Radial Glia-like Research Articles

Morphological classification of radial glia-like cells in the postnatal mouse subventricular zone.

The subventricular zone (SVZ) is one of the neurogenic regions of the adult mammalian brain. Neural stem cells (NSCs) in the SVZ have certain key features: they express glial fibrillary acidic protein (GFAP), proliferate slowly, have a radial glia-like (RG-L) morphology, and are in contact with the cerebrospinal fluid (CSF). NSCs have been isolated by FACS to analyse them, but their morphology has not been systematically examined. To address this knowledge gap, we sparsely labelled RG-L cells in the SVZ of neonatal mice by introducing via electroporation a plasmid expressing fluorescent protein under the control of the GFAP promoter. We then classified RG-L cells into three types (RG-L1, 2, and 3) based on their morphologies. RG-L1 cells had a basal process with some branches and numerous fine processes. RG-L2 cells had a basal process, but fewer branches and fine processes than RG-L1 cells. RG-L3 cells had one basal process that was almost free of branches and fine processes. Importantly, regardless of the cell type, about half of their somata resided on the basal side of the SVZ. Based on changes in their proportions during postnatal development and their expression of GFAP and cell proliferation markers at the adult stage, we speculated that NSCs change their morphologies during development/maturation and not all NSCs must always be in the apical SVZ or in contact with the CSF. Our results indicate that in addition to expression of markers for NSCs, the morphology is a critical feature to identify NSCs.

Glial Chloride Homeostasis Under Transient Ischemic Stress.

High water permeabilities permit rapid adjustments of glial volume upon changes in external and internal osmolarity, and pathologically altered intracellular chloride concentrations ([Cl–]int) and glial cell swelling are often assumed to represent early events in ischemia, infections, or traumatic brain injury. Experimental data for glial [Cl–]int are lacking for most brain regions, under normal as well as under pathological conditions. We measured [Cl–]int in hippocampal and neocortical astrocytes and in hippocampal radial glia-like (RGL) cells in acute murine brain slices using fluorescence lifetime imaging microscopy with the chloride-sensitive dye MQAE at room temperature. We observed substantial heterogeneity in baseline [Cl–]int, ranging from 14.0 ± 2.0 mM in neocortical astrocytes to 28.4 ± 3.0 mM in dentate gyrus astrocytes. Chloride accumulation by the Na+-K+-2Cl– cotransporter (NKCC1) and chloride outward transport (efflux) through K+-Cl– cotransporters (KCC1 and KCC3) or excitatory amino acid transporter (EAAT) anion channels control [Cl–]int to variable extent in distinct brain regions. In hippocampal astrocytes, blocking NKCC1 decreased [Cl–]int, whereas KCC or EAAT anion channel inhibition had little effect. In contrast, neocortical astrocytic or RGL [Cl–]int was very sensitive to block of chloride outward transport, but not to NKCC1 inhibition. Mathematical modeling demonstrated that higher numbers of NKCC1 and KCC transporters can account for lower [Cl–]int in neocortical than in hippocampal astrocytes. Energy depletion mimicking ischemia for up to 10 min did not result in pronounced changes in [Cl–]int in any of the tested glial cell types. However, [Cl–]int changes occurred under ischemic conditions after blocking selected anion transporters. We conclude that stimulated chloride accumulation and chloride efflux compensate for each other and prevent glial swelling under transient energy deprivation.

The transcription factor ZEB1 regulates stem cell self-renewal and cell fate in the adult hippocampus.

SummaryRadial glia-like (RGL) stem cells persist in the adult mammalian hippocampus, where they generate new neurons and astrocytes throughout life. The process of adult neurogenesis is well documented, but cell-autonomous factors regulating neuronal and astroglial differentiation are incompletely understood. Here, we evaluate the functions of the transcription factor zinc-finger E-box binding homeobox 1 (ZEB1) in adult hippocampal RGL cells using a conditional-inducible mouse model. We find that ZEB1 is necessary for self-renewal of active RGL cells. Genetic deletion of Zeb1 causes a shift toward symmetric cell division that consumes the RGL cell and generates pro-neuronal progenies, resulting in an increase of newborn neurons and a decrease of newly generated astrocytes. We identify ZEB1 as positive regulator of the ets-domain transcription factor ETV5 that is critical for asymmetric division.

