Filament Glial Fibrillary Acidic Protein Research Articles

The effect of activated microglia on astrogliosis parameters in astrocyte cultures

In the diseased central nervous system, astrogliosis is accompanied by microglial activation. Depending on the context of their activation, reactive astrocytes are involved in neuronal survival and regeneration in an either protective or impedimental way. Major reactive changes of astrocytes in vivo are the upregulation of the intermediate filaments GFAP (glial fibrillary acidic protein) and vimentin with accompanying cellular hypertrophy and/or hyperplasia. To examine the involvement of activated microglia in the onset and maintenance of astrogliosis, we used an in vitro model of purified cultures of astrocytes and assessed as parameters for astrogliosis GFAP, vimentin, astroglial hypertrophy and cell growth after treatment with medium conditioned by LPS (lipopolysaccarides)-stimulated microglia. Furthermore, IL-6 as a typically upregulated cytokine in proinflammatory processes in the brain was determined in treated astrocytes. GFAP, the classical marker for astrogliosis, was downregulated on its protein and in parallel with vimentin on its mRNA level. The expression of actin, another cytoskeleton protein used as control, remained unchanged. Ultrastructural studies of astroglial intermediate filaments supported these findings. No hypertrophy was found. Nevertheless, LPS-activated microglia stimulated astrocytes as demonstrated by an increased cell number and an enhanced mRNA expression of IL-6. Resting microglia did not change any of the determined parameters. Our results suggest that the role of activated microglia in astrogliotic processes following injury of the brain has to be reevaluated, as microglia in their activated state might support the onset of astrogliosis on the one hand, but might delay or reduce subsequent glial scar formation on the other hand.

The Alexander Disease–Causing Glial Fibrillary Acidic Protein Mutant, R416W, Accumulates into Rosenthal Fibers by a Pathway That Involves Filament Aggregation and the Association of αB-Crystallin and HSP27

Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed "Rosenthal fibers," contain the protein chaperones alpha B-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of alpha B-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease.

Glial Cell Reactivity in a Porcine Model of Retinal Detachment

Detachment of the neural retina from the pigment epithelium causes, in addition to photoreceptor deconstruction and neuronal cell remodeling, an activation of glial cells. It has been suggested that gliosis contributes to the impaired recovery of vision after reattachment surgery that may involve both formerly detached and nondetached retinal areas. Müller and microglial cell reactivity was monitored in a porcine model of rhegmatogenous retinal detachment, to determine whether gliosis is present in detached and nondetached retinal areas. Local detachment was created in the eyes of adult pigs by subretinal application of hyaluronate. Retinal slices were immunostained against glial intermediate filaments and K+ and water channel proteins (aquaporin-4, Kir4.1, Kir2.1), and P2Y receptor proteins. In retinal wholemounts, adenosine 5'-triphosphate (ATP)-induced intracellular Ca2+ responses of Müller cells were recorded, and microglial and immune cells were labeled with Griffonia simplicifolia agglutinin isolectin I-B4. K+ currents were recorded from isolated Müller cells. At 3 and 7 days after surgery, Müller cells in detached retinas showed a pronounced gliosis, as revealed by the increased expression of the intermediate filaments glial fibrillary acidic protein and vimentin, by the decrease of Kir4.1 immunoreactivity and of the whole-cell K+ currents, and by the increased incidence of cells that showed Ca2+ responses on stimulation of purinergic (P)2 receptors by ATP. By contrast, the immunohistochemical expression of Kir2.1 and aquaporin-4 were not altered after detachment. The increase in the expression of intermediate filaments, the decrease of the whole-cell K+ currents and of the Kir4.1 immunolabeling, and the increase in the Ca2+ responsiveness of Müller cells were also observed in attached retinal areas surrounding the focal detachment. The density of microglial-immune cells at the inner surface of the retinas increased in both detached and nondetached retinal areas. The immunoreactivities for P2Y1 and P2Y2 receptor proteins apparently increased only in detached areas. Reactive responses of Müller and microglial cells are not restricted to detached retinal areas but are also observed in nondetached regions of the porcine retina. The gliosis in the nondetached retina may reflect, or may contribute to, neuronal degeneration that may explain the impaired recovery of vision observed in human subjects after retinal reattachment surgery.

