Intermediate Filament Protein Vimentin Research Articles

Anaplasma phagocytophilum AptA modulates Erk1/2 signalling

Anaplasma phagocytophilum causes human granulocytic anaplasmosis, one of the most common tick-borne diseases in North America. This unusual obligate intracellular pathogen selectively persists within polymorphonuclear leucocytes. In this study, using the yeast surrogate model we identified an A. phagocytophilum virulence protein, AptA (A. phagocytophilum toxin A), that activates mammalian Erk1/2 mitogen-activated protein kinase. This activation is important for A. phagocytophilum survival within human neutrophils. AptA interacts with the intermediate filament protein vimentin, which is essential for A. phagocytophilum-induced Erk1/2 activation and infection. A. phagocytophilum infection reorganizes vimentin around the bacterial inclusion, thereby contributing to intracellular survival. These observations reveal a major role for the bacterial protein, AptA, and the host protein, vimentin, in the activation of Erk1/2 during A. phagocytophilum infection.

The Capsid Protein Encoded by U L 17 of Herpes Simplex Virus 1 Interacts with Tegument Protein VP13/14

The U(L)17 protein (pU(L)17) of herpes simplex virus 1 (HSV-1) likely associates with the surfaces of DNA-containing capsids in a heterodimer with pU(L)25. pU(L)17 is also associated with viral light particles that lack capsid proteins, suggesting its presence in the tegument of the HSV-1 virion. To help determine how pU(L)17 becomes incorporated into virions and its functions therein, we identified pU(L)17-interacting proteins by immunoprecipitation with pU(L)17-specific IgY at 16 h postinfection, followed by mass spectrometry. Coimmunoprecipitated proteins included cellular histone proteins H2A, H3, and H4; the intermediate filament protein vimentin; the major HSV-1 capsid protein VP5; and the HSV tegument proteins VP11/12 (pU(L)46) and VP13/14 (pU(L)47). The pU(L)17-VP13/14 interaction was confirmed by coimmunoprecipitation in the presence and absence of intact capsids and by affinity copurification of pU(L)17 and VP13/14 from lysates of cells infected with a recombinant virus encoding His-tagged pU(L)17. pU(L)17 and VP13/14-HA colocalized in the nuclear replication compartment, in the cytoplasm, and at the plasma membrane between 9 and 18 h postinfection. One possible explanation of these data is that pU(L)17 links the external face of the capsid to VP13/14 and associated tegument components.

Nestin is essential for mitogen-stimulated proliferation of neural progenitor cells

The intermediate filament (IF) protein nestin is a widely accepted molecular marker for neural progenitor cells (NPCs), but its function during neurogenesis remains largely unknown. We found that in embryonic cortical NPCs down-regulation of the expression of nestin, but not its co-polymer IF protein vimentin, resulted in a G1 cell-cycle arrest and a severe reduction in the generation of neurons. Furthermore, down-regulating nestin expression in cultured cortical NPCs markedly suppressed their colony-formation ability and blocked the elevation of the cyclin D1/E protein level in response to the treatment with bFGF. Interestingly, nestin down-regulation caused a marked suppression in the activation of the phosphoinositide 3-kinase (PI3K) pathway but not the mitogen-activated protein kinase (MAPK) pathway in these NPCs. Moreover, defects in the proliferation of cortical NPCs caused by nestin down-regulation could be prevented by up-regulating PI3K activity. Thus, nestin is essential for the proliferation of NPCs by promoting the activation of PI3K in response to mitogenic growth factors.

Divalent Cations Crosslink Vimentin Intermediate Filament Tail Domains to Regulate Network Mechanics

Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca 2+ and Mg 2+, showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, l P ≈ 0.5 μm, and Young's modulus, Y ≈ 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells.

Attenuation of Reactive Gliosis Does Not Affect Infarct Volume in Neonatal Hypoxic-Ischemic Brain Injury in Mice

BackgroundAstroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP−/−Vim−/−) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP−/−Vim−/− mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.Methodology/Principal FindingsWe subjected GFAP−/−Vim−/− and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP−/−Vim−/− and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN+ neurons in the ischemic and contralateral hemisphere was comparable between GFAP−/−Vim−/− and wild-type mice. In wild-type mice, the number of S100+ astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0±50.1 vs. 85.6±34.0, p<0.05). In the GFAP−/−Vim−/− mice, the number of S100+ astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP−/−Vim−/− mice showed an increase in NeuN+BrdU+ (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7±7.7; n = 29 versus 2.9±3.6; n = 28, respectively, p<0.05), but a comparable number of S100+BrdU+ (surviving newly born) astrocytes.Conclusions/SignificanceOur results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.

