As a result, approximately 42% RGCs are lost at 8 weeks check details after microbead injection in WT mice (Figures 4B and 4C). In these animals, at 7 days after microbead

injection, there was a marked increase in CHOP expression in RGCs assessed by immunostaining (Figure S4A). However, we failed to detect the spliced form of XBP-1 at all the time points studied (3, 5, and 7 days after microbead injection) (data not shown), suggesting that similar to optic nerve injury, IOP elevation triggers differential activation of different UPR pathways in RGCs. Importantly, both CHOP KO and XBP-1s overexpression significantly reduced RGC death. The combination Z-VAD-FMK purchase of CHOP KO and XBP-1s overexpression showed a trend of further protection, but the extent of the protection did not reach the level of statistical significance as compared to CHOP KO or XBP-1s overexpression alone ( Figures 4B and 4C). These protective effects are not due to the alteration of the IOP levels, because microbead injection induced similar degrees of IOP elevation in all experimental groups ( Figure S4B). Because brain-derived neurotrophic factor (BDNF) has been shown to be protective for RGCs ( Cohen-Cory and Fraser, 1994 and Mansour-Robaey et al., 1994), we simultaneously applied BDNF and XBP-1s to the eyes of animals that received

an optic nerve crush injury ( Figure S4C) or were subjected to IOP elevation ( Figure S4D). Although BDNF alone protected RGCs to some extent, it did not lead to a significant further enhancement of RGC survival in any of these models when it was combined with XBP-1s overexpression. The mechanistic interactions between UPR and neurotrophin pathways remain to be further elucidated. To mimic a clinically relevant scenario, we also examined whether a

delayed expression of XBP-1s can be protective for RGCs in the IOP-elevated model. We thus increased IOP by microbead injection followed by introduction of AAV-XBP-1s 1 or 7 days later. Because AAV-mediated gene expression in RGCs is normally peaked at 2 weeks after infection Mephenoxalone (Martin et al., 2002 and Park et al., 2008), XBP-1s expression in RGCs is likely to occur 2–3 weeks after IOP elevation. Interestingly, such delayed AAV-XBP-1s expression still showed significant protective effects on RGCs (Figures 4D and 4E), suggesting that forced XBP-1s expression might be a promising therapeutic approach for RGC degeneration in glaucoma. A predominant hypothesis holds that ER stress activates all UPR pathways, thereby simultaneously producing antagonistic outputs that can be both protective and harmful to cells; only unresolved ER stress results in cell death (Ron and Walter, 2007).