Xuejun Chai

Reelin Stabilizes the Actin Cytoskeleton of Neuronal Processes by Inducing n-Cofilin Phosphorylation at Serine3

The extracellular matrix protein Reelin, secreted by Cajal-Retzius cells in the marginal zone of the cortex, controls the radial migration of cortical neurons. Reelin signaling involves the lipoprotein receptors apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR), the adapter protein Disabled1 (Dab1), and phosphatidylinositol-3-kinase (PI3K). Eventually, Reelin signaling acts on the cytoskeleton; however, these effects on cytoskeletal organization have remained elusive. In Reelin-deficient mutant mice, most cortical neurons are unable to migrate to their destinations, suggesting a role for Reelin signaling in the dynamic cytoskeletal reorganization that is required for neurons to migrate. Here, we show that Reelin signaling leads to serine3 phosphorylation of n-cofilin, an actin-depolymerizing protein that promotes the disassembly of F-actin. Phosphorylation at serine3 renders n-cofilin unable to depolymerize F-actin, thereby stabilizing the cytoskeleton. We provide evidence for ApoER2, Dab1, Src family kinases (SFKs), and PI3K to be involved in n-cofilin serine3 phosphorylation. Phosphorylation of n-cofilin takes place in the leading processes of migrating neurons as they approach the Reelin-containing marginal zone. Immunostaining for phospho-cofilin in dissociated reeler neurons is significantly increased after incubation in Reelin-containing medium compared with control medium. In a stripe choice assay, neuronal processes are stable on Reelin-coated stripes but grow on control stripes by forming lamellipodia. These novel findings suggest that Reelin-induced stabilization of neuronal processes anchors them to the marginal zone which appears to be required for the directional migration process.

Reelin is a positional signal for the lamination of dentate granule cells.

Reelin is required for the proper positioning of neurons in the cerebral cortex. In the reeler mutant lacking reelin, the granule cells of the dentate gyrus fail to form a regular, densely packed cell layer. Recent evidence suggests that this defect is due to the malformation of radial glial processes required for granule cell migration. Here, we show that recombinant reelin in the medium significantly increases the length of GFAP-positive radial glial fibers in slice cultures of reeler hippocampus, but does not rescue either radial glial fiber orientation or granule cell lamination. However, rescue of radial glial fiber orientation and granule cell lamination was achieved when reelin was present in the normotopic position provided by wild-type co-culture, an effect that is blocked by the CR-50 antibody against reelin. These results indicate a dual function of reelin in the dentate gyrus, as a differentiation factor for radial glial cells and as a positional cue for radial fiber orientation and granule cell migration.

Recent progress in understanding the role of Reelin in radial neuronal migration, with specific emphasis on the dentate gyrus

Ten years following identification of Reelin as the product of the gene mutated in reeler mice, the signalling pathway activated by Reelin is being progressively unravelled with the identification of lipoprotein receptors as reelin receptors, of the Dab1 adapter and of some other proximal components in target cells. However, we are still a long way from understanding the action of this complex protein during brain development and maturation. The present review is organized in two parts. First, we summarize our present understanding of Reelin signalling. Then, we review critically some cell biological mechanisms for the action of Reelin based on recent studies on the development of the dentate gyrus, which has proved an extremely useful and tractable model system.

Emerging topics in Reelin function

Reelin signalling in the early developing cortex regulates radial migration of cortical neurons. Later in development, Reelin promotes maturation of dendrites and dendritic spines. Finally, in the mature brain, it is involved in modulating synaptic function. In recent years, efforts to identify downstream signalling events induced by binding of Reelin to lipoprotein receptors led to the characterization of novel components of the Reelin signalling cascade. In the present review, we first address distinct functions of the Reelin receptors Apoer2 and Vldlr in cortical layer formation, followed by a discussion on the recently identified downstream effector molecule n‐cofilin, involved in regulating actin cytoskeletal dynamics required for coordinated neuronal migration. Next, we discuss possible functions of the recently identified Reelin–Notch signalling crosstalk, and new aspects of the role of Reelin in the formation of the dentate radial glial scaffold. Finally, progress in characterizing the function of Reelin in modulating synaptic function in the adult brain is summarized. The present review has been inspired by a session entitled ‘Functions of Reelin in the developing and adult hippocampus’, held at the Spring Hippocampal Research Conference in Verona/Italy, June 2009.

Balance between Neurogenesis and Gliogenesis in the Adult Hippocampus: Role for Reelin

The extracellular matrix protein reelin is essential for the proper radial migration of cortical neurons. In reeler mice lacking reelin, there is a malformation of the radial glial scaffold required for granule cell migration. Immunostaining for glial fibrillary acidic protein (GFAP) reveals abundant radial glial cells with long fibers traversing the granular layer in the wild type, but almost exclusively astrocytes in the reeler mutant. With the concept that radial glial cells are precursors of neurons, we hypothesized that the balance between neurogenesis and gliogenesis is altered in the reeler mutant. To this end, adult reeler mutants and their wild-type littermates were injected with bromodeoxyuridine (BrdU), a marker of newly generated cells. When compared to wild-type animals, we found a reduction in the number of BrdU-labeled cells in the adult reeler dentate gyrus. Moreover, whereas there was a dramatic decrease in the number of newly generated granule cells identified by double labeling for BrdU and NeuN, the number of BrdU-labeled, GFAP-positive astrocytes had increased. Decreased neurogenesis in the adult reeler dentate gyrus was confirmed by immunostaining for doublecortin, a marker of newly generated neurons. These results indicate that adult neurogenesis is altered in the reeler dentate gyrus and that newly generated cells preferentially differentiate into astrocytes.

