Role of Rho GTPase in astrocyte morphology and migratory response during in vitro wound healing

Small Rho GTPases are key regulators of the cytoskeleton in a great variety of cells. Rho function mediates morphological changes as well as locomotor activity. Using astrocyte cultures established from neonatal mice we investigated the role of Rho in process formation during astrocyte stellation. Using a scratch-wound model, we examined the impact of Rho on a variety of morphological and functional variables such as stellation and migratory activity during wound healing. C3 proteins are widely used to study cellular Rho functions. In addition, C3 derived from Clostridium botulinum (C3bot) is considered selectively to promote neuronal regeneration. Because the latter requires a balanced activity of neurones and glial cells, the effects of C3 protein on glial cells such as astrocytes have to be considered carefully. Low nanomolar concentrations of C3 proteins significantly promoted process outgrowth and increased process branching. Besides enzymatic inactivation of Rho by ADP-ribosylation, changes in protein levels of the various Rho GTPases may also contribute to the observed effects. Furthermore, incubation of scratch-wounded astrocyte cultures with C3bot accelerated wound healing. By inhibiting the Rho downstream effector ROCK with the selective inhibitor Y27632 we were able to demonstrate that the accelerated wound closure resulted from both enhanced polarized process formation and increased migratory activity of astrocytes into the lesion site. These results suggest that Rho negatively regulates astrocytic process growth and migratory responses after injury and that its inactivation by C3bot in nanomolar concentrations promotes astrocyte migration.     

A comparative investigation of neuroglia in representative vertebrates: a silver carbonate study

The phylogenetic development of neuroglia (astrocytes, oligodendrocytes) was investigated in homologous cortical and subcortical forebrain regions of selected vertebrates. Microglia were not considered in the current study. Four to seven brains from each species were used. Scharenberg's modification for astroglia of del Rio Hortega's silver carbonate technique was used. The analysis of neuroglia cells was based on (1) the characteristic cellular morphology found in each species, (2) a comparison of the selected regions in each animal, (3) the interrelationships of astrocytes and their relations to neurons, blood vessels, and oligodendrocytes. The predominant type of neuroglia found in the fish, frog, and lizard was the ependymal cell; however, non-ependymal glial cells were also present. The bird represented a transitional phylogenetic stage from a predominance of ependymal glial to a predominance of non-ependymal glia. A progressive increase in the morphological relationships of glial cell bodies and processes to neurons was found with ascension of the phylogenetic scale from fish through primate. Interrelations were observed between adjacent astrocytic processes and cell bodies, and between astrocytes and oligodendrocytes. The processes of adjacent glial cells also appeared to show an increase in thickness at the point of approximation. A variety of astrocytes were observed ranging from small, round-oval shaped cells to large polygonal or stellate forms. Variations in the number of astrocytic processes, their thickness, and degree of secondary branching were described, and their possible functional significance was discussed.     

Meteorin: a secreted protein that regulates glial cell differentiation and promotes axonal extension

Glial cells are major components of the nervous system. The roles of these cells are not fully understood, however. We have now identified a secreted protein, designated Meteorin, that is expressed in undifferentiated neural progenitors and in the astrocyte lineage, including radial glia. Meteorin selectively promoted astrocyte formation from mouse cerebrocortical neurospheres in differentiation culture, whereas it induced cerebellar astrocytes to become radial glia. Meteorin also induced axonal extension in small and intermediate neurons of sensory ganglia by activating nearby satellite glia. These observations suggest that Meteorin plays important roles in both glial cell differentiation and axonal network formation during neurogenesis.     