Propionic Acid Induces Cytoskeletal Alterations in Cultured Astrocytes From Rat Cerebral Cortex

Severe neurological symptoms, cerebral edema, and atrophy are common features of the inherited metabolic disorder propionic acidemia. However, the pathomechanisms involved in the neuropathology of this disease are not well established. In this study, we investigate the effects of propionic acid (PA), a metabolite accumulating in this disorder, on cytoskeletal reorganization, on cell viability, and on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes from cerebral cortex of neonatal rats. We observed that the astrocytes changed their usual polygonal morphology when exposed to 5 mM PA for 72 h, leading to the appearance of fusiform or process-bearing cells, without elicit cell death. We also noticed that after 72 h treatment with 5 mM PA cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments and disorganized actin stress fibers, as revealed by immunocytochemistry. In addition, the morphological alterations were accompanied by increased in vitro 32P incorporation into GFAP and vimentin recovered into the high-salt Triton-insoluble cytoskeletal fraction. In conclusion, our results indicate that PA lead to cytoskeletal reorganization and to increased in vitro phosphorylation of Triton-insoluble GFAP and vimentin. On the basis of our results we could suppose that Triton-insoluble GFAP and vimentin hyperphosphorylation could be implicated in the reorganization of cellular structure and these findings could be involved in the brain damage characteristic of propionic acidemia patients.

Maturing neurons are selectively sensitive to human immunodeficiency virus type 1 exposure in differentiating human neuroepithelial progenitor cell cultures

Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neuronal injury manifested by dendritic pruning, aberrant neurofilament metabolism, and decreased synaptic density. The central nervous system (CNS) responds to neuronal injury by differentiating new neurons and astrocytes from resident populations of multipotent neuroepithelial progenitor cells (NEP) located in regions such as the subventricular zone or hippocampus. In vitro studies have demonstrated that the HIV-1 virion or envelope glycoprotein gp120 can injure differentiated human neurons and astrocytes, suggesting that HIV-1 proteins could similarly injure NEP or NEP-derived glial and neuronal lineage-committed precursor cells. To answer this question, human fetal brain-derived "neurospheres" containing NEP and NEP-derived precursor cells were cultured in low serum differentiation medium containing lymphotropic HIV-1(SF2), macrophage-tropic HIV-1(SF128A), or recombinant gp120SF2 from HIV-1(SF2). These experiments indicate that exposure to HIV-1 does not affect the ability of the NEP to differentiate into cells expressing either astrocyte-specific or neuron-specific cytoskeletal antigens. However prolonged exposure to HIV-1 does selectively decrease expression of neuronal antigens (microtubule beta-III-tubulin and intermediate filament neurofilament-L) but not astrocyte antigens (intermediate filament glial fibrillary acidic protein). The effects of continuous exposure to HIV-1 or gp120 may result from injury to developing neurons and/or impairment of the neuronal developmental process itself. By depressing neuronal microtubule and neurofilament protein expression, HIV-1 and gp120 exposure compromise the potential for postmitotic neuronal dendrite and axon development.

