Accumulation of amyloid-β (Aβ) peptide and the hyperphosphorylation of tau protein are major hallmarks of Alzheimer's disease (AD). The causes of AD are not well known but a number of environmental and dietary factors are suggested to increase the risk of developing AD. Additionally, altered metabolism of iron may have a role in the pathogenesis of AD. We have previously demonstrated that cholesterol-enriched diet causes AD-like pathology with iron deposition in rabbit brain. However, the extent to which chelation of iron protects against this pathology has not been determined. In this study, we administered the iron chelator deferiprone in drinking water to rabbits fed with a 2% cholesterol diet for 12 weeks. We found that deferiprone (both at 10 and 50 mg/kg/day) significantly decreased levels of Aβ40 and Aβ42 as well as BACE1, the enzyme that initiates cleavage of amyloid-β protein precursor to yield Aβ. Deferiprone also reduced the cholesterol diet-induced increase in phosphorylation of tau but failed to reduce reactive oxygen species generation. While deferiprone treatment was not associated with any change in brain iron levels, it was associated with a significant reduction in plasma iron and cholesterol levels. These results demonstrate that deferiprone confers important protection against hypercholesterolemia-induced AD pathology but the mechanism(s) may involve reduction in plasma iron and cholesterol levels rather than chelation of brain iron. We propose that adding an antioxidant therapy to deferiprone may be necessary to fully protect against cholesterol-enriched diet-induced AD-like pathology.
Accumulation of amyloid-beta (Abeta) peptide and deposition of hyperphosphorylated tau protein are two major pathological hallmarks of Alzheimer's disease (AD). We have shown that cholesterol-enriched diets and its metabolite 27-hydroxycholesterol (27-OHC) increase Abeta and phosphorylated tau levels. However, the mechanisms by which cholesterol and 27-OHC regulate Abeta production and tau phosphorylation remain unclear. Leptin, an adipocytokine involved in cell survival and in learning, has been demonstrated to regulate Abeta production and tau hyperphosphorylation in transgenic mice for AD. However, the involvement of leptin signaling in cholesterol and cholesterol metabolites-induced Abeta accumulation and tau hyperphosphorylation are yet to be examined. In this study, we determined the effect of high cholesterol diet and 27-OHC on leptin expression levels and the extent to which leptin treatment affects 27-OHC-induced AD-like pathology. Our results show that feeding rabbits a 2% cholesterol-enriched diet for 12 weeks reduces the levels of leptin by approximately 80% and incubating organotypic slices from adult rabbit hippocampus with 27-OHC reduced leptin levels by approximately 30%. 27-OHC induces a 1.5-fold increase in Abeta (40) and a 3-fold increase in Abeta (42) and in phosphorylated tau. Treatment with leptin reversed the 27-OHC-induced increase in Abeta and phosphorylated tau by decreasing the levels of BACE-1 and GSK-3beta respectively. Our results suggest that cholesterol-enriched diets and cholesterol metabolites induce AD-like pathology by altering leptin signaling. We propose that leptin administration may prevent the progression of sporadic forms of AD that are related to increased cholesterol and oxidized cholesterol metabolite levels.
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.
