Abstract
Background:
APOE4 is the strongest risk factor for Alzheimer’s disease (AD). However, limited information is currently available on APOE4 and the pathological role of plasma apolipoprotein E (ApoE) 4 remains unclear.
Objective:
The aims of the present study were to measure plasma levels of total ApoE (tE), ApoE2, ApoE3, and ApoE4 using mass spectrometry and elucidate the relationships between plasma ApoE and blood test items.
Methods:
We herein examined plasma levels of tE, ApoE2, ApoE3, and ApoE4 in 498 subjects using liquid chromatograph-mass spectrometry (LC-MS/MS).
Results:
Among 498 subjects, mean age was 60 years and 309 were female. tE levels were distributed as ApoE2/E3 = ApoE2/E4 >ApoE3/E3 = ApoE3/E4 >ApoE4/E4. In the heterozygous group, ApoE isoform levels were distributed as ApoE2 >ApoE3 >ApoE4. ApoE levels were not associated with aging, the plasma amyloid-β (Aβ) 40/42 ratio, or the clinical diagnosis of AD. Total cholesterol levels correlated with the level of each ApoE isoform. ApoE2 levels were associated with renal function, ApoE3 levels with low-density lipoprotein cholesterol and liver function, and ApoE4 levels with triglycerides, high-density lipoprotein cholesterol, body weight, erythropoiesis, and insulin metabolism.
Conclusion:
The present results suggest the potential of LC-MS/MS for the phenotyping and quantitation of plasma ApoE. Plasma ApoE levels are regulated in the order of ApoE2 >ApoE3 >ApoE4 and are associated with lipids and multiple metabolic pathways, but not directly with aging or AD biomarkers. The present results provide insights into the multiple pathways by which peripheral ApoE4 influences the progression of AD and atherosclerosis.
INTRODUCTION
Alzheimer’s disease (AD) is the most common cause of dementia and is characterized by the accumulation of amyloid-β (Aβ) and phosphorylated tau, which leads to neurodegeneration in the brain [1]. APOE4 is the strongest risk factor for sporadic AD [2], hypercholesterolemia, and atherosclerosis [3]. The APOE ɛ2, ɛ3, and ɛ4 alleles encode the major isoforms of apolipoprotein E (ApoE) 2, ApoE3, and ApoE4. The ɛ2 allele exerts protective effects against the development of AD [4]. In comparisons with ɛ3/ɛ3, the risk of developing AD is approximately 15-fold higher in individuals with ɛ4/ɛ4 and 0.6-fold higher in those with ɛ2/ɛ3 and ɛ2/ɛ2 [5]. The ɛ4 allele is associated with a more abundant amyloid pathology, the earlier onset of cognitive decline, and the faster clinical progression of AD [6, 7]. Recent studies implicated ApoE in tau neurofibrillary neurodegeneration, microglia and astrocyte responses, and disruption of the blood-brain barrier [8 –11]. Based on the role of ApoE in the pathogenesis of AD, several therapeutic approaches targeting ApoE are emerging in mouse models [8 , 12].
Emerging evidence indicates that peripheral ApoE, in addition to brain ApoE, directly or indirectly influences the progression of AD through multiple pathways [13]. The measurement of ApoE isoforms has recently been attracting increasing attention due to advances in mass spectrometry. This method successfully achieves linearity, reproducibility, and concordance with genetic testing as well as ELISA assays of ApoE isoforms in cerebrospinal fluid (CSF) and plasma [14 –17]. However, neither total ApoE (tE) nor ApoE isoforms in CSF are associated with the ɛ4 carrier status, Aβ status, or clinical diagnosis of dementia. Isoform-dependent differences in CSF ApoE concentrations have been observed in the order of ApoE2 <ApoE3 <ApoE4 in heterozygous subjects only [18]. The aim of the present study was to investigate the following: 1) isoform-dependent plasma ApoE concentrations and their natural course, 2) AD-associated differences in plasma ApoE isoforms, 3) the relationships between peripheral ApoE isoform levels and plasma Aβ levels, and 4) the effects of blood test parameters on plasma ApoE isoform levels. The results obtained will provide key insights for the development of therapeutic strategies targeting ApoE.