Branched-Chain α-Keto Acids Accumulating in Maple Syrup Urine Disease Induce Reorganization of Phosphorylated GFAP in C6-Glioma Cells

In this study we investigate the effects of the branched-chain keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV), and alpha-keto-beta-methylvaleric (KMV) acids, metabolites accumulating in maple syrup urine disease (MSUD), on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and cytoskeletal reorganization in C6-glioma cells. We observed that after 3 h treatment with KIC, KIV, or KMV cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments as revealed by immunocytochemistry. Western Blot analysis by anti-GFAP monoclonal antibody demonstrated that BCKA were not able to alter GFAP immunocontent in total cell homogenate, but the immunocontent as well as the in vitro (32)P incorporation into GFAP recovered into the high salt Triton-insoluble cytoskeletal fraction were significantly increased. Western Blot using monoclonal antiphosphoserine antibody showed that BCKA induced increased immunocontent of phosphoserine-containing amino acids in several proteins in total cell homogenate. In addition, the immunocontent of phosphoserine-containing amino acids was also greatly increased in GFAP recovered in the high-salt Triton insoluble cytoskeletal fraction, corresponding to the polymerized intermedite filament (IF) proteins present in the cell. In conclusion, our results indicate that KIC, KIV, or KMV increased the serine/threonine in vitro phosphorylation of GFAP leading to increased Triton-insoluble GFAP immunocontent and cytoskeletal reorganization. Considering IF networks can be regulated by phosphorylation of polypeptide subunits leading to reorganization of the IF filamentous structure, we could suppose that GFAP hyperphosphorylation and disorganization of cellular structure could be involved in the brain damage characteristic of MSUD patients.

S100b counteracts effects of the neurotoxicant trimethyltin on astrocytes and microglia

Central nervous system degenerative diseases are often characterized by an early, strong reaction of astrocytes and microglia. Both these cell types can play a double role, protecting neurons against degeneration through the synthesis and secretion of trophic factors or inducing degeneration through the secretion of toxic molecules. Therefore, we studied the effects of S100B and trimethyltin (TMT) on human astrocytes and microglia with two glial models, primary cultures of human fetal astrocytes and a microglia cell line. After treatment with 10(-5) M TMT, astrocytes showed morphological alterations associated with an increase in glial fibrillary acidic protein (GFAP) expression and changes in GFAP filament organization. Administration of S100B before TMT treatment prevented TMT-induced changes in morphology and GFAP expression. A decrease in inducible nitric oxide synthase expression was observed in astrocytes treated with TMT, whereas the same treatment induced iNOS expression in microglia. In both cases, S100B prevented TMT-induced changes. Tumor necrosis factor-alpha mRNA expression in astrocytes was not modified by TMT treatment, whereas it was increased in microglia cells. S100B pretreatment blocked the TMT-induced increase in TNF-alpha expression in microglia. To trace the mechanisms involved in S100B activity, the effect of BAY 11-7082, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and of PD98059, an inhibitor of MEK-ERK1/2, were investigated. Results showed that the protective effects of S100B against TMT toxicity in astrocytes depend on NF-kappaB, but not on ERK1/2 activation. These results might help in understanding the role played by glial cells in brain injury after exposure to chemical neurotoxicants and support the view that S100B may protect brain cells in case of injury. (c) 2005 Wiley-Liss, Inc.

1005. Replication and Anti-Tumor Efficacy of Conditionally Replicating Adenoviral Vectors for Glioma Treatment

In order to improve the efficacy of first generation adenoviral vectors for glioma treatment, we designed conditionally replicating adenoviruses (CRAd's) in which the E1A gene is under the control of the promoter of the glia specific intermediate filament Glial Fibrillary Acidic Protein (GFAP) and similar constructs containing additional glia-specific enhancers (GFAP 'super' promoters).

