Hyperhomocysteinemia (Hhcy), or increased levels of the excitatory amino acid homocysteine (Hcy), is implicated in glaucoma, a disease characterized by increased oxidative stress and loss of retinal ganglion cells (RGCs). Whether Hhcy is causative or merely a biomarker for RGC loss in glaucoma is unknown. Here we analyzed the role of NRF2, a master regulator of the antioxidant response, in Hhcy-induced RGC death in vivo and in vitro . By crossing Nrf2 −/− mice and two mouse models of chronic Hhcy ( Cbs +/- and Mthfr +/- mice), we generated Cbs +/- Nrf2 −/− and Mthfr +/- Nrf2 −/− mice and performed systematic analysis of retinal architecture and visual acuity followed by assessment of retinal morphometry and gliosis. We observed significant reduction of inner retinal layer thickness and reduced visual acuity in Hhcy mice lacking NRF2. These functional deficits were accompanied by fewer RGCs and increased gliosis. Given the key role of Müller glial cells in maintaining RGCs, we established an ex-vivo indirect co-culture system using primary RGCs and Müller cells. Hhcy-exposure decreased RGC viability, which was abrogated when cells were indirectly cultured with wildtype (WT) Müller cells, but not with Nrf2 −/− Müller cells. Exposure of WT Müller cells to Hhcy yielded a robust mitochondrial and glycolytic response, which was not observed in Nrf2 −/− Müller cells. Taken together, the in vivo and in vitro data suggest that deleterious effects of Hhcy on RGCs are likely dependent upon the health of retinal glial cells and the availability of an intact retinal antioxidant response mechanism.

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.

SUMMARYSpecific polyclonal antisera to human interferon-~l (HulFN-~I), human interferon-~2 (HulFN-~2) and human lymphoblastoid interferon (HulFN-ctLy Namalwa) have been raised in rabbits and sheep. The antisera raised against HulFN-~I and HulFN-~2 strongly neutralized the antiviral activity of their homologous IFN-~ subtypes, but were less active against the heterologous IFN-~ subtypes and preparations containing mixtures of IFN-~ subtypes, e.g. human leukocyte interferon (HulFN-aLe). Antisera raised against HulFN-aLy Namalwa strongly neutralized the antiviral activity of all IFN-~-containing preparations and showed weak cross-reactivity with human interferon-fl (HulFN-fl). Neither anti-HulFN-~l nor anti-HulFN-~2 could be demonstrated to neutralize the antiviral activity of HulFN-fl. A number of monoclonal antibodies to HulFN-a2 have been prepared and these were found to neutralize HulFN-~2 antiviral activity to varying degrees, but not to neutralize the heterologous subtype HulFN-~ 1, preparations containing mixtures of IFN-~ subtypes or HulFN-fl.