MATERIALS AND METHODS
Subjects
Four hundred and ninety-eight subjects were examined in the present study. Of these, 398 subjects were participants in the Iwaki Health Promotion Project (IHPP) conducted in 2014 [19]. Large datasets of health information, including blood tests and APOE genotypes, were referenced [20]. Since the ɛ2/ɛ3, ɛ2/ɛ4, and ɛ4/ɛ4 genotypes are less frequent APOE profiles in IHPP, these genotypes and the remaining ɛ3/ɛ3 and ɛ3/4 genotypes were randomly selected in up to 398 subjects. Another 75 subjects were participants in the Hirosaki Cohort for biomarker development for neurodegenerative diseases (HCBD) [21]. Among these subjects, 68 underwent a CSF examination and 22 were confirmed to have AD based on clinical criteria [22] with CSF tau and Aβ biomarkers [1]. The remaining 25 subjects were healthy and cognitively unimpaired Japanese employees of Soiken Holdings Inc. who were enrolled as controls (Soiken subjects).
Liquid chromatograph-mass spectrometry (LC-MS/MS) analysis
Recombinant ApoE2 and ApoE4 (PeproTech Inc., NJ, USA), heavy isotope-labeled peptides (Bio-Synthesis Inc., TX, USA), trypsin (Product Code: T8003; Merck KGaA Darmstadt, Germany), and dog serum (Kitayama Labes Co., Ltd., Nagano, Japan) were purchased. Other reagents were supplied by Kanto Chemical Co., Inc. (Tokyo, Japan) and Fuji Film Wako Pure Chemical Corporation (Osaka, Japan). Five signature peptides were selected as previously reported [14, 17]. They were LGADMEDVCGR (ApoE2/E3), LGADMEDVR (ApoE4 specific), LAVYQAGAR (ApoE3/E4), CLAVYQAGAR (ApoE2 specific), and LGPLVEQGR (common). Their heavy isotope-labeled peptides on R (Δ: +10) were used as internal standards in quantitative analyses. Calibration standards for quantitative analyses were prepared using dog serum, recombinant ApoE2 and ApoE4, and water with concentration ranges of 1–200μg/mL (2–400μg/mL for tE). Signature peptides were confirmed to be absent in dog ApoE in silico using a public database [23]. LC-MS/MS and data processing were based on previously reported methods [17]. Briefly, a plasma sample (10μL) was digested by trypsin, and 5 signature peptides and 5 corresponding heavy isotope-labeled peptides were analyzed by LC-MS/MS. Reversed phase high-performance liquid chromatography conditions and the parallel reaction monitoring detection mode were employed in the LC-MS/MS method. Phenotypes were identified by the detection patterns of 5 signature peptides. The peak area ratios of the signature peptides to the corresponding heavy isotope-labeled peptides were plotted against ApoE concentrations, and curves were fit by a linear least-squares regression method (a weighting factor of 1/X2). The concentrations of ApoE isoforms were calculated as previously reported [14, 17].
APOE genotyping, Aβ assay, and blood chemistry items
APOE genotypes were identified by Toshiba Corporation using the Japonica Array consisting of population-specific SNP markers designed from the 1070 whole-genome reference panel [19, 20]. An APOE promotor, SNP (rs405509 T>G alleles), that has been associated with plasma ApoE levels was also identified by the Japonica Array [24]. Plasma Aβ40 and Aβ42 levels were measured using the Human/Rat β Amyloid (40) ELISA Kit Wako II and Human/Rat β Amyloid (42) ELISA Kit Wako High-Sensitive (Wako Pure Chemical Industries, Ltd., Osaka, Japan) [20].
Standard protocol approvals, registrations, and patient consent
The present study was approved by the Ethical Committee of the Geriatrics Research Institute and Hospital (2019-78) and Hirosaki University (2014-014, 2015-377; 2016-028; 2017-026). All subjects provided written informed consent. All data were de-identified.
