Changes in the levels of cerebral and extracerebral sterols in the brain of patients with Alzheimer's disease
2004; Elsevier BV; Volume: 45; Issue: 1 Linguagem: Inglês
10.1194/jlr.m300320-jlr200
ISSN1539-7262
AutoresMaura Heverin, Nenad Bogdanović, Dieter Lütjohann, Thomas A. Bayer, Irina A. Pikuleva, Lionel Brétillon, Ulf Diczfalusy, Bengt Winblad, Ingemar Björkhem,
Tópico(s)Peroxisome Proliferator-Activated Receptors
Resumo24S-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. 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. Transport and turnover of cholesterol in the brain seems to be of importance for development of Alzheimer's disease (AD) (1Puglielli L. Tanzi R.E. Kovacs D.M. Alzheimer's disease: the cholesterol connection.Nat. Neurosci. 2003; 6: 345-351Crossref PubMed Scopus (686) Google Scholar). Apolipoprotein E (apoE) is involved in transport of cholesterol in the brain, and there is a strong association between the apoE4 allele and AD (2Strittmatter W.J. Saunders A.M. Schmechel D. Pericak Vance M. Enghild J. Salvesen S. Roses A.D. Apolipoprotein E: high-avidity binding to β-amyloid and increased frequency of Type 4 allele in late-onset familial Alzheimer disease.Proc. Natl. Acad. Sci. USA. 1993; 90: 1977-1981Crossref PubMed Scopus (3634) Google Scholar). Cholesterol loading or depletion affects deposition of amyloid-β protein both in vitro (3Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Cholesterol depletion inhibits the generation of β-amyloid in hippocampal regions.Proc. Natl. Acad. Sci. USA. 1998; 95: 6460-6464Crossref PubMed Scopus (1067) Google Scholar, 4Howland D.S. Trusko S.P. Savage M.J. Reaume A.G. Lang D.M. Hirsch J.D. Maeda N. Siman R. Greenberg B.D. Scott R.W. Flood D.G. Modulation of secreted β-amyloid precursor protein and amyloid β-peptide by cholesterol.J. Biol. Chem. 1998; 273: 16576-16582Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) and in vivo (5Refolo L.M. Pappolla M.A. Malester B. LaFrancois J. Bryant-Thomas T. Wang R. Tint G.S. Sambamurti K. Duff K. Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model.Neurobiol. Dis. 2000; 7: 321-331Crossref PubMed Scopus (868) Google Scholar). Clinical studies with HMG-CoA reductase inhibitors suggest that reduction of cholesterol synthesis may have a preventive effect on development of AD (6Wolozin B. Kellman W. Ruosseau P. Decreased prevalence of Alzheimer disease associated with HMG CoA reductase inhibitors.Arch. Neurol. 2000; 57: 1439-1443Crossref PubMed Scopus (1325) Google Scholar, 7Jick H. Zornberg G.L. Jick S. Seshadri S. Drachman A. Statins and the risk of dementia.Lancet. 2000; 356: 1627-1631Abstract Full Text Full Text PDF PubMed Scopus (1563) Google Scholar, 8Buxbaum J.D. Cullen E.I. Friedhoff L.T. Pharmacological concentrations of the HMG CoA reductase inhibitor lovastatin decrease the formation of the Alzheimer beta-amyloid peptide in vitro and in patients.Front. Biosci. 2002; 7: 50-59Crossref PubMed Google Scholar). Under normal conditions, with an intact blood-brain barrier, there is little or no exchange of cholesterol over the blood-brain barrier (9Dietschy J.M. Turley S.D. Cholesterol metabolism in the brain.Curr. Opin. Lipidol. 2001; 12: 105-112Crossref PubMed Scopus (706) Google Scholar, 10Snipes G.J. Suter U. Cholesterol and myelin.in: Bittman R. Subcellular Biochemistry. Vol. 28. Plenum Press, New York1997: 173-204Google Scholar). 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 (11Lütjohann D. Breuer O. Ahlborg G. Nennesmo I. Sidén Å. Diczfalusy U. Björkhem I. Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.Proc. Natl. Acad. Sci. USA. 1996; 93: 9799-9804Crossref PubMed Scopus (556) Google Scholar, 12Björkhem I. Lütjohann D. Breuer O. Sakinis A. Wennmalm A. Importance of a novel oxidative mechanism for elimination of brain cholesterol. Turnover of cholesterol and 24(S)-hydroxycholesterol in rat brain as measured with 18O2 techniques in vivo and in vitro.J. Biol. Chem. 1997; 272: 30178-30184Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 13Björkhem I. Lütjohann D. Diczfalusy U. Ståhle L. Ahlborg G. Wahren J. Cholesterol homeostasis in the human brain: turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation.J. Lipid Res. 1998; 39: 1594-1600Abstract Full Text Full Text PDF PubMed Google Scholar), 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 (14Lund E.G. Guileyardo J.M. Russell D.W. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain.Proc. Natl. Acad. Sci. USA. 1999; 16: 7238-7243Crossref Scopus (513) Google Scholar). 