Reelin Functions, Mechanisms of Action and Signaling Pathways During Brain Development and Maturation

During  embryonic  development  and  adulthood,  Reelin  exerts  several  important  functions  in  the  brain  including  the  regulation  of  neuronal  migration,  dendritic  growth  and  branching, dendritic spine formation, synaptogenesis and synaptic plasticity. As a consequence,  the Reelin signaling pathway has been associated with several human brain disorders such as  lissencephaly,  autism,  schizophrenia,  bipolar  disorder,  depression,  mental  retardation,  Alzheimer’s disease and epilepsy. Several elements of the signaling pathway are known. Core  components, such as the Reelin receptors very low‐density lipoprotein receptor (VLDLR) and  Apolipoprotein E receptor 2 (ApoER2), Src family kinases Src and Fyn, and the intracellular  adaptor Disabled‐1 (Dab1), are common to most but not all Reelin functions. Other downstream  effectors are, on the other hand, more specific to defined tasks. Reelin is a large extracellular  protein, and some aspects of the signal are regulated by its processing into smaller fragments.  Rather than being inhibitory, the processing at two major sites seems to be fulfilling important  physiological functions. In this review, I describe the various cellular events regulated by Reelin  and attempt to explain the current knowledge on the mechanisms of action. After discussing the  shared and distinct elements of the Reelin signaling pathway involved in neuronal migration,  dendritic growth, spine development and synaptic plasticity, I briefly outline the data revealing  the importance of Reelin in human brain disorders.

Chemistry of Reelin

Reelin is a large extracellular protein involved in several aspects of brain development, such as cell positioning, dendrite growth, synaptic plasticity, and memory, and may be implicated as a susceptibility factor in psychoses (Caviness and Rakic, 1978; Impagnatiello et al., 1998; Liu et al., 2001; Weeber et al., 2002; Jossin, 2004; Beffert et al., 2005; Fatemi, 2005). This wide array of functions indicates that Reelin is able to trigger different intracellular signaling pathways depending on the maturation state or the type of target cell that may express different receptors or intracellular signaling modules. In this chapter, I will review the current state of knowledge on the best established and some other putative partners of the Reelin pathwa

