Induction Of Glial Fibrillary Acidic Protein Research Articles

Desmin expression profile in reactive astrocytes in the 3-nitropropionic acid–lesioned striatum of rat: Characterization and comparison with glial fibrillary acidic protein and nestin

Desmin, a muscle-specific, type-III intermediate-filament protein, is reportedly expressed in astrocytes in the central nervous system. These cells become reactive astrocytes in response to brain injuries. To elucidate whether desmin is involved in this process, we examined the spatiotemporal expression profiles of desmin and their relationship with two astroglial intermediate filaments, glial fibrillary acidic protein (GFAP) and nestin, in the striatum of rats treated with the mitochondrial toxin 3-nitropropionic acid (3-NP). Weak, constitutive immunoreactivity for desmin was observed in astrocytes generally, and in reactive astrocytes in the peri-lesional area, its expression increased in parallel with that of GFAP over 3 d post-lesion and was maintained until at least day 28. Desmin, GFAP, and nestin showed characteristic time-dependent expression patterns in reactive astrocytes forming the astroglial scar; delayed and long-lasting induction of desmin and GFAP, and rapid but transient induction of nestin. In the lesion core, desmin was expressed in two categories of perivascular cells: nestin-negative and nestin-positive. These findings show that desmin, together with GFAP and nestin, is a dynamic component of intermediate filaments in activated astroglia, which may account for the dynamic structural changes seen in these cells in response to brain injuries.

HIV-1 Tat Induces Unfolded Protein Response and Endoplasmic Reticulum Stress in Astrocytes and Causes Neurotoxicity through Glial Fibrillary Acidic Protein (GFAP) Activation and Aggregation

HIV-1 Tat is a major culprit for HIV/neuroAIDS. One of the consistent hallmarks of HIV/neuroAIDS is reactive astrocytes or astrocytosis, characterized by increased cytoplasmic accumulation of the intermediate filament glial fibrillary acidic protein (GFAP). We have shown that that Tat induces GFAP expression in astrocytes and that GFAP activation is indispensable for astrocyte-mediated Tat neurotoxicity. However, the underlying molecular mechanisms are not known. In this study, we showed that Tat expression or GFAP expression led to formation of GFAP aggregates and induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in astrocytes. In addition, we demonstrated that GFAP up-regulation and aggregation in astrocytes were necessary but also sufficient for UPR/ER stress induction in Tat-expressing astrocytes and for astrocyte-mediated Tat neurotoxicity. Importantly, we demonstrated that inhibition of Tat- or GFAP-induced UPR/ER stress by the chemical chaperone 4-phenylbutyrate significantly alleviated astrocyte-mediated Tat neurotoxicity in vitro and in the brain of Tat-expressing mice. Taken together, these results show that HIV-1 Tat expression leads to UPR/ER stress in astrocytes, which in turn contributes to astrocyte-mediated Tat neurotoxicity, and raise the possibility of developing HIV/neuroAIDS therapeutics targeted at UPR/ER stress.

Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus.

BackgroundOuabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats.MethodsAdult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 μg/kg) or saline 20 minutes before LPS (200 μg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured.ResultsOUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1β, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain.ConclusionOur results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

MicroRNA 335 Is Required for Differentiation of Malignant Glioma Cells Induced by Activation of cAMP/Protein Kinase A Pathway

Glioma is the most common malignant cancer affecting the central nerve system, with dismal prognosis. Differentiation-inducing therapy is a novel strategy that has been preliminarily proved effective against malignant glioma. We have reported previously that activation of cAMP/protein kinase A (PKA) pathway is capable of inducing glioma cell differentiation, characterized by astrocyte-like shape and dramatic induction of astrocyte biomarker glial fibrillary acidic protein (GFAP). However, little progress has been made on molecular mechanisms related. Here we demonstrate that microRNA 335 (miR-335) is responsible for the glioma cell differentiation stimulated by activation of cAMP/PKA pathway. In the cAMP elevator cholera toxin-induced differentiation model of rat C6 glioma cells, miR-335 was significantly up-regulated, which was mimicked by other typical cAMP/PKA pathway activators (e.g., forskolin, dibutyryl-cAMP) and abolished by PKA-specific inhibitor (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i] [1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT5720). In an assay measuring gain and loss of miR-335 function, exogenetic miR-335 resulted in induction of GFAP, whereas miR-335 specific inhibitor antagomir-335 violently blocked cholera toxin-induced GFAP up-regulation. It is noteworthy that in human U87-MG glioma cells and human primary culture glioma cells, miR-335 also mediated cholera toxin-induced differentiation. Taken together, our findings suggest that miR-335 is potently required for differentiation of malignant glioma cells induced by cAMP/PKA pathway activation, and a single microRNA may act as an important fate determinant to control the differentiation status of malignant gliomas, which has provided a new insight into differentiation-inducing therapy against malignant gliomas.

