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EVOLUTIONARY HOMOLOGS part 2/2 | part 1/2

Dab1, Reelin and brain development

Although accurate long-distance neuronal migration is a cardinal feature of cerebral cortical development, little is known about control of this migration. The scrambler (scm) mouse shows abnormal cortical lamination that is indistinguishable from reeler. Genetic and physical mapping of scm identifies yeast artificial chromosomes containing an exon of mdab1, a homolog of Drosophila Disabled, which encodes a phosphoprotein that binds nonreceptor tyrosine kinases. mdab1 transcripts show abnormal splicing in scm homozygotes, with 1.5 kb of intracisternal A particle retrotransposon sequence inserted into the mdab1 coding region in antisense orientation, producing a mutated and truncated predicted protein. Therefore, mdab1 is most likely the scm gene, thus implicating nonreceptor tyrosine kinases in neuronal migration and lamination in developing cerebral cortex (Ware, 1997).

Formation of the mammalian brain requires choreographed migration of neurons to generate highly ordered laminar structures, such as those in the cortices of the forebrain and the cerebellum. These processes are severely disrupted by mutations in Reelin protein, which cause widespread misplacement of neurons and associated ataxia in reeler mutant mice. Reelin is a large extracellular protein secreted by pioneer neurons that coordinates cell positioning during neurodevelopment. Two new autosomal recessive mouse mutations, scramble and yotari have been described that exhibit a phenotype identical to reeler. scrambler and yotari arise from mutations in mdab1, a mouse gene related to the Drosophila gene Disabled (Dab). Both scrambler and yotari mice express mutated forms of mdab1 messenger RNA and little or no mDab1 protein. mDab1 is a phosphoprotein that appears to function as an intracellular adaptor in protein kinase pathways. mdab1 is expressed in neuronal populations exposed to Reelin. The similar phenotypes of reeler, scrambler, yotari and mdab1 null mice indicate that Reelin and mDab1 function as signaling molecules that regulate cell positioning in the developing brain (Sheldon, 1997).

During mammalian brain development, immature neurons migrate radially from the neuroectoderm to defined locations, giving rise to characteristic cell layers. Targeted disruption of the mouse disabled1 (mdab1) gene disturbs neuronal layering in the cerebral cortex, hippocampus and cerebellum. The gene encodes a cytoplasmic protein, mDab1 p80, which is expressed and tyrosine-phosphorylated in the developing nervous system. Most likely it is an adaptor protein, docking to others through its phosphotyrosine residues and protein-interacting domain. The mdab1 mutant phenotype is very similar to that of the reeler mouse mutant. The product of the reeler gene, Reelin, is a secreted protein that has been proposed to act as an extracellular signpost for migrating neurons. Because mDab1 is expressed in wild-type cortical neurons, and Reelin expression is normal in mdab1 mutants, mDab1 may be part of a Reelin-regulated or parallel pathway that controls the final positioning of neurons (Howell, 1997b).

A mouse homolog of the Drosophila Disabled (Dab) gene, disabled-1 (mdab1), encodes an adaptor molecule that functions in neural development. Targeted disruption of the mdab1 gene leads to anomalies in the development of the cerebrum, hippocampus, and cerebellum. A number of histologic abnormalities are described in the cerebellum of the mdab1-1 mouse. There is a complete absence of foliation, and most Purkinje cells are clumped in central clusters. However, lamination appears to develop normally in areas where the Purkinje cells and external granular layer are closely apposed. The granular layer forms a thin rind over most of the cerebellar surface, but is subdivided by both transverse and parasagittal boundaries. The Purkinje cells, identified by anti-zebrin II in the adult or anti-calbindin in the new born mdab1-1 mutant cerebellum, form a parasagittal banding pattern, similar to but distorted in comparison to the wild-type design. The data suggest that the development of the mdab1-1 cerebellum parallels the development of reeler. The reeler gene encodes an extracellular protein (Reelin) that is secreted by the external granular layer. Because Reelin expression is retained in the mdab1-1 mutant mouse, mDab1 p80 may act in a parallel pathway or downstream of Reelin, leading to the transformation of embryonic Purkinje cell clusters into the adult parasagittal bands (Gallagher, 1998).

