DISC1-related signaling pathways in adult neurogenesis of the hippocampus

Disrupted-in-schizophrenia 1 (DISC1) is a multifunctional scaffold protein which plays an important role in neurogenesis and neural development in the adult brain, especially in the dentate gyrus (DG) of the hippocampus. Accumulated research has unveiled the role of DISC1 in several aspects of neural development and neurogenesis, such as neuronal maturation, proliferation, migration, positioning, differentiation, dendritic growth, axonal outgrowth, and synaptic plasticity. Studies on the function of this protein have explored multiple facets, including variants and missense mutants in genetics, proteins interactivity and signaling pathways in molecular biology, and pathogenesis and treatment targets of major mental illness, and more. In this review, we present several signaling pathways discussed in recent research, such as the AKT signaling pathway, GABA signaling pathway, GSK3β signaling pathway, Wnt signaling pathway, and NMDA-R signaling pathway. DISC1 interacts, directly or indirectly, with these signaling pathways and they co-regulate the process of adult neurogenesis in the hippocampus.     

Interaction between FEZ1 and DISC1 in regulation of neuronal development and risk for schizophrenia

Disrupted-in Schizophrenia 1 (DISC1), a susceptibility gene for major mental disorders, encodes a scaffold protein that has a multifaceted impact on neuronal development. How DISC1 regulates different aspects of neuronal development is not well understood. Here, we show that Fasciculation and Elongation Protein Zeta-1 (FEZ1) interacts with DISC1 to synergistically regulate dendritic growth of newborn neurons in the adult mouse hippocampus, and that this pathway complements a parallel DISC1-NDEL1 interaction that regulates cell positioning and morphogenesis of newborn neurons. Furthermore, genetic association analysis of two independent cohorts of schizophrenia patients and healthy controls reveals an epistatic interaction between FEZ1 and DISC1, but not between FEZ1 and NDEL1, for risk of schizophrenia. Our findings support a model in which DISC1 regulates distinct aspects of neuronal development through its interaction with different intracellular partners and such epistasis may contribute to increased risk for schizophrenia.     

DISC1 and the aggresome

Disrupted in Schizophrenia 1 (DISC1) is a key susceptibility gene for major psychiatric disorders. DISC1 plays a role in key neuronal processes such as neuronal proliferation, migration, integration and function via DISC1's roles at the centrosome and synapse, and in the regulation of intracellular signaling pathways. Recently, the idea of protein aggregation as a disease mechanism for DISC1 has been suggested. In our recent paper we explore these DISC1 protein aggregates in cell lines and neurons and find they are recruited to the aggresome and cause disruption of DISC1 function in intracellular transport.     

PlexinD1 signaling controls morphological changes and migration termination in newborn neurons

Newborn neurons maintain a very simple, bipolar shape, while they migrate from their birthplace toward their destinations in the brain, where they differentiate into mature neurons with complex dendritic morphologies. Here, we report a mechanism by which the termination of neuronal migration is maintained in the postnatal olfactory bulb (OB). During neuronal deceleration in the OB, newborn neurons transiently extend a protrusion from the proximal part of their leading process in the resting phase, which we refer to as a filopodium‐like lateral protrusion (FLP). The FLP formation is induced by PlexinD1 downregulation and local Rac1 activation, which coincide with microtubule reorganization and the pausing of somal translocation. The somal translocation of resting neurons is suppressed by microtubule polymerization within the FLP. The timing of neuronal migration termination, controlled by Sema3E‐PlexinD1‐Rac1 signaling, influences the final positioning, dendritic patterns, and functions of the neurons in the OB. These results suggest that PlexinD1 signaling controls FLP formation and the termination of neuronal migration through a precise control of microtubule dynamics.     

CAMDI, a novel disrupted in schizophrenia 1 (DISC1)-binding protein, is required for radial migration

Centrosomes play a crucial role in the directed migration of developing neurons. However, the underlying mechanism is poorly understood. This study has identified a novel disrupted in schizophrenia 1 (DISC1)-interacting protein, named CAMDI after coiled-coil protein associated with myosin II and DISC1, which translocates to the centrosome in a DISC1-dependent manner. Knockdown of CAMDI by shRNA revealed severely impaired radial migration with disoriented centrosomes. A yeast two-hybrid screen identified myosin II as a binding protein of CAMDI. CAMDI interacts preferentially with phosphomyosin II and induces an accumulation of phosphomyosin II at the centrosome in a DISC1-dependent manner. Interestingly, one single nucleotide polymorphism of the CAMDI gene (R828W) is identified, and its gene product was found to reduce the binding ability to phosphomyosin II. Furthermore, mice with overexpression of R828W in neurons exhibit an impaired radial migration. Our findings indicate that CAMDI is required for radial migration probably through DISC1 and myosin II-mediated centrosome positioning during neuronal development.     