Alexander-disease mutation ofGFAPcauses filament disorganization and decreased solubility of GFAP

Alexander disease is a fatal neurological illness characterized by white-matter degeneration and the formation of astrocytic cytoplasmic inclusions called Rosenthal fibers, which contain the intermediate filament glial fibrillary acidic protein (GFAP), the small heat-shock proteins HSP27 and alphaB-crystallin, and ubiquitin. Many Alexander-disease patients are heterozygous for one of a set of point mutations in the GFAP gene, all of which result in amino acid substitutions. The biological effects of the most common alteration, R239C, were tested by expressing the mutated protein in cultured cells by transient transfection. In primary rat astrocytes and Cos-7 cells, the mutant GFAP was incorporated into filament networks along with the endogenous GFAP and vimentin, respectively. In SW13Vim(-) cells, which have no endogenous cytoplasmic intermediate filaments, wild-type human GFAP frequently formed filamentous bundles, whereas the R239C GFAP formed 'diffuse' and irregular patterns. Filamentous bundles of R239C GFAP were sometimes formed in SW13Vim(-) cells when wild-type GFAP was co-transfected. Although the presence of a suitable coassembly partner (vimentin or GFAP) reduced the potential negative effects of the R239C mutation on GFAP network formation, the mutation affected the stability of GFAP in cells in a dominant fashion. Extraction of transfected SW13Vim(-) cells with Triton-X-100-containing buffers showed that the mutant GFAP was more resistant to solubilization at elevated KCl concentrations. Both wild-type and R239C GFAP assembled into 10 nm filaments with similar morphology in vitro. Thus, although the R239C mutation does not appear to affect filament formation per se, the mutation alters the normal solubility and organization of GFAP networks.

284. Oncolytic Adenovirus E1B-19kD Deletion and E3B Retention Results in Both Enhanced Potency and Tumor Necrosis Factor - Mediated Selectivity

In order to improve the efficacy of first generation adenoviral vectors for glioma treatment, we designed conditionally replicating viruses in which the E1A gene is under the control of the promoter (gfa2) of the glia specific intermediate filament Glial Fibrillary Acidic Protein (GFAP) and similar constructs containing additional glial specific enhancers.

931. Specificity of gfa2 Derived Promoters in Adenoviral Vectors for Conditional Replication in Glioblastoma

4602 The limited spread of non-replicating adenoviral vectors after injection into glial tumors may contribute to the lack of significant tumor reduction in many human gene therapy trials. To enhance viral spread in the tumor while keeping it restricted to the the brain, we designed conditionally replicating viruses in which the E1A gene is under the control of the promoter of the glia specific intermediate filament Glial Fibrillary Acidic Protein (GFAP). Because of the moderate activity of the normal GFAP promoter (gfa2) compared to the CMV promoter, we also evaluated constructs containing additional glial specific enhancers: gfa2(B)3 and gfa2(ABD)3 promoters. The gfa2 derived promoters followed by a beta galactosidase reporter gene were cloned into the pShuttle plasmid, in an orientation inverse to wildtype E1 expression . Viral vectors were produced according to the Ad-Easy protocol and viruses giving rise to blue plaques upon infection of U251 but not A549 cells were selected and grown to high titers. To check the specificity and effectivity of the promoter constructs, a panel of human cell lines (glial and non-glial) was infected with the gfa2 (triple enhancer) promoter-beta galactosidase vectors. An adenoviral vector containing a CMV-beta galactosidase cassette was included to estimate cell entry. Expression of beta galactosidase was evaluated both in situ using X-gal staining and quantitatively using ONPG staining. Results: Of 11 glial cell lines tested, four were positive for GFAP on a Western blot incubated with anti-GFAP (U251, SNB19, U373 and GOS3). Furthermore, of 12 primary cultures of glial tumors one was positive for GFAP using immunohistochemistry. This positive primary culture was also GFAP positive on Western and was added to the panel of cell lines, as were fourteen non glial cell lines (all GFAP negative on Western). Of the three gfa2 derived promoters, the gfa2 (ABD)3-LacZ construct gives rise to high beta galactosidase expression levels in the GFAP positive cell lines. Beta galactosidase expression levels in some GFAP positive lines are at a level comparable to the CMV promoter construct. The ONPG ratio of GFAP positive cell lines to GFAP negative cell lines (both glial and non-glial) is best for the gfa2-(B)3 promoter containing virus. Preliminary results indicate that conditionally replicating adenovirus in which E1A is regulated by the gfa2-B3 promoter replicate better on the GFAP positive U251 cell line than on the GFAP negative Hep2 cells. Conclusion: The gfa2 promoter constructs are highly active in an adenoviral context and have retained their specificity. Constructs for conditionally replicating viruses using these promoters have been prepared and conditionally replicating virus is being produced.

