25-Hydroxycholesterol regulates lysosome AMP kinase activation and metabolic reprogramming to educate immunosuppressive macrophages.

Macrophages are critical to turn noninflamed "cold tumors" into inflamed "hot tumors". Emerging evidence indicates abnormal cholesterol metabolites in the tumor microenvironment (TME) with unclear function. Here, we uncovered the inducible expression of cholesterol-25-hydroxylase (Ch25h) by interleukin-4 (IL-4) and interleukin-13 (IL-13) via the transcription factor STAT6, causing 25-hydroxycholesterol (25HC) accumulation. scRNA-seq analysis confirmed that CH25Hhi subsets were enriched in immunosuppressive macrophage subsets and correlated to lower survival rates in pan-cancers. Targeting CH25H abrogated macrophage immunosuppressive function to enhance infiltrating T cell numbers and activation, which synergized with anti-PD-1 to improve anti-tumor efficacy. Mechanically, lysosome-accumulated 25HC competed with cholesterol for GPR155 binding to inhibit the kinase mTORC1, leading to AMPKα activation and metabolic reprogramming. AMPKα also phosphorylated STAT6 Ser564 to enhance STAT6 activation and ARG1 production. Together, we propose CH25H as an immunometabolic checkpoint, which manipulates macrophage fate to reshape CD8+ T cell surveillance and anti-tumor response.

Oxysterol Restraint of Cholesterol Synthesis Prevents AIM2 Inflammasome Activation.

Type I interferon restrains interleukin-1ß (IL-1ß)-driven inflammation in macrophages by upregulating cholesterol-25-hydroxylase (Ch25h) and repressing SREBP transcription factors. However, the molecular links between lipid metabolism and IL-1ß production remain obscure. Here, we demonstrate that production of 25-hydroxycholesterol (25-HC) by macrophages is required to prevent inflammasome activation by the DNA sensor protein absent in melanoma 2 (AIM2). We find that in response to bacterial infection or lipopolysaccharide (LPS) stimulation, macrophages upregulate Ch25h to maintain repression of SREBP2 activation and cholesterol synthesis. Increasing macrophage cholesterol content is sufficient to trigger IL-1ß release in a crystal-independent but AIM2-dependent manner. Ch25h deficiency results in cholesterol-dependent reduced mitochondrial respiratory capacity and release of mitochondrial DNA into the cytosol. AIM2 deficiency rescues the increased inflammasome activity observed in Ch25h-/-. Therefore, activated macrophages utilize 25-HC in an anti-inflammatory circuit that maintains mitochondrial integrity and prevents spurious AIM2 inflammasome activation.

The cholesterol metabolite 25-hydroxycholesterol restrains the transcriptional regulator SREBP2 and limits intestinal IgA plasma cell differentiation.

Diets high in cholesterol alter intestinal immunity. Here, we examined how the cholesterol metabolite 25-hydroxycholesterol (25-HC) impacts the intestinal B cell response. Mice lacking cholesterol 25-hydroxylase (CH25H), the enzyme generating 25-HC, had higher frequencies of immunoglobulin A (IgA)-secreting antigen-specific B cells upon immunization or infection. 25-HC did not affect class-switch recombination but rather restrained plasma cell (PC) differentiation. 25-HC was produced by follicular dendritic cells and increased in response to dietary cholesterol. Mechanistically, 25-HC restricted activation of the sterol-sensing transcription factor SREBP2, thereby regulating B cell cholesterol biosynthesis. Ectopic expression of SREBP2 in germinal center B cells induced rapid PC differentiation, whereas SREBP2 deficiency reduced PC output in vitro and in vivo. High-cholesterol diet impaired, whereas Ch25h deficiency enhanced, the IgA response against Salmonella and the resulting protection from systemic bacterial dissemination. Thus, a 25-HC-SREBP2 axis shapes the humoral response at the intestinal barrier, providing insight into the effect of high dietary cholesterol in intestinal immunity.

