Figure 7. siRNA to gadd153 reduces 27-OHC-induced increase in TNF-a levels. Western blot (a), densitometric analysis (b), and real- time RT-PCR (c) showing an increase in TNF-a expression levels with 27- OHC and a significant decrease in expression levels of TNF-a in slices treated with siRNA to gadd153. Levels of TNF-a released in media are also increased with 27-OHC treatment and these levels are reduced by siRNA to gadd153 (d). Values expressed are mean value 6 SEM from three different experiments. *p,0.05 and **p,0.01 versus controls; #p,0.05 and ##p,0.01 versus 27-OHC. doi:10.1371/journal.pone.0026420.g007 protein and regulates the quantity of iron in the cell [55]. Transferrin receptor (TfR) is a transmembrane protein that transports iron into cells and its density is regulated by intracellular iron levels [56–58]. Transferrin (Tf), with the support of TfR, mobilizes iron [59,60]. IRPs can sense iron levels in the cell and Gadd153 Mediates 27-OHC-Induced AD Pathology regulate the expression of proteins that are involved in iron metabolism [61–66]. Disruption of iron metabolism has been suggested to contribute to the pathogenesis of AD and other neurodegenerative diseases [67]. The iron accumulation we show with 27-OHC was associated with an increase in ferritin and a decrease in TfR as well as IRP-2 levels. An increase in level of ferritin was observed in AD compared to normal [68], varying depending on the brain regions [18]. Low levels of TfR have been observed in specific regions in the brain of AD in a study by Kalaria and colleagues [56]. The increase in the levels of iron with 27-OHC may have resulted from decreased expression of TfR, both at protein and mRNA levels, as TfR expression is strictly regulated by the intracellular iron levels and increased iron levels can lead to degradation of TfR mRNA [69]. Accumulation of ferritin and dysregulation of iron metabolism were observed in IRP-2 mutated mice [70] and alterations in the IRP-2 localization were reported in AD [71]. IRP-IRE binding regulates the iron levels in the cells. The high levels of iron in slices treated with 27- OHC is confirmed by REMSA assay that shows no band shift. A band shift would normally indicate that IRP did not bind to IRE probe. If the cells are overloaded with iron IRPs does not bind to IRE and the synthesis of ferritin and degradation of TfR increase. The increase in the levels of iron induced by 27-OHC can cause or exacerbate oxidative damage [72,73] and ER stress [74]. Remarkably, siRNA to gadd153 substantially reduced disturbanc- es in iron dyshomeostasis induced by 27-OHC. Activation of TNF-a may also potentially dysregulate iron homeostasis. Alteration in ferritin expression may result from TNF-a activation and may lead to the release of iron from ferritin stores, thereby increasing free iron levels. The increased levels of TNF-a with 27- OHC may also be a cause for the alterations in iron metabolism and neuronal death, as TNF-a can cause neuronal damage [75,76]. Inflammation and TNF are known to contribute to the progression of AD. Interestingly, silencing gadd153 in the slices with siRNA has reduced the increased levels of TNF-a by 27- OHC, thus potentially reducing inflammation, oxidative stress and iron dyshomeostasis. In summary, we demonstrate that the oxysterol 27-OHC induces AD-like pathology in organotypic slices from rabbit hippocampus. Therapeutic strategies for management of AD and other neurolog- ical disorders characterized by disturbed cholesterol and oxysterol metabolism have been suggested [77–79]. We also show that ER stress is an important event induced by the oxysterol 27-OHC. Interestingly, inhibition of downstream events to ER stress by silencing gene expression of the growth arrest and DNA-damage- inducible protein gadd153 markedly protects against 27-OHC- induced AD-like pathology. Several studies showing increased levels of 27-OHC in AD brains suggest that increased turn-over of cholesterol to 27-OHC may contribute to AD pathology. Our study is of seminal relevance to AD studies in that it adds new information on the role of gadd153 in underlying Ab production, phosphory- lated tau accumulation, oxidative stress generation and iron dyshomeostasis, which all are pathological hallmarks of AD. Preventing ER stress and silencing gadd153 expression may represent a strategy to prevent or reduce AD pathology. Supporting Information Figure S1 Western blot analyses showing that each of the three siRNA to gadd153 individually reduces gadd153 as well as BACE1 levels, thus confirming the specificity of the silencing of gadd153. *p,0.05, **p,0.01 and ***p,0.001 versus controls. ###p,0.001 versus 27-OHC. (TIF) PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e26420