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* Divisions of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden
Geriatrics, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden
Department of Clinical Pharmacology, University of Bonn, Bonn, Germany
** Department of Psychiatry, Division of Neurobiology, University of the Saarland, Homburg/Saar, Germany
Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX
Published, JLR Papers in Press, October 1, 2003. DOI 10.1194/jlr.M300320-JLR200
1 Present address of L. Bretillon: Lipid Nutrition Unit, National Institute for Research on Agronomy, Dijon, France. 
2 To whom correspondence should be addressed. e-mail: Ingemar.Bjorkhem{at}hs.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
Supplementary key words cerebral cholesterol • cholesterol 24S-hydroxylase • sterol 27-hydroxylase • oxysterols
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Under normal conditions, with an intact blood-brain barrier, there is little or no exchange of cholesterol over the blood-brain barrier (9, 10). We have defined a mechanism by which 6–7 mg of cholesterol is eliminated daily from the human brain in the form of a side-chain oxidized oxysterol, 24S-hydroxycholesterol (11–13), which is able to pass the intact blood-brain barrier. The enzyme responsible for the conversion of cholesterol into 24S-hydroxycholesterol is the cytochrome P450 species cholesterol 24S-hydroxylase (CYP46), which has been reported to be almost exclusively expressed in the neuronal cells in the normal human brain (14).
The neurodegeneration occurring in AD would be expected to eventually lead to loss of 24S-hydroxylase activity, with a resulting decrease in the flux of 24S-hydroxycholesterol from the brain into the circulation. In accordance with this, we found that patients with advanced AD had reduced plasma levels of 24S-hydroxycholesterol (15). A population of AD patients with less-advanced disease was, however, reported to have slightly increased levels of 24S-hydroxycholesterol, possibly due to an ongoing active neuronal destruction with increased liberation of cholesterol and 24S-hydroxycholesterol (16). Very recently, we studied the amount and distribution of CYP46 in autopsy brain material from AD and control patients (17). Neuronal cells from the frontal cortex of AD patients contained less CYP46 than did those of the controls. Surprisingly, a positive staining could be seen in the glial cells in AD brain but not in the controls. This indicates that in the normal state, the flux of 24S-hydroxycholesterol from the brain into the circulation is likely to be derived exclusively from the neurons, but possibly from both neurons and glial cells in patients with AD.
In a recent work (18), 24S-hydroxycholesterol was measured in the brains of transgenic mice carrying the Swedish mutation APP23, which causes typical AD-related pathological changes. The levels of this oxysterol were not significantly different between the transgenic mice and the controls. Because cholesterol 24S-hydroxylase has a broader organ distribution in mouse than in man, and is expressed to a considerable extent also in the liver, it is difficult to draw conclusions that are valid for humans. Interestingly, the levels of plant sterols were significantly increased in the brain of APP23 transgenic animals at the age of 12 and 18 months (18). Because these sterols are exclusively of dietary origin, the possibility must be considered that the APP23 transgenic mice had developed a defect in the blood-brain barrier. It is well established that patients with advanced Alzheimer's disease may have such defects (19, 20).
27-Hydroxycholesterol is another side-chain oxidized oxysterol, formed by sterol 27-hydroxylase (CYP27A1). In contrast to 24S-hydroxycholesterol, it is formed in most cells, and there is a constant flux of this oxysterol from extrahepatic tissues to the liver (21). There is, however, no net flux of 27-hydroxycholesterol from the brain, and the levels of 27-hydroxycholesterol in this organ are about 10-fold lower than those of 24S-hydroxycholesterol (11). In similarity with 24S-hydroxycholesterol, 27-hydroxycholesterol is likely to pass the blood-brain barrier, and an interchange between 27-hydroxycholesterol in the circulation and in the brain is likely to occur. Very recently, we showed that most of the 27-hydroxycholesterol present in human cerebrospinal fluid is of vascular origin (22).
