Targeted electroporation of defined lateral ventricular walls: a novel and rapid method to study fate specification during postnatal forebrain neurogenesis

<p>Abstract</p> <p>Background</p> <p>Postnatal olfactory bulb (OB) neurogenesis involves the generation of granule and periglomerular cells by neural stem cells (NSCs) located in the walls of the lateral ventricle (LV). Recent studies show that NSCs located in different regions of the LV give rise to different types of OB neurons. However, the molecular mechanisms governing neuronal specification remain largely unknown and new methods to approach these questions are needed.</p> <p>Results</p> <p>In this study, we refine electroporation of the postnatal forebrain as a technique to perform precise and accurate delivery of transgenes to NSCs located in distinct walls of the LV in the mouse. Using this method, we confirm and expand previous studies showing that NSCs in distinct walls of the LV produce neurons that invade different layers of the OB. Fate mapping of the progeny of radial glial cells located in these distinct LV walls reveals their specification into defined subtypes of granule and periglomerular neurons.</p> <p>Conclusions</p> <p>Our results provide a baseline with which future studies aiming at investigating the role of factors in postnatal forebrain neuronal specification can be compared. Targeted electroporation of defined LV NSC populations will prove valuable to study the genetic factors involved in forebrain neuronal specification.</p

Circulating human serum metabolites derived from the intake of a saffron extract (Safr’Inside™) protect neurons from oxidative stress: Consideration for depressive disorders

Increases in oxidative stress have been reported to play a central role in the vulnerability to depression, and antidepressant drugs may reduce increased oxidative stress in patients. Among the plants exerting anti-inflammatory and anti-oxidant properties, saffron, a spice derived from the flower of Crocus sativus, is also known for its positive effects on depression, potentially through its SSRI-like properties. However, the molecular mechanisms underlying these effects and their health benefits for humans are currently unclear. Using an original ex vivo clinical approach, we demonstrated for the first time that the circulating human metabolites produced following saffron intake (Safr’Inside™ ) protect human neurons from oxidative-stress-induced neurotoxicity by preserving cell viability and increasing BNDF production. In particular, the metabolites significantly stimulated both dopamine and serotonin release. In addition, the saffron’s metabolites were also able to protect serotonergic tone by inhibiting the expression of the serotonin transporter SERT and down-regulating serotonin metabolism. Altogether, these data provide new biochemical insights into the mechanisms underlying the beneficial impact of saffron on neuronal viability and activity in humans, in the context of oxidative stress related to depression

Biology of multiciliated cells

International audienceMulticiliated cells (MCCs) are specialized in fluid propulsion through directional beating of myriads of superficial motile cilia, which rest on modified centrioles named basal bodies. MCCs are found throughout metazoans, and serve functions as diverse as feeding and locomotion in marine organisms, as well as mucus clearance, cerebrospinal fluid circulation, and egg transportation in mammals. Impaired MCC differentiation or activity causes diseases characterized by severe chronic airway infections and reduced fertility. Through studies in Xenopus and mouse mainly, MCC biology has made significant progress on several fronts in recent years. The gene regulatory network that controls MCC specification and differentiation has been deciphered to a large extent. The enigmatic deuterosomes, which serve as centriole amplification platforms in vertebrate MCCs, have started to be studied at the molecular level. Principles of ciliary beating coordination within and between MCCs have been identified

Contrôle moléculaire de la neurogenèse postnatale chez la souris (étude du rôle du facteur de transcription NeuroD1 dans la différenciation neuronale par une nouvelle approche in vivo)