High level 27-HC impairs trophoblast cell invasion and migration via LXR in pre-eclampsia.

INTRODUCTION: To explore the potential impact of 27-hydroxycholesterol (27-HC) on trophoblast cell function in pre-eclampsia. RESULTS: The levels of 27-HC and the expression of CYP27A1 are upregulated in clinical samples of PE. Furthermore, high concentrations of 27-HC can inhibit the invasion and migration ability of trophoblast cells in vitro, and this inhibitory effect is weakened after LXR silencing. In HTR8/SVneo cells treated with 27-HC, the expression of ABCA1/ABCG1 are increased. Finally, we established a mouse model of PE using l-NAME (N-Nitro-l-Arginine Methyl Ester). We found an increase in the levels of 27-HC in the peripheral blood serum of the PE mouse model, and an upregulation of CYP27A1 and LXR expressions in the placenta of the PE mouse model. CONCLUSION: 27-HC inhibits the invasion and migration ability of trophoblast cells by activating the LXR signaling pathway, which is involved in the pathogenesis of Pre-eclampsia(PE).

Cholesterol neutralized vemurafenib treatment by promoting melanoma stem-like cells via its metabolite 27-hydroxycholesterol.

Vemurafenib has been used as first-line therapy for unresectable or metastatic melanoma with BRAFV600E mutation. However, overall survival is still limited due to treatment resistance after about one year. Therefore, identifying new therapeutic targets for melanoma is crucial for improving clinical outcomes. In the present study, we found that lowering intracellular cholesterol by knocking down DHCR24, the limiting synthetase, impaired tumor cell proliferation and migration and abrogated the ability to xenotransplant tumors. More importantly, administration of DHCR24 or cholesterol mediated resistance to vemurafenib and promoted the growth of melanoma spheroids. Mechanistically, we identified that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol synthesized by the enzyme cytochrome P450 27A1 (CYP27A1), reproduces the phenotypes induced by DHCR24 or cholesterol administration and activates Rap1-PI3K/AKT signaling. Accordingly, CYP27A1 is highly expressed in melanoma patients and upregulated by DHCR24 induction. Dafadine-A, a CYP27A1 inhibitor, attenuates cholesterol-induced growth of melanoma spheroids and abrogates the resistance property of vemurafenib-resistant melanoma cells. Finally, we confirmed that the effects of cholesterol on melanoma resistance require its metabolite 27HC through CYP27A1 catalysis, and that 27HC further upregulates Rap1A/Rap1B expression and increases AKT phosphorylation. Thus, our results suggest that targeting 27HC may be a useful strategy to overcome treatment resistance in metastatic melanoma.

25-Hydroxycholesterol in health and diseases.

Cholesterol is an essential structural component of all membranes of mammalian cells where it plays a fundamental role not only in cellular architecture, but also, for example, in signaling pathway transduction, endocytosis process, receptor functioning and recycling, or cytoskeleton remodeling. Consequently, intracellular cholesterol concentrations are tightly regulated by complex processes, including cholesterol synthesis, uptake from circulating lipoproteins, lipid transfer to these lipoproteins, esterification, and metabolization into oxysterols that are intermediates for bile acids. Oxysterols have been considered for long time as sterol waste products, but a large body of evidence has clearly demonstrated that they play key roles in central nervous system functioning, immune cell response, cell death, or migration and are involved in age-related diseases, cancers, autoimmunity, or neurological disorders. Among all the existing oxysterols, this review summarizes basic as well as recent knowledge on 25-hydroxycholesterol which is mainly produced during inflammatory or infectious situations and that in turn contributes to immune response, central nervous system disorders, atherosclerosis, macular degeneration, or cancer development. Effects of its metabolite 7α,25-dihydroxycholesterol are also presented and discussed.

Role of Cholesterol Metabolic Enzyme CYP46A1 and Its Metabolite 24S-Hydroxycholesterol in Ischemic Stroke.

