Maura Heverin

Crossing the barrier: net flux of 27-hydroxycholesterol into the human brain

Side chain oxidized oxysterols have a unique ability to traverse lipophilic membranes. We tested the hypothesis that there is a net flux of 27-hydroxycholesterol from the circulation into the brain using plasma samples collected from the internal jugular vein and an artery of healthy male volunteers. Two independent studies were performed, one in which total levels of 27-hydroxycholesterol were measured and one in which the free fraction of 27-hydroxycholesterol was measured. In the majority of subjects studied, the level of 27-hydroxycholesterol was higher in the artery than in the vein, and uptake from the circulation was calculated to be about 5 mg/24 h. The distribution of 27-hydroxycholesterol in human brain was found to be consistent with an extracerebral origin, with a concentration gradient from the white to the gray matter

Brain Cholesterol Synthesis in Mice Is Affected by High Dose of Simvastatin but Not of Pravastatin

On a global scale, there is an increasing tendency for a more aggressive treatment of hypercholesterolemia. Minor effects of statins on brain cholesterol metabolism have been reported in some in vivo animal studies, and it seems that this is due to a local effect of the drug. We treated male mice of the inbred strain C57/BL6 with a high daily dose of lipophilic simvastatin (100 mg/kg b.wt.) or hydrophilic pravastatin (200 mg/kg b.wt.) or vehicle (controls) by oral gavage for 3 days. To compare the impact of both statins on brain cholesterol synthesis and degradation, levels of cholesterol, its precursor lathosterol, and its brain metabolite 24(S)-hydroxycholesterol as well as statin concentrations were determined in whole-brain lipid extracts using mass spectrometry. The expression of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase mRNA and of other target genes were evaluated using real-time reverse transcription-polymerase chain reaction. In addition, analysis of liver and serum samples was performed. Similar levels of simvastatin and pravastatin were detected in whole-brain homogenates. Cholesterol contents in the brain, liver, and serum were not affected by high-dose statin treatment. Whereas brain cholesterol precursor levels were reduced in simvastatin-treated animals only, no effect was observed on the formation of the brain cholesterol metabolite, 24(S)-hydroxycholesterol. Polymerase chain reaction analysis revealed that mRNA expression of HMG-CoA reductase and ATP-binding cassette transporter A1 in the brain was significantly up-regulated in simvastatin-treated animals compared with pravastatin-treated or control animals. We conclude that, under the present experimental conditions, brain cholesterol synthesis is significantly affected by short-term treatment with high doses of lipophilic simvastatin, whereas whole-brain cholesterol turnover is not disturbed.

Oxysterols and Alzheimer's disease.

There is a clear link between cholesterol turnover and neurodegenerative diseases and hypercholesterolemia is an established risk factor for Alzheimers disease (AD). The failure to demonstrate a transfer of cholesterol from the circulation into the brain in humans and experimental animals makes it difficult to explain the link between hypercholesterolemia and AD. In contrast to cholesterol itself, side‐chain oxidized cholesterol metabolites such as 24S‐hydroxycholesterol and 27‐hydroxycholesterol are able to pass the blood–brain barrier (BBB). Formation of 24S‐hydroxycholesterol is the quantitatively most important mechanism for elimination of cholesterol from the brain and we recently demonstrated a significant net uptake of 27‐hydroxycholesterol by the brain from the circulation. We have also shown that patients with AD have increased brain levels of 27‐hydroxycholesterol, which may affect the production of β‐amyloid in the brain. The levels of 27‐hydroxycholesterol in the circulation are correlated with the levels of cholesterol and the possibility must be considered that the flux of 27‐hydroxycholesterol into the brain is the missing link between hypercholesterolemia and Alzheimers disease. Current knowledge about the role of the two oxysterols for cholesterol homeostasis in the brain as well as their diagnostic potential are reviewed.

