Astrocytic Glutamine Synthetase Research Articles

HIV-1 gp120 Induces Glutamate Excitotoxicity By Diminishing Glutamine Synthetase Expression in Astrocytes

Background： As the most abundant subtype of glial cells, astrocytes have an important role in maintaining homeostasis in the central nervous system(CNS) by regulating neurotransmitters. Glutamate is an excitatory neurotransmitter released from neurons, while astrocytes take up glutamate and mediate the conversion to glutamine, which is known as the glutamate-glutamine cycle between neurons and astrocytes. Glutamine synthetase (GS) is found primarily in astrocytes and is a key player in the glutamate-glutamine cycle. GS synthesizes glutamine using glutamate and contributes to the maintenance of glutamate homeostasis between neurons and astrocytes by facilitating glutamate clearance. In addition, glutamate excitotoxicity is a key pathological factor in neurodegenerative pathologies such as HIV-1-associated neurocognitive disorder (HAND) and Alzheimer's disease. Although the importance of GS in the glutamate-glutamine cycle has been demonstrated, the relationship between gp120 in regulating astrocyte glutamate metabolism and GS activity is unclear. Methods: To evaluate the glutamate clearance capacity, glutamate uptake assay was undertaken by using the glutamate colorimetric assay kit. Astrocytes (u251) were incubated for 24 hours with different concentrations of full-length HIV-1 gp120 and medium in each dish was replaced with serum-free HBSS that containing 2 mM glutamate. After 10, 30, 60, or 120 min in the incubator, 200 μL culture supernatants of the astrocytes were collected and mixed with the corresponding reagents provided in the kit as required by the protocol, then 100 μL of the mixed solution were transferred to each well of the 96-well culture plates. The absorbance of the mixed solution was measured at 340 nm using a microplate reader. The glutamate content of each sample was calculated according to the formula provided in the instruction manual. The expressions of GS in astrocytes which were incubated for 24 hours with different concentrations of full-length HIV-1 gp120 were detected by Western blot. Student's t-tests and ANOVA were applied to determine statistical significance. Results： The glutamate uptake assay reflects whether the glutamate uptake capacity of astrocytes is impaired by assessing the extracellular glutamate content. First, changes in extracellular glutamate content were detected by treating astrocytes with different concentrations of HIV-1 gp120 for 24h. As expected, the extracellular glutamate content increased relative to the blank control, indicating that HIV-1 gp120 impaired the glutamate uptake ability of astrocytes. The optimal experimental point to test the glutamate uptake capacity was 30 min after the addition of serum-free HBSS that containing 2 mM glutamate by time-point experiments. In addition, the results of western blot showed that GS expression in astrocytes was significantly decreased by HIV-1 gp120 treatment. The GS expression levels and glutamate uptake assay results followed the same trend. Conclusion: In conclusion, this study explored the specific mechanisms of glutamatergic excitotoxicity in the brains of HAND patients. HIV-1 gp120 could impair the glutamate uptake capacity of astrocytes by diminishing GS expression. ( Acknowledgements: National Natural Science Foundation of China, No. 82172259 to H.C.; Corresponding author: Hong Cao, gzhcao@smu.edu.cn )

Increased NMDARs in neurons and glutamine synthetase in astrocytes underlying autistic-like behaviors of Gabrb1−/− mice

Mutations of the GABA-A receptor subunit β1 (GABRB1) gene are found in autism patients. However, it remains unclear how mutations in Gabrb1 may lead to autism. We generated Gabrb1-/- mouse model, which showed autistic-like behaviors. We carried out RNA-seq on the hippocampus and found glutamatergic pathway may be involved. We further carried out single-cell RNA sequencing on the whole brain followed by qRT-PCR, immunofluorescence, electrophysiology, and metabolite detection on specific cell types. We identified the up-regulated Glul/Slc38a3 in astrocytes, Grin1/Grin2b in neurons, glutamate, and the ratio of Glu/GABA in the hippocampus. Consistent with these results, increased NMDAR-currents and reduced GABAAR-currents in the CA1 neurons were detected in Gabrb1-/- mice. NMDAR antagonist memantine or Glul inhibitor methionine sulfoximine could rescue the abnormal behaviors in Gabrb1-/- mice. Our data reveal that upregulation of the glutamatergic synapse pathway, including NMDARs at neuronal synapses and glutamine exported by astrocytes, may lead to autistic-like behaviors.