BACKGROUND: For several decades, it has been recognized that overactivation of the glutamate-gated N-methyl-D-aspartate receptors (NMDARs) and subsequent Ca2+ toxicity play a critical role in ischemic brain injury. 24S-hydroxycholesterol (24S-HC) is a major cholesterol metabolite in the brain, which has been identified as a potent positive allosteric modulator of NMDAR in rat hippocampal neurons. We hypothesize that 24S-HC worsens ischemic brain injury via its potentiation of the NMDAR, and reducing the production of 24S-HC by targeting its synthetic enzyme CYP46A1 provides neuroprotection. METHODS: We tested this hypothesis using electrophysiological, pharmacological, and transgenic approaches and in vitro and in vivo cerebral ischemia models. RESULTS: Our data show that 24S-HC potentiates NMDAR activation in primary cultured mouse cortical neurons in a concentration-dependent manner. At 10 µmol/L, it dramatically increases the steady-state currents by 51% and slightly increases the peak currents by 20%. Furthermore, 24S-HC increases NMDA and oxygen-glucose deprivation-induced cortical neuronal injury. The increased neuronal injury is largely abolished by NMDAR channel blocker MK-801, suggesting an NMDAR-dependent mechanism. Pharmacological inhibition of CYP46A1 by voriconazole or gene knockout of Cyp46a1 dramatically reduces ischemic brain injury. CONCLUSIONS: These results identify a new mechanism and signaling cascade that critically impacts stroke outcome: CYP46A1 → 24S-HC → NMDAR → ischemic brain injury. They offer proof of principle for further development of new strategies for stroke intervention by targeting CYP46A1 or its metabolite 24S-HC.

The mechanism of 25-hydroxycholesterol-mediated suppression of atrial ß1-adrenergic responses.

25-Hydroxycholesterol (25HC) is a biologically active oxysterol, whose production greatly increases during inflammation by macrophages and dendritic cells. The inflammatory reactions are frequently accompanied by changes in heart regulation, such as blunting of the cardiac ß-adrenergic receptor (AR) signaling. Here, the mechanism of 25HC-dependent modulation of responses to ß-AR activation was studied in the atria of mice. 25HC at the submicromolar levels decreased the ß-AR-mediated positive inotropic effect and enhancement of the Ca2+ transient amplitude, without changing NO production. Positive inotropic responses to ß1-AR (but not ß2-AR) activation were markedly attenuated by 25HC. The depressant action of 25HC on the ß1-AR-mediated responses was prevented by selective ß3-AR antagonists as well as inhibitors of Gi protein, Gßγ, G protein-coupled receptor kinase 2/3, or ß-arrestin. Simultaneously, blockers of protein kinase D and C as well as a phosphodiesterase inhibitor did not preclude the negative action of 25HC on the inotropic response to ß-AR activation. Thus, 25HC can suppress the ß1-AR-dependent effects via engaging ß3-AR, Gi protein, Gßγ, G protein-coupled receptor kinase, and ß-arrestin. This 25HC-dependent mechanism can contribute to the inflammatory-related alterations in the atrial ß-adrenergic signaling.

25-hydroxycholesterol promotes proliferation and metastasis of lung adenocarcinoma cells by regulating ERß/TNFRSF17 axis.

