The Guinea Pig as a model for sporadic Alzheimer's Disease (AD): the impact of cholesterol intake on expression of AD-related genes

Extent: 12p.We investigated the guinea pig, Cavia porcellus, as a model for Alzheimer’s disease (AD), both in terms of the conservation of genes involved in AD and the regulatory responses of these to a known AD risk factor - high cholesterol intake. Unlike rats and mice, guinea pigs possess an Aβ peptide sequence identical to human Aβ. Consistent with the commonality between cardiovascular and AD risk factors in humans, we saw that a high cholesterol diet leads to up-regulation of BACE1 (β-secretase) transcription and down-regulation of ADAM10 (α-secretase) transcription which should increase release of Aβ from APP. Significantly, guinea pigs possess isoforms of AD-related genes found in humans but not present in mice or rats. For example, we discovered that the truncated PS2V isoform of human PSEN2, that is found at raised levels in AD brains and that increases γ-secretase activity and Aβ synthesis, is not uniquely human or aberrant as previously believed. We show that PS2V formation is up-regulated by hypoxia and a high-cholesterol diet while, consistent with observations in humans, Aβ concentrations are raised in some brain regions but not others. Also like humans, but unlike mice, the guinea pig gene encoding tau, MAPT, encodes isoforms with both three and four microtubule binding domains, and cholesterol alters the ratio of these isoforms. We conclude that AD-related genes are highly conserved and more similar to human than the rat or mouse. Guinea pigs represent a superior rodent model for analysis of the impact of dietary factors such as cholesterol on the regulation of AD-related genes.Mathew J. Sharman, Seyyed H. Moussavi Nik, Mengqi M. Chen, Daniel Ong, Linda Wijaya, Simon M. Laws, Kevin Taddei, Morgan Newman, Michael Lardelli, Ralph N. Martins, Giuseppe Verdil

Gadd153 and NF-κB crosstalk regulates 27-hydroxycholesterol-induced increase in BACE1 and β-amyloid production in human neuroblastoma SH-SY5Y cells.

β-amyloid (Aβ) peptide, accumulation of which is a culprit for Alzheimer's disease (AD), is derived from the initial cleavage of amyloid precursor protein by the aspartyl protease BACE1. Identification of cellular mechanisms that regulate BACE1 production is of high relevance to the search for potential disease-modifying therapies that inhibit BACE1 to reduce Aβ accumulation and AD progression. In the present study, we show that the cholesterol oxidation product 27-hydroxycholesterol (27-OHC) increases BACE1 and Aβ levels in human neuroblastoma SH-SY5Y cells. This increase in BACE1 involves a crosstalk between the two transcription factors NF-κB and the endoplasmic reticulum stress marker, the growth arrest and DNA damage induced gene-153 (gadd153, also called CHOP). We specifically show that 27-OHC induces a substantial increase in NF-κB binding to the BACE1 promoter and subsequent increase in BACE1 transcription and Aβ production. The NF-κB inhibitor, sc514, significantly attenuated the 27-OHC-induced increase in NF-κB-mediated BACE1 expression and Aβ genesis. We further show that the 27-OHC-induced NF-κB activation and increased NF-κB-mediated BACE1 expression is contingent on the increased activation of gadd153. Silencing gadd153 expression with siRNA alleviated the 27-OHC-induced increase in NF-κB activation, NF-κB binding to the BACE1 promoter, and subsequent increase in BACE1 transcription and Aβ production. We also show that increased levels of BACE1 in the triple transgenic mouse model for AD is preceded by gadd153 and NF-κB activation. In summary, our study demonstrates that gadd153 and NF-κB work in concert to regulate BACE1 expression. Agents that inhibit gadd153 activation and subsequent interaction with NF-κB might be promising targets to reduce BACE1 and Aβ overproduction and may ultimately serve as disease-modifying treatments for AD

Silencing GADD153/CHOP gene expression protects against Alzheimer's disease-like pathology induced by 27-hydroxycholesterol in rabbit hippocampus.

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 β-amyloid (Aβ) 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 Aβ production by mechanisms involving reduction in levels of β-amyloid precursor protein (APP) and β-secretase (BACE1), the enzyme that initiates cleavage of APP to yield Aβ peptides. Additionally, 27-OHC-induced tau phosphorylation, ROS generation, TNF-α 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

Primers designed and used for RT-PCR, EMSA, and ChIP analyses.

