Reelin binds alpha3beta1 integrin and inhibits neuronal migration

Mice that are mutant for Reelin or Dab1, or doubly mutant for the VLDL receptor (VLDLR) and ApoE receptor 2 (ApoER2), show disorders of cerebral cortical lamination. How Reelin and its receptors regulate laminar organization of cerebral cortex is unknown. We show that Reelin inhibits migration of cortical neurons and enables detachment of neurons from radial glia. Recombinant and native Reelin associate with alpha3beta1 integrin, which regulates neuron-glia interactions and is required to achieve proper laminar organization. The effect of Reelin on cortical neuronal migration in vitro and in vivo depends on interactions between Reelin and alpha3beta1 integrin. Absence of alpha3beta1 leads to a reduction of Dab1, a signaling protein acting downstream of Reelin. Thus, Reelin may arrest neuronal migration and promote normal cortical lamination by binding alpha3beta1 integrin and modulating integrin-mediated cellular adhesion.     

Strange bedfellows: Reelin and Notch signaling interact to regulate cell migration in the developing neocortex

Neuronal cell migration in the developing neocortex relies on the Reelin signaling cascade to establish the characteristic "inside out" lamination pattern. In this issue of Neuron, Rakic and colleagues report that Notch signaling, long known to regulate neural stem cells, also plays a critical role in mediating the effects of Reelin on neuronal cell migration.     

Direct binding of Reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation

The large extracellular matrix protein Reelin is produced by Cajal-Retzius neurons in specific regions of the developing brain, where it controls neuronal migration and positioning. Genetic evidence suggests that interpretation of the Reelin signal by migrating neurons involves two neuronal cell surface proteins, the very low density lipoprotein receptor (VLDLR) and the apoE receptor 2 (ApoER2) as well as a cytosolic adaptor protein, Disabled-1 (Dab1). We show that Reelin binds directly and specifically to the ectodomains of VLDLR and ApoER2 in vitro and that blockade of VLDLR and ApoER2 correlates with loss of Reelin-induced tyrosine phosphorylation of Disabled-1 in cultured primary embryonic neurons. Furthermore, mice that lack either Reelin or both VLDLR and ApoER2 exhibit hyperphosphorylation of the microtubule-stabilizing protein tau. Taken together, these findings suggest that Reelin acts via VLDLR and ApoER2 to regulate Disabled-1 tyrosine phosphorylation and microtubule function in neurons.     

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.     

Reelin sets the pace of neocortical neurogenesis

Migration of neurons during cortical development is often assumed to rely on purely post-proliferative reelin signaling. However, Notch signaling, long known to regulate neural precursor formation and maintenance, is required for the effects of reelin on neuronal migration. Here, we show that reelin gain-of-function causes a higher expression of Notch target genes in radial glia and accelerates the production of both neurons and intermediate progenitor cells. Converse alterations correlate with reelin loss-of-function, consistent with reelin controlling Notch signaling during neurogenesis. Ectopic expression of reelin in isolated clones of progenitors causes a severe reduction in neuronal differentiation. In mosaic cell cultures, reelin-primed progenitor cells respond to wild-type cells by further decreasing neuronal differentiation, consistent with an increased sensitivity to lateral inhibition. These results indicate that reelin and Notch signaling cooperate to set the pace of neocortical neurogenesis, a prerequisite for proper neuronal migration and cortical layering.     

Reelin activates SRC family tyrosine kinases in neurons

BACKGROUND: Reelin is a large signaling molecule that regulates the positioning of neurons in the mammalian brain. Transmission of the Reelin signal to migrating embryonic neurons requires binding to the very-low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor-2 (apoER2). This induces tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1), which interacts with a shared sequence motif in the cytoplasmic tails of both receptors. However, the kinases that mediate Dab1 tyrosine phosphorylation and the intracellular pathways that are triggered by this event remain unknown. RESULTS: We show that Reelin activates members of the Src family of non-receptor tyrosine kinases (SFKs) and that this activation is dependent on the Reelin receptors apoER2 and VLDLR and the adaptor protein Dab1. Dab1 is tyrosine phosphorylated by SFKs, and the kinases themselves can be further activated by phosphorylated Dab1. Increased Dab1 protein expression in fyn-deficient mice implies a response to impaired Reelin signaling that is also observed in mice lacking Reelin or its receptors. However, fyn deficiency alone does not compound the neuronal positioning defect of vldlr- or apoer2-deficient mice, and this finding suggests functional compensation by other SFKs. CONCLUSIONS: Our results show that Dab1 is a physiological substrate as well as an activator of SFKs in neurons. Based on genetic evidence gained from multiple strains of mutant mice with defects in Reelin signaling, we conclude that activation of SFKs is a normal part of the cellular Reelin response.     

