Intermediate filaments and stress

Before we can explain why so many closely related intermediate filament genes have evolved in vertebrates, while maintaining such dramatically tissue specific expression, we need to understand their function. The best evidence for intermediate filament function comes from observing the consequences of mutation and mis-expression, primarily in human tissues. Mostly these observations suggest that intermediate filaments are important in allowing individual cells, the tissues and whole organs to cope with various types of stress, in health and disease. Exactly how they do this is unclear and many aspects of cell dysfunction have been associated with intermediate filaments to date. In particular, it is still not clear whether the non-mechanical functions now being attributed to intermediate filaments are primary functions of these structural proteins, or secondary consequences of their function to respond to mechanical stress. We discuss selected situations in which responses to stress are clearly influenced by intermediate filaments.     

Effects of Ionizing Radiation and Aluminum Chloride on Protein of Glial Intermediate Filaments in the Rat Brain

We studied the effects of irradiation with X-rays (the total dose of 0.0129 C/kg was attained over 7, 14, or 21 days), increased entry of Al³⁺ into the organism (0.2% AlCl₃ in drinking water), and the combined influence of these factors for 21 days on the contents of the soluble and filamentous forms of glial fibrillary acidic protein (GFAP) in the tissues of the hippocampus, cerebellum, and neocortex of albino rats. After irradiation for 7 days, a clear trend toward drops in the GFAP contents in the structures under study was observed, while irradiation in the same dose, but for 14 or 21 days, resulted in increases in the contents of both GFAP forms (within a range of 13-29%, as compared with the control). Entry of aluminum chloride with water also resulted in an increase in the GFAP contents in all studied structures; changes in the filamentous form were more intensive. The combined influence of irradiation and Al³⁺ resulted in more intensive shifts in the GFAP levels; the content of its filamentous form increased in all structures by about 50%, while shifts of the soluble form were somewhat smaller.     

Increased Cell Proliferation and Neurogenesis in the Hippocampal Dentate Gyrus of Old GFAP<Superscript>−/−</Superscript>Vim<Superscript>−/−</Superscript> Mice

In response to central nervous system (CNS) injury, and more discretely so also during aging, astrocytes become reactive and increase their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Studies of mice deficient in astrocytic intermediate filaments have provided insights into the function of reactive gliosis. Recently we demonstrated robust integrationof retinal transplants (1) and increased posttraumatic synaptic regeneration (2) in GFAP^–/–Vim^–/– mice, suggesting that modulation of astrocyte activity affects the permissiveness of the CNS environment for regeneration. Neurogenesis in the adult mammalian CNS is restricted to essentially two regions, the hippocampus and the subventricular zone. Here, we assessed neurogenesis in the hippocampus of 18-month-old GFAP^–/–Vim^–/– mice. In the granular layer of the dentate gyrus, cell proliferation/survival was 34% higher and neurogenesis 36% higher in GFAP^–/–Vim^–/– mice than in wildtype controls. These findings suggest that the adult hippocampal neurogenesis in healthy old mice can be increased by modulating astrocyte reactivity.Special issue dedicated to Lawrence. F. Eng.     

Effect of Vitamin E on the Content and Polypeptide Composition of Glial Fibrillary Acidic Protein from the Rat Brain under Conditions of Aluminum Chloride Intoxication

We studied the effects of isolated and combined chronic (21 days) introductions of aluminum chloride and vitamin E (α-tocopherol) on the polypeptide composition and content of glial fibrillary acidic protein (GFAP) in different brain structures of rats. Injections of AlCl₃ solution (12 mg/kg, i.p., daily) caused the appearance of low-molecular (47 to 38 kdalton) polypeptides and an increase in the content of GFAP in cytoskeletal fractions to 160 to 220%, as compared with the control. Introduction of vitamin E within the same interval provided significant normalization of the GFAP content in the brain of animals injected with AlCl₃ and to a considerable extent prevented the appearance of degraded polypeptides in the GFAP composition. We discuss the prospects of using vitamin E as an antioxidant for the correction of Al³⁺-induced pathological processes in the CNS.Neirofiziologiya/Neurophysiology, Vol. 37, No. 1, pp. 15–20, January–February, 2005.     