The reelin (reln) and disabled 1 (dab1) genes both ensure correct neuronal positioning during brain development. The intracellular Dab1 protein receives a tyrosine phosphorylation signal from extracellular Reln protein. Genetic analysis shows that reln function depends on dab1, and vice versa, as would be expected if both genes are in the same pathway. To investigate whether there is a worsening of phenotype in dab1::reln double mutants, double heterozygous dab1/+ reln/+ animals were generated and interbred. Double homozygous mutant progeny have been obtained at expected frequency and resemble the single homozygotes. The cerebral cortex loses lamination; the hippocampal scrolls are split, and the granule cells of the dentate gyrus are intermingled. One characteristic of the neocortex in dab1 and reln homozygotes is invasion of the marginal zone by neurons that are normally found deep in the cortex. Increased neuron numbers are also detected in the marginal zone of compound homozygotes, but there is no significant quantitative difference between the marginal zone population in single and double homozygotes. The cerebella of the double mutant mice are also indistinguishable from the single mutants. Mutant cerebella are unfoliated and small. A molecular layer is present but the granule cells are reduced in number. Furthermore, the Purkinje cells, which normally express Dab1, are found clustered in central regions. In contrast, the Purkinje cells in a wild-type cerebellum form a monolayer above a dense granule cell layer. The compound mutants are also indistinguishable behaviorally from the single mutants. Thus it seems unlikely that either Reln or Dab1 has residual function in the absence of the other protein. Dab1 is expressed at a higher level, yet phosphorylated at a lower level, in reln mutant embryo brains. In primary neuronal cultures, Dab1 tyrosine phosphorylation is stimulated by exogenous Reln. These results show that Dab1 and Reln act on the same pathway to control neuronal positioning in the developing brain. The kinetics of tyrosine phosphorylation suggest that Reln interacts directly with a receptor on neurons that express Dab1, and the effects of a generic phosphotyrosine inhibitor suggest that the Reln receptor is linked to a tyrosine kinase. Reln may also stimulate other signaling pathways that may act in parallel to Dab1 tyrosine phosphorylation, but the genetics suggest that Dab1 tyrosine phosphorylation is one step in an essential, nonredundant pathway that regulates neuron position. Because Reln is also involved in the ingrowth of entorhinal afferents into the hippocampus, and Dab1 is in growth cones, a Reln-Dab1 pathway may also regulate growth cone migration. Increased tyrosine phosphorylation of Dab1 is expected to potentiate interactions with proteins that contain SH2 domains or other phosphotyrosine-dependent domains. These interactions may arrest neuronal migration by modulating the actin cytoskeleton, membrane flow, or adhesion complexes. (Howell, 1999a).

The extracellular protein Reelin (Reln) controls neuronal migrations in parts of the cortex, hippocampus and cerebellum. In vivo, absence of Reln correlates with up-regulation of the docking protein Dab1 and decreased Dab1 tyrosine phosphorylation. Loss of the Reln receptor proteins, apolipoprotein receptor 2 and very low density lipoprotein receptor, results in a Reln-like phenotype accompanied by increased Dab1 protein expression. Complete loss of Dab1, however, recapitulates the Reln phenotype. To determine whether Dab1 tyrosine phosphorylation affects Dab1 protein expression and positioning of embryonic neurons, Dab1 tyrosine phosphorylation sites were identifed. Mice were generated in which the Dab1 protein had all the potential tyrosine phosphorylation sites mutated. This mutant protein is not tyrosine phosphorylated during brain development and is not upregulated to the extent observed in the Reln or the apoER2 and VLDLR receptor mutants. Animals expressing the non-phosphorylated Dab1 protein have a phenotype similar to the dab1-null mutant. It is concluded that Dab1 is downregulated by the Reln signal in neurons in the absence of tyrosine phosphorylation. Dab1 tyrosine phosphorylation sites, and not the downregulation of Dab1 protein, are required for Reln signaling (Howell, 2000).

