Oxidized Cholesterol Metabolites Research Articles

Associations between lifestyle, health, and clinical characteristics and circulating oxysterols and cholesterol precursors in women diagnosed with breast cancer: a cross-sectional study

Despite increasing evidence that cholesterol precursors and oxysterols, oxidized cholesterol metabolites, play a role in numerous pathological processes and diseases including breast cancer, little is known about correlates of these sterols in women with breast cancer. In this study, 2282 women with breast cancer and blood draw post diagnosis were included and cross-sectional associations between circulating levels of 15 sterols/oxysterols and (a) lifestyle, anthropometric, reproductive characteristics, (b) comorbidities and medication use, and (c) breast cancer tumor and treatment characteristics were calculated using generalized linear models. Obesity was strongly associated with circulating levels of 7-dehydrocholesterol (DC) (body mass index ≥ 30 vs. 18.5–24.9 kg/m2: 51.7% difference) and 7-ketocholesterol (KC) (40.0% difference). After adjustment for BMI, comorbidities such as cardiovascular disease were associated with higher levels of 7-DC (26.1% difference) and lower levels of desmosterol (− 16.4% difference). Breast cancer tumor characteristics including hormone receptor status, tumor stage, and endocrine therapy were associated with lanosterol, 24-DHLan, 7b-HC, and THC (e.g., THC; tumor stage IIIa vs. I: 36.9% difference). Weaker associations were observed for lifestyle characteristics and for any of the other oxysterols. The findings of this study suggest that cholesterol precursors are strongly associated with metabolic factors, while oxysterols are associated with breast cancer tumor characteristics, warranting further investigation into the role of cholesterol precursors and oxysterols in women with breast cancer and other populations.

24S-Hydroxycholesterol in Neuropsychiatric Diseases: Schizophrenia, Autism Spectrum Disorder, and Bipolar Disorder.

Neuropsychiatric diseases (NPDs) are severe, debilitating psychiatric conditions that affect the nervous system. These are among the most challenging disorders in medicine. Some examples include Alzheimer's, anxiety disorders, autism spectrum disorder, bipolar disorder, and schizophrenia. NPDs represent an ever-increasing burden on public health and are prevalent throughout the world. For most of these diseases, the particular etiopathogeneses are still enigmatic. NPDs are also associated with structural and functional changes in the brain, along with altered neurotransmitter and neuroendocrine systems.Approximately 25% of the total human body cholesterol is located in the brain. Its involvement in neuronal functions starts in the early growth stages and remains important throughout adulthood. It is also an integral part of the neuronal membrane, ensuring membrane lipid organization and regulating membrane fluidity. The main mechanism for removing cholesterol from the brain is cholesterol 24-hydroxylation by cytochrome P450 46A1 (CYP46A1), an enzyme specifically found in the central nervous system. Although research on 24S-OHC and its role in neuropsychiatric diseases is still in its early stages, this oxidized cholesterol metabolite is thought to play a crucial role in the etiology of NPDs. 24S-OHC can affect neurons, astrocytes, oligodendrocytes, and vascular cells. In addition to regulating the homeostasis of cholesterol in the brain, this oxysterol is involved in neurotransmission, oxidative stress, and inflammation. The role of 24S-OHC in NPDs has been found to be controversial in terms of the findings so far. There are several intriguing discrepancies in the data gathered so far regarding 24S-OHC and NPDs. In fact, 24S-OHC levels were reported to have decreased in a number of NPDs and increased in others.Hence, in this chapter, we first summarize the available data regarding 24S-OHC as a biomarker in NPDs, including schizophrenia, autism spectrum disorder, and bipolar disorder. Then, we present a brief synopsis of the pharmacological targeting of 24S-OHC levels through the modulation of CYP46A1 activity.