CHANGES IN THE NEUROGENIC NICHE OF THE RAT HIPPOCAMPUS UNDER HYPOXIC EXPOSURE

One of the most vulnerable brain structures to hypoxia is the hippocampus. Maintenance of the neurogenic niche cell pool in the subgranular zone of the hippocampus (SGZ) is provided by adaptive mechanisms. Among them are changes in the functional activity of mitochondrial respiratory chain complexes and the reaction of astroglia, which provides metabolic support for neurons. In order to study the dynamics of adaptive changes in neurons and glia in the dentate gyrus of the hippocampus under hypoxic conditions on a model of intermitten hypobaric hypoxia (5000 m, equivalent to 10.5% O2), with a single (60 min) and multiple (8 and 20 episodes) exposure in low-resistant rats, immunomorphological methods revealed the features of localization and content in the neurons of complex IV mitochondrial respiratory chain (MTCO1), astrocyte marker proteins glutamine synthetase (GS) and GFAP, and doublecortin (DCX) in immature neurons. With a single hypoxia, the content of MTCO1 in neurons significantly increased, and after eight exposures, the amount of glutamine synthetase (GS) in astrocytes of the dentate gyrus of the hippocampus increased. Changes in the content of GS were most pronounced in the processes of astrocytes, which indicates a redistribution of GS. The number of DCX+ neurons in the SGZ significantly decreased after 20 episodes of hypoxia. At the same time, DCX+ cells of glial morphology were found in the polymorphic layer, and staining for GFAP showed an increase in the number of astrocytes. This may be due to a shift in the direction of cell differentiation in the neurogenic niche. Thus, in hypoxia, at the initial stage, a functional restructuring of the respiratory chain of neurons of the granular layer occurs. Subsequently, it is noted by the activation of astrocytes that modulate glutamate metabolism. The presence of a relationship between the dynamics of adaptive reactions of energy exchange in neurons and glia and changes in neurogenesis during 20 episodes of hypoxia suggests that during long-term hypoxia, the differentiation of neural precursors of SGZ in the direction of astroglia occurs, however, this issue requires further study in order to more accurately determine the nature of DCX+ cells.

Age-Associated Upregulation of Glutamate Transporters and Glutamine Synthetase in Senescent Astrocytes In Vitro and in the Mouse and Human Hippocampus.

Aging is marked by complex and progressive physiological changes, including in the glutamatergic system, that lead to a decline of brain function. Increased content of senescent cells in the brain, such as glial cells, has been reported to impact cognition both in animal models and human tissue during normal aging and in the context of neurodegenerative disease. Changes in the glutamatergic synaptic activity rely on the glutamate-glutamine cycle, in which astrocytes handle glutamate taken up from synapses and provide glutamine for neurons, thus maintaining excitatory neurotransmission. However, the mechanisms of glutamate homeostasis in brain aging are still poorly understood. Herein, we showed that mouse senescent astrocytes in vitro undergo upregulation of GLT-1, GLAST, and glutamine synthetase (GS), along with the increased enzymatic activity of GS and [3H]-D-aspartate uptake. Furthermore, we observed higher levels of GS and increased [3H]-D-aspartate uptake in the hippocampus of aged mice, although the activity of GS was similar between young and old mice. Analysis of a previously available RNAseq dataset of mice at different ages revealed upregulation of GLAST and GS mRNA levels in hippocampal astrocytes during aging. Corroborating these rodent data, we showed an increased number of GS + cells, and GS and GLT-1 levels/intensity in the hippocampus of elderly humans. Our data suggest that aged astrocytes undergo molecular and functional changes that control glutamate-glutamine homeostasis upon brain aging.

Retina mirrors brain pathology and response to GA immunotherapy in advanced stage AD-model mice.