Two novel mouse mutations, scrambler and yotari exhibit remarkable behavioral and histopathological similarities to reeler. Molecular genetic studies reveal that scrambler and yotari arose from independent mutations in the disabled-1 (Dab1) gene coding for a phosphotyrosine-interacting/phosphotyrosine-binding domain protein that exhibits some degree of similarity to the Drosophila Disabled gene. It is an intracellular protein that becomes phosphorylated on tyrosine residues during embryonic development and it can bind to the protein tyrosine kinases Src, Abl and Fyn. Based on its biochemical properties, Dab1 is thought to function as an adapter molecule in the transduction of protein kinase signals. Mutation of either reelin (Reln) or Dab1 results in widespread abnormalities in laminar structures throughout the brain and ataxia in reeler and scrambler mice. Both exhibit the same neuroanatomical defects, including cerebellar hypoplasia with Purkinje cell ectopia and disruption of neuronal layers in the cerebral cortex and hippocampus. Despite these phenotypic similarities, Reln and Dab1 have distinct molecular properties. Reln is a large extracellular protein secreted by Cajal-Retzius cells in the forebrain and by granule neurons in the cerebellum. In contrast, Dab1 is a cytoplasmic protein that has properties of an adapter protein that functions in phosphorylation-dependent intracellular signal transduction. Dab1 is shown to participate in the same developmental process as Reln. In scrambler mice, neuronal precursors are unable to invade the preplate of the cerebral cortex and consequently, they do not align within the cortical plate. During development, cells expressing Dab1 are located next to those secreting Reln at critical stages of formation of the cerebral cortex, cerebellum and hippocampus, before the first abnormalities in cell position become apparent in either reeler or scrambler. In reeler, the major populations of displaced neurons contain elevated levels of Dab1 protein, although they express normal levels of Dab1 mRNA. This suggests that Dab1 accumulates in the absence of a Reln-evoked signal. Taken together, these results indicate that Dab1 functions downstream of Reln in a signaling pathway that controls cell positioning in the developing brain (Rice, 1998).

Layering of neurons in the cerebral cortex and cerebellum requires Reelin, an extracellular matrix protein, and mammalian Disabled (mDab1), a cytosolic protein that activates tyrosine kinases. A molecular pathway that regulates the migration of neurons along the radial glial fiber network involves the large extracellular protein Reelin. In the cortex, this modular protein is either associated with the extracellular matrix or with the surface of a special class of neurons that produce it in the outermost layer just beneath the pial surface. These so-called Cajal-Retzius neurons are formed during the early stages of neural development. In the reeler strain of mice, the gene encoding Reelin is defective. As a result, migratory neurons in these mice apparently do not receive a critical cue that informs them of their position, leading to an inversion of the cortical layers. These layers normally form from the inside out, with later-born neurons migrating past older ones to form progressively more superficial, and thus younger, layers of the neocortex. In the cerebellum, Reelin is required for the Purkinje cells to migrate outward, where they form a well-defined cortical plate through which postmitotic granule cells migrate inward to form the internal granular layer. Both of these laminated structures do not form in the reeler mouse. The cytoplasmic adaptor protein mDab1 is related to the Drosophila disabled gene product. It is predominantly expressed in neurons and has been shown to function downstream of Reelin. mDab1-deficient mice (identified as a naturally occurring strain called scrambler and also generated by gene knockout) develop a phenotype indistinguishable from reeler. Furthermore, in reeler mice, mDab1 protein expression is greatly increased even after the end of the migratory period, indicating a failure of neurons to adjust mDab1 expression due to lack of Reelin signal input. mDab1 contains a protein interaction domain that binds to NPxY motifs in the cytoplasmic tails of receptors. mDab1 can undergo tyrosine phosphorylation and subsequently interact with nonreceptor tyrosine kinases of the Abl and Src family, suggesting that the Reelin signaling pathway involves coupling of the signal, via mDab1, to intracellular kinase pathways. However, the cell surface receptors that mediate transmission of the signal across the neuronal plasma membrane are unknown. The requirement for two other proteins has been documented. These are cell surface receptors termed very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). The LDL receptor gene family comprises a group of structurally related multifunctional cell surface receptors that mediate endocytosis of extracellular ligands. The five known mammalian members of the family are the LDL receptor, LRP, Megalin, VLDLR, and ApoER2. The role of the LDL receptor in the regulation of cholesterol homeostasis is well understood. LRP and Megalin are both multifunctional and bind a diverse spectrum of ligands, including lipoproteins, proteases and their inhibitors, peptide hormones, and carrier proteins for vitamins. The VLDLR and ApoER2, like all members of the family, can bind ApoE, but the relevance of this interaction is unclear and their true physiological functions are unknown. Both receptors can bind mDab1 on their cytoplasmic tails and are expressed in cortical and cerebellar layers adjacent to layers that express Reelin. mDab1 expression is upregulated in knockout mice that lack both VLDLR and ApoER2. Inversion of cortical layers and absence of cerebellar foliation in these animals precisely mimic the phenotype of mice lacking Reelin or mDab1. These findings suggest that VLDLR and ApoER2 participate in transmitting the extracellular Reelin signal to intracellular signaling processes initiated by mDab1 (Trommsdorff, 1999).

