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. 2021 May 27;18(5):e1003615. doi: 10.1371/journal.pmed.1003615

Bile acid synthesis, modulation, and dementia: A metabolomic, transcriptomic, and pharmacoepidemiologic study

Vijay R Varma 1,, Youjin Wang 2,, Yang An 3, Sudhir Varma 4, Murat Bilgel 3, Jimit Doshi 5, Cristina Legido-Quigley 6, João C Delgado 7, Anup M Oommen 8, Jackson A Roberts 1, Dean F Wong 9, Christos Davatzikos 5, Susan M Resnick 3, Juan C Troncoso 10, Olga Pletnikova 10, Richard O’Brien 11, Eelko Hak 12, Brenda N Baak 12, Ruth Pfeiffer 2, Priyanka Baloni 13, Siamak Mohmoudiandehkordi 14, Kwangsik Nho 15, Rima Kaddurah-Daouk 14, David A Bennett 16, Shahinaz M Gadalla 2, Madhav Thambisetty 1,*
Editor: Carol Brayne17
PMCID: PMC8158920  PMID: 34043628

Abstract

Background

While Alzheimer disease (AD) and vascular dementia (VaD) may be accelerated by hypercholesterolemia, the mechanisms underlying this association are unclear. We tested whether dysregulation of cholesterol catabolism, through its conversion to primary bile acids (BAs), was associated with dementia pathogenesis.

Methods and findings

We used a 3-step study design to examine the role of the primary BAs, cholic acid (CA), and chenodeoxycholic acid (CDCA) as well as their principal biosynthetic precursor, 7α-hydroxycholesterol (7α-OHC), in dementia. In Step 1, we tested whether serum markers of cholesterol catabolism were associated with brain amyloid accumulation, white matter lesions (WMLs), and brain atrophy. In Step 2, we tested whether exposure to bile acid sequestrants (BAS) was associated with risk of dementia. In Step 3, we examined plausible mechanisms underlying these findings by testing whether brain levels of primary BAs and gene expression of their principal receptors are altered in AD.

  • Step 1: We assayed serum concentrations CA, CDCA, and 7α-OHC and used linear regression and mixed effects models to test their associations with brain amyloid accumulation (N = 141), WMLs, and brain atrophy (N = 134) in the Baltimore Longitudinal Study of Aging (BLSA). The BLSA is an ongoing, community-based cohort study that began in 1958. Participants in the BLSA neuroimaging sample were approximately 46% male with a mean age of 76 years; longitudinal analyses included an average of 2.5 follow-up magnetic resonance imaging (MRI) visits. We used the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (N = 1,666) to validate longitudinal neuroimaging results in BLSA. ADNI is an ongoing, community-based cohort study that began in 2003. Participants were approximately 55% male with a mean age of 74 years; longitudinal analyses included an average of 5.2 follow-up MRI visits. Lower serum concentrations of 7α-OHC, CA, and CDCA were associated with higher brain amyloid deposition (p = 0.041), faster WML accumulation (p = 0.050), and faster brain atrophy mainly (false discovery rate [FDR] p = <0.001–0.013) in males in BLSA. In ADNI, we found a modest sex-specific effect indicating that lower serum concentrations of CA and CDCA were associated with faster brain atrophy (FDR p = 0.049) in males.

  • Step 2: In the Clinical Practice Research Datalink (CPRD) dataset, covering >4 million registrants from general practice clinics in the United Kingdom, we tested whether patients using BAS (BAS users; 3,208 with ≥2 prescriptions), which reduce circulating BAs and increase cholesterol catabolism, had altered dementia risk compared to those on non-statin lipid-modifying therapies (LMT users; 23,483 with ≥2 prescriptions). Patients in the study (BAS/LMT) were approximately 34%/38% male and with a mean age of 65/68 years; follow-up time was 4.7/5.7 years. We found that BAS use was not significantly associated with risk of all-cause dementia (hazard ratio (HR) = 1.03, 95% confidence interval (CI) = 0.72–1.46, p = 0.88) or its subtypes. We found a significant difference between the risk of VaD in males compared to females (p = 0.040) and a significant dose–response relationship between BAS use and risk of VaD (p-trend = 0.045) in males.

  • Step 3: We assayed brain tissue concentrations of CA and CDCA comparing AD and control (CON) samples in the BLSA autopsy cohort (N = 29). Participants in the BLSA autopsy cohort (AD/CON) were approximately 50%/77% male with a mean age of 87/82 years. We analyzed single-cell RNA sequencing (scRNA-Seq) data to compare brain BA receptor gene expression between AD and CON samples from the Religious Orders Study and Memory and Aging Project (ROSMAP) cohort (N = 46). ROSMAP is an ongoing, community-based cohort study that began in 1994. Participants (AD/CON) were approximately 56%/36% male with a mean age of 85/85 years. In BLSA, we found that CA and CDCA were detectable in postmortem brain tissue samples and were marginally higher in AD samples compared to CON. In ROSMAP, we found sex-specific differences in altered neuronal gene expression of BA receptors in AD. Study limitations include the small sample sizes in the BLSA cohort and likely inaccuracies in the clinical diagnosis of dementia subtypes in primary care settings.

Conclusions

We combined targeted metabolomics in serum and amyloid positron emission tomography (PET) and MRI of the brain with pharmacoepidemiologic analysis to implicate dysregulation of cholesterol catabolism in dementia pathogenesis. We observed that lower serum BA concentration mainly in males is associated with neuroimaging markers of dementia, and pharmacological lowering of BA levels may be associated with higher risk of VaD in males. We hypothesize that dysregulation of BA signaling pathways in the brain may represent a plausible biologic mechanism underlying these results. Together, our observations suggest a novel mechanism relating abnormalities in cholesterol catabolism to risk of dementia.


Vijay Varma and co-workers study cholesterol and bile acid metabolism in dementia.

Author summary

Why was this study done?

  • Hypercholesterolemia is associated with increased risk of Alzheimer disease (AD) and vascular dementia (VaD).

  • However, cholesterol is impermeable to the blood–brain barrier (BBB), and it is unclear how peripheral cholesterol mediates risk of dementia.

  • While prior research has examined the relationship between de novo cholesterol biosynthesis and dementia, few studies have assessed the role of cholesterol catabolism and its principal breakdown products, oxysterols, and primary bile acids (BAs) in dementia.

What did the researchers do and find?

  • We examined the role of cholesterol catabolism in dementia pathogenesis by first testing the association between serum oxysterols and BAs and neuroimaging markers of dementia. We also tested whether exposure to bile acid sequestrants (BAS) was associated with risk of dementia in a large, real-world clinical dataset. Finally, we tested plausible mechanisms underlying these associations by examining whether primary BAs and mRNA of their receptors were altered in the brain in dementia.

  • We found that lower serum levels of 7α-hydroxycholesterol (7α-OHC) and primary BAs were associated with higher brain amyloid deposition, faster WML accumulation, and faster brain atrophy mainly in males. Consistent with this finding, we observed a sex difference in the association between use of BAS and risk of VaD.

  • We found that primary BAs were detectable in the brain, and levels of gene expression of BA receptors were altered in AD mainly in males.

What do these findings mean?

  • Our findings suggest that cholesterol catabolism and BA synthesis may impact dementia progression through sex-specific effects on signaling pathways in the brain.

  • These results set the stage for experimental studies to test whether BA signaling in the brain may be a novel therapeutic target in dementia.

Introduction

Accumulating evidence suggests that dementias such as Alzheimer disease (AD) and vascular dementia (VaD) may be the terminal consequences of metabolic abnormalities, such as hypercholesterolemia, manifesting several years prior to the onset of cognitive impairment and functional decline.

While hypercholesterolemia is associated with increased risk of both AD and VaD [1,2], the precise molecular mechanisms underlying this association remain unclear. Moreover, as cholesterol itself is impermeable to the blood–brain barrier (BBB), the question of how increased levels of peripheral cholesterol may mediate greater risk of dementia remains to be answered. These questions also bear important clinical translational implications for understanding how therapeutic targeting of cholesterol-related metabolic pathways may impact risk of dementia. The relationship between cholesterol metabolism and vascular disease has been predominantly studied from the perspective of de novo cholesterol biosynthesis, while relatively little attention has focused on cholesterol catabolism. The principal catabolic fate of cholesterol is its conversion to the primary bile acids (BAs), cholic acid (CA), and chenodeoxycholic acid (CDCA) through BBB-permeable intermediate metabolites called oxysterols (S1 Fig).

In order to test whether dysregulation of cholesterol catabolism is associated with dementia pathogenesis, we applied a 3-step study design.

First (Step 1), we used targeted metabolomics assays of serum samples within a longitudinal observational study to test whether serum concentrations of metabolites related to cholesterol catabolism, including the de novo synthesis of primary BAs, are associated with early neuroimaging markers of dementia including brain amyloid accumulation, white matter lesions (WMLs), and brain atrophy. We validated the association of BA levels with longitudinal neuroimaging outcomes in an independent cohort. Second (Step 2), based on associations between serum BA concentrations and neuroimaging markers of dementia identified in Step 1, we hypothesized that drugs targeting de novo BA synthesis, i.e., bile acid sequestrants (BAS) would alter risk of dementia. We tested this hypothesis in a large real-world clinical dataset. Third (Step 3), we explored plausible molecular mechanisms underlying our findings by testing whether levels of primary BAs and gene expression of their receptors were altered in the brain in dementia.

Given prior evidence suggesting sex-specific differences in the serum lipidome as well as in the association between lipid levels and dementia risk [3,4], we performed sex-stratified analyses to test the relationship between cholesterol catabolism and dementia.

Materials and methods

Data used in our analyses were derived from the Baltimore Longitudinal Study of Aging (BLSA), the Alzheimer’s Disease Neuroimaging Initiative (ADNI), the Alzheimer’s Disease Metabolomics Consortium (ADMC), the Religious Orders Study and Memory and Aging Project (ROSMAP), and the Clinical Practice Research Datalink (CPRD). BLSA, ADNI, and ROSMAP are long-running, longitudinal cohorts established and prospectively followed to help address broad questions related to aging and disease. CPRD includes anonymized electronic medical record (EMR) data gathered from general practitioners in the United Kingdom. Specific analyses addressing focused hypotheses described herein were not included in prospective analysis plans in the original study protocols for these cohorts. BLSA, ADNI, and ROSMAP participants included in our analyses were a convenience sample available to researchers; power calculations to determine study size were not performed. Details on the analytic plan, including when specific plans were developed, are included in the Statistical methods section below. This study is reported as per the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines (S1 Table).

Ethics approval

The BLSA study protocol has ongoing approval from the Institutional Review Board (IRB) of the National Institute of Environmental Health Science, National Institutes of Health (“Early Markers of Alzheimer’s Disease (BLSA)”, IRB No. 2009–074). Informed written consent was obtained at each visit from all participants.

The ADNI study protocol was approved by the IRBs of all the participating institutions/study sites [5]. Informed written consent was obtained from all participants at each site. All ADNI studies are conducted according to Good Clinical Practice guidelines, the Declaration of Helsinki, and United States of America 21 CFR Part 50 (Protection of Human Subjects) and Part 56 (IRBs). Additional details can be found at adni.loni.usc.edu.

The ROSMAP study, including the parent study and substudies, was approved by the IRB of Rush University Medical Center. Informed written consent was obtained for all participants as well as an Anatomical Gift Act and a repository consent to share data and biospecimens.

CPRD data are anonymized, general practitioners do not need to seek patient consent when sharing data with CPRD, and patients have the option of opting out. Additional details can be found at https://www.cprd.com/public. This study was approved by the CPRD Independent Scientific Advisory Committee (ISAC; Protocol # Protocol 18_173) and exempted from full IRB review by the National Institutes of Health Office of Human Subject Research.

Step 1: Test associations between cholesterol catabolism (i.e., BA synthesis) and neuroimaging markers of dementia

We performed targeted metabolomics assays measuring the principal cholesterol breakdown products (i.e., CA and CDCA) as well as their principal biosynthetic precursor, (7α-hydroxycholesterol; 7α-OHC) in serum samples from participants in the Baltimore Longitudinal Study of Aging Neuroimaging (BLSA-NI) substudy who also underwent in vivo brain amyloid positron emission tomography (PET) and longitudinal structural magnetic resonance imaging (MRI).

In order to validate index results from BLSA, we used the ADNI sample to test associations between CA and CDCA and neuroimaging outcomes (note: 7α-OHC was not assayed in the ADNI serum samples).

Study participants

The BLSA is a prospective cohort study that began in 1958 and is administered by the National Institute on Aging (NIA) [6]. BLSA-NI substudy imaging and visit schedules have varied over time and have been described in detail previously [7] and included in S1 Text.

ADNI is an ongoing longitudinal study launched in 2003. The primary goal of ADNI has been to test whether longitudinal MRI, PET, and other biological markers can measure the progression of mild cognitive impairment (MCI) and early AD. ADMC performed serum BA assays in participants enrolled in ADNI, and data are publicly available. Study design details have been published previously [5] and are available at www.adni-info.org.

Quantitative serum metabolomics assays

Blood serum samples were collected from BLSA participants at each visit; details on collection and processing have been published previously [8] and included in S1 Text.

Blood serum samples were collected from ADNI participants at baseline; details on collection and processing have been published previously [9,10] and are described in detail in the Biospecimen Results section (link: AD Metabolomics Consortium Bile Acids Methods (PDF)–Version: January 21, 2016) at http://adni.loni.usc.edu. Data used in this study are available in the Biospecimen Results section (link: AD Metabolomics Consortium Bile Acids–Post Processed Data [ADNI1, GO, 2]–Version: June 28, 2018).

In vivo brain amyloid imaging, WMLs, and brain volumes

BLSA-NI participants underwent 11C-Pittsburgh compound-B (PiB) PET scans to assess brain amyloid-β burden. A detailed description of acquisition and preprocessing procedures has been published previously [11]. Individuals were characterized as amyloid +ve or amyloid −ve based on a mean cortical distribution volume ratio (cDVR) threshold of 1.066 [11]. Among amyloid +ve individuals, we examined mean cDVR, a weighted global average of brain amyloid deposition, and regional DVR in the precuneus, a region vulnerable to early amyloid deposition in AD [12]. The total sample included 141 individuals (66 male; 75 female) of whom 36 were amyloid +ve (21 male; 15 female).

BLSA brain MRI was performed on a 3T Philips Achieva scanner (Philips Healthcare, Netherlands) to quantify both global and regional brain volumes and WMLs. A detailed description is included in S1 Text.

We a priori defined a set of brain regions to examine brain atrophy over time based on prior work using BLSA-NI data suggesting that these regions were sensitive to age-related change [7]. These regions included global brain volumes: total brain, ventricular cerebrospinal fluid (CSF), total gray matter, and white matter; lobar volumes: temporal, parietal, and occipital white matter and gray matter; and additional regions sensitive to early neurodegeneration: hippocampus, entorhinal cortex, amygdala, parahippocampal gyrus, fusiform gyrus, and precuneus.

All BLSA MRI data, including brain volumes and WMLs, after onset of clinical symptoms among individuals who developed MCI or AD were excluded (21 visits). The total sample included 134 individuals (62 male; 72 female) with an average of 2.5 longitudinal MRI visits (male: 2.3; female: 2.6).

ADNI brain MRI was used to quantify both global and regional brain volumes. A detailed description of acquisition and preprocessing is included in S1 Text. As our analyses in the ADNI sample were performed to confirm index results from BLSA, we restricted these analyses to gray matter and subcortical brain regions described above, excluding all white matter regions based on the lack of associations in BLSA analyses.