Abstract 3070: Leveraging oxysterol sulfation by SULT2B1b sulfotransferase and liver X receptor inactivation to suppress advanced prostate cancer

Abstract Advanced prostate cancer is a heterogeneous disease driven by diverse genetic alterations, with metastasis and lethal progression as the inevitable outcome. We observed that a subset of clinical primary prostate cancer is markedly reduced for SULT2B1b (SULT2B) sulfotransferase, which is an oxysterol-sulfating enzyme. Metastasized castration-resistant prostate cancer (CRPC) uniformly lacks SULT2B. Oxysterols are oxidized cholesterol metabolites and in agonist function, they activate liver X receptors (LXRα/β), which are members of the nuclear receptor family and regulators of lipogenesis, cholesterol homeostasis and immune functions. LXR signaling is enzymatically inhibited by SULT2B activity since sulfated oxysterols are LXR-inert. We previously reported that genetic silencing of SULT2B led to escalated CRPC proliferation; enhanced xenograft tumor growth; increased cell migration, invasion; reduced stiffness, adhesiveness; and EMT activation. SULT2B-depleted CRPC cells showed robust upregulation of AKR1C3 aldo-keto reductase and AKR1C3-dependent activation of the ERK1/2 survival signal. AKR1C3 regulates intratumoral androgen biosynthesis, and it is known to be elevated in advanced prostate cancer. In the current study, we validate our previous result by showing that ectopic expression of SULT2B caused AKR1C3 reduction in CRPC cells. Mechanistically, we observed that enhanced AKR1C3 expression in SULT2B-depleted cells was prevented upon genetic or pharmacologic inactivation of LXR. In addition, ERRα, another non-steroid nuclear receptor, was reduced in LXR-inactivated cells and notably, AKR1C3 was reduced in CRPC cells upon ERRα degradation by an ERRα-selective PROTAC. The LXR dependence of ERRα and AKR1C3 expression was observed in both androgen receptor expressing and non-expressing CRPC cells. Conclusion & Translational Significance: Above results reveal a novel LXR-ERRα-AKR1C3-ERK axis which is activated in SULT2B-deficient CRPC cells irrespective of the androgen receptor status of these cells. Notably, elevated ERRα portends poor prognosis for prostate cancer and cholesterol is an ERRα agonist. Considering that statin can diminish ERRα activity (PMID 26777690) and AKR1C3 inhibitors are in clinical trials, this novel axis revealed from our study may be actionable for the clinical interception of the progression in SULT2B-low advanced prostate cancer. Citation Format: Bobae Park, Katarzyna Knapczyk-Stwora, Yaguang Liu, Chung S. Song, Bandana Chatterjee. Leveraging oxysterol sulfation by SULT2B1b sulfotransferase and liver X receptor inactivation to suppress advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3070.

27-hydroxycholesterol promotes oligodendrocyte maturation: Implications for hypercholesterolemia-associated brain white matter changes.

Oxidized cholesterol metabolite 27-hydroxycholesterol (27-OH) is a potential link between hypercholesterolemia and neurodegenerative diseases since unlike peripheral cholesterol, 27-OH is transported across the blood-brain barrier. However, the effects of high 27-OH levels on oligodendrocyte function remain unexplored. We hypothesize that during hypercholesterolemia 27-OH may impact oligodendrocytes and myelin and thus contribute to the disconnection of neural networks in neurodegenerative diseases. To test this idea, we first investigated the effects of 27-OH in cultured oligodendrocytes and found that it induces cell death of immature O4+ /GalC+ oligodendrocytes along with stimulating differentiation of PDGFR+ oligodendrocyte progenitors (OPCs). Next, transgenic mice with increased systemic 27-OH levels (Cyp27Tg) underwent behavioral testing and their brains were immunohistochemically stained and lysed for immunoblotting. Chronic exposure to 27-OH in mice resulted in increased myelin basic protein (MBP) but not 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) or myelin oligodendrocyte glycoprotein (MOG) levels in the corpus callosum and cerebral cortex. Intriguingly, we also found impairment of spatial learning suggesting that subtle changes in myelinated axons of vulnerable areas like the hippocampus caused by 27-OH may contribute to impaired cognition. Finally, we found that 27-OH levels in cerebrospinal fluid from memory clinic patients were associated with levels of the myelination regulating CNPase, independently of Alzheimer's disease markers. Thus, 27-OH promotes OPC differentiation and is toxic to immature oligodendrocytes as well as it subtly alters myelin by targeting oligodendroglia. Taken together, these data indicate that hypercholesterolemia-derived higher 27-OH levels change the oligodendrocytic capacity for appropriate myelin remodeling which is a crucial factor in neurodegeneration and aging.

