Shp2 knockdown reduces ocular specific endoplasmic reticulum stress in experimental glaucoma

Abstract

Purpose : Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of multiple retinal degenerative disorders. We have examined whether modulation of the Shp2 gene in the retinal ganglion cells (RGCs) is associated with changes in ER stress and to determine whether knockdown of Shp2 in RGCs rescues glaucoma in a rat model with elevated intraocular pressure. Furthermore, we have determined whether ER stress is identified in human glaucoma retinas and optic nerve (ON) specimens from the Eye Bank.

Methods : Murine Shp2 and Shp2-shRNA sequences were separately incorporated into the AAV2 vector under cytomegalovirus chicken β actin hybrid promoter linked with green fluorescent protein and were injected intravitreally in normal Sprague Dawley rats to overexpress and knockdown Shp2 in rodent RGCs (n=40). Anterior chamber microbead injection was utilised to induce experimental glaucoma and eyes were subjected to Shp2 knockdown, with scramble sequence expressing viral vector used as corresponding control (n=32). ON and eye tissues obtained from rat models were examined for ER stress markers by western blot and immunofluorescence. Apoptosis was detected by TUNEL assay.

Results : ER stress was significantly higher in experimental induced glaucoma eyes compared to the contralateral controls, and in human glaucoma eyes compared to age matched controls (p<0.001). ER stress markers (CHOP and p-PERK) were primarily localised in the RGCs and inner nuclear layer of retina. Animal ON lysates over-expressing Shp2 showed significantly increased Grp78, CHOP and P-PERK: mean±sd%; 78.96±11.04 vs 216.6±10.48; 84.23±15.77 vs 191.8±12.11 and 76.69±13.21 vs 200.7±15.43. Knockdown of Shp2 using AAV lead to a significant decline in the ER stress marker both in retina sections and ON lysates (p<0.001) and a reduction in TUNEL positive cells in the rat glaucomatous eyes.

Conclusions : This study demonstrated that ER stress is elevated in glaucoma conditions. Shp2 overexpression in AAV mediated model had positive ER effects on RGCs. Suppressing Shp2 in rat eyes had beneficial effects, limiting the extent of ER stress in an experimental glaucoma model. The beneficial effects imply that modulation of Shp2 could have application to understand human glaucoma and potentially other neurodegenerative disorders involving ER stress.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.