Intermediate vimentin filaments and their role in intracellular organelle distribution

Intermediate filaments (IF) represent one of three main cytoskeletal structures in most animal cells. The human IF protein family includes about 70 members divided into five main groups. The characteristic feature of IF is that in various cells and tissues they are formed by proteins of different groups. Structures of all IF proteins follow a unique scheme: a central α-helical part is flanked at the N and C ends by positively charged polypeptide chains devoid of a clear secondary structure. The central part is highly conserved for all proteins in all animals, whereas the N and C termini strongly differ both in size and amino acid composition. This review covers the broad spectrum of recent investigations of IF structure and diverse functions. Special attention is paid to the regulatory mechanisms of IF functions, mainly to phosphorylation by different protein kinases whose role is well studied. The review gives examples of hereditary diseases associated with mutations of some IF proteins, which point to an important physiological role of these cytoskeletal structures.     

Roles of Rho-associated Kinase in Cytokinesis; Mutations in Rho-associated Kinase Phosphorylation Sites Impair Cytokinetic Segregation of Glial Filaments

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.     

Evolution of several cytoskeletal proteins of astrocytes in primary culture: Effect of prenatal alcohol exposure

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein -tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of -tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti - and anti--tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin.In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed in vivo.Special issue dedicated to Dr. Santiago Grisolia     

Nociceptive reaction-related state of glial intermediate filaments in the brain of rats with hyperfunction of the thyroid gland: An ontogenetic aspect

In experiments on Wistar rats of two age groups (5 weeks old and mature, 5 months old), we studied the content and polypeptide composition of glial fibrillary acidic protein (GFAP) in the cerebral cortex, hippocampus, thalamus, and brainstem under conditions of experimentally induced hyperfunction of the thyroid gland, as well as of combination of hyperthyroidism with the pain model (consequences of laparotomy). The hyperthyroid state was created by administration of L-thyroxine (initial dose, 10 μg/day) with food for 2 weeks; the dose was increased daily by 10 μg. At the end of experiment, we measured the level of thyroxine in the blood serum using an immunoenzymatic technique. We found that in the hyperthyroid state 5-week-old rats showed a significant increase in the content of filamentous GFAP fraction in the hippocampus, while when this influence was combined with the postoperation pain syndrome, an increase was observed in the brainstem (by 18 to 27%). Results of immunoblotting demonstrated that degraded polypeptides with a molecular mass of 47 to 45 kdalton were manifested under these conditions, which is indicative of intensification of cytoskeletal rearrangement of astroglia. In mature rats under the same conditions, we observed a drop in the level of insoluble polypeptides of intermediate filaments in astrocytes in the thalamus, hippocampus, and cerebral cortex. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 280–286, July–August, 2006.     

A novel human astrocyte cell line (A735) with astrocyte-specific neurotransmitter function

Summary Studies of brain cell function and physiology are hampered by the limited availability of imortal human brain-derived cell lines, as a result of the technical difficulties encountered in establishing immortal human cells in culture. In this study, we demonstrate the application of recombinant DNA vectors expressing SV40 T antigen for the development of immortal human cell cultures, with morphological, growth, and functional properties of astrocytes. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. One of these cultures gave rise to an immortal cell line, designated A735. All the human SV40-derived lines retained morphological features and growth properties of type 1 astrocytes. Immunohistochemical studies and Western blot analysis of the intermediate filament proteins and glutamine synthetase demonstrated a differentiated but immature astrocyte phenotype. Transport of γ-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines’ retaining aspects of normal glial function. We conclude that methods based on the use of SV40 T antigen can successfully immortalize human astrocytes, retaining key astrocyte functions, but T antigen-induced proliferation appeared to interfere with expression of glial fibrillary acidic protein. We believe A735 is the first documented nontumor-derived human glial cell line which is immortal.     

Alexander Disease: A Leukodystrophy Caused by a Mutation in GFAP

Alexander disease, a rare fatal disorder of the central nervous system, causes progressive loss of motor and mental function. Until recently it was of unknown etiology, almost all cases were sporadic, and there was no effective treatment. It was most common in an infantile form, somewhat less so in a juvenile form, and was rarely seen in an adult-onset form. A number of investigators have now shown that almost all cases of Alexander disease have a dominant mutation in one allele of the gene for glial fibrillary acidic protein (GFAP) that causes replacement of one amino acid for another. Only in very rare cases of the adult-onset form is the mutation present in either parent. Thus, in almost all cases, the mutation arises as a spontaneous event, possibly in the germ cell of one parent.     