Three models have been proposed to explain the phenotype of Reelin- and mDab1-deficient mice. One model suggests that Reelin acts as a repellent that induces the cortical preplate to split into marginal zone and subplate. The failure of the preplate to separate in reeler causes the cortical plate to develop ectopically underneath the subplate neurons. In another model, a Reelin gradient may selectively induce cortical plate neurons to migrate past the subplate neurons. In a third model, Reelin is thought to act as a stop signal that instructs migrating neurons to detach from their glial guidance fiber. The present findings are consistent with all these models, but they also indicate that neuronal migration along radial glia is more complex than these current models suggest. VLDLR and ApoER2 are both required for normal neuronal migration. Both receptors coordinate the development of the cortex and of the cerebellum; however, the phenotype of the VLDLR defect manifests itself mainly in the cerebellum, whereas deficiency in ApoER2 predominantly affects the development of the neocortex. Only the absence of both receptors causes an almost exact neuroanatomical phenocopy of the reeler and scrambler (mDab1-deficient) mutation. In the cortex, VLDLR and mDab1 are selectively expressed in the migrating neurons that are about to make contact with Reelin in the marginal zone, consistent with a role of VLDLR as a receptor for a migratory stop signal. Also consistent with such a model is the finding that in VLDLR-deficient cerebellum, Purkinje cells are ectopically located, apparently due to their inability to properly respond to the Reelin signal that emanates from the granule cells in the external granular layer. In contrast, ApoER2 is more ubiquitously expressed throughout the developing brain. Thus, other non-cell autonomous functions for this receptor cannot be ruled out. Absence of ApoER2 alone prevents a large portion of neurons from completing their migration and causes a partial inversion of the layers in the neocortex. As a result, the large and easily identifiable pyramidal neurons end up in a relatively ordered, but more superficial layer than the layer 5 in which they normally reside (Trommsdorff, 1999).

Furthermore, in VLDLR-deficient cortex, neurons are strictly radially aligned, and although they apparently have reached their normal assigned layer, they have failed to distribute within that layer. In contrast, in ApoER2-deficient cortex, there is no recognizable radial pattern; instead, neurons are packed into tight consecutive horizontal layers. In the absence of either VLDLR or ApoER2, neurons do not invade layer 1, in contrast to the double knockout and to reeler and mDab1 mutants (Trommsdorff, 1999 and references).

