Glutamine synthetase in brain: effect of ammonia

Glutamine synthetase (GS) in brain is located mainly in astrocytes. One of the primary roles of astrocytes is to protect neurons against excitotoxicity by taking up excess ammonia and glutamate and converting it into glutamine via the enzyme GS. Changes in GS expression may reflect changes in astroglial function, which can affect neuronal functions.Hyperammonemia is an important factor responsible of hepatic encephalopathy (HE) and causes astroglial swelling. Hyperammonemia can be experimentally induced and an adaptive astroglial response to high levels of ammonia and glutamate seems to occur in long-term studies. In hyperammonemic states, astroglial cells can experience morphological changes that may alter different astrocyte functions, such as protein synthesis or neurotransmitters uptake. One of the observed changes is the increase in the GS expression in astrocytes located in glutamatergic areas. The induction of GS expression in these specific areas would balance the increased ammonia and glutamate uptake and protect against neuronal degeneration, whereas, decrease of GS expression in non-glutamatergic areas could disrupt the neuron-glial metabolic interactions as a consequence of hyperammonemia.Induction of GS has been described in astrocytes in response to the action of glutamate on active glutamate receptors. The over-stimulation of glutamate receptors may also favour nitric oxide (NO) formation by activation of NO synthase (NOS), and NO has been implicated in the pathogenesis of several CNS diseases. Hyperammonemia could induce the formation of inducible NOS in astroglial cells, with the consequent NO formation, deactivation of GS and dawn-regulation of glutamate uptake. However, in glutamatergic areas, the distribution of both glial glutamate receptors and glial glutamate transporters parallels the GS location, suggesting a functional coupling between glutamate uptake and degradation by glutamate transporters and GS to attenuate brain injury in these areas.In hyperammonemia, the astroglial cells located in proximity to blood-vessels in glutamatergic areas show increased GS protein content in their perivascular processes. Since ammonia freely crosses the blood-brain barrier (BBB) and astrocytes are responsible for maintaining the BBB, the presence of GS in the perivascular processes could produce a rapid glutamine synthesis to be released into blood. It could, therefore, prevent the entry of high amounts of ammonia from circulation to attenuate neurotoxicity. The changes in the distribution of this critical enzyme suggests that the glutamate-glutamine cycle may be differentially impaired in hyperammonemic states.     

GLT1, glial glutamate transporter, is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain.

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high-affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin-like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin-positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development.     

GLT1, glial glutamate transporter,is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high‐affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin‐like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin‐positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 515–526, 1999     

Rapid activation of astrocyte-specific expression of GFAP-lacZ transgene by focal injury

Astrocytes form a key cellular component of the central nervous system. They respond vigorously to diverse neurologic insults by undergoing hypertrophy and increasing expression of the glial fibrillary acidic protein (GFAP) gene, but their functions are largely unknown. To analyze astrocytes in vivo we constructed a transgenic vector from GFAP gene sequences and monitored its efficiency by fusing it to lacZ. Injection of the GFAP-lacZ hybrid gene into the germline of mice yielded six different lines of transgenic mice. In all lines the expression of lacZ was astrocyte-specific. In unmanipulated transgenic animals beta-galactosidase activity was much more prominent in astrocytes of the hippocampal formation, selected white matter tracts, and glial limitans than in astrocytes of other areas. This pattern of expression illustrates the physiologic heterogeneity of astrocytes and probably reflects differences in functional demands placed on these cells in different brain regions. Upmodulation of transgene expression was used to determine the time frame within which astroglial activation and increased GFAP gene expression occur following a neurologic insult. Induction of GFAP-lacZ expression was detectable within 1 hour after focal mechanical trauma. This demonstrates that the response of astrocytes to neurologic injury is very rapid and implies that these cells could fulfill important early functions in wound healing within the central nervous system.     

Changes in the glial condition in different sections of the rat brain after stopping the systemic circulation]

The effect of ischemia on the expression of GFAP in astrocytes of cerebrum, hippocampus and cerebellum was studied on rat clinical death model. Cardiac arrest was induced by 10-or 15-min intrathoracic compression of the heart vascular bundle. Immunohistochemical staining showed that GFAP immunoreactivity significantly increased in the white matter, and GFAP-expressing astrocytes appeared in the gray matter. The reaction activity correlated with ischemia duration and phases of postresuscitation process. The obtained data are indicative of possible changes in the astrocytes condition in the absence of manifest lesions of neurons. This brings up the question of the role of glia homeostasis derangements in the formation of brain postresuscitation pathology.     

