Radial Glia-like Stem Cells Research Articles

Aging Modulates the Ability of Quiescent Radial Glia-Like Stem Cells in the Hippocampal Dentate Gyrus to be Recruited into Division by Pro-neurogenic Stimuli.

A delicate balance between quiescence and division of the radial glia-like stem cells (RGLs) ensures continuation of adult hippocampal neurogenesis (AHN) over the lifespan. Transient or persistent perturbations of this balance due to a brain pathology, drug administration, or therapy can lead to unfavorable long-term outcomes such as premature depletion of the RGLs, decreased AHN, and cognitive deficit. Memantine, a drug used for alleviating the symptoms of Alzheimer's disease, and electroconvulsive seizure (ECS), a procedure used for treating drug-resistant major depression or bipolar disorder, are known strong AHN inducers; they were earlier demonstrated to increase numbers of dividing RGLs. Here, we demonstrated that 1-month stimulation of quiescent RGLs by either memantine or ECS leads to premature exhaustion of their pool and altered AHN at later stages of life and that aging of the brain modulates the ability of the quiescent RGLs to be recruited into the cell cycle by these AHN inducers. Our findings support the aging-related divergence of functional features of quiescent RGLs and have a number of implications for the practical assessment of drugs and treatments with respect to their action on quiescent RGLs at different stages of life in animal preclinical studies.

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.

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.

Radial glial cells in the adult dentate gyrus: what are they and where do they come from?

Adult neurogenesis occurs in the dentate gyrus in the mammalian hippocampus. These new neurons arise from neural precursor cells named radial glia-like cells, which are situated in the subgranular zone of the dentate gyrus. Here, we review the emerging topic of precursor heterogeneity in the adult subgranular zone. We also discuss how this heterogeneity may be established during development and focus on the embryonic origin of the dentate gyrus and radial glia-like stem cells. Finally, we discuss recently developed single-cell techniques, which we believe will be critical to comprehensively investigate adult neural stem cell origin and heterogeneity.

Morphological study of a connexin 43-GFP reporter mouse highlights glial heterogeneity, amacrine cells, and olfactory ensheathing cells.

Connexin 43 (Cx43) is the main astrocytic connexin and forms the basis of the glial syncytium. The morphology of connexin-expressing cells can be best studied in transgenic mouse lines expressing cytoplasmic fluorescent reporters, since immunolabeling the plaques can obscure the shapes of the individual cells. The Cx43kiECFP mouse generated by Degen et al. (FASEBJ 26:4576, 2012) expresses cytosolic ECFP and has previously been used to establish that Cx43 may not be expressed by all astrocytes within a population, and this can vary in a region-dependent way. To establish this mouse line as a tool for future astrocyte and connexin research, we sought to consolidate reporter authenticity, studying cell types and within-region population heterogeneity. Applying anti-GFP, all cell types related to astroglia were positive-namely, protoplasmic astrocytes in the hippocampus, cortex, thalamus, spinal cord, olfactory bulb, cerebellum with Bergmann glia and astrocytes also in the molecular layer, and retinal Müller cells and astrocytes. Labeled cell types further comprise white matter astrocytes, olfactory ensheathing cells, radial glia-like stem cells, retinal pigment epithelium cells, ependymal cells, and meningeal cells. We furthermore describe a retinal Cx43-expressing amacrine cell morphologically reminiscent of ON-OFF wide-field amacrine cells, representing the first example of a mammalian CNS neuron-expressing Cx43 protein. In double staining with cell type-specific markers (GFAP, S100ß, glutamine synthetase), Cx43 reporter expression in the hippocampus and cortex was restricted to GFAP+ astrocytes. Altogether, this mouse line is a highly reliable tool for studies of Cx43-expressing CNS cells and astroglial cell morphology. © 2017 Wiley Periodicals, Inc.

Neurotransmitter-Regulated Regeneration in the Zebrafish Retina.