Rescue of ataxia and preplate splitting by ectopic expression of Reelin in reeler mice

The gene mutated in reeler (reelin) encodes a protein secreted by neurons in the developing brain that controls laminar positioning of migrating cells in the CNS by an unknown mechanism. To investigate Reelin function, we used the nestin promoter to express Reelin ectopically in the ventricular zone and other brain regions in transgenic mice. In the presence of the endogenous protein, ectopic Reelin did not alter cell migration in the neocortex or the cerebellum. However, in the reeler background, ectopic Reelin induced tyrosine phosphorylation of Dab-1 in the ventricular zone and rescued some, but not all, of the neuroanatomic and behavioral abnormalities characteristic of reeler. These results indicate that Reelin does not function simply as a positional signal. Rather, it appears to participate in multiple events critical for neuronal migration and cell positioning.     

Notch signaling coordinates the patterning of striatal compartments

Numerous lines of evidence suggest that Notch signaling plays a pivotal role in controlling the production of neurons from progenitor cells. However, most experiments have relied on gain-of-function approaches because perturbation of Notch signaling results in death prior to the onset of neurogenesis. Here, we examine the requirement for Notch signaling in the development of the striatum through the analysis of different single and compound Notch1 conditional and Notch3 null mutants. We find that normal development of the striatum depends on the presence of appropriate Notch signals in progenitors during a critical window of embryonic development. Early removal of Notch1 prior to neurogenesis alters early-born patch neurons but not late-born matrix neurons in the striatum. We further show that the late-born striatal neurons in these mutants are spared as a result of functional compensation by Notch3. Notably, however, the removal of Notch signaling subsequent to cells leaving the germinal zone has no obvious effect on striatal organization and patterning. These results indicate that Notch signaling is required in neural progenitor cells to control cell fate in the striatum, but is dispensable during subsequent phases of neuronal migration and differentiation.     

An In Silico Agent-Based Model Demonstrates Reelin Function in Directing Lamination of Neurons during Cortical Development

The characteristic six-layered appearance of the neocortex arises from the correct positioning of pyramidal neurons during development and alterations in this process can cause intellectual disabilities and developmental delay. Malformations in cortical development arise when neurons either fail to migrate properly from the germinal zones or fail to cease migration in the correct laminar position within the cortical plate. The Reelin signalling pathway is vital for correct neuronal positioning as loss of Reelin leads to a partially inverted cortex. The precise biological function of Reelin remains controversial and debate surrounds its role as a chemoattractant or stop signal for migrating neurons. To investigate this further we developed an in silico agent-based model of cortical layer formation. Using this model we tested four biologically plausible hypotheses for neuron motility and four biologically plausible hypotheses for the loss of neuron motility (conversion from migration). A matrix of 16 combinations of motility and conversion rules was applied against the known structure of mouse cortical layers in the wild-type cortex, the Reelin-null mutant, the Dab1-null mutant and a conditional Dab1 mutant. Using this approach, many combinations of motility and conversion mechanisms can be rejected. For example, the model does not support Reelin acting as a repelling or as a stopping signal. In contrast, the study lends very strong support to the notion that the glycoprotein Reelin acts as a chemoattractant for neurons. Furthermore, the most viable proposition for the conversion mechanism is one in which conversion is affected by a motile neuron sensing in the near vicinity neurons that have already converted. Therefore, this model helps elucidate the function of Reelin during neuronal migration and cortical development.     

Human Neural Cells Transiently Express Reelin during Olfactory Placode Development

Reelin, an extracellular glycoprotein is essential for migration and correct positioning of neurons during development. Since the olfactory system is known as a source of various migrating neuronal cells, we studied Reelin expression in the two chemosensory olfactory systems, main and accessory, during early developmental stages of human foetuses/embryos from Carnegie Stage (CS) 15 to gestational week (GW) 14. From CS 15 to CS 18, but not at later stages, a transient expression of Reelin was detected first in the presumptive olfactory and then in the presumptive vomeronasal epithelium. During the same period, Reelin-positive cells detach from the olfactory/vomeronasal epithelium and migrate through the mesenchyme beneath the telencephalon. Dab 1, an adaptor protein of the Reelin pathway, was simultaneously expressed in the migratory mass from CS16 to CS17 and, at later stages, in the presumptive olfactory ensheathing cells. Possible involvements of Reelin and Dab 1 in the peripheral migrating stream are discussed.     

