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Journal of Lipid Research logoLink to Journal of Lipid Research
. 2024 Oct 10;65(11):100667. doi: 10.1016/j.jlr.2024.100667

Apolipoprotein B gene expression and regulation in relation to Alzheimer’s disease pathophysiology

Gabriel Aumont-Rodrigue 1,2,3, Cynthia Picard 1,2, Anne Labonté 1,2, Judes Poirier 1,2,3,∗
PMCID: PMC11602985  PMID: 39395793

Abstract

Apolipoprotein B (APOB), a receptor-binding protein present in cholesterol-rich lipoproteins, has been implicated in Alzheimer's disease (AD). High levels of APOB-containing low-density lipoproteins (LDL) are linked to the pathogenesis of both early-onset familial and late-onset sporadic AD. Rare coding mutations in the APOB gene are associated with familial AD, suggesting a role for APOB-bound lipoproteins in the central nervous system. This research explores APOB gene regulation across the AD spectrum using four cohorts: BRAINEAC (elderly control brains), DBCBB (controls, AD brains), ROSMAP (controls, MCI, AD brains), and ADNI (control, MCI, AD clinical subjects). APOB protein levels, measured via mass spectrometry and ELISA, positively correlated with AD pathology indices and cognition, while APOB mRNA levels showed negative correlations. Brain APOB protein levels are also correlated with cortical Aβ levels. A common coding variant in the APOB gene locus affected its expression but didn't impact AD risk or brain cholesterol concentrations, except for 24-S-hydroxycholesterol. Polymorphisms in the CYP27A1 gene, notably rs4674344, were associated with APOB protein levels. A negative correlation was observed between brain APOB gene expression and AD biomarker levels. CSF APOB correlated with Tau pathology in presymptomatic subjects, while cortical APOB was strongly associated with cortical Aβ deposition in late-stage AD. The study discusses the potential link between blood-brain barrier dysfunction and AD symptoms in relation to APOB neurobiology. Overall, APOB's involvement in lipoprotein metabolism appears to influence AD pathology across different stages of the disease.

Supplementary key words: Alzheimer’s disease, Apolipoproteins, Brain Lipids, Cholesterol, Lipoproteins/Receptors, Oxysterol, Transcription

Apolipoprotein B (APOB) is the main structural and receptor-binding component of atherogenic lipoproteins in the very low-density lipoprotein (VLDL) pathway, including low density lipoprotein (LDL) (1, 2). It is also a ligand for several members of LDL receptor family of receptors, many of which internalize cholesterol-rich lipoproteins via the endosomal-lysosomal pathway such as the LDLR (3). The human APOB gene is on chromosome 2 and spans 43 kbp, with 29 exons and 28 introns. Expression of APOB is not primarily regulated at the level of gene transcription and the regulation of APOB synthesis and secretion also occurs at the post-transcriptional level. This is remarkable, given that the APOB protein (4,536 amino acids) and mRNA (14.1 kbp) are large and therefore energetically expensive to synthesize constitutively if the protein is not required. Nevertheless, APOB secretion from peripheral cells can vary considerably (as much as 10-fold) without change in the cellular level of APOB mRNA (4). Up until recently, it was believed that APOB was exclusively expressed in peripheral tissues, mainly the liver and intestine, but its synthesis in glial cells has been confirmed in the central nervous system (CNS) by RNA sequencing (5). Although the enzyme responsible for the post-transcriptional modification of APOB-100 into APOB-48, APOBEC1, has been measured in microglia, (6) only APOB-100 has been detected in the present study. However, it is impossible to exclude the possibility that APOB-48 might also be present. APOB has also been detected in human cerebrospinal and was found to correlate extensively with tau protein pathology in Alzheimer’s disease (AD) and, neurotoxicity in amyotrophic lateral sclerosis (7, 8).

Alzheimer’s disease is characterized by the formation of neurofibrillary tangles, the accumulation of β-amyloid plaques immunopositive for APOB (9) and severe neuronal loss in the brain. High serum total cholesterol in midlife represents a significant risk factor for AD and other dementias in later life (10, 11, 12). It is also associated with increased dementia mortality (13). Recent mendelian randomization analyses revealed that circulating levels of APOB but not LDL shortens health span and increases the risk of AD (5). Similarly, Skoog and colleagues (14) found that total cholesterol decline trajectory over the years is associated with relatively poorer cognition in a population-based representative sample of individuals aged 70 and older, not on lipid-lowering medications. Also, brain oxysterol levels, such as 24-S-hydroxycholesterol and 27-hydroxycholesterol, are altered in the presence of AD (15, 16). These cholesterol species linked to AD are also known modulators of APOB gene expression in periphery (17).

While the exact role these oxysterols play in this neurodegenerative disease remains controversial, they have been associated with compromised blood–brain barrier integrity (18). Altogether, these findings support the claim that cholesterol metabolic dysregulation is associated with cognitive decline in old age.