Characteristic signs of Alzheimer's disease (AD) are increasingly reported in the neurosensory retina of human patients and animal models, however examination of the retino-cerebral relationship, especially at late disease stages and in response to therapy, is scarce. Transgenic models of AD (APPSWE /PS1∆E9 ; ADtg mice) treated with glatiramer acetate (GA) immunomodulation showed disease alleviation in pre- and early-symptomatic disease stages. Here, we explored the link between retinal and cerebral AD-related biomarkers, including response to GA immunization, in cohorts of old, advanced-stage ADtg mice. These mice were aged to become more clinically relevant to AD. To determine the extent of amyloid β-protein (Aβ) burden in retinal and brain tissues from treated and untreated ADtg mice, we developed a new protocol for extracting, unfolding and quantifying Aβ levels utilizing a Meso Scale Discovery biochemical assay, which recognizes human and mouse Aβ1-42 , Aβ1-40 , and Aβ1-38 proteins. Homogenates from these tissues were additionally analyzed by mass spectrometry for detailed global protein levels. Immunohistochemical staining for Aβ plaques, inflammation, infiltrating myeloid cells, and synapses were conducted on brain sections to reveal cerebral effects of GA immunotherapy. Levels of synaptotoxic Aβ1-42 , angiopathic Aβ1-40 , non-amyloidogenic Aβ1-38 , and Aβ42 /Aβ40 ratios tightly correlated between paired retinas derived from oculus sinister (OS) and oculus dexter (OD) eyes, and between left and right brain hemispheres. Brains and retinas displayed significant, one-side dominant lateralization of Aβ burden. Importantly, OS and OD retinal Aβ levels correlated with their cerebral counterparts, in advanced disease stage and following GA immunotherapy, with stronger contralateral correlations. Moreover, immunomodulation in old ADtg mice provoked reductions in vascular and parenchymal Aβ deposits in the brain, especially of large, dense-core plaque subtypes, and alleviation of microgliosis and astrocytosis. Immunization further enhanced cerebral recruitment of peripheral myeloid cells and synaptic preservation. Global proteome profiling by mass spectrometry revealed novel molecular parallels in retino-cerebral AD-related pathology and response to GA immunization, including restoration of homeostatic astrocytic glutamine synthetase expression. Overall, our results support the sustainability of immunomodulation in provoking CNS repair, even in advanced-stage AD-model mice, while revealing similarities in retino-cerebral responses to therapy, providing incentives to explore retinal AD biomarkers.

Saul Brusilow: Understanding and treating diseases of ammonia toxicity

This article reviews the scientific career and accomplishments of the late Dr. Saul Brusilow, Professor of Pediatrics at Johns Hopkins. Dr. Brusilow's career was focused on diseases involving hyperammonemia. He and his colleagues developed a set of drugs that could lower ammonia levels in patients with genetic disorders of the urea cycle by providing alternative pathways for the synthesis of excretable nitrogenous molecules. Those drugs and their derivatives represent one of the earliest and most successful drug therapies for genetic diseases. Turning their attention to brain swelling caused by liver disease, Dr. Brusilow and colleagues developed the Osmotic Gliopathy Hypothesis to help explain the mechanism of ammonia toxicity, postulating that high ammonia drives glutamine synthetase in astrocytes to produce elevated levels of glutamine that act as a potent osmolyte, drawing water into the cell and causing cerebral edema. This hypothesis suggests that inhibiting glutamine synthetase with its well-characterized inhibitor, methionine sulfoximine, might prove therapeutic in cases of hepatic encephalopathy, a conclusion supported by their subsequent studies in animals. But although the drugs developed to treat hyperammonemia resulting from urea cycle disorders were successfully developed and approved by the FDA, the compound suggested as a treatment for hepatic encephalopathy was unable to attract sufficient interest and investment to be tested for use in humans.

Modulation of Astrocytic Glutamine Synthetase by Endocannabinoid 2-Arachidonoylglycerol in JNK-Independent Pathway.