Activation of a pro-survival pathway IL-6/JAK2/STAT3 contributes to glial fibrillary acidic protein induction during the cholera toxin-induced differentiation of C6 malignant glioma cells

Differentiation-inducing therapy has been proposed to be a novel potential approach to treat malignant gliomas. Glial fibrillary acidic protein (GFAP) is a well-known specific astrocyte biomarker and acts as a tumor suppressor gene (TSG) in glioma pathogenesis. Previously we reported that a traditional biotoxin cholera toxin could induce malignant glioma cell differentiation characterized by morphologic changes and dramatic GFAP expression. However, the molecular mechanisms underlying GFAP induction are still largely unknown. Here we demonstrate that an oncogenic pathway interleukin-6/janus kinase-2/signal transducer and activator of transcription 3 (IL-6/JAK2/STAT3) cascade mediates the cholera toxin-induced GFAP expression. Cholera toxin dramatically stimulated GFAP expression at the transcriptional level in C6 glioma cells. Meanwhile, phosphorylation of STAT3 and JAK2 was highly induced in a time-dependent manner after cholera toxin incubation, whereas no changes of STAT3 and JAK2 were observed. Furthermore, the IL-6 gene was quickly induced by cholera toxin and subsequent IL-6 protein secretion was stimulated. Importantly, exogenous recombinant rat IL-6 can also induce phosphorylation of STAT3 concomitant with GFAP expression while JAK2 specific inhibitor AG490 could effectively block both cholera toxin- and IL-6-induced GFAP expression. Given that the methylation of the STAT3 binding element can suppress GFAP expression, we detected the methylation status of the critical recognition sequence of STAT3 in the promoter of GFAP gene (−1518 ∼ −1510) and found that it was unmethylated in C6 glioma cells. In addition, neither DNA methyltransferase1 (DNMT1) inhibitor 5-Aza-2′-deoxycytidine (5-AZa-CdR) nor silencing DNMT1 can stimulate GFAP expression, indicating that the loss of GFAP expression in C6 cells is not caused by its promoter hypermethylation. Taken together, our findings suggest that activation of a pro-survival IL-6/JAK2/STAT3 cascade contributes to cholera toxin-induced GFAP expression, which implies that a survival-promoting signal may also play a differentiation-supporting role in malignant gliomas.

Histopathological changes of the hippocampus neurons in brain injury.

The glial fibrillary acidic protein (GFAP) is known as a peculiar marker of mature astrocytes of the central nervous system (CNS). However, we found distinct immunopositivity to a monoclonal anti-GFAP reagent in the hippocampus neurons in head injury fatalities. The present study investigated the neuronal and neuroglial GFAP-immunopositivity in the hippocampus in a series of head injury cases, which included acute and subacute/delayed deaths (n=17 and n=73, respectively), and acute cardiac death (n=13), delayed death due to multiple organ failure from non-head injury (n=6), and pneumonia (n=9) cases were examined as controls. GFAP-immunopositivity in the neurons was frequently observed in CA4, CA3 and CA2 regions in cases of subacute/delayed head injury death that showed marked brain swelling accompanied by secondary brain stem hemorrhages, showing an inverse relationship to that in astrocytes. These findings suggest possible induction of GFAP or a related protein in hippocampus neurons depending on the severity of brain swelling following head injury.

