24S-hydroxycholesterol is a side-chain oxidized oxysterol formed in the brain that is continuously crossing the blood-brain barrier to reach the circulation. There may be an opposite flux of 27-hydroxycholesterol, which is formed to a lower extent in the brain than in most other organs. Here we measured cholesterol, lathosterol, 24S-and 27-hydroxycholesterol, and plant sterols in four different brain areas of deceased Alzheimer's disease (AD) patients and controls. 24S-hydroxycholesterol was decreased and 27-hydroxycholesterol increased in all the brain samples from the AD patients. The difference was statistically significant in four of the eight comparisons. The ratio of 27-hydroxycholesterol to 24S-hydroxycholesterol was significantly increased in all brain areas of the AD patients and also in the brains of aged mice expressing the Swedish Alzheimer mutation APP751. Cholesterol 24S-hydroxylase and 27-hydroxylase protein was not significantly different between AD patients and controls. A high correlation was observed between the levels of 24S-hydroxycholesterol and lathosterol in the frontal cortex of the AD patients but not in the controls. Most probably the high levels of 27-hydroxycholesterol are due to increased influx of this steroid over the blood-brain barrier and the lower levels of 24S-hydroxycholesterol to decreased production.The high correlation between lathosterol and 24-hydroxycholesterol is consistent with a close coupling between synthesis and metabolism of cholesterol in the frontal cortex of the AD brain.

Historically understudied, cholesterol in the retina is receiving more attention now because of genetic studies showing that several cholesterol-related genes are risk factors for age-related macular degeneration (AMD) and because eye pathology studies showing high cholesterol content of drusen, aging Bruch's membrane, and newly found subretinal lesions. The challenge before us is determining how the cholesterol-AMD link is realized. Meeting this challenge will require an excellent understanding these genes’ roles in retinal physiology and how chorioretinal cholesterol is maintained. In the first half of this review, we will succinctly summarize physico-chemical properties of cholesterol, its distribution in the human body, general principles of maintenance and metabolism, and differences in cholesterol handling in human and mouse that impact on experimental approaches. This information will provide a backdrop to the second part of the review focusing on unique aspects of chorioretinal cholesterol homeostasis, aging in Bruch's membrane, cholesterol in AMD lesions, a model for lesion biogenesis, a model for macular vulnerability based on vascular biology, and alignment of AMD-related genes and pathobiology using cholesterol and an atherosclerosis-like progression as unifying features. We conclude with recommendations for the most important research steps we can take towards delineating the cholesterol-AMD link.

One of the major oxysterols in the human circulation is 4␤-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4␤-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7␣-, 27-, and 24-hydroxycholesterol to be ϳ0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4␤-hydroxycholesterol. The apparent half-life of deuteriumlabeled 4␤-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4␤-Hydroxycholesterol was converted into acidic products at a much slower rate than 7␣-hydroxycholesterol in primary human hepatocytes, and 4␤-hydroxycholesterol was 7␣-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4␤-hydroxycholesterol. These results suggest that the high plasma concentration of 4␤-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7␣-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4␤-hydroxycholesterol as a ligand for the nuclear receptor LXR.4␤-Hydroxycholesterol is one of the quantitatively most important oxysterols in human circulation (1). We have recently shown that it is formed by the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) 1 (1). Preliminary experiments showed that the formation of this oxysterol by human liver microsomes was relatively slow. The high plasma levels of the oxysterol are therefore surprising, and we hypothesized that this may be a consequence of slow metabolism. Therefore, in this work, we determined the rate of elimination of deuteriumlabeled 4␤-hydroxycholesterol from plasma. Oxysterols are generally degraded to bile acids, and the rate-limiting step in this conversion is the introduction of a hydroxyl group in the 7␣-position of the steroid. Alternative pathways for bile acid biosynthesis start with oxidation of the steroid side chain by CYP27A1 and CYP46. Therefore, we have studied the possibility that these cytochromes are active toward 4␤-hydroxycholesterol. The metabolism of 4␤-hydroxycholesterol was studied in human primary hepatocytes, control, and transfected cells and by incubations with recombinant enzymes. In addition, fecal samples from three untreated subjects and one subject treated with carbamazepine were analyzed for 4␤-hydroxylated bile acids. Based on these experiments, we present evidence that 4␤-hydroxycholesterol has an unusually long halflife in plasma and that this is the result of slow elimination, particularly slow 7␣-hydroxylation that is the rate-limiting step for further conversion into bile acids.