Immunohistochemical analysis of Disc1 expression in the developing and adult hippocampus

In recent years, Disrupted-In-Schizophrenia 1 (DISC1) has emerged as one of the most promising candidate genes whose disruption confers an increased risk for schizophrenia. Cell biology studies have implicated DISC1 in key neurodevelopmental processes including neurite outgrowth and neuronal migration. In situ hybridization analysis has revealed that Disc1 is expressed in the hypothalamus, olfactory bulbs, the developing cerebral cortex and the hippocampus. The hippocampus is of particular interest because abnormalities in hippocampal volume and function have been consistently reported in schizophrenics. Moreover, DISC1 mutations have been associated with abnormal activation of the hippocampus in humans. Given the involvement of the hippocampus in the pathophysiology of schizophrenia, there is an intriguing possibility that disruption of DISC1 may increase schizophrenia susceptibility by altering the normal development and function of the hippocampus. In order to contribute to our understanding of DISC1's role in the hippocampus, we have performed a detailed analysis of the Disc1 expression pattern in the mouse hippocampus throughout development. We report that Disc1 is expressed throughout the hippocampus during embryonic development, with expression becoming increasingly specialized in Ammon's horn and dentate gyrus granule cells within the first postnatal week. This expression pattern remains consistent into adulthood, with a noted decrease in Disc1 expression in the adult CA1. Disc1 is also expressed in proliferating cells in the adult subgranular zone, as well as in a subset of GABAergic interneurons. Our results are the first report of a detailed immunohistochemical analysis of the ontogeny of Disc1 expression within the hippocampus.     

MACF1 regulates the migration of pyramidal neurons via microtubule dynamics and GSK-3 signaling

Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons.     

Human immunodeficiency virus type-1 vulnerates nascent neuronal cells

Macrophages or microglial cells are the major target cells for HIV-1 infection in the brain. The infected cells release neurotoxic factors that may cause severe neuronal cell damage, especially in the basal ganglia and hippocampus. In this study, we used rat OHC to examine the region-specific neuronal cell damage caused by HIV-1-infected macrophages. When OHC was cocultured with HIV-1-infected MDM, we found that neuronal cells at the GCL of the DG were preferentially killed via apoptosis, and that projection of MF from GCL to PCL of the CA3 region was severely disturbed. We marked precursor cells around the DG region by using an EGFP-expressing retrovirus vector and found that these cells lost the ability to differentiate into neurons when exposed to HIV-1-infected MDM. In the DG, new neurons are normally incorporated into GCL or PCL, while in the presence of HIV-1-infected MDM, mature neurons failed to be incorporated into those layers. These data indicate that the neurotoxic factor(s) released from HIV-1-infected macrophages impede(s) neuronal cell repair in brain tissue. This suggests that DG is the region of the hippocampus most vulnerable to neuronal damage caused by HIV-1 infection, and that its selective vulnerability is most likely due to the highly active neurogenesis that takes place in this region.     

Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain

Adult neurogenesis occurs throughout life in discrete regions of the adult mammalian brain. Little is known about the mechanism governing the sequential developmental process that leads to integration of new neurons from adult neural stem cells into the existing circuitry. Here, we investigated roles of Disrupted-In-Schizophrenia 1 (DISC1), a schizophrenia susceptibility gene, in adult hippocampal neurogenesis. Unexpectedly, downregulation of DISC1 leads to accelerated neuronal integration, resulting in aberrant morphological development and mispositioning of new dentate granule cells in a cell-autonomous fashion. Functionally, newborn neurons with DISC1 knockdown exhibit enhanced excitability and accelerated dendritic development and synapse formation. Furthermore, DISC1 cooperates with its binding partner NDEL1 in regulating adult neurogenesis. Taken together, our study identifies DISC1 as a key regulator that orchestrates the tempo of functional neuronal integration in the adult brain and demonstrates essential roles of a susceptibility gene for major mental illness in neuronal development, including adult neurogenesis.     