The switchable integrin adhesome

Adhesion sites, which interconnect cells with their neighbors or with the extracellular matrix (ECM), are large multiprotein complexes that provide mechanical coupling as well as a means for cells to sense the chemical and physical properties of their environment ([Bershadsky et al., 2003][1]; [Chen

Regulation of cell adhesion to collagen via β1 integrins is dependent on interactions of filamin A with vimentin and protein kinase C epsilon

Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by β1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate β1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A–vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of β1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase Cɛ bind to repeats 1–8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1–8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of β1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase Cε is an essential regulatory step for the trafficking and activation of β1 integrins and cell spreading on collagen.

Intermediate Filament Carbonylation During Acute Acrolein Toxicity in A549 Lung Cells: Functional Consequences, Chaperone Redistribution, and Protection by Bisulfite

Extensive protein carbonylation accompanies cellular exposure to acrolein, a ubiquitous smoke constituent implicated in life-threatening pulmonary edema in fire victims, a condition involving rapid erosion of the "watertight" properties of respiratory epithelium. Since the identities of lung epithelial proteins that sustain carbonylation by acrolein are unknown, we sought to identify significant targets in subcellular fractions from A549 cells after 30 min exposure to either subtoxic or acutely toxic acrolein concentrations (60 or 360 fmol acrolein/cell). The lower concentration mainly modified cytosolic proteins while the higher concentration also damaged nuclear, membrane, and cytoskeletal proteins. The multifunctional intermediate filament proteins vimentin, keratin-18, keratin-7 and keratin-8, were conspicuous targets. Consistent with their mechanical functions, a loss of cellular adhesive strength accompanied adduction of the two most abundant intermediate filaments in A549 cells, keratins-8 and -18. Acrolein also elicited redistribution of several chaperones (Hsp40, -70, -90, and -110) to intermediate filament fractions, suggesting chaperone-mediated autophagy contributes to the triage of acrolein-adducted proteins. The carbonyl scavenger bisulfite suppressed acrolein toxicity, intermediate filament adduction, vimentin cross-linking, Hsp90 redistribution, and loss of cellular adhesive strength, while also suppressing vimentin hyperphosphorylation. These novel observations identify intermediate filaments as key targets for the reactive smoke constituent acrolein.

Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers.

IntroductionDevelopment of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes.MethodsWe searched for markers using the differential in-gel electrophoresis proteomic technology to compare proteins of bovine nucleus pulposus cells, phenotypically similar to mature human nucleus cells, with those of bovine articular chondrocytes. In the cohort of the differentially expressed proteins identified by mass spectrometry, cytokeratin 8 (CK8) was further validated by immunostaining of freshly isolated cells and frozen tissue sections using monoclonal antibodies.ResultsWe identified a set of 14 differentially expressed proteins. Immunohistochemistry showed that only a subset of cells (approximately 10%) was positive for one of these proteins, CK8, an intermediate filament protein present in epithelial but not mesenchymal cells. In tissue sections, CK8-positive cells were seen in all discs examined and appeared as small isolated clusters surrounded by gelatinous matrix. Notochordal nucleus pulposus cells from pig, phenotypically similar to human infant nucleus pulposus cells, were all CK8-positive. The mesenchymal intermediate filament protein vimentin was present in all bovine and porcine nucleus pulposus cells.ConclusionsThe notochordal cell population is reported to disappear from the nucleus pulposus of bovine discs before birth and from human discs in childhood. However our finding of the co-expression of vimentin and CK8 in small isolated clusters of the bovine nucleus pulposus cells indicates that a subpopulation of notochordal-like cells remains in the mature bovine disc. This finding agrees with reports in the literature on co-expression of cytokeratins and vimentin in adult human discs. As notochordal cells produce factors that promote matrix production, the CK8-positive subpopulation could have important implications for activity and survival of the nucleus pulposus, and should be considered in development of cell therapies for disc repair. In addition, the finding of differential expression of proteins in the cell population of nucleus pulposus has implications with regard to the search for specific markers.

