Gadd153 Mediates 27-OHC-Induced AD Pathology Figure 1. siRNA to gadd153 reduces 27-OHC-induced alteration in ER protein levels in organotypic slices from rabbit hippocampus. 27-OHC significantly increased protein levels (a,b) as well as mRNA (c) of gadd153 and treatment with siRNA to gadd153 markedly reduced the 27- OHC-induced increase in expression levels of gadd153. Treatment with siRNA also reduced the increase in levels of the ER chaperones grp 78 and and grp 94 induced by 27-OHC (d–f). *p,0.05, **p,0.01 and ***p,0.001 versus controls; #p,0.05 and ###p,0.001 versus 27-OHC. doi:10.1371/journal.pone.0026420.g001 gadd153 markedly reduced 27-OHC-induced increase in APP and BACE1 to control levels. These results suggest that gadd153 regulates Ab production by primarily controlling BACE1 levels. siRNA to gadd153 reversed 27-OHC-induced phosphorylation of tau The phosphorylation of tau protein was determined with PHF-1 and CP13, antibodies that detects tau phosphorylated at Ser396/ 404 and Ser202 respectively. Our results show that 27-OHC- induced about a two-fold increase in phosphoylated tau as detected by both PHF-1 and CP13 antibodies (Fig. 2 f–h). siRNA to gadd153 did not affect basal levels of phosphorylated tau but significantly reduced the increase in tau phosphorylation induced by 27-OHC. These results suggest that siRNA to gadd153 reduces tau phosphorylation in conditions where there is an abnormal increase in the phosphorylation of tau but not in the basal state. siRNA to gadd153 reduces 27-OHC-induced alteration in apoptosis-regulatory proteins A growing body of evidence suggests an active role for the ER in the regulation of apoptosis by mechanisms that involve regulation of apoptosis-regulatory protein levels [15]. We determined the effect of siRNA to gadd153 on levels of Bax, caspase 3 and Bcl-2. Our data shows that 27-OHC significantly increased level of the pro-apoptotic proteins Bax and the effector of apoptosis caspase 3 (Fig. 3). While siRNA to gadd153 didn’t affect basal levels of Bax and caspase 3, it significantly reduced the increase in levels of these proteins induced by 27-OHC. On the other hand, levels of the anti-apoptotic protein Bcl-2 were markedly reduced by 27-OHC treatment and incubation of slices with siRNA to gadd153 in presence or absence of 27-OHC markedly increased levels of Bcl-2 beyond control levels. These latter results suggest that gadd153 may regulate the basal expression levels of Bcl-2 (Fig. 3). siRNA to gadd153 protects from 27-OHC-induced oxidative stress ER stress is known to induce oxidative stress and oxidative stress can also cause ER stress [16]. We determined the extent to which 27-OHC-induced ER stress is associated with ROS generation, isoprostane formation and glutathione depletion, which are all markers for oxidative stress. Our results show that the 27-OHC increased levels of ROS, H2O2 and 8-iso-PGF2a, and reduced levels of glutathione in rabbit hippocampus organotypic slices (Fig. 4). While treatment with siRNA to gadd153 alone didn’t alter PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26420