Dab2ip Regulates Neuronal Migration and Neurite Outgrowth in the Developing Neocortex

Dab2ip (DOC-2/DAB2 interacting protein) is a member of the Ras GTPase-activating protein (GAP) family that has been previously shown to function as a tumor suppressor in several systems. Dab2ip is also highly expressed in the brain where it interacts with Dab1, a key mediator of the Reelin pathway that controls several aspects of brain development and function. We found that Dab2ip is highly expressed in the developing cerebral cortex, but that mutations in the Reelin signaling pathway do not affect its expression. To determine whether Dab2ip plays a role in brain development, we knocked down or over expressed it in neuronal progenitor cells of the embryonic mouse neocortex using in utero electroporation. Dab2ip down-regulation severely disrupts neuronal migration, affecting preferentially late-born principal cortical neurons. Dab2ip overexpression also leads to migration defects. Structure-function experiments in vivo further show that both PH and GRD domains of Dab2ip are important for neuronal migration. A detailed analysis of transfected neurons reveals that Dab2ip down- or up-regulation disrupts the transition from a multipolar to a bipolar neuronal morphology in the intermediate zone. Knock down of Dab2ip in neurons ex-vivo indicates that this protein is necessary for proper neurite development and for the expression of several major neuronal microtubule associated proteins (MAPs), which are important for neurite growth and stabilization. Thus, our study identifies, for the first time, a critical role for Dab2ip in mammalian cortical development and begins to reveal molecular mechanisms that underlie this function.     

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.     

Patterns of neurogenesis and amplitude of Reelin expression are essential for making a mammalian-type cortex

The mammalian neocortex is characterized as a six-layered laminar structure, in which distinct types of pyramidal neurons are distributed coordinately during embryogenesis. In contrast, no other vertebrate class possesses a brain region that is strictly analogous to the neocortical structure. Although it is widely accepted that the pallium, a dorsal forebrain region, is specified in all vertebrate species, little is known of the differential mechanisms underlying laminated or non-laminated structures in the pallium. Here we show that differences in patterns of neuronal specification and migration provide the pallial architectonic diversity. We compared the neurogenesis in mammalian and avian pallium, focusing on subtype-specific gene expression, and found that the avian pallium generates distinct types of neurons in a spatially restricted manner. Furthermore, expression of Reelin gene is hardly detected in the developing avian pallium, and an experimental increase in Reelin-positive cells in the avian pallium modified radial fiber organization, which resulted in dramatic changes in the morphology of migrating neurons. Our results demonstrate that distinct mechanisms govern the patterns of neuronal specification in mammalian and avian pallial development, and that Reelin-dependent neuronal migration plays a critical role in mammalian type corticogenesis. These lines of evidence shed light on the developmental programs underlying the evolution of the mammalian specific laminated cortex.     

Divergent roles of ApoER2 and Vldlr in the migration of cortical neurons

Reelin, its lipoprotein receptors [very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (ApoER2; also known as Lrp8)], and the cytoplasmic adaptor protein disabled 1 (Dab1) are important for the correct formation of layers in the cerebral cortex. Reeler mice lacking the reelin protein show altered radial neuronal migration resulting in an inversion of cortical layers. ApoER2 Vldlr double-knockout mutants and Dab1 mutants show a reeler-like phenotype, whereas milder phenotypes are found if only one of the two lipoprotein receptors for reelin is absent. However, the precise role of the individual reelin receptors in neuronal migration remained unclear. In the study reported here, we performed fate mapping of newly generated cortical neurons in single and double receptor mutants using bromodeoxyuridine-labeling and layer-specific markers. We present evidence for divergent roles of the two reelin receptors Vldlr and ApoER2, with Vldlr mediating a stop signal for migrating neurons and ApoER2 being essential for the migration of late generated neocortical neurons.     

