Glutamate Transporter Expression In Astrocytes Research Articles

Tsc2 gene inactivation causes a more severe epilepsy phenotype than Tsc1 inactivation in a mouse model of Tuberous Sclerosis Complex

Tuberous Sclerosis Complex (TSC) is an autosomal dominant, multi-system disorder, typically involving severe neurological symptoms, such as epilepsy, cognitive deficits and autism. Two genes, TSC1 and TSC2, encoding the proteins hamartin and tuberin, respectively, have been identified as causing TSC. Although there is a substantial overlap in the clinical phenotype produced by TSC1 and TSC2 mutations, accumulating evidence indicates that TSC2 mutations cause more severe neurological manifestations than TSC1 mutations. In this study, the neurological phenotype of a novel mouse model involving conditional inactivation of the Tsc2 gene in glial-fibrillary acidic protein (GFAP)-positive cells (Tsc2(GFAP1)CKO mice) was characterized and compared with previously generated Tsc1(GFAP1)CKO mice. Similar to Tsc1(GFAP1)CKO mice, Tsc2(GFAP1)CKO mice exhibited epilepsy, premature death, progressive megencephaly, diffuse glial proliferation, dispersion of hippocampal pyramidal cells and decreased astrocyte glutamate transporter expression. However, Tsc2(GFAP1)CKO mice had an earlier onset and higher frequency of seizures, as well as significantly more severe histological abnormalities, compared with Tsc1(GFAP1)CKO mice. The differences between Tsc1(GFAP1)CKO and Tsc2(GFAP1)CKO mice were correlated with higher levels of mammalian target of rapamycin (mTOR) activation in Tsc2(GFAP1)CKO mice and were reversed by the mTOR inhibitor, rapamycin. These findings provide novel evidence in mouse models that Tsc2 mutations intrinsically cause a more severe neurological phenotype than Tsc1 mutations and suggest that the difference in phenotype may be related to the degree to which Tsc1 and Tsc2 inactivation causes abnormal mTOR activation.

Modulation of astrocyte glutamate transporters decreases seizures in a mouse model of Tuberous Sclerosis Complex

Astrocyte dysfunction may contribute to epileptogenesis and other neurological deficits in Tuberous Sclerosis Complex (TSC). In particular, decreased expression and function of astrocyte glutamate transporters have been implicated in causing elevated extracellular glutamate levels, neuronal death, and epilepsy in a mouse model of TSC ( Tsc1 GFAPCKO mice), involving inactivation of the Tsc1 gene primarily in astrocytes. Here, we tested whether pharmacological induction of astrocyte glutamate transporter expression can prevent the neurological phenotype of Tsc1 GFAPCKO mice. Early treatment with ceftriaxone prior to the onset of epilepsy increased expression of astrocyte glutamate transporters, decreased extracellular glutamate levels, neuronal death, and seizure frequency, and improved survival in Tsc1 GFAPCKO mice. In contrast, late treatment with ceftriaxone after onset of epilepsy increased glutamate transporter expression, but had no effect on seizures. These results indicate that astrocyte glutamate transporters contribute to epileptogenesis in Tsc1 GFAPCKO mice and suggest novel therapeutic strategies for epilepsy in TSC directed at astrocytes.

Microglia in ALS: The Good, The Bad, and The Resting

Inflammation, including microglial activation and T cell infiltration, is a neuropathological hallmark of amyotrophic lateral sclerosis (ALS), a rapidly progressing neurodegenerative disease. The identification of mutations in the gene for Cu2+/Zn2+ superoxide dismutase (SOD1) from patients with an inherited form of ALS enabled the creation of transgenic mice overexpressing mutant forms of SOD1 (mSOD1) which develop a motoneuron disease that resembles the disease seen in ALS patients. These transgenic mice display similar inflammatory reactions at sites of motoneuron injury as detected in ALS patients, enabling the observation that this inflammation is not simply a late consequence of motoneuron degeneration, but actively contributes to the balance between neuroprotection and neurotoxicity. The microglial and T cell activation states influence the rate of disease progression. Initially, microglia and T cells can slow disease progression, while they may later contribute to the acceleration of disease. Accumulation of intracellular and extracellular misfolded mSOD1 may be key events regulating the transformation from neuroprotective alternatively activated M2 microglia to cytotoxic classically activated M1 microglia. Intracellular and extracellular mSOD1 utilizing different pathways may enhance the production and release of reactive oxygen species (ROS) and augment the inflammatory cytokine cascade from microglia. These ROS and cytokines may increase the susceptibility of motoneurons to glutamate toxicity and inhibit the function and expression of astrocytic glutamate transporters resulting in further neurotoxicity. Thus, the cumulative evidence suggests that inflammation plays a central role in ALS and manipulating these microglial effector functions may potentially modify the outcome of this devastating disease.