Parallel lines of evidence have shown that circulating LDL levels play an active role in the pathogenesis of early-onset familial Alzheimer's disease (EOAD) as they do for sporadic AD (19). Genetic association studies performed in familial AD have shown the presence of rare coding mutations in the APOB gene, suggesting a pathophysiological role for APOB-bound lipoproteins found in the CNS (19). Independently, CSF APOB protein levels were found to strongly correlate with total tau and p(181) tau concentrations as well as with tau deposition in vulnerable brain areas such as the entorhinal cortex and fusiform gyrus in “at-risk” but asymptomatic subjects (7). No associations were detected between APOB levels and Aβ42 neither in the CSF nor with amyloid deposition using PET imaging (7). CSF APOB levels are elevated in sporadic AD relative to age-matched cognitively unaffected individuals (7). A post-mortem study of pathologically confirmed, definite, AD patients showed that peripheral LDL and APOB levels positively correlate with brain Aβ42 levels (20) whereas APOB was found to be structurally bound to β-amyloid plaques and tangles in autopsied AD brain (9, 21).

Together, these findings suggested an early involvement of brain APOB in the pre-symptomatic phase of the disease, particularly in sync with the initiation of tau pathology. Later, as cognitive deficits emerge, APOB appears to interact with amyloid pathology in a manner that is reminiscent of apolipoprotein E neurobiology; facilitating amyloid deposition (22). Interestingly, it is also at that moment that the first signs of blood–brain dysfunction are emerging (23). This article examines how APOB is regulated in normal and AD brains and what could be the pathophysiological role of this important CNS apolipoprotein as cognitive deficits surface.

Materials and Methods

Cohorts

Brain eQTL Almanac (BRAINEAC cohort)

Publicly available cohort dataset composed of central nervous system (CNS) tissue originating from 134 cognitively unaffected control individuals was collected by the Medical Research Council Sudden Death Brain and Tissue Bank, (24) and the Sun Health Research Institute (SHRI) an affiliate of Sun Health Corporation (25). Anatomical regions of interest were sampled from brain coronal slices on dry ice. A detailed description of the samples used in the study, tissue processing, and dissection is provided in Trabzuni et al. (26) All samples had fully informed consent for retrieval and were authorized for ethically approved scientific investigation (Research Ethics Committee number 10/H0716/3).

Genotyping

Genomic DNA was extracted from sub-dissected samples of human post-mortem brain tissue using Qiagen’s DNeasy Blood & Tissue Kit (Qiagen). All samples were genotyped on the Illumina Infinium Omni1-Quad BeadChip and the Immunochip, a custom genotyping array designed for the fine-mapping of auto-immune disorders (27, 28). After standard quality controls (removal of suspected non-European descent individuals, samples with call rate <95% and checks on reported sex status, cryptic relatedness, autosomal heterozygosity rate check, monomorphic SNPs or call rate <95%, no genomic position info or redundant SNPs, P-value for deviation from HWE < 0.0001, genotyping call rate <95%, less than 2 heterozygotes present, mismatching alleles 1000G even after allowing for strand), imputation was performed using MaCH (29, 30). This resulted in ∼5.88 million SNPs and ∼577 thousand indels with good post-imputation quality (Rsq > 0.50) and minor allele frequency of at least 5%.

Microarray

Total RNA was isolated from human post-mortem brain tissues based on the single-step method of RNA isolation (31) using the miRNeasy 96 kit (Qiagen). The quality of total RNA was evaluated by the 2,100 Bioanalyzer (Agilent) and RNA 6000 Nano Kit (Agilent) before processing with the Ambion® WT Expression Kit and Affymetrix GeneChip Whole Transcript Sense Target Labeling Assay, and hybridization to the Affymetrix Exon 1.0 ST Arrays following the manufacturer’s protocols. Further details regarding RNA isolation, quality control, and processing are reported in Trabzuni et al (26). Gene-level expression was estimated for 26 thousand genes by calculating the Winzorised mean (below 10% and above 90%) signal of all probe sets corresponding to each gene. The resulting expression data was adjusted for brain bank, gender, and batch effects in Partek’s Genomics Suite v6.6 (Partek Incorporated). APOB-100 mRNA levels were calculated using the Winsorised mean over probesets (t2543163) for 10 brain regions.

Douglas-Bell Canada brain bank study (DBCBB cohort)

The Douglas-Bell Canada Brain Bank based and operating at our academic institution conforms to the Code of Ethics of the World Medical Association, abides by the Declaration of Helsinki, and was approved by the Ethics Board of the Douglas Hospital Research Centre. Every participant, or his or her legal tutor, signed an informed consent statement. Patients’ demographic characteristics are summarized in Table 1. Definite diagnosis of AD (n = 68) was based on histopathological confirmation of AD according to NINCDS-ADRDA criteria (32), whereas controls (n = 31) had to be free of neurological or psychiatric diseases and of brain structural lesions (tangle and plaque indices reading < 20 ⁄mm3 and < 10 ⁄mm2, respectively).

Table 1.