ADNI is enriched for individuals with MCI and AD at baseline, and all data across baseline diagnoses (control (CON), MCI, and AD) were included in analyses. Similar to our primary analyses in BLSA, MRI data from all CON individuals in ADNI, after onset of clinical symptoms among individuals who subsequently developed MCI or AD, were excluded (200 visits). The total sample included 1,666 individuals (918 male; 748 female) with an average of 5.2 longitudinal MRI visits (male: 5.3; female: 5.1).

Statistical methods

Step 1 of the analytic plan using BLSA data were developed in January 2018 prior to starting analyses in June 2019. The inclusion of ADNI BA and neuroimaging data to validate significant BLSA findings and sensitivity analyses (i.e., adding statin as a covariate) were performed in June 2020 in response to reviewer recommendations.

Similar to prior work in BLSA [8], metabolite concentrations above the upper limit of quantification (ULOQ) were excluded, concentrations below the limit of detection (LOD) were imputed as the threshold LOD/2, and resulting concentrations were natural log transformed. Outliers ± 3 × interquartile range (IQR) were excluded.

For ADNI, data processing steps for serum BA concentrations have been described in detail previously [9,10]. Metabolite concentrations below the LOD were imputed as LOD/2, and all samples were log2 transformed. Outliers ± 3 × IQR were excluded.

To test for group differences between amyloid +ve and amyoid −ve individuals, we examined associations between serum concentrations of metabolites (i.e., 7α-OHC, CA, and CDCA) and brain amyloid deposition, in overall and sex-stratified linear regression models with metabolites as the dependent variable and the binary brain amyloid variable (i.e., amyloid +ve/amyloid −ve) as the main predictor. Covariates included mean-centered age and sex in the overall model and mean-centered age only in the sex-stratified model. We next tested the association between metabolite concentrations and mean cDVR (BLSA) and precuneus DVR (BLSA) in amyloid +ve individuals only. We used similar linear regression models for the continuous DVR predictors. The significance threshold was uncorrected and set at p = 0.05 to accommodate the limited sample size.

To test the association between serum concentrations of metabolites (i.e., 7α-OHC, CA, and CDCA) and longitudinal changes in (1) regional brain volumes and (2) WMLs, we used total and sex-stratified linear mixed models with brain regions of interest (ROIs) volumes and WMLs as the dependent variable (i.e., outcome) and metabolite concentration as the predictor. We first performed analyses in BLSA and then validated results in ADNI. The statistical significance threshold for both BLSA and ADNI was set at a false discovery rate (FDR)-corrected p = 0.05. Additional details on model specifications are included in S1 Text.

In sensitivity analyses, we explored the effect on associations of adding statin drug use as a covariate in BLSA models.

Step 2: Test whether pharmacological modulation of BAs alters dementia risk in a large, real-world clinical dataset

In order to extend findings from our Step 1 analysis relating BA levels with brain amyloid accumulation, rates of brain atrophy, and progression of WMLs, we next tested whether pharmacological modulation of de novo BA synthesis influences dementia risk. As BAS are lipid-modifying treatments that are known to decrease the circulating pool of BAs [1315] and promote the breakdown of cholesterol, we hypothesized that exposure to these drugs would alter the risk of dementia. We therefore tested associations between exposure to BAS and dementia risk using data from the UK’s CPRD, an anonymized electronic health record (EHR) covering more than 4 million active registrants from the UK general practice clinics. Our results from Step 1 suggested a plausible sex difference in the effect of BAS on dementia risk, a hypothesis that we tested using the CPRD.

Data source and study population

The CPRD is a primary care database covering >four million active registrants from >650 general practice clinics and is representative of the broader UK population in terms of age and sex [16].

From the August 2018 CPRD data release, we identified patients ≥18 years old who had a first prescription record (i.e., new users) for BAS (colestipol, colesevelam, and cholestyramine) or non-statin lipid-modifying therapies (LMT; fibrate, cholesterol absorption inhibitor, nicotinic acid derivative, and probucol) between January 1, 1995 and August 1, 2018. BAS are often used as a second-line treatment independently or in combination with statins, and therefore, we selected non-statin LMT users as an active comparator group. In both groups (BAS or non-statin LMTs), we allowed for prior statin use in combination with either BAS or LMTs. Individuals who only had a prescription record of statin use were excluded from this study.

The index date was defined as the date of the first BAS or LMT prescription. For each BAS user, we selected up to five LMT users matched on sex, year of birth (±5 year), region, year of clinic registration (±2 year), and year of first prescription (±2 years). Analysis was restricted to those with at least 12 months of clinical registration prior to the index date (to allow for covariate evaluation). We restricted BAS users to those aged ≥50 years and to those with two or more BAS/LMT prescriptions. The final analysis included 3,208 (1,083 male; 2,125 female) new BAS users and 23,483 (8,977 male; 14,506 female) new LMT users (Fig 1).

Fig 1. Flowchart of study participants included in the CPRD analyses.

Fig 1

BAS, bile acid sequestrants including colestipol, colesevelam, and cholestyramine; CPRD, Clinical Practice Research Datalink; LMT, lipid-modifying therapies including fibrate, cholesterol absorption inhibitor, nicotinic acid derivative, and probucol.

The outcomes of interest were all-cause dementia, and its subtypes: AD, VaD, and other dementia not otherwise specified (NOS). We used the last reported dementia diagnosis to identify the disease subtype (read codes are available upon request). The significance threshold was set at p = 0.05 considering that each outcome of interest was a priori specified.

Statistical methods

Step 2 of the analytic plan using CPRD data was developed in January 2018 prior to starting data analyses in June 2019. We added a comparison of patient characteristics across outcome categories (i.e., dementia subtypes) based on reviewer recommendations.

We compared patient characteristics and their comorbidity profiles across dementia subtypes (AD, VaD, and NOS) as well as drug use (BAS and LMT) using the chi-squared test for categorical variables and Wilcoxon rank-sum tests for continuous variables.

For multivariable analyses, we used Cox proportional hazard models to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) comparing dementia risk (all-cause and subtypes) in BAS versus LMT users in the overall and sex-stratified samples. Our sex-specific analyses were a priori specified and based on findings from Step 1. We also tested the dose–effect relationship between dementia risk and drugs of interest using the number of prescriptions. Models were adjusted for factors that were significantly different between BAS and LMT groups to account for potential confounding by indication (since patient comorbidity profiles can lead to a BAS versus LMT prescription decision). Models were also adjusted for statin use during follow-up (until one year prior to exit date) using a time-varying covariate to account for its impact on dementia (26% versus 80% of BAS and LMT users, respectively, were prescribed statins in the 12 months before the index date and the majority (63%) continued its use for all or part of the follow-up). See S1 Text for details on model specifications.

Step 3: Test plausible molecular mechanisms relating BA signaling in the brain to dementia pathogenesis using targeted metabolomics and transcriptomics

Given our findings that peripheral levels of the primary BAs, CA, and CDCA are associated with neuroimaging markers of dementia (Step 1) and that their pharmacological manipulation influences dementia risk (Step 2), we hypothesized that alterations in brain BA-mediated signaling may be a plausible biological mechanism underlying these findings. We first tested whether concentrations of CA and CDCA were detectable in the brain and whether they were altered in AD in participants from the BLSA autopsy program. We then tested whether gene expression of BA receptors was altered in AD using single-cell RNA sequencing (scRNA-Seq) data from the ROSMAP autopsy program.

Study participants

The autopsy program of the BLSA was initiated in 1986 and has been described previously [17]. See S1 Text for additional details. Tissue samples from AD (n = 16) and CON (n = 13) from the inferior temporal gyrus (ITG) and middle frontal gyrus (MFG), regions representing areas of early neurofibrillary (i.e., tau) and neuritic plaque (i.e., amyloid) accumulation, respectively [18,19], as well as the cerebellum (CB) were included in these analyses.

scRNA-Seq gene expression data [20] from ROSMAP [21] were downloaded from Synapse (https://www.synapse.org/#!Synapse:syn18485175) under the doi 10.7303/syn18485175; code used to run analyses presented in Mathys and colleagues [20] was requested from coauthors. Data came from postmortem participants in ROSMAP including 46 individuals: 32 individuals (18 male and 14 female) in the AD category and 14 individuals (5 male and 9 female) in the CON category. The AD category included individuals with a clinical diagnosis of AD, including individuals with AD and no other condition contributing to cognitive impairment and AD and another condition contributing to cognitive impairment, as well as individuals with a clinical diagnosis of MCI and no other condition contributing to cognitive impairment. The CON category included individuals with a clinical diagnosis of no cognitive impairment.

Tissue was profiled from the prefrontal cortex (Brodmann area 10) across eight major cell types in the aged dorsolateral prefrontal cortex including inhibitory neurons, excitatory neurons, astrocytes, oligodendrocytes, microglia, oligodendrocyte progenitor cells, endothelial cells, and pericytes. Additional details are provided in the index paper [20]. We identified BA receptor genes (including receptors involved in BA homeostasis) using a literature search [2224] and include the full list in S2 Table. There were 21 BA receptor genes that had available data in the ROSMAP dataset: Nuclear Receptor Subfamily 1 Group I Member 3 (NR1I3); Retinoid X Receptor Gamma (RXRG); Nuclear Receptor Subfamily 5 Group A Member 2 (NR5A2); Cholinergic Receptor Muscarinic 3 (CHRM3); G Protein-Coupled Bile Acid Receptor 1 (GPBAR1); Peroxisome Proliferator Activated Receptor Gamma (PPARG); Nuclear Receptor Subfamily 1 Group I Member 2 (NR1I2); Kinase Insert Domain Receptor (KDR); Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1); Retinoid X Receptor Beta (RXRB); Peroxisome Proliferator Activated Receptor Delta (PPARD); Cholinergic Receptor Muscarinic 2 (CHRM2); Retinoid X Receptor Alpha (RXRA); Nuclear Receptor Subfamily 1 Group H Member 3 (NR1H3); Vitamin D Receptor (VDR); Nuclear Receptor Subfamily 1 Group H Member 4 (NR1H4); Retinoic Acid Receptor Alpha (RARA); Hepatocyte Nuclear Factor 4 Alpha (HNF4A); Nuclear Receptor Subfamily 1 Group H Member 2 (NR1H2); Formyl Peptide Receptor 1 (FPR1); Peroxisome Proliferator Activated Receptor Alpha (PPARA).

Quantitative brain metabolomics assays

Quantitative metabolomics assays were performed on brain tissue samples to measure concentrations of the primary BAs, including CA and CDCA, using the Biocrates Bile Acids kit (Biocrates Life Sciences AG, Austria). Details on both assay kits, as well as calibration steps, have been published previously [25]. Additional details regarding the use of internal standards are included in S1 Text.

Statistical methods

Step 3 of the analytic plan using BLSA data was developed in January 2020 in order to address a plausible molecular mechanism explaining findings from Step 1 and Step 2. The inclusion of scRNA-Seq data from ROSMAP occurred in June 2020 in response to reviewer recommendations to use non-array, non-bulk tissue-based gene expression data.

In order to assess whether primary BAs were present in the brain, we visualized CA and CDCA concentrations in AD and CON samples in the ITG, MFG, and CB using dot plots. Concentrations above and below LOD were indicated. We used tobit regression models to determine whether mean metabolite concentrations were significantly different between AD and CON samples. We set the lower limit as the metabolite-specific LOD threshold and included covariates age and sex (mean centered). In brain regions where metabolite concentrations were all below LOD (i.e., CB), we used chi-squared tests to determine whether percentage of samples below LOD was significantly different between AD and CON samples. Due to a small number of individuals with BA metabolite values above LOD, we were not able to sex-stratify these analyses. Additionally, the statistical significance threshold was set at p = 0.05 to accommodate the limited sample size.

For gene expression data, we scaled each sample to have the same total read count. To test differences between AD and CON, we used the Wilcoxon rank-sum test in the total and sex-stratified samples. Similar to the index paper [20], each single-cell–specific sample from a participant was treated as an independent sample. We summarized age- and sex-corrected fold changes (total sample) as well as sex-specific fold changes indicating whether genes were differentially expressed in AD versus CON samples. We additionally visualized results for significant associations using a heatmap. The significance threshold was set at an FDR-corrected p = 0.05.

Results

Step 1: Test associations between cholesterol catabolism (i.e., BA synthesis) and neuroimaging markers of dementia

Participant demographic details are included in S3A and S3B Table. Results of cross-sectional analyses testing associations between serum metabolite concentrations and amyloid status in BLSA are included in S4 Table, and associations between serum metabolite concentrations and brain amyloid-β burden among amyloid +ve individuals are included in Table 1. Brain amyloid +/−ve status was not significantly associated with serum concentrations of 7α-OHC, CDCA, or CA in the total or sex-stratified samples. In the total sample and in males only, serum 7α-OHC concentration, representing the rate-limiting biosynthetic precursor of the primary BAs [26,27], was significantly, negatively associated with mean cDVR (p = 0.034 and p = 0.041, respectively) (Fig 2) and precuneus DVR (p = 0.033 and p = 0.022, respectively), indicating that lower serum concentration of 7α-OHC was associated with higher levels of global and precuneus brain amyloid-β deposition. We observed no significant associations in the female-only sample.

Table 1. Associations between serum metabolite concentrations and brain amyloid-β deposition, rates of brain atrophy, and longitudinal changes in global brain WML burden–BLSA.

Global DVR (amyloid-β deposition) − amyloid +ve sample
Total (n = 36) Male (n = 21) Female (n = 15)
coef pval coef pval coef pval
7α-OHC −1.439 0.034 −1.568 0.041 −1.007 0.57
CDCA 2.91 0.063 2.518 0.176 4.241 0.245
CA 3.198 0.05 3.209 0.104 2.993 0.446
Precuneus DVR (amyloid-β deposition)amyloid +ve sample
Total (n = 36) Male (n = 21) Female (n = 15)
coef pval coef pval coef pval
7α-OHC −1.197 0.033 −1.447 0.022 0.09 0.948
CDCA 2.418 0.061 2.06 0.186 4.766 0.082
CA 1.966 0.152 1.994 0.236 2.062 0.501
Brain atrophy
Total (n = 134) Male (n = 62) Female (n = 72)
coef pval pval (FDR) coef pval pval (FDR) coef pval pval (FDR)
CDCA (Parietal GM) 0.034 0.426 0.719 0.289 <0.001 0.003 −0.112 0.014 0.119
CDCA (Precuneus) 0.019 0.1 0.491 0.094 <0.001 <0.001 −0.022 0.101 0.299
CA (Parietal GM) 0.022 0.577 0.797 0.207 0.001 0.013 −0.154 0.001 0.038
CA (Precuneus) 0.016 0.139 0.602 0.069 <0.001 <0.001 −0.031 0.02 0.119
WML
Total (n = 134) Male (n = 62) Female (n = 72)
coef pval coef pval coef pval
7α-OHC 0.015 0.088 −0.01 0.421 0.031 0.01
CDCA −0.001 0.892 −0.009 0.05 0.005 0.342
CA −0.001 0.769 −0.001 0.763 0 0.992

Significance threshold set at p = 0.05 for amyloid-β deposition and WMLs and FDR-corrected p = 0.05 for brain atrophy. Coefficients in green indicate that lower serum concentration of the metabolite is significantly associated with higher levels of brain amyloid-β, faster accumulation of WML accumulation, or faster brain atrophy. Coefficients in red indicate that lower serum concentration of the metabolite is significantly associated with lower levels of brain amyloid-β, slower accumulation of WML, or slower brain atrophy. Coef and pval highlighted in gray were not statistically significant.

7α-OHC, 7α-hydroxycholesterol; BLSA, Baltimore Longitudinal Study of Aging; CA, cholic acid; CDCA, chenodeoxycholic acid; cDVR, mean cortical DVR; coef, coefficient from linear regression model or mixed effects model; DVR, distribution volume ratio; FDR, false discovery rate (Benjamini–Hochberg) corrected p-value; GM, gray matter; pval, p-value; WML, white matter lesion.