Background and Objective: The glutamine synthetase (GS), an astrocyte-specific enzyme, plays an important role in neuroprotection through the glutamate/glutamine shuttle and can be modulated by endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) through extracellular signal-regulated protein kinase ½ (ERK1/2) and p38 signaling pathways. However, the role of c-Jun N-terminal kinase (JNK) signaling pathway in the modulation of GS in astrocytes by 2-AG is not clear.Materials and Methods: The expression of GS and JNK in astrocytes following the exposure to lipopolysaccharide (LPS) was examined with Western blotting and immunochemistry.Results: The results revealed that short-term exposure to LPS activated GS and increased phosphorylation of JNK in astrocytes in a time-dependent manner. Treatment with 2-AG reversed the changes in GS but had no effect on the activation of JNK.Conclusions: These findings suggest that the activation of JNK induced by LPS is not involved in the modulation of astrocytic GS by 2-AG.

Dysembryoplastic neuroepithelial tumors: A single-institutional series with special reference to glutamine synthetase expression

Dysembryoplastic neuroepithelial tumors (DNT) is a benign (World Health Organisation, WHO, grade I) glioneuronal tumor and it represent one of the most frequent neoplasm in patient affected by seizures. The epileptic neuronal activity can be determined by abnormal synchronization, excessive glutamate excitation and\\or inadequate GABA inhibition. Increasing evidence suggests that the astrocytes might be involved in this process even if neurons play a relevant role. In particular astrocytes promote the clearance of glutamate, a potent excitatory neurotransmitter of the central nervous system. Indeed, elevated concentrations of extracellular glutamate may determine iper-excitability and seizures as well as other neurological disorders. So, astrocytes, converting glutamate into glutamine via the enzyme glutamine synthetase (GS), could play a protective anti-seizures role. In the present study, we analyzed the immunohistochemical expression of GS in 20 DNTs specimens documenting a constant immunoistochemical expression of GS in astrocytes of the lesional tissue and of the cerebral cortex.

Glutamate Dehydrogenase Is Important for Ammonia Fixation and Amino Acid Homeostasis in Brain During Hyperammonemia.

Impaired liver function may lead to hyperammonemia and risk for hepatic encephalopathy. In brain, detoxification of ammonia is mediated mainly by glutamine synthetase (GS) in astrocytes. This requires a continuous de novo synthesis of glutamate, likely involving the action of both pyruvate carboxylase (PC) and glutamate dehydrogenase (GDH). An increased PC activity upon ammonia exposure and the importance of PC activity for glutamine synthesis has previously been demonstrated while the importance of GDH for generation of glutamate as precursor for glutamine synthesis has received little attention. We therefore investigated the functional importance of GDH for brain metabolism during hyperammonemia. To this end, brain slices were acutely isolated from transgenic CNS-specific GDH null or litter mate control mice and incubated in aCSF containing [U-13C]glucose in the absence or presence of 1 or 5 mM ammonia. In another set of experiments, brain slices were incubated in aCSF containing 1 or 5 mM 15N-labeled NH4Cl and 5 mM unlabeled glucose. Tissue extracts were analyzed for isotopic labeling in metabolites and for total amounts of amino acids. As a novel finding, we reveal a central importance of GDH function for cerebral ammonia fixation and as a prerequisite for de novo synthesis of glutamate and glutamine during hyperammonemia. Moreover, we demonstrated an important role of the concerted action of GDH and alanine aminotransferase in hyperammonemia; the products alanine and α-ketoglutarate serve as an ammonia sink and as a substrate for ammonia fixation via GDH, respectively. The role of this mechanism in human hyperammonemic states remains to be studied.

Astroglial Glutamine Synthetase and the Pathogenesis of Mesial Temporal Lobe Epilepsy.

The enzyme glutamine synthetase (GS), also referred to as glutamate ammonia ligase, is abundant in astrocytes and catalyzes the conversion of ammonia and glutamate to glutamine. Deficiency or dysfunction of astrocytic GS in discrete brain regions have been associated with several types of epilepsy, including medically-intractable mesial temporal lobe epilepsy (MTLE), neocortical epilepsies, and glioblastoma-associated epilepsy. Moreover, experimental inhibition or deletion of GS in the entorhinal-hippocampal territory of laboratory animals causes an MTLE-like syndrome characterized by spontaneous, recurrent hippocampal-onset seizures, loss of hippocampal neurons, and in some cases comorbid depressive-like features. The goal of this review is to summarize and discuss the possible roles of astroglial GS in the pathogenesis of epilepsy.