Dysregulation in brain iron homeostasis and subsequent oxidative stress are major hallmarks of Alzheimer's disease (AD) pathology as is the accumulation of β-amyloid (Aβ) peptide. Hypercholesterolemia causes disturbances in iron metabolism, increases levels of Aβ peptide and triggers oxidative stress in brains from rabbits fed a cholesterol-rich diet. However, because of the impermeability of the blood brain barrier (BBB) to circulating cholesterol, the mechanisms by which high cholesterol levels in the blood triggers AD-like pathology are not understood. Conversely to cholesterol, the oxidized cholesterol metabolite (oxysterol) 27-hydroxycholesterol (27-OHC) has the ability to cross the BBB into the brain. We speculated that the turnover of cholesterol to 27-OHC increases following hypercholesterolemia, leading to the entrance into the brain of higher levels of this oxysterol. Increased levels of 27-OHC in the brain would dysregulate iron homeostasis, increase Aβ levels and cause oxidative damage. Hippocampal organotypic slices from adult rabbits were incubated with 25 μM 27-OHC and harvested 72 hrs later. Iron-regulatory proteins, apoptosis and Aβ metabolism were determined using Western blot analyses and confocal microscopy. 27-OHC causes a decrease in the expression levels of transferrin receptor and iron regulatory protein (IRP)-2, and an increase in expression levels of ferritin light chain. 27-OHC also induces apoptosis by activating the growth arrest-and DNA damage-inducible gene 153 (gadd153), upregulating levels of the pro-apoptotic Bax and Bid proteins and downregulating levels of the anti-apoptotic protein Bcl-2. Levels of Aβ42, APP and BACE1 were also increased by 27-OHC treatment. Our results demonstrate that the oxysterol 27-OHC triggers pathological changes associated with AD pathology. These results strongly suggest that 27-OHC represents the link between high levels of circulating cholesterol and the triggering of AD hallmarks in the brain. As cholesterol metabolism is tightly regulated, disturbances in this process may put the brain at risk for AD.
Abstract Background Alzheimer's disease (AD) and age-related macular degeneration (AMD) share several pathological hallmarks including β-amyloid (Aβ) accumulation, oxidative stress, and apoptotic cell death. The causes of AD and AMD are likely multi-factorial with several factors such as diet, environment, and genetic susceptibility participating in the pathogenesis of these diseases. Epidemiological studies correlated high plasma cholesterol levels with high incidence of AD, and feeding rabbits with a diet rich in cholesterol has been shown to induce AD-like pathology in rabbit brain. High intake of cholesterol and saturated fat were also long been suspected to increase the risk for AMD. However, the extent to which cholesterol-enriched diet may also cause AMD-like features in rabbit retinas is not well known. Methods Male New Zealand white rabbits were fed normal chow or a 2% cholesterol-enriched diet for 12 weeks. At necropsy, animals were perfused with Dulbecco's phosphate-buffered saline and the eyes were promptly removed. One eye of each animal was used for immunohistochemistry and retina dissected from the other eye was used for Western blot, ELISA assays, spectrophotometry and mass spectrometry analyses. Results Increased levels of Aβ, decreased levels of the anti-apoptotic protein Bcl-2, increased levels of the pro-apoptotic Bax and gadd153 proteins, emergence of TUNEL-positive cells, and increased generation of reactive oxygen species were found in retinas from cholesterol-fed compared to normal chow-fed rabbits. Additionally, astrogliosis, drusen-like debris and cholesterol accumulations in retinas from cholesterol-fed rabbits were observed. As several lines of evidence suggest that oxidized cholesterol metabolites (oxysterols) may be the link by which cholesterol contributes to the pathogenesis of AMD, we determined levels of oxysterols and found a dramatic increase in levels of oxysterols in retinas from cholesterol-fed rabbits. Conclusions Our results suggest that cholesterol-enriched diets cause retinal degeneration that is relevant to AMD. Furthermore, our data suggests high cholesterol levels and subsequent increase in the cholesterol metabolites as potential culprits to AMD.
Purpose/aim of the study: Disturbances in cholesterol metabolism and increased levels of cholesterol oxidation products (oxysterols) in retina may contribute to age-related macular degeneration (AMD). The role of oxysterols or of their target receptors liver X receptors (LXRs) and estrogen receptors (ERs) in the pathogenesis of MD is ill-known. The purpose of this study is to determine the extent to which the oxysterols 27-hydroxycholesterol (27-OHC), 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-KC) affect the transcriptional activity of LXR and ER.Materials and methods: ARPE-19 cells, untreated or incubated with 27-OHC, 25-OHC or 7-KC for 24 h were harvested. We used Western blot analyses for detecting ERs and LXRs expression, dual luciferase assays for measuring LXRs and ERs transcriptional activity, cytotox-ONE homogeneous membrane integrity assay for measuring cytotoxicity, JC-1 method for measuring mitochondrial membrane potential changes and ELISA for measuring cytokine levels.Results: Both LXRs and ERs are expressed and are transcriptionally active in ARPE-19 cells. 27-OHC, 25-OHC and 7-KC inhibited ER-mediated transcriptional activity, whereas 27-OHC and 25-OHC increased LXR-mediated transcription. E2 reduced 25-OHC and 27-OHC-induced cytotoxicity, mitochondrial permeability potential decline, and cytokine secretion. The LXR agonist GW3965 or the LXR antagonist 5α-6α-epoxycholesterol-3-sulfate (ECHS) did not offer protection against either 27-OHC and 25-OHC or 7-KC.Conclusions: Increased levels of oxysterols can decrease ER and increase LXR signaling. ER agonists can offer protection against cytotoxic effects of 27-OHC and 25-OHC, two oxysterols derived by enzymatic reactions. Although they exert similar toxicity, the cellular mechanisms involved in the toxic effects of oxysterols whether derived by enzymatic or autoxidation reactions appear to be different.