Participants characteristics

Cohorts Sex APOE Genotype
BRAINEAC F (n = 35) M (n = 99) APOE4- (n = 95) APOE4+ (n = 39)
 Age at death (yrs ± SEM) 63.5 ± 3.5 56.7 ± 1.8 61.2 ± 1.9 51.9 ± 2.8∗
 APOB mRNA levels (mean ± SEM) 3.88 ± 0.01 3.88 ± 0.01 3.88 ± 0.01 3.88 ± 0.01
ROSMAP: CTL F (n = 123) M (n = 78) APOE4- (n = 168) APOE4+ (n = 33)
 Age at death >83 >81 >83 >83
 APOB mRNA levels (mean ± SEM) 0.018 ± 0.001 0.012 ± 0.001 0.017 ± 0.001 0.016 ± 0.003
ROSMAP: MCI F (n = 106) M (n = 62) APOE4- (n = 135) APOE4+ (n = 32)
 Age at death >86 >83 >85 >83
 APOB mRNA levels (mean ± SEM) 0.017 ± 0.001 0.016 ± 0.001 0.017 ± 0.001 0.016 ± 0.002
ROSMAP: AD F (n = 173) M (n = 83) APOE4- (n = 160) APOE4+ (n = 96)
 Age at death >87 >85 >86 >85
 APOB mRNA levels (mean ± SEM) 0.015 ± 0.001 0.014 ± 0.001 0.015 ± 0.001 0.014 ± 0.001
DBCBB: CTL F (n = 11) M (n = 20) APOE4- (n = 22) APOE4+ (n = 9)
 Age at death (yrs ± SEM) 80.82 ± 2.15 75.5 ± 2.89 78.95 ± 2.61 73.56 + 2.76
 APOB mRNA levels (mean ± SEM) 4.38 ± 0.10 4.15 ± 0.09 4.22 ± 0.08 4.25 ± 0.13
DBCBB: AD F (n = 23) M (n = 32) APOE4- (n = 23) APOE4+ (n = 32)
 Age at death (yrs ± SEM) 79.78 ± 1.35 81.38 ± 1.12 82.09 ± 1.31 79.72 + 1.13
 APOB mRNA levels (mean ± SEM) 4.14 ± 0.08 4.15 ± 0.06 4.10 ± 0.07 4.19 ± 0.06
ADNI: CTL F (n = 35) M (n = 36) APOE4- (n = 54) APOE4+ (n = 16)
 Age at baseline (yrs ± SEM) 75.58 ± 0.87 75.08 ± 0.94 75.16 ± 0.69 75.91 + 1.56
ADNI: MCI F (n = 21) M (n = 49) APOE4- (n = 41) APOE4+ (n = 29)
 Age at baseline (yrs ± SEM) 72.95 ± 1.57 75.70 ± 1.00 76.24 ± 1.02 72.94 ± 1.40
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∗P ˂ 0.05.

Microarray

Total RNA was quantified using a NanoDrop Spectrophotometer ND – 1,000 (NanoDrop Technologies, Inc.) and its integrity was assessed using a 2,100 Bioanalyzer (Agilent Technologies). Sense-strand cDNA was synthesized from 10 ng of total RNA, and fragmentation and labeling were performed to produce ss DNA with the GeneChip® WT Pico Terminal Labeling Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). After fragmentation and labeling, 5 μg DNA target was hybridized on GeneChip® Clariom™ D human (Thermo Fisher Scientific) and incubated at 45°C in the Genechip® Hybridization oven 640 (Affymetrix) for 17 h at 60 rpm. GeneChips were then washed in a GeneChips® Fluidics Station 450 (ThermoFisher) using GeneChip Hybridization Wash and Stain kit according to the manufacturer’s instructions (ThermoFisher). The microarrays were finally scanned on a GeneChip® scanner 3,000 (ThermoFisher).

APOB protein levels in brain tissue

100 mg of brain samples were homogenized mechanically with the Bead Ruptor 24 (Omni International) in ice-cold phosphate-buffered saline with protease inhibitors. Two freeze-thaw cycles were performed, and homogenates were centrifuged at 4°C at 5,000 rpm for 5 min. Supernatants were stored at −80°C until further use. Total protein concentrations were measured with the bicinchoninic acid assay (Thermo Fisher). APOB protein levels were measured using the Singleplex Magnetic Immunoassay from Millipore (cat# APOMAg-62k, lot#3840529) and normalized against total protein concentration.

Amyloid-β measurements and tissue fractionation

To measure amyloid-β in different tissue fractions, a sequential extraction protocol was followed as previously described (33). 50–100 mg of brain tissue were thawed on ice and homogenized using a Biomasher (Omni International). After a 1 min centrifugation at 4°C and at 14 000 g, the pellet was resuspended in 200 μl of Tris-buffered saline (TBS, 50 mM Tris pH 8.0, 100 mM NaCl) containing EDTA-free protease inhibitors (Pierce). The homogenized samples were then transferred to ultracentrifuge tubes and centrifuged at 100 000 g for 30 min at 4°C. The supernatant was removed (TBS fraction) and the pellet was resuspended in 200 μl of 100 mM Na2CO3 pH 11.0 and kept on ice for 20 min. After a centrifugation at 100 000g for 30 min at 4°C, the supernatant was recovered as the Na2CO3 fraction. The remaining pellet was re-suspended with 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM bicine, pH 8.5 and centrifuged as before. The supernatant was retained as the urea fraction. Amyloid-β 40–42 were then measured using solid-phase enzyme immunoassay (Innotest #81585, lot#409271 for Aβ-40 and Innotest #81583, lot#410221 for Aβ-42, Fujirebio).

