Development and Characterization of Normal Tension Glaucoma Mouse Over Expressing Mutant of OPTN (E50K)

Abstract

Abstract: : Purpose: Recently optineurin (OPTN) was reported as responsible for primary open–angle glaucoma and normal tension glaucoma. This protein was previously characterized to interact with several proteins including small GTP–binding protein RAB8. We had previously reported (ARVO 2003) that one of the mutations in OPTN (E50K) had significant effect to alter the binding against purified recombinant RAB8 protein. To characterize mutant OPTN (E50K) in vivo, transgenic mouse over expressing this mutant was developed and characterized. Methods: Mouse OPTN was amplified by RT–PCR from C57BL/6 brain total RNA and cloned into XX vector to introduce E50K mutation and deletion of exon 5 by mutagenesis kit (Quick Change Multi Site–Directed Mutagenesis Kit, Stratagene). The mutated OPTN cDNA was removed and inserted into transgenic construction vector (pBROAD2, Invivogen). Transgenic mouse over expressing wild type OPTN, mutOPTN(E50K), and delOPTN (exon5) were developed by YS Institute Inc. All transgenic mice were examined by PCR method using genomic DNA extracted from mouse tail. Pathological examination using slit lamp with digital camera (SR–D7, Topcon) and histological characterization by immunohistochemistry and HE staining were performed. Results: Minimal difference was observed between transgenic mouse over expressing wild type OPTN and non–transgenic mouse. Transgenic mouse over expressing delOPTN(exon5) resulted as lethal expression. No apparent histological change in the cell layer of peripheral retina was observed in mutOPTN(E50K) transgenic mice. However, significant optic nerve cupping and developmental retinal ganglion cell death was observed. Conclusions: Transgenic mouse over expressing wild type, E50K, Exon5 deleted OPTN was developed and characterized. Significant effect in optic nerve head of E50K transgenic mouse was observed. Developmental abnormality and OPTN–RAB8 interaction is under investigation.

Keywords: transgenics/knock-outs • optic disc • protein structure/function