Ascorbic acid and its transporter SVCT2, affect radial glia cells differentiation in postnatal stages.

Radial glia (RG) cells generate neurons and glial cells that make up the cerebral cortex. Both in rodents and humans, these stem cells remain for a specific time after birth, named late radial glia (lRG). The knowledge of lRG and molecules that may be involved in their differentiation is based on very limited data. We analyzed whether ascorbic acid (AA) and its transporter SVCT2, are involved in lRG cells differentiation. We demonstrated that lRG cells are highly present between the first and fourth postnatal days. Anatomical characterization of lRG cells, revealed that lRG cells maintained their bipolar morphology and stem-like character. When lRG cells were labeled with adenovirus-eGFP at 1 postnatal day, we detected that some cells display an obvious migratory neuronal phenotype, suggesting that lRG cells continue generating neurons postnatally. Moreover, we demonstrated that SVCT2 was apically polarized in lRG cells. In vitro studies using the transgenic mice SVCT2+/- and SVCT2tg (SVCT2-overexpressing mouse), showed that decreased SVCT2 levels led to accelerated differentiation into astrocytes, whereas both AA treatment and elevated SVCT2 expression maintain the lRG cells in an undifferentiated state. In vivo overexpression of SVCT2 in lRG cells generated cells with a rounded morphology that were migratory and positive for proliferation and neuronal markers. We also examined mediators that can be involved in AA/SVCT2-modulated signaling pathways, determining that GSK3-ß through AKT, mTORC2, and PDK1 is active in brains with high levels of SVCT2/AA. Our data provide new insights into the role of AA and SVCT2 in late RG cells.

Role of vitamin C and SVCT2 in neurogenesis.

Different studies have established the fundamental role of vitamin C in proliferation, differentiation, and neurogenesis in embryonic and adult brains, as well as in in vitro cell models. To fulfill these functions, the cells of the nervous system regulate the expression and sorting of sodium-dependent vitamin C transporter 2 (SVCT2), as well as the recycling of vitamin C between ascorbic acid (AA) and dehydroascorbic acid (DHA) via a bystander effect. SVCT2 is a transporter preferentially expressed in neurons and in neural precursor cells. In developmental stages, it is concentrated in the apical region of the radial glia, and in adult life, it is expressed preferentially in motor neurons of the cerebral cortex, starting on postnatal day 1. In neurogenic niches, SVCT2 is preferentially expressed in precursors with intermediate proliferation, where a scorbutic condition reduces neuronal differentiation. Vitamin C is a potent epigenetic regulator in stem cells; thus, it can induce the demethylation of DNA and histone H3K27m3 in the promoter region of genes involved in neurogenesis and differentiation, an effect mediated by Tet1 and Jmjd3 demethylases, respectively. In parallel, it has been shown that vitamin C induces the expression of stem cell-specific microRNA, including the Dlk1-Dio3 imprinting region and miR-143, which promotes stem cell self-renewal and suppresses de novo expression of the methyltransferase gene Dnmt3a. The epigenetic action of vitamin C has also been evaluated during gene reprogramming of human fibroblasts to induced pluripotent cells, where it has been shown that vitamin C substantially improves the efficiency and quality of reprogrammed cells. Thus, for a proper effect of vitamin C on neurogenesis and differentiation, its function as an enzymatic cofactor, modulator of gene expression and antioxidant is essential, as is proper recycling from DHA to AA by various supporting cells in the CNS.

Radial glia and radial glia-like cells: Their role in neurogenesis and regeneration.

Radial glia is a cell type traditionally associated with the developing nervous system, particularly with the formation of cortical layers in the mammalian brain. Nonetheless, some of these cells, or closely related types, called radial glia-like cells are found in adult central nervous system structures, functioning as neurogenic progenitors in normal homeostatic maintenance and in response to injury. The heterogeneity of radial glia-like cells is nowadays being probed with molecular tools, primarily by the expression of specific genes that define cell types. Similar markers have identified radial glia-like cells in the nervous system of non-vertebrate organisms. In this review, we focus on adult radial glia-like cells in neurogenic processes during homeostasis and in response to injury. We highlight our results using a non-vertebrate model system, the echinoderm Holothuria glaberrima where we have described a radial glia-like cell that plays a prominent role in the regeneration of the holothurian central nervous system.

Toxoplasma gondii infection impairs radial glia differentiation and its potential to modulate brain microvascular endothelial cell function in the cerebral cortex.

