Neuron–Astroglial Interactions in Cell-Fate Commitment and Maturation in the Central Nervous System

Neuron–astroglia interactions play a key role in several events of brain development, such as neuronal generation, migration, survival, and differentiation; axonal growth; and synapse formation and function. While there is compelling evidence of the effects of astrocyte factors on neurons, their effects on astrocytes have not been fully determined. In this review, we will focus on the role of neurons in astrocyte generation and maturation. Further, we highlight the great heterogeneity and diversity of astroglial and neural progenitors such as radial glia cells, and discuss the importance of the variety of cellular interactions in controlling the structural and functional organization of the brain. Finally, we present recent data on a new role of astrocytes in neuronal maturation, as mediators of the action of biolipids in the cerebral cortex. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership, by briefly discussing the emerging view of how neuron-astrocyte dysfunctions might be associated with neurodegenerative diseases and neurological disorders.     

Astrocyte Mitochondria in White-Matter Injury

This review summarizes the diverse structure and function of astrocytes to describe the bioenergetic versatility required of astrocytes that are situated at different locations. The intercellular domain of astrocyte mitochondria defines their roles in supporting and regulating astrocyte-neuron coupling and survival against ischemia. The heterogeneity of astrocyte mitochondria, and how subpopulations of astrocyte mitochondria adapt to interact with other glia and regulate axon function, require further investigation. It has become clear that mitochondrial permeability transition pores play a key role in a wide variety of human diseases, whose common pathology may be based on mitochondrial dysfunction triggered by Ca²⁺ and potentiated by oxidative stress. Reactive oxygen species cause axonal degeneration and a reduction in axonal transport, leading to axonal dystrophies and neurodegeneration including Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington’s disease. Developing new tools to allow better investigation of mitochondrial structure and function in astrocytes, and techniques to specifically target astrocyte mitochondria, can help to unravel the role of mitochondrial health and dysfunction in a more inclusive context outside of neuronal cells. Overall, this review will assess the value of astrocyte mitochondria as a therapeutic target to mitigate acute and chronic injury in the CNS.

     

Low temperature induced de-differentiation of astrocytes

Radial glial cells are astrocyte precursors, which are transiently present in the developing central nervous system and transform eventually into astrocytes in the cerebral cortex and into Bergmann glia in the cerebellum. Previous reports indicate that the transformation from radial glia to astrocytes can be reversed by diffusible chemical signals derived from embryonic forebrain in vitro and by freezing injury in vivo. But there is no direct evidence proving that mature astrocytes can de-differentiate into radial glial cells. Here we show that purified astrocytes could de-differentiate into radial glial-like cells (RGLCs) in vitro with freeze-thaw stimulation. RGLCs had the expression of markers for radial glia including Nestin and Pax6, and astrocyte markers, the glial fibrillary acidic protein and Vimentin. Cortical neurons, when co-cultured with RGLCs, migrated along the processes of RGLCs at an average speed of 26.26 +/- 3.36 microm/h. Moreover, the proliferation of RGLCs was significantly promoted by epidermal growth factor (EGF) at the concentration of 10-30 ng/ml. These results reveal that low temperature induces astrocytes to de-differentiate into immature RGLCs, which provides an in vitro model to investigate mechanisms of astroglial cells de-differentiation.     

The monolayer formation of Bergmann glial cells is regulated by Notch/RBP-J signaling