Taken together, these findings suggest that VLDLR and ApoER2 function in a coordinated and partially overlapping fashion. Either receptor is apparently capable of interpreting the stop signal conferred by Reelin, thus allowing migrating neurons to detach from the radial guidance fiber and preventing them from invading layer 1. Consistent with this interpretation is the finding that mDab1 expression is only slightly increased in either of the single knockouts. The distinct neuroanatomical phenotypes of vldlr and apoER2 knockouts may indicate that both receptors further interact with specific sets of other matrix or glial surface components. Other adaptor proteins besides mDab1 may also be involved and control different stages of neuronal migration (Trommsdorff, 1999).

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). Reelin binds directly and specifically to the ectodomains of VLDLR and ApoER2 in vitro and 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 (Hiesberger, 1999).

The disabled 1 (Dab1) p80 protein is essential for reelin signaling during brain development. p80 has an N-terminal domain for association with reelin receptors, followed by reelin-dependent tyrosine phosphorylation sites and about 310 C-terminal residues of unknown function. Mutant mice were generated that express only a natural splice form of Dab1, p45, that lacks the C-terminal region of p80. The normal development of these mice implies that the receptor-binding region and tyrosine phosphorylation sites of p80 are sufficient for reelin signaling. However, a single copy of the truncated gene does not support normal development of the neocortex and hippocampus. The CA1 region of the hippocampus is split into two well-organized layers, while the marginal zone of the neocortex is invaded by late-born cortical plate neurons. The haploinsufficiency of the p45 allele of Dab1 implies that the C terminus of p80 affects the strength of reelin-Dab1 signaling, yet there is no apparent change in reelin-dependent tyrosine phosphorylation of p45 relative to p80. Therefore, it is suggested that the C-terminal region of Dab1 p80 is involved in signaling to downstream effector molecules. Furthermore, the presence of late-born cortical plate neurons in the marginal zone reveals a requirement for reelin-Dab1 signaling in late-born cortical plate neurons, and helps distinguish models for the cortical inversion in the reeler mutant mouse (Herrick, 2002).

In the neocortex, reelin, a secreted protein that controls the migration of many neurons, including Purkinje cells in the cerebellum and most glutamatergic excitatory neurons of the neocortex and hippocampus, is made by the Cajal-Retzius (CR) neurons in the outer leaf of the preplate. When early cortical plate (CP) neurons are created in the ventricular zone below the preplate, they migrate outward on radial glia and between the cells of the inner leaf of the preplate. Upon reaching the CR cells, the early CP neurons detach from the radial glia and differentiate. The early CP neurons thus split the inner from the outer leaves of the preplate, driving the reelin-secreting cells ahead of them and leaving the inner part of the preplate behind as the subplate. Later born CP neurons also migrate through the subplate and between the early CP neurons, settling only when they reach the CR cells. This age stratification within the CP is known as inside-out layering, because the first neurons are innermost, forming layers V-VI, and the last ones are outermost, forming layers II-IV. The CR neurons occupy the cell-poor marginal zone (MZ) or layer I (Herrick, 2002).

The disabled 1 (Dab1) p80 protein is essential for reelin signaling during brain development. p80 has an N-terminal domain for association with reelin receptors, followed by reelin-dependent tyrosine phosphorylation sites and about 310 C-terminal residues of unknown function. Mutant mice have been generated that express only a natural splice form of Dab1, p45, that lacks the C-terminal region of p80. The normal development of these mice implies that the receptor-binding region and tyrosine phosphorylation sites of p80 are sufficient for reelin signaling. However, a single copy of the truncated gene does not support normal development of the neocortex and hippocampus. The CA1 region of the hippocampus is split into two well-organized layers, while the marginal zone of the neocortex is invaded by late-born cortical plate neurons. The haploinsufficiency of the p45 allele of Dab1 implies that the C terminus of p80 affects the strength of reelin-Dab1 signaling, yet there is no apparent change in reelin-dependent tyrosine phosphorylation of p45 relative to p80. Therefore, it is suggested that the C-terminal region of Dab1 p80 is involved in signaling to downstream effector molecules. Furthermore, the presence of late-born cortical plate neurons in the marginal zone reveals a requirement for reelin-Dab1 signaling in late-born cortical plate neurons, and helps distinguish models for the cortical inversion in the reeler mutant mouse (Herrick, 2002).