DISC1 and the aggresome: a disruption to cellular function?

Disrupted in Schizophrenia 1 (DISC1) is a key susceptibility gene for major psychiatric disorders. DISC1 plays a role in key neuronal processes such as neuronal proliferation, migration, integration and function via DISC1's roles at the centrosome and synapse, and in the regulation of intracellular signaling pathways. Recently, the idea of protein aggregation as a disease mechanism for DISC1 has been suggested. In our recent paper we explore these DISC1 protein aggregates in cell lines and neurons and find they are recruited to the aggresome and cause disruption of DISC1 function in intracellular transport.     

Reelin promotes hippocampal dendrite development through the VLDLR/ApoER2-Dab1 pathway

Reelin is a secreted glycoprotein that regulates neuronal positioning in cortical brain structures through the VLDLR and ApoER2 receptors and the adaptor protein Dab1. In addition to cellular disorganization, dendrite abnormalities are present in the brain of reeler mice lacking Reelin. It is unclear whether these defects are due primarily to cellular ectopia or the absence of Reelin. Here we examined dendrite development in the hippocampus of normal and mutant mice and in dissociated cultures. We found that dendrite complexity is severely reduced in homozygous mice deficient in Reelin signaling both in vivo and in vitro, and it is also reduced in heterozygous mice in the absence of cellular ectopia. Addition of Reelin interfering antibodies, receptor antagonists, and Dab1 phosphorylation inhibitors prevented dendrite outgrowth from normal neurons, whereas addition of recombinant Reelin rescued the deficit in reeler cultures. Thus, the same signaling pathway controls both neuronal migration and dendrite maturation.     

Interplay between DISC1 and GABA signaling regulates neurogenesis in mice and risk for schizophrenia

How extrinsic stimuli and intrinsic factors interact to regulate continuous neurogenesis in the postnatal mammalian brain is unknown. Here we show that regulation of dendritic development of newborn neurons by Disrupted-in-Schizophrenia 1 (DISC1) during adult hippocampal neurogenesis requires neurotransmitter GABA-induced, NKCC1-dependent depolarization through a convergence onto the AKT-mTOR pathway. In contrast, DISC1 fails to modulate early-postnatal hippocampal neurogenesis when conversion of GABA-induced depolarization to hyperpolarization is accelerated. Extending the period of GABA-induced depolarization or maternal deprivation stress restores DISC1-dependent dendritic regulation through mTOR pathway during early-postnatal hippocampal neurogenesis. Furthermore, DISC1 and NKCC1 interact epistatically to affect risk for schizophrenia in two independent case control studies. Our study uncovers an interplay between intrinsic DISC1 and extrinsic GABA signaling, two schizophrenia susceptibility pathways, in controlling neurogenesis and suggests critical roles of developmental tempo and experience in manifesting the impact of susceptibility genes on neuronal development and risk for mental disorders.     

Disabled-1-regulated adhesion of migrating neurons to radial glial fiber contributes to neuronal positioning during early corticogenesis

Disabled-1 regulates laminar organization in the developing mammalian brain. Although mutation of the disabled-1 gene in scrambler mice results in abnormalities in neuronal positioning, migratory behavior linked to Disabled-1 signaling is not completely understood. Here we show that newborn neurons in the scrambler cortex remain attached to the process of their parental radial glia during the entire course of radial migration, whereas wild-type neurons detach from the glial fiber in the later stage of migration. This abnormal neuronal-glial adhesion is highly linked to the positional abnormality of scrambler neurons and depends intrinsically on Disabled-1 Tyr220 and Tyr232, potential phosphorylation sites during corticogenesis. Importantly, phosphorylation at those sites regulates alpha3 integrin levels, which is critical for the timely detachment of migrating neurons from radial glia. Altogether, these results outline the molecular mechanism by which Disabled-1 signaling controls the adhesive property of neurons to radial glia, thereby maintaining proper neuronal positioning during corticogenesis.     