Interleukin‐6 is required for the early induction of glial fibrillary acidic protein in Schwann cells during Wallerian degeneration

Signal transducer and activator of transcription 3 (STAT3) regulates gene transcription in response to cytokines and growth factors. In the central nervous system, STAT3 plays a role in neuroprotection and reactive gliosis after lesions. During peripheral nerve regeneration, a nerve injury-induced up-regulation of cytokines and growth factors accompanies STAT3 activation in sensory neurons and Schwann cells (SCs) even though its molecular details and functions are unknown. We then analyzed the ligands and functions of STAT3 activation in RT4 schwannoma cells and adult SCs in vitro and in vivo. We have identified that interleukin-6 (IL-6), but not ciliary neurotrophic factor, leukemia inhibitory factor, or ligands for receptor tyrosine kinases, activates STAT3 in SCs. The IL-6/STAT3 signaling in primary SCs and RT4 cells induced the gene expression of glial fibrillary acidic protein (GFAP), which is known to be required for the proper regeneration of the injured nerves. Finally, the GFAP induction in the sciatic nerves after injury was significantly delayed in IL-6-deficient mice. These findings indicate that IL-6 plays an important role in STAT3-dependent GFAP induction in SCs during peripheral nerve regeneration.

Live imaging of amyotrophic lateral sclerosis pathogenesis: Disease onset is characterized by marked induction of GFAP in Schwann cells

Amyotrophic lateral sclerosis (ALS) is a late-onset neurological disease characterized by progressive loss of motor neurons. At present, the pathological events precipitating disease onset and the exact pattern of disease progression are not fully understood. Recent studies suggest that glial cells, in particular activated astrocytes, can release factors that can directly kill motor neurons. To further investigate the involvement of glial cells (astrocytes and Schwann cells) in the pathogenesis of ALS, we generated ALS-(GFAP-luciferase/SOD(G93A)) reporter mouse in which upregulation of glial fibrillary acidic protein (GFAP) can be visualized from live animals throughout the different stages of disease. Our results suggest that the disease in mice is initiated simultaneously in the spinal cord and in the peripheral nerves and is characterized by several cycles of GFAP upregulation. Immunohistochemical analysis confirmed that the induction GFAP bioluminescence signals were associated with the significant increases in GFAP immunoreactivity. The first pathological GFAP signals occurring at 25-30 days were asymptomatic and detectable at the level of lumbar spinal cord projections and at the periphery. These early events were then followed by GFAP promoter inductions that were associated with the distinct clinical symptoms. As expected, the onset of paralysis (112 days) was associated with the gradual and marked GFAP upregulation in the spinal cord. Interestingly, however, the disease onset (90 days) was characterized by sharp and synchronized induction of GFAP in peripheral nerve Schwann cells suggesting that peripheral nerves pathology/denervation and associated Schwann cell stress may play an important role in the ALS pathogenesis.

Fluctuations in astrocytic basic fibroblast growth factor in the cingulate cortex of cycling, ovariectomized and postpartum animals

In the current studies we investigated the timing of onset and the conditions needed for the maintenance of the upregulation of basic fibroblast growth factor (bFGF) and glial fibrillary acidic protein (GFAP) in the cingulate cortex area 2 (Cg2) that occurs in postpartum animals. We have previously shown that this upregulation is present from day 4 to day 24, and is not seen late in pregnancy (days 21–22). In the current studies, we demonstrate that bFGF and GFAP are both upregulated in Cg2 as early as 3 h postpartum, and are maintained until at least day 24 postpartum in animals deprived of pup stimulation for 8 days prior to perfusion. bFGF and GFAP immunoreactivity returns to prepartum levels by 5–6 weeks post-weaning, and the typical postpartum increase is not further enhanced in multiparous rats. We also show that, although there are significant changes in levels of bFGF immunoreactivity across the phases of the estrous cycle, peak cycling levels remain much lower than those observed in lactating rats. Possible stimuli involved in the induction of bFGF and GFAP in Cg2, and the potential relevance of these changes to the maternal state are discussed.

Aspirin-induced blockade of NF-kB activity restrains up-regulation of glial fibrillary acidic protein in human astroglial cells

The marked induction of glial fibrillary acidic protein (GFAP) has been observed in astrocytes during neuropathological processes accompanying reactive gliosis; however, the precise molecular mechanism(s) underlying this GFAP induction remains poorly resolved. Therefore, in this study, we examined whether the change of nuclear factor-kappa B (NF-κB) activity can influence GFAP expression levels. Aspirin, widely used to prevent NF-κB activity, reduced the levels of GFAP mRNA and protein in human astroglial cells including human glioblastoma A172 cells and primary human brain astrocyte cells (HBAs). Furthermore, aspirin inhibited the effects of hypoxic injury on the up-regulation of GFAP expression in HBAs. We confirmed the repressive effect of aspirin on GFAP transcription by GFAP promoter-driven reporter assay and found that one NF-κB binding site conserved in the mouse and human GFAP gene promoters is critical for this effect. To further delineate whether NF-κ Bi s directly involved in the regulation of GFAP gene expression, we transfected A172 cells with an expression vector encoding a super-repressor IκBα protein (IκBα-SR) to specifically inhibit NF-κB activity and found the marked reduction of GFAP protein levels in IκBα-SR-transfectant cells. Taken together, our results suggest that NF-κB may play pivotal roles in GFAP gene expression.