Heterogeneous immunoreactivity of glial cells in the mesencephalon of a lizard: A double labeling immunohistochemical study

Astrocytes and radial glia coexist in the adult mesencephalon of the lizard Gallotia galloti. Radial glia and star-shaped astrocytes express glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The same cell markers are also expressed by round or pear-shaped cells that are therefore astrocytes with unusual morphology. Other round or pear-shaped cells, also scattered in the tegmentum and the tectum, display only GS. Electron microscopy reveals that these cells may be oligodendrocytes. In this lizard, the GS is expressed in some oligodendrocytes while this does not occur in the central nervous system of mammals in situ. These results confirm that the cellular specificity of GS is different in various species and suggest that ependymal cells are also immunoreactive for GS but they do not contain GFAP. J. Morphol. 235:109–119, 1998. © 1998 Wiley-Liss, Inc.     

Subpopulation of nestin positive glial precursor cells occur in primary adult human brain cultures

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein considered to be the best astroglial marker. However, the predominant cell population in adult human brain tissue cultures does not express GFAP; these cells have been termed “glia-like” cells. The basic question about histological origin of adult human brain cultures remains unanswered. Some authors showed that “glia-like” cells in adult human brain cultures might be of non-glial origin. We examined primary explant tissue cultures derived from 70 adult human brain biopsies. Within first 5–10 days approximately 5–10% of the small explants became attached. Outgrowing cells were mostly flat cells. These cells formed confluent layer over 3–6 weeks in culture. At confluence the cultures contained 2–5% of microglial cells, 0.1% GFAP-positive astrocytes, less than 0.01% oligodendrocytes and 95–98% GFAP-negative “glia-like” cells. This population of flat “glia-like” cells was positively stained for vimentin, fibronectin, and 20–30% of these cells stained for nestin. Our findings revealed that 1 mM dibutyryl-cAMP addition, in serum free conditions, induced a reversible stellation in 5-10% of the flat “glia-like” cells but did not induce the expression of GFAP or nestin in morphologically changed stellate cells. These results demonstrate that “glia-like” cells in primary adult human brain cultures constitute heterogeneous cell populations albeit with similar morphological features. Two distinct subpopulations have been shown: (i) the one immunostained for nestin; and (ii) the other reactive for dibutyryl-cAMP treatment.     

A Model to Induce Low Temperature Trauma for <Emphasis Type="Italic">in vitro</Emphasis> Astrogliosis Study

Astrogliosis is an inevitable and rapid response of astrocytes to physical, chemical and pathological injuries. To study astrogliosis, we developed a reproducible in vitro model in which low temperature injury to cultured astrocytes could be induced by placing the culture dish onto a copper pipe pre-cooled by liquid nitrogen. Using this model, the relationship between the temperature decline and the severity of cellular damage was analyzed. An increase in the expression of some known injury-related proteins, such as glial fibrillary acidic protein (GFAP), immediate early response genes (IEGs), and heat shock proteins 70 (HSP70), was demonstrated in astrocytes after low temperature trauma. With the use of this low temperature trauma model, the flexibility in the temperature control and injury area may allow researchers to evaluate cryotherapy and cryosurgery, which could be applicable to future development of quality health care.Special issue dedicated to Lawrence F. Eng.     

Natural fluctuation and gonadal hormone regulation of astrocyte immunoreactivity in dentate gyrus