Forebrain astroglial plasticity is induced following withdrawal from repeated cocaine administration.

Astrocytes actively participate in synaptic plasticity and respond to insult or metabotropic glutamate receptor activation with increased expression of the intermediate filament glial fibrillary acidic protein (GFAP). Extended withdrawal from repeated cocaine administration induces many forms of neuroplasticity. The present study with rats utilized a 3-week withdrawal period from daily cocaine administration (i.p.; 7 days) to investigate whether astrocytes participate in cocaine-mediated plasticity observed in brain nuclei associated with addiction. Following the 3-week withdrawal period, immunoblotting revealed increased GFAP expression in the prefrontal cortex (PFC) and in the shell and core compartments of the nucleus accumbens (NAshell and NAcore). Upregulation of GFAP did not occur in the striatum or in any brain region tested following shorter withdrawal times from repeated cocaine (24 h or 1 week) or following 2-h withdrawal from an acute cocaine injection (30 mg/kg i.p.). However, GFAP expression increased following a 3-week withdrawal from a single cocaine injection selectively in the NAshell. Cell counts revealed that astrocyte cell number increased only in the NAcore while immunoblots of a marker for immature or reactive astrocytes, vimentin, showed an increase only in the PFC following the 3-week withdrawal. Taken together, these results suggest that altered intermediate filament expression within forebrain astrocytes may be a significant part of the plasticity occurring during withdrawal from repeated cocaine. Furthermore, the increase in GFAP may arise from regionally distinct mechanisms, with the NAcore relying more on cell proliferation while the PFC relies on a larger reactive astrocyte population.

Protective Role of Phosphorylation in Turnover of Glial Fibrillary Acidic Protein in Mice

Glial fibrillary acidic protein (GFAP), the principal intermediate filament (IF) protein of mature astrocytes in the CNS, plays specific roles in astrocyte functions. GFAP has multiple phosphorylation sites at its N-terminal head domain. To examine the role of phosphorylation at these sites, we generated a series of substitution mutant mice in which phosphorylation sites (Ser/Thr) were replaced by Ala, in different combinations.<i>Gfap</i>^{<i>hm3/hm3</i>} mice carrying substitutions at all five phosphorylation sites showed extensive decrease in both filament formation and amounts of GFAP.<i>Gfap^hm1/hm1</i> and<i>Gfap^hm2/hm2</i> mice, which carry substitutions at three of five sites and in different combinations, showed differential phenotypes. Although<i>Gfap^hm3/hm3</i> mice retained GFAP filaments in Bergmann glia in the cerebellum, the (<i>Gfap^hm3/hm3:Vim^−/−</i>) mice lacked GFAP filaments. Pulse-chase experiments of cultured astrocytes indicated that the Hm3-GFAP encoded by<i>Gfap^hm3</i> was unstable particularly in the absence of vimentin, another IF protein. These results revealed the role of phosphorylation in turnover of GFAP and a synergistic role of GFAP and vimentin in the dynamics of glial filaments. The data further suggest that each of the phosphorylated sites has a distinct impact on the dynamics of GFAP.

Protective role of phosphorylation in turnover of glial fibrillary acidic protein in mice.