27‐hydroxycholesterol promotes oligodendrocytic maturation: Implications for Alzheimer’s disease

Midlife hypercholesterolemia is an established risk factor for developing Alzheimer's disease (AD), still peripheral cholesterol does not pass the blood-brain-barrier (BBB). A proposed factor linking elevated systemic cholesterol levels and AD is the oxidized cholesterol metabolite 27-hydroxycholesterol (27-OH) that can pass the BBB. Abnormal myelin structure, resulting in the disconnection of neural networks, is an early phenomenon in AD. It is known that high levels of 27-OH has a negative impact on the brain, yet its effect on oligodendrocytes remains unexplored. The aim of this project was to determine whether 27-OH affects brain myelination. Effects of 27-OH treatment was investigated both in a human cell line and primary cell cultures from mice using mono- and co-culture settings. Behavioural testing of transgenic mice with increased systemic 27-OH levels (CYP27A1TG) compared to wild type mice was performed and oligodendrocytic markers were examined by immunohistochemical staining and Western blot. Finally, cerebrospinal fluid (CSF) samples from subjective controls (SCI), mild cognitive impairment (MCI) and AD memory clinic patients were analysed for associations between MMSE and RAVLT scores, AD biomarkers, 27-OH, cholesterol, and myelin proteins. High concentrations of 27-OH induce cell death in monocultured human oligodendroglioma cells and primary oligodendrocytes. In 3D co-cultures from embryonic hippocampus and cortex, 27-OH treatment stimulated oligodendrocyte maturation and not proliferation. CYP27A1TG exhibit cognitive impairment and levels of Myelin Basic Protein (MBP) were increased in brain sections from TG mice compared to wild type, still overall myelin structure was unaltered. Patient CSF levels of the myelination regulating enzyme CNPase was associated to levels of 27-OH and AD biomarkers. CSF levels of CNPase were increased in MCI compared to SCI but not to AD. The cholesterol metabolism associated 27-OH alters the rate of differentiation from oligodendrocyte progenitor cells into mature oligodendrocytes without increasing proliferation rate, suggesting that 27-OH may consequently reduce oligodendrocytic ability for appropriate remodelling in the aging brain. Maintaining or restoring a functional population of oligodendrocyte progenitor cells could possibly diminish the changes in white matter integrity seen in AD, and furthermore support axonal survival.

7-ketocholesterol enhances autophagy via the ROS-TFEB signaling pathway in osteoclasts

Oxysterols play a critical role in human health and diseases associated with high cholesterol and oxidative stress. Given that a positive correlation was observed between cholesterol and collagen type 1 fragment (CTX-1) or serum reactive oxygen species (ROS) in humans, we hypothesized that oxidized cholesterol metabolites may participate in cholesterol-induced bone loss. Therefore, this study aimed to identify the metabolite responsible for cholesterol-associated bone loss and evaluate its effect on osteoclasts (OCs) leading to bone loss. An atherogenic diet in mice increased the levels of the oxysterol, 7-ketocholesterol (7-KC) in bone, as well as serum ROS. 7-KC increased the number and activity of OCs by enhancing autophagy via the ROS-transcription factor EB signaling pathway. These findings suggest that 7-KC acts as a cholesterol metabolite and is at least partially responsible for cholesterol-induced bone loss by inducing autophagy in OCs.

Sterol auto-oxidation adversely affects human motor neuron viability and is a neuropathological feature of amyotrophic lateral sclerosis

Aberrant cholesterol homeostasis is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal neuromuscular disease that is due to motor neuron (MN) death. Cellular toxicity from excess cholesterol is averted when it is enzymatically oxidized to oxysterols and bile acids (BAs) to promote its removal. In contrast, the auto oxidation of excess cholesterol is often detrimental to cellular survival. Although oxidized metabolites of cholesterol are altered in the blood and CSF of ALS patients, it is unknown if increased cholesterol oxidation occurs in the SC during ALS, and if exposure to oxidized cholesterol metabolites affects human MN viability. Here, we show that in the SOD1G93A mouse model of ALS that several oxysterols, BAs and auto oxidized sterols are increased in the lumbar SC, plasma, and feces during disease. Similar changes in cholesterol oxidation were found in the cervical SC of sporadic ALS patients. Notably, auto-oxidized sterols, but not oxysterols and BAs, were toxic to iPSC derived human MNs. Thus, increased cholesterol oxidation is a manifestation of ALS and non-regulated sterol oxidation likely contributes to MN death. Developing therapeutic approaches to restore cholesterol homeostasis in the SC may lead to a treatment for ALS.

