Abstract
Purpose: :
Autophagy is a physiological mechanism enabling cells to digest their own cytosol, remove toxic protein aggregates, and eliminate defective or surplus organelles. This cytoplasmic homeostasis pathway allows cells to survive nutrient depletion or the absence of growth factors; however, under specific conditions, may also be linked to cell death. This study aimed to explore autophagy in an experimental rat model of glaucoma.
Methods: :
IOP elevation was induced in rats by hypertonic saline injections into episcleral veins. Enriched samples of RGCs and astrocytes were isolated through the two-step immunomagnetic cell selection process. Ocular hypertensive and control samples were collected by pooling from rat eyes matched for the cumulative IOP exposure. Protein expression was complementarily analyzed by quantitative LC-MS/MS followed by quantitative Western blot analysis and immunohistochemical analysis using specific antibodies.
Results: :
Quantitative LC-MS/MS analysis of cell-specific samples identified thousands of proteins with high confidence, which exhibited up-regulated or down-regulated expression in ocular hypertensive samples. Significantly up-regulated proteins in ocular hypertensive samples relative to normotensive controls (Mann-Whitney Rank Sum test; p<0.05) included various molecules involved in the autophagy signaling, such as immunity-related GTPase (IRG, a mediator of autophagy), mammalian target of rapamycin (mTOR, an upstream negative regulator of autophagy signaling), and autophagy-related proteins (Atgs, involved in the execution stages of autophagy). The MS/MS data and the findings of Western blot analysis and tissue immunolabeling collectively supported up-regulation of IRG and various Atgs in both ocular hypertensive RGCs and astrocytes. However, based on immunolabeling with a phosphorylation site-specific antibody, phosphorylation-mediated activation of mTOR was detectable only in ocular hypertensive astrocytes that predominantly exhibited co-activation of NF-ΚB. Despite increased expression of IRG and Atgs, no increase was detectable in phospho-mTOR expression in ocular hypertensive samples of RGCs.
Conclusions: :
Our findings highlighting autophagy signaling in experimental glaucoma for the first time motivate further research to clarify the importance and regulation of autophagy-related pathways in glaucoma. Whether NF-ΚB-dependent activation of mTOR controls the balance between activator and inhibitor pathways of autophagy in astrocytes, or whether autophagy (broadly associated with innate and adaptive immunity) may contribute to antigen presentation and/or autoantibody generation in glaucoma, seem to be particularly interesting to further pursue.
Keywords: astrocyte • ganglion cells • neuroprotection