Glial fibrillary acidic protein (GFAP), the principal intermediate filament (IF) protein of mature astrocytes in the CNS, plays specific roles in astrocyte functions. GFAP has multiple phosphorylation sites at its N-terminal head domain. To examine the role of phosphorylation at these sites, we generated a series of substitution mutant mice in which phosphorylation sites (Ser/Thr) were replaced by Ala, in different combinations. Gfap(hm3/hm3) mice carrying substitutions at all five phosphorylation sites showed extensive decrease in both filament formation and amounts of GFAP. Gfap(hm1/hm1) and Gfap(hm2/hm2) mice, which carry substitutions at three of five sites and in different combinations, showed differential phenotypes. Although Gfap(hm3/hm3) mice retained GFAP filaments in Bergmann glia in the cerebellum, the (Gfap(hm3/hm3):Vim(-/-)) mice lacked GFAP filaments. Pulse-chase experiments of cultured astrocytes indicated that the Hm3-GFAP encoded by Gfap(hm3) was unstable particularly in the absence of vimentin, another IF protein. These results revealed the role of phosphorylation in turnover of GFAP and a synergistic role of GFAP and vimentin in the dynamics of glial filaments. The data further suggest that each of the phosphorylated sites has a distinct impact on the dynamics of GFAP.

Glial fibrillary acidic protein expression and promoter activity in the inner ear of developing and adult mice.

The intermediate filament glial fibrillary acidic protein (GFAP) is a classic marker for several types of glial cells, including astrocytes and nonmyelinating Schwann cells. The pattern of expression of GFAP in the postnatal murine inner ear, from postnatal day 3 (P3) to P38, was studied by anti-GFAP immunostaining in wild-type mice as well as in two lines of transgenic mice expressing either beta-galactosidase (LacZ) or green fluorescent protein (GFP) under the control of the GFAP promoter. Analysis of protein and promoter activity shows that several classes of supporting cells in the sensory epithelia, as well as Schwann cells and satellite cells express GFAP. Early after birth, all cochlear supporting cells express GFAP, in a gradient decreasing in intensity from base to apex. After P15, GFAP expression in the organ of Corti is mostly restricted to supporting cells of the inner hair cell area (i.e., inner border and inner phalangeal cells) and outer hair cell area (i.e., Deiters' cells). A small population of limbic cells also showed expression in a base-to-apex gradient. In the vestibular organs, high expression was detected in supporting cells in extrastriolar regions of the utricular macula and in the canal ampullae, with weaker staining in the saccular macula. These results suggest that supporting cells of the inner ear have important similarities to glial cells and may play roles similar to those of astrocytes or Schwann cells in supporting the normal development and maintenance of neurons and sensory cells of the inner ear.

A 1.8kb GFAP-promoter fragment is active in specific regions of the embryonic CNS.

The intermediate filament glial fibrillary acidic protein (GFAP) constitutes the major cytoskeletal protein in astrocytes (J. Neuroimmunol. 8 (1985) 203) and is traditionally referred to as a specific marker for astrocytes. To identify early glial precursors, we created GFAPpromoter-lacZ transgenic mice, using a 1.8kb 5' fragment of human GFAP. The expression of the transgene was first detected in the neuroepithelium at embryonic day 9.5. It was further found in the ventricular zone of the developing telencephalon, in the cerebellar primordium, trigeminal ganglia, and radial glia. Later, scattered beta-gal+ cells were seen in pons, brain stem and glia limitans. The results indicate that GFAP activity is regulated in a region-specific manner during central nervous system (CNS) development and that the gene is turned on in different cell types independently.

Effect of elevated K(+), hypotonic stress, and cortical spreading depression on astrocyte swelling in GFAP-deficient mice.