Estrogen and tamoxifen reverse manganese‐induced glutamate transporter impairment in astrocytes

Chronic exposure to manganese (Mn) can cause manganism, a neurodegenerative disorder similar to Parkinson's disease. The toxicity of Mn includes impairment of astrocytic glutamate transporters. 17beta-Estradiol (E2) has been shown to be neuroprotective in various neurodegenerative diseases including Parkinson's disease and Alzheimer's disease, and some selective estrogen receptor modulators, including tamoxifen (TX), also possess neuroprotective properties. We have tested our hypothesis that E2 and TX reverse Mn-induced glutamate transporter impairment in astrocytes. The results established that E2 and TX increased glutamate transporter function and reversed Mn-induced glutamate uptake inhibition, primarily via the up-regulation of glutamate/aspartate transporter (GLAST). E2 and TX also increased astrocytic GLAST mRNA levels and attenuated the Mn-induced inhibition of GLAST mRNA expression. In addition, E2 and TX effectively increased the expression of transforming growth factor beta1, a potential modulator of the stimulatory effects of E2/TX on glutamate transporter function. This effect was mediated by the activation of MAPK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. These novel findings suggest, for the first time, that E2 and TX enhance astrocytic glutamate transporter expression via increased transforming growth factor beta1 expression. Furthermore, the present study is the first to show that both E2 and TX effectively reverse Mn-induced glutamate transport inhibition by restoring its expression and activity, thus offering a potential therapeutic modality in neurodegenerative disorders characterized by altered glutamate homeostasis.

Reversal of axonal loss and disability in a mouse model of progressive multiple sclerosis

Axonal degeneration is an important determinant of progressive neurological disability in multiple sclerosis (MS). Thus, therapeutic approaches promoting neuroprotection could aid the treatment of progressive MS. Here, we used what we believe is a novel water-soluble fullerene derivative (ABS-75) attached to an NMDA receptor antagonist, which combines antioxidant and anti-excitotoxic properties, to block axonal damage and reduce disease progression in a chronic progressive EAE model. Fullerene ABS-75 treatment initiated after disease onset reduced the clinical progression of chronic EAE in NOD mice immunized with myelin-oligodendrocyte glycoprotein (MOG). Reduced disease progression in ABS-75-treated mice was associated with reduced axonal loss and demyelination in the spinal cord. Fullerene ABS-75 halted oxidative injury, CD11b+ infiltration, and CCL2 expression in the spinal cord of mice without interfering with antigen-specific T cell responses. In vitro, fullerene ABS-75 protected neurons from oxidative and glutamate-induced injury and restored glutamine synthetase and glutamate transporter expression in astrocytes under inflammatory insult. Glutamine synthetase expression was also increased in the white matter of fullerene ABS-75-treated animals. Our data demonstrate the neuroprotective effect of treatment with a fullerene compound combined with a NMDA receptor antagonist, which may be useful in the treatment of progressive MS and other neurodegenerative diseases.

Differential involvement of TNFα in hypoxic suppression of astrocyte glutamate transporters

Transporter-mediated glutamate uptake is a principal function of astrocytes. Our previous studies have shown that this process is compromised under hypoxic conditions through the NF-kappaB mediated inhibition of expression of the glutamate transporters EAAT-1 and EAAT-2. Here, we demonstrate that identical conditions of hypoxia (1% O(2), 24 h) lead to a dramatic increase in TNFalpha production from astrocytes without altering their viability. This hypoxia-evoked production of TNFalpha was prevented in the presence of any of three mechanistically distinct NF-kappaB inhibitors. Exogenous application of TNFalpha was without effect on EAAT-1 expression as determined by Western blotting, but mimicked the effects of hypoxia to suppress expression of EAAT-2. Furthermore thalidomide, which prevents TNFalpha production, was without effect on hypoxic suppression of EAAT-1 but prevented hypoxic suppression of EAAT-2. These data indicate that regulation of glutamate transporter expression in astrocytes by hypoxia is subtype specific. Regulation of both EAAT-1 and EAAT-2 is mediated by NF-kappaB, and this transcriptional regulator is also required for increased production of TNFalpha. However, while TNFalpha is essential for hypoxic suppression of EAAT-2, hypoxic modulation of EAAT-1 expression is unaffected by this cytokine.

