Disruption of embryonic blood-CSF barrier in chick embryos reveals the actual importance of this barrier to control E-CSF composition and homeostasis in early brain development

In vertebrates, early brain development takes place at the expanded anterior end of the neural tube. After closure of the anterior neuropore, the brain wall forms a physiologically sealed cavity that encloses embryonic cerebrospinal fluid (E-CSF), a complex and protein-rich fluid that is initially composed of trapped amniotic fluid. E-CSF has several crucial roles in brain anlagen development. Recently, we reported the presence of transient blood-CSF barrier located in the brain stem lateral to the ventral midline, at the mesencephalon and prosencephalon level, in chick and rat embryos by transporting proteins, water, ions and glucose in a selective manner via transcellular routes. To test the actual relevance of the control of E-CSF composition and homeostasis on early brain development by this embryonic blood-CSF barrier, we block the activity of this barrier by treating the embryos with 6-aminonicotinamide gliotoxin (6-AN). We demonstrate that 6-AN treatment in chick embryos blocks protein transport across the embryonic blood-CSF barrier, and that the disruption of the barrier properties is due to the cease transcellular caveolae transport, as detected by CAV-1 expression cease. We also show that the lack of protein transport across the embryonic blood-CSF barrier influences neuroepithelial cell survival, proliferation and neurogenesis, as monitored by neurepithelial progenitor cells survival, proliferation and neurogenesis. The blockage of embryonic blood-CSF transport also disrupts water influx to the E-CSF, as revealed by an abnormal increase in brain anlagen volume. These experiments contribute to delineate the actual extent of this blood-CSF embryonic barrier controlling E-CSF composition and homeostasis and the actual important of this control for early brain development, as well as to elucidate the mechanism by which proteins and water are transported thought transcellular routes across the neuroectoderm, reinforcing the crucial role of E-CSF for brain development.

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

Influence of the in vivo deregulation of the expression of ntrk3 (trkc) on cortical development of a murine model of panic/anxiety disorder

Alteracions durant el desenvolupament cerebral produirien canvis en la connectivitat neuronal i la bioquímica cel•lular que podrien resultar en una disfunció cognitiva i/o emocional, desembocant a trastorns psiquiàtrics. Les neurotrofines intervenen en els processos del neurodesenvolupament i en la funcionalitat del cervell adult i, conseqüentment, serien bons candidats com a factors de predisposició en diverses malalties mentals. S’ha suggerit la implicació del receptor de la neurotrofina 3, TrkC, en el trastorn de pànic. Nosaltres proposem que la sobreexpressió del gen NTRK3 (TrkC) és un mediador comú dels desencadenants genètics i ambientals d’aquest trastorn. Concretament, la seva desregulació podria produir canvis estructurals i funcionals a l’escorça cerebral dels pacients pel seu paper durant l’establiment dels circuïts corticals i la neuroplasticitat a l’adult, probablement esdevenint elements de predisposició a patir atacs de pànic. Els objectius principals d’aquest treball han estat: 1/determinar la contribució específica del gen NTRK3 a les alteracions de l’escorça cerebral observades en pacients, utilitzant un model murí modificat genèticament (TgNTRK3), i 2/analitzar l’impacte específic de la sobreexpressió de NTRK3 sobre la corticogènesi durant estadis embrionaris o postnatals estudiant la neurogènesi i la neuritogènesi. Els resultats indiquen que la sobreexpressió de NTRK3 als ratolins produeix una reducció del gruix de l’escorça frontal, recapitulant la hipofrontalitat dels pacients, que comportaria una menor inhibició dels nuclis subcorticals del sistema límbic com l’amígdala, i alteracions citoarquitectòniques a l’escorça prefrontal medial que recolzen la hipòtesi del seu mal funcionament. Tanmateix, els ratolins TgNTRK3 presenten canvis estructurals a l’escorça somatosensorial, suggerint que el processament de la informació sensorial podria estar alterat, el que encara no s’ha explorat en pacients. La sobreexpressió de NTRK3 també afecta la neuritogènesi en cultius primaris corticals i modifica la resposta de les neurones a l’estimulació amb neurotrofines. Per tant, el fenotip cortical adult dels TgNTRK3 podria dependre d’alteracions durant la corticogènesi.