Migration defects by DISC1 knockdown in C57BL/6, 129X1/SvJ, and ICR strains via in utero gene transfer and virus-mediated RNAi

Disrupted-in-Schizophrenia 1 (DISC1) is a promising genetic risk factor for major mental disorders. Many groups repeatedly reported a role for DISC1 in brain development in various strains of mice and rats by using RNA interference (RNAi) approach. Nonetheless, due to the complexity of its molecular disposition, such as many splice variants and a spontaneous deletion in a coding exon of the DISC1 gene in some mouse strains, there have been debates on the interpretation on these published data. Thus, in this study, we address this question by DISC1 knockdown via short-hairpin RNAs (shRNAs) against several distinct target sequences with more than one delivery methodologies into several mouse strains, including C57BL/6, ICR, and 129X1/SvJ. Here, we show that DISC1 knockdown by in utero electroporation of shRNA against exons 2, 6, and 10 consistently results in neuronal migration defects in the developing cerebral cortex, which are successfully rescued by co-expression of full-length DISC1. Furthermore, lentivirus-mediated shRNA also led to migration defects, which is consistent with two other methodologies already published, such as plasmid-mediated and retrovirus-mediated ones. The previous study by Song’s group also reported that, in the adult hippocampus, the phenotype elicited by DISC1 knockdown with shRNA targeting exon 2 was consistently seen in both C57BL/6 and 129S6 mice. Taken together, we propose that some of DISC1 isoforms that are feasible to be knocked down by shRNAs to exon 2, 6, and 10 of the DISC1 gene play a key role for neuronal migration commonly in various mouse strains and rats.     

Mouse disabled 1 regulates the nuclear position of neurons in a Drosophila eye model

Nucleokinesis has recently been suggested as a critical regulator of neuronal migration. Here we show that Disabled 1 (Dab1), which is required for neuronal positioning in mammals, regulates the nuclear position of postmitotic neurons in a phosphorylation-site dependent manner. Dab1 expression in the Drosophila visual system partially rescues nuclear position defects caused by a mutation in the Dynactin subunit Glued. Furthermore, we observed that a loss-of-function allele of amyloid precursor protein (APP)-like, a kinesin cargo receptor, enhanced the severity of a Dab1 overexpression phenotype characterized by misplaced nuclei in the adult retina. In mammalian neurons, overexpression of APP reduced the ability of Reelin to induce Dab1 tyrosine phosphorylation, suggesting an antagonistic relationship between APP family members and Dab1 function. This is the first evidence that signaling which regulates Dab1 tyrosine phosphorylation determines nuclear positioning through Dab1-mediated influences on microtubule motor proteins in a subset of neurons.     

High expression of GLT-1 in hippocampal CA3 and dentate gyrus subfields contributes to their inherent resistance to ischemia in rats

It is well known that neurons in the CA3 and dentate gyrus (DG) subfields of the hippocampus are resistant to short period of ischemia which is usually lethal to pyramidal neurons in hippocampal CA1 subfield. The present study was undertaken to clarify whether the inherent higher resistance of neurons in CA3 and DG to ischemia is associated with glial glutamate transporter-1 (GLT-1) in rats. Western blot analysis and immunohistochemistry assay showed that the basal expressions of GLT-1 in both CA3 and DG were much higher than that in CA1 subfield. Mild global brain ischemia for 8 min induced delayed death of almost all CA1 pyramidal neurons and marked GLT-1 down-regulation in the CA1 subfield, but it was not lethal to the neurons in either CA3 or DG and induced GLT-1 up-regulation and astrocyte activation showed normal soma and aplenty slender processes in the both areas. When the global brain ischemia was prolonged to 25 min, neuronal death was clearly observed in CA3 and DG accompanied with down-regulation of GLT-1 expression and abnormal astrocytes represented with hypertrophic somas, but shortened processes. After down-regulating of GLT-1 expression and function by its antisense oligodeoxynucleotides or inhibiting GLT-1 function by dihydrokainate, an inhibitor of GLT-1, the mild global brain ischemia for 8 min, which usually was not lethal to CA3 and DG neurons, induced the neuronal death in CA3 and DG subfields. Taken together, the higher expression of GLT-1 in the CA3 and DG contributes to their inherent resistance to ischemia.     

