Expression of small heat shock proteins and intermediate filaments in the human optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro.

The small heat shock proteins (sHSP), alpha B-crystallin and Hsp27 are chaperone molecules that maintain the integrity of intermediate filament (IF) network and prevent unfolding of cellular proteins induced by stress. In the optic nerve head (ONH) of eyes with glaucoma, reactive astrocytes expressed Hsp27, perhaps in response to stress related to elevated intraocular pressure. In this study, we determined the effect of elevated hydrostatic pressure (HP) in the synthesis, distribution and co-localization of alpha B-crystallin and Hsp27 with IF in cultured ONH astrocytes. Astrocyte monolayers were pressurized to 60 mm Hg (92% air 8% CO(2)) and incubated at 37 degrees C for 6, 24 or 48 hr. Controls were exposed to ambient pressure. Cells were analyzed by immunocytochemistry, Western blot and immunoprecipitation using antibodies to Hsp27, alpha B-crystallin, vimentin or GFAP. Control astrocytes seemed flat, polygonal with short processes. alpha B-crystallin appeared granular in the perinuclear area and filamentous in the cell periphery. Fine granular Hsp27 was distributed throughout the cytoplasm. GFAP and vimentin co-localized with Hsp27 in the cytoplasm. Astrocytes exposed to HP were star-shaped with long processes. Hsp27 was condensed in large granules around the nucleus. GFAP and vimentin co-localized with Hsp27 and alpha B-crystallin in the perinuclear area. Western blot and metabolic labeling detected increased synthesis of Hsp27, GFAP and vimentin but no change in alpha B-crystallin. These results indicated that GFAP and vimentin associate with Hsp27 and alpha B-crystallin in ONH astrocytes. HP affected the integrity of the cytoskeleton consistent with morphological changes. Small HSP may reinforce and maintain IF integrity in response to HP.

Drosophila rab GDI mutants disrupt development but have normal Rab membrane extraction.

Rab GTPases are essential for vesicular transport. Rab GDP dissociation inhibitor (GDI) binds to GDP-bound rabs, removes rabs from acceptor membranes and delivers rabs to donor membranes. We isolated lethal GDI mutations in Drosophila and analyzed their developmental phenotypes. To learn how these mutations affect GDI structure, the crystal structure of Drosophila GDI was determined by molecular replacement to a resolution of 3.0 A. Two hypomorphic, missense mutations are located in domain II of GDI at highly conserved positions, but not in previously identified sequence conserved regions. The mutant GDIs were tested for ability to extract rabs from membranes and showed wild-type levels of rab membrane extraction. The two missense alleles showed intragenic complementation, indicating that domain II of GDI may have two separable functions. This study indicates that GDI function is essential for development of a complex, multicellular organism and that puparium formation and pole cell formation are especially dependent on GDI function.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human optic nerve head astrocytes.

Glaucomatous optic neuropathy is a common blinding disease characterized by remodeling of the extracellular matrix (ECM) and loss of retinal ganglion cell (RGC) axons at the level of the optic nerve head (ONH). Astrocytes, the major cell type in ONH, may participate in this process by production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). In normal and glaucomatous ONH, we detected MMP and TIMP expression by immunohistochemistry. Cultured astrocytes were used to characterize expression of MMPs and TIMPs by zymography, Western blot, and RNase protection assay. MMP production was stimulated with phorbol 12-myristate 13-acetate (PMA). Astrocytes expressed MMP1, MT1-MMP, MMP2, TIMP1, and TIMP2 in normal and glaucomatous ONH. MMP2, TIMP1, and TIMP2 localized to RGCs and their axons. Increased MMP1 and MT1-MMP expression was demonstrated in glaucoma. Cultured astrocytes constitutively expressed MMP2, MT1-MMP, TIMP1, and TIMP2, whereas MMP3, MMP7, MMP9, and MMP12 were not detectable in tissues or in cultured astrocytes. Our findings demonstrate the presence of specific MMPs and TIMPs in the ONH that may participate in the homeostasis and remodeling of the ECM in glaucoma. Expression of the same MMPs and TIMPs in cultured ONH astrocytes will allow further studies on the mechanisms regulating these enzymes.

Expression of myocilin/TIGR in normal and glaucomatous primate optic nerves.