Une neurogenèse fournissant des interneurones au bulbe olfactif persiste dans le cerveau de la souris tout au long de la vie. Dans ce système, les différentes étapes de la neurogenèse embryonnaire sont récapitulées. La séparation spatiale qui rend accessibles les différentes étapes de la neurogenèse fait de ce modèle un modèle de choix pour étudier le contrôle moléculaire de la neurogenèse. Ainsi, des cribles à grande échelle peuvent être réalisés pour isoler des candidats impliqués dans une étape particulière. Cependant, les approches expérimentales permettant de manipuler le système présentes certaines limites. Au cours de la première partie de ma thèse, j ai mis au point un protocole d électroporation permettant le transfert de gène dans le cerveau antérieur de la souris postnatale. Cette méthode permet de manipuler la neurogenèse bulbaire depuis la cellule souche jusqu aux interneurones différenciés. Un des grands avantages de cette technique est qu elle est relativement facile a mettre en oeuvre et permet de générer rapidement et de manière reproductible de large séries à analyser. De plus, elle permet d avoir une approche à l échelle cellulaire, contrôlée dans l espace de la neurogenèse postnatale. La combinaison de cette approche avec des moyens moléculaires et technologiques avancés devrait permettre d aborder plusieurs types de questions. La deuxième partie de mon travail de thèse à consisté en l analyse du rôle d un gène candidat isolé lors d un crible spécifique d expression génique entre des cellules différenciées et leurs précurseurs immédiats. Pour cela j ai réalisé des expériences de gain de fonction par électroporation. J ai pu montrer grâce à une analyse résolutive, que le facteur de transcription NeuroD1 un signal très fort qui, d une part, ne permet pas le maintien du statut de cellule souche et, d autre part, provoque une différenciation immédiate et ectopique en cellules présentant des caractéristiques morphologiques et moléculaires spécifiques de neurones. Les résultats obtenus permettent d élargir les possibilités d approches fonctionnelles de la neurogenèse postnatale. Par ailleurs, le travail que j ai réalisé sur le facteur de transcription NeuroD1 est un premier pas vers la compréhension des mécanismes transrationnels régulant la formation d un neurone.In postnatal and adult mammals, the subventricular zone (SVZ) lining the lateral wall of the lateral ventricle contains stem cells that generate transit amplifying precursors that, in turn, give rise to neuroblasts. These cells migrate along a specific pathway, the rostral migratory stream (RMS) to reach the olfactory bulb (OB) where they differentiate into interneurons. This system recapitulates the different step of neurogenesis. Over the past years this system attracted considerable attention. First, the demonstration that new neurons can be generated and integrated into the existing circuitry fundamentally changed our view of brain development and function. Second, the discovery of comparable neural stem cells and progenitors in humans raised hope for the use of this adult neurogenesis for brain repair either by transplantation of adult derived progenitors or via the activation and recruitment of the intrinsic neurogenetic pool. In addition, the postnatal SVZ-RMS-OB system became an important model to study the molecular and cellular mechanisms that underlie the regulation of neurogenesis in general. During my thesis, I developed a new approach that allows a rapid and reliable analysis of gene function in this system. This technology is based on the injection of DNA or RNA into the lateral ventricle of postnatal mice followed by electroporation. I demonstrated that gene transfer by electroporation is highly efficient and reproducible and that the transfected cells present normal proliferation, migration and differentiation. This method will allow rapid Gain-of-function and loss-of-function to analyze the function of candidate genes on cellular proliferation, migration and differentiation. In a second part of my work, I investigated the role of the bHLH transcription factor NeuroD1 in neurogenesis. I used in vivo electroporation of the postnatal forebrain to investigate the function of this gene and show that overexpression of NeuroD1 alone is not compatible with the maintenance of stem cell status. Furthermore its expression is sufficient to induce immediate morphological differentiation and the expression of neuronal markers such as NeuN and MAP2 already at the ventricular level. In the future, this work is the basis of a detailed analysis of the role of this promising gene will help to better understand the differentiation process increasing the possibility of cell manipulation for transplantation in therapeutic approach.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Contribution à l'étude clinique du syndrome de Landry post-grippal

Contient une table des matièresAvec mode text

NCAM expression induces neurogenesis in vivo.

Neural cell adhesion molecule (NCAM) plays an important role during neural development and in the adult brain, whereby most functions of NCAM have been ascribed to its unique polysialic acid (PSA) modification. Recently we presented evidence suggesting that expression of NCAM in vivo interferes with the maintenance of forebrain neuronal stem cells. We here aimed at investigating the fate of cells generated from NCAM-overexpressing stem cells in postnatal mouse brain and at elucidating the functional domains of NCAM mediating this effect. We show that ectopic expression of the NCAM140 isoform in radial glia and type C cells induces an increase in cell proliferation and consequently the presence of additional neuronal type A cells in the rostral migratory stream. A mutant NCAM protein comprising only fibronectin type III repeats and immunoglobulin-like domain 5 was sufficient to induce this effect. Furthermore, we show that the neurogenic effect is independent of PSA, as transgenic NCAM is not polysialylated in radial glia and type C cells. These results suggest that heterophilic interactions of NCAM with other components of the cell membrane must be involved

