Molecules In Astrocytes Research Articles

Drug developmental strategies based on functional analysis of pain-regulating molecules in astrocytes under chronic pain

Spinal cord astrocytes are activated in chronic pain models, especially under conditions of prolonged pain. Hence, targeting spinal cord astrocytes for the development of useful analgesics has attracted much attention. In the CNS, connexin43 (Cx43), a membrane protein expressed and functioning exclusively in astrocytes, is well known to be involved in intercellular signaling as a component of gap junction, but also interacts with intracellular molecules via its characteristically long C-terminal region, thereby affecting cellular function. Previously, we found that Cx43 expression was markedly reduced in spinal dorsal horn astrocytes from a mouse model of neuropathic pain. In order to investigate the relationship between reduced Cx43 expression in spinal astrocytes and the onset of pain, we showed that reduced Cx43 expression altered the expression of pain-related molecules such as the glutamate transporter GLT-1 and the pro-inflammatory cytokine interleukin-6 (IL-6). In particular, we focused on the regulation of IL-6 expression by reduced Cx43 expression in both in vivo and in vitro analyses, and found that IL-6 expression is increased through the Akt- glycogen synthase kinase-3β (GSK-3β) signaling system driven by reduced Cx43 expression during neuropathic pain, which in turn triggers pain. These findings suggest that astrocyte Cx43 is involved in pain prolongation by regulating gene expression of nociceptive factors through interactions with intracellular signaling molecules, which is different from its previously known function, and thus raises expectations for its potential as a new drug target for chronic pain.

Drug Discovery Research for Traumatic Brain Injury Focused on Functional Molecules in Astrocytes.

Traumatic brain injury (TBI) is severe damage to the head caused by traffic accidents, falls, and sports. Because TBI-induced disruption of the blood-brain barrier (BBB) causes brain edema and neuroinflammation, which are major causes of death or serious disabilities, protection and recovery of BBB function may be beneficial therapeutic strategies for TBI. Astrocytes are key components of BBB integrity, and astrocyte-derived bioactive factors promote and suppress BBB disruption in TBI. Therefore, the regulation of astrocyte function is essential for BBB protection. In the injured cerebrum of TBI model mice, we found that the endothelin ETB receptor, histamine H2 receptor, and transient receptor potential vanilloid 4 (TRPV4) were predominantly expressed in reactive astrocytes. We also showed that repeated administration of an ETB receptor antagonist, H2 receptor agonist, and TRPV4 antagonist alleviated BBB disruption and brain edema in a TBI mouse model. Furthermore, these drugs decreased the expression levels of astrocyte-derived factors promoting BBB disruption and increased the expression levels of astrocyte-derived protective factors in the injured cerebrum after TBI. These results suggest that the ETB receptor, H2 receptor, and TRPV4 are molecules that regulate astrocyte function, and might be attractive candidates for the development of therapeutic drugs for TBI.

Drug developmental strategies based on functional analysis of pain-regulating molecules in astrocytes under chronic pain

Drug developmental strategies based on functional analysis of pain-regulating molecules in astrocytes under chronic pain

Hypercapnia Modulates the Activity of Adenosine A1 Receptors and mitoK+ATP-Channels in Rat Brain When Exposed to Intermittent Hypoxia.

The mechanisms and signaling pathways of the neuroprotective effects of hypercapnia and its combination with hypoxia are not studied sufficiently. The study aims to test the hypothesis of the potentiating effect of hypercapnia on the systems of adaptation to hypoxia, directly associated with A1-adenosine receptors and mitochondrial ATP-dependent K+ -channels (mitoK+ATP-channels). We evaluated the relative number of A1-adenosine receptors and mitoK+ATP-channels in astrocytes obtained from male Wistar rats exposed to various respiratory conditions (15 times of hypoxia and/or hypercapnia). In addition, the relative number of these molecules in astrocytes was evaluated on an in vitro model of chemical hypoxia, as well as in the cerebral cortex after photothrombotic damage. This study indicates an increase in the relative number of A1-adenosine receptors in astrocytes and in cells next to the stroke region of the cerebral cortex in rats exposed to hypoxia and hypercapnic hypoxia, but not hypercapnia alone. Hypercapnia and hypoxia increase the relative number of mitoK+ATP-channels in astrocytes and in cells of the peri-infarct region of the cerebral cortex in rats. In an in vitro study, hypercapnia mitigates the effects of acute chemical hypoxia observed in astrocytes for A1-adenosine receptors and mitoK+ATP-channels. Hypercapnia, unlike hypoxia, does not affect the relative number of A1 receptors to adenosine. At the same time, both hypercapnia and hypoxia increase the relative number of mitoK+ATP-channels, which can potentiate their protective effects with combined exposure.

