Purpose: The aim of this study was to explore the roles of crystallins in the context of aging in glaucoma and potential mechanisms of neuroprotection in an experimental animal model of glaucoma.
Methods: Intraocular pressure (IOP) was significantly elevated for 8 weeks in animals at different ages (10 days, 12 weeks, and 44 weeks) by episcleral vein cauterization. Retinal ganglion cells (RGCs) were quantified by anti-Brn3a immunohistochemical staining (IHC). Proteomics using ESI-LTQ Orbitrap XL-MS was used to analyze the presence and abundance of crystallin isoforms the retinal samples, respectively. Neuroprotective property and localization of three selected crystallins CRYAB, CRYBB2, and CRYGB as most significantly changed in retina and retinal layers were determined by IHC. Their expressions and endocytic uptakes into Müller cells were analyzed by IHC and Western blotting. Müller cell secretion of neurotrophic factors into the supernatant following CRYAB, CRYBB2, and CRYGB supplementation in vitro was measured via microarray.
Results: IOP elevation resulted in significant RGC loss in all age groups (P < 0.001). The loss increased with aging. Proteomics analysis revealed in parallel a significant decrease of crystallin abundance - especially CRYAB, CRYBB2, and CRYGB. Significant neuroprotective effects of CRYAB, CRYBB2, and CRYGB after addition to retinal cultures were demonstrated (P < 0.001). Endocytic uptake of CRYAB, CRYBB2, and CRYGB was seen in Müller cells with subsequent increased secretion of various neurotrophic factors into the supernatant, including nerve growth factor, clusterin, and matrix metallopeptidase 9.
Conclusions: An age-dependent decrease in CRYAB, CRYBB2, and CRYGB abundance is found going along with increased RGC loss. Addition of CRYAB, CRYBB2, and CRYGB to culture protected RGCs in vitro. CRYAB, CRYBB2, and CRYGB were uptaken into Müller cells. Secretion of neurotrophic factors was increased as a potential mode of action.