Statistical analysis
tE levels followed a normal distribution on a histogram, and the level of every ApoE isoform within subjects divided by phenotypes also followed a normal distribution [25]. Multiple comparison tests using the Bonferroni method were used to compare tE levels among each phenotype group. A linear regression model was used to clarify the relationships among ApoE isoform levels, age, Aβ levels, and Mini-Mental State Examination (MMSE) scores. To investigate the relationships between ApoE levels and individual health information, each value was dichotomized based on quartiles or laboratory abnormalities (total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), creatinine, blood urea nitrogen, and brain natriuretic peptide), and an analysis of variance or the t-test was performed [26]. tE and ApoE isoform levels were also compared between the AD group and the cognitively unimpaired group matched for age and ApoE phenotypes using propensity scores [27, 28] by paired t-tests. A t-test or ANOVA was used to analyze whether plasma ApoE isoforms and tE values varied with APOE promotor SNP (GG, GT or TT). Analyses were performed with R 4.1.2 version [29] and IBM SPSS Statistics version 24 for Windows (IBM, Armonk, NY, USA).
RESULTS
Demographic and clinical characteristics
Table 1 summarizes demographic and clinical characteristics, including cholesterol levels, plasma Aβ levels, and the distribution of ApoE phenotypes. The mean (standard deviation (SD)) age and age range of 498 subjects were 60.2 (12.4) years and between 23 and 91 years, respectively. There were 309 (62%) women. Due to missing blood test data in HCBD and Soiken subjects, an association study among ApoE isoform levels and blood test data was conducted on the 398 subjects who were participants in IHPP. In HCBD subjects, CSF tau (p < 0.001) and the CSF Aβ40/42 ratio (p = 0.001) were significantly higher in the AD group (n = 22) than in the non-AD group (n = 46) (Table 2). In comparisons between AD and non-AD subjects, the percentages of ApoE4 carriers were 63.6 and 28.3%, respectively.
Demographic and clinical characteristics and plasma ApoE levels
Aβ, amyloid-β; IHPP, Iwaki Health Promotion Project subjects; HCBD, Hirosaki Cohort for biomarker development for neurodegenerative diseases subjects; Soiken, healthy Soiken controls; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Total ApoE (tE), total cholesterol, LDL-cholesterol, HDL-cholesterol; ApoE, apolipoprotein E; Age, mean (SD); Sex, numbers; Total ApoE, mean (SD) μg/mL; total cholesterol, LDL-cholesterol and HDL-cholesterol: mean (SD) mg/mL; plasma Aβ40 and Aβ42, mean (SD) pg/mL; Aβ40/42 ratio, mean (SD); ApoE phenotype, numbers (%).
CSF levels of Aβ and tau as well as ApoE phenotypes in HCBD subjects
CSF, cerebrospinal fluid; HCBD, Hirosaki Cohort for biomarker development for neurodegenerative diseases; N, number; AD, Alzheimer’s disease; Aβ, amyloid-β; ApoE, apolipoprotein E. CSF Aβ40, Aβ42, and tau, mean (SD) pg/mL; Aβ40/42 ratio, mean (SD).
Accuracy of LC-MS/MS
The validation of the LC-MS/MS analysis showed no interfering peaks for selectivity or carry-over, fewer relative errors for both matrix effects and the calibration curve (relative error (RE): –3.4 to 3.1%; coefficient of variation (CV): 6.1 to 7.9%; and the calibration curve (RE: –10. to 13.5%)), and good accuracy and precision for both the intra-batch (RE: –18 to 7.2%; CV: 0.7 to 11.5%) and inter-batch (RE: –11.7 to 5.3%; CV: 2.5 to 9.5%). The autosampler was stable for 48 hours with a residual ratio of 87.9 to 110.4%. The CV of batch sizes was 1.3 to 3.7% and a visually normal single peak was observed with an abnormal waveform. Among 398 subjects who were participants in IHPP, 398 (100%) matched genotype findings by Japonica array analyses with phenotyping by LC-MS/MS [14 , 20]. The mean (SD) level of tE corresponded to previously reported values [14, 16]. Measured tE levels were equal to the calculated sum of the corresponding ApoE isoform levels. Therefore, LC-MS/MS profiles suggest its practical relevance to the phenotyping and quantitation of plasma ApoE.