By converting cholesterol to 24S-hydroxycholesterol, cytochrome P450 46A1 (CYP46A1) initiates the major pathway for cholesterol removal from the brain. Two crystal structures of CYP46A1 were determined. First is the 1.9-Å structure of CYP46A1 complexed with a high-affinity substrate cholesterol 3-sulfate (CH-3S). The second structure is that of the substrate-free CYP46A1 at 2.4-Å resolution. CH-3S is bound in the productive orientation and occupies the entire length of the banana-shaped hydrophobic active-site cavity. A unique helix B -C loop insertion (residues 116 -120) contributes to positioning cholesterol for oxygenation catalyzed by CYP46A1. A comparison with the substrate-free structure reveals substantial substrate-induced conformational changes in CYP46A1 and suggests that structurally distinct compounds could bind in the enzyme active site. In vitro assays were performed to characterize the effect of different therapeutic agents on cholesterol hydroxylase activity of purified full-length recombinant CYP46A1, and several strong inhibitors and modest coactivators of CYP46A1 were identified. Structural and biochemical data provide evidence that CYP46A1 activity could be altered by exposure to some therapeutic drugs and potentially other xenobiotics.cholesterol metabolism ͉ monooxygenase ͉ drug interactions ͉ cholesterol 3-sulfate A ccumulating evidence indicates that neurodegeneration and development of neurological disorders such as Alzheimer's disease (AD) are associated with disturbances in cholesterol homeostasis in the brain (1-5). It is also becoming increasingly clear that the conversion of cholesterol to 24S-hydroxycholesterol (24OH-CH) is an important mechanism that controls cholesterol turnover in the central nervous system (6-8). Cholesterol 24-hydroxylation is carried out by cytochrome P450 46A1 (CYP46A1) and represents the first step in the major pathway for cholesterol elimination from the brain (9-11). Unlike cholesterol, 24OH-CH can cross the blood-brain barrier and be delivered to the liver for further degradation to bile acids.Studies of CYP46A1-knockout mice indicate that the continued synthesis and turnover of cerebral cholesterol via 24-hydroxylation are necessary for memory and learning (12). There appears to be a link between CYP46A1 and AD; the CYP46A1 expression pattern in the brain and levels of 24OH-CH in the serum and cerebrospinal f luid are different in healthy people and those affected by AD (13-17). The association between polymorphisms in the CYP46A1 gene and susceptibility to AD was also demonstrated in a number of studies (www.alzforum.org). However, this association has not yet been unambiguously proven and is still under investigation. Evidence has also been obtained in cultured cells and mice that supports a role for side-chain oxysterols, including 24OH-CH, as endogenous ligands for liver X receptors, that regulate the expression of genes involved in fatty acid and cholesterol metabolism (7). 24OH-CH was also shown to inhibit the formation of amyloid ␤-peptides, a ha...

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 mg/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 C 27 steroidal acid 7a-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7a-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7a-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.

Cytochrome P450 46A1 (CYP46A1 or cholesterol 24-hydroxylase) controls cholesterol elimination from the brain and plays a role in higher order brain functions. Genetically enhanced CYP46A1 expression in mouse models of Alzheimer’s disease mitigates the manifestations of this disease. We enhanced CYP46A1 activity pharmacologically by treating 5XFAD mice, a model of rapid amyloidogenesis, with a low dose of the anti-HIV medication efavirenz. Efavirenz was administered from 1 to 9 months of age, and mice were evaluated at specific time points. At one month of age, cholesterol homeostasis was already disturbed in the brain of 5XFAD mice. Nevertheless, efavirenz activated CYP46A1 and mouse cerebral cholesterol turnover during the first four months of administration. This treatment time also reduced amyloid burden and microglia activation in the cortex and subiculum of 5XFAD mice as well as protein levels of amyloid precursor protein and the expression of several genes involved in inflammatory response. However, mouse short-term memory and long-term spatial memory were impaired, whereas learning in the context-dependent fear test was improved. Additional four months of drug administration (a total of eight months of treatment) improved long-term spatial memory in the treated as compared to the untreated mice, further decreased amyloid-β content in 5XFAD brain, and also decreased the mortality rate among male mice. We propose a mechanistic model unifying the observed efavirenz effects. We suggest that CYP46A1 activation by efavirenz could be a new anti-Alzheimer’s disease treatment and a tool to study and identify normal and pathological brain processes affected by cholesterol maintenance.

BackgroundThe retina is a light-sensitive tissue lining the inner surface of the eye and one of the few human organs whose cholesterol maintenance is still poorly understood. Challenges in studies of the retina include its complex multicellular and multilayered structure; unique cell types and functions; and specific physico-chemical environment.Methodology/Principal FindingsWe isolated specimens of the neural retina (NR) and underlying retinal pigment epithelium (RPE)/choroid from six deceased human donors and evaluated them for expression of genes and proteins representing the major pathways of cholesterol input, output and regulation. Eighty-four genes were studied by PCR array, 16 genes were assessed by quantitative real time PCR, and 13 proteins were characterized by immunohistochemistry. Cholesterol distribution among different retinal layers was analyzed as well by histochemical staining with filipin. Our major findings pertain to two adjacent retinal layers: the photoreceptor outer segments of NR and the RPE. We demonstrate that in the photoreceptor outer segments, cholesterol biosynthesis, catabolism and regulation via LXR and SREBP are weak or absent and cholesterol content is the lowest of all retinal layers. Cholesterol maintenance in the RPE is different, yet the gene expression also does not appear to be regulated by the SREBPs and varies significantly among different individuals.Conclusions/SignificanceThis comprehensive investigation provides important insights into the relationship and spatial distribution of different pathways of cholesterol input, output and regulation in the NR-RPE region. The data obtained are important for deciphering the putative link between cholesterol and age-related macular degeneration, a major cause of irreversible vision loss in the elderly.

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