Fig 2. Association between serum 7α-OHC concentration and brain amyloid-β burden in amyloid +ve individuals.

Fig 2

Weighted global average of brain amyloid-β burden (mean cDVR); analyses restricted to amyloid +ve individuals (n = 36). 7α-OHC, 7α-hydroxycholesterol; cDVR, cortical distribution volume ratio.

Results of longitudinal analyses in BLSA testing associations between serum metabolite concentrations and brain atrophy are shown in Table 1. In males, lower serum CDCA and CA concentrations were associated with faster rates of atrophy in the parietal gray matter (CDCA: FDR p = 0.003; CA: FDR p = 0.013) and precuneus (CDCA: FDR p < 0.001; CA: FDR p < 0.001). In females, lower serum CA concentration was associated with slower total gray matter atrophy (FDR p = 0.038).

Sensitivity analyses including statin drug use as a covariate are included in S5 Table; results were not substantially altered.

Results of longitudinal analyses in ADNI testing associations between serum metabolite concentrations and brain trophy are shown in Table 2. In the total sample, lower serum CDCA and CA concentrations were associated with faster rates of atrophy in the entorhinal cortex (CDCA: FDR p = 0.032; CA: FDR p = 0.009), frontal gray matter (CDCA: FDR p = 0.045; CA: FDR p = 0.005), fusiform gyrus (CDCA: FDR p = 0.012; CA: FDR p = 0.001), total gray matter (CDCA: FDR p = 0.030; CA: FDR p = 0.003), hippocampus (CDCA: FDR p = 0.030; CA: FDR p = 0.012), parahippocampal gyrus (CDCA: FDR p = 0.012; CA: FDR p = 0.009), temporal gray matter (CDCA: FDR p = 0.016; CA: FDR p = 0.002), and ventricles (CDCA: FDR p = 0.030; CA: FDR p = 0.008). Lower CA was also associated with faster rates of atrophy in the amygdala (FDR p = 0.030), occipital gray matter (FDR p = 0.012), parietal gray matter (FDR p = 0.016), and precuneus (FDR p = 0.030). In males, lower serum CDCA and CA concentrations were associated with faster rates of atrophy in the parahippocampal gyrus (CDCA: FDR p = 0.049; CA: FDR p = 0.049). Lower serum CDCA was associated with faster rates of atrophy in the ventricles (CDCA: FDR p = 0.049), and lower serum CA was associated with faster rates of atrophy in the entorhinal cortex, frontal gray matter, fusiform gyrus, total gray matter, hippocampus, and temporal gray matter (FDR p = 0.049). In females, CDCA was not significantly associated with rates of brain atrophy; lower CA was associated with faster rates of atrophy in only the fusiform gyrus and temporal gray matter (FDR p = 0.039).

Table 2. Associations between serum metabolite concentrations and rates of brain atrophy and longitudinal changes in global brain WML burden–ADNI.

Brain atrophy
Total (n = 1,666) Male (n = 918) Female (n = 748)
coef pval pval (FDR) coef pval pval (FDR) coef pval pval (FDR)
CDCA (Ent. cortex) 2.341 0.025 0.032 2.802 0.059 0.115 1.735 0.229 0.262
CDCA (Frontal GM) 46.678 0.037 0.045 45.613 0.126 0.191 52.38 0.122 0.177
CDCA (Fus. gyrus) 7.382 0.005 0.012 6.302 0.063 0.115 9.188 0.024 0.083
CDCA (Total GM) 152.971 0.022 0.03 124.115 0.159 0.201 191.975 0.06 0.12
CDCA (Hipp) 2.439 0.023 0.03 2.244 0.134 0.191 2.613 0.086 0.148
CDCA (Parahipp. gyrus) 3.262 0.005 0.012 3.841 0.016 0.049 2.574 0.125 0.177
CDCA (Temporal GM) 43.824 0.009 0.016 31.99 0.143 0.191 59.941 0.021 0.083
CDCA (Ventricle) −64.575 0.022 0.03 −92.627 0.018 0.049 −30.85 0.438 0.438
CA (Amygdala) 1.005 0.02 0.03 1.103 0.049 0.108 0.801 0.244 0.266
CA (Ent. cortex) 3.524 0.003 0.009 3.891 0.014 0.049 2.898 0.097 0.155
CA (Frontal GM) 83.879 0.001 0.005 85.173 0.007 0.049 85.187 0.037 0.098
CA (Fus. gyrus) 11.923 <0.001 0.001 10.066 0.005 0.049 15.384 0.002 0.039
CA (Total GM) 264.396 <0.001 0.003 231.124 0.013 0.049 320.791 0.009 0.055
CA (Hipp) 3.367 0.005 0.012 3.772 0.018 0.049 2.662 0.153 0.194
CA (Occipital GM) 31.538 0.005 0.012 21.78 0.137 0.191 47.546 0.008 0.055
CA (Parahipp. gyrus) 3.928 0.002 0.009 4.188 0.013 0.049 3.591 0.078 0.144
CA (Parietal GM) 35.172 0.008 0.016 26.732 0.106 0.181 48.186 0.029 0.088
CA (Precuneus) 8.589 0.022 0.03 6.543 0.178 0.203 11.897 0.044 0.105
CA (Temporal GM) 71.579 <0.001 0.002 58.711 0.012 0.049 93.493 0.003 0.039
CA (Ventricle) −101.731 0.002 0.008 −92.725 0.028 0.068 −116.852 0.018 0.083
WML
Total (n = 875) Male (n = 456) Female (n = 419)
coef pval coef pval coef pval
CDCA 11.714 0.326 5.344 0.77 18.075 0.216
CA 17.236 0.215 13.627 0.496 23.858 0.198

Coefficients in green indicate that lower serum concentration of the metabolite is significantly associated with faster accumulation of WML accumulation or faster brain atrophy. Coefficients in red indicate that lower serum concentration of the metabolite is significantly associated with slower accumulation of WML or slower brain atrophy. Coef and pval highlighted in gray were not statistically significant.

ADNI, Alzheimer’s Disease Neuroimaging Initiative; CA, cholic acid; CDCA, chenodeoxycholic acid; coef, coefficient from linear regression model or mixed effects model; FDR, false discovery rate (Benjamini–Hochberg) corrected p-value. Significance threshold was set at an FDR-corrected p = 0.05; Fus. gyrus, fusiform gyrus; GM, gray matter; Hipp, Hippocampus; Parahipp gyrus, Parahippocampal gyrus; pval, p-value; WML, white matter lesion.

Results of longitudinal analyses in BLSA testing associations between serum metabolite concentrations and WML are shown in Table 1. In males, lower serum CDCA concentration was associated with faster accumulation of WML (p = 0.050), and in females, lower serum 7α-OHC was associated with slower accumulation of WML (p = 0.010).

Results of longitudinal analyses in ADNI testing associations between serum metabolite concentrations and WML are shown in Table 2. In ADNI, we did not observe significant associations between serum metabolite concentrations of BAs (i.e., CA and CDCA) and WML in the total male or female samples.

Step 2: Test whether pharmacological modulation of BAs alters dementia risk in a large, real-world clinical dataset

S6 Table summarizes characteristics of BAS and LMT users. LMT users were more likely to be overweight or obese compared with BAS users (73% versus 57%, respectively). In the 12 months prior to index date, LMT users were more likely to have used statins (80% versus 26%, respectively) or metformin (15% versus 7%) and had a record of coronary artery disease (7% versus 3%), type 2 diabetes (7% versus 3%), or dyslipidemia (25% versus 5%). BAS users were more likely to have a prior record of cancer (16% versus 8%).

Table 3 summarizes results from Cox proportional hazard models. During the median follow-up of 4.9 years, 809 incident dementia cases occurred (N = 72 for BAS versus 737 for LMT) corresponding to crude incidence rates of 4.8 (95% CI = 3.8 to 6.1) and 5.5 per 1,000 person-years (95% CI = 5.1 to 5.9) among BAS and LMT users, respectively. In multivariable adjusted models including all patients and compared to LMT use of > = two prescriptions, BAS use was not statistically significantly associated with risk of all-cause dementia (Table 3) or with its subtypes (any dementia: HR = 1.03, 95% CI = 0.72 to 1.46, p = 0.88; AD: HR = 1.24, 95% CI = 0.72 to 2.14, p = 0.43; VaD: HR = 1.27, 95% CI = 0.70 to 2.31, p = 0.43; other dementia: HR = 0.50, 95% CI = 0·22 to 1.15, p = 0.10).

Table 3. Association between BAS use and the risk of dementia in individuals who received at least two BAS or LMT prescriptions with at least one year of follow-up after second prescription.

Cause-specific analysis
Any dementia AD Vascular disease Other dementia, NOS1
Analysis Exposure N events/total HR (95% CI)2 p N events/total HR (95% CI)2 p N events/total HR (95% CI)2 p N events/total HR (95% CI)2 p
Overall
≥2 RXs BAS use3 72/3,208 1.03 (0.72–1.46) 0.88 30/3,208 1.24 (0.72–2.14) 0.43 31/3,208 1.27 (0.70–2.31) 0.43 11/3,208 0.50 (0.22–1.15) 0.10
LMT use3 737/23,483 Reference 302/23,483 Reference 260/23,483 Reference 175/23,483 Reference
Cumulative BAS number of RXs BAS 2 RXs 16/810 0.95 (0.45–1.99) 5/810 0.56 (0.14–2.20) 7/810 2.12 (0.53–8.57) 4/810 0.70 (0.21–2.34)
BAS 3–5 RXs 18/1,140 1.07 (0.50–2.30) 7/1,140 1.20 (0.39–3.69) 8/1,140 1.43 (0.40–5.09) 3/1,140 0.48 (0.05–4.49)
BAS ≥6 RXs 38/1,744 1.04 (0.67–1.63) 18/1,744 1.60 (0.80–3.20) 16/1,744 1.09 (0.52–2.26) 4/1,744 0.37 (0.10–1.33)
LMT ≥2 RXs 737/23,483 Reference 302/23,483 Reference 260/23,483 Reference 175/23,483 Reference
P for trend 0.84 0.23 0.62 0.09
Male
≥2 RXs BAS use4 25/1,083 1.20 (0.63–2.28) 0.58 6/1,083 0.72 (0.19–2.78) 0.64 14/1,083 2.89 (0.96–8.68) 0.06 5/1,083 0.52 (0.14–1.98) 0.34
LMT use4 240/8,977 Reference 91/8,977 Reference 90/8,977 Reference 59/8,977 Reference
Cumulative BAS number of RXs BAS 2 RXs 7/261 1.74 (0.58–5.21) 2/261 4.03 (0.28–57.44) 2/261 1.43 (0.1118.75) 3/261 1.02 (0.19–5.38)
BAS 3–5 RXs 4/367 0.52 (0.10–2.69) 1/367 3/367 2.41 (0.2919.80) 0/367
BAS ≥6 RXs 14/591 1.25 (0.54–2.91) 3/591 0.69 (0.12–3.87) 9/591 3.85 (0.9315.94) 2/591 0.33 (0.03–3.16)
LMT ≥2 RXs 240/8,977 Reference 91/8,977 Reference 90/8,977 Reference 59/8,977 Reference
P for trend 0.71 0.47 0.045 0.23
Female
≥2 RXs BAS use4 47/2,125 0.99 (0.65–1.52) 0.98 24/2,125 1.35 (0.73–2.48) 0.33 17/2,125 1.00 (0.46–2.15) >0.99 6/2,125 0.43 (0.13–1.37) 0.15
LMT use4 497/14,506 Reference 211/14,506 Reference 170/14,506 Reference 116/14,506 Reference
Cumulative BAS number of RXs BAS 2 RXs 9/549 0.64 (0.23–1.83) 3/549 0.17 (0.02–1.49) 5/549 2.95 (0.5017.49) 1/549 0.31 (0.03–3.23)
BAS 3–5 RXs 14/773 1.53 (0.64–3.67) 6/773 2.05 (0.60–7.02) 5/773 1.42 (0.28–7.07) 3/773 1.12 (0.11–11.64)
BAS ≥6 RXs 24/1,153 0.97 (0.57–1.65) 15/1,153 1.85 (0.84–4.07) 7/1,153 0.69 (0.26–1.83) 2/1,153 0.36 (0.07–1.83)
LMT ≥2 RXs 497/14,506 Reference 211/14,506 Reference 170/14,506 Reference 116/14,506 Reference
P for trend 0.93 0.12 0.66 0.18

1 Not otherwise specified.

2 Models were adjusted for smoking status, BMI, alcohol consumption close to index date, metformin use one year prior to index date, coronary artery diseases, type 2 diabetes and dyslipidemia record one year prior to index date, prior cancer history, time-varying statins use status during follow-up (until one year before exit date), and stratified on matched set.

3 ≥two RXs use.

Findings reported in the paper are indicated in bold.

AD, Alzheimer disease; BAS, bile acid sequestrants; BMI, body mass index; CI, confidence interval; HR, hazard ratio; LMT, non-statin lipid-modifying therapies; RX, prescription.

In analyses stratified by sex, we observed a significant (p = 0.040) difference between the HR of VaD in males compared to females, indicating a sex difference in the relationship between BAS and risk of VaD. BAS use was associated with nonsignificantly elevated risk of VaD in males (HR = 2.89, 95% CI = 0.96 to 8.68, p = 0.06). We identified a statistically significant dose–response relationship between BAS and risk of VaD in males. Specifically, risk of VaD was higher with the increased number of BAS prescriptions (p-trend = 0.045) (Table 3). There was no statistically significant association with VaD in females (overall or by number of prescriptions). Differences in patient characteristics across outcome categories are included in S7 Table.

Step 3: Test plausible molecular mechanisms relating BA signaling in the brain to dementia pathogenesis using targeted metabolomics and transcriptomics

Participant demographic details for the BLSA autopsy study are included in S8 Table. Demographic details of ROSMAP participants included in scRNA-Seq analyses have been published previously [20].

The primary BAs, CDCA, and CA were detectable in postmortem brain tissue samples, although the majority were below the LOD (i.e., <LOD) (Fig 3, S9 Table). Tobit regression models indicated marginally higher (nonsignificant) concentrations of the primary BAs in AD samples compared to CON samples in the ITG and MFG. Chi-squared models indicated significantly more participants with metabolite concentrations above LOD in AD compared to CON in the CB. Due to a small number of individuals with BA metabolite values above LOD, we were not able to sex-stratify these analyses.

Fig 3. Differences in brain primary BA concentrations between AD and CON.

Fig 3

Dots in gray indicate concentrations below the LOD; dots in blue and red indicate AD and CON brain tissue sample metabolite concentrations, respectively. p-Values indicate differences between AD and CON from a tobit regression model; the lower limit in the model is set at the metabolite-specific LOD (indicated by the dashed line). The p-value for CDCA in the CB is derived from a chi-squared test because there were no detectable concentrations above the LOD in the CON sample. AD, Alzheimer disease; BA, bile acid; CB, cerebellum; CDCA, chenodeoxycholic acid; CON, control; ITG, inferior temporal gyrus; LOD, limit of detection; MFG, middle frontal gyrus.