Diverse Immunoregulatory Roles of Oxysterols—The Oxidized Cholesterol Metabolites

Intermediates of both cholesterol synthesis and cholesterol metabolism can have diverse roles in the control of cellular processes that go beyond the control of cholesterol homeostasis. For example, oxidized forms of cholesterol, called oxysterols have functions ranging from the control of gene expression, signal transduction and cell migration. This is of particular interest in the context of immunology and immunometabolism where we now know that metabolic processes are key towards shaping the nature of immune responses. Equally, aberrant metabolic processes including altered cholesterol homeostasis contribute to immune dysregulation and dysfunction in pathological situations. This review article brings together our current understanding of how oxysterols affect the control of immune responses in diverse immunological settings.

Cholestenoic acid analogues as inverse agonists of the liver X receptors

Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action.

Estimating fetal cholesterol synthesis rates by cord blood analysis in intrauterine growth restriction and normally grown fetuses

BackgroundCholesterol is an essential component in human development. In fetuses affected by intrauterine growth restriction (IUGR), fetal blood cholesterol levels are low. Whether this is the result of a reduced materno-fetal cholesterol transport, or due to low fetal de novo synthesis rates, remains a matter of debate. By analyzing cholesterol interbolites and plant sterols we aimed at deeper insights into transplacental cholesterol transport and fetal cholesterol handling in IUGR with potential targets for future therapy. We hypothesized that placental insufficiency results in a diminished cholesterol supply to the fetus.MethodsVenous umbilical cord sera were sampled post-partum from fetuses delivered between 24 weeks of gestation and at full term. IUGR fetuses were matched to 49 adequate-for-age delivered preterm and term neonates (CTRL) according to gestational age at delivery. Cholesterol was measured by gas chromatography-flame ionization detection using 5a-cholestane as internal standard. Cholesterol precursors and synthesis markers, such as lanosterol, lathosterol, and desmosterol, the absorption markers, 5α-cholestanol and plant sterols, such as campesterol and sitosterol, as well as enzymatically oxidized cholesterol metabolites (oxysterols), such as 24S- or 27-hydroxycholesterol, were analyzed by gas chromatography-mass spectrometry, using epicoprostanol as internal standard for the non-cholesterol sterols and deuterium labeled oxysterols for 24S- and 27-hydroxycholesterol.ResultsMean cholesterol levels were 25% lower in IUGR compared with CTRL (p < 0.0001). Lanosterol and lathosterol to cholesterol ratios were similar in IUGR and CTRL. In relation to cholesterol mean, desmosterol, 24S-hydroxycholesterol, and 27-hydroxycholesterol levels were higher by 30.0, 39.1 and 60.7%, respectively, in IUGR compared to CTRL (p < 0.0001). Equally, 5α-cholestanol, campesterol, and β-sitosterol to cholesterol ratios were higher in IUGR than in CTRL (17.2%, p < 0.004; 33.5%, p < 0.002; 29.3%, p < 0.021).ConclusionsCholesterol deficiency in IUGR is the result of diminished fetal de novo synthesis rates rather than diminished maternal supply. However, increased oxysterol- and phytosterol to cholesterol ratios suggest a lower sterol elimination rate. This is likely caused by a restricted hepatobiliary function. Understanding the fetal cholesterol metabolism is important, not only for neonatal nutrition, but also for the development of strategies to reduce the known risk of future cardiovascular diseases in the IUGR fetus.

Cellular hormetic response to 27-hydroxycholesterol promotes neuroprotection through AICD induction of MAST4 abundance and kinase activity

The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer’s Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.

Abstract C153: New approaches to target mevalonate biosynthesis in cancer.