Anti-oxidants in astrocytes as target of neuroprotection for Parkinson's disease

Recently, it has been reported that dysfunction of astrocytes is involved vulnerability of neuronal cells in several neurological disorders. Glutathione (GSH) is the most abundant intrinsic antioxidant in the central nervous system, and its substrate cysteine is readily becomes the oxidized dimeric cystine. Since neurons lack a cystine transport system, neuronal GSH synthesis depends on cystine uptake via the cystine/glutamate exchange transporter (xCT), GSH synthesis and release in/from surrounding astrocytes. The expression and release of the zinc-binding protein metallothionein (MT) in astrocytes, which is a strong antioxidant, is induced and exerts neuroprotective in the case of dopaminergic neuronal damage. In addition, the transcription factor Nrf2 induces expression of MT-1 and GSH related molecules. We previously revealed that several antiepileptic drugs, serotonin 5-HT1A receptor agonists, plant-derived chemicals (phytochemicals) increased xCT expression, Nrf2 activation, GSH or MT expression and release in/from astrocytes, and exerted a neuroprotective effect against dopaminergic neurodegeneration in Parkinson's disease model. Our serial studies on neuroprotection via antioxidant defense mechanism of astrocytes have found three target molecular systems of astrocytes for neuroprotection: (1) xCT-GSH synthetic system, (2) Nrf2 system and (3) 5-HT1A receptor-Nrf2-MT system, 5-HT1A-S100β system. In this article, possible neuroprotective strategy for Parkinson's disease has been reviewed targeting antioxidative molecules in astrocytes.

Ginsenoside Rg1-induced activation of astrocytes promotes functional recovery via the PI3K/Akt signaling pathway following spinal cord injury

AimsTo determine whether ginsenoside Rg1 is involved in scratch wound healing through altered expression of related molecules in astrocytes and improved functional recovery after spinal cord injury (SCI). Materials and methodsAstrocytes were isolated from rats, followed by Rg1 treatment. The wound healing test was performed to observe the scratch wound healing in different groups. The expression of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and components of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway were detected by western blot. Reverse transcription–polymerase chain reaction (RT-PCR) was used to measure the altered expression of laminin (LN) and fibronectin (FN). A revised Allen's method for the SCI model was performed, followed by Rg1 treatment. Then, functional scoring was conducted to evaluate the functional recovery. Hematoxylin-eosin (HE) staining showed changes in the void area. Finally, western blot assessed the expression of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs). Key findingsRg1 mediated scratch wound healing through inducing an increased release of LN, FN, NGF, GDNF, and bFGF in vitro. Additionally, Rg1 activated the PI3K/Akt signaling pathway and promoted the functional recovery of hindlimb movement in rats. Furthermore, Rg1 significantly reduced the void area and downregulated the expression of GFAP and CSPGs. SignificanceRg1 not only enhanced the scratch wound repair in vitro through the release of astroglial neurotrophic factors, adhesion factors, and inhibitory factors, but it also improved the functional recovery in vivo following SCI.

Region-Specific Neuroprotective Features of Astrocytes against Oxidative Stress Induced by 6-Hydroxydopamine.

In previous studies, we found regional differences in the induction of antioxidative molecules in astrocytes against oxidative stress, postulating that region-specific features of astrocytes lead region-specific vulnerability of neurons. We examined region-specific astrocytic features against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) as an oxidative stress using co-culture of mesencephalic neurons and mesencephalic or striatal astrocytes in the present study. The 6-OHDA-induced reduction of mesencephalic dopamine neurons was inhibited by co-culturing with astrocytes. The co-culture of midbrain neurons with striatal astrocytes was more resistant to 6-OHDA than that with mesencephalic astrocytes. Furthermore, glia conditioned medium from 6-OHDA-treated striatal astrocytes showed a greater protective effect on the 6-OHDA-induced neurotoxicity and oxidative stress than that from mesencephalic astrocytes. The cDNA microarray analysis showed that the number of altered genes in both mesencephalic and striatal astrocytes was fewer than that changed in either astrocyte. The 6-OHDA treatment, apparently up-regulated expressions of Nrf2 and some anti-oxidative or Nrf2-regulating phase II, III detoxifying molecules related to glutathione synthesis and export in the striatal astrocytes but not mesencephalic astrocytes. There is a profound regional difference of gene expression in astrocytes induced by 6-OHDA. These results suggest that protective features of astrocytes against oxidative stress are more prominent in striatal astrocytes, possibly by secreting humoral factors in striatal astrocytes.