Immunocytochemical Detection of Tissue Antigens after Prolonged Storage of Specimens in Methylsalicylate

The aim of the present work was to verify the possibility of detecting various tissue antigens using standard immunocytochemical methods in paraffin sections of specimens stored in methylsalicylate for two years and longer before embedding in paraffin. Using the immunocytochemical reaction for the intermediate filament proteins nestin, vimentin, and GFAP, the neuron marker NeuN, neuron-specific enolase, and doublecortin as examples, these experiments showed that prolonged storage of dehydrated specimens in methylsalicylate does not prevent detection of antigens using immunocytochemical reactions.

Filamin A is required for vimentin-mediated cell adhesion and spreading

Cell adhesion and spreading are regulated by complex interactions involving the cytoskeleton and extracellular matrix proteins. We examined the interaction of the intermediate filament protein vimentin with the actin cross-linking protein filamin A in regulation of spreading in HEK-293 and 3T3 cells. Filamin A and vimentin-expressing cells were well spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, cells treated with small interfering RNA (siRNA) to knock down filamin A or vimentin were poorly spread; both of these cell populations exhibited >50% reductions of cell adhesion, cell surface beta1 integrin expression, and beta1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions, whereas knockdown of filamin and/or vimentin inhibited the formation of cell extensions. Reduced vimentin phosphorylation, cell spreading, and beta1 integrin surface expression, and activation were phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase C-epsilon. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins, we found an association between filamin A and vimentin. Filamin A also associated with protein kinase C-epsilon, which was enriched in cell extensions. These data indicate that filamin A associates with vimentin and to protein kinase C-epsilon, thereby enabling vimentin phosphorylation, which is important for beta1 integrin activation and cell spreading on collagen.

Identification of a novel vimentin promoter and mRNA isoform

The intermediate filament protein vimentin is involved in a variety of cellular functions both during the normal developmental processes and in human malignancies. We here describe the identification of an alternative vimentin transcript initiating upstream for the canonical vimentin gene promoter and spliced using the vimentin promoter sequence as an intron. Expression analysis showed that the alternative vimentin promoter had the same expression profile as the canonical vimentin gene promoter. The presented data suggest that alternative promoter usage and alternative splicing could be regulatory mechanisms participating in vimentin gene regulation.

HPV16 E7 oncogene expression in normal human epithelial cells causes molecular changes indicative of an epithelial to mesenchymal transition

Cancer-associated epithelial to mesenchymal transition (EMT) is crucial for invasion and metastasis. Molecular hallmarks of EMT include down-regulation of the epithelial adhesion protein E-cadherin and de-novo expression of N-cadherin and the mesenchymal intermediate filament proteins vimentin and fibronectin. Expression of HPV16 E7 in normal human epithelial cells caused increased levels of vimentin and fibronectin, whereas the epithelial adhesion protein E-cadherin was expressed at decreased levels. Similar expression patterns of vimentin, fibronectin and E-cadherin were also detected in cells expressing HPV16 E6 and E7 or the entire HPV16 early transcriptional unit. HPV16 E6 and E7 were each able to induce N-cadherin expression. Interestingly, these changes in expression levels of EMT-associated proteins are not similarly reflected at the level of mRNA expression, suggesting that HPV16 oncoproteins also modulate EMT through non-transcriptional mechanisms. Hence, HPV16 oncoproteins may contribute to malignant progression through EMT induction.

Intermediate filaments exchange subunits along their length and elongate by end-to-end annealing

Actin filaments and microtubules lengthen and shorten by addition and loss of subunits at their ends, but it is not known whether this is also true for intermediate filaments. In fact, several studies suggest that in vivo, intermediate filaments may lengthen by end-to-end annealing and that addition and loss of subunits is not confined to the filament ends. To test these hypotheses, we investigated the assembly dynamics of neurofilament and vimentin intermediate filament proteins in cultured cells using cell fusion, photobleaching, and photoactivation strategies in combination with conventional and photoactivatable fluorescent fusion proteins. We show that neurofilaments and vimentin filaments lengthen by end-to-end annealing of assembled filaments. We also show that neurofilaments and vimentin filaments incorporate subunits along their length by intercalation into the filament wall with no preferential addition of subunits to the filament ends, a process which we term intercalary subunit exchange.

