Comparison of Mouse Models of Autosomal Dominant Retinitis Pigmentosa Due to the P23H Mutation of Rhodopsin.

The need for robust and reliable animal models is a crucial step in studying any disease. This certainly applies to inherited retinal degenerative diseases, in which mutations of retinal specific genes result in photoreceptor cell death and subsequent visual loss. Animal models of retinal gene mutations have proven valuable to our understanding of disease mechanisms and as tools to evaluate therapeutic intervention strategies. Notable among these models are mice with a mutation of the rhodopsin gene at amino acid 23 in which proline is substituted for histidine (Rho-P23H). The RHO-P23H mutation is the most common cause of autosomal dominant retinitis pigmentosa. Here, we provide a brief review of the Rho-P23H mouse models currently available for research.

Evaluation of the role of Sigma 1 receptor and Cullin3 in retinal photoreceptor cells.

Sigma 1 receptor (Sig1R), a pluripotent modulator of cell survival, is neuroprotective in models of retinal degeneration when activated by the high-affinity, high-specificity ligand (+)-pentazocine ((+)-PTZ). The molecular mechanisms of Sig1R-mediated retinal neuroprotection are under investigation. We previously reported that the antioxidant regulatory transcription factor Nrf2 may be involved in Sig1R-mediated retinal photoreceptor cell (PRC) rescue. Cullin 3 (Cul3) is a component of the Nrf2-Keap1 antioxidant pathway and facilitates Nrf2 ubiquitination. Our earlier transcriptome analysis revealed decreased Cul3 in retinas lacking Sig1R. Here, we asked whether Sig1R activation can modulate Cul3 expression in 661 W cone PRCs. Proximity ligation and co-immunoprecipitation (co-IP) showed that Cul3 resides closely to and co-IPs with Sig1R. Activation of Sig1R using (+)-PTZ significantly increased Cul3 at the gene/protein level; silencing Sig1R decreased Cul3 gene/protein levels. Experiments in which Cul3 was silenced in cells exposed to tBHP resulted in increased oxidative stress, which was not attenuated with Sig1R activation by (+)-PTZ, whereas cells transfected with scrambled siRNA (and incubated with tBHP) responded to (+)-PTZ treatment by decreasing levels of oxidative stress. Assessment of mitochondrial respiration and glycolysis revealed significantly improved maximal respiration, spare capacity and glycolytic capacity in oxidatively-stressed cells transfected with scrambled siRNA and treated with (+)-PTZ, but not in (+)-PTZ treated, oxidatively-stressed cells in which Cul3 had been silenced. The data provide the first evidence that Sig1R co-localizes/interacts with Cul3, a key player in the Nrf2-Keap1 antioxidant pathway. The data suggest that the preservation of mitochondrial respiration/glycolytic function and reduction of oxidative stress observed upon activation of Sig1R occur in part in a Cul3-dependent manner.

Sigma 1 receptor activation improves retinal structure and function in the RhoP23H/+ mouse model of autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of devastating inherited retinal diseases that leads to visual impairment and oftentimes complete blindness. Currently no cure exists for RP thus research into prolonging vision is imperative. Sigma 1 receptor (Sig1R) is a promising small molecule target that has neuroprotective benefits in retinas of rapidly-degenerating mouse models. It is not clear whether Sig1R activation can provide similar neuroprotective benefits in more slowly-progressing RP models. Here, we examined Sig1R-mediated effects in the slowly-progressing RhoP23H/+ mouse, a model of autosomal dominant RP. We characterized the retinal degeneration of the RhoP23H/+ mouse over a 10 month period using three in vivo methods: Optomotor Response (OMR), Electroretinogram (ERG), and Spectral Domain-Optical Coherence Tomography (SD-OCT). A slow retinal degeneration was observed in both male and female RhoP23H/+ mice when compared to wild type. The OMR, which reflects visual acuity, showed a gradual decline through 10 months. Interestingly, female mice had more reduction in visual acuity than males. ERG assessment showed a gradual decline in scotopic and photopic responses in RhoP23H/+ mice. To investigate the neuroprotective benefits of Sig1R activation in the RhoP23H/+ mouse model, mutant mice were treated with a high-specificity Sig1R ligand (+)-pentazocine ((+)-PTZ) 3x/week at 0.5 mg/kg and examined using OMR, ERG, SD-OCT. A significant retention of visual function was observed in males and females at 10 months of age, with treated females retaining ∼50% greater visual acuity than non-treated mutant females. ERG revealed significant retention of scotopic and photopic b-wave amplitudes at 6 months in male and female RhoP23H/+ mice treated with (+)-PTZ. Further, in vivo analysis by SD-OCT revealed a significant retention of outer nuclear layer (ONL) thickness in male and female treated RhoP23H/+ mice. Histological studies showed significant retention of IS/OS length (∼50%), ONL thickness, and number of rows of photoreceptor cell nuclei at 6 months in (+)-PTZ-treated mutant mice. Interestingly, electron microscopy revealed preservation of OS discs in (+)-PTZ treated mutant mice compared to non-treated. Taken collectively, the in vivo and in vitro data provide the first evidence that targeting Sig1R can rescue visual function and structure in the RhoP23H/+ mouse. These results are promising and provide a framework for future studies to investigate Sig1R as a potential therapeutic target in retinal degenerative disease.

Effect of long-term chronic hyperhomocysteinemia on retinal structure and function in the cystathionine-ß-synthase mutant mouse.

