Silencing GADD153/CHOP Gene Expression Protects against Alzheimer’s Disease-Like Pathology Induced by 27-Hydroxycholesterol in Rabbit Hippocampus Jaya R. P. Prasanthi, Tyler Larson, Jared Schommer, Othman Ghribi* Department of Pharmacology, Physiology and Therapeutics, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota, United States of America Abstract Endoplasmic reticulum (ER) stress is suggested to play a key role in the pathogenesis of neurodegenerative diseases including Alzheimer’s disease (AD). Sustained ER stress leads to activation of the growth arrest and leucine zipper transcription factor, DNA damage inducible gene 153 (gadd153; also called CHOP). Activated gadd153 can generate oxidative damage and reactive oxygen species (ROS), increase b-amyloid (Ab) levels, disturb iron homeostasis and induce inflammation as well as cell death, which are all pathological hallmarks of AD. Epidemiological and laboratory studies suggest that cholesterol dyshomeostasis contributes to the pathogenesis of AD. We have previously shown that the cholesterol oxidized metabolite 27-hydroxycholesterol (27-OHC) triggers AD-like pathology in organotypic slices. However, the extent to which gadd153 mediates 27-OHC effects has not been determined. We silenced gadd153 gene with siRNA and determined the effects of 27-OHC on AD hallmarks in organotypic slices from adult rabbit hippocampus. siRNA to gadd153 reduced 27-OHC-induced Ab production by mechanisms involving reduction in levels of b-amyloid precursor protein (APP) and b-secretase (BACE1), the enzyme that initiates cleavage of APP to yield Ab peptides. Additionally, 27-OHC-induced tau phosphorylation, ROS generation, TNF-a activation, and iron and apoptosis-regulatory protein levels alteration were also markedly reduced by siRNA to gadd153. These data suggest that ER stress-mediated gadd153 activation plays a central role in the triggering of AD pathological hallmarks that result from incubation of hippocampal slices with 27-OHC. Our results add important insights into cellular mechanisms that underlie the potential contribution of cholesterol metabolism in AD pathology, and suggest that preventing gadd153 activation protects against AD related to cholesterol oxidized products. Citation: Prasanthi JRP, Larson T, Schommer J, Ghribi O (2011) Silencing GADD153/CHOP Gene Expression Protects against Alzheimer’s Disease-Like Pathology Induced by 27-Hydroxycholesterol in Rabbit Hippocampus. PLoS ONE 6(10): e26420. doi:10.1371/journal.pone.0026420 Editor: Sergio T. Ferreira, Federal University of Rio de Janeiro, Brazil Received May 9, 2011; Accepted September 26, 2011; Published October 14, 2011 Copyright: 2011 Prasanthi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the National Institutes of Health (NIH) (RO1ES014826). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: othman.ghribi@med.und.edu Introduction Alzheimer’s disease (AD), the most common neurodegenerative disorder, is histopathologically characterized by the accumulation of b-amyloid (Ab) peptide and the hyperphosphorylation of tau protein. In addition to increased levels of Ab and phosphorylated tau, oxidative stress, inflammation, and cell death contribute to the neurodegenerative features of AD [1]. Accumulation of Ab peptides in soluble and insoluble forms is suggested to be the trigger of the neurodegenerative processes that lead to the development of AD. Currently, there is no consensus as to what are the factors or cellular mechanisms that lead to Ab accumulation in the brain. We have shown that cholesterol-enriched diets increase Ab production and oxidative damage involving the activation of the growth and arrest DNA damage protein gadd153 [2]. Gadd153 (also called CHOP) is activated by endoplasmic reticulum (ER) stress and can cause cellular damage by mechanisms that include induction of oxidative stress, generation of reactive oxygen species (ROS), triggering of apoptosis, and disturbing iron homeostasis [3–5]. The ability of gadd153 to increase ROS generation can lead to increased production of b-secretase (BACE1), the rate limiting enzyme that cleaves b-APP to yield Ab, thus leading to increased Ab levels [6,7]. ER stress-induced gadd153 activation may thus triggers AD-like pathology by generating oxidative damage and increasing Ab production. Silencing gadd153 gene expression would therefore represent a potential strategy to reduce ROS generation, BACE1 activation, and Ab accumulation and ultimately protect against AD. We have recently shown that the cholesterol oxidized metabolite (oxysterol) 27-hydroxycholesterol (27-OHC) causes AD-like pathology in human neuroblastoma cells and in organotypic slices from adult rabbit hippocampus [8–10]. In this study, we incubated organotypic slices from adult rabbit hippocampus with 27-OHC, in the presence or absence of siRNA to gadd153, and determined the effects on levels of Ab, phosphorylated tau, ROS, oxidative and ER stress, iron homeostasis and apoptosis-regulatory proteins, which are all relevant to AD pathology. The organotypic slice system has many advantages in that connectivity between neurons, interneurons and glia is maintained. In addition, rabbits have a phylogeny closer to humans than rodents [11], and their Ab sequence, unlike that of rodents, is similar to the Ab sequence of the human [12]. We found that siRNA to gadd153 dramatically reduces the generation of a wide range of events that are relevant to AD. PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26420