Mechanism of action of Reelin in the developing cerebral cortex

La voie de signalisation de la Reelin joue un rôle clé dans le développement du système nerveux central. Dans le cortex, où la majorité des neurones migrent radialement vers la plaque corticale, la Reelin est secrétée par les cellules de Cajal-Retzius de la zone marginale et se lie à deux récepteurs, VLDLR et ApoER2, exprimés par les neurones en migration. Ceci induit la phosphorylation de résidus tyrosine de la protéine adaptatrice Dab1. Après sécrétion, la Reelin est clivée en trois fragments par une métalloprotéinase, mais la fonction de ce clivage est inconnue. En plus de son action sur le développement architectonique, certains travaux suggèrent que la Reelin influence la guidance axonale, puisque les souris reeler déficientes en Reelin accusent un retard des connexions afférentes dans l'hippocampe. Dans ce travail, nous avons tout d'abord étudié l'action de la Reelin sur la croissance axonale. Nous avons montré que ni la Reelin clivée, ni la Reelin entière n'ont d'effet attractif ou répulsif significatif. Ces résultats suggèrent que les effets publiés de la Reelin sur la guidance axonale sont indirects, secondaires aux perturbations architectoniques qui résultent de la déficience en Reelin, et que l'effet des cellules de Cajal-Retzius sur la connectivité est indépendant de la Reelin. La deuxième partie du travail est concentrée sur l'activité des différents produits de clivage de la Reelin. Nous avons montré que le fragment central est nécessaire et suffisant pour la liaison aux récepteurs et pour l'activation du signal, reflété par l'induction de la phosphorylation de Dab1, et que ce même fragment corrige partiellement le phénotype reeler dans un système organotypique original de culture de tranches de cerveau embryonnaire. La correction du phénotype n'est pas observée par des anticorps stimulants dirigés contre VLDLR et anti-ApoER2, ce qui indique que la partie du signal découlant de la phosphorylation de Dab1 ne suffit pas à traduire tout les effets de la Reelin. La dernière partie du travail a consisté à investiguer le rôle de quelques grandes voies de signalisation intracellulaire sur des cultures de tranches de cerveaux embryonnaires, à l'aide d'inhibiteurs de petit poids moléculaire. L'inhibition de la famille des Src kinases, des PKCs, de PI3K, d'Akt ou de mTor induit un phenotype semblable à reeler: perturbation du positionnement des neurones corticaux, inversion du gradient de maturation de « dedans-en-dehors » de ces cellules et d'une absence de la division de la préplaque. Ces expériences ont démontré que les Src kinases sont bien responsables de la phosphorylation de Dab1, et que les autres kinases (PKCs, PI3K, Akt et mTor) agissent en aval ou en parallèle de Dab1.The Reelin signaling pathway plays a key role in the architectonic development of the central nervous system. In the cortex, where a majority of neurons migrate radially to form the cortical plate, Reelin is secreted by Cajal-Retzius cells, early-born neurons in the marginal zone. Reelin binds to two receptors of the lipoprotein receptor family, VLDLR and ApoER2, expressed on migrating neurons, and induces tyrosine phosphorylation of the adaptor Dab1. After secretion, Reelin is cleaved into three fragments by a metalloproteinase but the function of processing is unknown. In addition to its action on architectonic development, some studies suggest that Reelin may influence axonal guidance, as reeler mice lacking Reelin show a delayed growth of hippocampal afferents. In this work, we first addressed the action of Reelin on axonal growth. We showed that neither cleaved nor full-length Reelin exhibited any significant attraction or repulsion on cortical axons. Our results suggest that the reported effects of Reelin on axonal pathways are indirect, secondary to the architectonic disturbances that result from Reelin deficiency, and that the reported effects of CajalRetzius cells on connectivity are independent of Reelin. The second part of our work addressed the activity of the different cleavage products of Reelin. We showed that the central cleavage fragment is necessary and sufficient for receptor binding and signal activation as reflected in Dab1 phosphorylation, and rescues the reeler phenotype in a new organotypic slice culture assay. Stimulating antibodies to the VLDLR and ApoER2 receptors did not rescue the phenotype in slices, suggesting that the Dab1 branch of the Reelin pathway is not sufficient to transmit the entire signal. The last part of our work consisted in probing intracellular transduction pathways in slice cultures, using small molecular weight inhibitors. We showed that inhibition of Src kinases, PKCs, PI3K, Akt or mTor induced a reeler-like phenotype defined by a disruption of cortical neuronal positioning, impaired inside-out layering of cortical cells and absence of preplate splitting. Our data indicate that Src kinases are responsible for Dab1 phosphorylation, whereas the other kinases (PKCs, PI3K, Akt and mTor) function further downstream or in parallel to the Dab1 branch of Reelin signaling.Thèse de doctorat en sciences biomédicales (neurobiologie du débeloppement) (SBIM 3)--UCL, 200

Neuronal migration and the role of reelin during early development of the cerebral cortex.

During development, neurons migrate to the cortex radially from periventricular germinative zones as well as tangentially from ganglionic eminences. The vast majority of cortical neurons settle radially in the cortical plate. Neuronal migration requires an exquisite regulation of leading edge extension, nuclear translocation (nucleokinesis), and retraction of trailing processes. During the past few years, several genes and proteins have been identified that are implicated in neuronal migration. Many have been characterized by reference to known mechanisms of neuronal and non-neuronal cell migration in culture; however, probably the most interesting have been identified by gene inactivation or modification in mice and by positional cloning of brain malformation genes in humans and mice. Although it is impossible to provide a fully integrated view, some patterns clearly emerge and are the subject of this article. Specific emphasis is placed on three aspects: first, the role of the actin treadmill, with cyclic formation of filopodial and lamellipodial extensions, in relation to surface events that occur at the leading edge of radially migrating neurons; second, the regulation of microtubule dynamics, which seems to play a key role in nucleokinesis; and third, the mechanisms by which the extracellular protein Reelin regulates neuronal positioning at the end of migration

Molecular mechanisms of cell polarity in a range of model systems and in migrating neurons.