<p>Primers designed and used for RT-PCR, EMSA, and ChIP analyses.</p

27-OHC-induced increase in the levels of intracellular as well as secreted forms of Aβ42 and Aβ40 is contingent on NF-κB activation.

<p>(a–d) ELISA immunoassay demonstrates that 27-OHC increases the levels of intracellular and secreted Aβ42 and Aβ40, while the NF-κB inhibitor sc514 mitigates the 27-OHC-induced increase in both intracellular and secreted Aβ42 and Aβ40. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). *p<0.05, **p<0.01, and ***p<0.001 versus control, ^†† p<0.01 and ^††† p<0.001 versus 27-OHC.</p

Schematic representation of the molecular mechanisms involved in the 27-OHC-induced increase in BACE1 expression.

<p>27-OHC activates gadd153 (1) which evokes the phosphorylation of the IKK-complex (2). Phosphorylation IKK complex (3) results in the phosphorylation and subsequent proteasomal degradation of IκB (4), thereby releasing the p65-p50 NF-κB dimer from inhibitory sequestration in the cytosol and allowing the p65-p50 NF-κB dimer to translocate to the nucleus (5). The translocated p65-p50 NF-κB dimer binds to distinct κB sites in the BACE1 promoter region and upregulates BACE1 expression (5). Increased gadd153 expression by 27-OHC may also induce NF-κB activation, nuclear translocation, and subsequently increase BACE1 expression by other mechanism that are yet to be elucidated (6). siRNA to gadd153 reduces the 27-OHC-induced nuclear translocation of NF-κB and thereby attenuates the increase in BACE1(7). The NF-κB inhibitor sc514 also decreases the 27-OHC-induced increase in BACE1 expression by inhibiting the nuclear translocation of NF-κB and subsequent increase in NF-κB-mediated transcription of BACE1 (8).</p

27-OHC-induced NF-κB-mediated increase in BACE1 transcription and expression is contingent on gadd153 expression.

<p>(a–c) siRNA to gadd153 mitigates the 27-OHC-induced increase in BACE1 protein levels and mRNA expression. (d) EMSA shows that 27-OHC increases the binding of NF-κB to the exogenous DNA sequence that corresponds to the NF-κB binding site in the BACE1 promoter; silencing gadd153 expression reduces the increase in the binding of NF-κB. (e) ChIP analysis shows that siRNA to gadd153 decreases the 27-OHC-induced increase in binding of NF-κB to the BACE1 promoter. (f,g) Dual luciferase assay demonstrates that siRNA to gadd153 decreases the 27-OHC-induced increase in NF-κB transcriptional activity and BACE1 promoter activity. (h) Levels of gadd153 are detectable in hippocampus of 3, 6, and 12 month-old 3xTg-AD, while NF-κB levels are detectable in hippocampus of 6 and 12month-old, and BACE1 levels increasing only in 12 month-old 3xTg-AD. Data in a-g is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). *p<0.05, **p<0.01, and ***p<0.001 versus control, ^†† p<0.01 and ^††† p<0.001 versus 27-OHC.</p

27-OHC-induced nuclear translocation of NF-κB is contingent on gadd153 activation.

<p>(a) siRNA to gadd153 suppresses the 27-OHC-induced increase in gadd153 protein levels. (b–d) siRNA to gadd153 attenuates the 27-OHC-induced increase in p65 and p50 subunit translocation to the nucleus. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). ***p<0.001 versus control, ^†† p<0.01 and ^††† p<0.001 versus 27-OHC.</p

27-OHC increases the activation of NF-κB via the phosphorylation of IKKα/β which is dependent on gadd153 expression.

<p>27-OHC induces a significant increase in the phosphorylation of IKKα/β, and siRNA to gadd153 reverses the 27-OHC-induced increase in phosphorylation of IKKα/β (a,b). 27-OHC induces a significant decrease in IKBα levels and a significant increase in the phosphorylation of IKBα; siRNA to gadd153 reverses the 27-OHC-induced alterations in IKBα and p- IKBα levels (c,d). Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). ***p<0.001 versus control; ^†† p<0.01 versus 27-OHC and ^††† p<0.001 versus 27-OHC.</p