Lionel Bretillon

Antiepileptic Drugs Increase Plasma Levels of 4β-Hydroxycholesterolin Humans EVIDENCE FOR INVOLVEMENT OF CYTOCHROME P450 3A4

The major cholesterol oxidation products in the human circulation are 27-hydroxycholesterol, 24-hydroxycholesterol, and 7α-hydroxycholesterol. These oxysterols are formed from cholesterol by specific cytochrome P450 enzymes, CYP27, CYP46, and CYP7A, respectively. An additional oxysterol present in concentrations comparable with 7α- and 24-hydroxycholesterol is 4β-hydroxycholesterol. We now report that patients treated with the antiepileptic drugs phenobarbital, carbamazepine, or phenytoin have highly elevated levels of plasma 4β-hydroxycholesterol. When patients with uncomplicated cholesterol gallstone disease were treated with ursodeoxycholic acid, plasma 4β-hydroxycholesterol increased by 45%. Ursodeoxycholic acid, as well as the antiepileptic drugs, are known to induce cytochrome P450 3A. Recombinant CYP3A4 was shown to convert cholesterol to 4β-hydroxycholesterol, whereas no conversion was observed with CYP1A2, CYP2C9, or CYP2B6. The concentration of 4α-hydroxycholesterol in plasma was lower than the concentration of 4β-hydroxycholesterol and not affected by treatment with the antiepileptic drugs or ursodeoxycholic acid. Together, these data suggest that 4β-hydroxycholesterol in human circulation is formed by a cytochrome P450 enzyme.

Nutritional omega-3 deficiency abolishes endocannabinoid-mediated neuronal functions

The corollaries of the obesity epidemic that plagues developed societies are malnutrition and resulting biochemical imbalances. Low levels of essential n-3 polyunsaturated fatty acids (n-3 PUFAs) have been linked to neuropsychiatric diseases, but the underlying synaptic alterations are mostly unknown. We found that lifelong n-3 PUFAs dietary insufficiency specifically ablates long-term synaptic depression mediated by endocannabinoids in the prelimbic prefrontal cortex and accumbens. In n-3–deficient mice, presynaptic cannabinoid CB1 receptors (CB1Rs) normally responding to endocannabinoids were uncoupled from their effector Gi/o proteins. Finally, the dietary-induced reduction of CB1R functions in mood-controlling structures was associated with impaired emotional behavior. These findings identify a plausible synaptic substrate for the behavioral alterations caused by the n-3 PUFAs deficiency that is often observed in western diets.

Effects of conjugated linoleic acid isomers on the hepatic microsomal desaturation activities in vitro

The influence of individual conjugated linoleic acid (CLA) isomers on the Δ6 desaturation of linoleic and α-linolenic acids and on the Δ9 desaturation of stearic acid was investigated in vitro, using rat liver microsomes. The Δ6 desaturation of 18∶2n−6 was decreased from 23 to 38% when the ratio of 9cis,11trans-18∶2 to 18∶2n−6 increased from 0.5 to 2. The compound 10trans,12cis-18∶2 exhibited a similar effect only at the highest concentration. The Δ6 desaturation of α-linolenic acid was slightly affected by the presence of CLA isomers. The sole isomer to induce an inhibitory effect on the Δ9 desaturation of stearic acid was 10trans,12cis-18∶2.

On the turnover of brain cholesterol in patients with Alzheimer's disease. Abnormal induction of the cholesterol-catabolic enzyme CYP46 in glial cells

Evidence is accumulating for a link between cerebral cholesterol metabolism and Alzheimers disease (AD). Here we focus on a possible relationship between AD and a newly discovered mechanism for cholesterol efflux from the brain, involving conversion of brain cholesterol into 24S-hydroxycholesterol by the neuronal oxidative enzyme CYP46. There was a marked difference in the distribution of CYP46 in brains of control and AD patients. The neuronal cells were less stained in AD brains than in controls while marked positive staining was found in glial cells in AD but not in controls. The dynamic changes in the mechanisms for cholesterol efflux from the brain are of interest in relation to the link between brain cholesterol and amyloid beta-protein in AD.

Plasma levels of 24S-hydroxycholesterol in patients with neurological diseases.

