Glial Signals Research Articles

Reduced Expression of P2Y1Receptors in Connexin43-Null Mice Alters Calcium Signaling and Migration of Neural Progenitor Cells

Glial calcium signals play important roles during CNS development. Calcium transients induced by ATP, acting on purinergic receptors, stimulate DNA synthesis, increase astrocytic and neural stem cell proliferation, and are prominent during the differentiation of radial glia. We have shown previously that expression of P2Y receptors in astrocytes is altered when connexin43 (Cx43) is downregulated. To evaluate the consequences of Cx43 deletion on calcium signaling during neural progenitor development, studies were performed on neurospheres derived from embryonic striatum. After adhesion, cells migrating from wild-type (WT) and Cx43-null neurospheres displayed spontaneous calcium oscillations. Such activity was blunted by apyrase, 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS-2179), and suramin, suggesting that ATP released by neural cells acts on purinergic receptors to induce calcium oscillations. The amplitudes of Ca2+ transients induced by P2Y but not P2X receptor agonists were larger in WT than in Cx43-null progenitors, suggesting that these two cell populations express different P2 receptors. Suramin, a nonselective P2 receptor antagonist, and MRS-2179, a P2Y1 receptor-selective antagonist, reduced the proliferation rate and the migration of WT progenitor cells to levels similar to those of Cx43-null cells. Conversely, exogenous expression of P2Y1 receptors in Cx43-null cells restored their migration pattern to levels seen in WT progenitors. However, treatment with P2 receptor antagonists did not alter the ratio of nestin to GFAP expression in WT neural progenitors. These data show that altered autocrine-paracrine communication attributable to reduced levels of P2Y1 receptors in neural progenitor cells lacking Cx43 affects proliferation and migration but not cell differentiation during early CNS development.

PTHrP expression is predictive of glial tumor local control

Purpose/Objective: Parathyroid Hormone Related Peptide (PTHrP) expression modulates cell survival in a number of human solid tumors. A role for PTHrP expression in glial signal transduction after irradiation has not been reported.

Heparin facilitates glial cell line-derived neurotrophic factor signal transduction.

Glial cell-line neurotrophic factor (GDNF), a neurotrophic factor with heparin binding affinity, promotes the survival and differentiation of a variety of neuronal cells including dopaminergic neuron. The effect of heparin on GDNF signaling was investigated based on the expression of the tyrosine hydroxyrase (TH) gene in neurobalstoma cells. Up-regulation of TH gene mRNA by GDNF was enhanced by co-administration of heparin. This facilitation by heparin was particularly evident at suboptimal levels of GDNF, which was consistent with the luciferase assay using TH gene promoter. Pretreatment with heparitinase decreased TH promoter activity in the absence of heparin. Phosphorylation of extracellular regulated kinase was increased in the presence of heparin, although tyrosine phosphorylation of Ret receptor tyrosine kinase was not affected by heparin. Expression of early response genes such as c-fos or Egr1 increased and sustained in the presence of heparin more than that without heparin. These results indicate that interaction with glycosaminoglycans such as heparin affects GDNF signal transduction positively.

Chromaffin cell mitogenesis by neurturin and glial cell line-derived neurotrophic factor

Neurturin and glial cell line-derived neurotrophic factor are novel mitogens for normal adult rat chromaffin cells in vitro. These neurotrophic factors differ from the previously described adult chromaffin cell mitogens, nerve growth factor and basic fibroblast growth factor, in that their effects are potentiated by depolarization and activation of protein kinase C. Neurturin and glial cell line-derived neurotrophic factor signal via the receptor tyrosine kinase, ret, but may also act independently of ret. Both depolarization and phorbol esters act synergistically with neurturin to up-regulate ret protein expression in chromaffin cell cultures, suggesting a mechanism for potentiation of mitogenesis. However, a direct role for ret in mitogenesis has not been established. Stimulation by neurturin causes increased phosphorylation of extracellular signal-regulated kinases 1 and 2 in cultured chromaffin cells, and mitogenesis is prevented by inhibitors of their phosphorylation. Inhibitors of phosphatidylinositol 3-kinase also prevent mitogenesis. The present findings suggest the hypothesis that neurotrophic factors and neurally derived signals might cooperatively regulate chromaffin cell proliferation in vivo in the rat. In addition, trans-synaptic stimulation might provide a route by which epigenetic factors could influence the development of adrenal medullary hyperplasia in humans with hereditary multiple endocrine neoplasia syndromes 2A and 2B by affecting expression and/or activation of ret.