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 (15Bretillon L. Sidén Å. Wahlund L-O. Minthon L. Crisby M. Hillert J. Groth C-G. Diczfalusy U. Björkhem I. Plasma levels of 24S-hydroxycholesterol in patients with neurological diseases.Neurosci. Lett. 2000; 293: 87-90Crossref PubMed Scopus (128) Google Scholar). 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 (16Lütjohann D. Papassotiropoulos A. Björkhem I. Locatelli S. Bagli M. Rao M.L. von Bergmann K. Heun R. 24S-hydroxycholesterol (cerebrosterol) and Alzheimer's disease.J. Lipid Res. 2000; 41: 195-198Abstract Full Text Full Text PDF PubMed Google Scholar). Very recently, we studied the amount and distribution of CYP46 in autopsy brain material from AD and control patients (17Bogdanovic N. Bretillon L. Lund E. Diczfalusy U. Lannfelt L. Winblad B. Russell D. Björkhem I. On the turnover of brain cholesterol in patients with Alzheimer's disease. Abnormal induction of the cholesterol-catabolic enzyme CYP46 in glial cells.Neurosci. Lett. 2001; 314: 45-48Crossref PubMed Scopus (152) Google Scholar). 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 (18Lütjohann D. Brzezinka A. Barth E. Abramowski D. Staufenbiel M. von Bergmann K. Beyreuther K. Multhaup G. Bayer T.A. Profile of cholesterol-related sterols in aged amyloid precursor protein transgenic mouse brain.J. Lipid Res. 2002; 43: 1078-1085Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), 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 (18Lütjohann D. Brzezinka A. Barth E. Abramowski D. Staufenbiel M. von Bergmann K. Beyreuther K. Multhaup G. Bayer T.A. Profile of cholesterol-related sterols in aged amyloid precursor protein transgenic mouse brain.J. Lipid Res. 2002; 43: 1078-1085Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). 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 (19Blennow K. Wallin A. Fredman P. Karlsson I. Gottfries C.G. Svennerholm L. Blood brain barrier disturbance in patients with Alzheimer's disease is related to vascular factors.Acta Neurol. Scand. 1990; 81: 323-326Crossref PubMed Scopus (154) Google Scholar, 20McKhann G. Drachman D. Folstein M. Katzman R. Price D. Stadlan E.M. Clinical diagnosis of Alzheimer disease.Neurology. 1984; 34: 939-944Crossref PubMed Google Scholar). 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 (21Lund E. Andersson O. Zhang J. Babiker A. Ahlborg G. Diczfalusy U. Einarsson K. Sjövall J. Björkhem I. Importance of a novel oxidative mechanism for elimination of intracellular cholesterol in humans.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 208-212Crossref PubMed Scopus (145) Google Scholar). 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 (11Lütjohann D. Breuer O. Ahlborg G. Nennesmo I. Sidén Å. Diczfalusy U. Björkhem I. Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation.Proc. Natl. Acad. Sci. USA. 1996; 93: 9799-9804Crossref PubMed Scopus (556) Google Scholar). 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 (22Leoni M. Masterman T. Patel P. Meaney S. Diczfalusy U. Björkhem I. Side-chain oxidized oxysterols in cerebrospinal fluid and integrity of the blood-brain barrier.J. Lipid Res. 2003; 44: 793-799Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). 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 (23Björkhem I. Diczfalusy U. Lütjohann D. Removal of cholesterol from extrahepatic sources by oxidative mechanisms.Curr. Opin. Lipidol. 1999; 10: 161-165Crossref PubMed Scopus (120) Google Scholar). 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. The rabbit antipeptide antibodies toward human CYP46 were a kind gift from Prof. D. W. Russell (Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX) (14Lund E.G. Guileyardo J.M. Russell D.W. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain.Proc. Natl. Acad. Sci. USA. 1999; 16: 7238-7243Crossref Scopus (513) Google Scholar). Polyclonal rabbit antibodies toward the whole sterol 27-hydroxylase protein were used in the Western blotting of CYP27A1. All organic solvents used were of gas chromatography or high performance liquid chromatography grade. 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 (24Mirra S.S. Heyman A. McKeel D. Sumi S.M. Crain B.J. Brownlee L.M. Vogel F.S. Hughes J.P. van Belle G. Berg L. The consortium to establish a register for Alzheimer's disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease.Neurology. 1991; 41: 479-486Crossref PubMed Google Scholar) and the neuropathological criteria for “definite” AD (25Bogdanovic N. Morris J.H. Diagnostic criteria for Alzheimer's disease in multicentre brain banking.in: Cruz-Sanchez F.F. Ravid R. Cuzner M.L. Neuropathological Diagnostic Criteria for Brain Banking. Biomedical and Health Research Series. IOS Press, Amsterdam, The Netherlands1995: 20-29Google Scholar). 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. 