Cholesterol and oxysterol derivatives

For analysis, 100 mg of human brain tissue was homogenized in 1 ml of 10% methanol/water. An internal standard mix consisting of 3 deuterated sterols (d6 25-hydroxycholesterol, d6 desmosterol, d6 campesterol) was added to 100uL of the tissue homogenates. Samples were then further processed by adding 500uL of 3:1 butanol/methanol combined with KOH to make a final KOH concentration of 0.2 N and then saponified for 1.5 h at 37C. The butanol and methanol (BUME) method was then carried out as described by adding 500uL of 3:1 heptane/ethyl acetate and 1% acetic acid (34). Samples were brought to dryness and run on a Waters Acquity UPLC interfaced with an AB Sciex 6500 QTrap mass spectrometer equipped with an APCI probe. A Phenomenex Kinetex C18 1.7uM 2.1 mm × 150 mm column was used for chromatographic separation using a step gradient over 10 min. The individual sterol species were identified by mass spectrometry using MRM (Multiple Reaction Monitoring). Standard curves were obtained in parallel using identical conditions. Data analysis was performed with Analyst and Mulitquant software packages (AB Sciex). In total, 17 MRMs were monitored.

Religious order study and the Memory and Aging Project (ROSMAP cohort)

The ROSMAP study combines two publicly available datasets, the Religious Orders Study (ROS) and the Memory and Aging Project (MAP). Participants from the cohorts were cognitively normal upon enrolment, agreed to annual blood tests and cognitive evaluations, and consented to organ donation. Upon death, post-mortem evaluations are performed to assess AD pathology (CERAD and Braak staging) (35).

Genotyping

Genotype data were generated from peripheral blood mononuclear cells or frozen brain tissues using Affymetrix or the Illumina Omniquad express gene chips, with imputation performed by Sanger Imputation Service (https://imputation.sanger.ac.uk). Pre-phasing was performed with SHAPEIT2 (36) and PBWT (37) was used for imputation with the 1,000 Genomes (phase 3) selected as reference panel. Only post-imputed SNPs with an info score >0.7 were kept.

RNA-sequencing

The ROSMAP study provides RNA-Seq data from the dorsolateral prefrontal cortex (38). The Broad Institutes’ Genomics Platform performed RNA-Seq library preparation using the strand-specific dUTP method with poly-A selection (39). Sequencing was performed on the Illumina HiSeq. Quantile normalization method was applied to FPKM first and combat package was used to remove the potential batch effect. The ROS-MAP cortical RNA-Seq consolidated dataset is available directly from Research Resource Sharing Hub at https://www.radc.rush.edu/home.htm and at synapse.org/syn# 23564903. For APOB mRNA levels, we selected the full-length transcript ENST00000233242.

Proteomics

Protein levels were measured in the prefrontal cortex by a mass spectrometry-based protein quantification approach using isobaric multiplex tandem mass tags (TMT) mass spectrometry as described previously by Ping et al. (40) Briefly, TMT labeling with synchronous precursor selection (SPS)-MS3 for reporter ion quantitation was used to achieve comprehensive global quantitation of 100 mg (wet tissue weight) pre-frontal cortex from healthy controls and AD cases. In total, 127,321 total unique peptides were identified from >1.5 million peptide spectral matches (PSMs), which mapped to 11,840 unique protein groups, representing 10,230 gene symbols, that map to ≈65% of the protein-coding genes in the brain (40). Here, we used P04114 which corresponds to APOB-100. Proteomics TMT consolidated datasets are available directly from Synapse (ROSMAP) upon request at synapse.org; metadata file syn21323404.

Expression quantitative trait loci (eQTL) analyses

For quantitative trait loci analyses (BRAINEAC and ROSMAP datasets), regression statistics were calculated with PLINK v1.09. (41) The eQTL analysis was run in R (http://www.R-project.org) using the MatrixEQTL package (42).

Alzheimer Disease Neuroimaging Initiative (ADNI cohort)

ADNI CSF and genetic datasets were downloaded from the ADNI website (www.loni.ucla.edu/ADNI).

CSF measurements

The CSF multiplex MRM mass spectrometry panel consists of 567 peptides representing 221 proteins, and for each peptide two or more mass transitions were monitored. 290 unique ADNI-1 baseline subjects are represented: 87 healthy control (CTL) subjects, as well as 66 with AD dementia and 136 with MCI. Two distinct peptides were quantified for APOB: IAELSATAQEIIK (APOB-100) and SVSLPSLDPASAK (common to APOB-48 and APOB-100). For a detailed methodology of the MRM mass spectrometry analyses used for CSF, refer to Spellman et al. (42). The Biomarker Consortium CSF Proteomics MRM consolidated dataset (CSFMRM.csv) is available directly from ADNI upon request at http://adni.loni.usc.edu/

Statistical analysis

Correlational analysis adjusted for age, sex, and APOE ε4 status was performed in SPSS using ANOVA.

Results

Table 1 summarizes the demographic characteristics of the different cohorts used to examine cortical APOB gene expression levels. Brain APOB mRNA prevalence is not affected by sex or APOE genotype in any of the cohorts.

Brain APOB protein and mRNA levels display opposite associations with clinical diagnoses and indices of AD pathology

In brain tissue from the ROSMAP cohort spanning the entire Alzheimer spectrum, cortical APOB protein levels, measured by TMT mass spectrometry, positively associated with both clinical and pathological indices of the disease. Braak stages [associated with the progression of Tau pathology, (R2 = 0.026; P = 0.009)], CERAD [associated with the progression of the amyloid deposition (R2 = 0.031; P = 0.01)] and clinical diagnosis (final Dx: R2 = 0.013; P = 0.03) are all significantly and positively associated with cortical APOB protein levels. Cognition (assessed with the Mini-Mental State Examination (P < 0.05, N.S.) is not. In sharp contrast, APOB mRNA levels negatively associate with the same traits: Braak (R2 = 0.014; P = 0.01), CERAD (R2 = 0.013; P = 0.04), cognition (R2 = 0.018; P = 0.003) and clinical diagnosis (R2 = 0.010; P = 0.02) (Fig. 1).

