Human Pro370Leu Mutant Myocilin Induces the Phenotype of Open-Angle Glaucoma in Transgenic Mice

To investigate the characteristics of mutation myocilin proteins and glaucoma pathological phenotype in transgenic mice with full-length human Pro370Leu mutant myocilin gene (Tg-MYOC^P370L). Tg-MYOC^P370L mice were established using the CRISPR/Cas9 system. Long-term intraocular pressure (IOP) was measured, myocilin protein expressions in anterior chamber angle, retina, optic nerve tissues and aqueous humor were detected by western blot. RBPMS, myocilin, Iba-1 and GFAP expression were visualized by immunofluorescence. H&E staining was applied to assess the ocular angle and retinal morphology. Aqueous humor dynamics were visualized by Gadolinium magnetic resonance imaging (Gd-MRI). TUNEL assay was used to evaluate the specific cell apoptosis in trabecular meshwork and retina. Optomotor and electroretinography tests were employed to evaluate the visual function in Tg-MYOC^P370L and wild-type (WT) mice. Homozygous myocilin mutation at position 503 (C > T) was identified by PCR and sequencing in Tg-MYOC^P370L mice. Myocilin protein expression was overexpressed in eye tissues of Tg-MYOC^P370L mice with reduced myocilin secretion in aqueous humor. H&E staining showed normal histological morphology of anterior chamber angle whereas decreased thickness and nuclei in ganglion cell layer were found (P < 0.05). Gd signals were significantly increased in the anterior chamber of Tg-MYOC^P370L compared with WT eyes (P < 0.05). IOP was elevated in Tg-MYOC^P370L mice starting at 5 months of age, with significant RGC loss (P < 0.05). Upregulation of caspase-3 and caspase-9 expressions and increased TUNEL-positive cells were found in eyes of Tg-MYOC^P370L mice. Excessive activation of retinal glial cells and impaired visual function were detected in Tg-MYOC^P370L mice. Tg-MYOC^P370L mice can induce the phenotype of open-angle glaucoma, featured as IOP elevation, activated retinal glial cells, loss of RGCs and impaired visual function. These pathologic changes may arise from the abnormal mutant myocilin protein accumulation in the trabecular meshwork and injured aqueous humor drainage. Therefore, Tg-MYOC^P370L mice model can serve as an effective animal model for glaucoma research, especially for glaucoma-associated myocilin mutation studies.