Intraocular Sustained Release of EPO-R76E Mitigates Glaucoma Pathogenesis by Activating the NRF2/ARE Pathway

Abstract

Erythropoietin (EPO) is neuroprotective in multiple models of neurodegenerative diseases, including glaucoma. EPO-R76E retains the neuroprotective effects of EPO but diminishes the effects on hematocrit. Treatment with EPO-R76E in a glaucoma model increases expression of antioxidant proteins and is neuroprotective. A major pathway that controls the expression of antioxidant proteins is the NRF2/ARE pathway. This pathway is activated endogenously after elevation of intraocular pressure (IOP) and contributes to the slow onset of pathology in glaucoma. In this study, we explored if sustained release of EPO-R76E in the eye would activate the NRF2/ARE pathway and if this pathway was key to its neuroprotective activity. Treatment with PLGA.EPO-E76E prevented increases in retinal superoxide levels in vivo, and caused phosphorylation of NRF2 and upregulation of antioxidants. Further, EPO-R76E activates NRF2 via phosphorylation by the MAPK pathway rather than the PI3K/Akt pathway, used by the endogenous antioxidant response to elevated IOP.

Keywords: NRF2; antioxidant; erythropoietin; glaucoma; neurodegeneration; neuroprotection; oxidative stress; retinal ganglion cell.

Figure 1

(A) IOP levels over time in mice intravitreally injected with PBS, PLGA or PLGA.EPO-R76E. (B) Retina EPO levels in PLGA or PLGA.EPO-R76E injected mice at 1, 2, 3, 4, and 6 weeks post-intravitreal injection. (C) Quantification of PhNR amplitude of the ERG at 2 and 5 weeks post-IOP elevation for all groups, ** p = 0.01, *** p < 0.001. (D) Quantification of b_max amplitude of the ERG at 2 and 5 weeks post-IOP elevation for all groups, * p < 0.05. (E) Quantification of N1 amplitude of the VEP at 2 and 5 weeks post-IOP elevation for all groups, * p < 0.05, ** p = 0.01, **** p < 0.0001. For all electrophysiological data, comparisons were made between all 2 week groups separately from all 5 week groups; unless otherwise indicated, asterisks indicate differences between PLGA.EPO-R76E group and both PBS and PLGA groups. (F) Representative micrographs of optic nerves at 2 and 5 weeks post-IOP elevation for all groups. Line represents 20 μm. (G,H) Quantification of total and degenerative axons, respectively, in optic nerves at 2 and 5 weeks post-IOP elevation for all groups, * p < 0.05, ** p = 0.01. For all optic nerve data, comparisons were made between all 2 week groups separately from all 5 week groups; unless otherwise indicated, asterisks indicate differences between PLGA.EPO-R76E group and both PBS and PLGA groups.

Figure 2

(A) Representative fundus images of DHE fluorescence at 1 and 2 weeks post-IOP elevation in empty PLGA controls and PLGA.EPO-R76E injected mice. (B) Quantification of DHE fluorescence at 1–4 weeks post-IOP elevation, *** p < 0.001, **** p < 0.0001. (C–F) Quantification of peroxiredoxin-related genes, thioredoxin-related genes, glutathione-related genes and oxygen-related genes shown as fold change over PLGA at 1 and 2 weeks post-IOP elevtaion, respectively. (G,H) Quantification of antioxidant gene transcription shown as fold change over PLGA at 1 and 2 weeks post-IOP elevation, respectively.

Figure 3

(A) Representative Western blot for pNRF2 and NRF2 in PLGA and PLGA.EPO-R76E injected mice at 2 weeks post-IOP elevation. (B) Quantification of pNRF2 levels normalized to total NRF2 levels at 1 week post-IOP elevation, * p < 0.05. (C) Representative fundus images of mice injected with AAV2/2.ARE 2 weeks prior to IOP elevation, imaged at both 1 and 2 weeks post-IOP elevation in mice treated with PLGA or PLGA.EPO-R76E. (D) Quantification of tdTomato fluorescence in mice injected with AAV2/2.ARE at 1 and 2 weeks post-IOP elevation, ** p = 0.01.

Figure 4

(A) Representative Western blot and quantification for pPI3K levels normalized to total PI3K levels. (B) Representative Western blot and quantification for pAkt levels normalized to total Akt levels. (C) Representative Western blot and quantification for pGSK3β levels normalized to total GSK3β levels. (D) Representative Western blot and quantification for pMAPK levels normalized to total MAPK levels, * p < 0.05.

Figure 5

(A) IOP levels over time in mice intravitreally injected with PLGA or PLGA.EPO-R76E and then 1 week later, injected with either vehicle or BIRB 796. (B) Quantification of PhNR amplitude of the ERG at 2 post-IOP elevation for all groups, ** p = 0.01, *** p < 0.001. (C,D) Quantification of antioxidant gene transcription shown as fold change over actin at 2 weeks post-IOP elevation in mice injected with vehicle or BIRB 796, respectively. (E) Representative Western blots for pNRF2, NRF2, PRDX6, and β-actin. pNRF2 was normalized to total NRF2 and PRDX6 was normalized to β-actin. (F) Quantification of pNRF2 and PRDX6 appropriately normalized as stated in (E), * p < 0.05, ** p = 0.01. (G) Representative micrographs of optic nerves at 2 and 5 weeks post-IOP elevation for all groups. (H,I) Quantification of total and degenerative axons, respectively, in optic nerves at 2 weeks post-IOP elevation for all groups, * p < 0.05, ** p = 0.01.