Differential Change in Hippocampal Radial Astrocytes and Neurogenesis in Shorebirds With Contrasting Migratory Routes.

Little is known about environmental influences on radial glia-like (RGL) α cells (radial astrocytes) and their relation to neurogenesis. Because radial glia is involved in adult neurogenesis and astrogenesis, we investigated this association in two migratory shorebird species that complete their autumnal migration using contrasting strategies. Before their flights to South America, the birds stop over at the Bay of Fundy in Canada. From there, the semipalmated sandpiper (Calidris pusilla) crosses the Atlantic Ocean in a non-stop 5-day flight, whereas the semipalmated plover (Charadrius semipalmatus) flies primarily overland with stopovers for rest and feeding. From the hierarchical cluster analysis of multimodal morphometric features, followed by the discriminant analysis, the radial astrocytes were classified into two main morphotypes, Type I and Type II. After migration, we detected differential changes in the morphology of these cells that were more intense in Type I than in Type II in both species. We also compared the number of doublecortin (DCX)-immunolabeled neurons with morphometric features of radial glial–like α cells in the hippocampal V region between C. pusilla and C. semipalmatus before and after autumn migration. Compared to migrating birds, the convex hull surface area of radial astrocytes increased significantly in wintering individuals in both C. semipalmatus and C. pusilla. Although to a different extent we found a strong correlation between the increase in the convex hull surface area and the increase in the total number of DCX immunostained neurons in both species. Despite phylogenetic differences, it is of interest to note that the increased morphological complexity of radial astrocytes in C. semipalmatus coincides with the fact that during the migratory process over the continent, the visuospatial environment changes more intensely than that associated with migration over Atlantic. The migratory flight of the semipalmated plover, with stopovers for feeding and rest, vs. the non-stop flight of the semipalmated sandpiper may differentially affect radial astrocyte morphology and neurogenesis.

The polarity and properties of radial glia-like neural stem cells are altered by seizures with status epilepticus: Study using an improved mouse pilocarpine model of epilepsy.

In the adult mouse hippocampus, new neurons are produced by radial glia-like (RGL) neural stem cells in the subgranular zone, which extend their apical processes toward the molecular layer, and express the astrocyte marker glial fibrillary acidic protein, but not the astrocyte marker S100β. In rodent models of epilepsy, adult hippocampal neurogenesis was reported to be increased after acute and mild seizures, but to be decreased by chronic and severe epilepsy. In the present study, we investigated how the severity of seizures affects neurogenesis and RGL neural stem cells in acute stages of epilepsy, using an improved mouse pilocarpine model in which pilocarpine-induced hypothermia was prevented by maintaining body temperature, resulting in a high incidence rate of epileptic seizures and low rate of mortality. In mice that experienced seizures without status epilepticus (SE), the number of proliferating progenitors and immature neurons were significantly increased, whereas no changes were observed in RGL cells. In mice that experienced seizures with SE, the number of proliferating progenitors and immature neurons were unchanged, but the number of RGL cells with an apical process was significantly reduced. Furthermore, the processes of the majority of RGL cells extended inversely toward the hilus, and about half of the aberrant RGL cells expressed S100β. These results suggest that seizures with SE lead to changes in the polarity and properties of RGL neural stem cells, which may direct them toward astrocyte differentiation, resulting in the reduction of neural stem cells producing new granule cells. This also suggests the possibility that cell polarity of RGL stem cells is important for maintaining the stemness of adult neural stem cells.

ProNGF Is a Cell-Type-Specific Mitogen for Adult Hippocampal and for Induced Neural Stem Cells.

The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR , as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223-1237.

