Gadd153 Mediates 27-OHC-Induced AD Pathology Figure 3. siRNA to gadd153 gene reverses 27-OHC-induced apoptosis. Treatment of slices with 27-OHC while increases levels of the pro- apoptotic Bax and of the active caspase 3, this treatment reduces levels of the anti-apoptotic Bcl-2 (a–d). siRNA to gadd153 reduces the increase in Bax as well as caspase 3 levels and markedly increases levels of Bcl-2 in presence or absence of 27-OHC. *p,0.05 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.g003 glutathione induced by 27-OHC (Fig. 4). Altogether, these results show that silencing the expression of gadd153 protects against the oxidative damage that can be triggered by 27-OHC. 27-OHC altered iron metabolism and increased TNF-a levels, effects reversed by siRNA to gadd153 Dyshomeostasis of iron metabolism has been suggested to mediate the neurodegenerative processes that characterize AD [17–21]. We determined levels of transferrin receptor (TfR) which regulates iron uptake and ferritin light and heavy chains that regulate iron storage. While treatment with 27-OHC reduced levels and expression of TfR, expression levels of ferritin light chain and ferritin heavy chain were significantly increased in slices treated with 27-OHC compared to untreated slices (Fig. 5a–d). Treatment with siRNA to gadd153, alone or in presence of 27- OHC, increased expression levels of TfR. siRNA to gadd153 significantly increased the 27-OHC-induced decrease in expres- sion levels of TfR (Fig. 5a,b). On the other hand, 27-OHC increased expression levels of both ferritin light and heavy chain (Fig. 5a,c,d). Treatment with siRNA to gadd153 didn’t affect basal levels of these proteins but reduced the increase in their expression levels induced by 27-OHC. The decrease in TfR and increase in ferritin levels resulting from 27-OHC treatment was associated with cellular accumulation of iron, as evidenced by the intense staining of phen-green SK, a sensitive fluorescence probe for free iron (Fig. 5e). The 27-OHC-induced iron accumulation was reduced with siRNA to gadd153. We also determined protein levels of the iron-regulatory proteins IRP-1 and IRP-2 that regulate levels of ferritin post-transcriptionally. Levels of IRP-1 and IRP-2 were significantly decreased with 27-OHC treatment compared to controls (Fig. 6a–c). Treatment with siRNA to gadd153 alone didn’t alter levels of these proteins but reversed the decrease induced by 27-OHC. REMSA assay (Fig. 6d) shows a clear shift in bands in control as well as in gadd153 siRNA alone-treated slices indicating the binding of IRP to IRE. In the contrary, no shift in band was observed in the slices treated with 27-OHC alone, demonstrating that 27-OHC inhibits binding of IRP to IRE. A significant band shift was observed in slices treated with both siRNA to gadd153 and 27-OHC showing that siRNA to gadd153 partially, but significantly, restores the binding of IRP to IRE. As activation of TNF-a has been shown to induce the expression of ferritin in a variety of cell lines [22,23] and thus can dysregulate iron homeostasis, we determined levels of TNF-a, which is also a prominent marker of inflammation. Levels of TNF- a in tissue (a,b) and in media (d) were significantly increased with 27-OHC treatment compared to control levels (Fig. 7). Treatment with siRNA to gadd153, while didn’t alter basal levels of TNF-a, it reduced the 27-OHC-induced increase in TNF-a levels. Real-time RT-PCR analysis also showed that siRNA to gadd153 reduced 27- OHC-induced increase in TNF-a expression (Fig. 7c). Discussion In the present study we show that treatment of organotypic slices with the oxysterol 27-OHC causes ER stress as evidenced with increased levels of the ER-specific proteins gadd153, grp 78 and grp 94. Stress in the ER is associated with increased Ab production, phosphorylated tau levels, apoptosis, oxidative stress, and iron dyshomeostasis, which are all pathological hallmarks of PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26420