Bianka Brunne

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.

Origin, maturation, and astroglial transformation of secondary radial glial cells in the developing dentate gyrus

The dentate gyrus is a brain region where neurons are continuously born throughout life. In the adult, the role of its radial glia in neurogenesis has attracted much attention over the past years; however, little is known about the generation and differentiation of glial cells and their relationship to radial glia during the ontogenetic development of this brain structure. Here, we combine immunohistochemical phenotyping using antibodies against glial marker proteins with BrdU birthdating to characterize the development of the secondary radial glial scaffold in the dentate gyrus and its potential to differentiate into astrocytes. We demonstrate that the expression of brain lipid‐binding protein, GLAST, and glial fibrillary acidic protein (GFAP) characterizes immature differentiating cells confined to an astrocytic fate in the early postnatal dentate gyrus. On the basis of our studies, we propose a model where immature astrocytes migrate radially through the granule cell layer to adopt their final positions in the molecular layer of the dentate gyrus. Time‐lapse imaging of acute hippocampal slices from hGFAP‐eGFP transgenic mice provides direct evidence for such a migration mode of differentiating astroglial cells in the developing dentate gyrus.

Role for Reelin in Neurotransmitter Release

The extracellular matrix molecule Reelin is known to control neuronal migration during development. Recent evidence suggests that it also plays a role in the maturation of postsynaptic dendrites and spines as well as in synaptic plasticity. Here, we aimed to address the question whether Reelin plays a role in presynaptic structural organization and function. Quantitative electron microscopic analysis of the number of presynaptic boutons in the stratum radiatum of hippocampal region CA1 did not reveal differences between wild-type animals and Reelin-deficient reeler mutant mice. However, additional detailed analysis showed that the number of presynaptic vesicles was significantly increased in CA1 synapses of reeler mutants. To test the hypothesis that vesicle fusion is altered in reeler, we studied proteins known to control transmitter release. SNAP25, a protein of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, was found to be significantly reduced in reeler mutants, whereas other SNARE complex proteins remained unaltered. Addition of recombinant Reelin to organotypic slice cultures of reeler hippocampi substantially rescued not only SNAP25 protein expression levels but also the number of vesicles per bouton area indicating a role for Reelin in presynaptic functions. Next, we analyzed paired-pulse facilitation, a presynaptic mechanism associated with transmitter release, and observed a significant decrease at CA1 synapses of reeler mutants when compared with wild-type animals. Together, these novel findings suggest a role for Reelin in modulating presynaptic release mechanisms.

Rescue of the reeler phenotype in the dentate gyrus by wild‐type coculture is mediated by lipoprotein receptors for reelin and disabled 1

Reelin is a positional signal for the lamination of the dentate gyrus. In the reeler mutant lacking Reelin, granule cells are scattered all over the dentate gyrus. We have recently shown that the reeler phenotype of the dentate gyrus can be rescued in vitro by coculturing reeler hippocampal slices with slices from wild‐type hippocampus. Here we studied whether Reelin from other brain regions can similarly induce this rescue effect and whether it is mediated via the Reelin receptors apolipoprotein E receptor 2 (ApoER2) and very‐low‐density lipoprotein receptor (VLDLR). We found that coculturing reeler hippocampal slices with slices from wild‐type olfactory bulb, cerebellum, and neocortex rescued the reeler phenotype as seen before with hippocampal slices, provided that the Reelin‐synthesizing cells of these regions were placed near the marginal zone of the reeler hippocampal slice. However, coculturing wild‐type hippocampal slices with hippocampal slices from mutants deficient in ApoER2 and VLDLR did not rescue the reeler‐like phenotype in these cultures. Similarly, no rescue of the reeler‐like phenotype was observed in slices from mutants lacking Disabled 1 (Dab1), an adapter protein downstream of Reelin receptors. Conversely, reeler hippocampal slices were rescued by coculturing them with slices from Dab1−/− mutants or ApoER2−/−/VLDLR−/− mice. These findings show that Reelin from other brain regions can substitute for the loss of hippocampal Reelin and that rescue of the reeler phenotype observed in our coculture studies is mediated via lipoprotein receptors for Reelin and Dab1. J. Comp. Neurol. 495:1–9, 2006.

Role of the postnatal radial glial scaffold for the development of the dentate gyrus as revealed by reelin signaling mutant mice

During dentate gyrus development, the early embryonic radial glial scaffold is replaced by a secondary glial scaffold around birth. In contrast to neocortical and early dentate gyrus radial glial cells, these postnatal glial cells are severely altered with regard to position and morphology in reeler mice lacking the secreted protein Reelin. In this study, we focus on the functional impact of these defects. Most radial glial cells throughout the nervous system serve as scaffolds for migrating neurons and precursor cells for both neurogenesis and gliogenesis. Precursor cell function has been demonstrated for secondary radial glial cells but the exact function of these late glial cells in granule cell migration and positioning is not clear. No data exist concerning the interplay between granule neurons and late radial glial cells during dentate gyrus development. Herein, we show that despite the severe morphological defects in the reeler dentate gyrus, the precursor function of secondary radial glial cells is not impaired during development in reeler mice. In addition, selective ablation of Disabled‐1, an intracellular adaptor protein essential for Reelin signaling, in neurons but not in glial cells allowed us to distinguish effects of Reelin signaling on radial glial cells from possible secondary effects based on defective granule cells positioning. GLIA 2013;61:1347–1363

Reelin together with ApoER2 regulates interneuron migration in the olfactory bulb.