Role of Reelin in the development and maintenance of cortical lamination

Reelin is a large extracellular matrix molecule, synthesized by early generated Cajal–Retzius cells in the marginal zone of the cortex. It plays an important role in the migration of cortical neurons and the development of cortical lamination. We recently discovered that Reelin is required not only for the formation of cortical layers during development but also for their maintenance in adulthood. Thus, decreased Reelin expression in a mouse model of epilepsy and in epileptic patients was accompanied by a loss of granule cell lamination, called granule cell dispersion, in the dentate gyrus of the hippocampal formation. Moreover, antibody blockade of Reelin in normal, adult mice resulted in granule cell dispersion. Collectively these findings point to a role for Reelin in the formation and maintenance of a laminated cortical structure. How does Reelin act on the cytoskeleton in the migration process of cortical neurons? It has been shown that Reelin signalling involves the lipoprotein receptors apolipoprotein E receptor 2 and very low density lipoprotein receptor, the adapter protein Disabled1, and phosphatidylinositol-3-kinase, but it has remained unclear how activation of the Reelin signalling cascade controls cytoskeletal reorganization. Here, we provide evidence that Reelin signalling leads to serine3 phosphorylation of cofilin, an actin-depolymerizing protein that promotes the disassembly of F-actin. Phosphorylation at serine3 renders cofilin unable to depolymerize F-actin, thereby stabilizing the cytoskeleton. Phosphorylation of cofilin in the leading processes of migrating neurons anchors them to the marginal zone containing Reelin. Our results indicate that Reelin-induced stabilization of the neuronal cytoskeleton is an important component of Reelin’s function in the development and maintenance of cortical architecture.

Structural plasticity of hippocampal mossy fiber synapses as revealed by high-pressure freezing

Despite recent progress in fluorescence microscopy techniques, electron microscopy (EM) is still superior in the simultaneous analysis of all tissue components at high resolution. However, it is unclear to what extent conventional fixation for EM using aldehydes results in tissue alteration. Here we made an attempt to minimize tissue alteration by using rapid high‐pressure freezing (HPF) of hippocampal slice cultures. We used this approach to monitor fine‐structural changes at hippocampal mossy fiber synapses associated with chemically induced long‐term potentiation (LTP). Synaptic plasticity in LTP has been known to involve structural changes at synapses including reorganization of the actin cytoskeleton and de novo formation of spines. While LTP‐induced formation and growth of postsynaptic spines have been reported, little is known about associated structural changes in presynaptic boutons. Mossy fiber synapses are assumed to exhibit presynaptic LTP expression and are easily identified by EM. In slice cultures from wildtype mice, we found that chemical LTP increased the length of the presynaptic membrane of mossy fiber boutons, associated with a de novo formation of small spines and an increase in the number of active zones. Of note, these changes were not observed in slice cultures from Munc13‐1 knockout mutants exhibiting defective vesicle priming. These findings show that activation of hippocampal mossy fibers induces pre‐ and postsynaptic structural changes at mossy fiber synapses that can be monitored by EM. J. Comp. Neurol. 520:2340–2351, 2012.

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.

Reelin Induces Branching of Neurons and Radial Glial Cells during Corticogenesis

Newborn neurons migrate along the processes of radial glial cells (RGCs) to reach their final positions in the cortex. Here, we visualized individual migrating neurons and RGCs using in utero electroporation. We show that branching of migrating neurons and RGCs is closely correlated spatiotemporally with the distribution of Reelin. Time-lapse imaging revealed that the leading processes of migrating neurons gave rise to increasingly more branches once their growth cones contacted the Reelin-containing marginal zone. This was accompanied by translocation of the nucleus and gradual shortening of the leading process. Absence of Reelin in reeler mice altered these processes resulting in misorientation, loss of bipolarity, and aberrant migration of cortical neurons. Moreover, in reeler, the branching of the basal processes of RGCs in the marginal zone was severely disrupted. Consistent with previous reports, we show that in dissociated reeler cortical cultures, exposure to recombinant Reelin enhanced dendritic complexity and glial branching. Our results suggest that Reelin induces branching of the leading processes of migrating neurons and that of basal processes of RGCs when they arrive at the Reelin-containing marginal zone. Branching of these processes may be crucial for the termination of nuclear translocation during the migratory process and for correct neuronal positioning.

Reelin acts as a stop signal for radially migrating neurons by inducing phosphorylation of n-cofilin at the leading edge

The extracellular matrix protein Reelin, secreted by Cajal-Retzius (CR) cells in the marginal zone (MZ) of the cerebral cortex, is important for neuronal migration during development. Two lipoprotein receptors for Reelin have been identified, apolipoprotein E receptor 2 (ApoER2) and the very low density lipoprotein receptor (VLDLR). The binding of Reelin to these receptors induces tyrosine phosphorylation of an adapter protein, disabled 1 (Dab1) by src family kinases (SFKs). In the Reelin-deficient mutant reeler, cortical lamination is inverted with many neurons invading the marginal zone and others that are unable to migrate to their destinations and accumulate underneath their predecessors, suggesting a role for Reelin signaling in dynamic cytoskeletal reorganization. At present these effects of Reelin are poorly understood. In our recent study, we showed that Reelin induces serine3 phosphorylation of n-cofilin, an actin-depolymerizing protein promoting the disassembly of F-actin. Phosphorylation of cofilin renders it unable to depolymerize F-actin, thus stabilizing the cytoskeleton. We provided evidence for ApoER2, Dab1, SFKs, and phosphatidylinositol-3-kinase (PI3K) to be involved in Reelin-induced cofilin phosphorylation. We found that phosphorylation of cofilin occurs in the leading processes of radially migrating neurons as they grow towards the Reelin-containing marginal zone. By cofilin phophorylation, Reelin may act as a stop signal for radially migrating neurons.