We observed that gene expression of several BA receptors was different in AD versus CON in the total and male samples, mainly within neurons (i.e., both inhibitory and excitatory neurons). The majority of differentially expressed genes showed lower expression in AD relative to CON. Results across all 8 major brain cell types are included in S10 Table. As indicated below in a heatmap visualizing sex-stratified differences in AD versus CON samples (Fig 4), for inhibitory neurons, in males, 10 out of 21 genes were significantly altered (FDR pval < 0.05); six had lower gene expression in AD compared to CON (AD<CON); and four had higher gene expression in AD compared to CON (AD>CON). In females, there were no differentially expressed BA receptor genes within inhibitory neurons. Within excitatory neurons, in males, 16 out of 21 genes were significantly altered (FDR pval < 0.05); 10 had lower gene expression in AD compared to CON (AD<CON); and 6 had higher gene expression in AD compared to CON (AD>CON). For females, four genes showed lower gene expression in AD compared to CON (AD<CON).

Fig 4. Differences in brain BA receptor gene expression between AD and CON.

Fig 4

Summary of differentially expressed BA receptor genes (including receptors involved in BA homeostasis) in neurons in AD compared to CON. Statistically significant (FDR-corrected p-value < 0.05) fold change differences are indicated in green or red shading. Green shading indicates that gene expression was significantly reduced in AD compared to CON. Red shading indicates that gene expression was significantly increased in AD compared to CON. Gray shading indicates gene expression was not significantly different between AD and CON. AD, Alzheimer disease; BA, bile acid; CHRM2, Cholinergic Receptor Muscarinic 2; CHRM3, Cholinergic Receptor Muscarinic 3; CON, control; FDR, false discovery rate; FPR1, Formyl Peptide Receptor 1; NR1H2, Nuclear Receptor Subfamily 1 Group H Member 2; NR1H3, Nuclear Receptor Subfamily 1 Group H Member 3; NR1H4, Nuclear Receptor Subfamily 1 Group H Member 4; NR1I2, Nuclear Receptor Subfamily 1 Group I Member 2; NR1I3, Nuclear Receptor Subfamily 1 Group I Member 3; NR3C1, Nuclear Receptor Subfamily 3 Group C Member 1; NR5A2, Nuclear Receptor Subfamily 5 Group A Member 2; PPARA, Peroxisome Proliferator Activated Receptor Alpha; PPARD, Peroxisome Proliferator Activated Receptor Delta; PPARG, Peroxisome Proliferator Activated Receptor Gamma; RARA, Retinoic Acid Receptor Alpha; RXRA, Retinoid X Receptor Alpha; RXRB, Retinoid X Receptor Beta; VDR, Vitamin D Receptor.

Discussion

We found that lower serum concentrations of the rate-limiting biosynthetic precursor of BA synthesis, i.e., 7α-OHC, as well as the primary BAs mainly in males, were associated with neuroimaging measures of dementia progression and that pharmacological lowering of BA levels was associated with higher risk of VaD in males. We hypothesize that disruption of BA signaling in the brain as reflected in altered levels of primary BAs and reduced neuronal gene expression of BA receptors may represent a plausible biological mechanism underlying these results. Together, our observations suggest a novel mechanism relating abnormalities in cholesterol catabolism to risk of dementia.

The role of hypercholesterolemia in the pathogenesis of dementia is well recognized but poorly understood. While the BBB ensures that brain concentrations of cholesterol are largely independent of peripheral tissues [28], the oxidative catabolism of cholesterol results in the generation of oxysterols that are permeable to the BBB and can both access the brain from the peripheral circulation, as well as efflux into the periphery from the brain (S1 Fig). Oxysterols, including 7α-OHC, are key biosynthetic precursors of the primary BAs, CA, and CDCA, which, in turn, represent the primary catabolic products of cholesterol.

We observed an association between serum concentration of 7α-OHC, representing the rate-limiting reaction in primary BA synthesis [2931], and global brain amyloid burden as well as that in the precuneus, an early site of amyloid deposition in AD [12] suggesting that impaired synthesis of primary BAs may be an important mediator of pathologic changes in AD. This relationship appears to be driven primarily by males suggesting a novel sex-specific association between BA synthesis and brain amyloid accumulation. It is important to note, however, that these cross-sectional analyses are not able to determine whether pathology, brain atrophy, or other dementia-associated endophenotypes may modify cholesterol catabolism.

We then examined the relationships between BA synthesis and both regional rates of brain atrophy as well as the accumulation of WMLs that are key vascular contributors to dementia [32]. Our results indicate that in males, lower serum CDCA and CA is associated with faster rates of brain atrophy and faster accumulation of brain WMLs in the BLSA. These findings were partially confirmed in ADNI where lower BA concentrations were associated with faster brain atrophy rates across several brain regions in males with far fewer associations in females. It is important to note, however, that the lack of sex-specific associations compared to the total sample in ADNI may be partially driven by sample size. Female participants in BLSA showed an opposite effect compared to males—lower serum concentrations of 7α-OHC and CA were associated with slower accumulation of brain WMLs and slower rates of brain atrophy. Our sex-specific WML associations in BLSA were not replicated in ADNI. This may be due, in part, to demographic differences: ADNI participants were younger at baseline and had a larger percentage of participants who were white. Additionally, ADNI participants represent later stages of disease progression compared to the BLSA sample with approximately 50% of participants at baseline being diagnosed as either MCI or AD.

To the best of our knowledge, these findings are among the first to demonstrate sex-specific associations between the rate-limiting step in primary BA synthesis and brain amyloid deposition as well as longitudinal changes in brain atrophy and accumulation of WML burden. A previous cross-sectional study by Nho and colleagues [9] in ADNI reported that lower plasma CA was associated with reduced hippocampal volume in a combined sample of AD, MCI, and CON participants and reported lower plasma CA levels in AD as well associations with increased risk of conversion from MCI to AD. These results, together with our current findings which included longitudinal markers of disease progression, suggest that the oxidative catabolism of cholesterol to BAs may impact both pathological changes in the brain preceding a diagnosis of dementia, as well as progression of clinical symptoms [33].

Given that our neuroimaging results revealed sex-specific associations between primary BA synthesis and measures of dementia-related pathology, we next tested whether the modulation of peripheral BA levels would alter the risk of incident dementia in a sex-specific manner. To test this hypothesis, we leveraged one of the world’s largest databases of primary care records, i.e., the UK CPRD, to examine whether exposure to BAS, a commonly used class of medicines to treat hyperlipidemia, would alter the risk of incident dementia. BAS are nonsystemic pharmacological agents that bind to BAs in the gastrointestinal tract, reducing their entry into the enterohepatic circulation. A lower pool of circulating BAs reduces feedback inhibition of the rate-limiting step in BA synthesis catalyzed by CYP7A1 [34], resulting in greater oxidative catabolism of cholesterol. We observed a significant positive association between the number of BAS prescriptions and risk of VaD in males and no association in females. We additionally observed a statistically significant sex difference in the association between BAS and VaD. These results, while suggestive, are consistent with our neuroimaging findings indicating that a lower circulating pool of BA is associated with neuroimaging markers of dementia progression mainly in males. Together, these results suggest that cholesterol catabolism through its enzymatic conversion to primary BAs is a biological mechanism associated with increased risk of VaD in males. These findings may provide novel insights into sex-specific interventions targeting this biochemical pathway in at-risk older individuals. Further exploration of the association between pharmacologic manipulation of BA levels and dementia outcomes in complementary population-based databases with distinct demographic and clinical characteristics is essential to validate our findings and assess their generalizability.

One plausible mechanism that may explain the association between dysregulated cholesterol catabolism and dementia pathogenesis is through altered BA signaling in the brain. Our findings are among the first to identify primary BAs (i.e., CA and CDCA) in the brain and report significant sex differences in neuronal gene expression of BA receptors in AD. A recent report by Mahmoudiandehkordi and colleagues reported a significant association between a higher ratio of the secondary BA, deoxycholic acid (DCA) to CA (DCA:CA) in both serum and brain tissue with severity of cognitive impairment in a combined sample of AD, MCI, and CON participants from the ROSMAP study [33]. Our scRNA-Seq results are also broadly consistent with a recent multi-cohort transcriptomic analysis in AD and CON brain tissue samples by Baloni and colleagues [22] that reported gene expression of several BA receptors. These included transcripts for RARA, RXRA, PPARA, and PPARG receptors that we find to be differentially expressed in AD brains in a sex-specific manner (Fig 4). Important differences between our current report and that by Baloni and colleagues include our use of scRNA-Seq compared to bulk tissue RNA-Seq as well as our sex-stratified analyses to probe differences in BA receptor transcript levels in AD. While the influx of BAs across the BBB from systemic circulation has been demonstrated [35], it is unclear whether de novo synthesis contributes substantially to the BA pool in the human brain [36]. Few previous studies have reported the existence of BAs in the human brain [37,38]. Pan and colleagues reported the presence of CDCA and CA in postmortem AD and CON brains but did not observe differences in their concentrations [37].

While BA receptors play a critical role in regulating hepatic BA synthesis by mediating feedback inhibition of CYP7A1, accumulating evidence also points to their importance in signaling pathways in the brain [39]. These include regulation of vascular risk factors including glucose, lipid, and energy homeostasis [40] as well as modulation of GABAergic and NMDA receptor–mediated neurotransmission [41]. Our results raise the possibility that dysregulation of cholesterol catabolism and BA synthesis in the periphery may impact early features of dementia pathogenesis through their effects on neuronal signaling pathways in the brain. This hypothesis merits evaluation in future experimental studies and may pave the way toward testing novel disease-modifying treatments in dementia targeting BA receptor–mediated signaling in the brain.

While we have not addressed the precise mechanisms underlying sex-specific associations between BA metabolism and dementia pathogenesis, prior evidence suggests important sex differences in lipid metabolism that impact risk of cardiovascular disease [42,43]. It is important to consider these findings together with animal studies that have also shown sex–specific differences in BA homeostasis during aging and suggest that these may be mediated by differences in expression of BA transporters as well as CYP7A1, the rate-limiting enzyme in BA synthesis [44]. It is likely that such differences are relevant in other biological pathways as well. Our own prior work has uncovered striking sex differences in the systemic inflammatory response in preclinical AD that is related to neurodegeneration [45] as well as differences in glucose metabolism that are associated with AD pathology [46]. These findings may have implications for testing targeted treatment interventions that take into consideration sex-specific differences in molecular mechanisms underlying AD pathogenesis. Understanding sex differences in biological pathways related to AD risk and progression may also have important implications in our understanding of descriptive epidemiological estimates of dementia that reveal sex-specific longitudinal differences in both prevalence and incidence of dementia in diverse cohorts [47]. It is also worth noting in this context that sex as a biological variable (SABV) has been largely ignored in neuroscience and dementia research [48,49].

Our study design represents an approach to identify biological mechanisms of risk associated with dementia as well as to discover potential targets for disease-modifying treatments. First, the use of targeted metabolomics and transcriptomics within longitudinal observational studies in combination with established neuroimaging markers of disease progression (e.g., amyloid accumulation, brain atrophy, and WMLs) enables the identification of specific biochemical pathways that may present plausible drug targets. Second, the use of large, real-world clinical datasets with dementia outcomes enables testing drugs that may impact such targets.

The strengths of our study include the use of a well-characterized population of older individuals with serial neuroimaging in the BLSA-NI and ADNI and testing the clinical implications of our findings in a large real-world clinical dataset.

Limitations of our study include the relatively small sample sizes in the BLSA-NI and autopsy samples. Additionally, we were unable to sex-stratify analyses of brain tissue BA concentration due to a limited number of individuals with BA metabolite concentration values above LOD. However, our inclusion of sc-RNASeq data comparing AD and CON samples from ROSMAP did allow us to sex-stratify gene expression analyses and correct for multiple comparisons. Additional limitations include a likely inaccuracy in clinical diagnoses of dementia subtypes in primary care settings. We have previously analyzed data from more than 20 million Medicare fee-for-service beneficiaries in the USA and reported that accurate subtyping of dementia in such datasets may be challenging [50]. While our matched cohort design with an active drug comparator group and adjustment for common comorbidities may have addressed some of the limitations associated with pharmacoepidemiologic analyses, our findings merit confirmation in other independent studies. It is also important to note that large longitudinal studies have consistently reported that mixed brain pathologies account for the majority of dementia cases with considerable overlap between AD neuropathology and vascular brain injury including macroscopic, lacunar, and microscopic infarcts [51,52]. Additionally, particularly in the oldest old, “single neuropathological entities” [53] may be less relevant compared to mixed pathologies including AD and vascular disease [5456].

In summary, we have combined targeted metabolomic assays of serum with in vivo amyloid PET and MRI of the brain to identify cholesterol catabolism through BA synthesis as a biological pathway involved in neuropathological changes prior to dementia onset. We then extended these findings by analyzing a large real-world clinical dataset to show that BA modulation alters the trajectory of VaD in males. Our transcriptomics results suggest that alterations in BA signaling through their neuronal receptors may mediate some of these associations. Our findings suggest that future experimental studies may provide insight into modulation of BA levels as a plausible therapeutic target in dementia.

Supporting information

S1 Fig. Catabolism of cholesterol into primary BAs.

The oxidative catabolism of cholesterol occurs through 3 enzymatically catalyzed biochemical pathways: the classic/neutral pathway in the liver accounts for the majority of BA synthesis in humans and begins with the oxidation of cholesterol to 7α-OHC by microsomal CYP7A1, the rate-limiting enzyme of the pathway. The alternative or acidic pathway is responsible for synthesis of a smaller proportion of the BA pool; cholesterol is oxidized to 27-OHC, catalyzed by mitochondrial CYP27A1 in both liver and extra-hepatic tissues. Both the classic/neutral and acidic pathways of BA synthesis ultimately generate the primary BAs, CA, and CDCA which are the principal catabolic products of cholesterol. A third, neuron-specific pathway of cholesterol breakdown in the brain is catalyzed by CYP46A1-mediated conversion of cholesterol to 24S-OHC which effluxes into the peripheral circulation for further conversion into the primary BAs in the liver [31,40,57]. 7α-OHC, 7α-hydroxycholesterol; 24S-OHC, 24S-hydroxycholesterol; 27-OHC, 27-hydroxycholesterol; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid.

(TIFF)

S1 Text. Supporting information text.

(DOCX)

S1 Table. STROBE checklist.

STROBE, Strengthening the Reporting of Observational studies in Epidemiology.

(DOCX)

S2 Table. ROSMAP scRNA-Seq BA receptor gene expression data availability.

Indicates data availability in the scRNA-Seq ROSMAP dataset. BA receptor genes that are indicated as “Not Available” either did not have sufficient counts or did not have any data available in the ROSMAP scRNA-Seq dataset. BA, bile acid; CHRM2, Cholinergic Receptor Muscarinic 2; CHRM3, Cholinergic Receptor Muscarinic 3; FGF19, Fibroblast Growth Factor 19; FPR1, Formyl Peptide Receptor 1; GPBAR1, G Protein-Coupled Bile Acid Receptor 1; HNF4A, Hepatocyte Nuclear Factor 4 Alpha; KDR, Kinase Insert Domain Receptor; NR0B2, Nuclear Receptor Subfamily 0 Group B Member 2; NR1H2, Nuclear Receptor Subfamily 1 Group H Member 2; NR1H3, Nuclear Receptor Subfamily 1 Group H Member 3; NR1H4, Nuclear Receptor Subfamily 1 Group H Member 4; NR1I2, Nuclear Receptor Subfamily 1 Group I Member 2; NR1I3, Nuclear Receptor Subfamily 1 Group I Member 3; NR3C1, Nuclear Receptor Subfamily 3 Group C Member 1; NR5A2, Nuclear Receptor Subfamily 5 Group A Member 2; PPARA, Peroxisome Proliferator Activated Receptor Alpha; PPARD, Peroxisome Proliferator Activated Receptor Delta; PPARG, Peroxisome Proliferator Activated Receptor Gamma; RARA, Retinoic Acid Receptor Alpha; ROSMAP, Religious Orders Study and Memory and Aging Project; RXRA, Retinoid X Receptor Alpha; RXRB, Retinoid X Receptor Beta; RXRG, Retinoid X Receptor Gamma; S1PR2, Sphingosine-1-Phosphate Receptor 2; scRNA-Seq, single-cell RNA sequencing; VDR, Vitamin D Receptor.

