Current Alzheimer Research (www.bentham.org/car), 2004, 1, 71-77
Bentham Science Publishers Ltd.(www.bentham.org)

The Effects of Gender and CYP46 and Apo E Polymorphism on 24S-Hydroxycholesterol Levels in Alzheimer’s Patients Treated with Statins

Gloria Lena Vega1,4,6,*, Myron Weiner2, 3,5, Heike Kölsch8, Klaus von Bergmann7, Reinhard Heun8, Dieter Lutjohan7, Anh Nguyen4 and Carol Moore5

The Departments of 1Clinical Nutrition, 2Psychiatry and 3Neurology, and the Center for Human 4Nutrition, 5the Alzheimer’s Disease Center of the University of Texas Southwestern Medical Center, 6the Nutrition and Metabolism Laboratory of the Metabolic Unit at the Veterans Affairs Medical Center in Dallas, Texas and 7the Departments of Clinical Pharmacology and 8Department of Psychiatry and Psychotherapy, University of Bonn, Bonn, Germany

*Address correspondence to this author at the University of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-9052, USA; Tel: 214 648 2869; Fax: 214 648 4837; E-mail: Gloria.Vega@utsouthwestern.edu

Abstract: To examine the effect of gender and polymorphisms of CYP46 and apo E on plasma levels of 24S-hydroxycholesterol in Alzheimer’s disease (AD) patients and to determine whether these factors contribute to the variability in responses to statin treatment

Fifty-three AD patients had measurement of plasma levels of 24S-hydroxycholesterol , plasma and lipoprotein cholesterol and genotyping of CYP46 and apo E. Thirty-nine of the subjects subsequently participated in a statin trial for 6 weeks, and had a repetition of the baseline measurements.

Baseline levels of 24S-hydroxycholesterol were higher in women than in men. There was a positive and significant correlation of plasma oxysterol levels with levels of total plasma cholesterol (women: r = .72, P < .0001; men: r = .47, P = .02) and non-HDL cholesterol (women: r = .68, P < .0001; men: r = 0.51, P = .01) (and LDL cholesterol) but not HDL cholesterol levels. There was no association of CYP46 or apo E polymorphisms with plasma levels of 24S-hydroxycholesterol. AD subjects treated with statins had a similar percent reduction in lathosterol, 24S-hydroxycholesterol, total cholesterol and non-HDL (and LDL) cholesterol regardless of gender and polymorphisms of CYP46. Subjects with the e4/4 polymorphism had less reduction in the ratios of 24S-hydroxycholesterol-LDL cholesterol.

Women with AD had higher levels of plasma 24S-hydroxycholesterol levels than men. Women also showed a very strong correlation of plasma levels of 24S-hydroxycholesterol-to-total and non-HDL cholesterol. This may suggest that the oxysterol may be an important marker of AD risk instead of total cholesterol, as suggested by others. Polymorphisms of CYP46 or apo E do not explain levels of oxysterol or non-HDL cholesterol or the responsiveness to statin treatment in this study

Keywords: 24S-hydroxycholesterol, CYP46 and apo E polymorphisms, statins, plasma cholesterol.

Introduction

Retrospective studies suggest that levels of plasma cholesterol may impart a risk for Alzheimer’s Disease (AD) [1, 2] and that statin therapy may reduce risk of AD [3, 4]. Although these statistical associations have not been proven in prospective clinical trials, the observations have engendered a great deal of interest in brain cholesterol metabolism and on the potential of statins in prevention and treatment of AD. Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A, the rate-limiting enzyme in de novo synthesis of cholesterol. The sterol is linked to the pathology of AD. A widely accepted hypothesis regarding the etiology of AD is that b-amyloid protein forms neuritic plaques that lead to neurodegeneration [6, 7]. The metabolism of amyloid protein precursor (APP), a transmembrane protein, can be regulated by altering the metabolism of cholesterol [5 - 7]. For example, inhibition of cholesterol biosynthesis by statins reduces production of b-amyloid protein [5, 7]; lowering the levels of cholesterol also reduces levels of b-amyloid protein in the central nervous system [5]. In humans, simvastatin treatment reduces levels of b-amyloid protein in the cerebrospinal fluid and plasma [5, 6]. These studies lend some support to the possibility that statin therapy may be beneficial to subjects at risk for AD.

The main product of brain cholesterol metabolism is the oxysterol 24S-hydroxycholesterol [8]. In humans, about 90% of the plasma oxysterol is derived from brain cholesterol, and it has been suggested that plasma levels of 24S-hydroxycholesterol can be used as a measure of brain cholesterol metabolism [9]. Brain cholesterol is converted to 24S-hydroxycholesterol by CYP46, an enzyme that is uniquely expressed in brain tissue [10]. Recent studies indicate that 24S-hydroxycholesterol is elevated in plasma of AD patients [11] and that patients with advanced AD seem to have low plasma levels of 24S-hydoxycholesterol [12]. It also has been shown that CYP46 gene polymorphisms impart risk for late-onset Alzheimer’s disease [13, 19]. Another established risk for late onset AD is the apo e4 allele [14, 15].