In view of the contrasting information obtained from in vivo studies in humans and the studies with the transgenic mouse model, we considered it to be of interest to measure the levels of the oxysterols 24S- and 27-hydroxycholesterol in autopsy materials from patients with Alzheimer's disease and from controls. In addition to oxysterols, the levels of cholesterol, lathosterol, and plant sterols were also measured. For reasons of comparison, we also assayed 27-hydroxycholesterol in the brains of APP23 transgenic mice. Attempts were made to measure cholesterol 24S-hydroxylase and sterol 27-hydroxylase protein in the brain material in order to investigate possible correlations between the protein expression and steroid levels. There may be a close coupling between cholesterol synthesis and cholesterol elimination by the CYP46 mechanism in the brain (23). In view of this, it was of interest to see if a correlation could be found between the levels of lathosterol (a marker for cholesterol synthesis) and levels of 24S-hydroxycholesterol (a marker for cholesterol elimination) in the two groups of patients.
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MATERIALS AND METHODS |
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Patients
Formalin-fixed autopsy tissue from the frontal cortex, occipital cortex, basal ganglia, and pons was obtained from 15 controls (age 61–88 years, mean age 73 years, male-to-female ratio 9:6) and 15 AD patients (age 66–92 years, mean age 82 years, male-to-female ratio 5:10). The control samples were obtained from assumed-healthy subjects who had died as a result of road traffic accidents. The AD patients fulfilled the clinical criteria for "probable" AD (24) and the neuropathological criteria for "definite" AD (25). The AD patients died as a result of the disease. These samples were collected for lipid extraction and sterol analysis only. Although all brain samples from all patients were analyzed, the great differences in age and female-to-male ratio necessitated the selection of a subset of subjects from each group. Eight of the AD patients and eight of the controls were paired together to form a gender- and age-matched group, with a female-to-male ratio of 5:3 and a mean age of 78 years (range 63–86 years) for the controls and 79 years (range 66–89 years) for the AD patients. The maximum difference of age in the pairs was 3 years.
In addition to the above materials, frozen brain samples from the same four brain regions were obtained from five additional AD patients and from five age- and gender-matched controls for Western blot analysis. Part of this material was also used for analyses of the sterols.
Experiments with APP23 mice
The experiments with transgenic mice expressing the Swedish mutation APP751 under the control of the murine Thy1 promoter (APP23) and wild-type littermate control mice have been described in detail previously (18). In short, groups of five wild-type and five APP transgenic mice were killed at the age of 3, 6, 9, and 12 months and 10 wild-type and 11 APP transgenic mice at 18 months. Brain homogenates were prepared for sterol extraction essentially as previously described. In the previous publication, results of measurements of cholesterol, cholesterol precursors, and 24S-hydroxycholesterol have been reported. The complementary analysis made here was the assay of 27-hydroxycholesterol by isotope dilution-mass spectrometry as described below.
Ethical aspects
Permission to create and utilize the brain bank at Huddinge University Hospital was obtained from the local ethical committee of the hospital. The animal experiments were performed in accordance with the German animal protection laws.
Assay of cholesterol, lathosterol, plant sterols, and oxysterols
Small pieces of the brains (
400 mg) were shock frozen in liquid nitrogen for 10 min and pulverized in a microdismembrator. The solid powder was weighed, and the lipid components were extracted with chloroform-methanol (2:1; v/v) by stirring overnight at room temperature under argon gas. The volume of this solution was adjusted to 10 ml, and aliquots were taken for analysis of cholesterol and oxysterols. Cholesterol and lathosterol were assayed by isotope dilution-mass spectrometry with the use of deuterium-labeled cholesterol and lathosterol, respectively, as internal standard (26, 27). The plant sterols campesterol and sitosterol were assayed in a similar fashion using trideuterated 24-hydroxycholesterol as internal standard and selected-ion monitoring at m/z 472 and m/z 486 for campesterol and sitosterol, respectively. Oxysterols were also analyzed by isotope dilution-mass spectrometry with the use of an individual deuterium-labeled oxysterol in each case as previously described (28).