Focal adhesion molecules regulate astrocyte morphology and glutamate transporters to suppress seizure-like behavior

Astrocytes are important regulators of neural circuit function and behavior in the healthy and diseased nervous system. We screened for molecules in Drosophila astrocytes that modulate neuronal hyperexcitability and identified multiple components of focal adhesion complexes (FAs). Depletion of astrocytic Tensin, β-integrin, Talin, focal adhesion kinase (FAK), or matrix metalloproteinase 1 (Mmp1), resulted in enhanced behavioral recovery from genetic or pharmacologically induced seizure. Overexpression of Mmp1, predicted to activate FA signaling, led to a reciprocal enhancement of seizure severity. Blockade of FA-signaling molecules in astrocytes at basal levels of CNS excitability resulted in reduced astrocytic coverage of the synaptic neuropil and expression of the excitatory amino acid transporter EAAT1. However, induction of hyperexcitability after depletion of FA-signaling components resulted in enhanced astrocyte coverage and an approximately twofold increase in EAAT1 levels. Our work identifies FA-signaling molecules as important regulators of astrocyte outgrowth and EAAT1 expression under normal physiological conditions. Paradoxically, in the context of hyperexcitability, this pathway negatively regulates astrocytic process outgrowth and EAAT1 expression, and their blockade leading to enhanced recovery from seizure.

Activation of FXR pathway does not alter glial cell function

BackgroundThe nuclear receptor farnesoid-X-receptor (FXR; NR1H4) is expressed not only in the liver, gut, kidney and adipose tissue but also in the immune cells. FXR has been shown to confer protection in several animal models of inflammation, including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). FXR agonists are currently tested in clinical trials for treatment of human metabolic diseases. The beneficial effect of FXR agonists in EAE suggests that FXR might represent a potential target in inflammatory-demyelinating CNS diseases, such as MS. In MS, oligodendrocytes not only undergo cell death but also contribute to remyelination. This repair mechanism is impaired due to a differentiation block of oligodendroglial progenitor cells. Activation of other nuclear receptors that heterodimerize with FXR promote oligodendroglial differentiation. Therefore, we wanted to address the functional relevance of FXR for glial cells, especially for oligodendroglial differentiation.MethodsWe isolated primary murine oligodendrocytes from FXR-deficient (FXR Ko) and wild-type (WT) mice and determined the effect of FXR deficiency and activation on oligodendroglial differentiation by analysing markers of oligodendroglial progenitor cells (OPCs) and mature oligodendrocytes (OLs) using qRT-PCR and immunocytochemistry. Additionally, we determined whether FXR activation modulates the pro-inflammatory profile of astrocytes or microglia and whether this may subsequently modulate oligodendroglial differentiation. These in vitro studies were complemented by histological analyses of oligodendrocytes in FXR Ko mice.ResultsFXR is expressed by OPCs and mature oligodendrocytes. However, lack of FXR did not affect oligodendroglial differentiation in vitro or in vivo. Furthermore, activation of FXR using the synthetic agonist GW4064 did not affect oligodendroglial differentiation, remyelination in an ex vivo model or the expression of pro-inflammatory molecules in astrocytes or microglia. Concordantly, no effects of supernatants from macrophages cultured in the presence of GW4064 were observed regarding a possible indirect impact on oligodendroglial differentiation.ConclusionsOur data suggest that FXR is dispensable for oligodendroglial differentiation and that FXR agonists, such as GW4064, represent a potential therapeutic approach for MS which specifically targets peripheral immune cells including macrophages but not brain-resident cells, such as oligodendrocytes, astrocytes or microglia.

Effect of FTY720-phosphate on the expression of inflammation-associated molecules in astrocytes in vitro.