We have recently examined the effect of statins on plasma levels of 24S-hydroxycholesterol in patients with AD [16] and reported that levels of the oxysterol are reduced significantly by treatment with this class of drugs. Because some studies suggest that apo E genotype influences response to statin treatment, two questions were examined in this study. First, how do polymorphisms for apo E and CYP46 influence levels of the oxysterol and response to statin therapy and secondly, what is the relationship between levels of plasma cholesterol to 24S-hydroxycholesterol in AD patients.

Patients and Methods

Subjects met NINCDS/ADRDA criteria [17] for of probable AD. None had history of cardiovascular disease and none were taking hypolipidemic agents. Subjects were excluded if they had plasma total cholesterol < 160 mg/dL or if they had history of sensitivity to any of the drugs tested in this trial. Fifty three AD patients (30 women and 23 men) had measurements of plasma sterols, triglycerides and genotyping for CYP46 and apo E. The women were older than the men (women: 74 + 1 years old + SE and men: 68 + 2 years old; P =.04). The average age of onset of AD also was higher in the women than in men (women: 70 + 2 years and men: 63 + 2 years, P = .01). Of the fifty-three subjects, 49 participated in a lipid-lowering drug trial to test efficacy and safety of statins or nicotinic acid [16]. Thirty-nine of the patients took statins and 10 were treated with nicotinic acid for a period of six weeks [16]. These patients had a repetion of the baseline measurements after treatment with statins or nicotinic acid. Since the statins lowered the levels of oxysterol, the responsiveness of these patients is considered in more detailed in the current study.

The Institutional Review Board for Investigation in Humans of the University of Texas Southwestern Medical Center at Dallas, Texas, approved the study. All patients signed informed consent that was co-signed by a family member or legal representative. The Institutional Review Board for Investigation in Humans also approved the added analyses for the polymorphisms of CYP46 and apo E.

Measurement of sterols and oxysterols was previously detailed [16]. Apo E genotyping was carried out in Dallas using the Hixson et al. procedure [18]. Genotyping of the CYP46 IVS3+43CàT polymorphism was carried out in the laboratory of Dr. Heun as detailed previously [19]. Levels of 24S-hydroxycholesterol, lathosterol and campesterol were measured and were reported previously [16]. Levels of plasma total cholesterol, triglyceride and lipoprotein cholesterol were measured in Dallas as detailed previously [16].

Statistical analyses were carried out using the SAS- Statview statistical software. Data are summarized as means plus or minus standard error of the mean. An unpaired t-test was employed for comparison of means of independent variables. Pearson’s moment correlations were calculated for 24S-hydroxycholesterol to other variables.

Results

Levels of plasma 24S-hydroxycholesterol were significantly higher in women than in men (Fig. 1) as were the levels of HDL cholesterol (Fig. 1) and total cholesterol (women: 220 + 5 mg/dL and men: 200 + 7 mg/dL, P = .02).

Fig. (1). Levels of 24S-hydroxycholesterol, Non-HDL cholesterol and HDL-cholesterol in women and men with AD.

However, non-HDL cholesterol (sum of VLDL plus LDL cholesterol) levels were not significantly different (Fig. 1) between the two genders. The ratio of 24S-hydroxy-cholesterol-to-non-HDL cholesterol showed a trend towards a higher value in women (.41 + .02) than in men (.37 + .02) but it was not significantly different between the two genders (P = .18). Levels of lathosterol (women: .25 + .02 mg/dL; men: .25 + .02 mg/dL, P = .86) and campesterol (women: .25 + .02 mg/dL, men: .28 + .04 mg/dL; P = .61) and the ratio of lathosterol-campesterol (women: 1.29 + .17; men: 1.30 + .22, P = .97) were similar and there were no significant differences in levels of LDL cholesterol (women: 136 + 4 mg/dL; men: 132 + 6 mg/dL; P = .57).

Fig. (2). Plots of linear regression analyses of 24S-hydroxycholesterol versus Non-HDL cholesterol in women and men.

Plasma levels of 24S-hydroxycholesterol correlated significantly with levels of total cholesterol (r = .72, P < .0001), and non-HDL cholesterol in women and men (Fig. 2). The correlation coefficient of the oxysterol levels with LDL was significant in women (r = .68, P < .0001 but not significant in men (r = .4, P = .06). There were no significant correlations between the oxysterol and plasma levels of HDL cholesterol, triglycerides, lathosterol and campesterol in either gender (data not shown).