The Bergmann glia is a unipolar astrocyte in the cerebellar cortex, displaying a tight association with Purkinje cells. The cell bodies of Bergmann glia are located in a row around Purkinje cell somata; they extend radially arranged Bergmann fibers which enwrap the synapses on the Purkinje cell dendrites. It is well known that Bergmann glial somata migrate from the ventricular zone through the mantle zone, forming an epithelium-like lining in the Purkinje cell layer during development. However, the mechanism of the monolayer formation of Bergmann glia is poorly understood. Several reports have suggested that Notch signaling plays instructive roles in promoting the identities of several types of glial cells, including Bergmann glia. Moreover, Notch receptors are expressed in Bergmann glia during development. Here, we have deleted the Notch1, Notch2 and RBP-J genes in the Bergmann glia by GFAP-driven, Cre-mediated recombination, to study the role of Notch-RBP-J-signaling in the monolayer formation of Bergmann glia. Notch1/2- and RBP-J-conditional mutant mice showed disorganization of Bergmann fibers, irregularities of the Bergmann glial lining and aberrant localization of Bergmann glia in the molecular layer. Thus, Notch-RBP-J signaling plays crucial roles in the monolayer formation and morphogenesis of Bergmann glia.     

Beta1 integrin activates Rac1 in Schwann cells to generate radial lamellae during axonal sorting and myelination

Myelin is a multispiraled extension of glial membrane that surrounds axons. How glia extend a surface many-fold larger than their body is poorly understood. Schwann cells are peripheral glia and insert radial cytoplasmic extensions into bundles of axons to sort, ensheath, and myelinate them. Laminins and beta1 integrins are required for axonal sorting, but the downstream signals are largely unknown. We show that Schwann cells devoid of beta1 integrin migrate to and elongate on axons but cannot extend radial lamellae of cytoplasm, similar to cells with low Rac1 activation. Accordingly, active Rac1 is decreased in beta1 integrin-null nerves, inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells of transgenic mice delays axonal sorting and impairs myelination. Finally, expressing active Rac1 in beta1 integrin-null nerves improves sorting. Thus, increased activation of Rac1 by beta1 integrins allows Schwann cells to switch from migration/elongation to the extension of radial membranes required for axonal sorting and myelination.     

Development of midline glial populations at the corticoseptal boundary

Three midline glial populations are found at the corticoseptal boundary: the glial wedge (GW), glia within the indusium griseum (IGG), and the midline zipper glia (MG). Two of these glial populations are involved in axonal guidance at the cortical midline, specifically development of the corpus callosum. Here we investigate the phenotypic and molecular characteristics of each population and determine whether they are generated at the same developmental stage. We find that the GW is derived from the radial glial scaffold of the cortex. GW cells initially have long radial processes that extend from the ventricular surface to the pial surface, but by E15 loose their pial attachment and extend only part of the way to the pial surface. Later in development the radial morphology of cells within the GW is replaced by multipolar astrocytes, providing supportive evidence that radial glia can transform into astrocytes. IGG and MG do not have a radial morphology and do not label with the radial glial markers, Nestin and RC2. We conclude that the GW and IGG have different morphological and molecular characteristics and are born at different stages of development. IGG and MG have many phenotypic and molecular characteristics in common, indicating that they may represent a common population of glia that becomes spatially distinct by the formation of the corpus callosum.     

A unified hypothesis on the lineage of neural stem cells

For many years, it was assumed that neurons and glia in the central nervous system were produced from two distinct precursor pools that diverged early during embryonic development. This theory was partially based on the idea that neurogenesis and gliogenesis occurred during different periods of development, and that neurogenesis ceased perinatally. However, there is now abundant evidence that neural stem cells persist in the adult brain and support ongoing neurogenesis in restricted regions of the central nervous system. Surprisingly, these stem cells have the characteristics of fully differentiated glia. Neuroepithelial stem cells in the embryonic neural tube do not show glial characteristics, raising questions about the putative lineage from embryonic to adult stem cells. In the developing brain, radial glia have long been known to produce cortical astrocytes, but recent data indicate that radial glia might also divide asymmetrically to produce cortical neurons. Here we review these new developments and propose that the stem cells in the central nervous system are contained within the neuroepithelial --> radial glia --> astrocyte lineage.     

The effect of extrinsic Wnt/β‐catenin signaling in Muller glia on retinal ganglion cell neurite growth

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β‐catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co‐cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt‐dependent target proteins that may mediate the axonal growth.     

