In vitro and in vivo infection of neural cells by a recombinant measles virus expressing enhanced green fluorescent protein

This study focused on the in vitro infection of mouse and human neuroblastoma cells and the in vivo infection of the murine central nervous system with a recombinant measles virus. An undifferentiated mouse neuroblastoma cell line (TMN) was infected with the vaccine strain of measles virus (MVeGFP), which expresses enhanced green fluorescent protein (EGFP). MVeGFP infected the cells, and cell-to-cell spread was studied by virtue of the resulting EGFP autofluorescence, using real-time confocal microscopy. Cells were differentiated to a neuronal phenotype, and extended processes, which interconnected the cells, were observed. It was also possible to infect the differentiated neuroblastoma cells (dTMN) with MVeGFP. Single autofluorescent EGFP-positive cells were selected at the earliest possible point in the infection, and the spread of EGFP autofluorescence was monitored. In this instance the virus used the interconnecting processes to spread from cell to cell. Human neuroblastoma cells (SH-SY-5Y) were also infected with MVeGFP. The virus infected these cells, and existing processes were used to initiate new foci of infection at distinct regions of the monolayer. Transgenic animals expressing CD46, a measles virus receptor, and lacking interferon type 1 receptor gene were infected intracerebrally with MVeGFP. A productive infection ensued, and the mice exhibited clinical signs of infection, such as ataxia and an awkward gait, identical to those previously observed for the parental virus (Edtag). Mice were sacrificed, and brain sections were examined for EGFP autofluorescence by confocal scanning laser microscopy over a period of 6 h. EGFP was detected in discrete focal regions of the brain and in processes, which extended deep into the parenchyma. Collectively, these results indicate (i) that MVeGFP can be used to monitor virus replication sensitively, in real time, in animal tissues, (ii) that infection of ependymal cells and neuroblasts provides a route by which measles virus can enter the central nervous system in mouse models of encephalitis, and (iii) that upon infection, the virus spreads transneuronally.     

Redistribution of GFAP and αB-crystallin after thermal stress in C6 glioma cell line

Summary Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein αB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, αB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein αB-crystallin may play a functional role regulating the cytoarchitecture of GFAP.     

Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons

The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.     

Intermediate filament protein partnership in astrocytes.

Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.     

Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.     

Membrane Ig-cytoskeletal interactions. III. Receptor cross-linking results in the formation of extensive filamentous arrays of vimentin

The proposed function of intermediate filaments is to provide a cell type-specific structural framework that maintains cell shape and organelle distribution and mediates signal transduction through its connections with the plasma membrane and the nucleus. Vimentin is the intermediate filament protein expressed in B lymphocytes. Immunocytochemical analysis of the high salt-stable cytoskeletons from B cells stimulated with anti-Ig revealed an increased accumulation of vimentin in the cytoskeleton compared to nontreated controls. This increased accumulation of vimentin in the cytoskeleton was manifested by the organization of vimentin into extensive filamentous arrays (EFA) as viewed in the fluorescent microscope. In contrast to the effects of anti-Ig, activation of B cells with LPS did not induce the organization of vimentin into EFA. This suggested that signals unique to anti-Ig directed EFA formation. Immunocytochemical results were verified by biochemical analysis showing that vimentin was more abundant in isolated cytoskeletons from anti-Ig activated B cells, than cytoskeletons isolated from LPS-activated B cells. These observations established a relationship between increased content of vimentin in the cytoskeleton and the formation of EFA. By testing a wide variety of activating agents, we were able to correlate increased vimentin expression in the cytoskeleton to activating agents that cross-link membrane Ig. It appeared that treatment of B cells with LPS prohibited the induction of EFA by anti-Ig because cotreatment with both anti-Ig and LPS resulted in decreased vimentin accumulation in the cytoskeleton to a level less than that in resting cells. The significance of these results with regard to B cell biology is discussed.     

Microinjection of intermediate filament proteins into living cells with and without preexisting intermediate filament network

Human cells grown in monolayer culture were microinjected with intermediate filament subunit proteins. In fibroblasts with a preexisting vimentin network, injected porcine glial fibrillary acidic protein (GFAP) co-localized with the vimentin network within 24 hours. Phosphorylated GFAP variants were found to become dephosphorylated concomitantly with their incorporation into filamentous structures. After microinjection of either porcine GFAP or murine vimentin into human carcinoma cells lacking cytoplasmic intermediate filaments, we observed that different types of filament networks developed. Whereas vimentin was incorporated into short filaments immediately after injection, GFAP was found to aggregate into rodlike structures. This may indicate a differential filament forming ability of these intermediate filament proteins in vivo.     