Elevated levels of the excitatory amino acid homocysteine (Hcy) have been implicated in retinal diseases in humans including glaucoma and macular degeneration. It is not clear whether elevated Hcy levels are pathogenic. Models of hyperhomocysteinemia (Hhcy) have proven useful in addressing this including mice with deficiency in the enzyme cystathionine ß-synthase (CBS). Cbs+/- mice have a ∼two-fold increase in plasma and retinal Hcy levels. Previous studies of visual function and structure in Cbs+/- mice during the first 10 months of life revealed mild ganglion cell loss, but minimal electrophysiological alterations. It is not clear whether extended, chronic exposure to moderate Hhcy elevation will lead to visual function loss and retinal pathology. The present study addressed this by performing comprehensive analyses of retinal function/structure in 20 month Cbs+/- and Cbs+/+ (WT) mice including IOP, SD-OCT, scotopic and photopic ERG, pattern ERG (pERG), and visual acuity. Eyes were harvested for histology and immunohistochemical analysis of Brn3a (ganglion cells), dihydroethidium (oxidative stress) and GFAP (gliosis). The analyses revealed no difference in IOP between groups for age/strain. Visual acuity measured ∼0.36c/d for mice at 20 months in Cbs+/- and WT mice; contrast sensitivity did not differ between groups at either age. Similarly SD-OCT, scotopic/photopic ERG and pERG revealed no differences between 20 month Cbs+/- and WT mice. There was minimal disruption in retinal structure when eyes were examined histologically. Morphometric analysis revealed no significant differences in retinal layers. Immunohistochemistry revealed ∼5 RGCs/100 µm retinal length in both Cbs+/- and WT mice at 20 months. While there was greater oxidative stress and gliosis in older (20 month) mice versus young (4 month) mice, there was no difference in these parameters between the 20 month Cbs+/- and WT mice. We conclude that chronic, moderate Hhcy (at least due to deficiency of Cbs) is not accompanied by retinal structural/functional changes that differ significantly from age-matched WT littermates. Despite considerable evidence that severe Hhcy is toxic to retina, moderate Hhcy appears tolerated by retina suggesting compensatory cellular survival mechanisms.

Sigma 1 Receptor Co-Localizes with NRF2 in Retinal Photoreceptor Cells.

Sigma 1 receptor (Sig1R), a modulator of cell survival, has emerged as a novel target for retinal degenerative disease. Studies have shown that activation of Sig1R, using the high affinity ligand (+)-pentazocine ((+)-PTZ), improves cone function in a severe retinopathy model. The rescue is accompanied by normalization of levels of NRF2, a key transcription factor that regulates the antioxidant response. The interaction of Sig1R with a number of proteins has been investigated; whether it interacts with NRF2, however, is not known. We used co-immunoprecipitation (co-IP), proximity ligation assay (PLA), and electron microscopy (EM) immunodetection methods to investigate this question in the 661W cone photoreceptor cell line. For co-IP experiments, immune complexes were precipitated by protein A/G agarose beads and immunodetected using anti-NRF2 antibody. For PLA, cells were incubated with anti-Sig1R polyclonal and anti-NRF2 monoclonal antibodies, then subsequently with (-)-mouse and (+)-rabbit PLA probes. For EM analysis, immuno-EM gold labeling was performed using nanogold-enhanced labeling with anti-NRF2 and anti-Sig1R antibodies, and data were confirmed using colloidal gold labeling. The co-IP experiment suggested that NRF2 was bound in a complex with Sig1R. The PLA assays detected abundant orange fluorescence in cones, indicating that Sig1R and NRF2 were within 40 nm of each other. EM immunodetection confirmed co-localization of Sig1R with NRF2 in cells and in mouse retinal tissue. This study is the first to report co-localization of Sig1R-NRF2 and supports earlier studies implicating modulation of NRF2 as a mechanism by which Sig1R mediates retinal neuroprotection.

Comparison of Sigma 1 Receptor Ligands SA4503 and PRE084 to (+)-Pentazocine in the rd10 Mouse Model of RP.

Purpose: Sigma 1 receptor is a novel therapeutic target for retinal disease. Its activation, using a high-affinity, high-specificity ligand (+)-pentazocine ((+)-PTZ), rescues photoreceptor cells in the rd10 mouse model of RP. Here, we asked whether the robust retinal neuroprotective properties of (+)-PTZ are generalizable to SA4503 and PRE084, two other high-affinity sigma 1 receptor ligands. Methods: We treated 661W cells with SA4503 or PRE084. Cell viability, oxidative stress, and expression of Nrf2 and NRF2-regulated antioxidant genes (Nqo1, Cat, and Sod1) were assessed. Rd10 mice were administered SA4503 (1 mg/kg), PRE084 (0.5 mg/kg), or (+)-PTZ (0.5 mg/kg). Visual acuity, retinal architecture, and retinal electrophysiologic function were measured in vivo and retinal structure was assessed histologically. Results: Similar to (+)-PTZ, SA4503 and PRE084 improved cell viability, attenuated oxidative stress, and increased Nrf2, Nqo1 and Cat expression. Although treatment of rd10 mice with (+)-PTZ improved visual acuity, increased outer retinal thickness, and improved photopic a- and b-wave responses compared with nontreated rd10 mice, treatment with SA4503 or PRE084 did not. The number of photoreceptor nuclei/100 µm retinal length in SA4503- and PRE084-treated rd10 mice (approximately 11/100) did not differ significantly from nontreated rd10 mice, whereas (+)-PTZ-treated mice had significantly more nuclei (approximately 22/100 µm). Conclusions: Cell survival and gene regulation experiments yielded similar outcomes when SA4503, PRE084, or (+)-PTZ were conducted in vitro, however neither SA4503 or PRE084 afforded in vivo protection in the severe rd10 retinopathy model comparable to (+)-PTZ. Despite all three compounds demonstrating the potential to activate sigma 1 receptor, the retinal neuroprotective properties of the three ligands differ significantly.