Reelin controls progenitor cell migration in the healty and pathological adult brain

Understanding the signals that control migration of neural progenitor cells in the adult brain may provide new therapeutic opportunities. Reelin is best known for its role in regulating cell migration during brain development, but we now demonstrate a novel function for reelin in the injured adult brain. First, we show that Reelin is upregulated around lesions. Second, experimentally increasing Reelin expression levels in healthy mouse brain leads to a change in the migratory behavior of subventricular zone-derived progenitors, triggering them to leave the rostral migratory stream (RMS) to which they are normally restricted during their migration to the olfactory bulb. Third, we reveal that Reelin increases endogenous progenitor cell dispersal in periventricular structures independently of any chemoattraction but via cell detachment and chemokinetic action, and thereby potentiates spontaneous cell recruitment to demyelination lesions in the corpus callosum. Conversely, animals lacking Reelin signaling exhibit reduced endogenous progenitor recruitment at the lesion site. Altogether, these results demonstrate that beyond its known role during brain development, Reelin is a key player in post-lesional cell migration in the adult brain. Finally our findings provide proof of concept that allowing progenitors to escape from the RMS is a potential therapeutic approach to promote myelin repair.

Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the Zona Limitans Intrathalamica (ZLI) brain organizer

Background: The zona limitans intrathalamica (ZLI) and the isthmus organizer (IsO) are two major secondary organizers of vertebrate brain development. These organizers are located at the interface of the expression domains of key patterning genes (Fezf-Irx and Otx-Gbx, respectively). To gain insights into the evolutionary origin of the ZLI, we studied Fezf in bilaterians. Results: In this paper, we identified a conserved sequence motif (Fezf box) in all bilaterians. We report the expression pattern of Fezf in amphioxus and Drosophila and compare it with those of Gbx, Otx and Irx. We found that the relative expression patterns of these genes in vertebrates are fully conserved in amphioxus and flies, indicating that the genetic subdivisions defining the location of both secondary organizers in early vertebrate brain development were probably present in the last common ancestor of extant bilaterians. However, in contrast to vertebrates, we found that Irx-defective flies do not show an affected Fezf expression pattern. Conclusions: The absence of expression of the corresponding morphogens from cells at these conserved genetic boundaries in invertebrates suggests that the organizing properties might have evolved specifically in the vertebrate lineage by the recruitment of key morphogens to these conserved genetic locations.

Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the Zona Limitans Intrathalamica (ZLI) brain organizer

Background: The zona limitans intrathalamica (ZLI) and the isthmus organizer (IsO) are two major secondary organizers of vertebrate brain development. These organizers are located at the interface of the expression domains of key patterning genes (Fezf-Irx and Otx-Gbx, respectively). To gain insights into the evolutionary origin of the ZLI, we studied Fezf in bilaterians. Results: In this paper, we identified a conserved sequence motif (Fezf box) in all bilaterians. We report the expression pattern of Fezf in amphioxus and Drosophila and compare it with those of Gbx, Otx and Irx. We found that the relative expression patterns of these genes in vertebrates are fully conserved in amphioxus and flies, indicating that the genetic subdivisions defining the location of both secondary organizers in early vertebrate brain development were probably present in the last common ancestor of extant bilaterians. However, in contrast to vertebrates, we found that Irx-defective flies do not show an affected Fezf expression pattern. Conclusions: The absence of expression of the corresponding morphogens from cells at these conserved genetic boundaries in invertebrates suggests that the organizing properties might have evolved specifically in the vertebrate lineage by the recruitment of key morphogens to these conserved genetic locations.

Most of the Abundant Protein Fractions of Embryonic Cerebrospinal Fluid are Produced Out of the Brain Anlagen

The microenvironment of the central nervous system is important for neuronal function and development. During the early stages of embryo development the cephalic vesicles are filled by embryonic cerebrospinal fluid, a complex fluid containing different protein fractions, which contributes to the regulation of the survival, proliferation and neurogenesis of neuroectodermal stem cells. The protein content of embryonic cerebrospinal fluid from chick and rat embryos at the start of neurogenesis has already been determined. Most of the identified gene products are thought to be involved in the regulation of developmental processes during embryogenesis. However, due to the crucial roles played by embryonic cerebrospinal fluid during brain development, the embryological origin of the gene products it contains remains an intriguing question. According to the literature most of these products are synthesised in embryonic tissues other than the neuroepithelium. In this study we examined the embryological origin of the most abundant embryonic cerebrospinal fluid protein fractions by means of slot-blot analysis and by using several different embryonic and extraembryonic protein extracts, immunodetected with polyclonal antibodies. This first attempt to elucidate their origin is not based on the proteins identified by proteomic methods, but rather on crude protein fractions detected by SDS-PAGE analysis and to which polyclonal antibodies were specifically generated. Despite some of the limitations of this study, i.e. that one protein fraction may contain more than one gene product, and that a specific gene product may be contained in different protein fractions depending on post-translational modifications, our results show that most of the analysed protein fractions are not produced by the cephalic neuroectoderm but are rather stored in the egg reservoir; furthermore, few are produced by embryo tissues, thus indicating that they must be transported from their production or storage sites to the cephalic cavities, most probably via embryonic serum. These results raise the question as to whether the transfer of proteins from these two embryo compartments is regulated at this early developmental stage.