Regulation of astrocytic glutamate transporter expression by Akt: evidence for a selective transcriptional effect on the GLT‐1/EAAT2 subtype

In the nervous system, astrocytes express different ratios of the two glial glutamate transporters, glutamate transporter subtype 1 (GLT-1) and glutamate/aspartate transporter (GLAST), but little is known about the signaling pathways that independently regulate their expression. Treatment with dibutyryl-cAMP, epidermal growth factor (EGF) or other growth factors both induces expression of GLT-1 and increases expression of GLAST in astrocyte cultures. The induction of GLT-1 is correlated with morphological and biochemical changes that are consistent with astrocyte maturation. Pharmacological studies suggest that phosphatidylinositol 3-kinase (PI-3K) and the nuclear transcription factor-kappaB (NF-kappaB) may be involved in the induction of GLT-1 expression. In several signaling systems Akt, also known as protein kinase B (PKB), functions downstream of PI-3K. In these present studies we used lentiviral vectors engineered to express dominant-negative (DN), constitutively active (CA), or null variants of Akt to study the possible involvement of Akt in the regulation of GLT-1. Expression of DN-Akt attenuated the EGF-dependent induction of GLT-1. Expression of CA-Akt caused a dose- and time-dependent increase in GLT-1 protein, increased GLT-1 mRNA levels, increased dihydrokainate-sensitive (presumably GLT-1 mediated) transport activity, and caused a change in astrocyte morphology to a more stellate shape, but had no effect on GLAST protein levels. Finally, the expression of CA-Akt increased the expression of a reporter construct containing a putative promoter fragment from the human homolog of GLT-1, called EAAT2. From these studies, we conclude that Akt induces the expression of GLT-1 through increased transcription and that Akt can regulate GLT-1 expression without increasing GLAST expression in astrocytes.

Role of Mitochondrial Dysfunction and Oxidative Stress in the Pathogenesis of Selective Neuronal Loss in Wernicke's Encephalopathy

Thiamine deficiency results in Wernicke's encephalopathy and is commonly encountered in chronic alcoholism, gastrointestinal diseases, and HIV AIDS. The earliest metabolic consequence of thiamine deficiency is a selective loss in activity of the thiamine diphosphate-dependent enzyme alpha-ketoglutarate dehydrogenase (alpha-KGDH), a rate-limiting tricarboxylic acid cycle enzyme. Thiamine deficiency is characterized neuropathologically by selective neuronal cell death in the thalamus, pons, and cerebellum. The cause of this region-selective neuronal loss is unknown, but mechanisms involving cellular energy failure, focal lactic acidosis, and NMDA receptor-mediated excitotoxicity have classically been implicated. More recently, evidence supports a role for oxidative stress. Evidence includes increased endothelial nitric oxide synthase, nitrotyrosine deposition, microglial activation, and lipid peroxidation. Reactive oxygen species production results in decreased expression of astrocytic glutamate transporters and decreased activities of alpha-KGDH, resulting in an amplification of cell death mechanisms in thiamine deficiency.

Mild hypothermia in the prevention of brain edema in acute liver failure: mechanisms and clinical prospects.

Mild hypothermia (32 degrees C-35 degrees C) reduces intracranial pressure in patients with acute liver failure and may offer an effective adjunct therapy in the management of these patients. Studies in experimental animals suggest that this beneficial effect of hypothermia is the result of a decrease in blood-brain ammonia transfer resulting in improvement in brain energy metabolism and normalization of glutamatergic synaptic regulation. Improvement in brain energy metabolism by hypothermia may result from a reduction in ammonia-induced decrease of brain glucose (pyruvate) oxidation. Restoration of normal glutamatergic synaptic regulation by hypothermia may be the consequence of the removal of ammonia-induced decreases in expression of astrocytic glutamate transporters resulting in normal glutamate neurotransmitter inactivation in brain. Randomized controlled clinical trials of hypothermia are required to further evaluate its clinical impact.

Glutamate transporter expression in astrocytes of the rat dentate gyrus following lesion of the entorhinal cortex.

