Biological Studies of Glaucoma Gene Myocilin
thesisposted on 24.10.2013, 00:00 by Hongyu Ying
Glaucoma is a major blinding disease. Myocilin is the first candidate gene identified for the most common form of glaucoma, primary open angle glaucoma (POAG). The human myocilin gene encodes for an acidic glycoprotein of 504 amino acids. Mutations of myocilin such as Pro370Leu (P370L) and Gln368Stop (Q368X) were found in 2-4% of POAG patients. Its structure, function and regulation are still largely unknown. We conducted new investigations on myocilin in five aspects (parts). Part I is to examine the involvement of Wnt pathway in myocilin mediated phenotypes such as loss of actin stress fibers and focal adhesions, elevated protein kinase A activity and downregulated RhoA activity. Wnt signaling is a key pathway involved in many important cellular processes. We showed that Wnt signaling is a player in the above mentioned myocilin phenotypes using Wnt specific inhibitor and activator. Blocking Wnt signaling pathway may have therapeutic potential for myocilin glaucoma. Part II is to establish 3 dimensional culture of human trabecular meshwork (TM) cells, a cell type that plays an important role in regulation of bulk flow of the aqueous humor. Several 3D platforms such as hydrogels and inserts were tested. Human TM cells were grown in multilayers when plated in QGel and Alvetex scaffold, formed cell-cell communication and showed a more in vivo like pattern of gene expression. The 3D system can be used for an effective in vitro model to help portray activities, characteristics, and stress responses of TM cells in vivo. Part III is to establish Tet-on inducible wild-type or mutated (P370L or Q368X) myocilin expressing neuronal RGC5 cell lines. The Tet-on inducible RGC5 cells provide a new tool for exploring the effects of myocilin up-regulation and mutations at cellular and molecular levels. Part IV is to detect alterations in protein expression profile and identify novel downstream pathways induced by myocilin. Cutting-edge quantitative proteomics were used. RGC5 cell lines established in part III were induced to express wild-type or mutated myocilin-GFP. The global protein profiles of these cells were compared with non-induced controls. Differentially expressed protein profiles pinpointed molecular trafficking, cytoskeleton reorganization, autophagy, microtubule dynamics, organization of filaments, apoptosis, mitochondria dysfunction and several signaling pathways. The information obtained may provide clues as to the functions of myocilin and point to new directions for myocilin investigations. Finally, part V is to identify microRNAs (miRNAs) that regulate the expression of mouse myocilin gene. MiRNAs are short RNAs functioning as posttranslational regulators (mostly in translation silencing). MiRNA array was performed and the up- or down-regulated miRNAs in induced cells were identified. Along with miRNAs predicted by computer algorithms, miRNAs that target mouse myocilin were identified and validated. Since expression of mutated myocilin is likely to be under the same miRNA control as the wild-type myocilin, results from the proposed experiments may be applied to silence myocilin mutants to abrogate mutant phenotypes in myocilin glaucoma.