Wave–sea-ice interactions in a brittle rheological framework

As sea ice extent decreases in the Arctic, surface ocean waves have more time and space to develop and grow, exposing the marginal ice zone (MIZ) to more frequent and more energetic wave events. Waves can fragment the ice cover over tens of kilometres, and the prospect of increasing wave activity has sparked recent interest in the interactions between wave-induced sea ice fragmentation and lateral melting. The impact of this fragmentation on sea ice dynamics, however, remains mostly unknown, although it is thought that fragmented sea ice experiences less resistance to deformation than pack ice. Here, we introduce a new coupled framework involving the spectral wave model WAVEWATCH III and the sea ice model neXtSIM, which includes a Maxwell elasto-brittle rheology. This rheological framework enables the model to efficiently track and keep a “memory” of the level of sea ice damage. We propose that the level of sea ice damage increases when wave-induced fragmentation occurs. We used this coupled modelling system to investigate the potential impact of such a local mechanism on sea ice kinematics. Focusing on the Barents Sea, we found that the internal stress decrease of sea ice resulting from its fragmentation by waves resulted in a more dynamical MIZ, particularly in areas where sea ice is compact. Sea ice drift is enhanced for both on-ice and off-ice wind conditions. Our results stress the importance of considering wave–sea-ice interactions for forecast applications. They also suggest that waves likely modulate the area of sea ice that is advected away from the pack by the ocean, potentially contributing to the observed past, current and future sea ice cover decline in the Arctic

Expression and function of CXCR7 in the mouse forebrain

The chemokine CXCL12/CXCR4 signaling system is important for the regulation of neuron migration in the developing forebrain. In particular it is crucial for correct distribution of Cajal-Retzius cells and migration of cortical interneurons. Here we investigated the expression of CXCR7, the second receptor for CXCL12, in comparison to CXCR4. We found that shifts in the expression of both receptors in the above cited cell populations coincide with major changes in their migratory behavior. Furthermore, we demonstrated that postnatally generated olfactory interneuron precursors express CXCR7 but not CXCR4 and that their distribution in the rostral migratory stream is affected by CXCR7 downregulation. This suggests an involvement of CXCR7 in neuronal cell migration and indicates a possible action of CXCR7 independently of CXCR4 as a mediator of CXCL12 signaling

Efficient In Vivo Electroporation of the Postnatal Rodent

Functional gene analysis in vivo represents still a major challenge in biomedical research. Here we present a new method for the efficient introduction of nucleic acids into the postnatal mouse forebrain. We show that intraventricular injection of DNA followed by electroporation induces strong expression of transgenes in radial glia, neuronal precursors and neurons of the olfactory system. We present two proof-of-principle experiments to validate our approach. First, we show that expression of a human isoform of the neural cell adhesion molecule (hNCAM-140) in radial glia cells induces their differentiation into cells showing a neural precursor phenotype. Second, we demonstrate that p21 acts as a cell cycle inhibitor for postnatal neural stem cells. This approach will represent an important tool for future studies of postnatal neurogenesis and of neural development in general

Plexin-B2 regulates the proliferation and migration of neuroblasts in the postnatal and adult subventricular zone.

In the postnatal forebrain, the subventricular zone (SVZ) contains a pool of undifferentiated cells, which proliferate and migrate along the rostral migratory stream (RMS) to the olfactory bulb and differentiate into granule cells and periglomerular cells. Plexin-B2 is a semaphorin receptor previously known to act on neuronal proliferation in the embryonic brain and neuronal migration in the cerebellum. We show here that, in the postnatal and adult CNS, Plexin-B2 is expressed in the subventricular zone lining the telencephalic ventricles and in the rostral migratory stream. We analyzed Plxnb2(-/-) mice and found that there is a marked reduction in the proliferation of SVZ cells in the mutant. Plexin-B2 expression is downregulated in the olfactory bulb as interneurons initiate radial migration. BrdU labeling and GFP electroporation into postnatal SVZ, in addition to time-lapse videomicroscopy, revealed that neuroblasts deficient for Plexin-B2 migrate faster than control ones and leave the RMS more rapidly. Overall, these results show that Plexin-B2 plays a role in postnatal neurogenesis and in the migration of SVZ-derived neuroblasts