Loss of postnatal quiescence of neural stem cells through mTOR activation upon genetic removal of cysteine string protein-α

Neural stem cells continuously generate newborn neurons that integrate into and modify neural circuitry in the adult hippocampus. The molecular mechanisms that regulate or perturb neural stem cell proliferation and differentiation, however, remain poorly understood. Here, we have found that mouse hippocampal radial glia-like (RGL) neural stem cells express the synaptic cochaperone cysteine string protein-α (CSP-α). Remarkably, in CSP-α knockout mice, RGL stem cells lose quiescence postnatally and enter into a high-proliferation regime that increases the production of neural intermediate progenitor cells, thereby exhausting the hippocampal neural stem cell pool. In cell culture, stem cells in hippocampal neurospheres display alterations in proliferation for which hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway is the primary cause of neurogenesis deregulation in the absence of CSP-α. In addition, RGL cells lose quiescence upon specific conditional targeting of CSP-α in adult neural stem cells. Our findings demonstrate an unanticipated cell-autonomic and circuit-independent disruption of postnatal neurogenesis in the absence of CSP-α and highlight a direct or indirect CSP-α/mTOR signaling interaction that may underlie molecular mechanisms of brain dysfunction and neurodegeneration.

Fine processes of Nestin-GFP–positive radial glia-like stem cells in the adult dentate gyrus ensheathe local synapses and vasculature

Adult hippocampal neurogenesis relies on the activation of neural stem cells in the dentate gyrus, their division, and differentiation of their progeny into mature granule neurons. The complex morphology of radial glia-like (RGL) stem cells suggests that these cells establish numerous contacts with the cellular components of the neurogenic niche that may play a crucial role in the regulation of RGL stem cell activity. However, the morphology of RGL stem cells remains poorly described. Here, we used light microscopy and electron microscopy to examine Nestin-GFP transgenic mice and provide a detailed ultrastructural reconstruction analysis of Nestin-GFP-positive RGL cells of the dentate gyrus. We show that their primary processes follow a tortuous path from the subgranular zone through the granule cell layer and ensheathe local synapses and vasculature in the inner molecular layer. They share the ensheathing of synapses and vasculature with astrocytic processes and adhere to the adjacent processes of astrocytes. This extensive interaction of processes with their local environment could allow them to be uniquely receptive to signals from local neurons, glia, and vasculature, which may regulate their fate.

Heterogeneity of Radial Glia-Like Cells in the Adult Hippocampus.

Adult neurogenesis is tightly regulated by the neurogenic niche. Cellular contacts between niche cells and neural stem cells are hypothesized to regulate stem cell proliferation or lineage choice. However, the structure of adult neural stem cells and the contact they form with niche cells are poorly described. Here, we characterized the morphology of radial glia-like (RGL) cells, their molecular identity, proliferative activity, and fate determination in the adult mouse hippocampus. We found the coexistence of two morphotypes of cells with prototypical morphological characteristics of RGL stem cells: Type α cells, which represented 76% of all RGL cells, displayed a long primary process modestly branching into the molecular layer and type β cells, which represented 24% of all RGL cells, with a shorter radial process highly branching into the outer granule cell layer-inner molecular layer border. Stem cell markers were expressed in type α cells and coexpressed with astrocytic markers in type β cells. Consistently, in vivo lineage tracing indicated that type α cells can give rise to neurons, astrocytes, and type β cells, whereas type β cells do not proliferate. Our results reveal that the adult subgranular zone of the dentate gyrus harbors two functionally different RGL cells, which can be distinguished by simple morphological criteria, supporting a morphofunctional role of their thin cellular processes. Type β cells may represent an intermediate state in the transformation of type α, RGL stem cells, into astrocytes.

Hopx distinguishes hippocampal from lateral ventricle neural stem cells

In the adult dentate gyrus (DG) and in the proliferative zone lining the lateral ventricle (LV-PZ), radial glia-like (RGL) cells are neural stem cells (NSCs) that generate granule neurons. A number of molecular markers including glial fibrillary acidic protein (GFAP), Sox2 and nestin, can identify quiescent NSCs in these two niches. However, to date, there is no marker that distinguishes NSC origin of DG versus LV-PZ. Hopx, an atypical homeodomain only protein, is expressed by adult stem cell populations including those in the intestine and hair follicle. Here, we show that Hopx is specifically expressed in RGL cells in the adult DG, and these cells give rise to granule neurons. Assessed by non-stereological quantitation, Hopx-null NSCs exhibit enhanced neurogenesis evident by an increased number of BrdU-positive cells and doublecortin (DCX)-positive neuroblasts. In contrast, Sox2-positive, quiescent NSCs are reduced in the DG of Hopx-null animals and Notch signaling is reduced, as evidenced by reduced expression of Notch targets Hes1 and Hey2, and a reduction of the number of cells expressing the cleaved, activated form of the Notch1 receptor, the Notch intracellular domain (NICD) in Hopx-null DG. Surprisingly, Hopx is not expressed in RGL cells of the adult LV-PZ, and Hopx-expressing cells do not give rise to interneurons of the olfactory bulb (OB). These findings establish that Hopx expression distinguishes NSCs of the DG from those of the LV-PZ, and suggest that Hopx potentially regulates hippocampal neurogenesis by modulating Notch signaling.