Glial Fibrillary Acidic Protein-Expressing Glia in the Mouse Lung

Autonomic nerves regulate important functions in visceral organs, including the lung. The postganglionic portion of these nerves is ensheathed by glial cells known as non-myelinating Schwann cells. In the brain, glia play important functional roles in neurotransmission, neuroinflammation, and maintenance of the blood brain barrier. Similarly, enteric glia are now known to have analogous roles in gastrointestinal neurotransmission, inflammatory response, and barrier formation. In contrast to this, very little is known about the function of glia in other visceral organs. Like the gut, the lung forms a barrier between airborne pathogens and the bloodstream, and autonomic lung innervation is known to affect pulmonary inflammation and lung function. Lung glia are described as non-myelinating Schwann cells but their function is not known, and indeed no transgenic tools have been validated to study them in vivo. The primary goal of this research was, therefore, to investigate the relationship between non-myelinating Schwann cells and pulmonary nerves in the airways and vasculature and to validate existing transgenic mouse tools that would be useful for studying their function. We focused on the glial fibrillary acidic protein promoter, which is a cognate marker of astrocytes that is expressed by enteric glia and non-myelinating Schwann cells. We describe the morphology of non-myelinating Schwann cells in the lung and verify that they express glial fibrillary acidic protein and S100, a classic glial marker. Furthermore, we characterize the relationship of non-myelinating Schwann cells to pulmonary nerves. Finally, we report tools for studying their function, including a commercially available transgenic mouse line.     

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.     

Apolipoprotein E Attenuates β-Amyloid-Induced Astrocyte Activation

Abstract: A common feature of Alzheimer's disease pathology is an abundance of activated glia, indicative of an inflammatory reaction in the brain. The relationship between glial activation and neurodegeneration is not known, although several cytokines and inflammatory mediators produced by activated glia have the potential to initiate or exacerbate the progression of neuropathology. As β-amyloid (Aβ) is one of several stimuli that can activate glia, it is important to determine how Aβ-induced glial activation is influenced by other proteins present in the plaque, such as apolipoprotein E (apoE). We examined the effect of native preparations of apoE on activation of rat cortical astrocyte cultures by Aβ1–42. The apoE source was conditioned medium from human embryonic kidney 293 cells stably transfected with human apoE3 or apoE4 cDNA. By morphological criteria, apoE inhibited Aβ-induced astrocyte activation in three experimental paradigms: apoE pretreatment blocked subsequent Aβ-induced activation, Aβ aged in the presence of apoE did not activate astrocytes, and apoE addition to activated astrocytes transiently reversed the activated phenotype. No apoE isoform selectivity was observed. The effect of apoE appears to be specific to Aβ, as apoE did not attenuate cyclic AMP-induced astrocyte activation. These data suggest that apoE may modulate the ability of Aβ to induce inflammatory responses in the brain.     

Cdc42 and Gsk3 modulate the dynamics of radial glial growth, inter-radial glial interactions and polarity in the developing cerebral cortex

Polarized radial glia are crucial to the formation of the cerebral cortex. They serve as neural progenitors and as guides for neuronal placement in the developing cerebral cortex. The maintenance of polarized morphology is essential for radial glial functions, but the extent to which the polarized radial glial scaffold is static or dynamic during corticogenesis remains an open question. The developmental dynamics of radial glial morphology, inter-radial glial interactions during corticogenesis, and the role of the cell polarity complexes in these activities remain undefined. Here, using real-time imaging of cohorts of mouse radial glia cells, we show that the radial glial scaffold, upon which the cortex is constructed, is highly dynamic. Radial glial cells within the scaffold constantly interact with one another. These interactions are mediated by growth cone-like endfeet and filopodia-like protrusions. Polarized expression of the cell polarity regulator Cdc42 in radial glia regulates glial endfeet activities and inter-radial glial interactions. Furthermore, appropriate regulation of Gsk3 activity is required to maintain the overall polarity of the radial glia scaffold. These findings reveal dynamism and interactions among radial glia that appear to be crucial contributors to the formation of the cerebral cortex. Related cell polarity determinants (Cdc42, Gsk3) differentially influence radial glial activities within the evolving radial glia scaffold to coordinate the formation of cerebral cortex.     

Effect of audiogenic convulsions on the activity of n- and m-forms of lactate dehydrogenase in the neurons and neuroglia of different sections of central nervous system]

It was shown spectrophotometrically that in Krushinsky-Molodkina and Wistar rats the ratio of the activity of the aerobic H-forms of lactic dehydrogenase (LDH) to the activity of the anaerobic M-forms was higher in the neurons of the cerebral cortex and the Purkinje's cells of the cerebellum and in their glial cells-satellites than in the motor neurons of the anterior horns of the spinal cord and their perineuronal glia. In Krushinsky-Molodkina rats (with genetically-determined high sensitivity to audiogenic convulsions) epileptiform attacks under the effect of sound were accompanied by a marked activation of both the H- and the M-forms of LDH in the cortical neurons in the absence of any shifts in the perineuronal glia. On the contrary, the activity of all the forms of LDH was unchanged in the spinal motor neurons, whereas in the neuroglia cells surrounding these neurons there was a distinct increase in the activity of the H-forms of LDH. In the Purkinje's cells of the cerebellum an increase and in the glial cells- satellites -- a reduction of the activity of the M-forms of LDH in case of convulsions was seen.     