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 (18Lütjohann D. Brzezinka A. Barth E. Abramowski D. Staufenbiel M. von Bergmann K. Beyreuther K. Multhaup G. Bayer T.A. Profile of cholesterol-related sterols in aged amyloid precursor protein transgenic mouse brain.J. Lipid Res. 2002; 43: 1078-1085Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). 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. 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. 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 (26Björkhem I. Blomstrand R. Svensson L. Determinations of cholesterol by mass fragmentography.Clin. Chim. Acta. 1974; 54: 185-193Crossref PubMed Scopus (70) Google Scholar, 27Lund E. Sisfontes L. Reihner E. Björkhem I. Determination of serum levels of unesterified lathosterol by isotope dilution-mass spectrometry.Scand. J. Clin. Lab. Invest. 1989; 49: 165-171Crossref PubMed Scopus (63) Google Scholar). 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 (28Dzeletovic S. Breuer O. Lund E.G. Diczfalusy U. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry.Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (467) Google Scholar). 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. 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. No significant age- or gender-related effects on the different parameters were found within the relatively heterogeneous groups of 15 controls and 15 AD patients. In view of the relatively small size of the groups, such differences cannot be excluded. Because of this, and in order to utilize paired t-tests, we defined eight gender- and age-matched pairs from the 30 subjects. 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. As shown in Fig. 1A, the cholesterol content was slightly higher in the AD patients in three of the four regions. In the basal ganglia, this difference was statistically significant (P < 0.05). In the larger, more heterogeneous group, there were no consistent differences between the two groups in any brain area (results not shown). 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. 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.Fig. 3Correlation between lathosterol and 24S-hydroxycholesterol in frontal cortex (A) and occipital cortex (B) from the eight AD patients belonging to the age- and gender-matched group.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In theory, part of the differences observed in absolute concentrations of the oxysterols and cholesterol between the samples from the Alzheimer brains and the control brains may be due to differences in water content. To exclude this possibility, we measured the water content of samples from each area in age- and gender-matched samples from five control and five Alzheimer patients. The water content of the frontal cortex of the AD and control samples was 79 ± 1% and 79 ± 1%, respectively. The water content of the occipital cortex was 74 ± 1% and 75 ± 1% for AD and control samples, respectively. The water content of the basal ganglia was 77 ± 1% and 74 ± 6% for AD and control samples, respectively. The water content of the pons was 68 ± 2% and 68 ± 1% for AD and control samples, respectively (mean ± SEM). Thus, there was no significant difference between AD and control samples with respect to water content. 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 (14Lund E.G. Guileyardo J.M. Russell D.W. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain.Proc. Natl. Acad. Sci. USA. 1999; 16: 7238-7243Crossref Scopus (513) Google Scholar, 29Cali J.J. Hsieh C.L. Francke U. Russell D.W. Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis.J. Biol. Chem. 1991; 266: 7779-7783Abstract Full Text PDF PubMed Google Scholar), 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). Levels of cholesterol-related sterols in the brain of aging wild-type and APP transgenic mice were measured previously (18Lütjohann D. Brzezinka A. Barth E. Abramowski D. Staufenbiel M. von Bergmann K. Beyreuther K. Multhaup G. Bayer T.A. Profile of cholesterol-related sterols in aged amyloid precursor protein transgenic mouse brain.J. Lipid Res. 2002; 43: 1078-1085Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). 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. At the age of 12 and 18 months, respectively, the transgenic mice were found to have significantly higher brain levels of 27-hydroxycholesterol than the control mice (increase of 21% and 29%, respectively) (P < 0.01 and 0.001, respectively). The ratio of 27-hydroxycholesterol to 24S-hydroxycholesterol showed a pattern similar to that of the absolute levels of 27-hydroxycholesterol. The ratio of 27:24 wa
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