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. Reelin is shown to activate members of the Src family of non-receptor tyrosine kinases (SFKs) and this activation is dependent on the Reelin receptors apoER2 and VLDLR and the adaptor protein Dab1. Dab1 is shown to be 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. These 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, it is concluded that activation of SFKs is a normal part of the cellular Reelin response (Bock, 2003).

Disabled-1 is an intracellular adaptor protein that regulates migrations of various classes of neurons during mammalian brain development. Dab1 function depends on its tyrosine phosphorylation, which is stimulated by Reelin, an extracellular signaling molecule. Reelin increases the stoichiometry of Dab1 phosphorylation and downregulates Dab1 protein levels. Reelin binds to various cell surface receptors, including two members of the low-density lipoprotein receptor family that also bind to Dab1. Mutations in Dab1, its phosphorylation sites, Reelin, or the Reelin receptors cause a common phenotype. However, the molecular mechanism whereby Reelin regulates Dab1 tyrosine phosphorylation is poorly understood. Reelin-induced Dab1 tyrosine phosphorylation in neuron cultures is inhibited by acute treatment with pharmacological inhibitors of the Src family, but not Abl family, kinases. In addition, Reelin stimulates Src family kinases by a mechanism involving Dab1. The Dab1 protein level and tyrosine phosphorylation stoichiometry were analyzed by using brain samples and cultured neurons that were obtained from mouse embryos carrying mutations in Src family tyrosine kinases. Fyn is found to be required for proper Dab1 levels and phosphorylation in vivo and in vitro. When fyn copy number is reduced, src, but not yes, becomes important, reflecting a partial redundancy between fyn and src. It is concluded that Reelin activates Fyn to phosphorylate and downregulate Dab1 during brain development. The results were unexpected because Fyn deficiency does not cause the same developmental phenotype as Dab1 or Reelin deficiency. This suggests additional complexity in the Reelin signaling pathway (Arnaud, 2003).

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. 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; this phosphorylation 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 (Sanada, 2004).

Tyrosine residues 220 and 232 are critical sites for the ability of Dab1 to induce detachment of neurons from radial glial fibers. Since Tyr220 and Tyr232 are sites of Reelin-induced phosphorylation in the cortical neuronal culture, it is likely that Reelin-Dab1 signaling regulates adhesion molecules such as alpha3ß1 integrin in migrating neurons and that downregulation of alpha3ß1 integrin is important for the radial detachment of neurons as they migrate closer toward the marginal zone. These data do not exclude the possibility that additional adhesion molecules contribute to the regulation of Dab1-mediated neuronal-glial interaction. Notably, tyrosine phosphorylation of Dab1 is induced by the direct binding of Reelin to VLDLR and ApoER2 but not to alpha3ß1 integrin. This suggests that Dab1-dependent neuronal detachment from radial glia is likely initiated by Reelin-VLDLR/ApoER2 signaling and not by Reelin-alpha3ß1 integrin signaling (Sanada, 2004).