Studies on the Transcriptional Regulation of Cholesterol 24-Hydroxylase (CYP46A1) MARKED INSENSITIVITY TOWARD DIFFERENT REGULATORY AXES

Mammalian CNS contains a disproportionally large and remarkably stable pool of cholesterol. Despite an efficient recycling there is some requirement for elimination of brain cholesterol. Conversion of cholesterol into 24S-hydroxycholesterol by the cholesterol 24-hydroxylase (CYP46A1) is the quantitatively most important mechanism. Based on the protein expression and plasma levels of 24S-hydroxycholesterol, CYP46A1 activity appears to be highly stable in adults. Here we have made a structural and functional characterization of the promoter of the human CYP46A1 gene. No canonical TATA or CAAT boxes were found in the promoter region. Moreover this region had a high GC content, a feature often found in genes considered to have a largely housekeeping function. A broad spectrum of regulatory axes using a variety of promoter constructs did not result in a significant transcriptional regulation. Oxidative stress caused a significant increase in transcriptional activity. The possibility of a substrate-dependent transcriptional regulation was explored in vivo in a sterol-deficient mouse model (Dhcr24 null) in which almost all cholesterol had been replaced with desmosterol, which is not a substrate for CYP46A1. Compared with heterozygous littermates there was no statistically significant difference in the mRNA levels of Cyp46a1. During the first 2 weeks of life in the wild-type mouse, however, a significant increase of Cyp46a1 mRNA levels was found, in parallel with an increase in 24S-hydroxycholesterol level and a reduction of cholesterol synthesis. The failure to demonstrate a significant transcriptional regulation under most conditions is discussed in relation to the turnover of brain and neuronal cholesterol.

Novel route for elimination of brain oxysterols across the blood-brain barrier: Conversion into 7α-hydroxy-3-oxo-4-cholestenoic acid

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4–5 mg/day. As the steady-state levels of this sterol are only 1–2 μg/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C27 steroidal acid 7α-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7α-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7α-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human. Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.

Studies on the Cholesterol-Free Mouse: Strong Activation of LXR-Regulated Hepatic Genes When Replacing Cholesterol With Desmosterol

Objective— Characterization of cholesterol homeostasis in male mice with a genetic inactivation of 3&bgr;-hydroxysteroid-&Dgr;24-reductase, causing replacement of almost all cholesterol with desmosterol. Methods and Results— There was an increase in hepatic sterol synthesis and markedly increased fecal loss of neutral sterols. Fecal excretion of bile acids was similar in knockout mice and in controls. The composition of bile acids was changed, with reduced formation of cholic acid. It was shown that both Cyp7a1 and Cyp27a1 are active toward desmosterol, consistent with the formation of normal bile acids from this steroid. The levels of plant sterols were markedly reduced. Hepatic mRNA levels of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase, Srebp-1c, Srebp-2, Cyp7a1, Abcg5, Abcg8, and Fas were all significantly increased. Conclusions— The changes in hepatic mRNA levels in combination with increased biliary and fecal excretion of neutral steroids, reduced tissue levels of plant sterols, increased plasma levels of triglyceride-rich VLDL, are consistent with a strong activation of LXR-targeted genes. The markedly increased fecal loss of neutral sterols may explain the fact that the Dhcr24−/− mice do not accumulate dietary cholesterol. The study illustrates the importance of the integrity of the cholesterol structure—presence of a double bond in the steroid side-chain is compatible with life but is associated with serious disturbances in sterol homeostasis.

Side Chain-oxidized Oxysterols Regulate the Brain Renin-Angiotensin System through a Liver X Receptor-dependent Mechanism

Disturbances in cholesterol metabolism have been associated with hypertension and neurodegenerative disorders. Because cholesterol metabolism in the brain is efficiently separated from plasma cholesterol by the blood-brain barrier (BBB), it is an unsolved paradox how high blood cholesterol can cause an effect in the brain. Here, we discuss the possibility that cholesterol metabolites permeable to the BBB might account for these effects. We show that 27-hydroxycholesterol (27-OH) and 24S-hydroxycholesterol (24S-OH) up-regulate the renin-angiotensin system (RAS) in the brain. Brains of mice on a cholesterol-enriched diet showed up-regulated angiotensin converting enzyme (ACE), angiotensinogen (AGT), and increased JAK/STAT activity. These effects were confirmed in in vitro studies with primary neurons and astrocytes exposed to 27-OH or 24S-OH, and were partially mediated by liver X receptors. In contrast, brain RAS activity was decreased in Cyp27a1-deficient mice, a model exhibiting reduced 27-OH production from cholesterol. Moreover, in humans, normocholesterolemic patients with elevated 27-OH levels, due to a CYP7B1 mutation, had markers of activated RAS in their cerebrospinal fluid. Our results demonstrate that side chain-oxidized oxysterols are modulators of brain RAS. Considering that levels of cholesterol and 27-OH correlate in the circulation and 27-OH can pass the BBB into the brain, we suggest that this cholesterol metabolite could be a link between high plasma cholesterol levels, hypertension, and neurodegeneration.