(DOCX)

S3 Table

(A) Demographic characteristics of BLSA-NI sample. APOE4, e4 allele of the Apolipoprotein E gene; BLSA, Baltimore Longitudinal Study of Aging; MRI, magnetic resonance imaging; NI, neuroimaging; PiB, Pittsburgh compound B; SD, standard deviation; WML, white matter lesion. (B) Demographic characteristics of ADNI sample. ADNI, Alzheimer’s Disease Neuroimaging Initiative; MRI, magnetic resonance imaging; NI, neuroimaging; SD, standard deviation; WML, white matter lesion

(DOCX)

S4 Table. Associations between serum metabolite concentrations and PiB/amyloid status.

coef, coefficient from linear regression model; PiB, Pittsburgh compound B; pval, p-value.

(DOCX)

S5 Table. Sensitivity analyses: Associations between serum metabolite concentrations and brain amyloid-β deposition, longitudinal changes in global brain WML burden, and rates of brain atrophy–BLSA.

Sensitivity analyses after including statin use as a covariate. BLSA, Baltimore Longitudinal Study of Aging; coef, coefficient from linear regression model or mixed effects model; FDR, false discovery rate (Benjamini–Hochberg) corrected p-value; pval, p-value; WML, white matter lesion.

(DOCX)

S6 Table. Characteristics of participants who received at least 2 BAS or LMT prescriptions with at least 1 year of follow-up after second prescription.

Wilcoxon rank-sum test. 1 Chi-squared test. 2 1 year prior to index date. 3 During study follow-up. BAS, bile acid sequestrants; LMT, lipid-modifying therapies.

(DOCX)

S7 Table. Characteristics of participants with incident dementia events during follow-up.

1 1 year prior to index date.

(DOCX)

S8 Table. Demographic characteristics of BLSA autopsy sample.

AD, Alzheimer disease; APOE4, apolipoprotein E allele epsilon 4; BLSA, Baltimore Longitudinal Study of Aging; CON, control; PMI, postmortem interval (hours).

(DOCX)

S9 Table. Differences in brain primary BA concentrations between AD and CON.

* In the CON sample in the CB, all concentrations were below LOD; we therefore tested for differences in the number of concentrations below LOD comparing AD to CON using the chi-squared test and present the associated p-value. AD, Alzheimer disease; BA, bile acid; coef, coefficient for disease (AD vs. CON) from the tobit model including mean-centered age and sex where the lower limit is set as the metabolite specific LOD; CON, control; LOD, limit of detection; pval, p-value.

(DOCX)

S10 Table. Differences in brain BA receptor gene expression (including receptors involved in BA homeostasis) in AD compared to CON.

AD, Alzheimer disease; BA, bile acid; CHRM2, Cholinergic Receptor Muscarinic 2; CHRM3, Cholinergic Receptor Muscarinic 3; CON, control; FDR, false discovery rate (Benjamini–Hochberg) corrected p-value; FPR1, Formyl Peptide Receptor 1; GPBAR1, G Protein-Coupled Bile Acid Receptor 1; HNF4A, Hepatocyte Nuclear Factor 4 Alpha; KDR, Kinase Insert Domain Receptor; NR1H2, Nuclear Receptor Subfamily 1 Group H Member 2; NR1H3, Nuclear Receptor Subfamily 1 Group H Member 3; NR1H4, Nuclear Receptor Subfamily 1 Group H Member 4; NR1I2, Nuclear Receptor Subfamily 1 Group I Member 2; NR1I3, Nuclear Receptor Subfamily 1 Group I Member 3; NR3C1, Nuclear Receptor Subfamily 3 Group C Member 1; NR5A2, Nuclear Receptor Subfamily 5 Group A Member 2; PPARA, Peroxisome Proliferator Activated Receptor Alpha; PPARD, Peroxisome Proliferator Activated Receptor Delta; PPARG, Peroxisome Proliferator Activated Receptor Gamma; pval: p-value; RARA, Retinoic Acid Receptor Alpha; RXRA, Retinoid X Receptor Alpha; RXRB, Retinoid X Receptor Beta; RXRG, Retinoid X Receptor Gamma; VDR, Vitamin D Receptor.

(XLSX)

Acknowledgments

We are grateful to BLSA and ROSMAP participants for their invaluable contributions. The authors thank Emily Carver, BS, and David Ruggieri, BS, both from Information Management Services (Calverton, Maryland, United States of America), for their important contributions to database management. We would like to additionally thank Dr. Li-Huei Tsai, Dr. Hansruedi Mathys, Dr. Manolis Kellis, and Dr. Jose Davila Valderrain for their support with scRNA-Seq data acquisition and analysis. We are thankful for support from the ADMC and the ADNI studies. Investigators within the ADMC and ADNI contributed to the design and implementation of ADMC/ADNI and/or provided data but did not participate in the 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_Authorship_List.pdf. A complete listing of ADMC investigators can be found at https://sites.duke.edu/adnimetab/team/.

Disclaimers: This study is based on data from the CPRD GOLD database August 2018 release, obtained under license from the UK Medicines and Healthcare Products Regulatory Agency. All rights reserved. The interpretation and conclusions contained in this study are those of the authors alone.

Abbreviations

7α-OHC

7α-hydroxycholesterol

AD

Alzheimer disease

ADMC

Alzheimer’s Disease Metabolomics Consortium

ADNI

Alzheimer’s Disease Neuroimaging Initiative

BA

bile acid

BAS

bile acid sequestrants

BBB

blood–brain barrier

BLSA

Baltimore Longitudinal Study of Aging

BLSA-NI

Baltimore Longitudinal Study of Aging Neuroimaging

CA

cholic acid

CB

cerebellum

CDCA

chenodeoxycholic acid

cDVR

cortical distribution volume ratio

CHRM2

Cholinergic Receptor Muscarinic 2

CHRM3

Cholinergic Receptor Muscarinic 3

CI

confidence interval

CON

control

CPRD

Clinical Practice Research Datalink

CSF

cerebrospinal fluid

DCA

deoxycholic acid

EHR

electronic health record

EMR

electronic medical record

FDR

false discovery rate

FPR1

Formyl Peptide Receptor 1

GPBAR1

G Protein-Coupled Bile Acid Receptor 1

HNF4A

Hepatocyte Nuclear Factor 4 Alpha

HR

hazard ratio

IQR

interquartile range

IRB

Institutional Review Board

ISAC

Independent Scientific Advisory Committee

ITG

inferior temporal gyrus

KDR

Kinase Insert Domain Receptor

LMT

lipid-modifying therapies

LOD

limit of detection

MCI

mild cognitive impairment

MFG

middle frontal gyrus

MRI

magnetic resonance imaging

NIA

National Institute on Aging

NOS

not otherwise specified

NR1H2

Nuclear Receptor Subfamily 1 Group H Member 2

NR1H3

Nuclear Receptor Subfamily 1 Group H Member 3

NR1H4

Nuclear Receptor Subfamily 1 Group H Member 4

NR1I2

Nuclear Receptor Subfamily 1 Group I Member 2

NR1I3

Nuclear Receptor Subfamily 1 Group I Member 3

NR3C1

Nuclear Receptor Subfamily 3 Group C Member 1

NR5A2

Nuclear Receptor Subfamily 5 Group A Member 2

PET

positron emission tomography

PiB

11C-Pittsburgh compound-B

PPARA

Peroxisome Proliferator Activated Receptor Alpha

PPARD

Peroxisome Proliferator Activated Receptor Delta

PPARG

Peroxisome Proliferator Activated Receptor Gamma

RARA

Retinoic Acid Receptor Alpha

ROI

region of interest

ROSMAP

Religious Orders Study and Memory and Aging Project

RXRA

Retinoid X Receptor Alpha

RXRB

Retinoid X Receptor Beta

RXRG

Retinoid X Receptor Gamma

SABV

sex as a biological variable

scRNA-Seq

single-cell RNA sequencing

STROBE

Strengthening the Reporting of Observational studies in Epidemiology

ULOQ

upper limit of quantification

VaD

vascular dementia

VDR

Vitamin D Receptor

WML

white matter lesion

Data Availability

Baltimore Longitudinal Study of Aging (BLSA) data are available to researchers and can be requested at https://www.blsa.nih.gov/researchers. Rush Memory and Aging Project (ROSMAP) data can be requested at https://www.radc.rush.edu; ROSMAP data used in this study are available at: https://www.synapse.org/#!Synapse:syn18485175 under the doi 10.7303/syn18485175 Clinical Practice Research Datalink (CPRD) data are available to researchers and can be requested at https://www.cprd.com/public. Alzheimer’s Disease Neuroimaging Network (ADNI) data are available to researchers at http://adni.loni.usc.edu.