Myocilin/TIGR was the first molecule discovered to be linked with primary open angle glaucoma (POAG), a blinding disease characterized by progressive loss of retinal ganglion cells. Mutations in myocilin/TIGR have been associated with age of disease onset and severity. The function of myocilin/TIGR and its role in glaucoma is unknown. Myocilin/TIGR has been studied in the trabecular meshwork to determine a role in regulation of intraocular pressure. The site of damage to the axons of the retinal ganglion cells is the optic nerve head (ONH). The myocilin/TIGR expression was examined in fetal through adult human optic nerve as well as in POAG. Myocilin/TIGR was expressed in the myelinated optic nerve of children and normal adults but not in the fetal optic nerve before myelination. Also examined was the expression in monkeys with experimental glaucoma. The results demonstrate that optic nerve head astrocytes constitutively express myocilin/TIGR in vivo in primates. Nevertheless, myocilin/TIGR is apparently reduced in glaucomatous ONH. The colocalization of myocilin/TIGR to the myelin suggests a role of myocilin/TIGR in the myelinated optic nerve.

Selective expression of neural cell adhesion molecule (NCAM)-180 in optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro.

Glaucomatous optic neuropathy is usually associated with elevated intraocular pressure. Optic nerve head astrocytes may respond to intraocular pressure by stimulation of pressure-sensitive mechanoreceptors on the cell surface. Neural cell adhesion molecule (NCAM) a transmembrane protein, mediates cell adhesion and migration. The NCAM 180 isoform increases in astrocytes of glaucomatous optic nerve head. We characterized the relative expression of NCAM isoforms in human optic nerve head astrocytes grown under elevated hydrostatic pressure. Astrocytes cultured from normal human optic nerve heads were exposed to either atmospheric or continuous hydrostatic pressure of 60 mm Hg, and analyzed at 6-48 h. Changes in cell shape, immunoreactivity, and distribution of GFAP, actin and NCAM were observed in pressure-treated cultures. Newly synthesized (35)S-labeled NCAM protein immunoprecipitated from cell lysates was increased 2-fold within 24 h after exposure to elevated pressure compared to control. The increase in NCAM synthesis was primarily due to the NCAM 180 isoform. A significant increase in NCAM 180 mRNA levels was detected by RT-PCR and Northern blots in cultured optic nerve head astrocytes within 6 h after exposure to elevated pressure. NCAM 180 mRNA and protein synthesis decreased after 24 h and returned to control levels by 48 h. Our data indicate that NCAM 180 transcription and synthesis in astrocytes is stimulated by elevated hydrostatic pressure. Because NCAM 180 interacts with the cytoskeleton through an extended cytoplasmic tail, a selective and transient increase in NCAM 180 in optic nerve head astrocytes exposed to elevated pressure may be relevant to the migration and interactions of reactive astrocytes in glaucoma.

Transforming growth factor beta isoforms in human optic nerve heads.

AIM: To determine if the isoforms of transforming growth factor beta (TGF-beta) are present in fetal, normal adult, and glaucomatous optic nerve heads. METHODS: To localise cells synthesising TGF-beta, optic nerve heads were stained using antibodies to TGF-beta 1, TGF-beta 2, and TGF-beta 3. To demonstrate synthesis, human optic nerve heads from fetal, glaucomatous, and normal age matched subjects were explanted, cultured overnight, and the culture supernatant was assayed for the presence of TGF-beta 1 and TGF-beta 2 by bioassay. In addition, semiquantitative RT-PCR was performed to determine the gene expression pattern of TGF-beta 2. RESULTS: Immunohistochemistry of glaucomatous samples revealed the presence of intense staining for TGF-beta 2 primarily in astrocytes, whereas TGF-beta 1 was localised to blood vessels. No TGF-beta 3 immunoreactivity was observed. There was little or no expression of TGF-beta in normal optic nerve heads. Optic nerve heads from glaucomatous eyes released 70-100-fold more TGF-beta 2 than normal age matched optic nerve heads. Fetal optic nerve heads released 90-100-fold more TGF-beta 2 than normal adult optic nerve heads. TGF-beta 1 was undetectable by bioassay in all samples tested. There was no apparent increase in TGF-beta 2 gene expression in glaucomatous and fetal eyes, suggesting post-transcriptional regulatory mechanisms. CONCLUSIONS: These results demonstrate that TGF-beta 2 is produced in high levels in the fetal and glaucomatous optic nerve heads, perhaps by a mechanism of post-transcriptional regulation. TGF-beta may be important during development of the optic nerve head and, in glaucoma, TGF-beta 2 may be a mediator of astrocyte reactivation and extracellular matrix remodelling in the lamina cribrosa.