Table 1.     Association of 24S-Hydroxycholesterol with Polymorphisms of CYP46 and apo E

The T allele for CYP46 was present in 22/27 women and 10/14 men (Table 1). Eleven women and 6 men were homozygous for the TT polymorphism and 5/27 women and 4/14 men were homozygous for the CC polymorphism for the CYP46 gene (Table 1). There were no significant differences in levels of 24S-hydroxycholesterol, or the ratios of 24S-hydroxycholesterol-total cholesterol or lathosterol-campesterol in either men or women grouped according to the CYP46 polymorphisms (Table 1). However, the levels of 24S-hydroxycholesterol were significantly higher in women compared to men regardless of the CYP46 polymorphism (Table 1).

Seven subjects (5 women and 2 men) had e4/4; 27 subjects had e3/4 (18 women and 9 men); 14 subjects had e3/3 (3 women and 11 men), two women had e2/4 and another 2 had e2/3 polymorphism for apo E; one man had an e2/2. The apo E polymorphisms were not associated with differences in levels of plasma 24S-hydroxycholesterol, or the ratios of 24S-hydroxycholesterol-total cholesterol (or LDL cholesterol) or campesterol-lathosterol in either gender (Table 1). However, the gender differences in the levels of 24S-hydroxycholesterol were persistently observed in the subjects grouped according to CYP46 or apo E polymorphisms (Table 1).

Table 2.     Responsiveness to Statin Therapy Based on Gender and on Polymorphisms of CPY46 and apo E

Percent change in levels of sterols during treatment with statins is summarized in (Table 2). There were no significant differences in the percent change of 24S-hydroxycholesterol, non-HDL cholesterol, lathosterol or the ratio of 24S-hydroxycholesterol - non HDL cholesterol (data not shown) in women versus men or in subjects classified according to polymorphism of the CYP46 gene (Table 2). Polymorphism of the apo E gene also did no seem to affect the percent change in levels of 24S-hydroxycholesterol, non-HDL cholesterol or lathosterol in the subjects (Table 2). However, subjects with the e4/4 had significantly lower ratios of 24S-hydroxycholesterol-non-HDL cholesterol (Table 2).

Discussion

AD was initially linked to cholesterol metabolism when it was shown that the e4 allele of apolipoprotein E imparts a risk for AD [14, 15]. Since then, several workers have shown that apo E may play a role in the metabolism of b-amyloid protein and of tau [20 - 23]. Apo E may be a chaperone protein for the formation of intracellular microtubules by tau [24] and the secretion of b-amyloid to the extracellular matrix [20 - 23]. The apo E gene has at least three alleles; the most common is e3 followed by e2 and e4. e4 is prevalent in AD and it imparts a high risk for AD [14, 15]. Apo E4 interferes with the function of b-amyloid protein and of tau [20 - 24].

More recently, it has been shown that brain cholesterol is converted to 24S-hyrdoxycholesterol by the enzyme CYP46 [25]. The oxysterol is then transported to the plasma where it is incorporated into lipoproteins and is taken up by the liver and converted to bile acids for excretion [26]. Some workers have shown that 24S-hydroxy-cholesterol is elevated in plasma of subjects with AD [27] and others have shown that the oxysterol is also elevated in the cerebrospinal fluid of AD [28].

A number of studies suggested that total cholesterol is a risk for AD [1, 2]. However, total cholesterol is the sum of non-HDL cholesterol and HDL cholesterol. These two classes of lipoproteins have different functions and metabolic patterns. Therefore, it is important to determine whether either of these components, non-HDL cholesterol or HDL cholesterol, is better associated with AD risk. In the current study a strong association between the oxysterol and non-HDL cholesterol with 24S-hydroxycholesterol was observed. There was no correlation of the oxysterol with HDL cholesterol. It is possible that the reported associations of total cholesterol with AD risk reflect an association of the oxysterol with risk rather than total cholesterol itself. This possibility is worth exploring in future epidemiologic studies.

In the current study, it was also found that levels of 24S-hydroxycholesterol differ in women and men with AD. It is important to determine whether there is a gender-specific difference in levels of oxysterol and whether the oxysterol is a risk marker in women but not in men. This question deserves attention in larger, epidemiologic studies.

Another important question regarding levels of 24S-hydroxycholesterol is the impact of polymorphisms of genes encoding for key proteins on sterol metabolism. One such gene is CYP46. It encodes for the CYP46 enzyme. The gene is located in chromosome 14q32.1 in humans. The gene consists of 15 exons and 14 introns. Polymorphisms of the CYP46 gene have been described recently [13,19]. An AàG polymorphism in intron 2,150 base pairs upstream of exon 3 and a CàT polymorphism (IVS3+43CàT) in intron 3, 43 base pairs downstream of exon 3 have been reported. The functional significance of these CYP46 polymorphisms is not understood. Kölsch et al [19] reported that the C-allele of the CàT polymorphism is associated with an increased risk for AD and that AD patients who were carriers of the CC genotype presented with increased CSF ratios of 24S-hydroxycholesterol to cholesterol. Papasotiropoulos et al [13] reported that carriers of the AA genotype of the AàG polymorphism (also designated CàT polymorphism [19]) presented with an increased risk of AD and with increased levels of b-amyloid protein in the cerebrospinal fluid of AD patients.