One pathway regulating the migration of neurons during development of the mammalian cortex involves the extracellular matrix protein Reelin. Reelin and components of its signaling cascade, the lipoprotein receptors ApoER2 and Vldlr and the intracellular adapter protein Dab1 are pivotal for a correct layer formation during corticogenesis. The olfactory bulb (OB) as a phylogenetically old cortical region is known to be a prominent site of Reelin expression. Although some aspects of Reelin function in the OB have been described, the influence of Reelin on OB layer formation has so far been poorly analyzed. Here we studied animals deficient for either Reelin, Vldlr, ApoER2 or Dab1 as well as double-null mutants. We performed organotypic migration assays, immunohistochemical marker analysis and BrdU incorporation studies to elucidate roles for the different components of the Reelin signaling cascade in OB neuroblast migration and layer formation. We identified ApoER2 as being the main receptor responsible for Reelin mediated detachment of neuroblasts and correct migration of early generated interneurons within the OB, a prerequisite for correct OB lamination.

Role for Reelin‐induced cofilin phosphorylation in the assembly of sympathetic preganglionic neurons in the murine intermediolateral column

Sympathetic preganglionic neurons (SPNs) are located in the intermediolateral column (IMLC) of the spinal cord. This specific localization results from primary and secondary migratory processes during spinal cord development. Thus, following neurogenesis in the neuroepithelium, SPNs migrate first in a ventrolateral direction and then, in a secondary step, dorsolaterally to reach the IMLC. These migratory processes are controlled, at least in part, by the glycoprotein Reelin, which is known to be important for the development of laminated brain structures. In reeler mutants deficient in Reelin, SPNs initially migrate ventrolaterally as normal. However, most of them then migrate medially to become eventually located near the central canal. Here, we provide evidence that in wild‐type animals this aberrant medial migration towards the central canal is prevented by Reelin‐induced cytoskeletal stabilization, brought about by phosphorylation of cofilin. Cofilin plays an important role in actin depolymerization, a process required for the changes in cell shape during migration. Phosphorylation of cofilin renders it unable to depolymerize F‐actin, thereby stabilizing the cytoskeleton. Using immunostaining for phosphorylated cofilin (p‐cofilin), we demonstrate that SPNs in wild‐type animals, but not in reeler mutants and other mutants of the Reelin signalling cascade, are immunoreactive for p‐cofilin. These findings suggest that Reelin near the central canal induces cofilin phosphorylation in SPNs, thereby preventing them from aberrant migration towards the central canal. The results extend our previous studies on cortical neurons in which Reelin in the marginal zone was found to stabilize the leading processes of migrating neurons and terminate the migration process.

Proteolytic cleavage of transmembrane cell adhesion molecule L1 by extracellular matrix molecule Reelin is important for mouse brain development

The cell adhesion molecule L1 and the extracellular matrix protein Reelin play crucial roles in the developing nervous system. Reelin is known to activate signalling cascades regulating neuronal migration by binding to lipoprotein receptors. However, the interaction of Reelin with adhesion molecules, such as L1, has remained poorly explored. Here, we report that full-length Reelin and its N-terminal fragments N-R2 and N-R6 bind to L1 and that full-length Reelin and its N-terminal fragment N-R6 proteolytically cleave L1 to generate an L1 fragment with a molecular mass of 80 kDa (L1-80). Expression of N-R6 and generation of L1-80 coincide in time at early developmental stages of the cerebral cortex. Reelin-mediated generation of L1-80 is involved in neurite outgrowth and in stimulation of migration of cultured cortical and cerebellar neurons. Morphological abnormalities in layer formation of the cerebral cortex of L1-deficient mice partially overlap with those of Reelin-deficient reeler mice. In utero electroporation of L1-80 into reeler embryos normalised the migration of cortical neurons in reeler embryos. The combined results indicate that the direct interaction between L1 and Reelin as well as the Reelin-mediated generation of L1-80 contribute to brain development at early developmental stages.

Trajectory analysis unveils Reelin's role in the directed migration of granule cells in the dentate gyrus

Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler, but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP-expressing dentate granule cells in slice cultures from reeler, reeler-like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler-like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer. SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the present study, we took advantage of the expression of proopiomelanocortin-EGFP by newly generated, migrating granule cells to analyze their migratory trajectories in hippocampal slice cultures from wild-type mice and mutants deficient in Reelin signaling. We show that the compartmentalized presence of Reelin is essential for the directionality, but not the actual migratory process or speed, of migrating granule cells leading to their characteristic lamination in the dentate gyrus.