Rbx2 regulates neuronal migration through different cullin 5-RING ligase adaptors.
ABSTRACT: 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. Here we provide 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. Our results suggest that CRL5 adaptor expression is spatiotemporally regulated to modulate Reelin signaling and ensure normal neuron positioning in the developing brain.
Project description:The laminated structure of the retina is fundamental for the organization of the synaptic circuitry that translates light input into patterns of action potentials. However, the molecular mechanisms underlying cell migration and layering of the retina are poorly understood. Here, we show that RBX2, a core component of the E3 ubiquitin ligase CRL5, is essential for retinal layering and function. RBX2 regulates the final cell position of rod bipolar cells, cone photoreceptors and Muller glia. Our data indicate that sustained RELN/DAB1 signaling, triggered by depletion of RBX2 or SOCS7 - a CRL5 substrate adaptor known to recruit DAB1 - causes rod bipolar cell misposition. Moreover, whereas SOCS7 also controls Muller glia cell lamination, it is not responsible for cone photoreceptor positioning, suggesting that RBX2, most likely through CRL5 activity, controls other signaling pathways required for proper cone localization. Furthermore, RBX2 depletion reduces the number of ribbon synapses and disrupts cone photoreceptor function. Together, these results uncover RBX2 as a crucial molecular regulator of retina morphogenesis and cone photoreceptor function.
Project description:The dentate gyrus (DG) is an essential part of the hippocampal formation and participates in the majority of hippocampal functions. The DG is also one of the few structures in the mammalian central nervous system that produces adult-born neurons and, in humans, alterations in adult neurogenesis are associated with stress and depression. Given the importance of DG in hippocampal function, it is imperative to understand the molecular mechanisms driving DG development and homeostasis. The E3 ubiquitin ligase Cullin-5/RBX2 (CRL5) is a multiprotein complex involved in neuron migration and localization in the nervous system, but its role during development and in the adult DG remain elusive. Here, we show that CRL5 participates in mossy fiber pruning, DG layering, adult neurogenesis, and overall physical activity in mice. During DG development, RBX2 depletion causes an overextension of the DG mossy fiber infrapyramidal bundle (IPB). We further demonstrate that the increased activity in Reelin/DAB1 or ARF6 signaling, observed in RBX2 knockout mice, is not responsible for the lack of IPB pruning. Knocking out RBX2 also affects granule cell and neural progenitor localization and these defects were rescued by downregulating the Reelin/DAB1 signaling. Finally, we show that absence of RBX2 increases the number neural progenitors and adult neurogenesis. Importantly, RBX2 knockout mice exhibit higher levels of physical activity, uncovering a potential mechanism responsible for the increased adult neurogenesis in the RBX2 mutant DG. Overall, we present evidence of CRL5 regulating mossy fiber pruning and layering during development and opposing adult neurogenesis in the adult DG.
Project description:Neuronal migration is critical for establishing neocortical cell layers and migration defects can cause neurological and psychiatric diseases. Recent studies show that radially migrating neocortical neurons use glia-dependent and glia-independent modes of migration, but the signaling pathways that control different migration modes and the transitions between them are poorly defined. Here, we show that Dab1, an essential component of the reelin pathway, is required in radially migrating neurons for glia-independent somal translocation, but not for glia-guided locomotion. During migration, Dab1 acts in translocating neurons to stabilize their leading processes in a Rap1-dependent manner. Rap1, in turn, controls cadherin function to regulate somal translocation. Furthermore, cell-autonomous neuronal deficits in somal translocation are sufficient to cause severe neocortical lamination defects. Thus, we define the cellular mechanism of reelin function during radial migration, elucidate the molecular pathway downstream of Dab1 during somal translocation, and establish the importance of glia-independent motility in neocortical development.
Project description:The Reelin signaling pathway plays a crucial role in regulating neocortical development. However, little is known about how Reelin controls the cytoskeleton during neuronal migration. Here, we identify CLASP2 as a key cytoskeletal effector in the Reelin signaling pathway. We demonstrate that CLASP2 has distinct roles during neocortical development regulating neuron production and controlling neuron migration, polarity, and morphogenesis. We found downregulation of CLASP2 in migrating neurons leads to mislocalized cells in deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. We discovered that Reelin regulates several phosphorylation sites within the positively charged serine/arginine-rich region that constitute consensus GSK3? phosphorylation motifs of CLASP2. Furthermore, phosphorylation of CLASP2 regulates its interaction with the Reelin adaptor Dab1 and this association is required for CLASP2 effects on neurite extension and motility. Together, our data reveal that CLASP2 is an essential Reelin effector orchestrating cytoskeleton dynamics during brain development.