Aspirin-induced blockade of NF-κB activity restrains up-regulation of glial fibrillary acidic protein in human astroglial cells

The marked induction of glial fibrillary acidic protein (GFAP) has been observed in astrocytes during neuropathological processes accompanying reactive gliosis; however, the precise molecular mechanism(s) underlying this GFAP induction remains poorly resolved. Therefore, in this study, we examined whether the change of nuclear factor-kappa B (NF-κB) activity can influence GFAP expression levels. Aspirin, widely used to prevent NF-κB activity, reduced the levels of GFAP mRNA and protein in human astroglial cells including human glioblastoma A172 cells and primary human brain astrocyte cells (HBAs). Furthermore, aspirin inhibited the effects of hypoxic injury on the up-regulation of GFAP expression in HBAs. We confirmed the repressive effect of aspirin on GFAP transcription by GFAP promoter-driven reporter assay and found that one NF-κB binding site conserved in the mouse and human GFAP gene promoters is critical for this effect. To further delineate whether NF-κB is directly involved in the regulation of GFAP gene expression, we transfected A172 cells with an expression vector encoding a super-repressor IκBα protein (IκBα-SR) to specifically inhibit NF-κB activity and found the marked reduction of GFAP protein levels in IκBα-SR-transfectant cells. Taken together, our results suggest that NF-κB may play pivotal roles in GFAP gene expression.

Neuronal Activation of NF-κB Contributes to Cell Death in Cerebral Ischemia

The transcription factor NF-kappaB is a key regulator of inflammation and cell survival. NF-kappaB is activated by cerebral ischemia in neurons and glia, but its function is controversial. To inhibit NF-kappaB selectively in neurons and glial cells, we have generated transgenic mice that express the IkappaBalpha superrepressor (IkappaBalpha mutated at serine-32 and serine-36, IkappaBalpha-SR) under transcriptional control of the neuron-specific enolase (NSE) and the glial fibrillary acidic protein (GFAP) promoter, respectively. In primary cortical neurons of NSE-IkappaBalpha-SR mice, NF-kappaB activity was partially inhibited. To assess NF-kappaB activity in vivo after permanent middle cerebral artery occlusion (MCAO), we measured the expression of NF-kappaB target genes by real-time polymerase chain reaction (PCR). The induction of c-myc and transforming growth factor-beta2 by cerebral ischemia was inhibited by neuronal expression of IkappaBalpha-SR, whereas induction of GFAP by MCAO was reduced by astrocytic expression of IkappaBalpha-SR. Neuronal, but not astrocytic, expression of the NF-kappaB inhibitor reduced both infarct size and cell death 48 hours after permanent MCAO. In summary, the data show that NF-kappaB is activated in neurons and astrocytes during cerebral ischemia and that NF-kappaB activation in neurons contributes to the ischemic damage.

Downregulation of osteoblast markers and induction of the glial fibrillary acidic protein by oncostatin M in osteosarcoma cells require PKCdelta and STAT3.