FTY720 is a new oral immunomodulatory therapy for the treatment of multiple sclerosis (MS). There is strong evidence that FTY720 has direct effects on brain resident cells such as astrocytes acting via sphingosine‑1‑phosphate (S1P) receptors. In the present study, the mRNA expression of S1P receptors as well as selected cytokines, chemokines and growth factors were investigated in primary murine astrocytes under inflammatory conditions in the presence or absence of the phosphorylated form of FTY720 (FTY720‑P). Following stimulation with either the pro‑inflammatory cytokine tumor necrosis factor‑α (TNF‑α) or with bacterial lipopolysaccharide, there was an increased expression of the receptors S1P1 and S1P3, the cytokines and chemokines interleukin (IL)‑1β, chemokine (C‑C‑motif) ligand 2 (CCL‑2), CCL‑20 and chemokine (C‑X‑C‑motif) ligand 12 as well as the growth factors insulin‑like growth factor‑1, ciliary neurotrophic factor and glial cell line‑derived neurotrophic factor (GDNF). FTY720‑P led to an increased expression of IL‑1β and GDNF at distinct time points following co‑stimulation with TNF‑α compared with TNF‑α treatment alone. However, the presence of FTY720‑P did not have any further significant effects on the expression of S1P receptors, cytokines or growth factors, suggesting that the regulation of these target genes in astrocytes is not likely to be a major mechanism underlying the effect of FTY720‑P in diseases such as MS.

The selective anti-IL17A monoclonal antibody secukinumab (AIN457) attenuates IL17A-induced levels of IL6 in human astrocytes

The family of interleukin 17 receptors (IL17Rs), subtypes IL17RA-IL17RE, is targeted by the group of pro-inflammatory IL17 cytokines (IL17A-F) and moreover the newly developed anti-IL17A antibody secukinumab (AIN457) has shown promise in Phase II trials in multiple sclerosis. Here, we show that human astrocytes, isolated from a fetal cerebral cortex, express IL17RA and IL17RC and in vitro treatment with IL17A increases protein levels of IL6 in human astrocytes, which is enhanced in the presence of TNFα, as determined by homogeneous time resolved fluorescence. Studies on acutely isolated mouse astrocytes are comparable to human astrocytes although the protein levels of IL6 are lower in mouse astrocytes, which also show a lower response to IL17F and IL1β in promoting IL6 levels. In human astrocytes, IL17A and TNFα also induce mRNA expression of IL6, IL8 and the Th17 cytokines CXCL1, CXCL2, and CCL20, with little effect on Th1 cytokines CXCL9, CXCL10, and CXCL11. The effects of IL17A are associated with nuclear translocation of the NF-κB transcription factor, as determined by immunocytochemistry, where treatment of human astrocytes with the inhibitors of the NF-κB pathway and with secukinumab inhibits the IL17A and IL17A/TNFα-induced increase in nuclear translocation of NF-κB and levels of IL6. Taken together the data shows that IL17A signaling plays a key role in regulating the levels of cytokines, such as IL6, in human astrocytes via a mechanism that involves NF-κB signaling and that selective inhibition of IL17A signaling attenuates levels of pro-inflammatory molecules in astrocytes.

P2 porin and loop L7 from Haemophilus influenzae modulate expression of IL-6 and adhesion molecules in astrocytes

During neuropathological conditions such as infections and degenerative diseases, astrocytes can be activated by infiltrating immune cells. Activated astrocytes can produce chemokines, cytokines and adhesion molecules. In this study, the production of IL-6 and adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1) and E-selectin by human astroglioma cells stimulated with Gram-negative surface components was investigated. Haemophilus influenzae type b porin P2 and its selected active peptide, loop L7, were found to induce MEK1-MEK2/ mitogen-activated protein kinase phosphorylation in U87-MG cells as demonstrated by ELISA, and up-regulate cellular adhesion molecule and interleukin-6 (IL-6) production as shown by RT-PCR and ELISA. Using two potent and selective inhibitors of MEK activation by Raf-1 (PD-098059) and p38 (SB-203580), it was also demonstrated that both ERK1/2 and p38 pathways play key roles in the production of IL-6 as well as in ICAM-1, VCAM-1 and E-selectin expression by Hib porin.