The brain is the exclusive or almost exclusive site of formation of 24S-hydroxycholesterol and we have shown that the circulating level of 24S-hydroxycholesterol is dependent upon the relation between cerebral production and hepatic clearance. In the present work we determined plasma levels of 24S-hydroxycholesterol in patients with various neurological diseases. Eleven subjects with brain death occurring 6-10 h before collection of the plasma samples had markedly reduced circulating levels of 24S-hydroxycholesterol (-43%, P<0.001). Patients with advanced Alzheimers disease and cerebral inflammatory diseases had slightly lower levels of 24S-hydroxycholesterol in plasma when compared to matched controls. Patients with acute ischemic stroke, multiple sclerosis and primary brain tumors had levels not significantly different from those of controls. The conditions leading to reduced plasma levels of 24S-hydroxycholesterol had no significant effect on plasma levels of another side-chain oxidized oxysterol, 27-hydroxycholesterol. Except for conditions characterized by very marked destruction of the central nervous system, different severe neurological diseases seem to have relatively small effects on the flux of 24S-hydroxycholesterol from the brain.

Short-Term Long Chain Omega3 Diet Protects from Neuroinflammatory Processes and Memory Impairment in Aged Mice

Regular consumption of food enriched in omega3 polyunsaturated fatty acids (ω3 PUFAs) has been shown to reduce risk of cognitive decline in elderly, and possibly development of Alzheimers disease. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the most likely active components of ω3-rich PUFAs diets in the brain. We therefore hypothesized that exposing mice to a DHA and EPA enriched diet may reduce neuroinflammation and protect against memory impairment in aged mice. For this purpose, mice were exposed to a control diet throughout life and were further submitted to a diet enriched in EPA and DHA during 2 additional months. Cytokine expression together with a thorough analysis of astrocytes morphology assessed by a 3D reconstruction was measured in the hippocampus of young (3-month-old) and aged (22-month-old) mice. In addition, the effects of EPA and DHA on spatial memory and associated Fos activation in the hippocampus were assessed. We showed that a 2-month EPA/DHA treatment increased these long-chain ω3 PUFAs in the brain, prevented cytokines expression and astrocytes morphology changes in the hippocampus and restored spatial memory deficits and Fos-associated activation in the hippocampus of aged mice. Collectively, these data indicated that diet-induced accumulation of EPA and DHA in the brain protects against neuroinflammation and cognitive impairment linked to aging, further reinforcing the idea that increased EPA and DHA intake may provide protection to the brain of aged subjects.

Differences in meibomian fatty acid composition in patients with meibomian gland dysfunction and aqueous-deficient dry eye

Aims: To evaluate the differences in meibomian fatty acid composition in healthy subjects and in patients suffering from meibomian gland dysfunction or aqueous-deficient dry eye. Methods: We collected meibomian oil using a sterile Schirmer paper in healthy individuals (n = 20), dry eye patients (aqueous-deficient) (n = 32) and meibomian gland dysfunction (MGD) patients (n = 25) after gentle massage of the lid margin. Meibomian fatty acids were directly transmethylated and analysed using gas chromatography (GC) and GC mass spectrometry. Results: Meibomian fatty acids were similar in healthy individuals and in dry eye patients but were different in MGD patients, who showed significantly higher levels of branched-chain fatty acids (29.8% vs 20.2%) (p<0.0001) and lower levels of saturated fatty acids (9.3 vs 24.6%) (p<0.0001), in particular lower levels of palmitic (C16) and stearic (C18) acids. Conclusion: The increase in branched-chain fatty acids may reflect greater quantities of wax and cholesterol esters and triglycerides in meibomian gland excreta. Since wax and cholesterol esters are the main lipids of meibum, these differences may have physical consequences for tear-film lipid-layer fluidity and stability. Meibomian fatty acid composition and particularly the increase in branched chains could be a marker for meibomian gland dysfunction.