Fig. 1.

Fig. 1

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Brain APOB protein and mRNA levels exhibit opposite associations with clinical and pathophysiological makers of AD. Bar graph depicting brain APOB protein and mRNA levels taken from ROSMAP cohort. A: Protein levels were normalized by a log2 transformation. B: Messenger RNA levels were measured by fragments per kilobase of transcript per million mapped reads (FPKM). Quantitative measures of AD pathology (Braak and CERAD) were based on pathologic criteria for AD. Error bars represent one standard error.

Non-degenerative and AD-related conditions exhibit similar cis-regulation of brain APOB

Cis-expression quantitative trait loci (eQTL) analysis of the APOB gene region identified 5 polymorphisms that reached locus-wide significance in the BRAINEAC cohort which is composed of 134 brains free of neurodegeneration (Fig. 2). The same analysis was performed in the cortex of 152 unaffected subjects from the ROSMAP cohort. It identified several SNPs that reached high levels of association with APOB expression upstream of the APOB gene (dots in dark blue). The most significant variants common to both cohorts were selected for subsequent in-depth analyses: namely rs934197 and rs1367117. The latter polymorphism is of special interest as it is a coding variant located in the N-terminal of the APOB protein known to be genetically associated with high circulating LDL-cholesterol levels (P = 7 × 10−125) (43).

Fig. 2.

Fig. 2

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Brain APOB cis-regulation in unaffected subjects from BRAINEAC and ROSMAP. APOB brain mRNA level associated with genetic variants in the APOB gene region on chromosome 2. The strength of association with the BRAINEAC cohort is represented on the Y axis while the strength of association with subjects without AD from ROSMAP is represented with color. The locus-wide significance level is established at -log10(P) = 3.9.

Brain tissue but not CSF APOB protein levels are modulated by trans-regulating elements

Trans-protein QTL analysis of cortical APOB protein levels was performed in ROSMAP cohort subjects free of clinical symptoms and pathological alterations. The main objective here is to examine APOB gene expression before any significant damage to the blood-brain barrier because of the emergence of neurodegeneration and cognitive deficits (7). Manhattan plot reveals a cluster of major modulating genetic polymorphisms in CYP27A1 gene (P = 9.36x10-7); with rs4674344 displaying the strongest association (Fig. 3A). A subsequent analysis contrasting APOB protein levels in frontal cortex tissue (ROSMAP) and in the CSF (ADNI) of asymptomatic subjects reveals an intriguing divergence. Cortical tissues levels of the protein, but not of the mRNA, are markedly affected by the presence of the CYP27A1 rs4674344 variant in ROSMAP control subjects (Fig. 3B, C) whereas CSF APOB concentrations are not affected by the CYP27A1 variant in the CSF in ADNI’s control group (Fig. 3D).

Fig. 3.

Fig. 3

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Trans-regulating element modulates brain APOB protein levels but not APOB CSF protein or brain mRNA levels. A: Manhattan plot representing SNPs associating with APOB protein levels in brains without clinical AD from the ROSMAP cohort. The suggestive significance threshold is set at -log10(P) = 5. B: Bar graph depicting levels of cortical APOB protein level, in arbitrary units, for all genotypes of the rs4674344 SNP in CYP27A1. C: Bar graph depicting levels of cortical APOB mRNA level, in fragments per kilobase of transcript per million mapped reads (FPKM), for all genotypes of the rs4674344 SNP in CYP27A1. D: Bar graph depicting APOB protein levels, in arbitrary units, measured by mass spectrometry and using two different peptides in the CSF of control subjects of the ADNI cohort. All error bars represent one standard error of the mean.

Insoluble Aβ40 and Aβ42 associated with APOB protein levels in the cortical area

Using frontal cortices from 79 brains from the DBCBB cohort (68 AD and 31 controls), Aβ40 and Aβ42 were measured by ELISA in two distinct biochemical fractions. In Na2CO3 fraction (used to solubilize membranes and vesicles, not plaques), both forms of amyloid associate with APOB protein levels (R2 = 0.08; P < 0.01, R2 = 0.10; P < 0.005, respectively, Fig. 4). In the urea fraction (used to solubilize plaques deposits), Aβ40 and Aβ42 also display a significant association with APOB protein levels (Aβ40 R2 = 0.10; P = 0.003, Aβ42 R2 = 0.07; P < 0.02).

Fig. 4.

Fig. 4

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DBCBB cohort APOB protein level correlates with Aβ40 and Aβ42 in different brain fractions. Scatterplot with linear regression of APOB protein levels in pg/μg transformed in log10 for normalization. β-amyloid peptides were solubilized in a Na2CO3 solution and in a urea solution. The Na2CO3 solution solubilizes membranes and external parts of Aβ plaques while the urea fraction solubilizes the core of the plaques. Aβ measurements units are represented in pg/mg. The data was transformed using the Johnson Sb transformation to obtain a normal distribution. APOB and Aβ were quantified using ELISA.