Plasma levels of ApoE isoforms
The mean (SD) levels of tE and ApoE isoforms for each phenotype are shown in Table 3. tE levels were significantly higher in the ApoE2/E3 and ApoE2/E4 groups than in the ApoE3/E3, ApoE3/E4, and ApoE4/E4 groups (all p values <0.001). No significant differences were observed in tE levels between the ApoE3/E3 and ApoE3/E4 groups (p > 0.9), between the ApoE3/E3 and ApoE4/E4 groups (p = 0.1148), or between the ApoE3/E4 and ApoE4/E4 groups (p = 0.3865) (Fig. 1A). In comparisons between homozygous groups, tE levels were significantly higher in the ApoE3/E3 group than in the ApoE4/E4 group (p = 0.0178) (Fig. 1B). Plasma tE levels showed a stepwise decrease in the order of ApoE2/E3 = ApoE2/E4 >ApoE3/E3 = ApoE3/E4 >ApoE4/E4. ApoE2 levels were significantly higher than ApoE3 levels in the ApoE2/E3 group (p < 0.001); ApoE2 levels were significantly higher than ApoE4 levels in the ApoE2/E4 group (p < 0.001); ApoE3 levels were significantly higher than ApoE4 levels in the ApoE3/E4 group (p < 0.001) (Fig. 1C). Therefore, the plasma levels of ApoE isoforms were consistently sustained in the order of ApoE2 >ApoE3 >ApoE4 in the homo- and heterozygous groups.
Plasma ApoE levels of participants
ApoE, apolipoprotein E; N, number.
Total ApoE and ApoE isoform levels in plasma. A) Plasma levels of total ApoE (tE) for each ApoE phenotype. Adjusted p-values (by the method of Bonferroni) are indicated between groups that showed significant differences. tE levels in the ApoE2/E3 and ApoE2/E4 groups did not significantly differ (p > 0.9). tE levels were significantly higher in the ApoE2/E3 and ApoE2/E4 groups than in the ApoE3/E3, ApoE3/E4, and ApoE4/E4 groups (all p values <0.001). No significant differences were observed in tE levels between the ApoE3/E3 and ApoE3/E4 groups (>0.9), between the ApoE3/E3 and ApoE4/E4 groups (p = 0.1148), or between the ApoE3/E4 and ApoE4/E4 groups (p = 0.3865). B) Plasma ApoE levels for the homozygous ApoE phenotypes. Since only one case had the ApoE2/E2 phenotype, a significance test cannot be performed. tE levels were significantly higher in the ApoE3/E3 group than in the ApoE4/E4 group (p = 0.0171). C) Plasma levels of ApoE isoforms within groups of heterozygous ApoE phenotypes. In the ApoE2/E3 phenotype, ApoE2 levels were significantly higher than ApoE3 levels. In the ApoE2/E4 phenotype, ApoE2 levels were significantly higher than ApoE4 levels. In the ApoE3/E4 phenotype, ApoE3 levels were significantly higher than ApoE4 levels. Therefore, the plasma levels of the ApoE isoforms were consistently sustained in the order of ApoE2 >ApoE3 >ApoE4 in the homo- and heterozygous groups.
Age-related alterations in ApoE isoforms
Total ApoE3 and ApoE4 levels in the homozygous ApoE3/E3 and ApoE4/E4 groups were not significantly affected by age (Fig. 2A, B). Slight age-related increases in ApoE3 levels were observed in the ApoE2/E3 group (p = 0.0376; R2 = 0.05865; Fig. 2C). The levels of the other ApoE isoforms, including those of ApoE2 in the ApoE2/E3 group, ApoE2 and ApoE4 in the ApoE2/E4 group, and ApoE4 in the ApoE3/E4 group, were not significantly affected by age (Fig. 2D, E).
Age-related alterations in plasma levels of ApoE isoforms. A) Age-related alterations in the plasma levels of ApoE in ApoE homozygote phenotypes. ApoE levels did not significantly change with aging in the ApoE3/E3 and ApoE4/E4 phenotypes. B) Age-related alterations in the plasma levels of total ApoE and ApoE isoforms in ApoE heterozygote phenotypes. In the ApoE2/E3 phenotype, ApoE3 levels slightly increased with aging (p = 0.0376). The p-value indicates the significance of the slope of the regression line. Five outliers of plasma Aβ40 and Aβ42 levels were excluded using Grubbs’ statistical method [51 –53].