Lung adenocarcinoma is the main type of lung cancer in women. Our previous findings have evidenced that 25-hydroxycholesterol (25-HC) promotes migration and invasion of lung adenocarcinoma cells (LAC), during which LXR as a 25-HC receptor plays an important role. Estrogen receptor beta (ERß) is a receptor of 27-hydroxycholesterol that is structurally analogous to 25-HC, but its role in the functional actions of 25-HC remained largely unknown. In this study, we demonstrated that 25-HC treatment triggered ERß expression in LAC. Knockdown of ERß inhibited 25-HC-mediated proliferation, migration and invasion, and reduced 25-HC-induced LAC metastasis in vivo. Further investigation revealed that ERß knockdown restrained the expression of TNFRSF17 (BCMA). In vivo experiments also confirmed that ERß knockdown blocked 25-HC-induced TNFRSF17 expression. TNFRSF17 knockdown also restrained 25-HC-induced proliferation, migration and invasion. Bioinformatic analysis showed that the levels of ERß and TNFRSF17 were elevated in lung adenocarcinoma, and were closely related to tumor stages and nodal metastasis status. These results suggested that 25-HC promoted the proliferation and metastasis of LAC by regulating ERß/TNFRSF17 axis.

27-Hydroxycholesterol activates the GSK-3ß/ß-catenin signaling pathway resulting in intestinal fibrosis by inducing oxidative stress: effect of dietary interventions.

OBJECTIVE: Intestinal fibrosis, a common and serious complication of inflammatory bowel disease (IBD), results from chronic inflammation. A high-cholesterol diet may be a risk factor for IBD and 27-hydroxylcholesterol (27HC) is the main human cholesterol metabolite. This study investigated whether 27HC can induce intestinal fibrosis. METHODS: The effects of cholesterol and 27HC on intestinal fibrosis were assessed in zebrafish and human intestinal epithelial Caco-2 cells. RESULTS: Cholesterol and 27HC induced intestinal inflammation and collagen deposition, inhibited E-cadherin (E-ca) expression in the intestinal epithelium, and promoted nuclear translocation of ß-catenin in zebrafish. Cholesterol and 27HC up-regulated expression of COL-1, α-SMA, CTGF, TIMP1, N-cadherin, vimentin, glycogen synthesis kinase-3ß (GSK-3ß) and ß-catenin, but inhibited E-ca, in Caco-2 cells. The expression of these proteins was inhibited by CYP27A1 knockdown and ß-catenin knockdown. 27HC-induced nuclear translocation of ß-catenin occurs in Caco-2 cells. p38, ERK, and AKT activate ß-catenin and thereby participate in 27HC-induced epithelia-mesenchymal transition (EMT) and fibrosis. 27HC-increased oxidative stress and the fibrosis and EMT markers, the nuclear translocation of ß-catenin, and the up-regulation of p-cell kinase proteins promoted by 27HC were inhibited by N-acetyl-L-cysteine (NAC). Folic acid (FA), resveratrol (RES), and NAC all ameliorated the 27HC-induced effects in Caco-2 cells and zebrafish. CONCLUSION: A high-cholesterol diet caused intestinal fibrosis in zebrafish, mediated by a major cholesterol metabolite, 27HC. 27HC increased oxidative stress and activated p38, ERK, AKT, and ß-catenin, leading to EMT of epithelial cells and intestinal fibrosis. FA and RES both ameliorated intestinal fibrosis by restraining 27HC-induced ß-catenin activation.

25-hydroxycholesterol triggers antioxidant signaling in mouse atria.

Oxysterol, 25-hydroxycholesterol (25HC), is a potent regulator of immune reactions, its synthesis greatly increases by macrophages during inflammation. We hypothesize that 25HC can have cardioprotective effects by limiting consequences of excessive ß-adrenoceptor (ßAR) stimulation, particularly reactive oxygen species (ROS) production, in mouse atria. Isoproterenol, a ßAR agonist, increased extra- and intracellular levels of ROS. This enhancement of ROS production was suppressed by NADPH oxidase antagonists as well as 25HC. Inhibition of ß3ARs, Gi protein and protein kinase Cε prevented the effect of 25HC on isoproterenol-dependent ROS synthesis. Furthermore, 25HC suppressed isoproterenol-induced lipid peroxidation and mitochondrial ROS generation as well as ROS-dependent component of positive inotropic response to isoproterenol. Additionally, 25HC decreased mitochondrial ROS production and lipid peroxidation induced by antimycin A, a mitochondrial poison. Thus, 25HC exerts antioxidant properties alleviating mitochondrial dysfunction-induced and ßAR-dependent cardiac oxidative damage. In the latter case, 25HC can act via signaling mechanism engaging ß3ARs, Gi protein and protein kinase Cε.

