Abstract
Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1β, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1β.
Highlights
The UPR, known as the endoplasmic reticulum (ER) stress response, is a series of evolutionarily conserved signaling pathways aimed at restoring homeostasis under conditions of ER stress.[2]
We have demonstrated that the progression of autosomal dominant retinitis pigmentosa (ADRP) is associated with an upregulation of UPR markers, and that ER dysregulation and the onset or progression of retinal degeneration are linked.[8]
The impact of UPR activation in photoreceptors was monitored by photoreceptor-derived a-wave amplitudes of the scotopic ERG, SD-OCT-assessed averaged thickness of the outer nuclear layer (ONL) and by performing histological analysis to count the number of photoreceptor nuclei rows
Summary
A persistently activated UPR promotes loss of photoreceptor function and retinal structure. We analyzed the physiological response of retinas to Tn injection (0.01 μg per eye) by ERG and found a loss of photoreceptor function at 10 and 30 days after treatment (Figure 1b and Supplementary Table S1). ERG analysis of mice injected with 0.001 μg of Tn demonstrated no alterations in the scotopic ERG a- and b-wave responses at 10 or 30 days post treatment, suggesting that the mild or transient ER stress did not induce retinal degeneration in the wild-type retina. Histological analysis of retinal sections confirmed our OCT findings and revealed that Tn-injected retinas lost ~ 36% of their photoreceptors at 30 days post treatment (Figure 1d). Our results demonstrated that Tn-induced UPR activation in photoreceptors promotes progressive retinal degeneration culminating in photoreceptor cell death within the context of the wild-type retina. Photoreceptors have been reported to express cytokines Cx3cl[1], Mcp-1, Rantes, Il-1β and Tnf-α in response to Relative TNF-
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