Relationships among ApoE isoforms, Aβ40, and Aβ42
The results obtained are summarized in Fig. 3A–C. ApoE3 levels positively correlated with Aβ40 (p < 0.0005, R2 = 0.05441) and Aβ42 (p = 0.0009, R2 = 0.04932), but not with the Aβ40/42 ratio, in the ApoE3/E3 group. A negative relationship was observed between ApoE4 and Aβ40 and between ApoE4 and Aβ42, while a positive relationship was noted between ApoE4 and the Aβ ratio; however, these relationships were not significant in the ApoE4/E4 group. No correlations were found among ApoE isoforms and Aβ species in the ApoE2/E3 or ApoE2/E4 group. In the ApoE3/E4 group, only ApoE3 levels correlated with Aβ40 levels (p = 0.0471; R2 = 0.02298).
Relationships between ApoE isoforms and Aβ species in plasma. Alterations in ApoE isoform levels by the plasma levels of Aβ40 (A), Aβ42 (B), the Aβ40/42 ratio (C), and Mini-Mental State Examination (MMSE) scores (D) in each ApoE phenotype. In the ApoE3/E3 phenotype, plasma levels of ApoE increased with elevations in the plasma levels of Aβ40 and Aβ42. In the ApoE3/E4 phenotype, plasma levels of ApoE3 slightly increased with elevations in the plasma level of Aβ40. However, these slopes were minor (A–C). High levels of ApoE3 in the ApoE3/E3 phenotype and ApoE2 in the ApoE2/E3 phenotype were associated with high scores of MMSE (D). The p-value indicates the significance of the slope of the regression line. β indicates the slope of the regression line.
Relationships between ApoE isoforms and health information and blood tests
The results of the association study are shown in Table 4 and Supplementary Tables 1–4. High tE levels were associated with female sex, a short height, low body weight, and high levels of ALT, γ-GTP, total cholesterol, high-density lipoprotein (HDL)-cholesterol, calcium, and free fatty acids. High ApoE2 levels were associated with high total cholesterol, high free fatty acids, and renal function (low creatinine and cystatin C). ApoE3 levels were associated with a short height and high levels of AST, ALT, γ-GTP, total cholesterol, HDL- and low-density lipoprotein (LDL)-cholesterol, blood sugar, free fatty acids, and cystatin C. High ApoE4 levels were associated with male sex, a high height and weight, high levels of ALT, γ-GTP, uric acid, total cholesterol, triglycerides, LDL-cholesterol, free fatty acids, cystatin C, erythropoiesis (high hemoglobin and ferritin), and insulin metabolism (high C-peptide and low glycoalbumin) as well as low levels of HDL-cholesterol. Total cholesterol levels showed a consistently strong relationship with ApoE2, ApoE3, and ApoE4 levels. ApoE3 levels were associated with LDL-cholesterol levels and ApoE4 levels with triglyceride and HDL-cholesterol levels. Regarding lipid-related HDL-cholesterol, LDL-cholesterol, total cholesterol, and triglycerides, we performed a regression analysis with plasma ApoE levels. A positive correlation was observed between tE and total cholesterol, HDL-cholesterol, and triglycerides (Fig. 4A, C, E, G). Total cholesterol positively correlated with plasma ApoE2, ApoE3, and ApoE4 (Fig. 4B), HDL-cholesterol positively correlated with plasma ApoE3 and negatively correlated with ApoE4 (Fig. 4D). Plasma ApoE3 positively correlated with LDL (Fig. 4F). Triglycerides positively correlated with plasma ApoE2, ApoE3, and ApoE4 (Fig. 4H).
Relationships between ApoE and health information, including blood test items
AST, aspartate aminotransferase; ALT, alanine aminotransferase; γ-GTP, γ-glutamyl transpeptidase; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Asterisks indicate low p-values in the t-test or analysis of variance: each p-value is *: 0.05∼0.01, **: 0.01∼0.001, and ***: <0.001.