CYP46 polymorphisms did not seem to affect levels of plasma 24S-hydroxy-cholesterol in the current study. This is in agreement with previous observations [19]. The response to statins also was unaffected by the CYP46 polymorphism in the current patients. The functional significance of the intronic polymorphism of the CYP46 remains to be elucidated. This is of major importance since the CYP46 TT allele has been associated with a significant risk for some forms of AD [13] and also it has been implicated in the levels of b-amyloid protein in the brain and cerebrospinal fluid of AD patients.

We have shown recently, that statins (lovastatin, simvastatin, and pravastatin) lower levels of plasma 24S-hydroxycholesterol and other sterols [16]. Statins are currently being employed in multi-center trials to examine the effects of this class of drugs on the course of AD. Statins were approved in the late 80’s to treat hypercholesterolemia in subjects having a high risk of mortality from coronary heart disease (CHD). Since then, several multi-center trials have been conducted in secondary and primary prevention that have shown reduction in CHD risk [29]. The drugs have been shown to be efficacious in lowering plasma levels of low-density lipoproteins and they have also been shown to be relatively safe when used appropriately [30]. Statins are targeted primarily to the liver where they are effectively extracted in the first pass. Statins also vary in their ability to traverse the blood-brain barrier [31]. More recently, there has been an increasing concern in the use of these drugs, particularly at high doses because of complaints of muscle weakness [32]. Other workers have reported cases of polyneuropathies associated with statin treatment [33]. Nonetheless, statins have a good track of relative safety when used appropriately [30].

Some studies suggest that apo E gene polymorphisms influence the LDL-lowering effect of statins [34 - 36]. It has been shown that subjects with at least one e4 allele do not respond as well as those with_ e2 or e3 alleles. However, other studies did not find significant differences in the cholesterol lowering effects of statins in subjects with different apo E genotypes [36 - 41]. In the current study, apo E genotype did not appear to influence levels of 24S-hydroxycholesterol. This observation is in agreement with the report of Lütjohann et al [28]. However, it has been clearly demonstrated in a number of studies that apo E alleles influence levels of LDL cholesterol. Subjects with the e4 allele tend to have higher levels of LDL than those with e3 or e2. In turn, subjects with e2 have the lowest LDL cholesterol levels. The differences in the LDL cholesterol levels can be explained on the basis of changes in function due to the amino acid substitutions of apo E. The apo E isoforms differ in cysteine and arginine content at positions 112 and 158: apoE2 (Cys112, Cys158), apoE3 (Cys112, Arg158), and apoE4 (Arg112, Arg158) [42, 43]. The amino acid substitutions impart different conformations to the isoproteins and thereby affect their function in lipid transport, receptor-mediated uptake of cholesterol and interaction with b-amyloid and tau [44]. In the current study, subjects with the e4/4 allele tended to have the lowest ratio of 24S-hydroxycholesterol-LDL cholesterol despite the fact that these individuals did not differ in the percent lowering of 24S-hydroxycholesterol during statin treatment. The ratio likely reflects the lesser reduction of LDL cholesterol during statin treatment that may be associated with the e4 allele.

 This study has several important clinical implications that warrant further exploration. The strong correlation of 24S-hydroxycholesterol to non-HDL cholesterol levels (and total cholesterol levels) suggests that the latter may actually be a surrogate marker for the plasma oxysterol, and may also explain the strong association reported between total cholesterol and AD. The stronger correlation between the oxysterol and non-HDL cholesterol in women may point to a gender-related vulnerability in women.

Acknowledgements

The authors express their appreciation for the excellent technical assistance of Kevin Vo, and Biman Pramanik in Dallas. Beverley Huet, Biostatistician of the General Clinical Research Center was consulted for statistical analysis of the data. The study was supported partially by the Veterans Affairs Medical Center Merit Review Grant, The Center for Human Nutrition, the Moss Heart Foundation, the Wallace, Barbara and Kelly King Charitable Foundation Trust, and The National Institute on Aging Grant no. P-30-AG12300; grant no. BMFT 01EC9402 from the Bundesministerium für Forschung und Technologie in Germany supported the sterol measurements, and the General Clinical Research Center grant (NIH # M01-RR00633) supported the statistical consultation, and American Alzheimer Association IIRG-98-142 supported the CYP46 polymorphism analyses.

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