Vimentin Coil 1A—A Molecular Switch Involved in the Initiation of Filament Elongation

Interestingly, our previously published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric α-helical coil instead of the expected dimeric coiled coil. However, the 39-amino-acid-long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim of investigating the molecular criteria that are needed to stabilize a dimeric coiled-coil structure. We have analysed the biophysical properties of the mutants by circular dichroism spectroscopy, analytical ultracentrifugation and X-ray crystallography. All four mutants exhibited an increased stability over the wild type as indicated by a rise in the melting temperature (Tm). At a concentration of 0.1 mg/ml, the Tm of the peptide with the single point mutation Y117L increased dramatically by 46 °C compared with the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or a d position did induce the formation of a stable dimer as demonstrated by sedimentation equilibrium experiments. The dimeric oligomerisation state of the Y117L peptide was furthermore confirmed by X-ray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when this mutation was introduced into full-length vimentin, filament assembly was completely arrested at the unit-length filament (ULF) level, both in vitro and in cDNA-transfected cultured cells. Therefore, the low propensity of the wild-type coil 1A to form a stable two-stranded coiled coil is most likely a prerequisite for the end-to-end annealing of ULFs into filaments. Accordingly, the coil 1A domains might “switch” from a dimeric α-helical coiled coil into a more open structure, thus mediating, within the ULFs, the conformational rearrangements of the tetrameric subunits that are needed for the intermediate filament elongation reaction.

Nestin-positive microglia in adult rat cerebral cortex

Nestin is a class VI intermediate filament protein, which was first identified in the early developmental stages of the nervous system. It is widely used as a stem or progenitor cell marker. In the adult mammalian brain, nestin is expressed not only in germinal cells in the neurogenic regions but also in non-germinal cells, such as reactive astrocytes, endothelial cells and pericytes. In the present study, we found another nestin-positive cell type within the adult rat cerebral cortex. We immunohistochemically analyzed which types of cells exhibit immunoreactivity for nestin, and through the use of co-immunostaining with Iba1, CD11b and GLUT5, which are known to be specific for microglia, identified these cells as microglia. Approximately > 20% of the microglia were immunoreactive for nestin in the rat cerebral cortex under normal conditions. Nestin signals were not widely distributed in the microglial cytoplasm, but were restricted to the perikaryon and to parts of the cell processes. Nestin-positive microglia were also immunoreactive for the intermediate filament protein vimentin. These observations demonstrate that a subpopulation of microglia in a resting state has nestin-containing intermediate filament networks. Therefore, nestin in conjunction with vimentin might have roles in maintaining the structural integrity of the microglia.

Desmin and Vimentin Intermediate Filament Networks: Their Viscoelastic Properties Investigated by Mechanical Rheometry

We have investigated the viscoelastic properties of the cytoplasmic intermediate filament (IF) proteins desmin and vimentin. Mechanical measurements were supported by time-dependent electron microscopy studies of the assembly process under similar conditions. Network formation starts within 2 min, but it takes more than 30 min until equilibrium mechanical network strength is reached. Filament bundling is more pronounced for desmin than for vimentin. Desmin filaments (persistence length l p ≈ 900 nm) are stiffer than vimentin filaments ( l p ≈ 400 nm), but both IFs are much more flexible than microfilaments. The concentration dependence of the plateau modulus G 0 ∼ c α is much weaker than predicted theoretically for networks of semiflexible filaments. This is more pronounced for vimentin (α = 0.47) than for desmin (α = 0.70). Both networks exhibit strain stiffening at large shear deformations. At the transition from linear to nonlinear viscoelastic response, only desmin shows characteristics of nonaffine network deformation. Strain stiffening and the maximum modulus occur at strain amplitudes about an order of magnitude larger than those for microfilaments. This is probably attributable to axial slippage within the tetramer building blocks of the IFs. Network deformation beyond a critical strain γ max results in irreversible damage. Strain stiffening sets in at lower concentrations, is more pronounced, and is less sensitive to ionic strength for desmin than for vimentin. Hence, desmin exhibits strain stiffening even at low-salt concentrations, which is not observed for vimentin, and we conclude that the strength of electrostatic repulsion compared to the strength of attractive interactions forming the network junctions is significantly weaker for desmin than for vimentin filaments. These findings indicate that both IFs exhibit distinct mechanical properties that are adapted to their respective cellular surroundings [i.e., myocytes (desmin) and fibroblasts (vimentin)].