The normalizing effects of the CYP46A1 activator efavirenz on retinal sterol levels and risk factors for glaucoma in Apoj-/- mice.

Apolipoprotein J (APOJ) is a multifunctional protein with genetic evidence suggesting an association between APOJ polymorphisms and Alzheimer's disease as well as exfoliation glaucoma. Herein we conducted ocular characterizations of Apoj-/- mice and found that their retinal cholesterol levels were decreased and that this genotype had several risk factors for glaucoma: increased intraocular pressure and cup-to-disk ratio and impaired retinal ganglion cell (RGC) function. The latter was not due to RGC degeneration or activation of retinal Muller cells and microglia/macrophages. There was also a decrease in retinal levels of 24-hydroxycholesterol, a suggested neuroprotectant under glaucomatous conditions and a positive allosteric modulator of N-methyl-D-aspartate receptors mediating the light-evoked response of the RGC. Therefore, Apoj-/- mice were treated with low-dose efavirenz, an allosteric activator of CYP46A1 which converts cholesterol into 24-hydroxycholesterol. Efavirenz treatment increased retinal cholesterol and 24-hydroxycholesterol levels, normalized intraocular pressure and cup-to-disk ratio, and rescued in part RGC function. Retinal expression of Abcg1 (a cholesterol efflux transporter), Apoa1 (a constituent of lipoprotein particles), and Scarb1 (a lipoprotein particle receptor) was increased in EVF-treated Apoj-/- mice, indicating increased retinal cholesterol transport on lipoprotein particles. Ocular characterizations of Cyp46a1-/- mice supported the beneficial efavirenz treatment effects via CYP46A1 activation. The data obtained demonstrate an important APOJ role in retinal cholesterol homeostasis and link this apolipoprotein to the glaucoma risk factors and retinal 24-hydroxycholesterol production by CYP46A1. As the CYP46A1 activator efavirenz is an FDA-approved anti-HIV drug, our studies suggest a new therapeutic approach for treatment of glaucomatous conditions.

27-Hydroxycholesterol inhibits trophoblast fusion during placenta development by activating PI3K/AKT/mTOR signaling pathway.

Trophoblast cell syncytialization is essential for placental and fetal development. Abnormal trophoblast cell fusion leads to pregnancy pathologies, such as preeclampsia (PE), intrauterine growth restriction (IUGR), and miscarriage. 27-hydroxycholesterol (27-OHC) is the most abundant oxysterol in human peripheral blood synthesized by sterol 27-hydroxylase (CYP27A1) and is considered a critical mediator between hypercholesterolemia and a variety of related disorders. Gestational hypercholesterolemia was associated with spontaneous preterm delivery and low birth weight (LBW) in term infants, yet the mechanism is unclear. In this study, two trophoblast cell models and CD-1 mice were used to evaluate the effects of 27-OHC on trophoblast fusion during placenta development. Two different kinds of trophoblast cells received a dosage of 2.5, 5, or 10 uM 27-OHC. Three groups of pregnant mice were randomly assigned: control, full treatment (E0.5-E17.5), or late treatment (E13.5-E17.5). All mice received daily intraperitoneal injections of saline (control group) and 27-OHC (treatment group; 5.5 mg/kg). In vitro experiments, we found that 27-OHC inhibited trophoblast cell fusion in primary human trophoblasts (PHT) and forskolin (FSK)-induced BeWo cells. 27-OHC up-regulated the expression of the PI3K/AKT/mTOR signaling pathway-related proteins. Moreover, the PI3K inhibitor LY294002 rescued the inhibitory effect of 27-OHC. Inhibition of trophoblast cell fusion by 27-OHC was also observed in CD-1 mice. Furthermore, fetal weight and placental efficiency decreased and fetal blood vessel development was inhibited in pregnant mice treated with 27-OHC. This study was the first to prove that 27-OHC inhibits trophoblast cell fusion by Activating PI3K/AKT/mTOR signaling pathway. This study reveals a novel mechanism by which dyslipidemia during pregnancy results in adverse pregnancy outcomes.

