the developing mouse hippocampus by using the RNA interference (RNAi) technology and an in utero electroporation system. Disc1 suppression was found to impair migration of the CA1 pyramidal neurons. This effect was especially apparent while the majority of the transfected neurons were passing through the stratum pyramidale of the developing hippocampus. The migration of neurons was restored by expression of an RNAi-resistant wild-type mouse Disc1, indicating that the migration defect was caused by specific suppression of Disc1. In the mature hippocampus, the migration defect resulted in malposition and disarray of the pyramidal neurons. These findings indicate that Disc1 is required for migration and layer formation by the CA1 pyramidal neurons during hippocampal development.