Cholesterol-24S-hydroxylase (CYP46A1) Is Specifically Expressed in Neurons of the Neural Retina

Increasing biological findings argue for the importance of cholesterol-24S-hydroxylase (CYP46A1) in cholesterol homeostasis in cerebral structures. Based on the similarity between the brain and the neural retina, the aim of the current study was to evaluate the expression of CYP46A1 in the mammalian retina. RT-PCR analysis of CYP46A1 in bovine samples revealed the highest expression in the neural retina. The retinal pigment epithelium expressed CYP46A1 gene at a low level while the ciliary body showed no expression. Immunohistochemical evaluation of the posterior pole of rat retina showed that the protein is specifically expressed in neurons, whereas cone-rods photoreceptors were negative for CYP46A1 staining. The metabolite produced by CYP46A1, 24S-hydroxycholesterol, was almost exclusively found in neural retina, the concentration therein being more than 10-fold higher than in the retinal pigment epithelium or the ciliary body. The results of the current study are consistent with our primary hypothesis: the neural retina specifically expresses cholesterol-24S-hydroxylase, a metabolizing enzyme responsible for the removal of cholesterol in neurons. Based on the link between cholesterol-24S-hydroxylase, 24S-hydroxycholesterol, and neurologic disorders, CYP46A1 may be a valuable gene candidate for retinal pathologies like age-related macular degeneration or glaucomas, and 24S-hydroxycholesterol may be involved in the onset of the degenerative processes in these diseases.

The ins and outs of cholesterol in the vertebrate retina

The vertebrate retina has multiple demands for utilization of cholesterol and must meet those demands either by synthesizing its own supply of cholesterol or by importing cholesterol from extraretinal sources, or both. Unlike the blood-brain barrier, the blood-retina barrier allows uptake of cholesterol from the circulation via a lipoprotein-based/receptor-mediated mechanism. Under normal conditions, cholesterol homeostasis is tightly regulated; also, cholesterol exists in the neural retina overwhelmingly in unesterified form, and sterol intermediates are present in minimal to negligible quantities. However, under certain pathological conditions, either due to an inborn error in cholesterol biosynthesis or as a consequence of exposure to selective inhibitors of enzymes in the cholesterol pathway, the ratio of sterol intermediates to cholesterol in the retina can rise dramatically and persist, in some cases resulting in progressive degeneration that significantly compromises the structure and function of the retina. Although the relative contributions of de novo synthesis versus extraretinal uptake are not yet known, herein we review what is known about these processes and the dynamics of cholesterol in the vertebrate retina and indicate some future avenues of research in this area.

Oxysterols Induced Inflammation and Oxidation in Primary Porcine Retinal Pigment Epithelial Cells

Purpose: Aging is associated with an accumulation of cholesterol esters in the Bruch membrane. Cholesterol esters are prone to undergo oxidation and generate oxysterols that have cytotoxic and proinflammatory properties. We investigated the effects of three oxysterols on mitochondrial dysfunctions, inflammation, and oxidative stress in primary cultures of porcine retinal pigment epithelial (RPE) cells. Methods: RPE cells were incubated with oxysterols (50 μ M of 24-hydroxycholesterol, 25-hydroxycholesterol, or 7-ketocholesterol) for 24 hr and 48 hr. Oxysterol content was determined in cells and in corresponding media by gas chromatography. Mitochondrial activity was measured by mitochondrial dehydrogenase activity. The intracellular formation of reactive oxygen species in RPE cells was detected by using the fluorescent probe DCFH-DA. IL-8 was assayed in the supernatants by ELISA, and the corresponding cellular transcripts were semiquantified by RT-PCR. Results: Analyses of the oxysterols content in the RPE cells and corresponding media suggested a high rate of cellular uptake, although some differences were observed between 7-ketocholesterol on the one hand and 24-hydroxycholesterol and 25-hydroxycholesterol on the other hand. All oxysterols induced slight mitochondrial dysfunctions but a significant 2- to 4-fold increase in reactive oxygen species (ROS) production compared with the control. They also enhanced IL-8 gene expression and IL-8 protein secretion in the following decreasing order: 25-hydroxycholesterol > 24-hydroxycholesterol > 7-ketocholesterol. Conclusions: We conclude that in confluent primary porcine RPE cells, 24-hydroxycholesterol, 25-hydroxycholesterol, and 7-ketocholesterol are potent inducers of oxidation and inflammation.