Glial Fibrillary Acidic Protein Expression in Rat Brain and in Radial Glia Culture Is Delayed by Prenatal Ethanol Exposure

Abstract: The alterations in astrocyte proliferation and differentiation induced by prenatal exposure to alcohol (PEA) suggest that ethanol exposure affects the radial glial cells, the main astrocytic precursors. We have investigated the effects of ethanol on the early stages of astrogliogenesis by analyzing the developmental pattern of vimentin and glial fibrillary acidic protein (GFAP) immunoreactivity and their mRNA levels during embryonic/fetal brain development and in radial glia in primary culture. GFAP appeared late in gestation and at day 5 of culture of radial glial, whereas GFAP mRNA was first detected on fetal day 15 and increased in content on fetal day 21. In contrast, the levels of vimentin and its mRNA were high at fetal day 15 but decreased on day 21. Alcohol exposure delays the appearance of GFAP and its mRNA and significantly decreases the GFAP expression in fetal brain and in primary culture of radial glial. In addition, some morphological alterations were observed in PEA glial cells in culture. These results demonstrate that astroglial precursor cells are damaged by prenatal exposure to ethanol and suggest that abnormalities in the astrogliogenesis may underlie the disruption in neuronal migration and other CNS alterations observed after prenatal ethanol exposure.     

Six3 is required for ependymal cell maturation

Ependymal cells are part of the neurogenic niche in the adult subventricular zone of the lateral ventricles, where they regulate neurogenesis and neuroblast migration. Ependymal cells are generated from radial glia cells during embryonic brain development and acquire their final characteristics postnatally. The homeobox gene Six3 is expressed in ependymal cells during the formation of the lateral wall of the lateral ventricles in the brain. Here, we show that Six3 is necessary for ependymal cell maturation during postnatal stages of brain development. In its absence, ependymal cells fail to suppress radial glia characteristics, resulting in a defective lateral wall, abnormal neuroblast migration and differentiation, and hydrocephaly.     

Tissue-specific neuro-glia interactions determine neurite differentiation in ganglion cells

Guided formation and extension of axons versus dendrites is considered crucial for structuring the nervous system. In the chick visual system, retinal ganglion cells (RGCs) extend their axons into the tectum opticum, but not into glial somata containing retina layers. We addressed the question whether the different glia of retina and tectum opticum differentially affect axon growth. Glial cells were purified from retina and tectum opticum by complement-mediated cytolysis of non-glial cells. RGCs were purified by enzymatic delayering from flat mounted retina. RGCs were seeded onto retinal versus tectal glia monolayers. Subsequent neuritic differentiation was analysed by immunofluorescence microscopy and scanning electron microscopy. Qualitative and quantitative evaluation revealed that retinal glia somata inhibited axons. Time-lapse video recording indicated that axonal inhibition was based on the collapse of lamellipodia- and filopodia-rich growth cones of axons. In contrast to retinal glia, tectal glia supported axonal extension. Notably, retinal glia were not inhibitory for neurons in general, because in control experiments axon extension of dorsal root ganglia was not hampered. Therefore, the axon inhibition by retinal glia was neuron type-specific. In summary, the data demonstrate that homotopic (retinal) glia somata inhibit axonal outgrowth of RGCs, whereas heterotopic (tectal) glia of the synaptic target area support RGC axon extension. The data underscore the pivotal role of glia in structuring the developing nervous system.     

Boundaries defined by adhesion molecules during development of the cerebral cortex: the J1/tenascin glycoprotein in the mouse somatosensory cortical barrel field