Cell polarity is defined as the asymmetric distribution of cellular components along an axis. Most cells, from the simplest single-cell organisms to highly specialized mammalian cells, are polarized and use similar mechanisms to generate and maintain polarity. Cell polarity is important for cells to migrate, form tissues, and coordinate activities. During development of the mammalian cerebral cortex, cell polarity is essential for neurogenesis and for the migration of newborn but as-yet undifferentiated neurons. These oriented migrations include both the radial migration of excitatory projection neurons and the tangential migration of inhibitory interneurons. In this review, I will first describe the development of the cerebral cortex, as revealed at the cellular level. I will then define the core molecular mechanisms - the Par/Crb/Scrib polarity complexes, small GTPases, the actin and microtubule cytoskeletons, and phosphoinositides/PI3K signaling - that are required for asymmetric cell division, apico-basal and front-rear polarity in model systems, including C elegans zygote, Drosophila embryos and cultured mammalian cells. As I go through each core mechanism I will explain what is known about its importance in radial and tangential migration in the developing mammalian cerebral cortex

Reelin does not directly influence axonal growth.

Reelin is a large extracellular glycoprotein involved in the development of architectonic patterns, particularly in the cerebral cortex and hippocampus, where it is synthesized primarily by Cajal-Retzius cells. In the hippocampus, Reelin also regulates the growth and/or distribution of afferent entorhinal and commissural axons. To assess further the possible action of Reelin on axonal growth, we used the three-dimensional collagen gel assay to measure axonal elongation from reeler cortical explants in the presence of Reelin. Because Reelin is proteolytically processed in vivo, normal explants and Reelin-transfected human embryonic kidney 293T cells were used, respectively, as sources of processed and full-length protein. The reliability of the assay was tested by demonstrating a clear repulsive action of semaphorin 3F (p < 0.0001). However, neither full-length nor processed Reelin exhibited any significant attraction or repulsion on cortical axons. Our results suggest that the reported effects of Reelin on axonal pathways are indirect, secondary to the architectonic disturbances that result from Reelin deficiency, and that the effects of Cajal-Retzius cells on connectivity are primarily independent of Reelin

Reelin signals through phosphatidylinositol 3-kinase and Akt to control cortical development and through mTor to regulate dendritic growth

Reelin is an extracellular matrix protein with various functions during development and in the mature brain. It activates different signaling cascades in target cells, one of which is the phosphatidylinositol 3-kinase (PI3K) pathway, which we investigated further using pathway inhibitors and in vitro brain slice and neuronal cultures. We show that the mTor (mammalian target of rapamycin)-S6K1 (S6 kinase 1) pathway is activated by Reelin and that this depends on Dab1 (Disabled-1) phosphorylation and activation of PI3K and Akt (protein kinase B). PI3K and Akt are required for the effects of Reelin on the organization of the cortical plate, but their downstream partners mTor and glycogen synthase kinase 3beta (GSK3beta) are not. On the other hand, mTor, but not GSK3beta, mediates the effects of Reelin on the growth and branching of dendrites of hippocampal neurons. In addition, PI3K fosters radial migration of cortical neurons through the intermediate zone, an effect that is independent of Reelin and Akt

Neuronal Polarity in the Embryonic Mammalian Cerebral Cortex

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Cloning, sequencing and characterisation of a Listeria monocytogenes gene encoding a fibronectin-binding protein.

Listeria monocytogenes is a gram-positive, non-sporulating food-borne pathogen of man and animals that is able to invade many eukaryotic cells. L. monocytogenes possesses several proteins that bind fibronectin. In this study, an L. monocytogenes DNA library in pUC19 was screened with fibronectin and a gene encoding a 24.6-kDa fibronectin-binding protein (Fbp) was isolated and sequenced. Transcripts of the fbp gene were found in wild-type, in deltaprfA, and PrfA-S183A strains, despite the presence of a 'PrfA-like' box around its ribosome-binding site. The fbp gene was found to be present in all tested isolates of the species L. monocytogenes and a homologous DNA fragment was amplified in L. welshimeri. No homologies between the fbp gene and its translation product with any other DNA or proteins deposited in databanks were found. Restriction endonuclease-PCR (RE-PCR) showed that the fbp gene displays a degree of allelic variation among isolates of L. monocytogenes, whereas the corresponding amplified fragment of L. welshimeri seems to be monomorphic among isolates of this species. RE-PCR with Hha I, Dde I or Taq I produced DNA banding profiles specific for each of these two species, allowing their identification