SummaryCurrent efforts to repair damaged or diseased mammalian retinas are inefficient and largely incapable of fully restoring vision. Conversely, the zebrafish retina is capable of spontaneous regeneration upon damage using Müller glia (MG)-derived progenitors. Understanding how zebrafish MG initiate regeneration may help develop new treatments that prompt mammalian retinas to regenerate. We show that inhibition of γ-aminobutyric acid (GABA) signaling facilitates initiation of MG proliferation. GABA levels decrease following damage, and MG are positioned to detect decreased ambient levels and undergo dedifferentiation. Using pharmacological and genetic approaches, we demonstrate that GABAA receptor inhibition stimulates regeneration in undamaged retinas while activation inhibits regeneration in damaged retinas.

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.

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.

Capture of Neuroepithelial-Like Stem Cells from Pluripotent Stem Cells Provides a Versatile System for In Vitro Production of Human Neurons

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) provide new prospects for studying human neurodevelopment and modeling neurological disease. In particular, iPSC-derived neural cells permit a direct comparison of disease-relevant molecular pathways in neurons and glia derived from patients and healthy individuals. A prerequisite for such comparative studies are robust protocols that efficiently yield standardized populations of neural cell types. Here we show that long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from 3 hESC and 6 iPSC lines in two independent laboratories exhibit consistent characteristics including i) continuous expandability in the presence of FGF2 and EGF; ii) stable neuronal and glial differentiation competence; iii) characteristic transcription factor profile; iv) hindbrain specification amenable to regional patterning; v) capacity to generate functionally mature human neurons. We further show that lt-NES cells are developmentally distinct from fetal tissue-derived radial glia-like stem cells. We propose that lt-NES cells provide an interesting tool for studying human neurodevelopment and may serve as a standard system to facilitate comparative analyses of hESC and hiPSC-derived neural cells from control and diseased genetic backgrounds.

Distinct stages of adult hippocampal neurogenesis are regulated by running and the running environment

Hippocampal neurogenesis continues into adulthood in mammalian vertebrates, and in experimental rodent models it is powerfully stimulated by exposure to a voluntary running wheel. In this study, we demonstrate that exposure to a running wheel environment, in the absence of running, is sufficient to regulate specific aspects of hippocampal neurogenesis. Adult mice were provided with standard housing, housing enriched with a running wheel or housing enriched with a locked wheel (i.e., an environment comparable to that of running animals, without the possibility of engaging in running). We found that mice in the running wheel and locked wheel groups exhibited equivalent increases in proliferation within the neurogenic niche of the dentate gyrus; this included comparable increases in the proliferation of radial glia-like stem cells and the number of proliferating neuroblasts. However, only running animals displayed increased numbers of postmitotic neuroblasts and mature neurons. These results demonstrate that the running wheel environment itself is sufficient for promoting proliferation of early lineage hippocampal precursors, while running per se enables newly generated neuroblasts to survive and mature into functional hippocampal neurons. Thus, both running-independent and running-dependent stimuli are integral to running wheel-induced hippocampal neurogenesis.

Glial cells in adult neurogenesis

Adult neurogenesis is an exceptional feature of the adult brain and in an intriguing way bridges between neuronal and glial neurobiology. Essentially, all classes of glial cells are directly or indirectly linked to this process. Cells with astrocytic features, for example, serve as radial glia-like stem cells in the two neurogenic regions of the adult brain, the hippocampal dentate gyrus and the subventricular zone of the lateral ventricles, producing new neurons, create a microenvironment permissive for neurogenesis, and are themselves generated alongside the new neurons in an associated but independently regulated process. Oligodendrocytes are generated from precursor cells intermingled with those generating neurons in an independent lineage. NG2 cells have certain precursor cell properties and are found throughout the brain parenchyma. They respond to extrinsic stimuli and injury but do not generate neurons even though they can express some preneuronal markers. Microglia have positive and negative regulatory effects as constituents of the "neurogenic niche". Ependymal cells play incompletely understood roles in adult neurogenesis, but under certain conditions might exert (back-up) precursor cell functions. Glial contributions to adult neurogenesis can be direct or indirect and are mediated by mechanisms ranging from gap-junctional to paracrine and endocrine. As the two neurogenic regions differ between each other and both from the non-neurogenic rest of the brain, the question arises in how far regionalization of both the glia-like precursor cells as well as of the glial cells determines site-specific "neurogenic permissiveness." In any case, however, "neurogenesis" appears to be an essentially glial achievement.