The number and the surface density of cells immunoreactive for the specific astrocytic marker glial fibrillary acidic protein (GFAP), were evaluated in both the hilus of the dentate gyrus and the granular layer of the vermis of the cerebellar cortex of adult female rats during the different phases of the estrous cycle, after ovariectomy and after the pharmacological administration of estradiol and/or progesterone to overiectomized rats. Although no significant differences were detected in the number of immunoreactive cells among the different experimental groups studied, their surface density showed significant changes in the hilus of the dentate gyrus. The surface density of immunoreactive cells was increased in the afternoon of proestrus and on the morning of estrus compared to the morning of proestrus, diestrus, and metestrus, was decreased after ovariectomy, and showed a dose-dependent increase in ovariectomized rats injected with 17β estradiol (1, 10, or 300 μg/rat), alone or in combination with progesterone (500 μg/rat). In contrast, it was not affected by the administration of 17β estradiol (300 μg/rat). The surface density of immunoreactive cells was significantly increased over control values by 5 h after the injection of 17β estradiol (300 μg/rat) and as early as 1 h after the administration of progesterone. The separate injection of either 17β estradiol or progesterone had smaller effects on the surface density of immunoreactive cells than did the administration of both hormones together. The surface density of GFAP-immunoreactive cells reached maximal values by 24 h after the administration of 17β estradiol and/or progesterone and returned to control levels by 48 h after the combined injection of progesterone and 17β estradiol, while in the rats that were injected with only one of the two hormones, the surface density of immunoreactive cells remained over control values for at least 9 days. No such hormonal effects on GFAP-immunoreactive cells were observed in the cerebellar cortex. © 1993 John Wiley & Sons, Inc.     

Motile Properties of Vimentin Intermediate Filament Networks in Living Cells

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.     

Complete separation of the triplet components of neurofilament by DE-52 column chromatography depends upon urea concentration

In the separation of the triplet components of neurofilament (P 200, P 160, and P 68) by DE-52 column chromatography in the presence of urea, it was revealed that the efficiency of separation depended upon urea concentration. When chromatography was performed in the presence of 8 m urea and a linearly increasing sodium phosphate concentration from 10 to 400 mm at pH 6.8, P 160 and P 68 were eluted in the same peak, although P 200 was eluted faster. P 160 and P 68 were partially separated with 6 m urea, and completely separated with 4 m urea. But, under these conditions, P 200 was eluted at the same position as contaminated glial fibrillary acidic protein (GFA). From these results, two methods were recommended for the complete separation of the triplet components of neurofilament and GFA by DE-52 column chromatography. In one method, chromatography was performed in the presence of 8 m urea at first, and then P 160 and P 68 were separated in the presence of 4 m urea. In the other method, chromatography was performed with a linearly decreasing urea concentration from 8 to 0 m.     

Developmental expression of the Glial fibrillary acidic protein (GFAP) gene in the mouse retina

1. In the nervous system, Glial fibrillary acidic protein (GFAP) is a well-known, cell type-specific marker for astrocytes. 2. In the mammalian retina, Muller cells, the major class of retinal glia, do not express GFAP or contain only low amounts of this protein. In retinas with photoreceptor degeneration, however, high levels of GFAP are found. It is possible that GFAP synthesis in these retinas could result from "dedifferentiation" of Muller cells as a consequence of disruption of normal neuron-glia interactions. 3. We have carried out immunocytochemical and in situ hybridization studies to examine whether GFAP or its mRNA is expressed by retinal cells early in embryonic development. 4. Our results show that GFAP-containing cells, which are probably astrocytes, are found only in the ganglion cell and nerve fiber layers and that these cells appear after postnatal day-1 (P-1) and continue to form until P-10. 5. Astrocyte formation starts from the optic disc and moves toward the periphery of the retina at a rate of approximately 160-200 microns per day. 6. An unexpected result from these studies is that GFAP mRNA levels are high in the first week of birth and decline rapidly as the animal develops. 7. Finally, we did not find either GFAP or GFAP mRNA in retinal cells other than astrocytes during normal development.     

αB-Crystallin is Associated with Intermediate Filaments in Astrocytoma Cells

B-crystallin, a major protein of the vertebrate lens and a member of the small heat shock protein family, is expressed in non-lenticular tissues, including the central nervous system, where it is found mainly in glia. In Rosenthal fibers (RF), astrocytic inclusions that accumulate in Alexander's Disease, B-crystallin is found with hsp27 and skeins of intermediate filaments (IF) of the GFAP and vimentin types. We have investigated the association between IF and B-crystallin in a human astrocytoma cell line, U-373MG, which expresses B-crystallin. Cytoskeletal preparations contained B-crystallin, and a filamentous pattern in which B-crystallin co-localized with GFAP and vimentin by double label immunofluorescence. Immuno-electronmicroscopy confirmed the localization to IF. GFAP isolated from bovine brain and re-assembled, was associated with B-crystallin. Thus, a proportion of B-crystallin in astroglia is associated with IF, and this association may be critical in the formation of RF.