Glial fibrillary acidic protein (GFAP) is the main component of intermediate filaments in astrocytes. To assess its function in astrocyte swelling, we compared astrocyte membrane properties and swelling in spinal cord slices of 8- to 10-day-old wild-type control (GFAP(+/+)) and GFAP-knockout (GFAP(-/-)) mice. Membrane currents and K(+) accumulation around astrocytes after a depolarizing pulse were studied using the whole-cell patch-clamp technique. In vivo cell swelling was studied in the cortex during spreading depression (SD) in 3 to 6-month-old animals. Swelling-induced changes of the extracellular space (ECS) diffusion parameters, i.e., volume fraction alpha and tortuosity lambda, were studied by the real-time iontophoretic tetramethylammonium (TMA(+)) method using TMA(+)-selective microelectrodes. Morphological analysis using confocal microscopy and quantification of xy intensity profiles in a confocal plane revealed a lower density of processes in GFAP(-/-) astrocytes than in GFAP(+/+) astrocytes. K(+) accumulation evoked by membrane depolarization was lower in the vicinity of GFAP(-/-) astrocytes than GFAP(+/+) astrocytes, suggesting the presence of a larger ECS around GFAP(-/-) astrocytes. Astrocyte swelling evoked by application of 50 mM K(+) or by hypotonic solution (HS) produced a larger increase in [K(+)](e) around GFAP(+/+) astrocytes than around GFAP(-/-) astrocytes. No differences in alpha and lambda in the spinal cord or cortex of GFAP(+/+) and GFAP(-/-) mice were found; however, the application of either 50 mM K(+) or HS in spinal cord, or SD in cortex, evoked a large decrease in alpha and an increase in lambda in GFAP(+/+) mice only. Slower swelling in GFAP(-/-) astrocytes indicates that GFAP and intermediate filaments play an important role in cell swelling during pathological states.

Mitotic Reorganization of the Intermediate Filament Protein Nestin Involves Phosphorylation by cdc2 Kinase

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.

Intermediate filament proteins define different glial subpopulations.

In certain species, specialized glial cells delineate cell domains in the central nervous system and assist in the elongation of axons by providing mechanical and chemical barriers. We showed previously, that the glial intermediate filament proteins vimentin and glial fibrillary acidic protein are extensively coexpressed in radial glia in the developing hindbrain, and that subsequently, the two proteins define distinct rhombomere domains: vimentin is localized in radial glia at the rhombomere boundaries and glial fibrillary acidic protein expression is restricted to the rhombomere centers (Yoshida and Colman [2000] J. Comp. Neurol. 424:47-57). The present study reveals that vimentin and glial fibrillary acidic protein continue to display distinct expression domains throughout the developing Xenopus central nervous system. Although the precise function of the two intermediate filaments in glial cells has yet to be revealed, the observations presented here suggests that glial intermediate filament proteins demarcate different populations of glial cells during nervous system development and that the existence of different glial populations may define glial boundaries.

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

Double-immunolabelling techniques were employed to investigate the distribution of smooth muscle alpha-actin (actin) in glial fibrillary acidic protein (GFAP)—positive cells in rat brain during early postnatal development and maturation and in glial primary culture derived from newborn rat brain. In addition the expression of desmin was studied in the glial primary cultures as a function of the differentiation of the cells. Comparison of the cultured astroglial cells at an early age with hepatic stellate cells derived from CCl 4-induced cirrhotic rat liver, revealed features of the astrocytic cytoskeleton characteristic of myofibroblastic cells, i.e., strong expression of both myofibroblastic markers, actin and desmin. In astroglial cells with an initial morphology reminiscent of fibroblasts the non-filamentous perinuclear immunoreaction of GFAP increased with time at the expense of actin and, partially, desmin. GFAP filaments were spread throughout the cytoplasm of the cells which acquired stellate morphology. The alterations in the morphology of the cells and the distribution and intensity of staining for GFAP and actin during the differentiation of astrocytes in culture were similar to those observed in astrocytes during the maturation of the brain. In astrocytes from a newborn brain as well as in cirrhotic hepatic stellate cells, the area of immunoreaction of GFAP was reduced and confined mainly to the nuclear region. In contrast, the cells expressed actin throughout the cytoplasm. These findings may hint at a similar function of these regionally specialized perivascular myofibroblastic cells in a normal brain and diseased liver and at inverse organ-specific functions which the cells fulfill under non-pathological conditions in vivo.