Fate Sealed by Age

Neurons and glia are derived from a common set of precursor stem cells. Morrow et al. used a cortical slice assay to examine the signals required to specify neuronal versus glial cell fate and differentiation. Dissociated embryonic neural stem cells from a green fluorescent protein (GFP)-expressing strain of mice were plated with cortical slices from mice of different ages (embryonic through postnatal). Cell fate and differentiation were monitored by changes in the morphology and expression of marker proteins for neurons and glia. Incubation of neural stem cells with embryonic cortical slices led to the development of mostly neuronal cells and incubation with postnatal cortical slices produced mostly glial cells, suggesting that different factors are produced at different stages of development and that the stem cells are poised to respond to either set of signals. Analysis of clones of the GFP-expressing cells showed that the signals were predominantly instructive and not trophic signals. The steps of glial cell fate specification and glial differentiation could be separated. The effects of postnatal cortical slices could be recapitulated by culture of the stem cells with fibroblast growth factor 2 (FGF2) and ciliary neurotrophic factor (CNTF), with FGF2 providing the signal for glial fate specification and CNTF providing the glial differentiation signal. T. Morrow, M-R. Song, A. Ghosh, Sequential specification of neurons and glia by developmentally regulated extracellular factors. Development 128 , 3585-3594 (2001). [Online Journal]

Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C.

Epithelial-mesenchymal tissue interactions regulate the formation of signaling centers that play a role in the coordination of organogenesis, but it is not clear how their activity leads to differences in organogenesis. We report that type XVIII collagen, which contains both a frizzled and an endostatin domain, is expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomes localized to the epithelial tips in the lung during the early stages of epithelial branching, while its expression in the kidney is confined to the epithelial stalk region and is lost from the nearly formed ureter tips, thus displaying the reverse pattern to that in the lung. In recombinants, between ureter bud and lung mesenchyme, type XVIII collagen expression pattern in the ureter bud shifts from the kidney to the lung type, accompanied by a shift in sonic hedgehog expression in the epithelium. The lung mesenchyme is also sufficient to induce ectopic lung surfactant protein C expression in the ureter bud. Moreover, the shift in type XVIII collagen expression is associated with changes in ureter development, thus resembling aspects of early lung type epigenesis in the recombinants. Respecification of collagen is necessary for the repatterning process, as type XVIII collagen antibody blocking had no effect on ureter development in the intact kidney, whereas it reduced the number of epithelial tips in the lung and completely blocked ureter development with lung mesenchyme. Type XVIII collagen antibody blocking also led to a notable reduction in the expression of Wnt2, which is expressed in the lung mesenchyme but not in that of the kidney, suggesting a regulatory interaction between this collagen and Wnt2. Respecification also occurred in a chimeric organ containing the ureter bud and both kidney and lung mesenchymes, indicating that the epithelial tips can integrate the morphogenetic signals independently. A glial cell line-derived neurotrophic factor signal induces loss of type XVIII collagen from the ureter tips and renders the ureter bud competent for repatterning by lung mesenchyme-derived signals. Our data suggest that differential organ morphogenesis is regulated by an intra-organ patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen.