Head and Rod 1 Interactions in Vimentin

We have used site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to identify residues 17 and 137 as sites of interaction between the head domain and rod domain 1A of the intermediate filament protein vimentin. This interaction was maximal when compared with the spin labels placed at up- and downstream positions in both head and rod regions, indicating that residues 17 and 137 were the closest point of interaction in this region. SDSL EPR characterization of residues 120-145, which includes the site of head contact with rod 1A, reveals that this region exhibits the heptad repeat pattern indicative of alpha-helical coiled-coil structure, but that this heptad repeat pattern begins to decay near residue 139, suggesting a transition out of coiled-coil structure. By monitoring the spectra of spin labels placed at the 17 and 137 residues during in vitro assembly, we show that 17-137 interaction occurs early in the assembly process. We also explored the effect of phosphorylation on the 17-137 interaction and found that phosphorylation-induced changes affected the head-head interaction (17-17) in the dimer, without significantly influencing the rod-rod (137-137) and head-rod (17-137) interactions in the dimer. These data provide the first direct evidence for, and location of, head-rod interactions in assembled intermediate filaments, as well as direct evidence of coiled-coil structure in rod 1A. Finally, the data identify changes in the structure in this region following in vitro phosphorylation.

The transcriptional repressor ZBP-89 and the lack of Sp1/Sp3, c-Jun and Stat3 are important for the down-regulation of the vimentin gene during C2C12 myogenesis

Currently, considerable information is available about how muscle-specific genes are activated during myogenesis, yet little is known about how non-muscle genes are down-regulated. The intermediate filament protein vimentin is known to be “turned off” during myogenesis to be replaced by desmin, the muscle-specific intermediate filament protein. Here, we demonstrate that vimentin down-regulation is the result of the combined effect of several transcription factors. Levels of the positive activators, Sp1/Sp3, which are essential for vimentin expression, decrease during myogenesis. In addition, c-Jun and Stat3, two additional positive-acting transcription factors for vimentin gene expression, are also down-regulated. Over-expression via adenoviral approaches demonstrates that the up-regulation of the repressor ZBP-89 is critical to vimentin down-regulation. Elimination of ZBP-89 via siRNA blocks the down-regulation of vimentin and Sp1/Sp3 expression. From these studies we conclude that the combinatorial effect of the down-regulation of positive-acting transcription factors such as Sp1/Sp3, c-Jun and Stat3 versus the up-regulation of the repressor ZBP-89 contributes to the “turning off” of the vimentin gene during myogenesis.

Cultured astrocytes derived from corpus callosum or cortical grey matter show distinct glutamate handling properties

While the astrocytic control of extracellular glutamate concentration at synaptic contacts is well characterized, little is known regarding the clearance of glutamate along axon tracts, even though local excitotoxic damage has been reported. Therefore, we have compared glutamate handling in astrocyte cultures derived from white matter (corpus callosum) and grey matter tissues (cortical structures). These populations of astrocytes showed clearly distinct phenotypes, adopting stellate or protoplasmic morphologies respectively. In addition, white matter astrocytes showed high densities of the intermediate filament proteins glial fibrillary acidic protein, vimentin and nestin. The glutamate-aspartate transporter and glutamate transporter-1, as well as glutamine synthetase, were found to be expressed at higher levels in white matter compared with grey matter astrocytes. Consistent with this aspartate uptake capacity was three to fourfold higher in white matter cells, and the use of specific inhibitors revealed a substantial activity of glutamate transporter-1, contrasting with grey matter cells where this transporter appeared poorly functional. In addition, expression of type 5 metabotropic glutamate receptors was considerably higher in white matter astrocytes where the agonist (S)-3,5-dihydroxyphenylglycine triggered a large release of intracellular calcium. Differences in these astrocyte cultures were also observed when exposed to experimental conditions that trigger glial activation. This study highlights typical features of cultured astrocytes derived from white matter tissues, which appear constitutively adapted to handle excitotoxic insults. Moreover, the expression and activity of the astroglial components involved in the control of glutamatergic transmission are reinforced when these cells are maintained under conditions mimicking a gliotic environment.