Disruption of interactions between immunocytes, glia and neurons in demyelinating diseases: a view from neuroscience

Microglia play a crucial role in the development of inflammatory demyelinating lesions in multiple sclerosis (MS). Microglia act on inflammatory lymphocytes as antigen presenting cells, and produce inflammatory cytokines, glutamate, and reactive oxygen species (ROS). Neurodegeneration, which is observed in the demyelinating lesions, affects the prognosis in MS. Neuritic beading, focal bead-like swellings of the dendrites and axons, is a neuropathological sign in the early phase of neurodegeneration in MS. Microglia-derived glutamate and ROS initiate beading formation. Microglia can exert neuroprotective effect by deprivation of dead cells and induction of neurotrophic factors, anti-inflammatory cytokines, and anti-oxidant enzyme in MS. Neurons are thought to be not merely passive targets of microglia but rather control microglial activity through various signals including cytokines and chemokines. Soluble fractalkine (sFKN), which is secreted from damaged neurons by glutamate, promotes microglial phagocytosis of neuronal debris, and induces the antioxidant enzyme heme oxygenase-1 in microglia. IL-34 secreted from neurons also induces microglial neuroprotection. Astrocytes exert neuroprotective effect. However, toll-like receptor ligands induce neurotoxic molecules in astrocytes. IL-33 produced by astrocytes induces microglial activation. Thus, disruption of beneficial interaction between glia and neurons is crucial for the pathogenesis of MS.

The interleukin‐1 signalling pathway in astrocytes: a key contributor to inflammation in the brain

A dysregulated inflammatory response in the central nervous system (CNS) lies at the heart of many neuropathological conditions such as multiple sclerosis and Alzheimer's disease. A key component of these inflammatory conditions is the accumulation of leukocytes in the CNS. The infiltration of leukocytes into the brain is dependent on the induction of leukocyte adhesion molecules and chemoattractant chemokines. Recent studies have suggested the astrocyte to be a key cell in mediating the inflammatory process in the brain and in expressing adhesion molecules and chemokines. Here I overview work in my laboratory and others that demonstrates interleukin-1 (IL-1) to be a key inducer of the expression of these molecules in astrocytes. The temporal expression is sustained in nature and this is due to prolonged activation of the transcription factor NFkappaB. The molecular basis to the sustained activation of NFkappaB is also discussed. The IL-1 signalling pathway thus emerges as a valuable therapeutic target in the treatment of presently incurable neuropathological conditions.

Zaprinast, an inhibitor of cGMP-selective phosphodiesterases, enhances the secretion of TNF-alpha and IL-1beta and the expression of iNOS and MHC class II molecules in rat microglial cells.

Proinflammatory cytokines produced by activated glial cells may in turn augment the immune/inflammatory reactions of glial cells through autocrine and paracrine routes. The NO/cGMP signaling represents one of the reactions of activated glial cells. We investigated whether the production of proinflammatory cytokines by glial cells is affected by NO-dependent downstream cGMP signaling. In primary cultures of mixed astrocytes and microglial cells, zaprinast (0.1 mM), an inhibitor of cGMP-selective phosphodiesterases, enhanced the basal and LPS (1.0 microg/ml)-induced secretion of TNF-alpha and IL-1beta. Zaprinast also enhanced NO production induced by LPS or IFN-gamma (100 U/ml), and in microglial cell cultures, but not in astrocyte cultures, zaprinast enhanced the basal and the IFN-gamma-induced production of the cytokines, TNF-alpha and IL-1beta, and of NO. This upregulation by zaprinast was partially inhibited by KT5823 (1.0 microM), an inhibitor of protein kinase G. The LPS-induced production of TNF-alpha, IL-1beta, and NO was inhibited by ODQ (50 microM), an inhibitor of soluble guanylyl cyclase, and by KT5823. Immunohistochemical analysis of mixed glial cell cultures showed that LPS/IFN-gamma-induced iNOS expression and the enhanced expression of iNOS by zaprinast were restricted to microglial cells. Zaprinast enhanced the IFN-gamma (200 U/ml)-induced expression of MHC Class II molecules in astrocytes and microglial cells in mixed cultures, but did not enhance this IFN-gamma-induced expression in pure astrocytes, which lacked paracrine TNF-alpha from microglial cells. Summarizing, zaprinast, which is associated with cGMP/protein kinase G signaling, may augment central immune/inflammatory reactions, possibly via the increased production of TNF-alpha and IL-1beta by activated microglial cells.

Down-regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type-1 receptor and nuclear factor-kappaB activation.

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin-1, ZO-1 and ZO-2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO-1 expression in astrocytes. TNF decreased occludin, but not ZO-1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down-regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor-kappaB (NF-kappaB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type-1 receptor (TNFR1), TNF type-2 receptor (TNFR2), or both, respectively, revealed that down-regulation was mediated entirely by TNFR1. ZO-1, which can interact with occludin, was found to co-precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down-regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF-kappaB is involved as a negative regulator.

The role of protein tyrosine phosphatase SHP-1 in the regulation of IFN-gamma signaling in neural cells.