Differential Control of Clustering of the Sodium Channels Na v1.2 and Na v1.6 at Developing CNS Nodes of Ranvier

Na v1.6 is the main sodium channel isoform at adult nodes of Ranvier. Here, we show that Na v1.2 and its β2 subunit, but not Na v1.6 or β1, are clustered in developing central nervous system nodes and that clustering of Na v1.2 and Na v1.6 is differentially controlled. Oligodendrocyte-conditioned medium is sufficient to induce clustering of Na v1.2 α and β2 subunits along central nervous system axons in vitro. This clustering is regulated by electrical activity and requires an intact actin cytoskeleton and synthesis of a non-sodium channel protein. Neither soluble- or contact-mediated glial signals induce clustering of Na v1.6 or β1 in a nonmyelinating culture system. These data reveal that the sequential clustering of Na v1.2 and Na v1.6 channels is differentially controlled and suggest that myelination induces Na v1.6 clustering.

Serotonin Neurons, Neuroplasticity, and Homeostasis of Neural Tissue

Homeostasis is the process by which the internal milieu of the body is able to maintain equilibrium in the face of constant insults from the external world. Endocrine, immune, and vascular systems play pivotal roles in adjusting internal biochemical reactions to counteract assaults from the outside. Despite the vast accumulation of data over the last 50 years, a role for serotonin in brain homeostasis has not been proposed. In this chapter I will review the plasticity and anatomy of serotonergic neurons in integrating external sensory and motor systems as well as internal endocrine, glial and vascular signals with the various cellular elements comprising neural tissue. Steroids and neuropeptides have both been shown to alter the morphology of serotonergic neurons. In turn, alterations in serotonin levels in the adult brain can change the morphology of its target cells. A pivotal role for serotonin in the homeostasis of neural tissue is consistent with the function of serotonin throughout evolution and explains the large number of biological systems, behavioral activities, and clinical diseases associated with serotonergic neurons.

Commissure formation in the embryonic CNS of Drosophila.

Most of the neurons of the ventral nerve cord send out long projecting axons which cross the midline. In the Drosophila central nervous system (CNS) cells of the midline give rise to neuronal and glial lineages with different functions during the establishment of the commissural pattern. Here we present evidence that beside the previously known NETRIN/FRAZZLED (DCC) signalling system an additional attractive system(s) is operating in the developing embryonic nervous system of Drosophila. Attractive cues appear to be provided by the midline neurons. We show that the glial cells present repulsive signals to the previously described ROUNDABOUT receptor in addition to a permissive contact-dependent signal helping commissural growth cones across the midline. A novel repulsive component is encoded by the karussell gene. Furthermore the midline glial cells separate anterior and posterior commissures. By genetic criteria we demonstrate that some of the genes we have identified are acting in the midline glia whereas other genes are required in the midline neurons. The results lead to a detailed model relating different cellular functions to axonal patterning at the midline.

Clustering of neuronal sodium channels requires contact with myelinating Schwann cells.

Efficient and rapid conduction of action potentials by saltatory conduction requires the clustering of voltage-gated sodium channels at nodes of Ranvier. This clustering results from interactions between neurons and myelinating glia, although it has not been established whether this glial signal is contact-dependent or soluble. To investigate the nature of this signal, we examined sodium channel clustering in co-cultures of embryonic rat dorsal root ganglion neurons and Schwann cells. Cultures maintained under conditions promoting or preventing myelination were immunostained with antibodies against the alpha subunit of the sodium channel and against ankyrin(G), a cytoskeletal protein associated with these channels. Consistent with previous in vivo studies (Vabnick et al., 1996), sodium channels and ankyrin G cluster at the onset of myelination. These clusters form adjacent to the ends of the myelinating Schwann cells and appear to fuse to form mature nodes. In contrast, sodium channels and ankyrin G do not cluster in neurons grown alone or in co-cultures where myelination is precluded by growing cells in defined media. Conditioned media from myelinating co-cultures also failed to induce sodium channel or ankyrin G clusters in cultures of neurons alone. Finally, no clusters develop in the amyelinated portions of suspended fascicles of dorsal root ganglia explants despite being in close proximity to myelinated segments in other areas of the dish. These results indicate that clustering of sodium channels requires contact with myelinating Schwann cells.

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.

Synaptic efficacy enhanced by glial cells in vitro.