Gadd45b Mediates Electroconvulsive Shock Induced Proliferation of Hippocampal Neural Stem Cells.

Electroconvulsive shock (ECS), also known as an electroconvulsive therapy (ECT), is an effective and safe treatment for neuropsychiatric disorders including pharmacoresistant major depressive disorder. Previous research in animal models suggests ECS efficacy is achieved by Gadd45b-mediated increases in adult hippocampal neurogenesis. The present study aims to delineate the role of Gadd45b in mediating proliferation of neural stem cell types including quiescent radial glia-like (RGL) and amplifying non-radial glia-like (non-RGL) neural precursors following ECS. RGL and non-RGL neural stem cell populations defined by co-localization of MCM2+ and nestin+ cells and morphologically by the presence of radial processes were stereologically analyzed. ECS increased hippocampal density of both quiescent RGLs and amplifying non-RGLs. Gadd45b mediates the action of ECS-induced proliferation through activation of quiescent neural stem cells.

Abstract WMP42: Remote Ischemic Stroke Decreases Quiescence and Increases Neurogenic Activation of Radial Glia-like Precursors in the Subgranular Zone of the Dentate Gyrus in Adult Mice

Adult neurogenesis occurs in two discrete neurogenic niches in the mammalian brain: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampal formation. Under physiologic conditions, neurogenesis in these regions is thought to be important for the maintenance and reorganization of interneurons in the olfactory bulb (SVZ), and the modulation and refinement of existing circuits in the dentate gyrus (SGZ). In rodents, stroke enhances neurogenesis in these regions, and stroke-induced neurogenesis appears to play a role in functional recovery. However, little is known about the behavior of quiescent vs. activated pools of neural stem cells in response to stroke, or the mechanisms of stroke-induced neurogenesis at the cellular level. Here we tested the hypothesis that stroke-induced neurogenesis involves the activation of quiescent stem cell pools, either alone or in combination with further differentiation of activated precursors. We used a genetic labeling strategy for in vivo lineage tracing of quiescent, nestin-expressing radial glia-like (RGL) precursors in the SGZ to elucidate the effect of remote focal ischemic infarction on RGLs at a clonal level. Unilateral infarction of the visual cortex by photothrombosis in transgenic mice caused an increase in the number of differentiated neuronal precursors and a decrease in the number of quiescent RGLs in the ipsilateral SGZ. Furthermore, among activated RGL clones, there was an increase in symmetric and neurogenic RGL divisions in response to stroke, with a concomitant loss of astrogliogenic RGL divisions. These preliminary results suggest that stroke may alter hippocampal neurogenesis from a distance by activating quiescent RGL pools in the SGZ through an as-of-yet unidentified mechanism and pushing them toward a neuronal fate. In turn, this may accelerate the time-dependent depletion of this RGL population. We speculate that this phenomenon contributes to cognitive changes associated with stroke and may represent a target for pharmacological intervention.

A circuit‐based gatekeeper for adult neural stem cell proliferation

Newborn neurons are generated in the adult hippocampus from a pool of self-renewing stem cells located in the subgranular zone (SGZ) of the dentate gyrus. Their activation, proliferation, and maturation depend on a host of environmental and cellular factors but, until recently, the contribution of local neuronal circuitry to this process was relatively unknown. In their recent publication, Song and colleagues have uncovered a novel circuit-based mechanism by which release of the neurotransmitter, γ-aminobutyric acid (GABA), from parvalbumin-expressing (PV) interneurons, can hold radial glia-like (RGL) stem cells of the adult SGZ in a quiescent state. This tonic GABAergic signal, dependent upon the activation of γ(2) subunit-containing GABA(A) receptors of RGL stem cells, can thus prevent their proliferation and subsequent maturation or return them to quiescence if previously activated. PV interneurons are thus capable of suppressing neurogenesis during periods of high network activity and facilitating neurogenesis when network activity is low.