The effects of OSM on proliferation and differentiation of osteosarcoma and nontransformed osteoblasts were analyzed. OSM downregulates osteoblast markers but induces the glial fibrillary acidic protein by the combined activation of PKCdelta and STAT3, offering new lines of therapeutic investigations. Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family implicated in embryonic development, differentiation, inflammation, and regeneration of various tissues, mainly the liver, bone, and the central nervous and hematopoietic systems. One particularity of OSM relies on its growth inhibitory and pro-differentiating effects on a variety of tumor cell lines such as melanoma, providing arguments for a therapeutic application of OSM. The objective of this study was to analyze the effects of OSM on osteosarcoma cell lines proliferation and differentiation. Proliferation was analyzed by 3H thymidine incorporation. Differentiation was analyzed by semiquantitative RT-PCR and immunocytochemistry for various markers. Alizarin red S staining was used to evaluate bone nodule formation. Morphological changes were studied by confocal and electron microscopy. Western blotting, kinases inhibitors, and dominant negative STAT3 were used to identified the signaling pathways implicated. OSM inhibits the growth of rat osteosarcoma cell lines as well as normal osteoblasts, in correlation with induction of the cyclin-dependent kinases inhibitor p21WAF1. However, OSM reduces osteoblast markers such as alkaline phosphatase, osteocalcin, and bone sialoprotein, leading to strong inhibition of mineralized nodule formation. This inhibitory effect is restricted to mature osteoblasts and differentiated osteosarcoma because OSM effectively stimulates osteoblast markers and bone nodule formation in early, but not late, bone marrow mesenchymal stem cell (BMSC) cultures. In osteosarcoma cells or BMSC, OSM induces expression of the glial fibrillary acidic protein (GFAP) as well as morphological and ultrastructural changes, for example, elongated shape and bundles of microfilaments in cell processes. Rottlerin (PKCdelta inhibitor), and to a lesser degree UO126 (MEK/ERK inhibitor), prevents the loss of osteoblastic markers by OSM, whereas dominant negative STAT3 prevents GFAP induction. These results highlight the particular gene expression profile of OSM-treated osteosarcoma cells and BMSCs, suggesting either a osteocytic or a glial-like phenotype. Together with the implication of PKCdelta, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.

Antagonists of the Neurokinin‐1 or Dopamine D1 Receptors Confer Protection from Methamphetamine on Dopamine Terminals of the Mouse Striatum

Methamphetamine (METH) is a highly addictive compound that induces toxicity of the dopamine (DA) terminals of the neostriatum. Exposure to METH induces long-term deficits in dopamine transporter (DAT) and tyrosine hydroxylase (TH) levels as well as induction of glial fibrillary acidic protein (GFAP) in the caudate putamen (CPu) and the nucleus accumbens (NAc). The primary effect of exposure to METH is elevation of the level of extracellular DA; therefore, we assessed the role of the DA D1 receptor (D1R) and neurokinin-1 receptor (NK-1R) on the expression of toxicity. METH was injected intraperitoneally (10 mg/kg) four times at 2-h intervals (an acute toxic dose), and the mice were sacrificed three days after the treatment. Exposure to METH resulted in marked reduction of DAT sites (reduced to 30 and 21% relative to control in medial and lateral aspects of the CPu) assessed by binding of [125I]RTI-121 by autoradiography or Western blot analysis. Pretreatment with the nonpeptide NK-1R antagonist WIN-51,708 (10 mg/kg) 30 min prior to the first and fourth injections of METH prevented the loss of DAT sites of the CPu. Moreover, pretreatment with WIN-51,708 also prevented the reduction of TH levels induced by METH as well as the induction of GFAP in astrocytes. Pretreatment with the D1R antagonist SCH-23390 (0.25 mg/kg) 30 min before the first and fourth injections of METH conferred partial protection on DAT sites of the CPu. These results demonstrate that receptors postsynaptic to the DA terminals of the CPu are needed in order to express the neurotoxic effects of METH on integral components of the DA terminals of the nigrostriatal projection.

Beta-naphthoflavone disturbs astrocytic differentiation of C6 glioma cells by inhibiting autocrine interleukin-6.

Regulation of astrocyte differentiation is a key process in the development of the central nervous system (CNS), and disturbance of the differentiation can lead to brain system dysfunction. Here we show that beta-naphthoflavone (betaNF), an agonist of the aryl hydrocarbon receptor (AhR), disturbed the cAMP-induced astrocytic differentiation of C6 glioma by inhibiting autocrine interleukin-6 (IL-6). Treatment of cells with betaNF reduced the induction of an astrocyte marker glial fibrillary acidic protein (GFAP). This was caused by the inactivation of its upstream transcription factor signal transducer and activator of transcription 3 (STAT3) by betaNF. In addition, betaNF attenuated the induction of the IL-6 gene, which leads to the activation of STAT3. Most importantly, the inhibitory effect of betaNF on GFAP promoter activity was recovered by the addition of recombinant IL-6. Taken together, these results indicate that the inhibitory effect of betaNF on IL-6 induction suppresses STAT3 activation. These processes subsequently lead to the attenuation of GFAP induction.