Single regression models of ApoE and cholesterol levels. A,C,E,G) Relationships between total ApoE and total cholesterol (A): Y = 0.1047*X + 18.69, p < 0.0001, HDL-cholesterol (C): Y = 0.1195*X + 32.21, p = 0.002, LDL-cholesterol (E): Y = 0.02229*X + 37.39, p = 0.2869, and triglycerides (G): Y = 0.06088*X + 34.35, p < 0.0001. B) Relationships between total cholesterol and ApoE isoforms. ApoE2: Y = 0.07241*X + 21.54, p = 0.008; ApoE3: Y = 0.1603*X - 3.099, p < 0.0001; ApoE4: Y = 0.04234*X + 3.636, p = 0.0003 (B). D) Relationships between HDL-cholesterol and ApoE isoforms. ApoE2: Y = 0.07188*X + 30.41, p = 0.2222; ApoE3: Y = 0.1067*X + 22.57, p = 0.006; ApoE4: Y = –0.08322*X + 17.75, p = 0.0003 (D). F) Relationships between LDL-cholesterol and ApoE isoforms. ApoE2: Y = 0.02237*X + 32.99, p = 0.4668; ApoE3: Y = 0.1261*X + 14.69, p < 0.0001; ApoE4: Y = 0.01938*X + 10.05, p = 0.1305 (F). H) Relationships between triglycerides and ApoE isoforms. ApoE2: Y = 0.04291*X + 30.72, p = 0.0003; ApoE3: Y = 0.03988*X + 25.63, p < 0.0001; ApoE4: Y = 0.04152*X + 7.896, p < 0.0001 (H).
Relationships among ApoE isoforms and MMSE
Of the HCBD and Soiken subjects, 42 did not receive the MMSE test. Therefore, 456 subjects were included in the statistical analysis. The results obtained are summarized in Fig. 3D. ApoE3 levels positively correlated with MMSE (p = 0.0357, R2 = 0.02264) in the ApoE3/E3 group. Positive relationships were observed between MMSE and tE (p = 0.0006, R2 = 0.1579) and Apo2 (p < 0.0001, R2 = 0.218) levels. No correlations were found among ApoE isoforms and MMSE in the ApoE4/E4, ApoE2/E4 or ApoE3/E4 group.
Plasma ApoE levels do not predict or diagnose AD
In the propensity score matching analysis, 17 AD subjects were successfully matched and 5 were excluded. No significant differences were observed in tE levels between AD and non-AD subjects matched by age and ApoE phenotype groups (Fig. 5A). A paired t-test of ApoE isoform levels between AD and non-AD subjects matched by age in each ApoE phenotype (ApoE3/E3, ApoE4/E4, and ApoE3/E4) showed no significant differences (Fig. 5B–F). Therefore, plasma ApoE levels are not a direct biomarker for the prediction or diagnosis of AD.
The paired t-test for ApoE values in AD and matched control subjects. A) Paired t-tests of total ApoE levels between AD (n = 17) and non-AD (n = 17) matched by age and the ApoE phenotype showed no significant differences. B,C) In the ApoE4/E4 and ApoE3/E3 groups, paired t-tests of total ApoE levels between AD and non-AD matched by age showed no significant differences. D, E) In the ApoE3/E4 group, the paired t-test showed no significant differences in ApoE3, ApoE4, or total ApoE levels between AD and age-matched non-AD. AD, Alzheimer’s disease; ns, not significant.
The association between APOE promotor SNP and plasma ApoE levels
Of the 398 subjects analyzed with the Japonica array, APOE promotor rs405509 T alleles analysis was available for 376 subjects. In ApoE2/E4 phenotype and ApoE4/E4 phenotype, since there was only a single allele type (only GT in ApoE2/E4 and GG in ApoE4/4), it was not possible to analyze the association with plasma ApoE levels. This SNP was not associated with plasma ApoE levels in ApoE2/E3 phenotype (N = 16 in GG and 44 in GT; p values were 0.2495 for ApoE2, 0.1464 for ApoE3 and 0.9529 for tE), ApoE3/E3 phenotype (N = 75 in TT, 71 in GT and 10 in GG; p value was 0.2455), and ApoE3/4 phenotype (N = 103 in TT and 38 in GT; p values were 0.5101 for ApoE3, 0.9802 for ApoE4, and 0.6475 for tE).
