Gadd153 Mediates 27-OHC-Induced AD Pathology Figure 6. Reduction in iron-regulatory protein levels by 27-OHC is prevented by siRNA to gadd153. While treatment with 27-OHC reduces levels of IRP-1 and IRP-2, siRNA to gadd153 reverses the effects of 27-OHC on IRP-1 and IRP-2 levels (a–c). REMSA assay shows a clear shift in bands resulting from binding of IRP to IRE in control (line 4) as well as in gadd153 siRNA alone- treated slices (line 6). No shift in band was observed in the slices treated with 27-OHC alone (line 5) demonstrating that 27-OHC inhibits binding of IRP to IRE. Treatment with siRNA to gadd153 in presence of 27-OHC causes a band shift indicating that siRNA to gadd153 partially, but significantly, restores the binding of IRP to IRE (line 7). Lanes 1–3 – positive controls; lane 1 - biotin labeled IRE probe, lane 2 - biotin labeled IRE probe+cytosolic liver extract, lane 3 - biotin labeled IRE probe+cytosolic liver extract+200 fold molar excess of unlabeled IRE RNA. *p,0.05 versus controls; #p,0.05 versus 27-OHC. doi:10.1371/journal.pone.0026420.g006 that siRNA to gadd153 markedly reduces basal levels of BACE1 enzyme. These results suggest that gadd153 regulates transcription activity of BACE1. Further studies are needed to determine the cellular mechanisms by which gadd153 regulate BACE1 expres- sion. There is evidence that BACE1 expression is transcriptionally regulated by NF-kB [37] and that gadd153 activated by ER stress regulates NF-kB signaling [38]. On the other hand, the ER has been shown to regulate Ab production by controlling the protein folding for APP and delaying the access for BACE1 to process APP [39]. Alterations in the ER function subsequently to stress would lead to alterations in the processing and regulation of Ab levels. Further studies are needed to determine the cellular mechanisms by which gadd153 regulate BACE1 expression in our system. The increase in the levels of isoprostanes and depletion in the glutathione system foster oxidative stress. Decreased levels of glutathione (GSH) are a marker for increased free radical levels in AD [40]. Free radicals increase production of APP, Ab as well as ROS [41,42]. Our results show that 27-OHC-induced increase in ROS, isoprostanes and glutathione depletion are greatly reduced by siRNA to gadd153. Of seminal relevance, siRNA to gadd153 alone increases levels of glutathione to levels higher than basal levels suggesting, as is the case for Bcl-2 and BACE1, that gadd153 regulates glutathione activity. It has been suggested that gadd153 may interfere with glutathione synthesis by regulating c-glutamyl cysteine synthase enzyme [3]. Effects of gadd153 on c-glutamy cysteine synthase enzyme and glutathione synthesis are still to be investigated. ER stress is associated with a number of diseases including neurodegenerative diseases, obesity, and atherosclerosis, [43–46]. Increased levels of gadd153 have been observed in PS-1 transgenic mice for AD [47]. In addition to increasing levels of gadd153, 27- OHC increases levels of ER molecular chaperone grp 78 and grp 94. Grp 78 has been shown to bind to b-APP in the ER and to reduce Ab secretion in human embryonic kidney 293 cells [48]. Grp 78 has also been demonstrated to stimulate Ab42 clearance in rat mixed glial cell cultures [49]. These results suggest that grp 78 facilitates the correct folding of APP in the ER and that alterations in grp 78 levels may cause the accumulation of extracellular Ab42 [49]. Regarding grp 94, the expression of this protein has been found to increase in the parietal cortex of AD patients [50]. We have found increased levels of both grp 78 and grp 94. While the significance of increased levels of these proteins to 27-OHC- induced apoptosis, oxidative stress and Ab accumulation is still to be elucidated, alterations in these levels is a direct indication of triggering of ER stress by 27-OHC. Gadd153 gene silencing reduced the increase in levels of these proteins. Our results show that 27-OHC also increased phosphorylation of tau protein, another important hallmark of AD. The exact mechanism by which gadd153 regulates tau phosphorylation is yet to be determined. siRNA to gadd153 may reduce levels and activities of enzymes responsible for phosphorylation of tau. It may also be possible that reduction in Ab by siRNA to gadd153 reduces phosphorylation of tau as tau phosphorylation is considered a downstream event to Ab accumulation [51,52]. Another impor- tant pathological hallmark observed in AD is disturbances in iron homeostasis. Iron is an essential element and participate in various toxic reactions and generate free radicals by Fenton reaction [2,53,54]. The iron homeostasis in the cells is maintained by interactions of iron regulatory proteins (IRP), ferritin, transferrin and transferrin receptor proteins. Ferritin is the iron storage PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26420