Determination of the water content of the brain samples
Formalin-fixed tissue (200 mg) from the four brain regions of five AD patients and five control patients was patted dry and weighed. The samples were then incubated overnight at 60°C, and the dehydrated samples weighed again. The difference in weight before and after drying was determined to be the water content of the brain samples.
Western blotting
For immunoblotting, frozen brain tissue from each brain area of the controls and AD patients was homogenized in 0.32 M sucrose medium and centrifuged to produce crude organelle fractions.
The nuclear and microsomal fractions, corresponding to 2 and 4 µg of total protein, respectively, were diluted in sample buffer and run on 7.5% polyacrylamide gel. The samples were transferred to Hybond® nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, UK) and blocked with 5% Biorad blocking agent in Tris-buffered saline containing 0.1% Tween (TBS-T). The membranes were washed with TBS-T and incubated with primary antibody to CYP46 or CYP27A1 overnight, followed by washing with TBS-T and incubation with peroxidase-conjugated goat anti-rabbit IgG (Sigma) for 4–5 h. The protein was visualized with an enhanced chemiluminescence kit (Amersham) according to the manufacturer's instructions, and films were exposed to the membranes for a few minutes. Band intensity was estimated using a Pharmacia Imagemaster system.
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RESULTS |
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In the following section, we show the detailed results obtained from the two smaller homogeneous groups (8 + 8 subjects, Fig. 1A) but also describe the results obtained with the two larger, more heterogeneous groups.
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Similarly to cholesterol, lathosterol levels were slightly higher in basal ganglia from AD patients than in the corresponding controls (Fig. 1B). The situation was similar in the larger groups (results not shown). The ratio of lathosterol to cholesterol, believed to be a marker for cholesterol synthesis, was, however, not significantly different between controls and AD patients in either of the patient groups (results not shown).
The levels of 24S-hydroxycholesterol were consistently lower in all areas of the brain of the AD patients compared with controls (Fig. 1C). Statistical significance was reached only in the case of occipital cortex. In the complete group, however, this difference was statistically different in all the areas (results not shown). The situation was similar with the cholesterol-related levels of 24S-hydroxycholesterol. Although the levels were decreased in all brain areas of the AD patients, the difference only reached statistical significance in the basal ganglia (Fig. 1D).
The levels of 27-hydroxycholesterol were significantly increased in all the different brain areas of the AD patients, with the exception of the pons (Fig. 1E). In the larger groups, the magnitude of the increase was greater in all areas, including the pons (results not shown).
The ratio of 27-hydroxycholesterol to cholesterol was higher in all brain areas of the AD patients (Fig. 1F), but this difference was not statistically significant. In contrast, this difference was found to be statistically significant in all areas of the brain in the larger groups.
The ratio of 27-hydroxycholesterol to 24S-hydroxycholesterol was found to be significantly increased in all areas of the AD brains, in both the smaller (Fig. 1G) and the larger groups.
The plant sterols campesterol and sitosterol were found to be slightly increased in all brain areas except the pons (Figs. 1H, I). This difference reached statistical significance in the case of sitosterol in frontal cortex and basal ganglia. The situation was similar in the larger groups. The same pattern was observed also in the ratios of sitosterol to cholesterol and campesterol to cholesterol.
Correlation analyses
Attempts were made to uncover correlations between the different parameters measured. The highest correlations were observed between the levels of 24S-hydroxycholesterol and 27-hydroxycholesterol in the frontal and occipital cortex of the AD patients (r 2 = 0.9 in both cases) (Fig. 2)
. This correlation was not evident in controls (r 2 = 0.2 and 0.6, respectively). Good correlations were also found between lathosterol and 24S-hydroxycholesterol in the frontal and occipital cortex of the AD patients (Fig. 3)
. Significant correlations were absent in the corresponding control patients (r 2 = 0.2 in both cases). The correlations between 24S-hydroxycholesterol and lathosterol, as well as between 24S-hydroxycholesterol and 27-hydroxycholesterol, were not due to the cholesterol levels, because the correlations were retained after correction for differences in cholesterol concentrations.