Reelin-induced tyrosine-phosphorylation of Dab1 is believed to be catalyzed by Src family tyrosine kinases such as Fyn and Src, and tyrosine-phosphorylated Dab1 is thought to act as an adaptor protein for the recruitment of various SH2 domain-containing proteins. Src family tyrosine kinases themselves are progressively activated in the presence of Dab1, possibly because tyrosine-phosphorylated Dab1 recruits additional SH2/PTB domain-containing tyrosine kinases that activate Src family tyrosine kinases. The sequence encompassing the tyrosine phosphorylation site Tyr220 and Tyr232 nicely fits a consensus motif for the binding of the SH2 domain of Abl, Crk, and NcK, which all prefer to bind to the Tyr(PO4)-X-X-Pro motif. On the basis of these results, it is hypothesized that Tyr220/Tyr232-phosphorylated Dab1 may recruit Abl, or its relative Arg, Crk, or NcK, and activate their respective signaling pathways in order to transcriptionally repress or degrade alpha3 integrin (and other adhesive molecules) in neurons, as they migrate closer toward the marginal zone. Consistent with this idea, Tyr220 and Tyr232 contribute in a phosphorylation-dependent manner to the association of Dab1 with Crk and Nckß. However, Dab1 also binds directly to the ß1 integrin cytoplasmic tail. The binding region within ß1 integrin encompasses two Asn-Pro-X-Tyr (NPXY, where X denotes any amino acids) motifs that are thought to be potential motifs for endocytosis and the targeting to the lysosome. Thus, it is conceivable that Tyr220/Tyr232-phosphorylated Dab1 may directly control the turnover rate of alpha3ß1 integrin through its association with the NPXY motif of ß1 integrin (Sanada, 2004).

In addition to integrins, Dab1 also interacts with several other cell surface proteins, including VLDLR, ApoER, amyloid precursor protein, and amyloid precursor-like proteins. Dab1 associates with the cytoplasmic tail of each of these receptors, through an NPXY motif. These various types of receptors may be clustered into a large complex at the cell surface of the migrating neuron, and upon binding of Reelin to VLDLR and ApoER2, the entire complex may be endocytosed and subsequently degraded. In this regard, it may be possible that the Reelin signal induces the tyrosine phosphorylation of Dab1 in the receptor complex, which could promote the endocytosis of the entire complex, including alpha3ß1 integrin, and lead to a regional reduction of alpha3ß1 integrin levels in the neocortex (Sanada, 2004).

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. This study used cultured primary embryonic neurons as a model to demonstrate that Reelin treatment targets Dab1 for proteolytic degradation by the ubiquitin-proteasome pathway. 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. These 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 (Bock, 2004).

Reelin and Disabled 1 (Dab1) are essential for positioning migrating neurons in the developing neocortex. Cell-autonomous RNA interference-mediated suppression of Dab1 in migrating neurons destined for layer 2/3 shifted the median position of these cells to deeper positions within the cortex. At the time of migration arrest [embryonic day 20 (E20) to E21], Dab1-suppressed cells were underrepresented in the upper approximately 40 microm of the cortex compared with controls, suggesting that Dab1 is essential for somal translocation through the cell-dense cortical plate. Closer examination of the morphology of Dab1-suppressed neurons at E20 revealed simplified leading processes that are less likely to contact the marginal zone (MZ), in which high levels of Reelin are expressed. Examination of Dab1-suppressed cells 3 d later (postnatal day 2) revealed simplified dendrites that are also less likely to contact the MZ. These data reveal a cell-autonomous role of Dab1 in dendritogenesis in the neocortex and suggest that remodeling of the leading process of a migrating neuron into a nascent dendrite by Reelin/Dab1 signaling plays an important role in cell positioning (Olson, 2006).