The glutamate transporters GLT-1 and GLAST localized in astrocytes are essential in limiting transmitter signalling and restricting harmful receptor overstimulation. To show changes in the expression of both transporters following lesion of the entorhinal cortex (and degeneration of the glutamatergic tractus perforans), quantitative microscopic in situ hybridization (ISH) using alkaline-phosphatase-labelled oligonucleotide probes was applied to the outer molecular layer of the hippocampal dentate gyrus of rats (termination field of the tractus perforans). Four groups of rats were studied: sham-operated controls, and animals 3, 14 and 60 days following unilateral electrolytic lesion of the entorhinal cortex. The postlesional shrinkage of the terminal field of the perforant path, ipsilateral to the lesion side, was determined and considered in the evaluation of quantitative ISH data. Statistical analysis revealed that ipsilateral to the lesion side there was a significant decrease of the GLT-1 mRNA at every postlesional time-point and of the GLAST mRNA at 14 and 60 days postlesion. The maximal decrease was approximately 45% for GLT-1 and approximately 35% for GLAST. In the terminal field of the perforant path contralateral to the lesion side, no significant changes of ISH labelling were measured. The results were complemented by immunocytochemical data achieved using antibodies against synthetic GLT-1 and GLAST peptides. In accordance with ISH results, there was an obvious decrease of GLT-1 and GLAST immunostaining in the terminal field of the perforant path ipsilateral to the lesion side. From these data we conclude that, following a lesioning of the entorhinal cortex, the loss of glutamatergic synapses in the terminal field of the perforant path resulted in a strong downregulation of glutamate transporters in astrocytes. The decrease of synaptically released glutamate or of other neuronal factors could be involved in this downregulation.

Glutamate transporter and receptor function in disorders of ammonia metabolism.

Disorders of ammonia metabolism including urea cycle enzymopathies, Reye Syndrome, and liver failure are associated with brain edema and severe neurological impairment. Excess blood-borne ammonia crosses the blood-brain barrier by diffusion as NH(3) where it interacts with various cellular processes involved in neurotransmission and brain energy metabolism. Ammonia exerts a potent effect on glutamate (AMPA) receptor-mediated neurotransmission. Ammonia also inhibits high affinity transport of glutamate by an action on astrocytic glutamate transporter expression, an action which results in increased extracellular concentrations of glutamate. Acute hyperammonemia directly activates the NMDA subclass of glutamate receptors resulting in increased intracellular Ca(2+) and increased synthesis of nitric oxide and cGMP. Chronic hyperammonemia, on the other hand, results in a loss of NMDA receptor sites. Activation of NMDA receptors in acute ammonia toxicity results in depletion of ATP in brain. Neuropathologic studies in experimental animals with congenital urea cycle disorders and severe hyperammonemia reveal evidence of neuronal cell death which is excitotoxic in nature. These findings suggest that overactivation of NMDA receptors is a significant feature of acute hyperammonemic syndromes and that antagonists of these receptors or of their signal transduction pathway enzymes such as nNOS could be beneficial in the treatment of the central nervous system manifestations (encephalopathy, brain edema) which are characteristic of hyperammonemic disorders.

Diagnosis of ascites

Astrocyte expression of metabotropic glutamate receptor 5 (mGluR5) is consistently observed in resected tissue from patients with epilepsy and is equally prevalent in animal models of epilepsy. However, little is known about the functional signaling properties or downstream consequences of astrocyte mGluR5 activation during epilepsy development. In the rodent brain, astrocyte mGluR5 expression is developmentally regulated and confined in expression/function to the first weeks of life, with similar observations made in human control tissue. Herein, we demonstrate that mGluR5 expression and function dramatically increase in a mouse model of temporal lobe epilepsy (TLE). Interestingly, in both male and female mice, mGluR5 function persists in the astrocyte throughout the process of epileptogenesis following status epilepticus. However, mGluR5 expression and function is transient in animals that do not develop epilepsy over an equivalent time period, suggesting that patterns of mGluR5 expression may signify continuing epilepsy development or its resolution. We demonstrate that during epileptogenesis, astrocytes re-acquire mGluR5-dependent calcium transients following agonist application or synaptic glutamate release—a feature of astrocyte-neuron communication absent since early development. Finally, we find that the selective and conditional knockout of mGluR5 signaling from astrocytes during epilepsy development slows the rate of glutamate clearance through astrocyte glutamate transporters under high-frequency stimulation conditions, a feature that suggests astrocyte mGluR5 expression during epileptogenesis may recapitulate earlier developmental roles in regulating glutamate transporter function. <b>SIGNIFICANCE STATEMENT</b> In development, astrocyte mGluR5 signaling plays a critical role in regulating structural and functional interactions between astrocytes and neurons at the tripartite synapse. Notably, mGluR5 signaling is a positive regulator of astrocyte glutamate transporter expression and function—an essential component of excitatory signaling regulation in hippocampus. After early development, astrocyte mGluR5 expression is downregulated, but re-emerges in animal models of TLE development and patient epilepsy samples. We explored the hypothesis that astrocyte mGluR5 re-emergence recapitulates earlier developmental roles during TLE acquisition. Our work demonstrates that astrocytes with mGluR5 signaling during TLE development perform faster glutamate uptake in hippocampus, revealing a previously unexplored role for astrocyte mGluR5 signaling in hypersynchronous pathology.