Camilla Eliasson

Absence of Glial Fibrillary Acidic Protein and Vimentin Prevents Hypertrophy of Astrocytic Processes and Improves Post-Traumatic Regeneration

The regenerative capacity of the CNS is extremely limited. The reason for this is unclear, but glial cell involvement has been suspected, and oligodendrocytes have been implicated as inhibitors of neuroregeneration (Chen et al., 2000, GrandPre et al., 2000; Fournier et al., 2001). The role of astrocytes in this process was proposed but remains incompletely understood (Silver and Miller, 2004). Astrocyte activation (reactive gliosis) accompanies neurotrauma, stroke, neurodegenerative diseases, or tumors. Two prominent hallmarks of reactive gliosis are hypertrophy of astrocytic processes and upregulation of intermediate filaments. Using the entorhinal cortex lesion model in mice, we found that reactive astrocytes devoid of the intermediate filament proteins glial fibrillary acidic protein and vimentin (GFAP-/-Vim-/-), and consequently lacking intermediate filaments (Colucci-Guyon et al., 1994; Pekny et al., 1995; Eliasson et al., 1999), showed only a limited hypertrophy of cell processes. Instead, many processes were shorter and not straight, albeit the volume of neuropil reached by a single astrocyte was the same as in wild-type mice. This was accompanied by remarkable synaptic regeneration in the hippocampus. On a molecular level, GFAP-/-Vim-/- reactive astrocytes could not upregulate endothelin B receptors, suggesting that the upregulation is intermediate filament dependent. These findings show a novel role for intermediate filaments in astrocytes and implicate reactive astrocytes as potent inhibitors of neuroregeneration.

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 astrocytesin 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.

Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally.

Glial fibrillary acidic protein (GFAP) is the main component of the intermediate filaments in cells of astroglial lineage, including astrocytes in the CNS, nonmyelin forming Schwann cells and enteric glia. To address the function of GFAP in vivo, we have disrupted the GFAP gene in mice via targeted mutation in embryonic stem cells. Mice lacking GFAP developed normally, reached adulthood and reproduced. We did not find any abnormalities in the histological architecture of the CNS, in their behavior, motility, memory, blood‐brain barrier function, myenteric plexi histology or intestinal peristaltic movement. Comparisons between GFAP and S‐100 immunohistochemical staining patterns in the hippocampus of wild‐type and mutant mice suggested a normal abundance of astrocytes in GFAP‐negative mice, however, in contrast to wild‐types, GFAP‐negative astrocytes of the hippocampus and in the white matter of the spinal cord were completely lacking intermediate filaments. This shows that the loss of GFAP intermediate filaments is not compensated for by the up‐regulation of other intermediate filament proteins, such as vimentin. The GFAP‐negative mice displayed post‐traumatic reactive gliosis, which suggests that GFAP up‐regulation, a hallmark of reactive gliosis, is not an obligatory requirement for this process.

Intermediate filaments regulate astrocyte motility.

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell‐type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.

Impaired induction of blood‐brain barrier properties in aortic endothelial cells by astrocytes from GFAB‐deficient mice

Cell culture models have been extensively used for studies of blood‐brain barrier (BBB) function. However, most in vitro models fail to reproduce the peculiar physiological and morphological properties of in situ brain microvascular endothelial cells. A recently developed, tridimensional and dynamic model of the BBB has permitted studies of glial‐endothelial interactions in hollow fibers exposed to intraluminal flow. We have taken advantage of this technique and have investigated the ability of glial fibrillary acidic protein (GFAP)‐deficient (GFAP−/−) astrocytes to induce BBB properties in aortic endothelial cells (BAEC) cultured in vitro. BAEC exposed to flow were seeded intraluminally in hollow fibers and co‐cultured with extraluminally seeded mouse astrocytes. Under these conditions, astrocytes have been shown to induce blood‐brain barrier properties in non‐brain endothelial cells. We followed induction of a BBB phenotype by measuring the transendothelial resistance, as well as endothelial permeability to potassium, theophylline, 8‐sulphophenyl‐theophylline (8‐SPT), sucrose, and Evans blue. Wild‐type mouse astrocytes induced BBB properties in aortic endothelial cells following 3–4 weeks of co‐culturing. Thus, these endothelial cells restricted passage of K+ ions into the extracapillary space and selectively excluded hydrophilic molecules, such as 8‐SPT and 14C‐sucrose. GFAP−/− astrocytes failed to induce a significant restriction to the passage of potassium and hydrophilic drugs (sucrose, 8‐SPT), failed to induce transendothelial resistance values comparable to control co‐cultures, but were capable of inducing exclusion of Evans blue by endothelial cells. These results suggest that GFAP (and intermediate filaments) may play a role in the induction of BBB properties in non‐BBB endothelial cells. GLIA 22:390–400, 1998.

Altered taurine release following hypotonic stress in astrocytes from mice deficient for GFAP and vimentin

Astrocytes maintain their volume in response to changes in osmotic pressure in their environment by an afflux/influx of ions and organic osmoequivalents. The initial swelling of an astrocyte transferred to a hypoosmotic medium is thus reversed within minutes. The mechanisms which trigger this process as well as the sensors for cell volume are largely unknown, however, the cytoskeleton appears to be involved. We have addressed the role of one component of the cytoskeleton, the intermediate filaments, in the maintenance of astrocytic cell volume. Astrocytes from wild type mice were compared with cells from mice deficient for either glial fibrillary acidic protein (GFAP-/-) or vimentin (vimentin-/-) and with astrocytes from mice deficient for both proteins (GFAP-/-vim-/-). Whereas GFAP-/- and vimentin-/- cultured or reactive astrocytes retain intermediate filaments, the GFAP-/-vim-/- astrocytes are completely devoid of these structures. The rate of efflux of the preloaded osmoequivalent 3H-taurine from primary and passaged cultures of astrocytes was monitored. A reduction of NaCl (25 mM) in the perfusion medium led to a 400-900% increase of 3H-taurine afflux in astrocytes from wild type mice. The stimulated efflux was not significantly affected in astrocytes from GFAP-/- or vimentin-/- mice. However, the efflux from astrocytes from GFAP-/-vim-/- mice was 25-46% lower than the wild type levels. The results strengthen the role of the cytoskeleton in astrocyte volume regulation and suggest an involvement of intermediate filaments in the process.

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 α and tortuosity λ, 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 α and λ 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 α and an increase in λ 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. GLIA 35:189–203, 2001.

Loss of GFAP expression in high-grade astrocytomas does not contribute to tumor development or progression

In astrocytic neoplasms, the number of cells expressing glial fibrillary acidic protein (GFAP) is inversely proportional to the extent of anaplasia. The loss of GFAP expression, the principal marker of astroglial cells, in these tumors has been proposed to constitute a step in their development and progression. To test this hypothesis, we crossed p53-negative (p53−/−) mice, which frequently develop astrocytomas after intrauterine exposure to ethylnitrosourea, with GFAP-negative (GFAP−/−) mice or GFAP+/+ controls. Brain tumors of glial origin were found in 12 of 35 GFAP+/+ p53−/− mice (34%) and in 11 of 27 GFAP−/− p53−/− mice (41%). The two groups did not differ in the age at which tumors were detected or in tumor histology or progression. Thus, the loss of GFAP expression frequently seen in high-grade astrocytomas does not constitute a step in tumor development. Rather, it may represent the undifferentiated state of these cells.