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Measurements of cholesterol 24S-hydroxylase and sterol 27-hydroxylase protein in brain samples from AD patients and controls by Western blotting
The levels of the two proteins were assessed by Western blotting. Initially, crude homogenates of the brain samples were used. The results obtained in these measurements were less than satisfactory. Considerably higher quality analyses could be performed by measuring cholesterol 24S-hydroxylase in isolated microsomal fractions and sterol 27-hydroxylase in isolated mitochondrial fractions from the frozen brain samples. In accordance with previous studies (14, 29), only one band with the expected molecular mass of 57 kDa was obtained in the analysis of CYP46, whereas two bands with molecular mass of 57 kDa and 60 kDa, respectively, were obtained in the analyses of CYP27. In both cases, no significant differences between AD and controls were observed with respect to enzyme levels in the different areas of the brain. In the analysis of the content of CYP46 in the frontal cortex from the AD patients and from the controls, the response was 84 ± 19 and 99 ± 5 arbitrary units, respectively (P > 0.05). In the analysis of the content of the sterol 27-hydroxylase protein in the same area, the response was 25 ± 2 and 22 ± 4 arbitrary units, respectively (P > 0.05). Similar results were also obtained in the other brain areas.
The levels of 24S-hydroxycholesterol and 27-hydroxycholesterol were also measured in the specific brain samples used for immunoblotting. No significant correlations were found between these levels and the levels of the corresponding enzyme protein (results not shown).
Measurement of 27-hydroxycholesterol and 27:24 ratio in APP23 transgenic mice
Levels of cholesterol-related sterols in the brain of aging wild-type and APP transgenic mice were measured previously (18). The levels of 27-hydroxycholesterol were not analyzed at that time. The results of the present measurements of 27-hydroxycholesterol in the brain of these mice are summarized in Fig. 4
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The levels of 24S-hydroxycholesterol were lower in all areas of AD brains compared with controls. This decrease is likely to reflect the lower number of CYP46-containing neuronal cells in AD. It is evident that the abnormal expression of CYP46 in glial cells in AD brains observed in a previous study (17) is not able to functionally compensate for the loss of the neuronal enzyme. The results are consistent with our observation that patients with advanced AD have significantly reduced plasma levels of 24S-hydroxycholesterol as compared with healthy controls (15). The fact that we could not demonstrate a significant reduction of CYP46 enzyme protein in our study may be due to the less-accurate Western blotting technique used, which is not well suited to detect relatively small changes. There was a tendency, however, toward lower levels in the frontal cortex of the AD brains.
A significant finding of the present study was the relatively high increase in 27-hydroxycholesterol levels in all areas of the brain tested. In view of our recent demonstration that most of the 27-hydroxycholesterol present in human cerebrospinal fluid originates in the circulation (22), it seems likely that a considerable part of the 27-hydroxycholesterol present in the brain is derived from extracerebral sources. Sterol 27-hydroxylase is present in the brain, but only at very low levels. In the present work, we did not find evidence of a difference in the levels of the enzyme in the brain of AD patients and controls. A defective blood-brain barrier would be expected to permit an increased flux of 27-hydroxycholesterol from the circulation into the brain. Abnormal blood-brain barrier function has, in fact, been reported in at least some populations of AD patients (19, 20). We have recently shown that a disturbed blood-brain function in some other neurological diseases leads to increased flux of 27-hydroxycholesterol from the circulation into the cerebrospinal fluid (22). Consistent with our observations in patients with AD, increased levels of 27-hydroxycholesterol were found also in the APP23 mice at the age of 12 and 18 months. It is of interest that there may be alterations in the blood-brain barrier of mice expressing both APP and presenilin-1 (31).
If there is an increased flux of 27-hydroxycholesterol from the circulation into the brain as a consequence of a disturbed blood-brain barrier, there may also be an increased flux of other steroids. Slightly higher levels of plant sterols were found in most areas of the AD brain studied, and in the APP23 mice, a statistically significant accumulation of the plant sterols sitosterol and campesterol has been reported (18). In the transgenic mice, there was a clear correlation between the levels of the two plant sterols and 27-hydroxycholesterol, supporting the contention that the increased flux of 27-hydroxycholesterol is related to a defect in the blood-brain barrier.