Enzymatically Formed Oxysterols and Cell Death.

The side-chain hydroxylation of cholesterol by specific enzymes produces 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and other products. These enzymatically formed side-chain oxysterols act as intermediates in the biosynthesis of bile acids and serve as signaling molecules that regulate cholesterol homeostasis. Besides these intracellular functions, an imbalance in oxysterol homeostasis is implicated in pathophysiology. Furthermore, growing evidence reveals that oxysterols affect cell proliferation and cause cell death. This chapter provides an overview of the pathophysiological role of side-chain oxysterols in developing human diseases. We also summarize our understanding of the molecular mechanisms underlying the induction of various forms of cell death by side-chain oxysterols.

Obesity-Associated Breast Cancer: Analysis of Risk Factors and Current Clinical Evaluation.

Several studies show that a significantly stronger association is obvious between increased body mass index (BMI) and higher breast cancer incidence. Additionally, obese and postmenopausal women are at higher risk of all-cause and breast cancer-specific mortality compared with non-obese women with breast cancer. In this context, increased levels of estrogens, excessive aromatization activity of the adipose tissue, overexpression of pro-inflammatory cytokines, insulin resistance, adipocyte-derived adipokines, hypercholesterolemia, and excessive oxidative stress contribute to the development of breast cancer in obese women. Genetic evaluation is an integral part of diagnosis and treatment for patients with breast cancer. Despite trimodality therapy, the four-year cumulative incidence of regional recurrence is significantly higher. Axillary lymph nodes as well as primary lesions have diagnostic, prognostic, and therapeutic significance for the management of breast cancer. In clinical setting, because of the obese population primary lesions and enlarged lymph nodes could be less palpable, the diagnosis may be challenging due to misinterpretation of physical findings. Thereby, a nomogram has been created as the "Breast Imaging Reporting and Data System" (BI-RADS) to increase agreement and decision-making consistency between mammography and ultrasonography (USG) experts. Additionally, the "breast density classification system," "artificial intelligence risk scores," ligand-targeted receptor probes," "digital breast tomosynthesis," "diffusion-weighted imaging," "18F-fluoro-2-deoxy-D-glucose positron emission tomography," and "dynamic contrast-enhanced magnetic resonance imaging (MRI)" are important techniques for the earlier detection of breast cancers and to reduce false-positive results. A high concordance between estrogen receptor (ER) and progesterone receptor (PR) status evaluated in preoperative percutaneous core needle biopsy and surgical specimens is demonstrated. Breast cancer surgery has become increasingly conservative; however, mastectomy may be combined with any axillary procedures, such as sentinel lymph node biopsy (SLNB) and/or axillary lymph node dissection whenever is required. As a rule, SLNB-guided axillary dissection in breast cancer patients who have clinically axillary lymph node-positive to node-negative conversion following neoadjuvant chemotherapy is recommended, because lymphedema is the most debilitating complication after any axillary surgery. There is no clear consensus on the optimal treatment of occult breast cancer, which is much discussed today. Similarly, the current trend in metastatic breast cancer is that the main palliative treatment option is systemic therapy.

27-Hydroxycholesterol contributes to hypercholesterolemia-associated aggravation of apical periodontitis in ovariectomized rats and raloxifene counteracts its action.