Distribution of aromatase activity in cultured neurons and glia cells

Aromatase plays a crucial role in the mechanism of action of testosterone in the central nervous system. Nevertheless, the exact cellular localization of this enzymatic complex within the different cell populations of the brain is still uncertain. In the experiments described here the presence of aromatase (assayed by the tritiated water method) has been evaluated in the two main cellular components of the brain: neurons and glia. Neurons, mixed glial cells, type 1 astrocytes, were obtained in cultures; oligodendrocytes were prepared by gradient ultracentrigugation. The results indicate that, among the different cells tested, only neurons possess a significant degree of aromatase activity, while the enzymatic activity is extremely low in mixed glial cell and in astrocyte preparations. Oligodendrocytes seem to be completely inactive in this respect.     

Glial Contributions to Neural Function and Disease

The nervous system consists of neurons and glial cells. Neurons generate and propagate electrical and chemical signals, whereas glia function mainly to modulate neuron function and signaling. Just as there are many different kinds of neurons with different roles, there are also many types of glia that perform diverse functions. For example, glia make myelin; modulate synapse formation, function, and elimination; regulate blood flow and metabolism; and maintain ionic and water homeostasis to name only a few. Although proteomic approaches have been used extensively to understand neurons, the same cannot be said for glia. Importantly, like neurons, glial cells have unique protein compositions that reflect their diverse functions, and these compositions can change depending on activity or disease. Here, I discuss the major classes and functions of glial cells in the central and peripheral nervous systems. I describe proteomic approaches that have been used to investigate glial cell function and composition and the experimental limitations faced by investigators working with glia.The nervous system is composed of neurons and glial cells that function together to create complex behaviors. Traditionally, glia have been considered to be merely passive contributors to brain function, resulting in a pronounced neurocentric bias among neuroscientists. Some of this bias reflects a paucity of knowledge and tools available to study glia. However, this view is rapidly changing as new tools, model systems (culture and genetic), and technologies have permitted investigators to show that glia actively sculpt and modulate neuronal properties and functions in many ways. Glia have been thought to outnumber neurons by 10:1, although more recent studies suggest the ratio in the human brain is closer to 1:1 with region-specific differences (1). There are many different types of glia, some of which are specific to the central nervous system (CNS),¹ whereas others are found only in the peripheral nervous system (PNS). The main types of CNS glia include astrocytes, oligodendrocytes, ependymal cells, radial glia, and microglia. In the PNS, the main glial cells are Schwann cells, satellite cells, and enteric glia. These cells differ and are classified according to their morphologies, distinct anatomical locations in the nervous system, functions, developmental origins, and unique molecular compositions. Among the different classes of glia there are additional subclasses that reflect further degrees of specialization. In this review, I will discuss the characteristics and functions of the major glial cell types including astrocytes, microglia, and the myelin-forming oligodendrocytes (CNS) and Schwann cells (PNS). Because of space limitations, it is impossible to give a complete accounting of all glia and what is known about each of these cell types. Therefore, I encourage the interested reader to refer to some of the many excellent reviews referenced below that focus on individual glial cell types. Finally, I will discuss proteomic studies of glial cell function and some of the unique challenges investigators face when working with these cells.     

Glial processes at the Drosophila larval neuromuscular junction match synaptic growth

Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.     

Neuroglia in ageing and disease

The proper operation of the mammalian brain requires dynamic interactions between neurones and glial cells. Various types of glial cells are susceptible to morpho-functional changes in a variety of brain pathological states, including toxicity, neurodevelopmental, neurodegenerative and psychiatric disorders. Morphological modifications include a change in the glial cell size and shape; the latter is evident by changes of the appearance and number of peripheral processes. The most blatant morphological change is associated with the alteration of the sheer number of neuroglia cells in the brain. Functionally, glial cells can undergo various metabolic and biochemical changes, the majority of which reflect upon homeostasis of neurotransmitters, in particular that of glutamate, as well as on defence mechanisms provided by neuroglia. Not only glial cells exhibit changes associated with the pathology of the brain but they also change with brain aging.     