DISCUSSION
We summarized these major reports of peripheral or CSF ApoE measurement in Table 5. The results of previous studies suggested that plasma tE levels differ among APOE genotypes [16 , 30–33] and that there may be a strong genetic influence on tE levels that vary with age and sex [34]. In addition, the results showed that brain volume and brain metabolism may be affected in an ApoE isoform-dependent manner [35], but no correlation was found between tE levels and cognitive function or AD diagnosis [36]. The present results are consistent with APOE genotype-dependent stepwise decreases in tE levels. However, age- and AD-related alterations in tE and ApoE isoform levels were interpreted as being negative. We found that the plasma levels of ApoE2, ApoE3, and ApoE4 were stably controlled at the following three step levels: ApoE2 >ApoE3 >ApoE4. This phenomenon may simply be interpreted as the cause of genotype-dependent stepwise decreases in tE levels. Since the frequency of ɛ4 increases in MCI and AD, overestimations of decreases in tE levels may occur. Therefore, the present results suggest that the plasma levels of ApoE isoforms were not affected by aging or AD.
Summarized major previous reports of peripheral or CSF ApoE measurement
LC-MS, liquid chromatograph-mass spectrometry; ELISA, enzyme-linked immuno sorbent assay; CSF, cerebrospinal fluid; AD, Alzheimer’s disease; ND, neurodegenerative disorders; ApoE, apolipoprotein E; LDL, low density lipoprotein; HDL, high-density lipoprotein.
CSF tE levels were reported to be elevated in amyloid-positive (A+) individuals; however, the levels of ApoE isoforms varied conversely in the order of ApoE4 >ApoE3 >ApoE2 in heterozygous individuals [18]. A negative finding of CSF ApoE has been suggested as a diagnostic biomarker for AD in addition to plasma ApoE [18 , 37]. This finding suggests different ApoE isoform-dependent turnover rates in the periphery and brain. Plasma ApoE turnover occurs in an isoform-dependent manner with a rank order of ApoE4 >ApoE3 >ApoE2. In contrast, brain ApoE turnover is 3- to 6-fold slower and is independent of the isoform [13]. Although ApoE is unable to cross the blood-brain barrier [13, 38], a recent study revealed that ɛ4 disrupted the blood-brain barrier [39, 40], induced severe cerebral amyloid angiopathy, and promoted the accumulation of insoluble Aβ40 and ApoE [41]. Furthermore, the breakdown of the blood-brain barrier has been implicated in both ɛ4-associated and AD pathology-independent cognitive decline [11, 42]. The targeting of ApoE4 has been proposed to ameliorate cerebral amyloid angiopathy and the amyloid pathology with the protection of cerebrovascular integrity and function [12, 43]. Based on these advances, the present results on the plasma levels of ApoE4, which were 10-fold higher than brain and CSF levels, indicate the necessity of modifying ApoE4 levels not only to attenuate cerebral amyloid angiopathy and the AD pathology, but also to avoid severe cardiovascular atherosclerosis.
The plasma Aβ42/40 ratio is a useful biomarker for brain Aβ amyloidosis in normal individuals [44, 45]. Age, APOE genotypes, and Aβ amyloidosis are specific factors that influence Aβ kinetics from the brain to plasma. The clearance efficiency of Aβ is regulated in the order of ɛ2 > ɛ3 > ɛ4 [46]. We previously identified age and ɛ4 as major factors affecting the plasma levels of Aβ42 and the Aβ40/42 ratio [20]. In the present study, plasma Aβ40 and Aβ42 levels, but not the Aβ40/42 ratio, correlated with ApoE3 levels in ApoE3/E3 homozygotes. The relationships between Aβ40, Aβ42, the Aβ ratio, and ApoE4 levels were inverted, but not significant in ɛ4/ɛ4 homozygotes. These results imply that plasma ApoE3 and ApoE4 levels are associated with differences in the Aβ clearance capacity and peripheral degradation rates, but not with brain Aβ amyloidosis levels. A large-scale analysis of ɛ4 homozygotes is warranted to reach more definite conclusions.
