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.     

Changes in reelin expression in the mouse olfactory bulb after chemical lesion to the olfactory epithelium

To explore the functional roles of Reelin in the adult olfactory system, we examined changes in the expression of reelin mRNA and Reelin protein in the olfactory bulb (OB) of adult mice after a chemical lesion to the olfactory epithelium. Following intranasal irrigation with 2% zinc sulphate solution, animals were perfused at various times between 5 and 40 days post-lesion. The expression of reelin mRNA in mitral cells in the OB was slightly reduced at 5 days post-lesion, completely abolished by 20 days, but restored almost to the normal level at 40 days post-lesion. Similarly, the expression of Reelin protein in mitral cells of the deafferented OB also recovered, although not to the normal level. No recovery of either reelin mRNA or Reelin immunoreactivity was seen in the periglomerular cells and external tufted cells. The expression profile of reelin mRNA and Reelin protein in the OB coincided with the time course of degeneration and regeneration of olfactory nerves, as indicated by anterograde labeling of olfactory nerves with WGA-HRP. These results suggest that expression of reelin mRNA in the adult OB is regulated by olfactory inputs.     

Lack of Reelin causes malpositioning of nigral dopaminergic neurons: evidence from comparison of normal and Reln(rl) mutant mice

The reeler gene (Reln(rl), formerly rl) product Reelin controls neuronal migration and positioning and thereby plays a key role in brain development. Mutation of Reln leads to widespread disruption of laminar cortical regions and ectopia in some brainstem nuclei. In the embryonic striatum of normal mice, a substantial expression of reelin mRNA has been documented; however, the anomalous positioning of neurons in the basal ganglia of reeler mice remains to be studied. We provide first evidence for a potential role of Reelin in the developmental formation of the substantia nigra. In reeler mutant mice lacking Reelin, dopaminergic neurons destined for the substantia nigra fail to migrate laterally and become anomalously clustered just lateral to the ventral tegmental area. Their axons appear to project to striatal patches forming "dopamine islands." Results from the normal mice show that, at the midembryonic stage, Reelin identified with CR-50 is highly concentrated in the ventral mesencephalon, where nigral dopaminergic neurons are in progress to migrate laterally to their eventual position of the adult brain. A combination of CR-50 labeling and anterograde axonal tracing provided evidence that embryonic striatal neurons may supply the ventral portion of the mesencephalon with Reelin through their axonal projections. We hypothesize that Reelin plays a role in the positioning of nigral dopaminergic neurons and that it can act as an environmental cue at a remote site far from its birthplace via a transaxonal delivery system.     

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 and the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promote dendritic Golgi translocation in hippocampal neurons

In the cerebral cortex of reeler mutant mice lacking reelin expression, neurons are malpositioned and display misoriented apical dendrites. Neuronal migration defects in reeler have been studied in great detail, but how misorientation of apical dendrites is related to reelin deficiency is poorly understood. In wild‐type mice, the Golgi apparatus transiently translocates into the developing apical dendrite of radially migrating neurons. This dendritic Golgi translocation has recently been shown to be promoted by reelin. However, the underlying signalling mechanisms are largely unknown. Here, we show that the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promoted translocation of Golgi cisternae into developing dendrites of hippocampal neurons. Reelin treatment further increased the αPIX‐dependent effect. In turn, overexpression of exchange activity‐deficient αPIX or dominant‐negative (dn) Cdc42 or dn‐Rac1 impaired dendritic Golgi positioning, an effect that was not compensated by reelin treatment. Together, these data suggest that αPIX may promote dendritic Golgi translocation, as a downstream component of a reelin‐modulated signalling pathway. Finally, we found that reelin promoted the translocation of the Golgi apparatus into the dendrite that was most proximal to the reelin source. The distribution of reelin may thus contribute to the selection of the process that becomes the apical dendrite.     