Alterations in hydroxylated cholesterol derivatives, but not cholesterol, are associated with AD and APOB coding variants

Tissue levels of total cholesterol, 24-S-hydroxycholesterol (24-OHC), and 27-hydroxycholesterol (27-OHC) were assayed by RT-UPLC/MS in 86 brains (frontal cortex of AD (n = 55) and pathology-free control (n = 31) subjects). Figure 5 illustrates the results obtained in the frontal cortex as a function of disease status (left panels), APOB rs1367117 polymorphism (center panels) and CYP27A1 rs4674344 (right panels). Consistent with literature reports using mass spectrometry methods, total cholesterol levels in the cortex are not affected by AD nor by the APOB and CYP27A1 variants found to normally affect APOB gene expression levels in the CNS. In contrast, 24-S-hydroxycholesterol levels are significantly lower in AD brains (R2 = 0.084; P = 0.0092, Fig. 5, center) and higher with the APOB rs1367117 allele dose (R2 = 0.135; P = 0.0046, Fig. 5, center). In contrast, 27-hydroxycholesterol levels are significantly higher in AD brains (R2 = 0.116; P = 0.0019) but remain relatively unaffected by the APOB and CYP27A1 variants. The distribution of the different genotypes (AA, AT, TT) in the AD and control groups are not significantly different from one another (not shown) and does not explain the elevated concentrations of 27-OHC observed in the AD cortex.

Fig. 5.

Fig. 5

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Brain cholesterol metabolites as a function of AD diagnostic, rs1367117 and rs4674344 genotypes. Mean cortical cholesterol, 24-hydroxycholesterol, and 27-hydroxycholesterol levels, in ng/mg wet weight, from the DBCBB cohort are contrasted with diagnosis, genotypes for APOB SNP rs1367117 and CYP27A1 SNP rs4674344. P-values are calculated by ANOVA.

Discussion

A meta-analysis of 3 different cohorts of early-onset AD subjects found a strong association between rare genetic coding variants of APOB and the familial early-onset form of AD, independent of APOE ε4 allele (19). Sporadic late-onset AD patients, on the other hand, typically exhibit increased blood levels of APOB and LDL along with decreased high-density lipoprotein (HDL) levels (44, 45) which positively correlate, in post-mortem studies, with brain tissue Aβ42 levels (20). These findings are consistent with the existing transgenic mice literature which shows that life-long exposure to high APOB protein levels in apob100 overexpressing animals leads to significant neurodegenerative changes in the brains (46) and hyperphosphorylation of tau protein in the absence of amyloid deposition. Extensive cortical and hippocampal neuronal apoptosis, marked reduction in the number and size of the dendritic spines in the hippocampal neurons and impaired hippocampal presynaptic function have also been reported in these apob100-deficient animals (47). Aging combined with apob100 overexpression induces significant cognitive decline in the Morris water maze at mid-life (2).

During the pre-symptomatic phase of the disease, CSF APOB protein levels in humans have demonstrated a strong correlation with CSF tau and phosphorylated-tau, but not with CSF Aβ42 levels or its deposition measured by PET (7). In sharp contrast, frontal cortex APOB protein levels in end-stage AD are associated with plaque-bound Aβ40 and Aβ42 (Fig. 4, urea fraction); consistent with previous reports of APOB-immunohistochemical staining in senile plaque amyloid, vascular amyloid and intracellular neurofibrillary tangles in humans and, with amyloid plaques in APP transgenic mice (21).

Using cross-sectional analyses of APOB mRNA and APOB protein levels in the frontal cortex in asymptomatic, MCI and AD patients from the ROSMAP cohort, we found an inverse relationship between mRNA and protein concentrations as a function of cognitive status (MMSE or Diagnosis, Fig. 1). The increase cortical levels of APOB protein in MCI and AD cases (Fig. 1A) is most likely due to the progressive deterioration of blood–brain barrier integrity (23) which facilitate peripheral APOB entry into the CNS. The maximally elevated concentration of cortical APOB protein in Braak stages IV-VI and CERAD stage 1 (definite) and, corresponding reduced mRNA prevalence, suggest a compensatory downregulation of gene expression in the glial cells compartment in response to a weak or porous blood-brain barrier. Alternatively, it is conceivable that reduced clearance and/or degradation of local APOB is also at play at this late stage of the neurodegenerative process. Consistent with this concept, we reported elevated proprotein convertase subtilisin/kexin type 9 (PCSK9) levels in hippocampal and cortical areas in autopsy-confirmed AD cases and, strong correlations between PCSK9 and APOB, as well as with phosphorylated-tau in the CSF in the asymptomatic PREVENT-AD cohort (48). PCSK9, which normally enhances LDLR catabolism and reduces APOB binding, internalization, and degradation in brain cells, could contribute to the observed APOB increase both in the CSF and cortical areas in the late stages of the disease.

This model is supported by recent evidence from the Swedish bioFINDER study showing a significant reduction of soluble LDLR protein levels in the CSF of AD subjects versus controls (49): an observation that we recently replicated in the CCNA cohort where both MCI and AD subjects display significantly lower levels of soluble LDLR in the CSF relative to cognitively unaffected aged subjects (supplemental Fig. S1A, P < 0.001).