Abstract To support rapid growth, tumors need to synthesize lipids and cholesterol, and stopping production of these metabolites can inhibit tumors. Statins are among the most prescribed and well-tolerated drugs in the world to treat cardiovascular disease by inhibiting cholesterol synthesis. Statins are known to be toxic to cancer cells and have attracted renewed attention because of the increased appreciation of the importance of the regulation of metabolism in tumors. Statins have not progressed beyond clinical trials because some patients significantly benefit, while others do not in a range of cancers. Our strategy is to improve our understanding of the mechanisms of tumor responses to statins and thereby enhance statin toxicity to cancer cells. We have found that combining statins with oxysterols synergistically enhances statin toxicity by 10-fold. Oxysterols are oxidized cholesterol metabolites that naturally occur during cholesterol synthesis. We hypothesize that oxysterols inhibit the expression of cholesterol synthesis through a master regulator of metabolism, SREBP-2, making cells more sensitive to statins. We find that oxysterols enhance statin induced apoptosis and suppress the expression of SREBP-2 along with SREBP-2 targets including HMGCR. We have tested a number of oxysterols that work with a range of efficacies in seven representative ovarian cancer cell lines. P53 status is not a major factor in mediating oxysterol activity. Oxysterols inhibit expression of SREBP-2 leading to lower expression of the statin target, HMGCR, along with lower expression of most of the mevalonate and cholesterol biosynthesis pathways. These observations suggests that the cell's response to statins and not initial expression levels may be most critical to determine which patients may benefit from statin therapy. Current studies are testing the combination in mouse models and evaluating the function and regulation of SREBP-2 in ovarian cancer. In summary, we have discovered an approach to suppress cancer cell's response to statins leading to greatly improved statin efficacy highlighting the importance of the mevalonate and cholesterol pathways in mediating growth and survival. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C153. Citation Format: Alexander Brodsky, Cinzia Casella. New approaches to target mevalonate biosynthesis in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C153.

Abstract A40: New approaches to target mevalonate biosynthesis

Abstract To support rapid growth, tumors need to synthesize lipids and cholesterol, and stopping production of these metabolites can inhibit tumors. Statins are among the most prescribed and well-tolerated drugs in the world to treat cardiovascular disease by inhibiting cholesterol synthesis. Statins are known to be toxic to cancer cells and have attracted renewed attention because of the increased appreciation of the importance of the regulation of metabolism in tumors. Statins have not progressed beyond clinical trials because some patients significantly benefit, while others do not in a range of cancers. Our strategy is to improve our understanding of the mechanisms of tumor responses to statins and thereby enhance statin toxicity to cancer cells. We have found that combining statins with oxysterols synergistically enhances statin toxicity by 10-fold. Oxysterols are oxidized cholesterol metabolites that naturally occur during cholesterol synthesis. We hypothesize that oxysterols inhibit the expression of cholesterol synthesis through a master regulator of metabolism, SREBP-2, making cells more sensitive to statins. We find that oxysterols enhance statin induced apoptosis and suppress the expression of SREBP-2 along with SREBP-2 targets including HMGCR. We have tested a number of oxysterols that work with a range of efficacies in seven representative ovarian cancer cell lines. P53 status is not a major factor in mediating oxysterol activity. Oxysterols inhibit expression of SREBP-2 leading to lower expression of the statin target, HMGCR, along with lower expression of most of the mevalonate and cholesterol biosynthesis pathways. These observations suggests that the cell's response to statins and not initial expression levels may be most critical to determine which patients may benefit from statin therapy. Current studies are testing the combination in mouse models and evaluating the function and regulation of SREBP-2 in ovarian cancer. In summary, we have discovered an approach to suppress cancer cell's response to statins leading to greatly improved statin efficacy highlighting the importance of the mevalonate and cholesterol pathways in mediating growth and survival. Citation Format: Cinzia Casella, Alexander S. Brodsky. New approaches to target mevalonate biosynthesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A40.

Differences in brain cholesterol metabolism and insulin in two subgroups of patients with different CSF biomarkers but similar white matter lesions suggest different pathogenic mechanisms

Investigate possible associations of white matter hyperintensities (WMHs) with the metabolism of cholesterol and insulin in two subgroups of patients with memory complaints and different CSF Aβ42 and CSF tau levels. 59 patients from the memory clinic at Karolinska Hospital were included. Degree of WMHs was rated using the ARWMC scale and the following biomarkers were measured in CSF and plasma: insulin, cholesterol, lanosterol, lathosterol, and oxidized cholesterol metabolites. The WMHs in CSF control-like group correlated with increased brain cholesterol synthesis and reduced efflux of oxysterols and insulin in CSF. In the CSF AD-like group, the WMHs correlated with increased peripheral cholesterol metabolism. Despite having similar appearance on FLAIR images, the pathogenic mechanisms of WMHS are likely to be different in the two groups investigated.