Baicalin combats glutamate excitotoxicity via protecting glutamine synthetase from ROS-induced 20S proteasomal degradation

BackgroundMany neuroprotective approaches targeting neurons in animal models fail to provide benefits for the treatment of ischemic stroke in clinic and glial cells have become the targets in some basic studies. Baicalin has neuroprotective effects but the mechanisms related to glial cells are not revealed. This study investigated whether and how baicalin can combat excitotoxicity via protecting the functions of astrocytes in early stage of ischemia/reperfusion (I/R) insult by focusing on glutamine synthetase (GS). Experimental approachThe role of baicalin was explored in primary astrocytes exposed to oxygen-glucose deprivation/reperfusion (OGD/R) and rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). Key resultsMitochondrial succinate dehydrogenase (SDH) activation led to an excessive production of reactive oxygen species (ROS) via reverse electron transport (RET) under conditions of OGD/R or I/R, which increased the carbonylation and proteasomal degradation of GS in astrocytes. Treatment of baicalin decreased the oxidative stress mediated by SDH and reduced the subsequent loss of GS. This effect increased the glutamate disposal by astrocytes and protected neurons from excitotoxicity in response to I/R insults. Conclusions and implicationsBaicalin inactivated SDH to suppress ROS production and protected GS protein stability against oxidative stress, contributing to the improvement of the glutamate disposal and decrease in excitotoxicity. These results suggest that protection of GS stability in astrocytes might be an effective strategy to prevent neuronal injury in acute ischemic stroke.

Ectopic expression of L1CAM ectodomain alters differentiation and motility, but not proliferation, of human neural progenitor cells

Adult human neural progenitor and stem cells have been implicated as a potential source of brain cancer causing cells, but specific events that might cause cells to progress towards a transformed phenotype remain unclear. The L1CAM (L1) cell adhesion/recognition molecule is expressed abnormally by human glioma cancer cells and is released as a large extracellular ectodomain fragment, which stimulates cell motility and proliferation. This study investigates the effects of ectopic overexpression of the L1 long ectodomain (L1LE; ˜180 kDa) on the motility, proliferation, and differentiation of human neural progenitor cells (HNPs). L1LE was ectopically expressed in HNPs using a lentiviral vector. Surprisingly, overexpression of L1LE resulted in reduced HNP motility in vitro, in stark contrast to the effects on glioma and other cancer cell types. L1LE overexpression resulted in a variable degree of maintenance of HNP proliferation in media without added growth factors but did not increase proliferation. In monolayer culture, HNPs expressed a variety of differentiation markers. L1LE overexpression resulted in loss of glutamine synthetase (GS) and β3-tubulin expression in normal HNP media, and reduced vimentin and increased GS expression in the absence of added growth factors. When co-cultured with chick embryonic brain cell aggregates, HNPs show increased differentiation potential. Some HNPs expressed p-neurofilaments and oligodendrocytic O4, indicating differentiation beyond that in monolayer culture. Most HNP-L1LE cells lost their vimentin and GFAP (glial fibrillary acidic protein) staining, and many cells were positive for astrocytic GS. However, these cells rarely were positive for neuronal markers β3-tubulin or p-neurofilaments, and few HNP oligodendrocyte progenitors were found. These results suggest that unlike for glioma cells, L1LE does not increase HNP cell motility, but rather decreases motility and influences the differentiation of normal brain progenitor cells. Therefore, the effect of L1LE on increasing motility and proliferation appears to be limited to already transformed cells.