Enhanced tenascin expression associated with reactive astrocytes in human optic nerve heads with primary open angle glaucoma.

Tenascin is a large extracellular matrix glycoprotein expressed in neural and non-neural tissues. In the central nervous system, tenascin is synthesized by astrocytes during development and wound healing, forming barriers and affecting neurite outgrowth. In this study we examined tenascin expression in optic nerve heads of normal and glaucomatous eyes and found that there is upregulation of tenascin mRNA and protein in reactive astrocytes from human glaucomatous optic nerve heads compared to normal age-matched controls. In the prelaminar region there was a band of tenascin immunoreactivity around the blood vessels of glaucomatous, but not in normal eyes. However, tenascin mRNA was only localized to astrocytes, suggesting that astrocytes are the cellular source of tenascin. In the lamina cribrosa, tenascin immunoreactivity and gene expression were localized to astrocytes in the cribriform plates and inside the nerve bundles. In the post-lamina region, tenascin immunoreactivity and gene expression were localized to astrocytes lining the pial septum immediately adjacent to the lamina cribrosa. In normal optic nerve heads, tenascin expression at the mRNA and protein levels was confined to clusters of astrocytes at the level of Bruch's membrane in the prelaminar optic nerve head. In glaucoma, enhanced expression of tenascin may be protective to the axons of the retinal ganglion cells by providing a barrier for humoral and/or blood-borne factors that may cause further neural damage. However, the precise role of tenascin in glaucomatous optic neuropathy is not yet elucidated.

Differential expression of neural cell adhesion molecule isoforms in normal and glaucomatous human optic nerve heads.

Type 1B astrocytes of the human optic nerve head (ONH) constitutively express neural cell adhesion molecule (NCAM) in vivo and in vitro. Increased synthesis of NCAM has been detected in reactive astrocytes in the glaucomatous ONH of human donor eyes. Several NCAM isoforms are generated through alternate RNA splicing in tissue- and disease-specific patterns. In this study, we analyzed expression of NCAM isoforms in ONH of normal donors at different ages and in glaucoma. Total RNA was extracted from ONH of fetal, normal adult and glaucomatous eyes, and cultured human ONH astrocytes, fetal brain astrocytes and an astrocytoma cell line, for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. To distinguish between NCAM 180 and 140 isoforms, exon-specific primer sets covering exons 13-19 were used. Isoform-specific riboprobes were used for in situ hybridization (ISH) in glaucomatous and in age-matched ONH. By RT-PCR, NCAM 140 was the predominant isoform in adult ONH as well as in all cultured cells. NCAM 180 mRNA was strongly expressed in glaucoma, whereas in normal adult tissues it was not detectable. ISH confirmed expression of NCAM in normal adult ONH and localized NCAM 140 mRNA to astrocytes. ISH demonstrated expression of NCAM 180 mRNA in reactive astrocytes in glaucomatous ONH. Our results demonstrate that the NCAM 180 isoform is induced in glaucoma. NCAM 180 may play a role in astrocyte interaction with extracellular matrix (ECM), vessels, axons and other astrocytes and, through its expanded cytoplasmic domain, serve as a signaling molecule for reactive astrocytes during remodeling of the ONH in glaucoma.

Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength x-ray diffraction on the selenomethionyl protein at 2.9-A resolution.

The crystal structure of the tetrameric DNA-binding domain of the single-stranded DNA binding protein from Escherichia coli was determined at a resolution of 2.9 A using multiwavelength anomalous dispersion. Each monomer in the tetramer is topologically similar to an oligomer-binding fold. Two monomers each contribute three beta-strands to a single six-stranded beta-sheet to form a dimer. Two dimer-dimer interfaces are observed within the crystal. One of these stabilizes the tetramer in solution. The other interface promotes a superhelical structure within the crystal that may reflect tetramer-tetramer interactions involved in the positive cooperative binding of the single-stranded DNA-binding protein to single-stranded DNA.

A conditional-lethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions.