AIM: The influence of hypercholesterolemia on the development of apical periodontitis (AP) is inconclusive. Recent studies revealed that cholesterol metabolite 27-hydoxycholesterol (27HC) can affect cellular responses to bacterial infections and oestrogen status and raloxifene may influence its action. Herein, we aimed to examine the impact of 27HC on production of inflammatory mediators by macrophages and the regulatory function of raloxifene. The contribution of 27HC to AP development and the therapeutic effect of raloxifene were evaluated in a rat model. METHODS: Murine macrophages J774 cells were used. The expression of inducible nitric oxide synthase (iNOS) was examined by Western blot. The concentrations of C-C motif chemokine ligand (CCL) 2 and 27HC were assessed by enzyme-linked immunosorbent assay. Colorimetric assay was used to evaluate cholesterol levels. Experimental AP was induced in ovariectomized (OVX) or un-operated rats receiving high-fat/high-cholesterol diet (HFHCD) or normal diet (ND). Micro-computed tomography and immunohistochemistry were employed to evaluate disease severity and the therapeutic effect of raloxifene. RESULTS: Cholesterol enhanced 27HC production in macrophages. 27HC induced iNOS and CCL2 synthesis by macrophages and estradiol suppressed the responses. In our animal model of AP, HFHCD plus OVX significantly augmented serum and lesion tissue levels of 27HC (p < .05 versus the ND group). Lesion size, infiltration of CD68+ cells, and iNOS+ monocytes were increased in parallel with 27HC accumulation. Raloxifene inhibited pro-inflammatory effects of 27HC on macrophages and suppressed AP progression in HFHCD/OVX rats (p < .05 versus the vehicle control group). CONCLUSIONS: Our results suggested that 27HC contributes to AP aggravation associated with hypercholesterolemia. Oestrogen deficiency may both enhance 27HC production and exacerbate its downstream action.

Cholesterol profiling reveals 7ß-hydroxycholesterol as a pathologically relevant peripheral biomarker of Alzheimer's disease.

AIM: Cholesterol homeostasis is associated with Alzheimer's disease (AD). Despite the multitude of cholesterol metabolites, little is known about which metabolites are directly involved in AD pathogenesis and can serve as its potential biomarkers. METHODS: To identify "hit" metabolites, steroid profiling was conducted in mice with different age, diet, and genotype and also in humans with normal cognition, mild cognitive impairment, and AD using gas chromatography-mass spectrometry. Then, using one of the "hit" molecules (7ß-hydroxycholesterol; OHC), molecular and histopathological experiment and behavioral testing were conducted in normal mice following its intracranial stereotaxic injection to see whether this molecule drives AD pathogenesis and causes cognitive impairment. RESULTS: The serum levels of several metabolites, including 7ß-OHC, were increased by aging in the 3xTg-AD unlike normal mice. Consistently, the levels of 7ß-OHC were increased in the hairs of patients with AD and were correlated with clinical severity. We found that 7ß-OHC directly affects AD-related pathophysiology; intrahippocampal injection of 7ß-OHC induced astrocyte and microglial cell activation, increased the levels of pro-inflammatory cytokines (TNF-alpha, IL-1ß, IL-6), and enhanced amyloidogenic pathway. Mice treated with 7ß-OHC also exhibited deficits in memory and frontal/executive functions assessed by object recognition and 5-choice serial reaction time task, respectively. CONCLUSIONS: Our results suggest that 7ß-OHC could serve as a convenient, peripheral biomarker of AD. As directly involved in AD pathogenesis, 7ß-OHC assay may help actualize personalized medicine in a way to identify an at-risk subgroup as a candidate population for statin-based AD treatment.

Plasma oxysterols are associated with serum lipids and dementia risk in older women.