The role for protein tyrosine phosphatase SHP-1 in controlling signal transduction by IFN-gamma in astrocytes was studied. IFN-gamma induced the gamma-activated factor (GAF) within 30 min and GAF subsequently declined by 8 h after treatment. However, treatment with IFN-gamma in the presence of protein tyrosine phosphatase inhibitor vanadate blocked the decrease in GAF activity. The increased stability of GAF in vanadate-treated cultures was similarly observed in astrocytes of motheaten mice, which specifically lack the protein tyrosine phosphatase SHP-1. Prolongation of GAF activity coincided with increased expression of the IFN-inducible transcription factor, IFN-regulatory factor-1 (IRF-1). Increased IRF-1 was coincident with increased expression of MHC class I molecules in astrocytes in accordance with the activity of IRF-1 in the promoter region. These data implicate an important role for protein tyrosine phosphatases, including SHP-1, in the regulation of IFN-gamma-signaling and IFN-gamma-inducible genes in neural cells.

Modulation of major histocompatibility complex class I genes by interferon-gamma and ganglioside GT 1b in astrocytes: involvement of protein tyrosine phosphatases.

We have previously reported that the polysialoganglioside GT1b suppresses the induction of major histocompatibility complex class I molecules by interferon-gamma in astrocytes. Suppression by GT1b depended on the presence of sialic acid moieties because asialo-GM1 was not suppressive. In the present report, GT1b was found to act transcriptionally to suppress class I genes because both the interferon-gamma induction of RNA and the activity of class I promoter constructs were inhibited. Furthermore, GT1b suppressed promoter activity through interferon regulatory factor elements, indicating an effect on the transcription activation factor, interferon regulatory factor 1. Interferon-gamma induced interferon regulatory factor 1 within 8 h, and GT1b suppressed this induction. The suppression of interferon regulatory factor 1 by GT1b correlated with the suppression of gamma-activated factor binding at the promoter of the interferon regulatory factor 1 gene. The suppression of gamma-activated factor by GT1b appeared to involve increased protein tyrosine phosphatase activity because treatment of the cells with pervanadate reversed the effect of GT1b on the gamma-activated factor and, correspondingly, phosphotyrosine content. In sum, GT1b displays specific effects on interferon-gamma signaling and negative feedback regulatory molecules in astrocytes.

Hypertrophy of the cerebral white matter in hemimegalencephaly

We morphometrically measured the cut surface of the cerebral hemisphere and localized epidermal growth factor (EGF)-like immunoreactivity in 4 patients with hemimegalencephaly. On the affected side, the area of cerebral white matter was more than twice as large as that on the unaffected side, while the area of the cerebral cortex on the affected side was relatively small. EGF-like immunoreactivity was demonstrated in cortical neurons (4 of 4 patients) and glial cells (3 of 4 patients), notably in astrocytes. Significant enlargement of the cerebral white matter compared to the cerebral cortex and the expression of EGF-like molecules in astrocytes suggest excessive proliferation in the white matter with hemimegalencephaly and the possible relevance of EGF to these events.

Effect of glucose deprivation on rat glutamine synthetase in cultured astrocytes.

Glutamine synthetase was purified from the cerebral cortex of adult rats and characterized. Polyclonal rabbit antibodies were raised against the enzyme, purified and their specific anti-(glutamine synthetase) activity determined. A primary astroglial culture was prepared from newborn Sprague-Dawley rats. Astrocytes at different ages of development were incubated in the presence and absence of glucose. In glucose-deprived conditions the specific activity of glutamine synthetase decreased. This decrease was more pronounced in 8-day-old than in 21-day-old cultures. Kinetic analysis demonstrated that the reduction in activity was mainly related to a decrease in Vmax. By immunoprecipitation, it was shown that the number of enzyme molecules in astrocytes was decreased in glucose-deprived conditions. On addition of glucose, a total recovery of glutamine synthetase was obtained after 36 h in 8-day-old culture. Rates of degradation and synthesis were investigated. When compared with an incubation in the presence of glucose, glucose deprivation increased enzyme turnover, as estimated from the first-order disappearance of radioactivity from glutamine synthetase. Synthesis rate was estimated from the incorporation of [35S]methionine during a 2 h incubation period and was decreased in glucose-deprived conditions. Trichloroacetate-precipitable proteins changed only slightly in the experimental conditions, and total protein did not vary significantly during the experimental period. A mathematical model is presented which attempts to integrate degradation and synthesis in our experimental model.