Expression of Wild Type and Truncated Myocilins in Trabecular Meshwork Cells: their Subcellular Localization

PURPOSE: To investigate subcellular localizations of wild type myocilin and trunctated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells. METHODS: GFP and GFP tagged truncated myocilin, full-length myocilin, and stromelysin were expressed in TM cells using adenoviral vectors, and their secretory properties were examined by western blotting. To determine the subcellular localizations of myocilins, cellular organelles of the infected TM cells were stained with antibodies or orgenelle specific fluorescent indicators and examined under confocal microscope. RESULTS: Wild type myocilin was expressed as discrete fine vesicles in the perinuclear region of TM cells as well as in ER, but not in microtubules or mitochondria. Colocalization of wild type and truncated myocilin, indicative of in vivo interaction of the proteins, was also observed in cells co-expressing the proteins. Truncated myocilin was found to be accumulated as aggregates in ER, and inhibited the secretion of normal myocilin. CONCLUSIONS: Our result suggests that intracellular accumulation of truncated myocilin may cause a dysfunction of the cells, resulting in alterations in structural compartmentalization of trabecular ECM and obstruction of aqueous outflow.

The Cytotoxicity of Truncated (Q368X) Myocilin in Trabecular Meshwork (TM) Cells

PURPOSE: To investigate the cytotoxicities of wild type myocilin and trunctated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells. METHODS: GFP tagged truncated myocilin and DsRED1 tagged wild type myocilin were expressed in TM cells using adenoviral vectors and observed colocalization by confocal microscope. Cytopathic effects in the cells were examined by light microscope and WST-1 cell proliferation assay. RESULTS: Colocalization of wild type and truncated myocilin was observed in cells co-expressing the proteins. Truncated myocilin was found to be toxic to cells, leading to deformed cellular morphology and diminished cell proliferation. CONCLUSIONS: The intracellular accumulation of truncated myocilin exhibited cytotoxicity in trabecular meshwork cells, and eventually resulted in diminished number and dysfunction of trabecular meshwork cells, which might be involved in the pathogenesis of glaucoma.

Stromelysin Gene Expression in Rat Eye Mediated by the Adenovirus Vector

The purpose of this study is to evaluate the eligibility of gene therapy in treating glaucoma by constructing a replication deficient recombinant adenovirus carrying cDNA of stromelysin and by observing its expression in the rat eye. Stromelysin cDNA was obtained by RT-PCR using total RNA extracted from the cultured human trabecular cells. The cDNA was cloned into TA vector and subsequently into p deltaA. CMV. pA. The adenovirus vector containing stromelysin cDNA was constructed by co-transfection of pJM17 and p delta A. CMV-str. pA into the 293 cells. The expression of stromelysin in the trabecular meshwork and uveoscleral outflow channel of the rat eyes was detected by in situ hybridization and immunohistochemistry. In conclusion, this study showed the possibility of gene therapy in glaucoma by showing the expression of stromelysin with recombinant adenovirus vector containing stromelysin cDNA in the rat eye.

Association of(G-T)n Dinucleotide Repeat Polymorphism of 5'-Flanking Region of TIGR/MYOC with Normal-Tension Glaucoma and Steroid-Induced Glaucoma

PURPOSE: We investigated whether TIGR/MYOC, a candidate gene for the primary open angle glaucoma(POAG) is also involved in the pathogenesis of normal tension glaucoma(NTG) and steroid-induced glau-coma(SIG). METHODS: Genomic DNA was extracted from the peripheral blood samples collected from 72 normal volunteers and 60 POAG, 47 NTG, 61 SIG patients. The genotype distribution of dinucleotide repeat polymorphism, (G-T)n microsatellite located 249 bp upstream of transcription start site was determined by direct sequencing of the Polymerase Chain Reaction(PCR) product. RESULTS: We found 6 alleles in the (G-T)n microsatellite of TIGR/MYOC ranging from 12 to 17, which differ slightly from that of previous reports. There was no obvious difference in the genotype distribution and allele frequency between the POAG group and the control group. However, a significant association of the microsatellite marker with SIG and, to a lesser extent, with NTG was observed. A significant increase in the frequency of (G-T)13/(G-T)13 genotype and a concomitant decrease in the frequency of (G-T)13/(G-T)14 genotype was seen in both the NTG and SIG group compared to that of the control group. In the SIG group, a significant decrease in the frequency of (G-T)14 allele was also observed compared to the control group, although the decrease did not contribute to the increase in the frequency of the allele. CONCLUSIONS: These findings suggest that a polymorphism in the 5 flanking region of the TIGR/MYOC is associated with patients with NTG and SIG.

Analysis of TIGR Gene Mutation in Glaucoma

In the present study, we have evaluated the mutations of the TIGR[Trabecular meshwork Inducible Glucocorticoid Response] gene, which has been reported to be associated with the glaucoma, in primary open angle glaucoma[POAG], normal tension glaucoma[NTG], and steroid-induced glaucoma[SIG]. We have analyzed the TIGR gene in 18 members of 3 families affected with POAG, 28 familially unrelated patients with POAG, 32 patients with NTG, 30 patients with SIG, and 45 normal subjects.DNA was extracted from the blood samples of each patient, exon 2 and exon 3 of the TIGR gene were amplified by PCR and DNA sequencing was performed.No mutation was found in familially unrelated patients with POAG.Two kinds of mutation[Ser341Pro, Gly367Arg]were found in 3 families affected with POAG.Another mutations, located in exon 3, were detected in one NTG patient and in one SIG patient, but they were silent substitution.Identification of TIGR gene mutation will provide early diagnosis of POAG before irreversible visual impairment develops in cases of positive family history of glaucoma.