Observations on the brainstem-spinal descending systems of normal and reeler mutant mice by the retrograde HRP method

Brainstem neurons which project to the lumbar spinal level were identified in both reeler mutant mice and normal controls (Balb/c mice) by the retrograde horseradish peroxidase (HRP) technique. In normal controls after HRP injection into the lumbar cord, retrogradely labelled neurons were observed in (1) the lateral vestibular nucleus, (2) the pontine and medullary reticular formations including the nucleus centralis caudalis pontis, nucleus gigantocellularis, nucleus paragigantocellularis, nucleus raphe magnus et pallidus, and nucleus centralis medullae oblongatae pars ventralis et dorsalis, and (3) the dorsal column nuclei, i.e., the nucleus gracilis and nucleus cuneatus medialis. In reeler mutant mice, labelled neurons were again seen in the nuclei referred to above, and their cellular type and distribution patterns within the corresponding nuclei were similar to those of the normal controls. These observations suggest that (1) the brainstem nuclei of reeler mutant mice which project to the lumbar spinal cord are cytoarchitecturally normal, (2) the reeler genetic locus (rl) does not affect the nonlaminated structures in the brainstem, at least those referred to above, and (3) the motor dysfunctions observed in the reeler, such as action tremor, dystonic posture, and reeling ataxic gait, are not attributable to the brainstem-spinal descending systems.     

Events governing organization of postmigratory neurons: Studies on brain development in normal and reeler mice

The purpose of the present work is to examine some of the mechanisms responsible for the early architectonic differentiation of the central nervous system, as well as for the abnormal development which occurs in certain hereditary malformations. In order to approach these questions, the embryonic development of the cerebral cortex, the cerebellum, the inferior olivary complex and the facial nerve nucleus has been studied in normal and reeler mutant mice, using morphological methods.The adult reeler phenotype is characterized not only by extreme laminar abnormalities of cell positioning in the telencephalic and cerebellar cortices, but also by relatively less extreme, though distinct abnormal architectonics in non-cortical structures such as the inferior olive and the facial nerve nucleus. Study of the embryonic development of these structures reveals that neurons are generated at the normal time and migrate along normal pathways. Moreover, the processes of directional axonal growth, differentiation of class specific features of neurons and glia, and synaptogenesis appear similar in both genotypes and are probably not directly affected by the reeler mutation. However, in all instances, the early architectonic organization achieved by reeler cortical, Purkinje, olivary or facial neurons at the end of their migration is consistently less regular than in normal embryos. In addition, these anomalies become amplified during the later developmental period.This evidence for the early appearance of abnormalities in reeler embryos indicates that the disposition of neurons at maturity cannot be exclusively regarded as secondary to the maturation of cells, neurites and connections, but is contingent upon a specific mechanism. One may infer that the presence of a normal allele at the reeler locus is necessary for the normal completion of this histogenetic step, which consequently is submitted to genetic control.Although the factor(s) responsible for the stable configuration of the early architectonics is unknown, various hypotheses are considered. Several lines of evidence are presented which argue against a major role being played by diffusible factors, mesodermal components and afferent fiber systems. Two mechanisms are considered particularly worth evaluating: (1) a diminution of relative adhesivity between neurons and radial glial fibers at the end of migration, and (2) a stabilization of neuronal configuration by selective recognition-adhesion among postmigratory neurons. The reeler gene could, directly or indirectly, affect these cell-cell interactions.A better definition of the mechanisms responsible for the early architectonic patterning is central to our understanding of brain development in normal as well as in pathological states.     

Reeler小鼠海马齿状回形态结构与组织化学研究

目的 分析 reelin 基因突变小鼠,即reeler小鼠海马锥体细胞和颗粒细胞的组织化学特征,了解Reelin缺乏对海马皮质发育的影响,为认识Reelin功能提供形态学证据. 方法 用免疫荧光双重标记法, 分别标记野生型和reeler 小鼠海马锥体细胞、颗粒细胞和苔藓细胞; 结果 Reeler小鼠海马皮质板发育明显障碍,表现为锥体层和颗粒层细胞扩散,海马片层状细胞构筑紊乱.颗粒层细胞明显增殖并向门区迁移,以致颗粒层与门区的界限消失,呈鼓槌状结构. 结论 Reelin作为神经细胞迁移的终止信号和细胞增殖的调控信号对神经细胞迁移、发育过程中皮质板的片层化,特别是对调节颗粒细胞的增殖有明显影响.     