Funding Statement

This research was supported in part by the intramural program of the National Institute on Aging (NIA) and the National Cancer Institute (NCI). ROSMAP is supported by NIA grants P30AG10161, R01AG15819, R01AG17917, and U01AG61356. The ADMC is supported by National Institute on Aging (NIA): grant R01AG046171, a component of the Accelerated Medicines Partnership for AD (AMP-AD) Target Discovery and Preclinical Validation Project; grant RF1 AG0151550, a component of the M2OVE-AD Consortium (Molecular Mechanisms of the Vascular Etiology of AD–Consortium; and RF1AG057452, R01AG059093, RF1AG058942, U01AG061359, U19AG063744 and FNIH: #DAOU16AMPA. Specific authors, indicated in parentheses, were supported by additional grants: NIA RF1 AG058942 and R01 AG057452 (RKD); NLM R01 LM012535 and NIA R03 AG054936 (KN). MT is grateful for funding support from the Andrew and Lillian A. Posey foundation to the Clinical and Translational Neuroscience Section, Laboratory of Behavioral Neuroscience, NIA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Appleton JP, Scutt P, Sprigg N, Bath PM. Hypercholesterolaemia and vascular dementia. Clin Sci (Lond). 2017;131(14):1561–78. Epub 2017/07/02. 10.1042/CS20160382 . [DOI] [PubMed] [Google Scholar]
  • 2.Polidori MC, Pientka L, Mecocci P. A review of the major vascular risk factors related to Alzheimer’s disease. J Alzheimers Dis. 2012;32(3):521–30. Epub 2012/07/28. 10.3233/JAD-2012-120871 . [DOI] [PubMed] [Google Scholar]
  • 3.Schilling S, Tzourio C, Soumare A, Kaffashian S, Dartigues JF, Ancelin ML, et al. Differential associations of plasma lipids with incident dementia and dementia subtypes in the 3C Study: A longitudinal, population-based prospective cohort study. PLoS Med. 2017;14(3):e1002265. Epub 2017/03/30. 10.1371/journal.pmed.1002265 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ancelin ML, Ripoche E, Dupuy AM, Barberger-Gateau P, Auriacombe S, Rouaud O, et al. Sex differences in the associations between lipid levels and incident dementia. J Alzheimers Dis. 2013;34(2):519–28. Epub 2012/12/21. 10.3233/JAD-121228 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Mueller SG, Weiner MW, Thal LJ, Petersen RC, Jack C, Jagust W, et al. The Alzheimer’s disease neuroimaging initiative. Neuroimaging Clin N Am. 2005;15(4):869–77, xi–xii. 10.1016/j.nic.2005.09.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ferrucci L. The Baltimore Longitudinal Study of Aging (BLSA): a 50-year-long journey and plans for the future. J Gerontol A Biol Sci Med Sci. 2008;63(12):1416–9. 10.1093/gerona/63.12.1416 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Armstrong NM, An Y, Beason-Held L, Doshi J, Erus G, Ferrucci L, et al. Sex differences in brain aging and predictors of neurodegeneration in cognitively healthy older adults. Neurobiol Aging. 2019;81:146–56. Epub 2019/07/08. 10.1016/j.neurobiolaging.2019.05.020 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Varma VR, Oommen AM, Varma S, Casanova R, An Y, Andrews RM, et al. Brain and blood metabolite signatures of pathology and progression in Alzheimer disease: A targeted metabolomics study. PLoS Med. 2018;15(1):e1002482. Epub 2018/01/26. 10.1371/journal.pmed.1002482 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Nho K, Kueider-Paisley A, MahmoudianDehkordi S, Arnold M, Risacher SL, Louie G, et al. Altered bile acid profile in mild cognitive impairment and Alzheimer’s disease: Relationship to neuroimaging and CSF biomarkers. Alzheimers Dement. 2019;15(2):232–44. Epub 2018/10/20. 10.1016/j.jalz.2018.08.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.St John-Williams L, Mahmoudiandehkordi S, Arnold M, Massaro T, Blach C, Kastenmuller G, et al. Bile acids targeted metabolomics and medication classification data in the ADNI1 and ADNIGO/2 cohorts. Sci Data. 2019;6(1):212. Epub 2019/10/19. 10.1038/s41597-019-0181-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bilgel M, An Y, Zhou Y, Wong DF, Prince JL, Ferrucci L, et al. Individual estimates of age at detectable amyloid onset for risk factor assessment. Alzheimers Dement. 2016;12(4):373–9. Epub 2015/11/22. 10.1016/j.jalz.2015.08.166 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Mintun MA, Larossa GN, Sheline YI, Dence CS, Lee SY, Mach RH, et al. [11C]PIB in a nondemented population: potential antecedent marker of Alzheimer disease. Neurology. 2006;67(3):446–52. Epub 2006/08/09. 10.1212/01.wnl.0000228230.26044.a4 . [DOI] [PubMed] [Google Scholar]
  • 13.Ast M, Frishman WH. Bile acid sequestrants. J Clin Pharmacol. 1990;30(2):99–106. Epub 1990/02/01. 10.1002/j.1552-4604.1990.tb03447.x . [DOI] [PubMed] [Google Scholar]
  • 14.Kobayashi M, Ikegami H, Fujisawa T, Nojima K, Kawabata Y, Noso S, et al. Prevention and treatment of obesity, insulin resistance, and diabetes by bile acid-binding resin. Diabetes. 2007;56(1):239–47. Epub 2006/12/29. 10.2337/db06-0353 . [DOI] [PubMed] [Google Scholar]
  • 15.Guyton JR, Goldberg AC. Bile Acid Sequestrants. In: Ballantyne CM, editor. Clinical Lipidology: a Comanion to Braunwald’s Heart Disease. Philadelphia PA: Saunders, an imprint of Elsevier Inc.; 2009. p. 281–7. [Google Scholar]
  • 16.Herrett E, Gallagher AM, Bhaskaran K, Forbes H, Mathur R, van Staa T, et al. Data Resource Profile: Clinical Practice Research Datalink (CPRD). Int J Epidemiol. 2015;44(3):827–36. Epub 2015/06/08. 10.1093/ije/dyv098 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.O’Brien RJ, Resnick SM, Zonderman AB, Ferucci L, Crain BJ, Pletnikova O, et al. Neuropathologic studies of the Baltimore Longitudinal Study on Aging (BLSA). J Alzheimers Dis. 2009;18(3):665–75-S89. 10.3233/JAD-2009-1179 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Buckley RF, Hanseeuw B, Schultz AP, Vannini P, Aghjayan SL, Properzi MJ, et al. Region-Specific Association of Subjective Cognitive Decline With Tauopathy Independent of Global beta-Amyloid Burden. JAMA Neurol. 2017;74(12):1455–63. Epub 2017/10/04. 10.1001/jamaneurol.2017.2216 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Li Y, Rinne JO, Mosconi L, Pirraglia E, Rusinek H, DeSanti S, et al. Regional analysis of FDG and PIB-PET images in normal aging, mild cognitive impairment, and Alzheimer’s disease. Eur J Nucl Med Mol Imaging. 2008;35(12):2169–81. Epub 2008/06/21. 10.1007/s00259-008-0833-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Mathys H, Davila-Velderrain J, Peng Z, Gao F, Mohammadi S, Young JZ, et al. Single-cell transcriptomic analysis of Alzheimer’s disease. Nature. 2019;570(7761):332–7. Epub 2019/05/03. 10.1038/s41586-019-1195-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Bennett DA, Buchman AS, Boyle PA, Barnes LL, Wilson RS, Schneider JA. Religious Orders Study and Rush Memory and Aging Project. J Alzheimers Dis. 2018;64(s1):S161–S89. Epub 2018/06/06. 10.3233/JAD-179939 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Baloni P, Funk CC, Yan J, Yurkovich JT, Kueider-Paisley A, Nho K, et al. Metabolic Network Analysis Reveals Altered Bile Acid Synthesis and Metabolism in Alzheimer’s Disease. Cell Rep Med. 2020;1(8):100138. Epub 2020/12/10. 10.1016/j.xcrm.2020.100138 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Fiorucci S, Biagioli M, Zampella A, Distrutti E. Bile Acids Activated Receptors Regulate Innate Immunity. Front Immunol. 2018;9:1853. Epub 2018/08/29. 10.3389/fimmu.2018.01853 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Schaap FG, Trauner M, Jansen PL. Bile acid receptors as targets for drug development. Nat Rev Gastroenterol Hepatol. 2014;11(1):55–67. Epub 2013/08/29. 10.1038/nrgastro.2013.151 . [DOI] [PubMed] [Google Scholar]
  • 25.Mahajan UV, Varma VR, Huang CW, An Y, Tanaka T, Ferrucci L, et al. Blood metabolite signatures of metabolic syndrome in two cross-cultural older adult cohorts. Int J Mol Sci. 2020; 21(4):1324. Epub 2020/02/16. 10.3390/ijms21041324 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Li T, Chiang JY. Regulation of bile acid and cholesterol metabolism by PPARs. PPAR Res. 2009;2009:501739. Epub 2009/07/29. 10.1155/2009/501739 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Javitt NB. Bile acid synthesis from cholesterol: regulatory and auxiliary pathways. FASEB J. 1994;8(15):1308–11. Epub 1994/12/01. 10.1096/fasebj.8.15.8001744 . [DOI] [PubMed] [Google Scholar]
  • 28.Loera-Valencia R, Goikolea J, Parrado-Fernandez C, Merino-Serrais P, Maioli S. Alterations in cholesterol metabolism as a risk factor for developing Alzheimer’s disease: Potential novel targets for treatment. J Steroid Biochem Mol Biol. 2019;190:104–14. Epub 2019/03/18. 10.1016/j.jsbmb.2019.03.003 . [DOI] [PubMed] [Google Scholar]
  • 29.Norlin M, Wikvall K. Enzymes in the conversion of cholesterol into bile acids. Curr Mol Med. 2007;7(2):199–218. Epub 2007/03/10. 10.2174/156652407780059168 . [DOI] [PubMed] [Google Scholar]
  • 30.Russell DW. The enzymes, regulation, and genetics of bile acid synthesis. Annu Rev Biochem. 2003;72:137–74. Epub 2003/01/25. 10.1146/annurev.biochem.72.121801.161712 . [DOI] [PubMed] [Google Scholar]
  • 31.Russell DW, Setchell KD. Bile acid biosynthesis. Biochemistry. 1992;31(20):4737–49. Epub 1992/05/26. 10.1021/bi00135a001 . [DOI] [PubMed] [Google Scholar]
  • 32.Alber J, Alladi S, Bae HJ, Barton DA, Beckett LA, Bell JM, et al. White matter hyperintensities in vascular contributions to cognitive impairment and dementia (VCID): Knowledge gaps and opportunities. Alzheimers Dement (N Y). 2019;5:107–17. Epub 2019/04/24. 10.1016/j.trci.2019.02.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.MahmoudianDehkordi S, Arnold M, Nho K, Ahmad S, Jia W, Xie G, et al. Altered bile acid profile associates with cognitive impairment in Alzheimer’s disease-An emerging role for gut microbiome. Alzheimers Dement. 2019;15(1):76–92. Epub 2018/10/20. 10.1016/j.jalz.2018.07.217 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Staels B. A review of bile acid sequestrants: potential mechanism(s) for glucose-lowering effects in type 2 diabetes mellitus. Postgrad Med. 2009;121(3 Suppl 1):25–30. Epub 2009/06/13. 10.3810/pgm.2009.05.suppl53.290 . [DOI] [PubMed] [Google Scholar]
  • 35.Kamp F, Hamilton JA, Kamp F, Westerhoff HV, Hamilton JA. Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers. Biochemistry. 1993;32(41):11074–86. Epub 1993/10/19. 10.1021/bi00092a017 . [DOI] [PubMed] [Google Scholar]
  • 36.Ogundare M, Theofilopoulos S, Lockhart A, Hall LJ, Arenas E, Sjovall J, et al. Cerebrospinal fluid steroidomics: are bioactive bile acids present in brain? J Biol Chem. 2010;285(7):4666–79. Epub 2009/12/10. 10.1074/jbc.M109.086678 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Pan X, Nasaruddin MB, Elliott CT, McGuinness B, Passmore AP, Kehoe PG, et al. Alzheimer’s disease-like pathology has transient effects on the brain and blood metabolome. Neurobiol Aging. 2016;38:151–63. Epub 2016/02/02. 10.1016/j.neurobiolaging.2015.11.014 . [DOI] [PubMed] [Google Scholar]
  • 38.Ferdinandusse S, Denis S, Faust PL, Wanders RJ. Bile acids: the role of peroxisomes. J Lipid Res. 2009;50(11):2139–47. Epub 2009/04/10. 10.1194/jlr.R900009-JLR200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Mertens KL, Kalsbeek A, Soeters MR, Eggink HM. Bile Acid Signaling Pathways from the Enterohepatic Circulation to the Central Nervous System. Front Neurosci. 2017;11:617. Epub 2017/11/23. 10.3389/fnins.2017.00617 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Kiriyama Y, Nochi H. The Biosynthesis, Signaling, and Neurological Functions of Bile Acids. Biomolecules. 2019;9(6). Epub 2019/06/19. 10.3390/biom9060232 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Schubring SR, Fleischer W, Lin JS, Haas HL, Sergeeva OA. The bile steroid chenodeoxycholate is a potent antagonist at NMDA and GABA(A) receptors. Neurosci Lett. 2012;506(2):322–6. Epub 2011/12/14. 10.1016/j.neulet.2011.11.036 . [DOI] [PubMed] [Google Scholar]
  • 42.Mittendorfer B. Sexual dimorphism in human lipid metabolism. J Nutr. 2005;135(4):681–6. Epub 2005/03/30. 10.1093/jn/135.4.681 . [DOI] [PubMed] [Google Scholar]
  • 43.Palmisano BT, Zhu L, Eckel RH, Stafford JM. Sex differences in lipid and lipoprotein metabolism. Mol Metab. 2018;15:45–55. Epub 2018/06/03. 10.1016/j.molmet.2018.05.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Fu ZD, Csanaky IL, Klaassen CD. Gender-divergent profile of bile acid homeostasis during aging of mice. PLoS ONE. 2012;7(3):e32551. Epub 2012/03/10. 10.1371/journal.pone.0032551 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Varma VR, Varma S, An Y, Hohman TJ, Seddighi S, Casanova R, et al. Alpha-2 macroglobulin in Alzheimer’s disease: a marker of neuronal injury through the RCAN1 pathway. Mol Psychiatry. 2017;22(1):13–23. 10.1038/mp.2016.206 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Demarest TG, Varma VR, Estrada D, Babbar M, Basu S, Mahajan UV, et al. Biological sex and DNA repair deficiency drive Alzheimer’s disease via systemic metabolic remodeling and brain mitochondrial dysfunction. Acta Neuropathol. 2020;140(1):25–47. Epub 2020/04/26. 10.1007/s00401-020-02152-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Wu YT, Beiser AS, Breteler MMB, Fratiglioni L, Helmer C, Hendrie HC, et al. The changing prevalence and incidence of dementia over time—current evidence. Nat Rev Neurol 2017;13(6):327–39. Epub 2017/05/13. 10.1038/nrneurol.2017.63 . [DOI] [PubMed] [Google Scholar]
  • 48.Waters A, Society for Women’s Health Research Alzheimer’s Disease N, Laitner MH. Biological sex differences in Alzheimer’s preclinical research: A call to action. Alzheimers Dement (N Y). 2021;7(1):e12111. Epub 2021/02/23. 10.1002/trc2.12111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Beery AK, Zucker I. Sex bias in neuroscience and biomedical research. Neurosci Biobehav Rev. 2011;35(3):565–72. Epub 2010/07/14. 10.1016/j.neubiorev.2010.07.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Goodman RA, Lochner KA, Thambisetty M, Wingo TS, Posner SF, Ling SM. Prevalence of dementia subtypes in United States Medicare fee-for-service beneficiaries, 2011–2013. Alzheimers Dement. 2017;13(1):28–37. Epub 2016/05/14. 10.1016/j.jalz.2016.04.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Matthews FE, Brayne C, Lowe J, McKeith I, Wharton SB, Ince P. Epidemiological pathology of dementia: attributable-risks at death in the Medical Research Council Cognitive Function and Ageing Study. PLoS Med. 2009;6(11):e1000180. Epub 2009/11/11. 10.1371/journal.pmed.1000180 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Kapasi A, DeCarli C, Schneider JA. Impact of multiple pathologies on the threshold for clinically overt dementia. Acta Neuropathol. 2017;134(2):171–86. Epub 2017/05/11. 10.1007/s00401-017-1717-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Gardner RC, Valcour V, Yaffe K. Dementia in the oldest old: a multi-factorial and growing public health issue. Alzheimers Res Ther. 2013;5(4):27. Epub 2013/07/03. 10.1186/alzrt181 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Schneider JA, Arvanitakis Z, Bang W, Bennett DA. Mixed brain pathologies account for most dementia cases in community-dwelling older persons. Neurology. 2007;69(24):2197–204. Epub 2007/06/15. 10.1212/01.wnl.0000271090.28148.24 . [DOI] [PubMed] [Google Scholar]
  • 55.Pierce AL, Kawas CH. Dementia in the oldest old: Beyond Alzheimer disease. PLoS Med. 2017;14(3):e1002263. Epub 2017/03/23. 10.1371/journal.pmed.1002263 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Middleton LE, Grinberg LT, Miller B, Kawas C, Yaffe K. Neuropathologic features associated with Alzheimer disease diagnosis: age matters. Neurology. 2011;77(19):1737–44. Epub 2011/10/28. 10.1212/WNL.0b013e318236f0cf [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Chiang JY. Bile acids: regulation of synthesis. J Lipid Res. 2009;50(10):1955–66. Epub 2009/04/07. 10.1194/jlr.R900010-JLR200 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Louise Gaynor-Brook

13 Feb 2020

Dear Dr Varma,

Thank you for submitting your manuscript entitled "Bile acid synthesis and modulation are associated with brain amyloid deposition, white matter lesions, neurodegeneration and risk of vascular dementia" for consideration by PLOS Medicine.

Your manuscript has now been evaluated by the PLOS Medicine editorial staff and I am writing to let you know that we would like to send your submission out for external peer review.

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Decision Letter 1

Emma Veitch

20 Jun 2020

Dear Dr. Varma,

Thank you very much for submitting your manuscript "Bile acid synthesis and modulation are associated with brain amyloid deposition, white matter lesions, neurodegeneration and risk of vascular dementia" (PMEDICINE-D-20-00371R1) for consideration at PLOS Medicine.

Your paper was evaluated by a senior editor and discussed among all the editors here. It was also discussed with an academic editor with relevant expertise, and sent to independent reviewers, including a statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

In light of these reviews, I am afraid that we will not be able to accept the manuscript for publication in the journal in its current form, but we would like to consider a revised version that addresses the reviewers' and editors' comments. Obviously we cannot make any decision about publication until we have seen the revised manuscript and your response, and we plan to seek re-review by one or more of the reviewers.

In revising the manuscript for further consideration, your revisions should address the specific points made by each reviewer and the editors. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

In addition, we request that you upload any figures associated with your paper as individual TIF or EPS files with 300dpi resolution at resubmission; please read our figure guidelines for more information on our requirements: http://journals.plos.org/plosmedicine/s/figures. While revising your submission, please upload your figure files to the PACE digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at PLOSMedicine@plos.org.

We expect to receive your revised manuscript by Jul 13 2020 11:59PM. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

We ask every co-author listed on the manuscript to fill in a contributing author statement, making sure to declare all competing interests. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. If new competing interests are declared later in the revision process, this may also hold up the submission. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT. You can see our competing interests policy here: http://journals.plos.org/plosmedicine/s/competing-interests.

Please use the following link to submit the revised manuscript:

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Your article can be found in the "Submissions Needing Revision" folder.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosmedicine/s/submission-guidelines#loc-methods.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

We look forward to receiving your revised manuscript.

Sincerely,

Emma Veitch, PhD

PLOS Medicine

On behalf of Clare Stone, PhD, Acting Chief Editor,

PLOS Medicine

plosmedicine.org

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Requests from the editors:

*We'd suggest revising the title according to PLOS Medicine's style; this should have the initial phrase outlining the study question and then the study design(s) in the second phrase after a colon (eg, (: "randomized controlled trial," "A retrospective study," "A modelling study," etc.)

*In the last sentence of the Abstract Methods and Findings section, please describe the main limitation(s) of the study's methodology.

*At this stage, we ask that you include a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract. Please see our author guidelines for more information: https://journals.plos.org/plosmedicine/s/revising-your-manuscript#loc-author-summary

*Please ensure the Methods section states whether the analysis plan followed in this paper for the 3 different studies was set out prospectively (ie prior to collection of data). Please state this (either way) early in the Methods section.

a) If a prospective analysis plan (from your funding proposal, IRB or other ethics committee submission, study protocol, or other planning document written before analyzing the data) was used in designing the study, please include the relevant prospectively written document with your revised manuscript as a Supporting Information file to be published alongside your study, and cite it in the Methods section. A legend for this file should be included at the end of your manuscript.

b) If no such document exists, please make sure that the Methods section transparently describes when analyses were planned, and when/why any data-driven changes to analyses took place.

c) In either case, changes in the analysis-- including those made in response to peer review comments-- should be identified as such in the Methods section of the paper, with rationale.

*Referencing callouts should ideally be sequential numerals in square brackets (ie [1], [2] etc). If referencing software was used then this should be fairly quick and easy.

*Please note the comments from one reviewer that the conclusions drawn in the paper should be more cautious, given the possible risks of multiple testing and the potential for false-positive findings (ie that some analyses may not survive correction for multiple testing).

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Comments from the reviewers:

Reviewer #1: "Bile acid synthesis and modulation are associated with brain amyloid deposition, white matter lesions, neurodegeneration and risk of vascular dementia" employs a three-step approach to investigate possible relationships between primary bile acids (BA) and various biomarkers of Alzheimer's Disease (AD) and vascular dementia (VaD). Step 1 was concerned with serum and neuroimaging markers, Step 2 with exposure to bile acid sequestrants (BAS), and Step 3 with assessment of autopsied brain tissue.

The major experimental findings were that there possibly exists a sex-specific association between lower serum BA, and faster accumulation of brain white matter lesions (WML)/faster rate of brain atrophy in males (opposite effect in females; Step 1), supported by large-scale primary care data analysis (again for males; Step 2). A possible mechanism was then proposed with evidence that the principal BA receptors were significantly more often expressed in postmortem AD brains, compared to control brains (Step 3).