Congenital toxoplasmosis is a parasitic disease that occurs due vertical transmission of the protozoan Toxoplasma gondii (T. gondii) during pregnancy. The parasite crosses the placental barrier and reaches the developing brain, infecting progenitor, glial, neuronal and vascular cell types. Although the role of Radial glia (RG) neural stem cells in the development of the brain vasculature has been recently investigated, the impact of T. gondii infection in these events is not yet understood. Herein, we studied the role of T. gondii infection on RG cell function and its interaction with endothelial cells. By infecting isolated RG cultures with T. gondii tachyzoites, we observed a cytotoxic effect with reduced numbers of RG populations together with decrease neuronal and oligodendrocyte progenitor populations. Conditioned medium (CM) from RG control cultures increased ZO-1 protein levels and organization on endothelial bEnd.3 cells membranes, which was impaired by CM from infected RG, accompanied by decreased trans-endothelial electrical resistance (TEER). ELISA assays revealed reduced levels of anti-inflammatory cytokine TGF-ß1 in CM from T. gondii-infected RG cells. Treatment with recombinant TGF-ß1 concomitantly with CM from infected RG cultures led to restoration of ZO-1 staining in bEnd.3 cells. Congenital infection in Swiss Webster mice led to abnormalities in the cortical microvasculature in comparison to uninfected embryos. Our results suggest that infection of RG cells by T. gondii negatively modulates cytokine secretion, which might contribute to endothelial loss of barrier properties, thus leading to impairment of neurovascular interaction establishment.

Radial Glia-endothelial Cells' Bidirectional Interactions Control Vascular Maturation and Astrocyte Differentiation: Impact for Blood-brain Barrier Formation.

BACKGROUND: In the developing cerebral cortex, Radial Glia (RG) multipotent neural stem cell, among other functions, differentiate into astrocytes and serve as a scaffold for blood vessel development. After some time, blood vessel Endothelial Cells (ECs) become associated with astrocytes to form the neurovascular Blood-Brain Barrier (BBB) unit. OBJECTIVE: Since little is known about the mechanisms underlying bidirectional RG-ECs interactions in both vascular development and astrocyte differentiation, this study investigated the impact of interactions between RG and ECs mediated by secreted factors on EC maturation and gliogenesis control. METHODS: First, we demonstrated that immature vasculature in the murine embryonic cerebral cortex physically interacts with Nestin positive RG neural stem cells in vivo. Isolated Microcapillary Brain Endothelial Cells (MBEC) treated with the conditioned medium from RG cultures (RG-CM) displayed decreased proliferation, reduction in the protein levels of the endothelial tip cell marker Delta-like 4 (Dll4), and decreased expression levels of the vascular permeability associated gene, plasmalemma vesicle-associated protein-1 (PLVAP1). These events were also accompanied by increased levels of the tight junction protein expression, zonula occludens-1 (ZO-1). RESULTS: Finally, we demonstrated that isolated RG cells cultures treated with MBEC conditioned medium promoted the differentiation of astrocytes in a Vascular Endothelial Growth Factor-A (VEGF-A) dependent manner. CONCLUSION: These results suggest that the bidirectional interaction between RG and ECs is essential to induce vascular maturation and astrocyte generation, which may be an essential cell-cell communication mechanism to promote BBB establishment.

Astrocytes and the TGF-ß1 Pathway in the Healthy and Diseased Brain: a Double-Edged Sword.

Transforming growth factor betas (TGF-ßs) are known as multifunctional growth factors that participate in the regulation of key events of development, disease, and tissue repair. In the brain, TGF-ß1 has been widely recognized as an injury-related cytokine, particularly associated with astrocyte scar formation in response to brain injury. In the last decade, however, evidence has indicated that in addition to its role in brain injury, TGF-ß1 might be a crucial regulator of cell survival and differentiation, brain homeostasis, angiogenesis, memory formation, and neuronal plasticity. In this review, we will discuss the emerging scenario of TGF-ß1 as a key regulator of astrocyte differentiation and function and the implications of TGF-ß1 as a novel mediator of cellular interactions in the central nervous system. First, we will discuss the cellular and molecular basis underlying the effect of TGF-ß on astrocyte generation and its impact on angiogenesis and blood-brain barrier function. Then, we will focus on the role of astrocytes in the development and remodeling of synapses and the role of TGF-ß1 as a new mediator of these events. Furthermore, we present seminal data that contributed to the emerging concept that astrocyte dysfunction might be associated with neurodegenerative diseases, with a special focus on Alzheimer's disease, and discuss the pros and cons of TGF-ß signaling deficits in these processes. Finally, we argue that understanding how astrocytic signals, such as TGF-ß1, regulate brain function might offer new insights into human learning, memory, and cognition, and ultimately, this understanding may provide new targets for the treatment of neurological diseases.

Radial Glia Cells Control Angiogenesis in the Developing Cerebral Cortex Through TGF-ß1 Signaling.