Abstract Background Activation of the liver × receptors (LXRs) by exogenous ligands stimulates the degradation of β-amyloid 1–42 (Aβ42), a peptide that plays a central role in the pathogenesis of Alzheimer's disease (AD). The oxidized cholesterol products (oxysterols), 24-hydroxycholesterol (24-OHC) and 27-hydroxycholesterol (27-OHC), are endogenous activators of LXRs. However, the mechanisms by which these oxysterols may modulate Aβ42 levels are not well known. Results We determined the effect of 24-OHC and/or 27-OHC on Aβ generation in SH-SY5Y cells. We found that while 27-OHC increases levels of Aβ42, 24-OHC did not affect levels of this peptide. Increased Aβ42 levels with 27-OHC are associated with increased levels of β-amyloid precursor protein (APP) as well as β-secretase (BACE1), the enzyme that cleaves APP to yield Aβ. Unchanged Aβ42 levels with 24-OHC are associated with increased levels of sAPPα, suggesting that 24-OHC favors the processing of APP to the non-amyloidogenic pathway. Interestingly, 24-OHC, but not 27-OHC, increases levels of the ATP-binding cassette transporters, ABCA1 and ABCG1, which regulate cholesterol transport within and between cells. Conclusion These results suggest that cholesterol metabolites are linked to Aβ42 production. 24-OHC may favor the non-amyloidogenic pathway and 27-OHC may enhance production of Aβ42 by upregulating APP and BACE1. Regulation of 24-OHC: 27-OHC ratio could be an important strategy in controlling Aβ42 levels in AD.
Evidence shows that the insulin-like growth factor-1 (IGF-1) and leptin reduce β-amyloid (Aβ) production and tau phosphorylation, two major hallmarks of Alzheimer's disease (AD). IGF-1 expression involves the JAK/STAT pathway and the expression of leptin is regulated by the mammalian target of rapamycin complex 1 (mTORC1). We have previously shown that Aβ reduces leptin by inhibiting the mTORC1 pathway and Aβ was also suggested to inhibit the JAK/STAT pathway, potentially attenuating IGF-1 expression. As IGF-1 can activate mTORC1 and leptin can modulate JAK/STAT pathway, we determined the extent to which IGF-1 and leptin can upregulate the expression of one another and protect against Aβ-induced downregulation.We demonstrate that incubation of organotypic slices from adult rabbit hippocampus with Aβ42 downregulates IGF-1 expression by inhibiting JAK2/STAT5 pathway. Leptin treatment reverses these Aβ42 effects on IGF-1 and treatment with the STAT5 inhibitor completely abrogated the leptin-induced increase in IGF-1. Furthermore, EMSA and ChIP analyses revealed that leptin increases the STAT5 binding to the IGF-1 promoter. We also show that IGF-1 increases the expression of leptin and reverses the Aβ42-induced attenuation in leptin expression via the activation of mTORC1 signaling as the mTORC1 inhibitor rapamycin completely precluded the IGF-1-induced increase in leptin expression.Our results demonstrate for the first time that Aβ42 downregulates IGF-1 expression and that leptin and IGF-1 rescue one another from downregulation by Aβ42. Our study provides a valuable insight into the leptin/IGF-1/Aβ interplay that may be relevant to the pathophysiology of AD.
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.
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