Persistent increase in ventral hippocampal long‐term potentiation by juvenile stress: A role for astrocytic glutamine synthetase

A traumatic childhood is among the most important risk factors for developing stress-related psychopathologies such as posttraumatic stress disorder or depression later in life. However, despite the proven role of astrocytes in regulating transmitter release and synaptic plasticity, the contribution of astrocytic transmitter metabolism to such stress-induced psychopathologies is currently not understood. In rodents, childhood adversity can be modeled by juvenile stress exposure, resulting in increased anxiety, and impaired coping with stress in adulthood. We describe that such juvenile stress in rats, regardless of additional stress in adulthood, leads to reduced synaptic efficacy in the ventral CA1 (vCA1) Schaffer collaterals, but increased long-term potentiation (LTP) of synaptic transmission after high-frequency stimulation. We tested whether the glutamate-glutamine-cycle guides the lasting changes on plasticity observed after juvenile stress by blocking the astrocytic glutamate-degrading enzyme, glutamine synthetase (GS). Indeed, the pharmacological inhibition of GS by methionine sulfoximine in slices from naïve rats mimics the effect of juvenile stress on vCA1-LTP, while supplying glutamine is sufficient to normalize the LTP. Assessing steady-state mRNA levels in the vCA1 stratum radiatum reveals distinct shifts in the expression of GS, astrocytic glutamate, and glutamine transporters after stress in juvenility, adulthood, or combined juvenile/adult stress. While GS mRNA expression levels are lastingly reduced after juvenile stress, GS protein levels are maintained stable. Together our results suggest a critical role for astrocytes and the glutamate-glutamine cycle in mediating long-term effects of juvenile stress on plasticity in the vCA1, a region associated with anxiety and emotional memory processing.

2-arachidonyl glycerol modulates astrocytic glutamine synthetase via p38 and ERK1/2 pathways

BackgroundThe glutamine synthetase (GS), an astrocyte-specific enzyme, is involved in lipopolysaccharide (LPS)-induced inflammation which activates the mitogen-activated protein kinase (MAPK) signaling. Endocannabinoid 2-arachidonyl glycerol (2-AG) has been described to serve as an endogenous mediator of analgesia and neuroprotection. However, whether 2-AG can directly influence astrocytic GS and MAPK expressions remains unknown.MethodsIn the present study, the effects of 2-AG on astrocytic GS expression, p38 and ERK1/2 expression, cell viability, and apoptosis following LPS exposure were investigated.ResultsThe results revealed that LPS exposure increased GS expression with p38 activation in the early phase and decreased GS expression with activation of ERK1/2, decrease of cell viability, and increase of apoptosis in the late phase. Inhibition of p38 reversed GS increase in the early phase while inhibition of ERK1/2 reversed GS decrease in the late phase induced by LPS exposure. 2-AG protected astrocytes from increase of apoptosis and decrease of cell viability induced by the late phase of LPS exposure. In the early phase of LPS exposure, 2-AG could suppress the increase of GS expression and activation of p38 signaling. In the late phase of LPS exposure, 2-AG could reverse the decrease of GS expression and activation of ERK1/2 induced by LPS.ConclusionThese findings suggest that 2-AG could maintain the GS expression in astrocytes to a relatively stable level through modulating MAPK signaling and protect astrocytes from LPS exposure.

Astrocytes and Glutamine Synthetase in Epileptogenesis.

The cellular, molecular, and metabolic mechanisms that underlie the development of mesial temporal lobe epilepsy are incompletely understood. Here we review the role of astrocytes in epilepsy development (a.k.a. epileptogenesis), particularly astrocyte pathologies related to: aquaporin 4, the inwardly rectifying potassium channel Kir4.1, monocarboxylate transporters MCT1 and MCT2, excitatory amino acid transporters EAAT1 and EAAT2, and glutamine synthetase. We propose that inhibition, dysfunction or loss of astrocytic glutamine synthetase is an important causative factor for some epilepsies, particularly mesial temporal lobe epilepsy and glioblastoma-associated epilepsy. We postulate that the regulatory mechanisms of glutamine synthetase as well as the downstream effects of glutamine synthetase dysfunction, represent attractive, new targets for antiepileptogenic interventions. Currently, no antiepileptogenic therapies are available for human use. The discovery of such interventions is important as it will fundamentally change the way we approach epilepsy by preventing the disease from ever becoming manifest after an epileptogenic insult to the brain.