CAMP-induced Astrocytic Differentiation of C6 Glioma Cells Is Mediated by Autocrine Interleukin-6

Elevation in the level of intracellular cAMP is known to induce the astrocytic differentiation of C6 glioma cells by unknown mechanisms. In this report, we show that cAMP-induced autocrine interleukin 6 (IL-6) promoted astrocytic differentiation of C6 cells. Treatment of cells with N(6),2'-O-dibutyryl cAMP (Bt(2)AMP) and theophylline caused the delayed phosphorylation of signal transducer and activator of transcription 3 (STAT3), as well as the expression of an astrocyte marker, glial fibrillary acidic protein (GFAP). Overexpression of the dominant-negative form of STAT3 leads to the suppression of GFAP promoter activity, suggesting that STAT3 activity was essential for cAMP-induced GFAP promoter activation. On the other hand, the IL-6 gene was quickly induced by Bt(2)AMP/theophylline, and subsequent IL-6 protein secretion was stimulated. In addition, recombinant IL-6 induced GFAP expression and STAT3 phosphorylation. Most importantly, treatment with IL-6-neutralizing antibody dramatically reduced the cAMP-induced GFAP expression and STAT3 phosphorylation and reversed the cellular morphological changes that had been caused by Bt(2)AMP/theophylline. Taken together, these results indicated that Bt(2)AMP/theophylline lead to delayed STAT3 activation via autocrine IL-6. These processes subsequently led to the induction of GFAP. IL-6 secretion is thus thought to be a key event in controlling the astrocytic differentiation of C6 cells.

Inducible nitric oxide synthase and vascular endothelial growth factor are colocalized in the retinas of human subjects with diabetes.

Nitric oxide (NO) mediates vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. This study was undertaken to study the cellular distribution of inducible nitric oxide synthase (iNOS) and VEGF in the retinas from human subjects with diabetes mellitus. In addition, glial reactivity and peroxynitrite generation were detected by immunolocalization of glial fibrillary acidic protein (GFAP) and nitrotyrosine, respectively. Eight post-mortem eyes from four consecutive subjects with diabetes mellitus and eight eyes from four subjects without diabetes and without known ocular disease were prospectively collected and examined. We used immunohistochemical techniques and antibodies directed against iNOS, VEGF, GFAP, and nitrotyrosine. In retinas from all subjects without diabetes, weak GFAP immunoreactivity was confined to nerve fibre and ganglion cell layers. There was no immunoreactivity for iNOS, nitrotyrosine, and VEGF. All diabetic retinas showed GFAP induction in Müller cells and GFAP upregulation in nerve fibre and ganglion cell layers. All diabetic retinas showed cytoplasmic immunoreactivity for iNOS, and VEGF in ganglion cells, cells in the inner nuclear layer, and glial cells. In serial sections, ganglion cells and cells in the inner nuclear layer expressing VEGF were localized in the same area of iNOS-expressing ganglion cells and cells in the inner nuclear layer. Six retinas from three subjects with diabetes showed immunoreactivity for nitrotyrosine in vascular endothelial cells in inner retinal layer. iNOS and VEGF are colocalized in diabetic retinas. Increased GFAP immunoreactivity is a pathological event in the retina during diabetes.

Behavioural changes related to olfactory mucosal metaplasia and bulbar glial fibrillary acidic protein (GFAP) induction in methylsulphonyl-dichlorobenzene-treated mice.