Oxysterols exert proinflammatory effects in placental trophoblasts via TLR4-dependent, cholesterol-sensitive activation of NF- B

Oxidized cholesterol metabolites (oxysterols) promote inflammation in a variety of cell types and are thought to be involved in a number of disease pathologies. Oxysterol concentrations are increased in pregnancy, together with systemic oxidative stress and inflammation. We tested the hypothesis that oxysterols 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-ketoC) promote placental trophoblast inflammation, and determined the mechanisms involved. Treatment of primary trophoblasts in culture with 25-OHC and 7-ketoC increased the production of proinflammatory cytokines (interleukin-6, macrophage inflammatory protein-1β and tumour necrosis factor-α) in a concentration-dependent fashion. Inhibition of TLR4 activation using selective inhibitors of TLR4 complex formation (OxPAPC) or signalling transmission (CLI095) prevented lipopolysaccharide (LPS)- and oxysterol-induced inflammatory cytokine production. Pretreatment of trophoblasts with selective inhibitors of I-kB kinase activity (parthenolide and TPCA-1) reduced oxysterol- and LPS-stimulated inflammatory responses, consistent with the involvement of the nuclear factor kappa B (NF-κB) pathway downstream of TLR4 signalling. Both oxysterols also increased the phosphorylation and nuclear localization of NF-κB subunit p65/RelA. Oxysterols are also known to activate liver X receptors (LXRs) which can inhibit inflammatory signalling, either directly or indirectly via membrane cholesterol reduction. Treatment with the LXR agonist, T0901317, exerted significant anti-inflammatory effects, reducing LPS- and oxysterol-driven cytokine production. Treatment with methyl-β-cyclodextrin to deplete membrane microdomain cholesterol and thereby disrupt TLR4 signalling, similarly abrogated their effects. Together, these findings indicate that although oxysterols likely activate both pro- and anti-inflammatory pathways in the placenta, the predominant effect is the promotion of placental inflammation via TLR4-dependent activation of NF-κB.

Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer's patients with the Swedish APP 670/671 mutation

There is a significant flux of the neurotoxic oxysterol 27-hydroxycholesterol (27OHC) from the circulation across the blood-brain barrier. Because there is a correlation between 27OHC and cholesterol in the circulation and lipoprotein-bound cholesterol does not pass the blood-brain barrier, we have suggested that 27OHC may mediate the effects of hypercholesterolemia on the brain. We previously demonstrated a modest accumulation of 27OHC in brains of patients with sporadic Alzheimer's disease (AD), consistent with a role of 27OHC as a primary pathogenetic factor. We show here that there is a 4-fold accumulation of 27OHC in different regions of the cortexes of patients carrying the Swedish amyloid precursor protein (APPswe) 670/671 mutation. The brain levels of sitosterol and campesterol were not significantly different in the AD patients compared with the controls, suggesting that the blood-brain barrier was intact in the AD patients. We conclude that accumulation of 27OHC is likely to be secondary to neurodegeneration, possibly a result of reduced activity of CYP7B1, the neuronal enzyme responsible for metabolism of 27OHC. We discuss the possibility of a vicious circle in the brains of the patients with familial AD whereby neurodegenerative changes cause an accumulation of 27OHC that further accelerates neurodegeneration.

Inhibition of endothelial- and neuronal-type, but not inducible-type, nitric oxide synthase by the oxidized cholesterol metabolite secosterol aldehyde: Implications for vascular and neurodegenerative diseases