Astrocytic glutamine synthetase is expressed in the neuronal somatic layers and down-regulated proportionally to neuronal loss in the human epileptic hippocampus.

Human mesial temporal lobe epilepsy (MTLE) features subregion-specific hippocampal neurodegeneration and reactive astrogliosis, including up-regulation of the glial fibrillary acidic protein (GFAP) and down-regulation of glutamine synthetase (GS). However, the regional astrocytic expression pattern of GFAP and GS upon MTLE-associated neurodegeneration still remains elusive. We assessed GFAP and GS expression in strict correlation with the local neuronal number in cortical and hippocampal surgical specimens from 16 MTLE patients using immunohistochemistry, stereology and high-resolution image analysis for digital pathology and whole-slide imaging. In the cortex, GS-positive (GS+) astrocytes are dominant in all neuronal layers, with a neuron to GS+ cell ratio of 2:1. GFAP-positive (GFAP+) cells are widely spaced, with a GS+ to GFAP+ cell ratio of 3:1-5:1. White matter astrocytes, on the contrary, express mainly GFAP and, to a lesser extent, GS. In the hippocampus, the neuron to GS+ cell ratio is approximately 1:1. Hippocampal degeneration is associated with a reduction of GS+ astrocytes, which is proportional to the degree of neuronal loss and primarily present in the hilus. Up-regulation of GFAP as a classical hallmark of reactive astrogliosis does not follow the GS-pattern and is prominent in the CA1. Reactive alterations were proportional to the neuronal loss in the neuronal somatic layers (stratum pyramidale and hilus), while observed to a lesser extent in the axonal/dendritic layers (stratum radiatum, molecular layer). We conclude that astrocytic GS is expressed in the neuronal somatic layers and, upon neurodegeneration, is down-regulated proportionally to the degree of neuronal loss.

Progressive neuronal activation accompanies epileptogenesis caused by hippocampal glutamine synthetase inhibition

Loss of glutamine synthetase (GS) in hippocampal astrocytes has been implicated in the causation of human mesial temporal lobe epilepsy (MTLE). However, the mechanism by which the deficiency in GS leads to epilepsy is incompletely understood. Here we ask how hippocampal GS inhibition affects seizure phenotype and neuronal activation during epilepsy development (epileptogenesis). Epileptogenesis was induced by infusing the irreversible GS blocker methionine sulfoximine (MSO) unilaterally into the hippocampal formation of rats. We then used continuous video-intracranial electroencephalogram (EEG) monitoring and c-Fos immunohistochemistry to determine the type of seizures and spatial distribution of neuronal activation early (1–5days postinfusion) and late (16–43days postinfusion) in epileptogenesis. Early in epileptogenesis, seizures were preferentially mild (stage 1–2), activating neurons in the entorhinal-hippocampal area, the basolateral amygdala, the piriform cortex, the midline thalamus, and the anterior olfactory area. Late in epileptogenesis, the seizures were generally more severe (stages 4–5) with neuronal activation extending to the neocortex, the bed nucleus of the stria terminalis, the mediodorsal thalamu\\s, and the central nucleus of the amygdala. Our findings demonstrate that inhibition of GS focally in the hippocampal formation triggers a process of epileptogenesis characterized by gradual worsening of seizure severity and involvement of progressively larger neuronal populations over a period of several weeks. Knowledge about the underlying mechanism of epileptogenesis is important because such knowledge may result in more specific and efficacious treatments of MTLE by moving away from large and poorly specific surgical resections to highly targeted surgical or pharmacological interventions of the epileptogenic process.