Rendimiento neuropsicológico y anomalías del cuerpo callosoen adolescentes con antecedentes de prematuridad

Existe una clara relación entre prematuridad y un bajo rendimiento cognitivo y escolar. Sin embargo, los efectos concretos del nacimiento prematuro sobre el funcionamiento cognitivo así como sobre el desarrollo cerebral a largo plazo son poco conocidos. Objetivos: Identificar las disfunciones cognitivas concretas en adolescentes que nacieron prematuros mediante una evaluación neuropsicológica exhaustiva, y relacionar los datos cognitivos con la posible afectación del cuerpo calloso. Metodología y Resultados: se comparó dos muestras de sujetos prematuros y sujetos nacidos a término. Se evaluó el rendimiento cognitivo general y específico, y se cuantificó la estructura cerebral del cuerpo calloso. Se realizaron varios análisis estadísticos y se redactaron diversos artículos presentando los resultados obtenidos. Resultados: adolescentes con antecedentes de prematuridad: a) presentan dificultades cognitivas y anormalidades estructurales, más relacionadas con la edad gestacional que con el peso al nacer; b) tienen déficits cognitivos específicos que pueden explicarse parcialmente por sus disfunciones en el rendimiento cognitivo general; c) la media de sus puntuaciones en el CI se sitúa en el rango normal; d) los subtests de las escalas Wechsler no presentan el mismo grado de sensibilidad; e) presentan una reducción de tamaño del cuerpo calloso, f) más acusada en el genu, posterior midbody y splenium; g) existe una asociación específica entre el genu y el menor rendimiento en funciones del lóbulo prefrontal; h) la edad gestacional presenta una clara relación con las anormalidades del cuerpo calloso y con el bajo rendimiento cognitivo general.

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse

The reelin gene encodes an extracellular protein that is crucial for neuronal migration in laminated brain regions. To gain insights into the functions of Reelin, we performed high-resolution in situ hybridization analyses to determine the pattern of reelin expression in the developing forebrain of the mouse. We also performed double-labeling studies with several markers, including calcium-binding proteins, GAD65/67, and neuropeptides, to characterize the neuronal subsets that express reelin transcripts. reelinexpression was detected at embryonic day 10 and later in the forebrain, with a distribution that is consistent with the prosomeric model of forebrain regionalization. In the diencephalon, expression was restricted to transverse and longitudinal domains that delineated boundaries between neuromeres. During embryogenesis,reelin was detected in the cerebral cortex in Cajal-Retzius cells but not in the GABAergic neurons of layer I. At prenatal stages, reelin was also expressed in the olfactory bulb, and striatum and in restricted nuclei in the ventral telencephalon, hypothalamus, thalamus, and pretectum. At postnatal stages, reelin transcripts gradually disappeared from Cajal-Retzius cells, at the same time as they appeared in subsets of GABAergic neurons distributed throughout neocortical and hippocampal layers. In other telencephalic and diencephalic regions,reelin expression decreased steadily during the postnatal period. In the adult, there was prominent expression in the olfactory bulb and cerebral cortex, where it was restricted to subsets of GABAergic interneurons that co-expressed calbindin, calretinin, neuropeptide Y, and somatostatin. This complex pattern of cellular and regional expression is consistent with Reelin having multiple roles in brain development and adult brain function.

The embryonic blood-CSF barrier has molecular elements for specific glucose transport and for the general transport of molecules via transcellular routes.

