Nanoceria with Grp78/Bip produce enhanced inhibition of retinal degeneration in tubby mice

Abstract

Purpose: Previously we demonstrated cerium oxide nanoparticles (nanoceria) catalytically scavenge Reactive Oxygen Species (ROS) and inhibit tubby retinal degeneration. We also showed that ER stress is involved in photoreceptor death in tubby mice. Here we test the hypothesis that nanoceria in combination with an ER stress regulator, Grp78/Bip, can synergistically or additively prevent inherited retinal degeneration in tubby mice.

Methods: Tubby pups at P7 were subretinally injected with 1µl of 1mM nanoceria and 8x10⁹ vector genome (vg) of AAV5-hGrp78/Bip viral particles. Wild type, uninjected tubby, AAV5-GFP injected, nanoceria injected or AAV5-hGrp78/Bip injected alone served as controls. At P37 the expression and localization of hGrp78/Bip protein was analyzed by immunocytochemistry and western blots. The expression of GFP was observed by fundoscopy whereas the expression of other ER stress markers and rod and cone opsin genes was evaluated by RT-PCR and western blots. Retinal structure and function were assessed by histology and ERG.

Results: Subretinal delivery of AAV5-GFP and AAV5-hGrp78/Bip under the direction of CBA promoter resulted in the exogenous transgene expressed in the retina. Immunocytochemistry demonstrated that transferred hGrp78/Bip was localized in the photoreceptors. Compared to GFP injected mice, the combination treatment with nanoceria and AAV5-hGrp78/Bip leads to effects which are additive and more than the individual treatment with either nanoceria or hGrp78/Bip alone. This included an increased expression of rhodopsin and M-opsin, increased Grp78/Bip and decreased CHOP expression, altered activation of PERK, IRE1 and ATF6, and improvement of retinal structure. Experiments to assess retinal function and to identify the underlying mechanism of the additive response are ongoing.

Conclusions: Despite nanoceria and Grp78/Bip having antioxidant roles and having different cellular mechanisms of action, their additive response indicated that they regulate the same downstream effects. It is possible that these reagents or the proteins they activate have access to distinct pools of ROS. These data suggest that combinations of nanoceria with multiple therapeutic agents will be more beneficial than any single agent.

Keywords: 608 nanomedicine • 538 gene transfer/gene therapy • 615 neuroprotection