The authors have largely acknowledged the relative strengths and limitations of this study in the discussion, in particular the relatively small sample sizes involved in Step 1 and Step 3 (although the number of dementia cases [N=72] amongst BAS users is also relatively small, for Step 2). Overall, the manuscript represents a comprehensive and ambitious effort to quantify and describe potential mechanisms behind bile acid synthesis/modulation and AD/VaD signs. However, there remain some issues that might be addressed:

1. It is summarized in the abstract that "We found that lower serum concentrations of 7α-OHC, CA and CDCA were associated with higher brain amyloid deposition, faster WML accumulation and faster brain atrophy in males. Opposite effects were observed in females". However, the actual data presented in Table 1 suggests that this description might be a simplification that possibly mischaracterizes the specifics. For example, it appears that no statistically-significant correlations between 7α-OHC/CA/CDCA and amyloid deposition was found for females, as was the case for CA/CDCA and amyloid deposition for males. Moreover, the Results section then states that "We observed no significant associations in the female only-sample" (line 298). The specific findings might thus be presented more precisely in the abstract.

2. Perhaps of greater concern is that when analyzing all subjects (male+female) in Step 1, it appears that only 7α-OHC shows statistically-significant correlations with amyloid deposition/brain atrophy, with the actual BAs (CA/CDCA) exhibiting no correlations at all. The BA correlations appear only when considering a male/female sex-specific stratification of the cohort.

As such, it might be clarified whether this sex-specific analysis was a priori defined in the initial study design from some theoretical motivation/prior work, or if it was a post-hoc discovery. This is of interest due to the relatively small sample size (N=134/141) involved in Step 1, and also previous similar cited work on AD [citation 8] not automatically considering sex-based stratification.

3. For Step 2, the authors might consider discussing the appropriateness of using matched LMT users as the control group for BAS users, in greater detail. In particular, what might be some considerations leading to a patient being prescribed LMT instead of BAS (or vice versa)? This is particularly since the matching was performed based on relatively few demographics (sex, year of birth, region, year of clinic registration/first prescription)

4. For Step 2, a correlation was found between BAS use and VaD for males, but not between BAS use and any dementia/AD/other dementia, even for males. From line 120, the hypothesis inspired from Step 1 was whether BAS would alter risk of (various types of) dementia. As such, it might be discussed further as to whether there are any characteristics particular to VaD that might have given rise to this correlation, all the more due to the acknowledged limitation of likely inaccuracy in clinical diagnoses of dementia subtypes in primary care settings (line 481).

5. For Step 3, given the major role played by sex-based stratification in the previous analyses, it might be appropriate for the relevant analyses and results (Figures 5 & 6, Supplementary Tables 7 & 8) for this step to be stratified by age too.

6. It is stated that "these findings are among the first to demonstrate sex-specific associations between the rate-limiting step in primary BA synthesis and brain amyloid deposition as well as longitudinal changes in brain WML burden" (line 411). Given the significance of the sex-specific analyses throughout, it might be appropriate to discuss any other work suggesting possible sex-specific associations/mechanisms. The existing discussion does not appear to propose any plausible explanation for the observed sex-specific associations through the underlying mechanism.

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Reviewer #2: In this manuscript, Varma VR. Et al aim to investigate the role of cholesterol catabolism in the pathogenesis of dementia.

The study is divided in 3 steps:

-Step 1: the authors measured levels of primary bile acids (cholic acid CA, Chenodeoxycholic acid CDCA) and one of the main BAs precursor, 7alpha-hydroxycholesterol (7a-OHC) in the Baltimore Longitudinal Study of Aging (BLSA) and analyzed their association with amyloid deposition and neuroimaging data. Main findings: lower concentration of 7a-OHC, CA and CDCA are associated with increase of amyloid deposition, faster WML accumulation and brain atrophy in males. The opposite was observed in females.

-Step 2: the authors tested whether patients using bile acid sequestrants (BAS) had altered risk of dementia, by using the Clinical Practice Research Datalink (CPRD) dataset. Main findings: exposure to BAS increases risk of vascular dementia in males and not in females.

-Step 3: the authors investigated levels of BAs and 7OHC in autopsy brains from BLSA cohort and used gene expression omnibus (GEO) data to test whether mRNA levels of BAs receptors were altered in Alzheimer's Disease and control patients. Main findings: receptors for BAs, as FXR and TGR5, as well as concentration of CA and CDCA, are higher in AD brains.

The current study is relevant, and the research question is important in the field of metabolism of cholesterol as a risk factor for different types of dementia. The results have the potential to provide an advance over existing knowledge and also important implications for developing new therapeutic targets. The rationale behind is strong but data as presented may not be enough strong to support the hypothesis of the authors. There are critical points that should be addressed before the manuscript can be accepted for publication.

STEP 1. There are previous published results on this same topic supporting the hypothesis that circulating BAs may contribute to AD pathogenesis (PMID:19288586), where it is shown that low levels of CA are associated to greater atrophy and to reduced glucose metabolism (PMID:30337152) and that BAs serum levels are decrease in AD patients from ADNI cohort (PMID: 30337151). When compared to these previous studies, the authors of this manuscript added a number of novelties, such as using another study cohort, white matter lesions measurements, Pib +/- subjects, sex stratification.

1) Were the samples collected from fasting patients? The information seems to be missing in the text.

2) As acknowledged by the authors the smaller N in this cohort may be a limitation and sex-specific analyses can further reduce power. It would be therefore very relevant to have data from other cohorts, ie ADNI, where some BAs were previously measured but sex stratification analyses were not provided (PMID: 30337152).

3) Were the analyses adjusted for use of medications for ie. cholesterol? This could also be very relevant and affect the analyses.

4) What are the levels of BAs in AD serum samples? Were the results from previous studies confirmed?

STEP 2

1) Users of statins were excluded, I wonder if it could be relevant to investigate them as well in this study?

STEP 3

1) LOD of BAs in measurement from autopsy brain may be a limitation. Would it be possible to measure amyloid beta levels as well? It would add important information.

2) Use of data from GEO datasets to prove hypothesis. The authors are strongly suggested to perform qPCR on autopsy brain from BLSA cohort to investigate and confirm the changes in gene expression observed in datasets from GEO. They should use for this the same regions used for BAs measurements (brain areas used for gene expression of FXR and TGR5 are not the same, nor the same cohort).

3) Gene expression levels of receptors may not be enough to support hypothesis on mechanisms and at least protein levels should be also measured.

4) The results from STEP 3 do not support sex differences observed in Step 1 and 2, which are not explored. The authors should run analyses in males and females and if possible, increase the N for this study.

In general:

1) The differences in male and females in STEP 1 and 2 are the main novel result, very interesting and should definitely be discussed more and investigated deeper. What could be the mechanisms behind? Is there literature that could support these sex-specific differences? What reported by the authors seems not exhaustive.

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Reviewer #3: Review comments on manuscript PMEDICINE-D-20-00371_R1

The manuscript by Varma et al explores the relationships between bile acid (BA) levels/synthesis and dementia related pathology, such as white matter lesions (WML) and amyloid deposition, as well as vascular dementia risk and sex related differences. To address these questions, the authors used three different experimental and/or analysis approaches: (1) a cross-sectional study to evaluate associations of serum bile acids with brain amyloid accumulation, WML and sex differences (BLSA cohort, n=141 - 134 subjects), (2) a subset from large clinical dataset (CPRD, UK) was used to test dementia risk in subjects using bile acid sequestrants (BAS) with n=3,208 BAS users and n=23,483 non-statin lipid modifying therapies (LMT) users, (3) cross-sectional study comparing concentrations of bile acids and mRNA expression levels of their receptors (FXR and TGR5) in control vs AD post-mortem brain samples (BLSA autopsy program, n=13 control and n=16 AD).

The question of whether there is a relationship between peripheral levels of bile acids and dementia incidence or risk is an interesting one, particularly as bile acids are products of cholesterol metabolism, which has been widely studied regarding its role in neurodegenerative diseases of ageing. It is an emerging area of interest with relatively few studies exploring the role of bile acids in dementia, so the study asks a relatively novel question.

My concerns with the current work are as follows:

(1) On the back of three experiments of varying design and mostly all low powered studies, the authors are making some fairly strong claims about dementia associations and mechanisms. However the data to support these claims are low powered (as they themselves recognize and point out within their limitations section), and the p values frequently borderline, and unlikely to survive correction for multiple testing (e.g., Table 1 showing associations of BA with brain volumetric measures; cDVR, precuneus DVR, WML, Table 2 testing association of BAS use and dementia risk; out of 24 tests, only one has a p value of < 0.05, which would not survive correction for multiple testing, Fig 5 testing brain BA concentration in control vs AD subjects; out of 6 tests only one has p<0.05). These data are at best suggestive, and the discussion should reflect this, rather than presenting strong and definitive claims about dementia associations and mechanisms. Having a limitations section is a good idea, but does not obviate the need for a measured discussion, which makes claims in proportion to the strength of the data.

(2) In several parts of the manuscript the authors claim to have used a "novel study design". However the three study designs they outline are commonly used study types. The authors can claim perhaps to have used 3 studies which each differ in design and/or methodology, but should avoid claiming/overstating the novelty of the design or methodology. Furthermore, I am uncertain as to whether combining three independent studies adds statistical power, or risks conflating potentially unrelated results? Using replication cohorts would have seemed a stronger approach, rather than using multiple small studies of orthogonal design. However a statistician would be better placed to address this question.

(3) The authors interpret the lower BA values they observe in males as a possible "mediator of early pathological changes in AD" (pg 18). However they do not canvas other possible explanations, such as secondary effects, resultant from neuronal cell death, brain atrophy or neuropathologies present. In the case of the post mortem tissue, ease of metabolite extraction from control vs AD tissue might be another contributing factor (were internal standards used for the assay?). Furthermore the negative associations of metabolites and brain atrophy in females are not discussed in any detail, nor why the marked difference between males and females, nor reasons for the heterogeneous associations of brain pathology and specific metabolite levels. The rational for stratification by sex is also not entirely clear, since, of the three metabolites assayed, only 7�-hydroxycholesterol had a significant p-value of 0.034 (without correction for multiple testing) for the "total" group (Table 1). Similarly in Table 2/supplementary Table 4 there are no significant differences in the "overall" analysis, so the rationale for stratification should be more clearly outlined.

My specific comments are as follows:

- Subject numbers should be shown within all tables and figures, both in the main text and supplementary section, and include the totals and numbers following sex stratification.

- Supplementary Table 2 shows that there are no significant associations between any of the three serum metabolite concentrations and PiB PET status, whereas Table 1 in the main text (pg 12) shows some significant associations between cDVR (amyloid-� deposition) and 7�-hydroxycholesterol in males. These apparently conflicting outcomes should be discussed, and some explanation offered.

- It is a little odd that the authors discuss their findings in the context of "early neuroimaging markers", "early neurodegeneration", "early pathology changes", etc, since the majority of their subjects are >65 years of age, and well into the range of late onset dementia.

- The authors do not make a clear statement as to whether the changes observed here are likely specific to a particular dementia subtype (they mention BA associations in both AD and vascular dementia), a particular neuropathology (amyloid deposition, WMH) or may just be an age related phenomenon, increasing in parallel with increase of neuropathological features with age.

- Table 2 and supplementary Table 4 appear to have the same data, but supplementary Table 4 has more detail. I suggest replacing Table 2 with supplementary Table 4 in the main manuscript.

- The Figure 3 flow chart shows that n=3,208 BAS users and 23,483 LMT users were available from the CPRD dataset. However were all these subjects used in the analyses presented here? It is a bit difficult to ascertain since subject numbers were not shown in most figures and tables (see previous comment - they should all be shown) - however the "N cases" in supplementary figure 4 have much smaller numbers. If fewer numbers were used for analysis than shown at the bottom of figure 3, then it would be useful to extend this flow chart to show the number of BAS and LMT users that were included in the analyses performed in the current study.

- The authors are using a non-conservative approach to data/results interpretation, frequently claiming that specific associations were established, when only uncorrected data have p<0.05 but correction for multiple testing /FDR values not are statistically significant. Occasionally they even claim to observe changes when the uncorrected p-value is ≥0.05. This approach is particularly evident on pg 14 (results section). While they could speak of suggestive changes or data trends, however data which does not survive correction for multiple testing, or has p-values ≥0.05 should be reported as not significant. There is otherwise a risk of overinterpreting weak data.

- Figure 6 would more clearly represent the level of difference between control and AD bile acid gene expression if the y-axes were presented as non-transformed data, rather than log2 data.

- The last paragraph on pg19 beginning "Our study design represents…." could be deleted, since a detailed outline of the three studies was provided earlier in the manuscript.

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Any attachments provided with reviews can be seen via the following link:

[LINK]

Decision Letter 2

Thomas J McBride

25 Nov 2020

Dear Dr. Varma,

Thank you very much for submitting your revised manuscript "Bile acid synthesis and modulation are associated with brain amyloid deposition, white matter lesions, neurodegeneration and risk of vascular dementia: a metabolic, neuroimaging, pharmacoepidemiologic and transcriptomic analysis" (PMEDICINE-D-20-00371R2) for consideration at PLOS Medicine.

Your revision was evaluated by a senior editor and discussed among the editors here. It was also discussed with an academic editor with relevant expertise, and sent to two of the original reviewers, including the statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

In light of these reviews, I am afraid that we still will not be able to accept the manuscript for publication in the journal in its current form, but we would like to consider a further revised version that addresses the reviewers' and editors' remaining comments. Obviously we cannot make any decision about publication until we have seen the revised manuscript and your response, and we plan to seek re-review.

In revising the manuscript for further consideration, your revisions should address the specific points made by each reviewer and the editors. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

In addition, we request that you upload any figures associated with your paper as individual TIF or EPS files with 300dpi resolution at resubmission; please read our figure guidelines for more information on our requirements: http://journals.plos.org/plosmedicine/s/figures. While revising your submission, please upload your figure files to the PACE digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at PLOSMedicine@plos.org.

We expect to receive your revised manuscript by Dec 16 2020 11:59PM. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

We ask every co-author listed on the manuscript to fill in a contributing author statement, making sure to declare all competing interests. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. If new competing interests are declared later in the revision process, this may also hold up the submission. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT. You can see our competing interests policy here: http://journals.plos.org/plosmedicine/s/competing-interests.

Please use the following link to submit the revised manuscript:

https://www.editorialmanager.com/pmedicine/

Your article can be found in the "Submissions Needing Revision" folder.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosmedicine/s/submission-guidelines#loc-methods.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

We look forward to receiving your revised manuscript.

Sincerely,

Thomas McBride, PhD

Senior Editor

PLOS Medicine

plosmedicine.org

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Requests from the editors:

1- Please report your study according to the relevant guideline(s), which can be found here: http://www.equator-network.org/ Please include the completed checklist as Supporting Information. When completing the checklist, please use section and paragraph numbers, rather than page numbers. Please add the following statement, or similar, to the Methods: "This study is reported as per the XXX guideline (S1 Checklist)." As this study uses multiple designs, I am leaving it to you to choose the appropriate guideline(s).

2- In your data statement, please include accession numbers or DOIs necessary for researchers to request the specific datasets used in this study.

3- Thank you for editing the reference call-outs. Please also remove the space between reference numbers (e.g., [1,2]).

4- Thank you for editing the title, however, please edit further to fit PLOS Medicine’s style. Titles cannot be declarative. Additionally, while we realize that this is a unique multi-step study design, but a briefer description of the study design would be preferred.

* Throughout the manuscript, please remove causal language (e.g., “increases risk of” at line 72 should be “was associated with”).

