Gadd153 Mediates 27-OHC-Induced AD Pathology Figure 4. siRNA to gadd153 gene reduces 27-OHC-induced oxidative stress. Silencing gadd153 gene expression reduces 27-OHC-induced increase in reactive oxygen species (ROS) generation, isoprostane formation and glutathione depletion. ROS generation was measured with increased fluorescence with the DCFH-DA assay (a), H2O2 levels were measured using Amplex red (b), isoprostane level were detected with formation of 8-iso- PGF2a (c) and glutathione depletion was assessed with reduction in glutathione (GSH): reduced glutathione (GSSG) ratio (d). *p,0.05, **p,0.01 and ***p,0.001 versus controls; #p,0.05, ##p,0.01 and ###p,0.001 versus 27-OHC. doi:10.1371/journal.pone.0026420.g004 AD. Remarkably, silencing gadd153 gene expression substantially reduced the generation of the AD hallmarks. Our data strongly suggest that increased levels of gadd153 plays an important role in the 27-OHC-induced effects. Disturbances in ER functions leads to activation of ER stress response that involves various pathways and proteins (see for review [24]). Gadd153 is a member of the C/EBP family of bZIP transcription factors, and is expressed at low levels in normal conditions and is highly expressed in response to sustained stress in the ER [3,4,25]. Various transcription factors that activate the unfolded protein response activate gadd153 gene transcription. Overexpression of gadd153 has been shown to trigger apoptosis and to contribute to cell death by downregulating the anti- apoptotic protein Bcl-2 [4,26]. Gadd153 deficiency protects against apoptosis in mice [27,28] and cultured macrophages [29–31]. The mechanisms by which gadd153 induces apoptosis are not well known but downregulation of the anti-apoptotic protein Bcl-2 may play an important role [15]. We demonstrate here that silencing gadd153 gene expression reversed the 27- OHC-induced reduction in levels of Bcl-2 and increased levels of the apoptotic proteins Bax and caspase 3. Interestingly, siRNA to gadd153 increases the basal levels of Bcl-2, suggesting that gadd153 regulate the transcription of Bcl-2 gene. Previous studies have also shown that gadd153 sensitizes cells to ER stress by downregulating Bcl-2 [3] and upregulating Bax [4,26]. It may also be possible that 27-OHC-induced accumulation of Ab may trigger apoptosis by mechanisms independent of gadd153 as accumula- tion of Ab is known to increase cell death by increasing Bax and decreasing Bcl-2 levels [32–34]. siRNA to gadd153 may reduce apoptosis indirectly by reducing BACE1 and APP, which are responsible for Ab production. Whether 27-OHC causes apoptosis through increased levels of Ab and/or directly by increasing levels of gadd153 is yet to be elucidated. In addition to promoting apoptosis, gadd153 can increase ROS production [3,16,27,35], leading to oxidative damage. Oxidative damage is considered as an early events in AD [36]. As both ER stress and oxidative damage are tightly linked and increased levels of ROS also triggers ER stress [16], the extent to which ER stress precedes oxidative damage or vice versa is not clear. Increased levels of ROS are known to enhance the activity of BACE1, the enzyme that initiates cleavage of b-APP to yield Ab, thereby causing Ab overproduction [6,7]. Interestingly, we demonstrate PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26420