Multifactorial Effects on Different Types of Brain Cells Contribute to Ammonia Toxicity

Effects of ammonia on astrocytes play a major role in hepatic encephalopathy, acute liver failure and other diseases caused by increased arterial ammonia concentrations (e.g., inborn errors of metabolism, drug or mushroom poisoning). There is a direct correlation between arterial ammonia concentration, brain ammonia level and disease severity. However, the pathophysiology of hyperammonemic diseases is disputed. One long recognized factor is that increased brain ammonia triggers its own detoxification by glutamine formation from glutamate. This is an astrocytic process due to the selective expression of the glutamine synthetase in astrocytes. A possible deleterious effect of the resulting increase in glutamine concentration has repeatedly been discussed and is supported by improvement of some pathologic effects by GS inhibition. However, this procedure also inhibits a large part of astrocytic energy metabolism and may prevent astrocytes from responding to pathogenic factors. A decrease of the already low glutamate concentration in astrocytes due to increased synthesis of glutamine inhibits the malate-aspartate shuttle and energy metabolism. A more recently described pathogenic factor is the resemblance between NH4+ and K+ in their effects on the Na+,K+-ATPase and the Na+,K+, 2 Cl- and water transporter NKCC1. Stimulation of the Na+,K+-ATPase driven NKCC1 in both astrocytes and endothelial cells is essential for the development of brain edema. Na+,K+-ATPase stimulation also activates production of endogenous ouabains. This leads to oxidative and nitrosative damage and sensitizes NKCC1. Administration of ouabain antagonists may accordingly have therapeutic potential in hyperammonemic diseases.

A sub-threshold dose of pilocarpine increases glutamine synthetase in reactive astrocytes and enhances the progression of amygdaloid-kindling epilepsy in rats.

The prognosis of patients exposed to a sub-threshold dose of a proconvulsant is difficult to establish. In this study, we investigated the effect of a single sub-threshold dose of the proconvulsant pilocarpine (PILO) on the progression of seizures that were subsequently induced by daily electrical stimulation (kindling) of the amygdaloid formation. Male Sprague–Dawley rats were each implanted with an electrode in the right basolateral amygdala and an indwelling cannula in the right ventricle. The animals were randomized into groups and were administered one of the following treatments: saline, PILO, saline+L-α-aminoadipic acid (L-AAA; one dosage tested), PILO+L-AAA, or PILO+L-methionine sulfoximine (three dosages tested). Amygdaloid stimulation and electroencephalography were performed once daily. We performed immunohistochemistry and western blot for glial fibrillary acidic protein and glutamine synthetase (GS). We also assayed the enzymic activity of GS in discrete brain regions. An intraperitoneal injection of a sub-threshold PILO dose enhanced the progression of amygdaloid-kindling seizures and was accompanied by an increase in reactive-astrocyte and GS (content and activity) in the hippocampus and piriform cortex. L-AAA and L-methionine sulfoximine, inhibitors of astrocytic and GS function, respectively, abolished the effect of PILO on amygdaloid-kindling seizures. We conclude that one sub-threshold dose of a proconvulsant may enhance the progression of subsequent epilepsy and astrocytic GS may play a role in this phenomenon. Thus, a future therapy for epilepsy could be inhibition of astrocytes and/or GS.

Inhibition of Astrocytic Glutamine Synthetase by Lead is Associated with a Slowed Clearance of Hydrogen Peroxide by the Glutathione System

Lead intoxication in humans is characterized by cognitive impairments, particularly in the domain of memory, where evidence indicates that glutamatergic neurotransmission may be impacted. Animal and cell culture studies have shown that lead decreases the expression and activity of glutamine synthetase (GS) in astrocytes, yet the basis of this effect is uncertain. To investigate the mechanism responsible, the present study exposed primary astrocyte cultures to a range of concentrations of lead acetate (0–330 μM) for up to 24 h. GS activity was significantly reduced in cells following 24 h incubation with 100 or 330 μM lead acetate. However, no reduction in GS activity was detected when astrocytic lysates were co-incubated with lead acetate, suggesting that the mechanism is not due to a direct interaction and involves intact cells. Since GS is highly sensitive to oxidative stress, the capacity of lead to inhibit the clearance of hydrogen peroxide (H2O2) was investigated. It was found that exposure to lead significantly diminished the capacity of astrocytes to degrade H2O2, and that this was due to a reduction in the effectiveness of the glutathione system, rather than to catalase. These results suggest that the inhibition of GS activity in lead poisoning is a consequence of slowed H2O2 clearance, and supports the glutathione pathway as a primary therapeutic target.