Regional difference of glutamate-induced swelling in cultured rat brain astrocytes

L-glutamate (glutamate) is an important neurotoxin as well as the major excitatory neurotransmitter. Extracellular glutamate levels are elevated following ischemia, hypoglycemia, and trauma. One consequence of elevated glutamate levels is cell swelling. Such swelling occurs primarily in astroglial cells. We characterized the regional difference in glutamate-induced swelling response of cultured astrocytes from rat cerebral cortex, hippocampus and cerebellum. Glutamate produced dose-dependent astrocytic swelling in both cerebral cortex and hippocampus, showing a maximal effect in 0.5 mM concentration, as measured by 3-O-methyl-D-[1-3H]glucose uptake. However, in cerebellum, glutamate did not produce astrocytic swelling. It has been suggested that Na+ -dependent glutamate uptake is a possible mechanism of glutamate-induced swelling. The Vmax for glutamate uptake into cerebellum astrocytes was significantly lower (6.7 nmol/mg protein/min) than those for cerebral cortex and hippocampus astrocytes (13.0 and 12.0 nmol/mg protein/min, respectively). In three regions, more than 90% of the cultured cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. Immunoreactivity of GLT, one of the markers of glutamate transporters, which is expressed at low levels in cultured astrocytes, did not show any differences in three regions. However, immunoreactivities of GLAST, the other astroglial glutamate transporter, and aquaporin4 (APQ4), a water transporter, were significantly higher in cerebral cortex and hippocampus than in cerebellum. These results may explain the regional difference of glutamate-induced astrocytic swelling.     

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy-induced apoptosis via modulation of retinal glia in vivo

Adenoviral-mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy-induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRalpha) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral-mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter-1 (GLAST-1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRalpha, pSTAT(3), GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection.     

GFAP-Deficient Astrocytes Are Capable of Stellationin VitroWhen Cocultured with Neurons and Exhibit a Reduced Amount of Intermediate Filaments and an Increased Cell Saturation Density

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein predominantly expressed in cells of astroglial origin. To allow for the study of the biological functions of GFAP we have previously generated GFAP-negative mice by gene targeting [Peknyet al.(1995)EMBO J.14, 1590–1598]. Astrocytes in culture, similar to reactive astrocytesin vivo,express three intermediate filament proteins: GFAP, vimentin, and nestin. Using primary astrocyte-enriched cultures from GFAP-negative mice, we now report on the effect of GFAP absence on (i) the synthesis of other intermediate filament proteins in astrocytes, (ii) intermediate filament formation, (iii) astrocyte process formation (stellation) in response to neurons in mixed cerebellar astrocyte/neuron cultures, and (iv) saturation cell densityin vitro.GFAP−/− astrocytes were found to produce both nestin and vimentin. At the ultrastructural level, the amount of intermediate filaments as revealed by transmission electron microscopy was reduced in GFAP−/− astrocytes compared to that in GFAP+/+ astrocytes. GFAP−/− astrocytes retained the ability to form processes in response to neurons in mixed astrocyte/neuron cultures from the cerebellum. GFAP−/− astrocyte-enriched primary cultures exhibited an increased final cell saturation density. The latter leads us to speculate that the loss of GFAP expression observed focally in a proportion of human malignant gliomas may reflect tumor progression toward a more rapidly growing and malignant phenotype.     

Further similarities between astrocytes and perisinusoidal stellate cells of liver (Ito cells): Colocalization of desmin and glial fibrillary acidic protein in astroglial primary cultures

The colocalization of desmin and glial fibrillary acidic protein (GFAP) in astrocytes was inferred from previous studies demonstrating a unique antigenic composition comprising GFAP, desmin and vimentin in perisinusoidal stellate cells (PSC) of liver which share several features with astrocytes. In the present study the colocalization of GFAP and desmin was investigated by double-immunolabeling experiments in 12-day-old rat astroglial primary cultures with antiserum against GFAP and two commercial monoclonal antibodies against desmin, antibodies of clone DEU-10 and clone DEB-5. These antibodies selectively decorated the perisinusoidal stellate cells (PSC) of liver for which desmin is known to be a marker. The results obtained with astroglial cells demonstrate that both GFAP and desmin are coexpressed in morphologically different types, process-bearing and process-lacking astrocytes. The expression of desmin was apparently more pronounced in process-lacking astrocytes and was considerably lower in process-bearing ones. In process-lacking astrocytes, in contrast to filamentous cytoplasmic staining for GFAP, the immunoreactivity for desmin was non-filamentous and was irregularly spread in the perinuclear cytoplasm of the cells, while in process-bearing astrocytes the pattern of staining for desmin was similar to that of GFAP. The variability in the intensity and pattern of staining for desmin in astrocytes might be due to transitional stages of differentiation for part of the cells. This interpretation was supported by the presence of GFAP in the cells weakly expressing smooth muscle alpha-actin and the absence of GFAP in the cells enriched with microfilaments.     

Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo.

Double-immunolabelling techniques were employed to investigate the distribution of smooth muscle alpha-actin (actin) in glial fibrillary acidic protein (GFAP)-positive cells in rat brain during early postnatal development and maturation and in glial primary culture derived from newborn rat brain. In addition the expression of desmin was studied in the glial primary cultures as a function of the differentiation of the cells. Comparison of the cultured astroglial cells at an early age with hepatic stellate cells derived from CCl4-induced cirrhotic rat liver, revealed features of the astrocytic cytoskeleton characteristic of myofibroblastic cells, i.e., strong expression of both myofibroblastic markers, actin and desmin. In astroglial cells with an initial morphology reminiscent of fibroblasts the non-filamentous perinuclear immunoreaction of GFAP increased with time at the expense of actin and, partially, desmin. GFAP filaments were spread throughout the cytoplasm of the cells which acquired stellate morphology. The alterations in the morphology of the cells and the distribution and intensity of staining for GFAP and actin during the differentiation of astrocytes in culture were similar to those observed in astrocytes during the maturation of the brain. In astrocytes from a newborn brain as well as in cirrhotic hepatic stellate cells, the area of immunoreaction of GFAP was reduced and confined mainly to the nuclear region. In contrast, the cells expressed actin throughout the cytoplasm. These findings may hint at a similar function of these regionally specialized perivascular myofibroblastic cells in a normal brain and diseased liver and at inverse organ-specific functions which the cells fulfill under non-pathological conditions in vivo.     

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy‐induced apoptosis via modulation of retinal glia in vivo

Adenoviral‐mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy‐induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRα) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral‐mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter‐1 (GLAST‐1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRα, pSTAT₃, GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Fibronectin is expressed by astrocytes cultured from embryonic and early postnatal rat brain

In early primary cultures from newborn rat brain, few glial fibrillary acidic protein (GFAP)-positive glial cells expressed intracytoplasmic immunoreactivity for fibronectin. After the second week in culture, however, fibronectin was expressed by a distinct population of GFAP-positive flat astrocytes, irrespective of which brain region was studied. In cerebellar cultures, these cells were more abundant than in cortical or neostriatal cultures and often formed a major population of the GFAP-positive cells. The difference in fibronectin expression between cerebellum and the other areas studied was statistically significant. When cultures were started from 9-day-old postnatal rat brain, fibronectin-positive astrocytes appeared earlier than in those from newborn animals, in all areas studied. Further, especially in the case of cerebellum, the number of fibronectin-positive astrocytes increased as a function of time in culture. In cultures started from whole brains of 12-day-old rat embryos, fibronectin was expressed within 24 h in culture by all the cells with morphology of flat astrocytes, positive for vimentin but negative for GFAP. These results indicate that astrocytes cultured from newborn and early postnatal rat brain are a heterogeneous population of cells: depending on the brain region studied and also depending on the age of brain tissue or the time in culture, less than 1-60% of the GFAP-positive flat astrocytes expressed fibronectin. This, together with the fact that fibronectin was present in early embryonic brain cells in culture, suggests that fibronectin may be a prerequisite for the development or interactions of brain cells.     

Distribution of Kiaa0319-like immunoreactivity in the adult mouse brain--a novel protein encoded by the putative dyslexia susceptibility gene KIAA0319-like

Kiaa0319L is a novel protein encoded by a recently discovered gene KIAA0319-like(L) that may be associated with reading disability. Little is known about the characteristics of this protein and its distribution in the brain. We investigated here expression of this protein in adult mice, using an antibody specific for human and rodent Kiaa0319L. In the brain, Kiaa0319L was localized strongly in the olfactory bulb, and strong expression was found in other regions, including hippocampus, cerebellum, diencephalon and the cerebral cortex. Immunohistochemistry confirmed expression in these brain regions, and showed further that the protein was expressed preferentially in neurons in layer IV and VI of the neocortex, CA1 and CA2 subfields of the hippocampus and a subpopulation of neurons in CA3 and dentate gyrus. Furthermore, the protein was confined to dendrites of CA1 neurons in the stratum radiatum, but not those in the stratum oriens, and in astrocytes within the hippocampus. In the cerebellum, the protein was observed in the molecular layer and a fraction of Purkinje neurons. These findings confirmed expression of Kiaa0319L in brain regions that are involved in reading performance, supporting its possible involvement in reading disability. The specific patterns of localization in the neocortex, hippocampus and cerebellum suggest further that this protein may be related to other biological processes in a subpopulation of neurons within these regions, eg. formation and maintenance of polarity in the neuron.