Project description:Reelin controls neuronal positioning in the developing brain by binding to the two lipoprotein receptors, very-low-density lipoprotein receptor and apolipoprotein E receptor 2, to stimulate phosphorylation of Disabled-1 (Dab1) by the Fyn and Src tyrosine kinases. Crk and Crk-like (CrkL) have been proposed to interact with tyrosine phosphorylated Dab1 to mediate downstream events in the Reelin pathway. However, these adaptor proteins are widely expressed, and they fulfill essential functions during embryonic development. To address their specific roles in Reelin-mediated neuronal migration, we generated mutant mice, by Cre-loxP recombination, lacking Crk and CrkL in most neurons. These animals displayed the major anatomic features of reeler including, cerebellar hypofoliation, failure of Purkinje cell migration, absence of preplate splitting, impaired dendritic development, and disruption of layer formation in the hippocampus and cerebral cortex. However, proximal signaling involving tyrosine phosphorylation and turnover of Dab1 occurred normally in the mutant mouse brain and in primary cortical neurons treated with Reelin. In contrast, two downstream signaling events, Reelin-induced phosphorylation of C3G and Akt, were not observed in the absence of Crk and CrkL in mouse embryonic cortical neurons. These findings place C3G and Akt phosphorylation downstream of Crk and CrkL, which play essential overlapping functions in the Reelin signaling pathway.
Project description:The Reelin ligand regulates a Dab1-dependent signaling pathway required for brain lamination and normal dendritogenesis, but the specific mechanisms underlying these actions remain unclear. We find that Stk25, a modifier of Reelin-Dab1 signaling, regulates Golgi morphology and neuronal polarization as part of an LKB1-Stk25-Golgi matrix protein 130 (GM130) signaling pathway. Overexpression of Stk25 induces Golgi condensation and multiple axons, both of which are rescued by Reelin treatment. Reelin stimulation of cultured neurons induces the extension of the Golgi into dendrites, which is suppressed by Stk25 overexpression. In vivo, Reelin and Dab1 are required for the normal extension of the Golgi apparatus into the apical dendrites of hippocampal and neocortical pyramidal neurons. This demonstrates that the balance between Reelin-Dab1 signaling and LKB1-Stk25-GM130 regulates Golgi dispersion, axon specification, and dendrite growth and provides insights into the importance of the Golgi apparatus for cell polarization.
Project description:Reelin and its receptor machinery are well known to be required for the migration and positioning of neocortical projection neurons. More recently, reelin has been shown both necessary and sufficient to determine the rate of neocortical neurogenesis. The molecular links underlying its seemingly distinct proliferative and post-proliferative functions remain unknown. Here we reveal an enriched expression of functional reelin receptors, largely of Apolipoprotein E Receptor 2 (ApoER2), in radial glia basal processes and intermediate progenitor cells during mid/late cortical development. In vivo, ApoER2 overexpression inhibits neuronal migration. In contrast, precluding excessive levels of ApoER2 in reelin-deficient cortices, by either ApoER2 knock-down or the transgenic expression of reelin in neural progenitor cells, improves neuronal migration and positioning. Our study provides groundwork for the highly orchestrated clearance of neocortical neurons from their birth site, suggesting that a reelin-dependent ApoER2 downregulation mechanism uncouples newborn neurons from progenitor cells, thereby enabling neurons to migrate.
Project description:The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.
Project description:The Reelin-Disabled 1 (Dab1) signaling pathway plays an important role in neuronal cell migration during brain development. Dab1, an intracellular adapter protein which is tyrosine phosphorylated upon Reelin stimulation, has been directly implicated in the transmission and termination of Reelin-mediated signaling. Two main forms of Dab1 have been identified in the developing chick retina, an early isoform (Dab1-E) expressed in progenitor cells and a late isoform (Dab1-L, a.k.a. Dab1) expressed in differentiated cells. Dab1-E is missing two Src family kinase (SFK) phosphorylation sites that are critical for Reelin-Dab1 signaling and is not tyrosine phosphorylated. We have recently demonstrated a role for Dab1-E in the maintenance of retinal progenitor cells. Here, we report that Dab1-E is phosphorylated at serine/threonine residues independent of Reelin. Cdk2, highly expressed in retinal progenitor cells, mediates Dab1-E phosphorylation at serine 475 which in turn promotes ubiquitination-triggered proteasome degradation of Dab1-E. Inhibition of protein phosphatase 1 and/or protein phosphatase 2A leads to increased Dab1-E instability. We propose that Dab1 turnover is regulated by both Reelin-independent serine/threonine phosphorylation and Reelin-dependent tyrosine phosphorylation.