The distribution of the 200/220 KDa J1 glycoprotein (J1-200/220), within the developing vibrissae-related barrel field of the mouse somatosensory cortex, was studied by immunocytochemistry using a monoclonal antibody. J1-200/220, a member of the L2/HNK-1 family of adhesion molecules, also appears to be the mouse homologue of tenascin. J1/tenascin-positive barrel-like structures are visible in the somatosensory cortex between 24 and 48 hr after birth, with the molecule present in prospective barrel boundaries. Immunoelectronmicroscopy reveals labeling that is associated with glial and neuronal plasma membranes, as well as glial end-feet on blood vessels. A possible major source of J1/tenascin expression at this time is astrocyte precursor cells and radial glia. In the putative astrocyte precursor cells, immunolabeling was observed within organelles including the Golgi apparatus. At P6-7 J1/tenascin is most prevalent within prospective interbarrel septae. J1/tenascin-positive barrel boundaries are barely visible on P9 and not observed on P16. The findings indicate that J1/tenascin represents a major component of previously described "hidden" boundaries that we have seen during development using other methodologies. The expression of adhesion molecule-rich boundaries during the critical stages of barrel field formation indicates roles for such molecules during specific cerebral cortical pattern formation events.     

Spatially discrete FGF-mediated signalling directs glial morphogenesis.

Neurons provide critical signals that regulate both the number and differentiation of glia. In addition, glia are attracted to and enwrap neuronal axonal processes. FGF-like signalling is thought to be one of the many potential axon-derived morphogenetic signals, however, the multiple roles of FGFs have made experimental tests of these signals difficult in vivo. In the Drosophila FGF receptor mutant heartless, glia migrate to axons, but fail to elongate around them. This study shows that in the similar but larger grasshopper CNS, FGF signalling is likely to mediate one step in the close interaction between glia and axons. FGF2-coated beads attract glia in the CNS and compete with axons for their resident, enwrapped glia. In addition, bath applied FGF2 causes mature axonal glia, which normally enwrap axon tracts, to round up. FGF2 activates the product of the grasshopper heartless FGF receptor gene and probably interferes with the normal function of an endogenous axon-associated FGF-like molecule. It is proposed that insect axons provide a critical spatially restricted FGF-like signal that induces glia to enwrap them.     

Radial glial cell development and transformation are disturbed in reeler forebrain

Radial glia are among the earliest cell types to differentiate in the developing mammalian forebrain. Glial fibers span the early cortical wall, forming a dense scaffold; this persists throughout corticogenesis, providing a cellular substrate which supports and directs the migration of young neurons. Although the mechanisms regulating radial glial cell development are poorly understood, a secreted cortical radial glial differentiation signal was recently identified in the embryonic mouse forebrain. This signal is abundant at the time radial glia function to support neuronal migration, and down-regulated perinatally, when radial glia are known to undergo transformation into astrocytes. Therefore, it seems that this signal functions as a radial glial maintenance factor, the availability of which regulates the phenotype of cortical astroglia. Here the differentiation signal is further characterized as RF60, a protein with a molecular weight of approximately 60 kD. In addition, the neurologic mutant mouse reeler provides a genetic model for analysis of RF60 function. Radial glia in reeler cortex are shown to be poorly differentiated and the radial scaffold is shown to be maintained for a shorter time than normal. Furthermore, although astroglial cells from normal cortex are induced to elaborate a radial phenotype by RF60, reeler astroglia show an impaired differentiation response to this. These findings suggest that an intrinsic defect in glial differentiation contributes to the phenotype of abnormal cortical lamination seen in reeler mouse, and indicate that RF60 may play a critical role in normal cortical patterning. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 459–472, 1997     

Lineage of radial glia in the chicken optic tectum.

In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following. (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor. (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7). Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce. (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives. (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.     

PTEN deletion in Bergmann glia leads to premature differentiation and affects laminar organization

Development of the central nervous system is controlled by both intrinsic and extrinsic signals that guide neuronal migration to form laminae. Although defects in neuronal mobility have been well documented as a mechanism for abnormal laminar formation, the role of radial glia, which provide the environmental cues, in modulating neuronal migration is less clear. We provide evidence that loss of PTEN in Bergmann glia leads to premature differentiation of this crucial cell population and subsequently to extensive layering defects. Accordingly, severe granule neuron migration defects and abnormal laminar formation are observed. These results uncover an unexpected role for PTEN in regulating Bergmann glia differentiation, as well as the importance of time-dependent Bergmann glia differentiation during cerebellar development.