The cholesterol ozonolysis products secosterol-A and its aldolization product secosterol-B were recently detected in human atherosclerotic tissues and brain specimens, and have been postulated to play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. We examined several oxidized cholesterol metabolites including secosterol-A, secosterol-B, 25-hydroxycholesterol, 5β,6β-epoxycholesterol and 7-ketocholesterol for their effects on the activities of three nitric oxide synthases. In contrast to other oxidized metabolites, secosterol-A was found to be a potent inhibitor against the neuronal- and endothelial-type, but not the inducible-type nitric oxide synthase, with IC50 values of 22 ± 1 and 50 ± 5 µM, respectively. The calmodulin-binding regions of the neuronal- and endothelial-nitric oxide synthases contain lysine residues which are not present in the inducible-type nitric oxide synthase. Secosterol-A modifies proteins through the formation of a Schiff base with the lysine epsilon-amino group. It is possible that secosterol-A modifies lysine residues of constitutive nitric oxide synthases, leading to the inhibition of enzymatic activities. As nitric oxide is a critical signaling molecule in vascular function and in long-term potentiation, its reduced production through inhibition of constitutive nitric oxide synthases by secosterol-A may contribute to the development of atherosclerosis and memory impairment in particular neurodegenerative diseases.

The effect of APOE genotype on brain levels of oxysterols in young and old human APOE ε2, ε3 and ε4 knock-in mice

Despite apolipoprotein E's important role in cholesterol transport and metabolism in the brain as well as its influence on Alzheimer's disease, the impact of the human APOE genotype on cholesterol metabolism in brain has not been fully examined. This study was carried out to investigate APOE genotype effects on oxysterols measured. In this study the measurement of cholesterol and several oxysterols in the brains of human APOE ε2, ε3 and ε4 knock-in mice at 8 weeks and 1 year of age using gas chromatography mass spectrometry (GC–MS) demonstrated no APOE genotype or age effect on total brain cholesterol and the oxysterol 24-hydroxycholesterol. The level of 27-hydroxycholesterol was elevated in 1 year old animals for all APOE genotypes. Interestingly, lathosterol an indicator of cholesterol synthesis was significantly reduced in the 1 year old animals for all APOE genotypes. APOE ε4 expressing mice exhibited statistically lower levels of lathosterol compared to APOE ε2 in both the young and old mice. Oxidized cholesterol metabolites were significantly lower in APOE ε2 mice compared to other genotypes at 8 weeks old. Although minimal differences were observed between APOE E3 and E4 knock-in (KI) mice, these findings indicate that there are some clear APOE genotype specific effects on brain cholesterol synthesis and associated metabolic pathways, particularly in APOE ε2 KI mice.

Leptin Reduces the Accumulation of Aβ and Phosphorylated Tau Induced by 27-Hydroxycholesterol in Rabbit Organotypic Slices

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.

Lovastatin ameliorates α-synuclein accumulation and oxidation in transgenic mouse models of α-synucleinopathies

Alpha-synuclein (α-syn) aggregation is a neuropathological hallmark of many diseases including Dementia with Lewy Bodies (DLB) and Parkinson's Disease (PD), collectively termed the α-synucleinopathies. The mechanisms underlying α-syn aggregation remain elusive though emerging science has hypothesized that the interaction between cholesterol and α-syn may play a role. Cholesterol has been linked to α-synucleinopathies by recent work suggesting cholesterol metabolites appear to accelerate α-syn fibrillization. Consistent with these findings, cholesterol-lowering agents have been demonstrated to reduce α-syn accumulation and the associated neuronal pathology in vitro. In this context, this study sought to investigate the in vivo effects of the cholesterol synthesis inhibitor lovastatin on α-syn aggregation in two different transgenic (Tg) mouse models that neuronally overexpress human α-syn. Lovastatin-treated mice displayed significantly reduced plasma cholesterol levels and levels of oxidized cholesterol metabolites in the brain in comparison to saline-treated controls. Immunohistochemical analysis demonstrated a significant reduction of neuronal α-syn aggregates and α-syn immunoreactive neuropil in the temporal cortex of lovastatin-treated Tg mice in comparison to saline-treated α-syn Tg controls. Consistently, immunoblot analysis of mouse brain homogenates showed a reduction in levels of total and oxidized α-syn in lovastatin-treated α-syn Tg mice in comparison to saline-treated α-syn Tg controls. The reduced α-syn accumulation in lovastatin-treated mice was associated with abrogation of neuronal pathology. The results from this study demonstrate that lovastatin administration can reduce α-syn aggregation and associated neuropathology and support the possibility that treatment with cholesterol-lowering agents may be beneficial for patients with PD and/or DLB.