In the developing nervous system, glial cells guide axons to their target areas, but it is unknown whether they help neurons to establish functional synaptic connections. The role of glial cells in synapse formation and function was studied in cultures of purified neurons from the rat central nervous system. In glia-free cultures, retinal ganglion cells formed synapses with normal ultrastructure but displayed little spontaneous synaptic activity and high failure rates in evoked synaptic transmission. In cocultures with neuroglia, the frequency and amplitude of spontaneous postsynaptic currents were potentiated by 70-fold and 5-fold, respectively, and fewer transmission failures occurred. Glial cells increased the action potential-independent quantal release by 12-fold without affecting neuronal survival. Thus, developing neurons in culture form inefficient synapses that require glial signals to become fully functional.

Glial Cell Line-derived Neurotrophic Factor Signals through the RET Receptor and Activates Mitogen-activated Protein Kinase

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta family of growth factors, was first identified by its ability to promote the survival of midbrain dopaminergic neurons in culture. We demonstrate that GDNF treatment of several neuroblastoma cell lines leads to dose-dependent tyrosine phosphorylation of the RET receptor and that other transforming growth factor-beta family members are not able to activate the RET receptor. GDNF treatment of neuroblastoma cells also results in increased transcription of an Elk luciferase reporter gene, suggesting that GDNF activates the mitogen-activated protein kinase signal transduction pathway.

GLIA-NEURON INTERCOMMUNICATIONS AND SYNAPTIC PLASTICITY

It is established that astrocytes are the intimate partner of neurons throughout their lifespan. However, astrocytes play different roles at different stages of the lifespan. During neurogenesis and early development, glial cells provide a scaffold for the correct migration of neurons and growth cones. They provide guidance cues and may also be involved in neuronal proliferation. In the adult, astrocytes maintain neuronal homeostasis and synaptic plasticity. This review discusses some of the cell-cell communication signals which are involved in the maintenance of synaptic plasticity. They are divided into: (a) glia-to-glia signaling, which involves non-synaptic communication by coupling of astrocytes. In this cell communication, cytoplasmic exchange of ions and small molecules among cells is accomplished through cell coupling of cells via cell-to-cell contacts, termed gap junctions; (b) neuron-to-glia signaling, which involves synaptic interactions. This cell-cell intercommunication has received considerable attention. Of special interest is the role of astrocytes in glutamic acid compartmentation and in preventing glutamic acid neurotoxicity. A glutamate-induced calcium signaling appears to be involved in this neuron-astrocyte interaction. The intriguing possibility is that neuronally induced astrocyte calcium signals may feed back to influence neuronal excitability or regulate synaptic transmission; (c) signals from astrocytes to neurons: a new concept of neuron-glia intercommunication. As mentioned, calcium appears to be the molecule in this glia-neuron signaling, although glial cell receptor-mediated signals are involved also. Receptor interactions on astrocytes through a cascade of events lead to modification in the extracellular concentration of glutamate. The role of astrocytes in synaptic plasticity is not as well understood during ageing and their role in neuronal cell death during ageing and neurodegeneration can only be speculated. However, astrocytes in the aged brain exhibit various receptors, including glutamate receptors. Thus, astrocytes can be expected to modify the expression of endogenous neurotoxins and thus contribute to synaptic plasticity in ageing. Synaptic plasticity continues to be a homeostatic relationship between neurons and glial cells. The possibility of signaling from astrocytes to neurons has opened new horizons for glial cell function and new challenges of research for gliobiologists. Copyright © 1996 Elsevier Science Ltd

Synaptic regulation of glial protein expression in vivo

We investigated signaling between individual nerve terminals and perisynaptic Schwann cells, the teloglial cells that cover neuromuscular junctions. When deprived of neuronal activity in vivo, either by motor nerve transection or tetrodotoxin injection, perisynaptic Schwann cells rapidly up-regulated glial fibrillary acidic protein. Addition of transcription or translation inhibitors to excised muscles prevented this increase. Stimulation of cut nerves prevented glial fibrillary acidic protein increases even when postsynaptic nicotinic receptors were blocked, but not when neurotransmitter release was blocked with w-conotoxin GVIA. We conclude that there is a nerve terminal to glial signal, requiring presynaptic neurotransmitter release, which regulates perisynaptic Schwann cell genes. This may be a general principle since many types of glia are sensitive to transmitters applied in vitro or released in situ.