Reelin induces EphB activation

The integration of newborn neurons into functional neuronal networks requires migration of cells to their final position in the developing brain, the growth and arborization of neuronal processes and the formation of synaptic contacts with other neurons. A central player among the signals that coordinate this complex sequence of differentiation events is the secreted glycoprotein Reelin, which also modulates synaptic plasticity, learning and memory formation in the adult brain. Binding of Reelin to ApoER2 and VLDL receptor, two members of the LDL receptor family, initiates a signaling cascade involving tyrosine phosphorylation of the intracellular cytoplasmic adaptor protein Disabled-1, which targets the neuronal cytoskeleton and ultimately controls the positioning of neurons throughout the developing brain. However, it is possible that Reelin signals interact with other receptor-mediated signaling cascades to regulate different aspects of brain development and plasticity. EphB tyrosine kinases regulate cell adhesion and repulsion-dependent processes via bidirectional signaling through ephrin B transmembrane proteins. Here, we demonstrate that Reelin binds to the extracellular domains of EphB transmembrane proteins, inducing receptor clustering and activation of EphB forward signaling in neurons, independently of the ''classical'' Reelin receptors, ApoER2 and VLDLR. Accordingly, mice lacking EphB1 and EphB2 display a positioning defect of CA3 hippocampal pyramidal neurons, similar to that in Reelin-deficient mice, and this cell migration defect depends on the kinase activity of EphB proteins. Together, our data provide biochemical and functional evidence for signal integration between Reelin and EphB forward signaling.     

Glycogen Synthase Kinase-3β Regulates Equilibrium Between Neurogenesis and Gliogenesis in Rat Model of Parkinson’s Disease: a Crosstalk with Wnt and Notch Signaling

Neurogenesis involves generation of functional newborn neurons from neural stem cells (NSCs). Insufficient formation or accelerated degeneration of newborn neurons may contribute to the severity of motor/nonmotor symptoms of Parkinson’s disease (PD). However, the functional role of adult neurogenesis in PD is yet not explored and whether glycogen synthase kinase-3β (GSK-3β) affects multiple steps of adult neurogenesis in PD is still unknown. We investigated the possible underlying molecular mechanism of impaired adult neurogenesis associated with PD. Herein, we show that single intra-medial forebrain bundle (MFB) injection of 6-hydroxydopamine (6-OHDA) efficiently induced long-term activation of GSK-3β and reduced NSC self-renewal, proliferation, neuronal migration, and neuronal differentiation accompanied with increased astrogenesis in subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Indeed, 6-OHDA also delayed maturation of neuroblasts in the DG as witnessed by their reduced dendritic length and arborization. Using a pharmacological approach to inhibit GSK-3β activation by specific inhibitor SB216763, we show that GSK-3β inhibition enhances radial glial cells, NSC proliferation, self-renewal in the SVZ, and the subgranular zone (SGZ) in the rat PD model. Pharmacological inhibition of GSK-3β activity enhances neuroblast population in SVZ and SGZ and promotes migration of neuroblasts towards the rostral migratory stream and lesioned striatum from dorsal SVZ and lateral SVZ, respectively, in PD model. GSK-3β inhibition enhances dendritic arborization and survival of granular neurons and stimulates NSC differentiation towards the neuronal phenotype in DG of PD model. The aforementioned effects of GSK-3β involve a crosstalk between Wnt/β-catenin and Notch signaling pathways that are known to regulate NSC dynamics.     

Roundabout 2 regulates migration of sensory neurons by signaling in trans

BACKGROUND: Roundabout (Robo) receptors and their ligand Slit are important regulators of axon guidance and cell migration. The development of Drosophila embryonic sense organs provides a neuronal migration paradigm where the in vivo roles of Slit and Robo can be assayed using genetics. RESULTS: Here we show that Slit-Robo signaling controls migration of Drosophila larval sensory neurons that are part of the Chordotonal (Cho) stretch receptor organs. We used live imaging to show that abdominal Cho organs normally migrate ventrally during development, whereas thoracic Cho organs do not. Robo2 overexpression in cis (in the sensory neurons) or in trans (on neighboring visceral mesoderm) transforms abdominal organs to a thoracic morphology and position by blocking migration, while loss of Slit-Robo signaling produces a reverse transformation in which thoracic organs migrate ectopically. Rescue and tissue-specific knockout experiments indicate that trans signaling by Robo2 contributes to the normal positioning of the thoracic Cho organs. The differential positioning of Cho organs between the thorax and abdomen is known to be regulated by Hox genes, and we show that the essential Hox cofactor Homothorax, represses Robo2 expression in the abdominal visceral mesoderm. CONCLUSIONS: Our results suggest that segment-specific neuronal migration patterns are directed through a novel signaling complex (the "Slit sandwich") in which Robo2 on the thoracic visceral mesoderm binds to Slit and presents it to Robo receptors on Cho neurons. The differential positioning of Cho organs between thorax and abdomen may be determined by Hox gene-mediated repression of robo2.