Phosphatidylinositol 3-kinase interacts with the adaptor protein Dab1 in response to Reelin signaling and is required for normal cortical lamination

Reelin is a large secreted signaling protein that binds to two members of the low density lipoprotein receptor family, the apolipoprotein E receptor 2 and the very low density lipoprotein receptor, and regulates neuronal positioning during brain development. Reelin signaling requires activation of Src family kinases as well as tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This results in activation of phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt, and the inhibition of glycogen synthase kinase 3beta, a protein that is implicated in the regulation of axonal transport. Here we demonstrate that PI3K activation by Reelin requires Src family kinase activity and depends on the Reelin-triggered interaction of Dab1 with the PI3K regulatory subunit p85alpha. Because the Dab1 phosphotyrosine binding domain can interact simultaneously with membrane lipids and with the intracellular domains of apolipoprotein E receptor 2 and very low density lipoprotein receptor, Dab1 is preferentially recruited to the neuronal plasma membrane, where it is phosphorylated. Efficient Dab1 phosphorylation and activation of the Reelin signaling cascade is impaired by cholesterol depletion of the plasma membrane. Using a neuronal migration assay, we also show that PI3K signaling is required for the formation of a normal cortical plate, a step that is dependent upon Reelin signaling.     

Reconstitution of the Reelin signaling pathway in fibroblasts demonstrates that Dab1 phosphorylation is independent of receptor localization in lipid rafts

The Reelin signaling pathway operates in migrating neurons and is indispensable for their correct positioning during embryonic brain development. Many biochemical and cell biological studies to dissect the Reelin pathway at the molecular level are hampered by the lack of a cell line harboring a functional Reelin signaling pathway. Here we present fibroblast cell lines in which all required functional components of the pathway have been reconstituted. These cells react upon Reelin treatment in the same way as primary neurons. We have subsequently used these cell lines to study the subcellular localization of ApoER2 and the VLDL receptor and could demonstrate that receptor-mediated Dab1 phosphorylation does not depend on lipid rafts and that phosphorylated Dab1 remains bound to the receptor tail when the pathway is activated by Reelin.     

Reelin is a ligand for lipoprotein receptors

A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association of Reelin with VLDLR. After binding to VLDLR on the cell surface, Reelin is internalized into vesicles. In dissociated neurons, apoE reduces the level of Reelin-induced tyrosine phosphorylation of Dab1. These data suggest that Reelin directs neuronal migration by binding to VLDLR and ApoER2.     

Reelin signals through phosphatidylinositol 3-kinase and Akt to control cortical development and through mTor to regulate dendritic growth

Reelin is an extracellular matrix protein with various functions during development and in the mature brain. It activates different signaling cascades in target cells, one of which is the phosphatidylinositol 3-kinase (PI3K) pathway, which we investigated further using pathway inhibitors and in vitro brain slice and neuronal cultures. We show that the mTor (mammalian target of rapamycin)-S6K1 (S6 kinase 1) pathway is activated by Reelin and that this depends on Dab1 (Disabled-1) phosphorylation and activation of PI3K and Akt (protein kinase B). PI3K and Akt are required for the effects of Reelin on the organization of the cortical plate, but their downstream partners mTor and glycogen synthase kinase 3beta (GSK3beta) are not. On the other hand, mTor, but not GSK3beta, mediates the effects of Reelin on the growth and branching of dendrites of hippocampal neurons. In addition, PI3K fosters radial migration of cortical neurons through the intermediate zone, an effect that is independent of Reelin and Akt.     

Apolipoprotein E receptors are required for reelin-induced proteasomal degradation of the neuronal adaptor protein Disabled-1

The cytoplasmic adaptor protein Disabled-1 (Dab1) is necessary for the regulation of neuronal positioning in the developing brain by the secreted molecule Reelin. Binding of Reelin to the neuronal apolipoprotein E receptors apoER2 and very low density lipoprotein receptor induces tyrosine phosphorylation of Dab1 and the subsequent activation or relocalization of downstream targets like phosphatidylinositol 3 (PI3)-kinase and Nckbeta. Disruption of Reelin signaling leads to the accumulation of Dab1 protein in the brains of genetically modified mice, suggesting that Reelin limits its own action in responsive neurons by down-regulating the levels of Dab1 expression. Here, we use cultured primary embryonic neurons as a model to demonstrate that Reelin treatment targets Dab1 for proteolytic degradation by the ubiquitin-proteasome pathway. We show that tyrosine phosphorylation of Dab1 but not PI3-kinase activation is required for its proteasomal targeting. Genetic deficiency in the Dab1 kinase Fyn prevents Dab1 degradation. The Reelin-induced Dab1 degradation also depends on apoER2 and very low density lipoprotein receptor in a gene-dose dependent manner. Moreover, pharmacological blockade of the proteasome prevents the formation of a proper cortical plate in an in vitro slice culture assay. Our results demonstrate that signaling through neuronal apoE receptors can activate the ubiquitin-proteasome machinery, which might have implications for the role of Reelin during neurodevelopment and in the regulation of synaptic transmission.