The "driving force" for the flux of 24S-hydroxycholesterol from the brain into the circulation is likely to be the concentration difference between the two compartments. The ratio of 24S-hydroxycholesterol to cholesterol is 20-fold higher in the brain than in the circulation, and a similar difference is present between the brain and most other organs (11). In contrast, the ratio of 27-hydroxycholesterol to cholesterol is of a similar magnitude in the circulation and the brain (0.1 ng/µg), whereas this ratio is higher in almost all other tissues and organs in the body. In the lung, the ratio is about 70-fold higher than in the circulation and the brain (32). In view of this, some net flux of 27-hydroxycholesterol into the brain seems likely to occur. A prerequisite for such a passage is a transport over the blood-brain barrier. It is well established that both 24S- and 27-hydroxycholesterol are able to pass lipophilic membranes at a much higher rate than cholesterol (33). The rate of transport of 27-hydroxycholesterol across such membranes is, however, significantly higher than that of 24S-hydroxycholesterol. This may be the explanation for our finding here that the ratio of 27-hydroxycholesterol to cholesterol was almost identical in all the brain areas studied (Fig. 1F). Considerably greater differences in the relative concentrations of 24S-hydroxycholesterol were found in the different brain areas (Fig. 1D).
Both 24S-hydroxycholesterol and 27-hydroxycholesterol are ligands for the nuclear receptor LXRß, which is present at relatively high levels in the brain. The ligand binding efficiency is, however, higher with 24S-hydroxycholesterol than with 27-hydroxycholesterol, and the activation efficiency toward LXRß is almost 10-fold higher (34). It is noteworthy that the expression patterns of cholesterol 24S-hydroxylase and LXRß are remarkably similar (35). In view of the role of liver X receptors as regulators of cholesterol and phospholipid export proteins (35), the possibility must be considered that a change in the ratio of 27- to 24S-hydroxycholesterol may reduce the activation of the receptor, with consequences for lipid homeostasis in the brain. It was recently reported that a knockout of the liver X receptors causes age-related neurodegeneration, with lipid deposits, proliferation of astrocytes, loss of neurons, and disorganized myelin sheaths (36).
Theoretically, the most important factors regulating the levels of cholesterol in the brain should be the rate of de novo synthesis and the rate of metabolism by the CYP46-mediated mechanism. Very recently, it was reported that a disruption of the CYP46 gene causes a marked reduction of cholesterol synthesis in mouse brain (37). Under steady-state conditions, a relatively constant relation would be expected between a marker for cholesterol synthesis (lathosterol) and a marker for cholesterol metabolism (24S-hydroxycholesterol). A high correlation was found between levels of these two steroids in the frontal and occipital cortex of the brains from the AD patients. A similar correlation has also been observed in APP23 transgenic mice (18). A high correlation between 24S-hydroxycholesterol and 27-hydroxycholesterol was also found in these two areas of the brains from the AD patients.
This is consistent with a relation between increased metabolism of cholesterol in the brain and a decreased blood-brain barrier function in AD. That the above correlations were most significant in the frontal cortex is consistent with a previous finding that this area of the brain seems to be more sensitive to changes in overall cholesterol homeostasis than most other areas of the brain, at least in mice (38). There was a distinct difference between the findings in the AD brains and the control brains, possibly as a consequence of the ongoing degeneration in the former.
To summarize, we have shown that the ratio of two oxysterols, one of cerebral and one of mainly extracerebral origin, is markedly changed in all brain areas of patients with Alzheimer's disease, as well as in brains of transgenic mice carrying the Swedish Alzheimer mutation. The results confirm and extend previous studies demonstrating disturbed cholesterol turnover in this disease.
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ACKNOWLEDGMENTS |
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Manuscript received July 22, 2003 and in revised form September 18, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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