The Cytoskeleton of Primary Astrocytes in Culture Contains Actin, Glial Fibrillary Acidic Protein, and the Fibroblast-Type Filament Protein, Vimentin

Abstract: Primary astrocytes were cultured from the forebrains of 1-day-old rats. Immunofluorescence microscopy showed that approximately 80% of the cells were positive for glial fibrillary acidic protein (GFAP) and >80% were stained with an antiserum to the molecular weight 58,000 fibroblast intermediate filament protein (vimentin). Gel electrophoresis of Triton-insoluble cytoskeleton preparations from these cultures revealed three major bands having molecular weights of 58,000, 51,000, and 42,000, together with some prominent lower-molecular-weight species. The protein of molecular weight 51,000 was not present in preparations from fibroblasts. Each of the three major astrocyte proteins was subjected to limited proteolysis, while two of the proteins were cleaved by cyanogen bromide. The electrophoretic peptide patterns of the 58,000 protein were similar to those of vimentin isolated from NIL-8 fibroblasts, and the patterns of the 51,000 protein were similar to those of GFAP isolated from rat spinal cord. The patterns of the protein of molecular weight 42,000 resembled those of muscle actin. Rocket immunoelectrophoresis showed that the 51,000 astrocyte protein reacted with an antiserum to bovine GFAP, but the 58,000 and 42,000 proteins failed to react. We conclude that the major proteins of cytoskeleton preparations from cultured primary astrocytes are vimentin (58,000), GFAP (51,000), and actin (42,000), and that our data show no obvious structural relationship among them.     

Reexpression of glial fibrillary acidic protein rescues the ability of astrocytoma cells to form processes in response to neurons

Astroglial cells play an important role in orchestrating the migration and positioning of neurons during central nervous system development. Primary astroglia, as well as astrocytoma cells will extend long stable processes when co-cultured with granule neurons. In order to determine the function of the glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astroglia and astrocytoma cells, we suppressed the expression of GFAP by stable transfection of an anti- sense GFAP construct in human astrocytoma U251MG cells. The resulting AS2-U251 cells can no longer extend stable processes in the presence of granule neurons. To show that this effect is due specifically to the absence of GFAP, we reintroduced a fully encoding rat brain GFAP cDNA into these AS2-U251 cells. The resulting rat GFAP appeared as a filamentous network and the reexpression of GFAP rescued the ability of these astrocytoma cells to form stable processes when co-cultured with neurons. From these results, it is clear that the glial specific intermediate filament protein, GFAP, is required for process extension of these astrocytoma cells in response to granule neurons.     

Semiquantitative Postembedding Characterization of Intermediate Filaments in Central Nervous System Lesions Using Immunoelectron Microscopy

Standardized postembedding immunoelectron microscopy was performed to demonstrate glial fibrillary acidic protein (GFAP) and vimentin in individual intermediate filaments to determine the diagnostic value of demonstrating ultrastructural and immunophenotypic characteristics of intermediate filaments in routine brain biopsy specimens. Dual expression of GFAP and vimentin was observed in the astroblastoma and astrocytes of Alexander's disease. The antigen availability for vimentin, however, was too low to allow reliable assessment of the GFAP:vimentin ratio in individual intermediate filaments and/or filament bundles. In meningioma, only vimentin positive intermediate filaments were found. GFAP positive intermediate filaments were present in all other specimens except the oligodendroglial components of the mixed glioma, which were devoid of intermediate filaments. GFAP positivity in the filamentous periphery and electron-dense core of Rosenthal fibers was demonstrated. Technical and tissue processing factors had a significant effect on particle density values obtained for individual specimens. Although the number, distribution, and density of glial intermediate filaments varies in different astroglial entities, correlation of particle density values determined by immunoelectron microscopy with relative GFAP concentrations in different lesions requires utmost caution. Nevertheless, application of the postembedding approach to routinely fixed biopsy specimens indicated an association of different entities with the exclusive presence of GFAP and/or vimentin in individual intermediate filaments, thus emphasizing the diagnostic value of intermediate filament typing for pathological characterization.     

Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed predominantly in astrocytes. The study of its expression in the astrocyte lineage during development and in reactive astrocytes has revealed an intricate relationship with the expression of vimentin, another intermediate filament protein widely expressed in embryonic development. these findings suggested that vimentin could be implicated in the organization of the GFAP network. To address this question, we have examined GFAP expression and network formation in the recently generated vimentin knockout (Vim-) mice. We show that the GFAP network is disrupted in astrocytes that normally coexpress vimentin and GFAP, e.g., those of the corpus callosum or the Bergmann glia of cerebellum. Furthermore, Western blot analysis of GFAP protein content in the cerebellum suggests that posttranslational mechanisms are implicated in the disturbance of GFAP network formation. The role of vimentin in this process was further suggested by transfection of Vim- cultured astrocytes with a vimentin cDNA, which resulted in the normal assembly of the GFAP network. Finally, we examined GFAP expression after stab wound-induced astrogliosis. We demonstrate that in Vim- mice, reactive astrocytes that normally express both GFAP and vimentin do not exhibit GFAP immunoreactivity, whereas those that normally express GFAP only retain GFAP immunoreactivity. Taken together, these results show that in astrocytes, where vimentin is normally expressed with GFAP fails to assemble into a filamentous network in the absence of vimentin. In these cells, therefore, vimentin appears necessary to stabilize GFAP filaments and consequently the network formation.     

GFAP-Deficient Astrocytes Are Capable of Stellationin VitroWhen Cocultured with Neurons and Exhibit a Reduced Amount of Intermediate Filaments and an Increased Cell Saturation Density

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein predominantly expressed in cells of astroglial origin. To allow for the study of the biological functions of GFAP we have previously generated GFAP-negative mice by gene targeting [Peknyet al.(1995)EMBO J.14, 1590–1598]. Astrocytes in culture, similar to reactive astrocytesin vivo,express three intermediate filament proteins: GFAP, vimentin, and nestin. Using primary astrocyte-enriched cultures from GFAP-negative mice, we now report on the effect of GFAP absence on (i) the synthesis of other intermediate filament proteins in astrocytes, (ii) intermediate filament formation, (iii) astrocyte process formation (stellation) in response to neurons in mixed cerebellar astrocyte/neuron cultures, and (iv) saturation cell densityin vitro.GFAP−/− astrocytes were found to produce both nestin and vimentin. At the ultrastructural level, the amount of intermediate filaments as revealed by transmission electron microscopy was reduced in GFAP−/− astrocytes compared to that in GFAP+/+ astrocytes. GFAP−/− astrocytes retained the ability to form processes in response to neurons in mixed astrocyte/neuron cultures from the cerebellum. GFAP−/− astrocyte-enriched primary cultures exhibited an increased final cell saturation density. The latter leads us to speculate that the loss of GFAP expression observed focally in a proportion of human malignant gliomas may reflect tumor progression toward a more rapidly growing and malignant phenotype.     

Analysis of Cyclic AMP-Dependent Changes in Intermediate Filament Protein Phosphorylation and Cell Morphology in Cultured Astroglia

Receptor agonists that increase cyclic AMP levels in cultured astroglia have been shown to increase 32P-labeling of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin in these cells. Experiments were designed to determine if the increase in 32P-labeling resulted from either an increase in the turnover or net number of phosphates associated with the intermediate filament proteins and if the phosphorylation of these proteins causally affected astroglial morphology. Time course experiments indicated that 6-8 h were required to reach steady-state 32P-labeling of both GFAP and vimentin. Treatment with forskolin (10 microM) after steady-state 32P-labeling increased GFAP and vimentin phosphorylation fourfold and twofold, respectively, and also induced a morphological change from polygonal to process-bearing cells within 20-30 min of drug addition. Cells incubated in media containing brain extract (30%) for 24 h at 37 degrees C and then 3 h at 23 degrees C underwent changes from polygonal to process-bearing cells with no apparent increase in the phosphorylation of either GFAP or vimentin. Treatment of process-bearing cells (induced by brain extract) or polygonal cells with 10 microM forskolin at 23 degrees C resulted in a three- to fourfold increase in GFAP phosphorylation without significant morphological changes. These results suggest that forskolin stimulation of GFAP and vimentin increases net number of phosphates associated with these intermediate filament proteins and that the resulting increase in phosphorylation can be dissociated from morphological changes.     

Synthesis and Turnover of Cytoskeletal Proteins in Cultured Astrocytes

Abstract: We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X-100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short-term incorporation experiments vimentin, GFAP, and actin in the Triton-insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast-decaying pool with a half-life of 12–18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half-life of about 8 days.