The desmosterolosis phenotype: spasticity, microcephaly and micrognathia with agenesis of corpus callosum and loss of white matter

Desmosterolosis is a rare autosomal recessive disorder of elevated levels of the cholesterol precursor desmosterol in plasma, tissue and cultured cells. With only two sporadic cases described to date with two very different phenotypes, the clinical entity arising from mutations in 24-dehydrocholesterol reductase (DHCR24) has yet to be defined. We now describe consanguineous Bedouin kindred with four surviving affected individuals, all presenting with severe failure to thrive, psychomotor retardation, microcephaly, micrognathia and spasticity with variable degree of hand contractures. Convulsions near birth, nystagmus and strabismus were found in most. Brain MRI demonstrated significant reduction in white matter and near agenesis of corpus callosum in all. Genome-wide linkage analysis and fine mapping defined a 6.75 cM disease-associated locus in chromosome 1 (maximum multipoint LOD score of six), and sequencing of candidate genes within this locus identified in the affected individuals a homozygous missense mutation in DHCR24 leading to dramatically augmented plasma desmosterol levels. We thus establish a clear consistent phenotype of desmosterolosis (MIM 602398).

On the regulatory role of side-chain hydroxylated oxysterols in the brain. Lessons from CYP27A1 transgenic and Cyp27a1−/− mice

The two oxysterols, 27-hydroxycholesterol (27OH) and 24S-hydroxycholesterol (24OH), are both inhibitors of cholesterol synthesis and activators of the liver X receptor (LXR) in vitro. Their role as physiological regulators under in vivo conditions is controversial, however. In the present work, we utilized a previously described mouse model with overexpressed human sterol 27-hydroxylase (CYP27A1). The levels of 27OH were increased about 12-fold in the brain. The brain levels of HMG-CoA reductase mRNA and HMG-CoA synthase mRNA levels were increased. In accordance with increased cholesterol synthesis, most of the cholesterol precursors were also increased. The level of 24OH, the dominating oxysterol in the brain, was decreased by about 25%, most probably due to increased metabolism by CYP27A1. The LXR target genes were unaffected or slightly changed in a direction opposite to that expected for LXR activation. In the brain of Cyp27−/− mice, cholesterol synthesis was slightly increased, with increased levels of cholesterol precursors but normal mRNA levels of HMG-CoA reductase and HMG-CoA synthase. The mRNA levels corresponding to LXR target genes were not affected. The results are consistent with the possibility that both 24OH and 27OH are physiological suppressors of cholesterol synthesis in the brain. The results do not support the contention that 27OH is a general activator of LXR target genes in this organ.

Differential diagnosis in patients with suspected bile acid synthesis defects

AIM To investigate the clinical presentations associated with bile acid synthesis defects and to describe identification of individual disorders and diagnostic pitfalls. METHODS Authors describe semiquantitative determination of 16 urinary bile acid metabolites by electrospray ionization-tandem mass spectrometry. Sample preparation was performed by solid-phase extraction. The total analysis time was 2 min per sample. Authors determined bile acid metabolites in 363 patients with suspected defects in bile acid metabolism. RESULTS Abnormal bile acid metabolites were found in 36 patients. Two patients had bile acid synthesis defects but presented with atypical presentations. In 2 other patients who were later shown to be affected by biliary atresia and cystic fibrosis the profile of bile acid metabolites was initially suggestive of a bile acid synthesis defect. Three adult patients suffered from cerebrotendinous xanthomatosis. Nineteen patients had peroxisomal disorders, and 10 patients had cholestatic hepatopathy of other cause. CONCLUSION Screening for urinary cholanoids should be done in every infant with cholestatic hepatopathy as well as in children with progressive neurological disease to provide specific therapy.