Elevated intraocular pressure impairs distribution of RNA-binding protein HuR in glaucomatous rat retina

INTRODUCTION: Glaucoma is a serious social problem as it may result in blindness. Most often, it is related to increased intraocular pressure, but the exact biologic mechanism is not known yet. RNA-binding proteins may be one of the pathogenetic factors for this disease. AIM(S): To evaluate impact of increased intraocular pressure (IOP) on HuR protein expression in retina and optic nerve in rat glaucoma model. METHOD(S): IOP was increased unilaterally using modified rat bead model. Fellow eye was used as a healthy control. Animals were sacrificed 1 day, 1‑, 4‑, 6‑ or 8‑weeks after beads injection. Retinas and optic nerves were collected and processed for Western blot (WB) analysis, mass spectrometry (MS), PCR and immunostainings. RESULTS: The loss of retinal ganglion cells (RGCs) was at the level of 36% after 8‑weeks of IOP elevation. The presence of HuR protein and its transcipt was confirmed in retinas and optic nerves using WB, MS and PCR analysis. Additionally, Gene Ontology enrichment analysis revealed that the most significant alterations in glaucoma retinas were linked to the molecular function of binding proteins. In fractionated WB of retinal homogenates, the level of cytoplasmic fraction of HuR was decreased approximately 3-times when compared with healthy tissue (p<0.05). This decrease was accompanied by alteration of the cytoplasmic level of HuR-regulated proteins, i.e. Hsp70 decrease in retina and p53 decrease in optic nerve. Stereological analysis of retinas revealed that some RGCs have lost visible HuR expression. Immunostaining of retinal and optic nerves cross sections showed decreased staining for HuR within RGCs and increased within optic nerve glia, with nuclear polarisation of HuR expression in glaucoma samples. CONCLUSIONS: Increased intraocular pressure results in alteration of RNA-binding protein HuR within retina and, subsequently, decreased expression of HuR-dependent stress-response regulatory proteins. This alterations might contribute to the development of glaucomatous degeneration.