Neuroangiogenesis in the developing central nervous system is controlled by interactions between endothelial cells (ECs) and radial glia (RG) neural stem cells, although RG-derived molecules implicated in these events are not fully known. Here, we investigated the role of RG-secreted TGF-ß1, in angiogenesis in the developing cerebral cortex. By isolation of murine microcapillary brain endothelial cells (MBECs), we demonstrate that conditioned medium from RG cultures (RG-CM) promoted MBEC migration and formation of vessel-like structures in vitro, in a TGF-ß1-dependent manner. These events were followed by endothelial regulation of GPR124 and BAI-1 gene expression by RG-CM. Proteome profile of RG-CM identified angiogenesis-related molecules IGFBP2/3, osteopontin, endostatin, SDF1, fractalkine, TIMP1/4, Ang-1, pentraxin3, and Cyr61, some of them modulated by TGF-ß1 induction. In vivo gain and loss of function assays targeting RG cells demonstrates a specific TGF-ß1-dependent control of blood vessels branching in the cerebral cortex. Together, our results point to TGF-ß1 signaling pathway as a potential mediator of the RG-EC interactions and shed light to the key role of RG in paving the brain vascular network.

Spinal Cord Stem Cells In Their Microenvironment: The Ependyma as a Stem Cell Niche.

The ependyma of the spinal cord is currently proposed as a latent neural stem cell niche. This chapter discusses recent knowledge on the developmental origin and nature of the heterogeneous population of cells that compose this stem cell microenviroment, their diverse physiological properties and regulation. The chapter also reviews relevant data on the ependymal cells as a source of plasticity for spinal cord repair.

Apical Polarization of SVCT2 in Apical Radial Glial Cells and Progenitors During Brain Development.

During brain development, radial glial (RG) cells and the different progenitor subtypes are characterized by their bipolar morphology that includes an ovoid cell body and one or two radial processes that span across the developing cerebral wall. Different cells transport the reduced form of vitamin C, ascorbic acid (AA), using sodium-dependent ascorbic acid cotransporters (SVCT1 or SVCT2). SVCT2 is mainly expressed in the nervous system (CNS); however, its localization in the central nervous system during embryonic development along with the mechanism by which RG take up vitamin C and its intracellular effects is unknown. Thus, we sought to determine the expression and localization of SVCT2 during CNS development. SVCT2 is preferentially localized in the RG body at the ventricular edge of the cortex during the neurogenic stage (E12 to E17). The localization of SVCT2 overexpressed by in utero electroporation of E14 embryos is consistent with ventricular polarization. A similar distribution pattern was observed in human brain tissue sections at 9 weeks of gestation; however, SVCT2 immunoreaction was also detected in the inner and outer subventricular zone (SVZ). Finally, we used C17.2 neural stem cell line, J1ES cells and primary cell cultures derived from the brain cortex to analyze functional SVCT2 activity, AA effects in progenitor cells bipolar morphology, and SVCT2 expression levels in different culture conditions. Our results indicate that basal RG cells and apical intermediate and subapical progenitors are the main cell types expressing SVCT2 in the lissencephalic brain. SVCT2 was mainly detected in the apical region of the ventricular zone cells, contacting the cerebrospinal fluid. In gyrencephalic brains, SVCT2 was also detected in progenitor cells located in the inner and outer SVZ. Finally, we defined that AA has a strong radializing (bipolar morphology) effect in progenitor cells in culture and the differentiation condition modulates SVCT2 expression.

Corrigendum: TGF-ß1 promotes cerebral cortex radial glia-astrocyte differentiation in vivo.

[This corrects the article on p. 393 in vol. 8, PMID: 25484855.].

TGF-ß1 promotes cerebral cortex radial glia-astrocyte differentiation in vivo.

The major neural stem cell population in the developing cerebral cortex is composed of the radial glial cells, which generate glial cells and neurons. The mechanisms that modulate the maintenance of the radial glia (RG) stem cell phenotype, or its differentiation, are not yet completely understood. We previously demonstrated that the transforming growth factor-ß1 (TGF-ß1) promotes RG differentiation into astrocytes in vitro (Glia 2007; 55:1023-33) through activation of multiple canonical and non-canonical signaling pathways (Dev Neurosci 2012; 34:68-81). However, it remains unknown if TGF-ß1 acts in RG-astrocyte differentiation in vivo. Here, we addressed the astrogliogenesis induced by TGF-ß1 by using the intraventricular in utero injection in vivo approach. We show that injection of TGF-ß1 in the lateral ventricles of E14,5 mice embryos resulted in RG fibers disorganization and premature gliogenesis, evidenced by appearance of GFAP positive cells in the cortical wall. These events were followed by decreased numbers of neurons in the cortical plate (CP). Together, we also described that TGF-ß1 actions are region-dependent, once RG cells from dorsal region of the cerebral cortex demonstrated to be more responsive to this cytokine compared with RG from lateral cortex either in vitro as well as in vivo. Our work demonstrated that TGF-ß1 is a critical cytokine that regulates RG fate decision and differentiation into astrocytes in vitro and in vivo. We also suggest that RG cells are heterogeneous population that acts as distinct targets of TGF-ß1 during cerebral cortex development.