Methylsulphonyl-2,6-dichlorobenzene [2,6-(diCl-MeSO(2)-B)], and its 2,5-chlorinated isomer [2,5-(diCl-MeSO(2)-B)] bind firmly in the olfactory mucosa of mice. Both isomers are also selectively localised in the olfactory bulb. Persistent olfactory mucosal metaplasia is induced by 2,6-(diCl-MeSO(2)-B) whereas 2,5-(diCl-MeSO(2)-B) has no effects. Furthermore, a strong induction of glial fibrillary acidic protein (GFAP) restricted to the olfactory bulb has been reported in 2,6-(diCl-MeSO(2)-B)-treated mice. To explore whether these lesions give rise to early or long-lasting changes in behaviour, spontaneous motor activity and radial arm maze (RAM) learning were examined at 1, 2, 4 and 12 weeks following an intraperitoneal injection of a single low (32 mg/kg) or high (65 mg/kg) dose of 2,6-(diCl-MeSO(2)-B). 2,5-(DiCl-MeSO(2)-B) (65 mg/kg) was used as a negative control. Hyperactivity was observed in all treatment groups while deficits in the RAM performance was only seen in the 2,6-(diCl-MeSO(2)-B)-treated groups. Alterations in motor activity and impaired performance in the RAM-test induced by 2,6-(diCl-MeSO(2)-B) persisted up to 2 weeks in the low-dose group and 12 weeks in the high-dose group. The low-dose group consistently showed a less pronounced effect than the high-dose group. The 2,5-(diCl-MeSO(2)-B)-induced changes in motor activity declined rapidly and did not remain after 2 weeks. As determined by immunohistochemistry, 2,6-(diCl-MeSO(2)-B)-induced GFAP immunoreactivity was mainly confined to the glomerular layer of the olfactory bulb. We propose that the behavioural deficits caused by 2,6-(diCl-MeSO(2)-B) result from a primary loss of sensory neurons in the olfactory mucosa with consequent astrocyte proliferation in the glomerular layer of the olfactory bulb. A targeted uptake of metabolites into the olfactory bulb could also contribute to the GFAP induction and/or behaviour response.

Equine estrogens induce apolipoprotein E and glial fibrillary acidic protein in mixed glial cultures

Premarin, which contains several equine estrogens, as well as estradiol (E2) as a minor component, is widely used for replacement therapy of estrogen deficits, but little is known of its direct actions on brain cells. In mixed glial cultures, apolipoprotein E (apoE) and glial fibrillary acidic protein (GFAP) are induced by estrogens. GFAP induction showed an inverted-U shape E2 dose response, with a maximum induction at 1 pM, whereas apoE mRNA induction was greatest at 100 pM. GFAP and ApoE mRNAs were induced by equine estrogens in the following order: E2=equilin>estrone>17α-dihydroequilenin. However, the induction of apoE secretion by 17α-dihydroequilenin was as effective as by the other estrogens. The greater response of apoE secretion than GFAP mRNA induction to 17α-dihydroequilenin might be therapeutically important because of the glial scarring during brain lesions, in which GFAP induction has a major role in inhibiting neurite outgrowth, whereas apoE secretion supports neurite outgrowth.

Sarin causes early differential alteration and persistent overexpression in mRNAs coding for glial fibrillary acidic protein (GFAP) and vimentin genes in the central nervous system of rats.

Neurotoxic effects of single dose of 0.5 x LD50 sarin (O-isopropylmethylphosphonoflouridate) on central nervous system (CNS) of male Sprague-Dawley rats were studied. We investigated the mRNA expression of the astroglial marker genes glial fibrillary acidic protein (GFAP) and vimentin to evaluate the fate of astroglial and neuronal cells, because reactive gliosis is very often used to assess the extent of CNS damage. Rats were treated with 50 microg/kg/ml of sarin and terminated at the time-points 1 and 2 hours and 1, 3, and 7 days post-treatment. Control rats were treated with normal saline. Total RNA was extracted and Northern blots were hybridized with cDNA probes for GFAP and vimentin, as well as 28S RNA (control). The data obtained indicate that a single dose of sarin (0.5 x LD50) showed induction in the transcript levels of GFAP and vimentin in the cortex, cerebellum, brainstem and midbrain, and spinal cord. The induction showed distinct spatial-temporal differences for each tissue studied. Both GFAP and vimentin were induced at 1 hour in all the tissues studied except brainstem, where moderate and high levels of GFAP induction were noted at 1 and 3 days. Overexpressed transcript levels of GFAP and vimentin remained high in more responsive tissues such as the brainstem and midbrain. Other tissues, such as the cortex, spinal cord, and cerebellum showed a more downward trend for either GFAP or vimentin, or both, transcript levels at 7 days. It is noteworthy that both cortex (318 +/- 12%) and spinal cord (368 +/- 12%) showed relatively higher induction of GFAP, whereas cortex alone showed the highest level of overexpressed vimentin transcript levels (284 +/- 11%). Overall it is also clear that both GFAP and vimentin are needed for the effective recovery involving co-ordinated alternating up- and downregulation of these two key astrocyte genes, depending on tissue specificity. The changes seen in the transcript levels of GFAP and vimentin may be the result of astrocyte dysfunction and loss, accompanied by compensatory proliferation and dedifferentiation of the astroglia. These changes could affect the neuronal cell types, thus altering the neuron-glia homeostasis.