INTRODUCTION: Apolipoprotein E4 (APOE4) carriers' tendency toward hypercholesterolemia may contribute to Alzheimer's disease (AD) risk through oxysterols, which traverse the blood-brain barrier. METHODS: Relationships between baseline plasma oxysterols, APOE status, serum lipids, and cognitive impairment risk were examined in 328 postmenopausal women from the Women's Health Initiative Memory Study. Women were followed for 25 years or until incident dementia or cognitive impairment. RESULTS: Levels of 24(S)-hydroxycholesterol (24-OHC), 27-hydroxycholesterol (27-OHC), and 24-OHC/27-OHC ratio did not differ by APOE status (p's > 0.05). Higher 24-OHC and 27-OHC were associated with higher total, low density lipoprotein (LDL), non-high density lipoprotein (HDL), remnant, LDL/HDL, and total/HDL cholesterol and triglycerides (p's < 0.05). Higher 24-OHC/27-OHC was associated with greater dementia risk (hazard ratio = 1.51, 95% confidence interval:1.02-2.22), which interaction analyses revealed as significant for APOE3 and APOE4+, but not APOE2+ carriers. DISCUSSION: Less favorable lipid profiles were associated with higher oxysterol levels. A higher ratio of 24-OHC/27-OHC may contribute to dementia risk in APOE3 and APOE4+ carriers.

Oxysterol Induces Expression of 60 kDa Chaperone Protein on Cell Surface of Microglia.

Microglia, essential immune cells in the brain, play crucial roles in neuroinflammation by performing various functions such as neurogenesis, synaptic pruning, and pathogen defense. These cells are activated by inflammatory factors like ß-amyloid (Aß) and oxysterols, leading to morphological and functional changes, including the secretion of inflammatory cytokines and the upregulation of MHC class II molecules. This study focused on identifying specific markers for microglial activation, with a particular emphasis on the roles of oxysterols in this process. We used the HMC3 human microglial cell line to investigate the induction of heat shock protein 60 (HSP60), a chaperonin protein by oxysterols, specifically in the presence of 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol). Our findings obtained by the proteomics approach revealed that these oxysterols significantly increased HSP60 expression on microglial cells. This induction was further confirmed using Western blot analysis and immunofluorescence microscopy. Additionally, Aß1-42 also promoted HSP60 expression, indicating its role as a microglial activator. HSP60 involved in protein folding and immune modulation was identified as a potential marker for microglial activation. This study underscores the importance of HSP60 in the inflammatory response of microglia, suggesting its utility as a target for new therapeutic approaches in neuroinflammatory diseases such as Alzheimer's disease (AD).

Lemon Flavonoid Extract Eriomin Improves Pro/Antioxidant Status and Interferes with Cholesterol Metabolism without Affecting Serum Cholesterol Levels in Aged Rats.

This study aimed to assess the antioxidant capacity of lemon flavonoid extract Eriomin® (LE) and its impact on cholesterol metabolism in the context of healthy aging. We orally treated 24-month-old male Wistar rats with an LE (40 mg/kg) suspended in 0.3 mL of sunflower oil. At the same time, control groups received an equal volume of sunflower oil (CON) or remained untreated (ICON) daily for 4 weeks. We examined LE's effects on superoxide dismutase and catalase- and glutathione-related enzyme activities, the concentration of lipid peroxides and protein carbonyls, total oxidant status (TOS) and antioxidant status (TAS), and oxidative stress index (OSI) in the liver, jejunum, and ileum. We also measured total cholesterol, its biosynthetic precursors (lanosterol, lathosterol, desmosterol), its degradation products (bile acid precursors) in the serum, liver, jejunum, and ileum, and serum phytosterols (intestinal absorption markers). LE reduced TOS, TAS, and OSI (p < 0.05) compared with control values, indicating its consistent antioxidant action in all examined organs. LE lowered hepatic desmosterol (p < 0.05) while also reducing 7α- and 24-hydroxycholesterol levels in the liver and ileum (p < 0.01). Serum cholesterol, hepatic gene expression, and the immunostaining intensity of CYP7A1 were unchanged. In conclusion, LE exerted non-enzymatic antioxidant effects and reduced cholesterol degradation, reducing its biosynthesis products, thereby maintaining serum cholesterol levels.