The inner lining of the reptilian brain: a heterogeneous cellular mosaic.

The ependymal layer is a preserved structure across vertebrates but its functional significance remains poorly understood. Modern studies emphasize the role played by radial glia (RG) as neurogenic progenitors. We speculated that the cells lining the prosencephalon ventricles of freshwater turtles may have retained key features of RG. To test this idea, we applied an approach that combined cellular, molecular, fine structural, and electrophysiological techniques. In the prosencephalon of juvenile turtles, we found cells with typical radial morphology that expressed four RG proteins: glial fibrillary acidic protein (GFAP), vimentin, S100/S100ß and brain lipid-binding protein (BLBP). Most of these cells expressed the transcription factor Sox2 but few co-expressed Pax6. One type of RG had their somata close to the ventricle lumen and bear multiple cilia. A second class with cell bodies far from the lumen was usually uniciliated. RGs had low input resistances, passive properties and were coupled via Cx43 at the level of the cell bodies and radial processes. A third kind of cell was uncoupled, expressed neuronal proteins (HuC/D and NeuN) and fired spikes. The differential expression of HuC/D and NeuN together with their electrophysiological properties suggested various maturational stages. The occurrence of ependymal patches with a high density of 5-bromo-2-deoxyuridine (BrdU) labeled cells provides evidence of the proliferative capability of ependymal RG. Our data support the view that RG have retained key properties of neuroepithelial cells. The maintenance of proliferating RG could be also related with the outstanding endogenous ability of lower vertebrates for self-repair after injury.

Typical and atypical stem cells in the brain, vitamin C effect and neuropathology

Stem cells are considered a valuable cellular resource for tissue replacement therapies in most brain disorders. Stem cells have the ability to self-replicate and differentiate into numerous cell types, including neurons, oligodendrocytes and astrocytes. As a result, stem cells have been considered the "holy grail" of modern medical neuroscience. Despite their tremendous therapeutic potential, little is known about the mechanisms that regulate their differentiation. In this review, we analyze stem cells in embryonic and adult brains, and illustrate the differentiation pathways that give origin to most brain cells. We also evaluate the emergent role of the well known anti-oxidant, vitamin C, in stem cell differentiation. We believe that a complete understanding of all molecular players, including vitamin C, in stem cell differentiation will positively impact on the use of stem cell transplantation for neurodegenerative diseases.

Nestin down-regulation of cortical radial glia is delayed in rats submitted to recurrent status epilepticus during early postnatal life / Atraso no desaparecimento da nestina na glia radial cortical de ratos submetidos a recorrentes status epilepticus durante o desenvolvimento pós-natal precoce

OBJECTIVE: Nestin is temporarily expressed in several tissues during development and it is replaced by other protein types during cell differentiation process. This unique property allows distinguishing between undifferentiated and differentiated cells. This study was delineated to analyze the temporal pattern of nestin expression in cortical radial glial cells of rats during normal development and of rats submitted to recurrent status epilepticus (SE) in early postnatal life (P). METHOD: Experimental rats were submitted to pilocarpine-induced SE on P7-9. The cortical temporal profile of nestin was studied by immunohistochemistry at multiple time points (P9, P10, P12, P16, P30 and P90). RESULTS: We observed delayed nestin down-regulation in experimental rats of P9, P10, P12 and P16 groups. In addition, few radial glial cells were still present only in P21 experimental rats. CONCLUSION: Our results suggested that SE during early postnatal life alters normal maturation during a critical period of brain development.

OBJETIVO: A nestina, temporariamente expressa em diversos tecidos durante o desenvolvimento, é substituída no processo de diferenciação celular, o que permite a distinção entre células diferenciadas e indiferenciadas. O objetivo deste estudo foi verificar o padrão temporal da expressão da nestina nas células da glia radial cortical de ratos durante o desenvolvimento normal e nos ratos submetidos a sucessivos status epilepticus (SE) no periodo pós-natal precoce (P). MÉTODO: Os animais foram submetidos ao SE induzido pela pilocarpina em P7-9. O perfil temporal da nestina foi estudado por imuno-histoquímica em P9, P10, P12, P16, P30 e P90. RESULTADOS: Nos ratos experimentais, observamos atraso no desaparecimento da nestina nos grupos P9, P10, P12 e P16. Ainda, encontramos algumas glias radiais corticais apenas em P21 experimental. CONCLUSÃO: Nossos resultados sugerem que o SE durante o desenvolvimento pós-natal precoce altera o processo de maturação durante um periodo crítico do desenvolvimento encefálico.