The Pessimist's and Optimist's Views of Adult Neurogenesis

The reports by Bonaguidi etal. (in this issue of Cell) and Encinas etal. (in Cell Stem Cell) come to differing conclusions about whether and how the proliferation of radial glia-like stem cells of the adult hippocampus impacts their long-term potential for neurogenesis.

Preservation of positional identity in fetus-derived neural stem (NS) cells from different mouse central nervous system compartments.

Neural stem (NS) cells are a self-renewing population of symmetrically dividing multipotent radial glia-like stem cells, characterized by homogeneous expansion in monolayer. Here we report that fetal NS cells isolated from different regions of the developing mouse nervous system behave in a similar manner with respect to self-renewal and neuropotency, but exhibit distinct positional identities. For example, NS cells from the neocortex maintain the expression of anterior transcription factors, including Otx2 and Foxg1, while Hoxb4 and Hoxb9 are uniquely found in spinal cord-derived NS cells. This molecular signature was stable for over 20 passages and was strictly linked to the developmental stage of the donor, because only NS cells derived from E14.5 cortex, and not those derived from E12.5 cortex, carried a consistent transcription factor profile. We also showed that traits of this positional code are maintained during neuronal differentiation, leading to the generation of electrophysiologically active neurons, even if they do not acquire a complete neurochemical identity.

Differential Recovery of Neural Stem Cells in the Subventricular Zone and Dentate Gyrus After Ionizing Radiation

Radiation therapy is a widely used treatment for malignant central nervous system tumors. Mature neurons are terminally differentiated, whereas stem and progenitor cells have a prominent proliferative capacity and are therefore highly vulnerable to irradiation. Our aim was to investigate how cranial radiation in young rats would affect stem/progenitor cells in the two niches of adult neurogenesis, the subventricular zone (SVZ) and the dentate gyrus of the hippocampal formation. Nine weeks after irradiation we found that in irradiated animals, hippocampal neurogenesis was reduced to 5% of control levels. Similarly, the numbers of actively proliferating cells and radial glia-like stem cells (nestin+/glial fibrillary acidic protein [GFAP]+) in the dentate gyrus were reduced to 10% and 15% of control levels, respectively. In the irradiated olfactory bulb, neurogenesis was reduced to 40% of control levels, and the number of actively proliferating cells in the SVZ was reduced to 53% of control levels. However, the number of nestin+/GFAP+ cells in the SVZ was unchanged compared with controls. To evaluate the immediate response to the radiation injury, we quantified the amount of proliferation in the SVZ and dentate gyrus 1 day after irradiation. We found an equal reduction in proliferating cells both in dentate gyrus and SVZ. In summary, we show an initial response to radiation injury that is similar in both brain stem cell niches. However, the long-term effects on stem cells and neurogenesis in these two areas differ significantly: the dentate gyrus is severely affected long-term, whereas the SVZ appears to recover with time.

A subpopulation of precursor cells in the mouse dentate gyrus receives synaptic GABAergic input

Fate choice decision and the initiation of neuronal development occur in precursor cells of the adult dentate gyrus. We here show that the earliest signs of neuronal function can be detected in a nestin-positive progenitor cell population, which is morphologically distinct from the putative radial glia-like stem cells. Whereas radial glia-like stem cells had a glial physiological phenotype, this other cell population was more heterogeneous including cells with the ability to generate action potentials. These cells had only plump horizontal processes or lacked processes. Some of these cells received spontaneous and evoked synaptic input, which was sensitive to GABA A, but not to glutamate receptor antagonists. In radial glia-like stem cells, such a response was not observed. However, both types of precursor cells expressed functional GABA A and glutamate receptors and glutamate transporters. This early establishment of GABAergic innervation in adult neurogenesis thus mimics the pattern described for brain development.