Impaired reelin processing and secretion by Cajal–Retzius cells contributes to granule cell dispersion in a mouse model of temporal lobe epilepsy

Cajal–Retzius cells play a crucial role during ontogeny in regulating cortical lamination via release of reelin. In adult brain, they comprise small calretinin‐positive interneurons located in the marginal zone of the cerebral cortex and in the hippocampal fissure. Alterations of reelin signaling or expression have been involved in major neurological disorders, and they underlie granule cell dispersion (GCD) in mesial temporal lobe epilepsy (TLE). Here, we investigated in a mouse model of TLE the contribution of Cajal–Retzius cells to reelin production in epileptic hippocampus and the molecular mechanisms underlying GCD. Following unilateral intrahippocampal Kainic acid injection in adult mice to induce an epileptic focus, we observed that Cajal–Retzius cells gradually became strongly immunopositive for reelin, due to intracellular accumulation. This phenotype resembled the morphology of Cajal–Retzius cells in reeler Orleans (reln^orl/orl) mice, which express a secretion‐deficient 310‐kDa reelin fragment. The possibility that GCD might result from abnormal reelin processing in Cajal–Retzius cells, leading to a lack of reelin secretion, was confirmed by KA injection in reln^orl/+ mice, which induced severe GCD. Furthermore, Western blot analysis in KA‐treated wildtype mice revealed increased production of ～300‐kDa reelin fragments, confirming abnormal proteolytic processing. This effect was not seen upon treatment with Botulinum neurotoxin E (BoNT/E), which prevents GCD in KA‐lesioned hippocampus by chronic blockade of synaptic transmission. Furthermore, BoNT/E blocked upregulation of TrkB in Cajal–Retzius cells, suggesting that production of truncated reelin in KA‐treated hippocampus is activity‐dependent and regulated by BDNF. Altogether, these data reveal that GCD results from abnormal reelin processing in Cajal–Retzius cells under the control of BDNF. Our findings highlight the critical role played by Cajal–Retzius cells for hippocampal neuronal reorganization in TLE. © 2010 Wiley‐Liss, Inc.     

Developmental distribution of reelin-positive cells and their secreted product in the rodent spinal cord

To date, only sympathetic and parasympathetic preganglionic neurons are known to migrate abnormally in reeler mutant spinal cord. Reelin, the large extracellular matrix protein absent in reeler, is found in wild-type neurons bordering both groups of preganglionic neurons. To understand better Reelin's function in the spinal cord, we studied its developmental expression in both mice and rats. A remarkable conservation was found in the spatiotemporal pattern of Reelin in both species. Numerous Reelin-expressing cells were found in the intermediate zone, except for regions containing somatic and autonomic motor neurons. A band of Reelin-positive cells filled the superficial dorsal horn, whereas only a few immunoreactive cells populated the deep dorsal horn and dorsal commissure. High levels of diffuse Reelin product were detected in the lateral marginal and ventral ventricular zones in both rodent species. This expression pattern was detected at all segmental spinal cord levels during embryonic development and remained detectable at lower levels throughout the first postnatal month. To discriminate between the cellular and secreted forms of Reelin, brefeldin A was used to block secretion in organotypic cultures. Such perturbations revealed that the high levels of secreted Reelin in the lateral marginal zone were derived from varicose axons of more medially located Reelin-positive cells. Thus, the laterally located secreted Reelin product may normally prevent the preganglionic neurons from migrating too far medially. Based on the strong evolutionary conservation of Reelin expression and its postnatal detection, Reelin may have other important functions in addition to its role in neuronal migration.