5- Please edit the Abstract Background section to replace the summary of the 3 step study design with a brief statement of the main study question. The study design should be introduced in the Abstract Methods and Findings section.

6- Abstract Methods and Findings:

* Please include the population and setting, years during which the study took place, length of follow up, and brief demographic details (e.g., age, sex) for each cohort included.

* Please quantify the main results (with 95% CIs and p values).

* Before describing the positive association at line 71, lease add a sentence describing the negative findings quoted around line 505.

* Please remove the last sentence (“Our findings merit confirmation…”) or move it to the Abstract Conclusions

7- The Abstract Conclusions are still a bit strong, given the limitations of this study. Please address the study implications without overreaching what can be concluded from the data; the phrase "In this study, we observed ..." may be useful.

* Please interpret the study based on the results presented in the abstract, emphasizing what is new without overstating your conclusions.

* Please remove the last sentence “Targeting brain primary BA signaling...”

8- Figure 2 can be removed, and Figure 1 can be a supplementary figure.

9- In the Methods section, please include a section on ethics approval, specify the ethics boards/committees that provided approval for each of the cohorts and studies included (rather than “The local Institutional Review Board...”, and specify how consent was obtained (or who waived consent, if that was the case).

10- Line 653, “(fig-xx)” is this a reference to a figure to be added?

11- Final sentence, lines 712-714, please be a bit more specific on the implications for clinical practice and drug discovery.

12- Please move the funding information from the main text to the Financial Disclosure section of the metadata.

13- At the end of the main text, please provide a list of all supplementary documents with titles and legends where applicable. Please also make sure all supplementary documents are referenced when relevant to the main text.

Comments from the reviewers:

Reviewer #1: We thank the authors for largely addressing our concerns from the previous review round. A fairly major concern however remains with the sex-specific associations claimed with CA and CDCA. In particular, with reference to the additional confirmatory ADNI analysis as presented in Table 1b, the impression gained at first glance is that the associations with CA and CDCA are universal (from all variables being significant in the "Total (n=1666)" column). Indeed, looking at the pvals, it appears likely that the non-significance of many variables once broken down into male/female subsets, might have arisen largely due to the reduced sample size.

Furthermore, while there appear to be far more CA/CDCA variables showing significance in Table 1b for males as compared to females, it is notable that all these nine variables have pval (FDR)=0.049, which appears to be just marginally below the significance threshold of p=0.05. In other words, were the significance threshold changed by merely 0.002, the conclusion reached would have been that neither CA nor CDCA variables had been shown to have had any associations with the rate of brain atrophy for males. Given that p=0.05 is a somewhat arbitrary, if traditional, measure (see for example "The reign of the p-value is over: what alternative analyses could we employ to fill the power vacuum?", L.G. Halsey, Biology Letters, 2019), it might remain slightly problematic to claim evidence of general CA/CDCA sex-specific associations as a major finding on this basis. Nonetheless, we believe the rest of the analysis to have merit.

Minor issue: in Line 179, "Religions Orders" might be "Religious Orders".

Reviewer #2: Dear authors,

Thank you for your detailed responses and additional data. It added considerable value to the manuscript.

I don't have any further comment.

Best regards

Reviewer

Any attachments provided with reviews can be seen via the following link:

[LINK]

Decision Letter 3

Richard Turner

23 Feb 2021

Dear Dr. Varma,

Thank you very much for re-submitting your manuscript "Bile acid synthesis, modulation and dementia: a metabolomic, transcriptomic and pharmacoepidemiologic study" (PMEDICINE-D-20-00371R3) for consideration at PLOS Medicine. We do apologize for the delay in sending you a response.

I have discussed the paper with our academic editor and it was also seen again by one reviewer. I am pleased to tell you that, provided the remaining editorial and production issues are dealt with, we expect to be able to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

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Please let me know if you have any questions, and we look forward to receiving the revised manuscript shortly.   

Sincerely,

Richard Turner PhD, for Thomas McBride, PhD

Senior Editor, PLOS Medicine

rturner@plos.org

------------------------------------------------------------

Requests from Editors:

In your data statement, please use "data are" consistently. Are all sources of data for your study quoted here (ADNI appears to be absent)? It appears that information on ADNI needs to be moved from the Acknowledgements section at the end of the paper.

At line 91, should "CN" be "CON"?

At line 133, please remove "Finally ..."; and we suggest adapting the wording to "... levels of gene expression of BA receptors were altered ...".

In your Introduction around lines 160-175, please integrate "STEP 1" and so on into the text in a single paragraph; e.g., "First, we used targeted metabolomics assays ... Finally, we explored plausible molecular mechanisms ...".

At line 539, we ask you to adapt "marginally increased risk" to "non-significantly elevated risk" (removing the subsequent "(non-significant)", noting the p value of 0.06.

In the paragraph summarizing the study findings at the start of your Discussion section, please make that "were/was associated" at lines 601/603.

Please remove the information on data access from the end of the main text. This information will appear in the article metadata, via entries in the submission form.

In the reference list, please ensure that journal names are abbreviated consistently (e.g., "J Alzheimers Dis." for reference 2 and others).

For reference 22 and any other preprints cited, please substitute "[preprint]" for the sentence about the absence of peer review.

For reference 44, please make the journal name "PLoS ONE".

Please break the STROBE checklist out into a separate attached file, labelled "S1_STROBE_Checklist" or similar and referred to by this label in your Methods section.

Comments from Academic Editor:

This study reports a nice multi-pronged approach to explore hypotheses from a variety of angles.

The authors should improve their discussion of what the implications of sex-specific pathways might be, the magnitude of possible differences, and how this might fit with our understanding of descriptive epidemiological estimates of dementia.

The authors might also want to consider making stronger statements about the fact that so much research doesn't report or address sex.

I'd like the authors to acknowledge literature that suggests their ideas of circumscribed AD, VaD are out of kilter with what's observed in usual dementia in the oldest age groups, thus contributing to the perpetuation of identifiable clean subtypes (reasonable for younger old, not for older old).

Comments from Reviewers:

*** Reviewer #1:

We thank the authors for clarifying our main concern about the FDR pvals, and reflecting a slightly more nuanced treatment in the Abstract and Discussion.

The claim however in the response that "the raw p-values for those comparisons are generally far lower in the male-only samples than the female-only samples" might however then be supported by these raw p-values, perhaps in the Supplementary material. Moreover, it might be explained as to why these raw p-values were not initially used for analysis in this case since they supposedly demonstrate sex-specific differences that much more clearly, and why the minimum alpha method of calculating false discovery rate was used instead.

***

Any attachments provided with reviews can be seen via the following link:

[LINK]

Decision Letter 4

Richard Turner

6 Apr 2021

Dear Dr Varma, 

On behalf of my colleagues and the Academic Editor, Dr Brayne, I am pleased to inform you that we have agreed to publish your manuscript "Bile acid synthesis, modulation and dementia: a metabolomic, transcriptomic and pharmacoepidemiologic study" (PMEDICINE-D-20-00371R4) in PLOS Medicine.

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Prior to final acceptance, we suggest amending the title to "... synthesis and modulation and risk of dementia: A metabolomic ..."; and removing "the" at line 50.

In the meantime, please log into Editorial Manager at http://www.editorialmanager.com/pmedicine/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process. 

PRESS

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Thank you again for submitting to PLOS Medicine. We look forward to publishing your paper. 

Sincerely, 

Richard Turner, PhD 

Senior Editor, PLOS Medicine

rturner@plos.org

Associated Data

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

    Supplementary Materials

    S1 Fig. Catabolism of cholesterol into primary BAs.

    The oxidative catabolism of cholesterol occurs through 3 enzymatically catalyzed biochemical pathways: the classic/neutral pathway in the liver accounts for the majority of BA synthesis in humans and begins with the oxidation of cholesterol to 7α-OHC by microsomal CYP7A1, the rate-limiting enzyme of the pathway. The alternative or acidic pathway is responsible for synthesis of a smaller proportion of the BA pool; cholesterol is oxidized to 27-OHC, catalyzed by mitochondrial CYP27A1 in both liver and extra-hepatic tissues. Both the classic/neutral and acidic pathways of BA synthesis ultimately generate the primary BAs, CA, and CDCA which are the principal catabolic products of cholesterol. A third, neuron-specific pathway of cholesterol breakdown in the brain is catalyzed by CYP46A1-mediated conversion of cholesterol to 24S-OHC which effluxes into the peripheral circulation for further conversion into the primary BAs in the liver [31,40,57]. 7α-OHC, 7α-hydroxycholesterol; 24S-OHC, 24S-hydroxycholesterol; 27-OHC, 27-hydroxycholesterol; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid.

    (TIFF)

    S1 Text. Supporting information text.

    (DOCX)

    S1 Table. STROBE checklist.

    STROBE, Strengthening the Reporting of Observational studies in Epidemiology.

    (DOCX)

    S2 Table. ROSMAP scRNA-Seq BA receptor gene expression data availability.

    Indicates data availability in the scRNA-Seq ROSMAP dataset. BA receptor genes that are indicated as “Not Available” either did not have sufficient counts or did not have any data available in the ROSMAP scRNA-Seq dataset. BA, bile acid; CHRM2, Cholinergic Receptor Muscarinic 2; CHRM3, Cholinergic Receptor Muscarinic 3; FGF19, Fibroblast Growth Factor 19; FPR1, Formyl Peptide Receptor 1; GPBAR1, G Protein-Coupled Bile Acid Receptor 1; HNF4A, Hepatocyte Nuclear Factor 4 Alpha; KDR, Kinase Insert Domain Receptor; NR0B2, Nuclear Receptor Subfamily 0 Group B Member 2; NR1H2, Nuclear Receptor Subfamily 1 Group H Member 2; NR1H3, Nuclear Receptor Subfamily 1 Group H Member 3; NR1H4, Nuclear Receptor Subfamily 1 Group H Member 4; NR1I2, Nuclear Receptor Subfamily 1 Group I Member 2; NR1I3, Nuclear Receptor Subfamily 1 Group I Member 3; NR3C1, Nuclear Receptor Subfamily 3 Group C Member 1; NR5A2, Nuclear Receptor Subfamily 5 Group A Member 2; PPARA, Peroxisome Proliferator Activated Receptor Alpha; PPARD, Peroxisome Proliferator Activated Receptor Delta; PPARG, Peroxisome Proliferator Activated Receptor Gamma; RARA, Retinoic Acid Receptor Alpha; ROSMAP, Religious Orders Study and Memory and Aging Project; RXRA, Retinoid X Receptor Alpha; RXRB, Retinoid X Receptor Beta; RXRG, Retinoid X Receptor Gamma; S1PR2, Sphingosine-1-Phosphate Receptor 2; scRNA-Seq, single-cell RNA sequencing; VDR, Vitamin D Receptor.

    (DOCX)

    S3 Table

    (A) Demographic characteristics of BLSA-NI sample. APOE4, e4 allele of the Apolipoprotein E gene; BLSA, Baltimore Longitudinal Study of Aging; MRI, magnetic resonance imaging; NI, neuroimaging; PiB, Pittsburgh compound B; SD, standard deviation; WML, white matter lesion. (B) Demographic characteristics of ADNI sample. ADNI, Alzheimer’s Disease Neuroimaging Initiative; MRI, magnetic resonance imaging; NI, neuroimaging; SD, standard deviation; WML, white matter lesion

    (DOCX)

    S4 Table. Associations between serum metabolite concentrations and PiB/amyloid status.

    coef, coefficient from linear regression model; PiB, Pittsburgh compound B; pval, p-value.

    (DOCX)

    S5 Table. Sensitivity analyses: Associations between serum metabolite concentrations and brain amyloid-β deposition, longitudinal changes in global brain WML burden, and rates of brain atrophy–BLSA.

    Sensitivity analyses after including statin use as a covariate. BLSA, Baltimore Longitudinal Study of Aging; coef, coefficient from linear regression model or mixed effects model; FDR, false discovery rate (Benjamini–Hochberg) corrected p-value; pval, p-value; WML, white matter lesion.

    (DOCX)

    S6 Table. Characteristics of participants who received at least 2 BAS or LMT prescriptions with at least 1 year of follow-up after second prescription.

    Wilcoxon rank-sum test. 1 Chi-squared test. 2 1 year prior to index date. 3 During study follow-up. BAS, bile acid sequestrants; LMT, lipid-modifying therapies.

    (DOCX)

    S7 Table. Characteristics of participants with incident dementia events during follow-up.

    1 1 year prior to index date.

    (DOCX)

    S8 Table. Demographic characteristics of BLSA autopsy sample.

    AD, Alzheimer disease; APOE4, apolipoprotein E allele epsilon 4; BLSA, Baltimore Longitudinal Study of Aging; CON, control; PMI, postmortem interval (hours).

    (DOCX)

    S9 Table. Differences in brain primary BA concentrations between AD and CON.

    * In the CON sample in the CB, all concentrations were below LOD; we therefore tested for differences in the number of concentrations below LOD comparing AD to CON using the chi-squared test and present the associated p-value. AD, Alzheimer disease; BA, bile acid; coef, coefficient for disease (AD vs. CON) from the tobit model including mean-centered age and sex where the lower limit is set as the metabolite specific LOD; CON, control; LOD, limit of detection; pval, p-value.

    (DOCX)

    S10 Table. Differences in brain BA receptor gene expression (including receptors involved in BA homeostasis) in AD compared to CON.

    AD, Alzheimer disease; BA, bile acid; CHRM2, Cholinergic Receptor Muscarinic 2; CHRM3, Cholinergic Receptor Muscarinic 3; CON, control; FDR, false discovery rate (Benjamini–Hochberg) corrected p-value; FPR1, Formyl Peptide Receptor 1; GPBAR1, G Protein-Coupled Bile Acid Receptor 1; HNF4A, Hepatocyte Nuclear Factor 4 Alpha; KDR, Kinase Insert Domain Receptor; NR1H2, Nuclear Receptor Subfamily 1 Group H Member 2; NR1H3, Nuclear Receptor Subfamily 1 Group H Member 3; NR1H4, Nuclear Receptor Subfamily 1 Group H Member 4; NR1I2, Nuclear Receptor Subfamily 1 Group I Member 2; NR1I3, Nuclear Receptor Subfamily 1 Group I Member 3; NR3C1, Nuclear Receptor Subfamily 3 Group C Member 1; NR5A2, Nuclear Receptor Subfamily 5 Group A Member 2; PPARA, Peroxisome Proliferator Activated Receptor Alpha; PPARD, Peroxisome Proliferator Activated Receptor Delta; PPARG, Peroxisome Proliferator Activated Receptor Gamma; pval: p-value; RARA, Retinoic Acid Receptor Alpha; RXRA, Retinoid X Receptor Alpha; RXRB, Retinoid X Receptor Beta; RXRG, Retinoid X Receptor Gamma; VDR, Vitamin D Receptor.

    (XLSX)

    Attachment

    Submitted filename: Summary_response_9.2.20.docx

    Attachment

    Submitted filename: Response_to_reviewers_R2_12.12.2.docx

    Attachment

    Submitted filename: Response_to_reviewers_R3_3.1.21.docx

    Data Availability Statement

    Baltimore Longitudinal Study of Aging (BLSA) data are available to researchers and can be requested at https://www.blsa.nih.gov/researchers. Rush Memory and Aging Project (ROSMAP) data can be requested at https://www.radc.rush.edu; ROSMAP data used in this study are available at: https://www.synapse.org/#!Synapse:syn18485175 under the doi 10.7303/syn18485175 Clinical Practice Research Datalink (CPRD) data are available to researchers and can be requested at https://www.cprd.com/public. Alzheimer’s Disease Neuroimaging Network (ADNI) data are available to researchers at http://adni.loni.usc.edu.


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