An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina.

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-1ß (IL-1ß), 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-1ß, 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-1ß was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1ß. 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-1ß.

Knockdown of wild-type mouse rhodopsin using an AAV vectored ribozyme as part of an RNA replacement approach.

PURPOSE: To develop a hammerhead ribozymes (Rz) that might be exploited in a "digest and replace" gene therapy strategy for autosomal dominant retinitis pigmentosa (ADRP) caused by mutations in the gene for rhodopsin (RHO). METHODS: A ribozyme (Rz397) was designed to hybridize with an accessible region in rhodopsin mRNA. It was tested in vitro to determine the kinetics of cleavage of a target oligonucleotide. Following transfection of cultured cells, reduction of rhodopsin mRNA in response to Rz397 was measured RT-PCR. The gene for the ribozyme (Rz397) was inserted in an adeno-associated virus (AAV2) vector and packaged in AAV2 capsids. The virus was injected subretinally in the eyes of C57BL/6J (RHO+/+) and rhodopsin knockout hemizygous (RHO+/-) mice at postnatal days 6 (P6) and 30 (P30). Mice were analyzed by full-field electroretinography (ERG). The reduction of opsin protein was measured by western blot analysis and visualized by immunocytochemistry. Reduction of rhodopsin mRNA was assessed using in situ hybridization. Morphometric microscopy of fluorescent antibody-antigen complexes and autoradiography of retinas were used to quantify levels of rhodopsin protein and mRNA, respectively. RESULTS: Transient co-transfection of HEK 293 cells with a wild-type rhodopsin cDNA and Rz397 resulted in an approximately 60% reduction of RHO mRNA one day after transfection. RHO+/- -mice injected with AAV2-Rz397 at P6 showed a 50% reduction in b-wave amplitudes in injected eyes relative to saline injected contralateral eyes. However, injection of RHO+/- -animals at one month and of RHO+/+-animals at either age had no impact on ERG. Nevertheless, we detected an 80% reduction of opsin protein in ribozyme-injected eyes of hemizygous mice (by western blot) and a 50% reduction in opsin content in RHO+/+ mice (by morphometry). These reductions were confirmed by in situ hybridization. CONCLUSIONS: AAV2-Rz397 led to significant (greater than or equal to 50%) reduction of rhodopsin mRNA and protein in mice. It affected ERG amplitudes only when injected in hemizygous RHO knockout pups. This RNA inhibitor may prove useful in treating animal models of ADRP as part of an RNA replacement approach.