Analysis of PCSK9 protein levels in the frontal cortex of AD and age-matched control subjects from the DBCBB cohort revealed the expected opposite alterations: increased cortical PCSK9 in AD versus control cases (supplemental Fig. S1B, P < 0.005): consistent with the proposed APOB-LDLR-PCSK9 catabolic regulation in the CNS. The increased PCSK9 level observed in cortical tissue clearly leads to enhanced LDLR catabolism, increased levels of partially degraded soluble LDLR in the CSF, and diminished APOB protein removal by local glial and neuronal cells. Therefore, APOB progressively accumulates in cortical structures in late-stage AD (Braak IV-VI) causing a concomitant compensatory reduction in APOB mRNA prevalence (Fig. 1).

To further clarify the role of the genomic regulation of APOB expression in the unaffected human brain, a trans-pQTL analysis was performed on asymptomatic patients from the ROSMAP cohort. Figure 3A illustrates the resulting Manhattan plot, identifying CYP27A1 as the most significant genomic markers associated with cortical APOB protein levels. The reduction in cortical APOB protein concentration follows a dose-dependent pattern where homozygous rs4674344 TT carriers display the lowest concentrations found in the human frontal cortex (Fig. 3B). In contrast, CYP27A1 rs4674344 TT carriers do not show alterations in cortical APOB mRNA prevalence in ROSMAP nor CSF APOB protein levels in asymptomatic control subjects from the ADNI cohort.

Altogether, these findings suggest a CYP27A1-dependent, post-translational regulation of APOB concentration that most likely implicates CYP27A1’s molecular cascade and the production of modulating cholesterol intermediates in the CNS such as the 27-OHC species. Further analyses of frontal cortex tissue cholesterol, 24-OHC, and 27-OHC concentrations in AD and age-matched control subjects revealed a marked increase of 27-OHC (R2 = 0.12, P < 0.002) but a significant reduction in 24-OHC (R2 = 0.084, P < 0.01) in the AD brain. No significant alteration of cortical cholesterol levels was detected in the AD brains (Fig. 5, left). These findings are consistent with previous reports on these cholesterol species in the CNS in AD (15, 16) using sensitive and reliable mass spectrometry methods.

Further stratification by CYP27A1 rs4674344 genotype revealed no association with any of the three cholesterol species (Fig. 5, right). In contrast, APOB rs1367117 AA variant which was identified by cis-QTL analyses using ROSMAP and BRAINEAC cohorts (Fig. 2) reveals a marked allele-dose effect on cortical brain levels of 24-OHC but not with cholesterol or 27-OHC (Fig. 5, Centre). Cholesterol enzymatic elimination products 27-OHC and 24-OHC were shown to influence amyloid production and accumulation via secretase activities (50). In experimental studies, 27-OHC has been shown to increase Aβ levels (51, 52) whereas 24-OHC induces the expression of the cholesterol efflux molecule ABCA1 with higher levels of ABCA1 known to be associated with increased Aβ production (53).

This cascade of molecular events is certainly consistent with the observed effects of APOB rs1367117 missense mutation on APOB protein and 24-OHC levels in cortical areas (Fig. 5) and, reported association with MMSE score alterations in the ROSMAP cohort and elevated serum cholesterol levels (from the Global Lipids Genetic Consortium, supplemental Table S1).

Similarly, CYP27A1 rs4674344 which significantly modulates cortical APOB brain expression level (Fig. 3B) has been previously associated with augmented circulating total cholesterol, LDL cholesterol, and non-HDL cholesterol (54) in population studies and, is believed to act as a susceptibility gene for hypercholesterolemia risk (55). Yet, the absence of a significant effect of the CYP27A1 rs4674344 TT genotype on all three cholesterol species in the CNS excludes cholesterol metabolism alterations as the putative intermediate for the CYP27A1-mediated reduction of APOB protein levels in the CNS.

The dichotomy between peripheral and CNS cholesterol metabolism is not surprising considering that cholesterol metabolism in the brain is independently regulated from the periphery and that cholesterol measured in the brain is mostly produced locally (56). Unlike cholesterol in other organs in the periphery, brain cholesterol is primarily derived from local de novo synthesis. The intact blood-brain barrier (BBB) prevents the uptake of lipoproteins, such as LDL, from the circulation; a situation that changes as cognitive symptoms emerge.

In this context, we propose that APOB's contribution to AD pathophysiology involves two distinct phases; the pre-symptomatic phase where in the absence of blood–brain barrier dysfunction, APOB acts as a disruptor of tau and phospho-tau homeostasis with little or no involvement with the amyloid metabolism (7) and, a second phase that corresponds to the emergence of cognitive deficit, enhanced amyloid deposition both in the neuropil and in the vascular walls, leading to significant alterations of the blood-brain barrier integrity (23). In the second phase as the transition toward full-blown AD pathology unfolds (Braak stages IV-VI, CERAD stage 1 (definite), cortical APOB protein markedly increases as mRNA levels decrease in response to PCSK9 upregulation and conceivably, LDL-derived APOB originating from the periphery. In response, 24-OHC is reduced, except in subjects who are carriers of the APOB rs1367177 TT somewhat protective variant that alleviates brain cholesterol metabolism perturbations. The increase in 27-OHC observed in the AD brain most likely originates from the circulation through the increasingly compromised blood–brain barrier (57). The complete lack of effect of APOB rs1367177 and CYP27A1 rs4674344 on cerebral 27-OHC is certainly consistent with this interpretation.