The Reelin signaling pathway controls neuronal positioning in human and mouse brain during development as well as modulation of long-term potentiation (LTP) and behavior in the adult. Reelin signals by binding to two transmembrane receptors, apolipoprotein E receptor 2 (Apoer2) and very-low-density lipoprotein receptor. After Reelin binds to the receptors, Disabled-1 (Dab1), an intracellular adaptor protein that binds to the cytoplasmic tails of the receptors, becomes phosphorylated on tyrosine residues, initiating a signaling cascade that includes activation of Src-family kinases and Akt. A line of mutant mice (Apoer2 EIG) has been created in which the Apoer2 NFDNPVY motif has been altered to EIGNPVY to disrupt the Apoer2-Dab1 interaction to further study Reelin signaling in development and adult brain. Using primary neuronal cultures stimulated with recombinant Reelin, it was found that normal Reelin signaling requires the wild-type NFDNPVY sequence and likely the interaction of Apoer2 with Dab1. Furthermore, examination of hippocampal, cortical, and cerebellar layering reveals that the NFDNPVY sequence of Apoer2 is indispensable for normal neuronal positioning during development of the brain. Adult Apoer2 EIG mice display severe abnormalities in LTP and behavior that are distinct from those observed for mice lacking Apoer2. In Apoer2 EIG slices, LTP degraded to baseline within 30 min, and this was prevented in the presence of Reelin. Together, these findings emphasize the complexity of Reelin signaling in the adult brain, which likely requires multiple adaptor protein interactions with the intracellular domain of Apoer2 (Beffert, 2006).

Many laminated regions of the mammalian brain develop by the migration of neuronal precursor cells, whose final positions are coordinated by signals from the secreted molecule Reelin. Early events in Reelin signaling have been identified, but the mechanism of signal down-regulation has been unclear. A possible source of negative feedback is the Reelin-induced degradation of the critical intracellular signaling component, Disabled-1 (Dab1). This study shows that degradation of Dab1 depends on Dab1 phosphorylation at specific tyrosine residues and on the E3 ubiquitin ligase component Cullin 5 (Cul5). Cul5 forms complexes with SOCS (suppressors of cytokine signaling) proteins, which bind to phosphorylated Dab1 and target it for degradation in tissue culture cells. Ablation of Cul5 in migrating neurons causes an accumulation of active Dab1 protein and a unique cortical layering defect, characterized by excess migration and buildup of neurons at the top of the cortical plate. The results implicate Cul5 and SOCS proteins in down-regulation of Dab1 in vivo and show that Cul5 plays an essential role in regulating neuron migrations during cortical development, possibly by opposing a promigratory effect of Dab1 (Feng, 2007).

Reelin signaling specifies the molecular identity of the pyramidal neuron distal dendritic compartment

The apical dendrites of many neurons contain proximal and distal compartments that receive synaptic inputs from different brain regions. These compartments also contain distinct complements of ion channels that enable the differential processing of their respective synaptic inputs, making them functionally distinct. At present, the molecular mechanisms that specify dendritic compartments are not well understood. This study reports that the extracellular matrix protein Reelin, acting through its downstream, intracellular Dab1 and Src family (see Drosophila Src64B) tyrosine kinase signaling cascade, is essential for establishing and maintaining the molecular identity of the distal dendritic compartment of cortical pyramidal neurons. Reelin signaling is required for the striking enrichment of HCN1 and GIRK1 channels in the distal tuft dendrites of both hippocampal CA1 and neocortical layer 5 pyramidal neurons, where the channels actively filter inputs targeted to these dendritic domains (Siegelbaum, 2014).

Rbx2 regulates neuronal migration through different cullin 5-RING ligase adaptors

Morphogenesis requires the proper migration and positioning of different cell types in the embryo. Much more is known about how cells start and guide their migrations than about how they stop when they reach their destinations. This study provides evidence that Rbx2, a subunit of the Cullin 5-RING E3 ubiquitin ligase (CRL5) complex, stops neocortical projection neurons at their target layers. Rbx2 mutation causes neocortical and cerebellar ectopias dependent on Dab1, a key signaling protein in the Reelin pathway. SOCS7, a CRL5 substrate adaptor protein, is also required for neocortical layering. SOCS7-CRL5 complexes stimulate the ubiquitylation and turnover of Dab1. SOCS7 is upregulated during projection neuron migration, and unscheduled SOCS7 expression stops migration prematurely. Cerebellar development requires Rbx2 but not SOCS7, pointing to the importance of other CRL5 adaptors. These results suggest that CRL5 adaptor expression is spatiotemporally regulated to modulate Reelin signaling and ensure normal neuron positioning in the developing brain (Simo, 2013).

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