Sodium channel density in hypomyelinated brain increased by myelin basic protein gene deletion

Trophic control over the expression and membrane distribution of voltage-dependent ion channels is one of the principal organizing events underlying the maturation of excitable cells. The myelin sheath is a major structural determinant of regional ion channel topography in central axons, but the exact molecular signals that mediate local interactions between the oligodendrocyte and axolemma are not known. We have found that large caliber fibre pathways in the brain of the mutant mouse shiverer (shi, gene on chromosome 18), whose developmental fate of myelination is averted by deletion of five exons in the myelin basic protein gene, have a striking excess of sodium channels. As cytoplasmic membranes of shiverer oligodendroglia still adhere to axons, the evidence indicates that myelin basic protein or a myelin basic protein-dependent glial transmembrane signal associated with compact myelin formation, rather than a simple glial-axon contact inhibition or an intrinsic genetic program of neuronal differentiation, could be critical in downregulating sodium channel density in axons. Here we use the shiverer mutant to show that mature central nervous system projection neurons with large caliber unmyelinated fibres sustain functional excitability by increasing sodium channel density. This axon plasticity, triggered by the absence of a single glial protein, contributes to the unexpectedly mild degree of neurological impairment in the mutant brain without myelin, and may be a potentially inducible mechanism determining the recovery of function from dysmyelinating disease.

Interneurons versus efferent neurons: Heterogeneity in their neurite outgrowth response to glia from several brain regions

We have studied in vitro the morphology of two populations of dopaminergic neurons from mouse embryos: the periglomerular interneurons from the olfactory bulb (DOBI) and the efferent neurons from the substantia nigra (DENN). The intrinsic potential of both neuronal types has been studied by comparing process outgrowth in a predominantly neuronal environment or in a glial environment that is endogenous or from other brain regions. Both populations exhibit in vitro different characteristics that reflect their phenotype in situ. In addition they greatly differ in their response to glial signals. DOBI maintain a constant stellate morphology with short processes under all culture conditions tested, whereas DENN exhibit a great plasticity and in particular respond to olfactory bulb glia with a striking increase in neurite length. The olfactory bulb glia differs from other brain region glia in two aspects: (a) in addition to type I astrocytes, common to all the glial monolayers that we have studied, it contains a population of fusiform astrocytes (GFAP +) that might represent the superficial glia ( Raisman, 1985); and (b) both astrocytes and fusiform cells produce large amounts of laminin that is secreted in a thick extracellular matrix. DENN outgrowth on olfactory bulb glia, however, is not blocked by antilaminin antibodies that block outgrowth on a laminin substrate. Our results demonstrate that two neuronal populations sharing the same neurotransmitter present intrinsic differences in the control of cell shape. The fact that glia harvested from different brain regions supports varying extent of DENN neurite outgrowth suggests a heterogeneity of environmental signals throughout the developing brain.

Neurotransmitters: past, present, and future directions.

As originally conceived, central neurotransmitters operated uniformly, exciting or inhibiting postsynaptic targets by receptors that activated passive ionic conductances. As the list of transmitter substances and their actions expanded, concepts of transmitter actions have broadened and grown more complex to include a variety of intramembranous and intracytoplasmic second messengers that can regulate both active and passive ionic conductances. Present-day research directions center on further expansion of the lists of identified transmitter candidates, and on the more precise characterization of their sites and mechanisms of receptor regulation and transduction. Current research is also illuminating the means by which neurotransmitters act in a coordinated fashion to regulate common synaptic targets. Future directions will likely include new forms of interneuronal, intraneuronal, and glial signals, including lipids, steroids, and as-yet-undiscovered superfamilies of peptides and receptors. Although recent advances in understanding specific transmitters have been achieved largely through in vitro electrophysiological analyses, it is hoped that future research will recast these events in the context of the intact functioning brain. Neurotransmitters are likely to remain a productive focus of future research.