The coronavirus spike glycoprotein (S) mediates both the attachment of virus to the host cell receptor and membrane fusion. We describe here the characterization of a temperature-sensitive mutant of the coronavirus mouse hepatitis virus A59 (MHV-A59) having multiple S protein-related defects. The most remarkable of these was that the mutant, designated Albany 18 (Alb18), assembled virions devoid of the S glycoprotein at the nonpermissive temperature. Alb18 also failed to bring about syncytia formation in cells infected at the nonpermissive temperature. Virions of the mutant assembled at the permissive temperature were much more thermolabile than wild type. Moreover, mutant S protein that was incorporated into virions at the permissive temperature showed enhanced pH-dependent thermolability in its ability to bind to the MHV receptor. Alb18 was found to have a single point mutation in S resulting in a change of serine 287 to isoleucine, and it was shown by revertant analysis that this was the lesion responsible for the phenotype of the mutant.

Site-directed mutagenesis of the genome of mouse hepatitis virus by targeted RNA recombination.

We have genetically characterized a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHV). This mutant, designated Alb4, is both temperature-sensitive and thermolabile, and its N protein is smaller than wild-type N. Sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. PCR analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five nucleotide insertion in the 3' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronaviral genomic RNA and a tailored, synthetic RNA species. To date, we have constructed three additional mutants by this procedure. For two of these, a second-site point mutation that reverts the Alb4 phenotype has been transduced into a wild type background, which does not contain the Alb4 deletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted RNA recombination.

The genetic characterization of a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHV) is described. The mutant, Albany 4 (Alb4), is both temperature sensitive and thermolabile. Analysis of the progeny of a mixed infection showed that the defective Alb4 allele is recessive to wild type, and its gene product is diffusible. The N protein of Alb4 was found to be smaller than its wild-type counterpart, and sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. Polymerase chain reaction analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five-nucleotide insertion in the 3' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronavirus genomic RNA and a tailored, synthetic RNA species.

Conformational change of the coronavirus peplomer glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell fusion.

We have obtained biochemical and electron microscopic evidence of conformational changes at pH 8.0 and 37 degrees C in the coronavirus spike glycoprotein E2 (S). The importance of these changes is reflected in the loss of virus infectivity, the aggregation of virions, and increased virus-induced cell fusion at the same pH. Coronavirus (MHV-A59) infectivity is exquisitely sensitive to pH. The virus was quite stable at pH 6.0 and 37 degrees C (half-life, approximately 24 h) but was rapidly and irreversibly inactivated by brief treatment at pH 8.0 and 37 degrees C (half-life, approximately 30 min). Virions treated at pH 8.0 and 37 degrees C formed clumps and large aggregates. With virions treated at pH 8.0 and 37 degrees C, the amino-terminal peptide E2N (or S1) was released from virions and the remaining peptide, E2C (S2), was aggregated. Viral spikes isolated from detergent-treated virions also aggregated at pH 8.0 and 37 degrees C. Loss of virus infectivity and E2 (S) aggregation at pH 8.0 and 37 degrees C were markedly enhanced in the presence of dithiothreitol. On the basis of the effects of dithiothreitol on the reactions of the peplomer, we propose that release of E2N (S1) and aggregation of E2C (S2) may be triggered by rearrangement of intramolecular disulfide bonds. The aggregation of virions and the isolated E2 (S) glycoprotein at pH 8.0 and 37 degrees C or following treatment with guanidine and urea at pH 6.0 and 37 degrees C indicate that an irreversible conformational change has been induced in the peplomer glycoprotein by these conditions. It is interesting that coronavirus-induced cell fusion also occurred under mildly alkaline conditions and at 37 degrees C. Some enveloped viruses, including influenza viruses and alphaviruses, show conformational changes of spike glycoproteins at a low pH, which correlates with fusion and penetration of those viruses in acidified endocytic vesicles. For coronavirus MHV-A59, comparable conformational change of the spike glycoprotein E2 (S) and cell fusion occurred at a mildly alkaline condition, suggesting that coronavirus infection-penetration, like that of paramyxoviruses and lentiviruses, may occur at the plasma membrane, rather than within endocytic vesicles.

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments.

In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell-fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities.

Isolation of the subunits of the coronavirus envelope glycoprotein E2 by hydroxyapatite high-performance liquid chromatography.

The coronavirus glycoprotein E2, which is responsible for virus attachment to cell receptors and virus-induced cell fusion, was purified by solubilization of virions with Triton X-114 and phase fractionation. Native E2 and tryptic subunits of the glycoprotein were separated by size-exclusion high-performance liquid chromatography (HPLC). Two distinct 90 kD E2 subunits, which had identical electrophoretic mobilities when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were separated by hydroxyapatite HPLC in the presence of sodium dodecyl sulfate.