Finally, a close examination of the APOB genetic variants identified above by QTL analyses showed no significant association with AD risk using recent large, published genome-wide association studies in sporadic AD (IGAP, ADGC, and IGAP-plus, supplemental Table S1). Altogether, these results show that in contrast to familial early-onset AD where APOB mutations are associated with AD risk, in sporadic AD, APOB contribution (and possible toxicity) is not genetic but metabolic, especially in the presymptomatic phase of the disease. However, when cognitive deficits emerge and the blood-brain barrier integrity becomes progressively compromised, APOB’s contribution to AD pathology changes and becomes particularly exacerbated in the late stages of the disease when amyloid and tangle deposition reach their peak. An intriguing recent observation from the ALS field reported that APOB alone suffices to recapitulate clinical and pathological outcomes in vivo and induce death of human induced pluripotent stem cell-derived motor neurons in vitro. Targeted removal in mice of APOB from sporadic amyotrophic lateral sclerosis CSF via filtration or immunodepletion successfully reduced the neurotoxic capacity of sporadic amyotrophic lateral sclerosis CSF to induce motor disability, motor neuron death, and TDP-43 translocation (8). The possibility that APOB may act as a neurotoxin that works in tandem with tau and phospho-tau to compromise the vascular and neuronal integrity in AD is currently being investigated by our team in aging apob transgenic animals.

Data availability

Data from public databases used in this study are available at: http://www.braineac.org/, https://www.radc.rush.edu/home.htm and http://adni.loni.usc.edu/. Data from our local DBCBB autopsied brain cohort is available upon request to Dr Judes Poirier, Douglas Mental Health University Institute, judes.poirier@mcgill.ca.

Supplemental data

Supplementary material is available at Journal of Lipid Research online. This article contains supplemental data (58, 59, 60, 61).

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Acknowledgments

Data used in preparation of this article were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database (adni.loni.usc.edu). As such, the investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or writing of this report. A complete listing of ADNI investigators can be found at: http://adni.loni.usc.edu/wp-content/uploads/how_to_apply/ADNI_Acknowledgement_List.pdf. The authors wish to thank Dr John Breitner, Dr Sylvia Villeneuve and Jennifer Tremblay-Mercier for their individual contribution at different stages of the project. The authors would also like to thank Dr Naguib Mechawar at the Douglas Institute/Bell Canada Brain Bank for providing human brain tissues from the Québec Founding Population.

Author contributions

A. L. data curation; C. P. project administration; C. P. investigation; C. P. conceptualization; G. A-R. writing–original draft; G. A-R. methodology; G. A-R. formal analysis; J. P. writing–review & editing; J. P. supervision; J. P. resources; J. P. funding acquisition.

Funding and additional information

Dr Poirier is supported by the Fonds de la Recherche en Santé du Québec (FRSQ), the Canadian Institute for Health Research (CIHR # PJT 153287 & PJT 178210), National Research Council of Canada (NSERC) and the J. L. Levesque Foundation. Data collection and sharing for this project was funded by the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (National Institutes of Health Grant U01 AG024904) and DOD ADNI (Department of Defense award number W81XWH-12-2-0012). ADNI is funded by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, and through generous contributions from the following: AbbVie, Alzheimer’s Association; Alzheimer’s Drug Discovery Foundation; Araclon Biotech; BioClinica, Inc.; Biogen; Bristol-Myers Squibb Company; CereSpir, Inc.; Cogstate; Eisai Inc.; Elan Pharmaceuticals, Inc.; Eli Lilly and Company; EuroImmun; F. Hoffmann-La Roche Ltd. and its affiliated company Genentech, Inc.; Fujirebio; GE Healthcare; IXICO Ltd.; Janssen Alzheimer Immunotherapy Research & Development, LLC.; Johnson & Johnson Pharmaceutical Research & Development LLC.; Lumosity; Lundbeck; Merck & Co., Inc.; Meso Scale Diagnostics, LLC.; NeuroRx Research; Neurotrack Technologies; Novartis Pharmaceuticals Corporation; Pfizer Inc.; Piramal Imaging; Servier; Takeda Pharmaceutical Company; and Transition Therapeutics. ADNI data are disseminated by the Laboratory for Neuro Imaging at the University of Southern California.

Supplementary data

Suppl Figure 1
mmc1.pdf (288.5KB, pdf)
Supplementary Table 1
mmc2.docx (17.8KB, docx)
Supplementary Table 3
mmc3.docx (14.9KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suppl Figure 1
mmc1.pdf (288.5KB, pdf)
Supplementary Table 1
mmc2.docx (17.8KB, docx)
Supplementary Table 3
mmc3.docx (14.9KB, docx)

Data Availability Statement

Data from public databases used in this study are available at: http://www.braineac.org/, https://www.radc.rush.edu/home.htm and http://adni.loni.usc.edu/. Data from our local DBCBB autopsied brain cohort is available upon request to Dr Judes Poirier, Douglas Mental Health University Institute, judes.poirier@mcgill.ca.

Articles from Journal of Lipid Research are provided here courtesy of American Society for Biochemistry and Molecular Biology

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