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.     

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.     

Roles of glia in the Drosophila nervous system

Glial cells have diverse functions that are necessary for the proper development and function of complex nervous systems. Various insects, primarily the fruit fly Drosophila melanogaster and the moth Manduca sexta, have provided useful models of glial function during development. The present review will outline evidence of glial contributions to embryonic, visual, olfactory and wing development. We will also outline evidence for non-developmental functions of insect glia including blood-brain-barrier formation, homeostatic functions and potential contributions to synaptic function. Where relevant, we will also point out similarities between the functions of insect glia and their vertebrate counterparts.     

TOPOCHEMICAL ASPECTS OF NUCLEIC ACID AND PROTEIN METABOLISM WITHIN THE NEURON-NEUROGLIA UNIT OF THE SPINAL CORD ANTERIOR HORN

Abstract— Using a two-wavelength modification of ultraviolet and visible cytospectro-photometric methods, the content of nucleic acids per cell was determined in neuronal cytoplasm and glial satellite cell-bodies from the spinal cord anterior horns in mice and rats. Mice which had been swimming for 3 and 4 h showed an increase in the content of RNA in the spinal motoneurons with no changes in the neuroglia. Stronger stimulation of the nervous system such as electrical skin irritation (20-40 V, approx. 40 impulses/min) for 5 min resulted in an increase of RNA in the motoneurons of rat spinal cord and a decrease in the surrounding glia. Exhausting actions upon the nervous system (60 min irritation of rat paws by the electrical current, acute clonic convulsions in rats injected with cardiazol (pentamethylenetetrazol, metrazol) or initial free motor activity after 3 weeks of restraint of mice) induced a marked decrease of RNA content throughout the whole neuron-neuroglia unit. After stimulation, return to normal amounts of RNA and protein was more rapid in glia than in neurons. After 1-3 days rest the level of RNA was normal in motoneurons, but a decrease in glial RNA was shown. These trace changes in the glia are believed to reflect an adaptation mechanism in the nervous system at the cellular level. The relationship between neuronal and glial compartments within the neuron-neuroglia unit is discussed; a supporting, homeostatic, secondary role of glial metabolism with respect to adequate reconstruction of neuronal metabolism is outlined.     

Cyclooxygenase-2 expression in astrocytes and microglia in human oligodendroglioma and astrocytoma

Cyclooxygenases (cox) are potent mediators of inflamation and two cox-izoenzymes, cox-1, cox-2, are described to date. Cox-2 is cytokine-inducible in inflammatory cells and enhanced cox-2 expression has been attributed a key role in the development of edema and immunomodulation in pathologically altered brain tissues. In normal cerebral cortex cox-2 is present only in neurons, but not in the glial or vascular endothelial cells. The function of microglia in glioma biology is unclear. Microglia have both neurotrophic and neurotoxic functions and have been shown to release a variety of cytokines. Our preliminary results showed that the expression pattern of cox-2 is predominantly neuronal although glial expression was observed with the correlation of high malignancy. In this study we aimed to assess the phenotypes (astrocyte, microglia) of the cox-2-expressing glial cells in various types of human gliomas and to compare their expression patterns. For this purpose we employed dual immunohistochemistry for cox-2 and GFAP (astrocyte) or LCA-MAC (microglia-macrophage) in archival formalin-fixed, paraffin embedded human tissue diagnosed as oligodendroglioma and/or astrocytoma. The results showed that cox-2 immunoreactivity is up-regulated in the neurons according to the tumor grade. Most of the cox-2 immunoreactive glia were GFAP-positive in anaplastic oligodendrogliomas and at lesser extend in glioblastomas. Cox-2 and LCA co-localization was detected in more glial cells in glioblastomas. It may be speculated that the induction of cox-2 in microglia may contribute to the deleterious effects of prostanoids in cerebral edema formation during the progression of oligodendrogliomas. The detection of cox-2 in astrocytes surrounding the necrotic areas might be important to develop new strategies, such as the usage of cox-2 inhibitors combine with chemotherapy and radiotherapy in the treatment of glioma patients.