On the other hand, we showed that MMSE scores were not associated with ApoE4 values but correlated with ApoE3 in the E3/E3 group and ApoE2 in the E2/E3 group. The APOE ɛ2 allele is thought to be protective against the development of dementia [4], and lower ApoE2 levels also may be associated with lower MMSE. However, the subjects in this study had skewed MMSE scores (because the study included a population-based cohort, many of whom had normal cognitive function), making accurate assessment difficult.
The rs405509 T allele reduces APOE expression and plasma ApoE levels [24]. However, in our cohort, there was no predominant association between this SNP and plasma ApoE levels. This may have been due to allelic variation in a number of subjects, and difficulties in correcting for lipids affecting plasma ApoE.
ApoE3 and ApoE2 are more closely associated with HDL particles, while ApoE4 preferentially associates with triglyceride-rich VLDL, leading to the downregulation of LDL receptors and, thus, the reduced clearance of LDL and increases in cholesterol levels. These differences in affinity increase the risk of atherosclerosis and stroke in ɛ4 carriers, as well as the risk of type III hyperlipoproteinemia in ɛ2 carriers because of the defective binding of ApoE2 LDL receptors, which impairs the clearance of triglyceride-rich lipoprotein remnant particles [9, 13]. In comparisons with tE, Copenhagen studies reported an inverted increase in total and LDL cholesterol, but a decrease in HDL cholesterol in the order of ɛ2/ɛ2, ɛ2/ɛ3, ɛ2/ɛ4, ɛ3/ɛ3, ɛ3/ɛ4, and ɛ4/ɛ4 [34]. The present results showed different relationships between ApoE isoform levels and blood lipid compositions. ApoE2 levels were associated with total cholesterol and free fatty acids levels, ApoE3 levels with total cholesterol, LDL-cholesterol, and free fatty acid levels, and ApoE4 levels with total cholesterol, triglyceride, HDL- and LDL-cholesterol, and free fatty acid levels. ApoE2 levels were associated with renal function, ApoE3 levels with height and liver function, and ApoE4 levels with female sex, body weight, erythropoiesis, and insulin metabolism. These results suggest that ApoE isoforms undertake multiple roles in human diseases, except for AD and atherosclerosis. We reported that the significant relationships between ApoE and insulin metabolism (C-peptide and glycoalbumin) were found only in ApoE4, not ApoE2 or ApoE3. ApoE4 impairs cerebral insulin signaling in an age-dependent manner in vivo. In humans, there is an involvement between plasma ApoE levels and glucose metabolism in the hippocampus [48]. Our study provides the possibility that ApoE4 may be involved in the regulation of insulin signaling outside the cerebrum. On the other hand, there is a report that ApoE3 levels, but not ApoE4, is involved in the process of controlling plasma glucose levels in an insulin-independent manner [49]. These conflicting results might have been caused by differences in measurement methods or in the characteristics of the included participants. Therefore, a reevaluation of the plasma levels of ApoE isoforms is critical for discovering the natural course and multiple functions of ApoE.
Several therapeutic approaches that target ApoE are emerging in mouse models [8 , 12]. The absence of ApoE does not cause cognitive impairment, except for atherosclerosis, in humans [50]. Forthcoming anti-Aβ immunotherapy for AD needs to avoid ɛ4-dependent amyloid-related imaging abnormalities [43]. Therefore, the significance of controlling the amount of ApoE based on the amounts of ApoE in the blood and brain remains to be clarified. Further information on the natural course of and alterations in plasma ApoE levels is needed.
Footnotes
ACKNOWLEDGMENTS
We thank Naoko Nakahata, Sakiko Narita, Kaoru Sato, and members of the Iwaki Health Promotion Project (IHPP) group for research assistance. IHPP was supported by Hirosaki University, Hirosaki city, Hirosaki City Medical Association, and Aomori Prefecture.
FUNDING
This work was supported by Grants-in-Aid for Scientific Research [grant numbers 18K07385, 19K07989] from the Ministry of Education, Science, and Culture of Japan; and the JST COI [grant number JPMJCE1302].
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
DATA AVAILABILITY
The information obtained from this study cannot be publicly disseminated due to ethical considerations. However, researchers who meet the criteria for accessing the data may request it from the Hirosaki University COI Program Institutional Data Access/Ethics Committee.