In vertebrates, early brain development takes place at the expanded anterior end of the neural tube, which is filled with embryonic cerebrospinal fluid (E-CSF). We have recently identified a transient blood-CSF barrier that forms between embryonic days E3 and E4 in chick embryos and that is responsible for the transport of proteins and control of E-CSF homeostasis, including osmolarity. Here we examined the presence of glucose transporter GLUT-1 as well the presence of caveolae-structural protein Caveolin1 (CAV-1) in the embryonic blood-CSF barrier which may be involved in the transport of glucose and of proteins, water and ions respectively across the neuroectoderm. In this paper we demonstrate the presence of GLUT-1 and CAV-1 in endothelial cells of blood vessels as well as in adjacent neuroectodermal cells, located in the embryonic blood-CSF barrier. In blood vessels, these proteins were detected as early as E4 in chick embryos and E12.7 in rat embryos, i.e. the point at which the embryonic blood-CSF barrier acquires this function. In the neuroectoderm of the embryonic blood-CSF barrier, GLUT-1 was also detected at E4 and E12.7 respectively, and CAV-1 was detected shortly thereafter in both experimental models. These experiments contribute to delineating the extent to which the blood-CSF embryonic barrier controls E-CSF composition and homeostasis during early stages of brain development in avians and mammals. Our results suggest the regulation of glucose transport to the E-CSF by means of GLUT-1 and also suggest a mechanism by which proteins are transported via transcellular routes across the neuroectoderm, thus reinforcing the crucial role of E-CSF in brain development.

Altered brain-derived neurotrophic factor blood levels and gene variability are associated with anorexia and bulimia

Murine models and association studies in eating disorder (ED) patients have shown a role for the brain-derived neurotrophic factor (BDNF) in eating behavior. Some studies have shown association of BDNF -270C/T single-nucleotide polymorphism (SNP) with bulimia nervosa (BN), while BDNF Val66Met variant has been shown to be associated with both BN and anorexia nervosa (AN). To further test the role of this neurotrophin in humans, we screened 36 SNPs in the BDNF gene and tested for their association with ED and plasma BDNF levels as a quantitative trait. We performed a family-based association study in 106 ED nuclear families and analyzed BDNF blood levels in 110 ED patients and in 50 sib pairs discordant for ED. The rs7124442T/rs11030102C/rs11030119G haplotype was found associated with high BDNF levels (mean BDNF TCG haplotype carriers = 43.6 ng/ml vs. mean others 23.0 ng/ml, P = 0.016) and BN (Z = 2.64; P recessive = 0.008), and the rs7934165A/270T haplotype was associated with AN (Z =-2.64; P additive = 0.008). The comparison of BDNF levels in 50 ED discordant sib pairs showed elevated plasma BDNF levels for the ED group (mean controls = 41.0 vs. mean ED = 52.7; P = 0.004). Our data strongly suggest that altered BDNF levels modulated by BDNF gene variability are associated with the susceptibility to ED, providing physiological evidence that BDNF plays a role in the development of AN and BN, and strongly arguing for its involvement in eating behavior and body weight regulation.

Podocalyxin is a novel polysialylated neural adhesion protein with multiple roles in neural development and synapse formation

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.

Different effects of hyperlipidic diets in human lactation and adulthood: growth versus the development of obesity

After birth, the body shifts from glucose as primary energy substrate to milk-derived fats, with sugars from lactose taking a secondary place. At weaning, glucose recovers its primogeniture and dietary fat role decreases. In spite of human temporary adaptation to a high-fat (and sugars and protein) diet during lactation, the ability to thrive on this type of diet is lost irreversibly after weaning. We could not revert too the lactating period metabolic setting because of different proportions of brain/muscle metabolism in the total energy budget, lower thermogenesis needs and capabilities, and absence of significant growth in adults. A key reason for change was the limited availability of foods with high energy content at weaning and during the whole adult life of our ancestors, which physiological adaptations remain practically unchanged in our present-day bodies. Humans have evolved to survive with relatively poor diets interspersed by bouts of scarcity and abundance. Today diets in many societies are largely made up from choice foods, responding to our deeply ingrained desire for fats, protein, sugars, salt etc. Consequently our diets are not well adjusted to our physiological needs/adaptations but mainly to our tastes (another adaptation to periodic scarcity), and thus are rich in energy roughly comparable to milk. However, most adult humans cannot process the food ingested in excess because our cortical-derived craving overrides the mechanisms controlling appetite. This is produced not because we lack the biochemical mechanisms to use this energy, but because we are unprepared for excess, and wholly adapted to survive scarcity. The thrifty mechanisms compound the effects of excess nutrients and damage the control of energy metabolism, developing a pathologic state. As a consequence, an overflow of energy is generated and the disease of plenty develops.

Podocalyxin is a novel polysialylated neural adhesion protein with multiple roles in neural development and synapse formation

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.