Isolation of Radial Glia-Like Neural Stem Cells from Fetal and Adult Mouse Forebrain via Selective Adhesion to a Novel Adhesive Peptide-Conjugate

Preferential adhesion of neural stem cells to surfaces covered with a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]) provided a unique, rapid procedure for isolating radial glia-like cells from both fetal and adult rodent brain. Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of the adult brain maintained several radial glia-specific features including nestin, RC2 immunoreactivity and Pax6, Sox2, Blbp, Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Continuous proliferation allowed isolating one-cell derived clones of radial glia-like cells. All clones generated neurons, astrocytes and oligodendrocytes under appropriate inducing conditions. Electrophysiological characterization indicated that passive conductance with large delayed rectifying potassium current might be a uniform feature of non-induced radial glia-like cells. Upon induction, all clones gave rise to GABAergic neurons. Significant differences were found, however, among the clones in the generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes.

In Vivo Clonal Analysis Reveals Self-Renewing and Multipotent Adult Neural Stem Cell Characteristics

Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage tracing invivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multilineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal but ultimately leads to terminal astrocytic differentiation and RGL depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into invivo properties of adult neural stem cells.

The Alzheimer's disease drug memantine increases the number of radial glia‐like progenitor cells in adult hippocampus

New neurons are continuously generated in the hippocampus of the adult mammalian brain, and N-methyl-D-aspartate receptor (NMDA-R) antagonists have been found to increase the number of newly generated neurons in the dentate gyrus (DG) of the adult hippocampus. In this study, we examined the effect of memantine, an NMDA-R antagonist that is clinically used for the treatment of Alzheimer's disease, on primary progenitor cells exhibiting a radial glia-like (RGL) morphology in the DG. We injected 3-month-old mice with memantine (50 mg/kg body weight, intraperitoneally [i.p.]); 3 days later, we injected the mice with 5-bromo-2-deoxyuridine (BrdU; 75 mg/kg body weight, i.p.). We then counted the number of BrdU-labeled RGL progenitor cells in the DG 1 or 7 days after the BrdU-injection. The number of BrdU-labeled RGL progenitor cells had increased significantly by 5.1-fold on day 1 and by 13.7-fold on day 7 after BrdU-injection. Immunohistochemical staining revealed that the BrdU-labeled RGL progenitor cells expressed two primary progenitor cell marker proteins, nestin and Sox2. These results clearly demonstrated that memantine promotes the proliferation of RGL progenitor cells. We also found that memantine increased the ratio of horizontally aligned RGL progenitor cells, which are probably produced by symmetric division. These findings suggest that memantine increases the proliferation of primary progenitor cells and expands the primary progenitor cell pool in the adult hippocampus by stimulating symmetric division.

Further characterization of embryonic stem cell‐derived radial glial cells

Previously, we showed that radial glia-like (RG) cells differentiated from embryonic stem (ES) cells after retinoic acid induction (Liour and Yu, 2003: Glia 42:109-117). In the present study, we demonstrate that the production of RG cells from ES cells is independent of the neural differentiation protocol used. These ES cell-derived RG (ES-RG) cells are similar in morphology to RG cells in vivo and express several characteristic RG cell markers. The processes of these ES-RG cells are organized into radial arrays similar to the RG scaffold in developing CNS. Expression of Pax6, along with other circumstantial data, suggests that at least some of these ES-RG cells are neural progenitors. The progression of neurogenesis into gliogenesis during the in vitro neural differentiation of ES cells recapitulates the in vivo developmental process. The identification of two cell surface markers, SSEA-1 and GM1, on both the native embryonic RG cells and ES-RG cells, may facilitate purification of radial glial cells for future studies and cell therapy. Overall, our study suggests that differentiation of radial glial cells is a common pathway during the neural differentiation of ES cells.