CSF ApoE predicts clinical progression in nondemented APOEε4 carriers

Accepted Manuscript CSF ApoE predicts clinical progression in non-demented APOEε4 carriers A.C. van Harten, M.D PhD., W. Jongbloed, PhD., C.E. Teunissen, PhD, P. Scheltens, MD PhD, R. Veerhuis, PhD, W.M. van der Flier, PhD PII:

S0197-4580(17)30120-3

DOI:

10.1016/j.neurobiolaging.2017.04.002

Reference:

NBA 9897

To appear in:

Neurobiology of Aging

Received Date: 27 June 2014 Revised Date:

2 April 2017

Accepted Date: 4 April 2017

Please cite this article as: van Harten, A.C., Jongbloed, W., Teunissen, C.E., Scheltens, P., Veerhuis, R., van der Flier, W.M., CSF ApoE predicts clinical progression in non-demented APOEε4 carriers, Neurobiology of Aging (2017), doi: 10.1016/j.neurobiolaging.2017.04.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT CSF ApoE predicts clinical progression in non-demented APOEεε4 carriers A.C. van Harten M.D PhD.a,b, W. Jongbloed PhD.c, C.E. Teunissen PhDc, P. Scheltens MD PhDa,b, R. Veerhuis PhDc,d, W.M. van der Flier PhDa,b,e

Center and Departments of bNeurology, cClinical Chemistry, and dPsychiatry,

eEpidemiology/Biostatistics,

Neuroscience Campus Amsterdam, VU University Medical

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aAlzheimer

Center, Amsterdam, the Netherlands.

* Corresponding author: A.C. van Harten. Alzheimer Center, VU University Medical Center,

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Fax nr: +31204448529. Email: [email protected]

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De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands. Telephone nr: +31204448527.

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ACCEPTED MANUSCRIPT ABSTRACT

Possible associations between cerebrospinal fluid (CSF) and plasma ApoE concentration and early clinical and pathophysiological manifestation of Alzheimer’s disease (AD) were studied in a large and well-defined population of non-demented patients. CSF and plasma

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ApoE concentrations were related to CSF Aβ42, Tau and pTau levels and clinical characteristics in patients with SCD (n=207) or MCI (n=213) aged 64.29.0 years, with a 2.5±1.5 year follow-up. A 1 standard deviation increase in log-transformed CSF ApoE

concentrations increased the risk of clinical progression in APOEε4 carriers 1.5 times [HR

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(95% CI) 1.5 (1.1-2.0)], while this was not the case in APOEε4 non-carriers [HR (95% CI) 1.0 (0.8-1.2)]. Plasma ApoE did not predict clinical progression. Using linear regression

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models, strong associations between CSF ApoE levels and CSF Tau (β 0.51 [0.38;0.65]) and pTau (β 0.53 [0.40: 0.60]) values were observed in APOEε4 carriers. We hypothesize CSF ApoE4 increases risk of clinical progression through its association with CSF Tau in APOEε4 carriers. Development of AD in APOEε4 non-carriers may be unrelated to ApoE

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concentration.

Keywords: Apolipoprotein E; Cerebrospinal fluid; plasma; Alzheimer’s disease;

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biomarkers; clinical progression.

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ACCEPTED MANUSCRIPT HIGHLIGHTS

High CSF ApoE predicts clinical progression in non-demented APOEε4 carriers.

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ApoE4 may be detrimental through its influence on amyloid-independent pathways.

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Alternative pathways are more likely to play a role in APOEε4 non-carriers.

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Plasma ApoE does not predict clinical progression in non-demented subjects.

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The effect of plasma ApoE on cognition may differ according to disease stage.

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ACCEPTED MANUSCRIPT INTRODUCTION

Apolipoprotein E (ApoE) is a glycoprotein with three major isoforms (ε2, ε3 and ε4). Its resulting proteins (ApoE2, ApoE3 and ApoE4) differ structurally at only two amino acids.

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The APOEε4 isoform is a major genetic risk factor for dementia due to Alzheimer's disease (AD) (Bertram et al., 2007). In non-demented subjects, presence of the APOEε4 isoform

predicted clinical progression to MCI or dementia due to AD (Blair et al., 2005; Boyle et al., 2010; Elias-Sonnenschein et al., 2011). Many functional differences between ApoE3 and ApoE4 have been proposed to explain the increased risk of dementia due to AD associated

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with APOEε4 genotype (Caselli e Reiman, 2013; Holtzman et al., 2012; Yu et al., 2014). Which of these differences influence this risk, and whether ApoE expression levels play a

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role in humans in vivo remains to be elucidated.

ApoE concentrations have previously been measured in CSF and plasma, but associations with AD have remained ambiguous. A meta-analysis revealed plasma ApoE concentrations are lower in patients with dementia due to AD than in controls (Wang et al., 2014), but

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several negative findings were not included (Martínez-Morillo et al., 2014; Scarabino et al., 2016; Simon et al., 2012; Song et al., 2012; Łojkowska et al., 2013). Two population-based studies recently suggested low plasma ApoE concentration predicts AD dementia (Rasmussen et al., 2015; Wolters et al., 2016), but others did not find so (Hye et al., 2014;

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Song et al., 2012; Toledo et al., 2014; Yasuno et al., 2012). CSF ApoE did not differ between controls and patients with AD dementia in a recent meta-analysis (Talwar et al., 2016).

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Nonetheless, CSF ApoE has been related to dementia severity at follow-up in a large cohort study including non-demented and demented subjects and to incident AD dementia in patients with MCI (Cruchaga et al., 2012; Toledo et al., 2014).

In this study we aimed to elucidate the association between plasma and CSF ApoE concentration and early manifestation of AD in non-demented patients with SCD or MCI. To this end we assessed whether ApoE concentration predicted clinical progression over time. Also, we assessed cross-sectional associations between ApoE concentrations and AD pathophysiology using classical CSF biomarkers Aβ42, Tau and pTau as endophenotype for AD.

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ACCEPTED MANUSCRIPT METHODS

Subjects Four-hundred-forty-eight non-demented patients with a baseline diagnosis of subjective cognitive decline (SCD) or Mild Cognitive Impairment (MCI) from our memory clinic based

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Amsterdam Dementia Cohort were eligible for this study, because APOE genotype had been determined and EDTA plasma and/or CSF samples were available for ApoE

concentration measurements and they underwent at least one follow-up. Eighteen patients were excluded, because they progressed to another form of dementia than dementia due to

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AD, resulting in inclusion of 430 patients available for analysis. Two hundred and seven were considered to have SCD at baseline and 223 had MCI (Petersen, 2004).

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Clinical evaluation at baseline and follow-up has been described in detail elsewhere (van Harten et al., 2013). All patients underwent a standardized dementia screening including neuropsychological, physical and neurologic examination as well as laboratory tests, electro-encephalography (EEG) and brain magnetic resonance imaging (MRI). Diagnoses were made in a multidisciplinary meeting, without knowledge of APOE genotype or CSF

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results. Patients were labelled as having SCD when they presented with cognitive complaints, but ancillary investigations were normal and criteria for MCI, dementia or any other neurological or psychiatric disorder known to cause cognitive complaints were not met (i.e. cognitively normal elderly). MCI was diagnosed according to Petersen's criteria

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(Petersen et al., 1999). In retrospect all MCI patients fulfilled NIA-AA core clinical criteria for the diagnosis of MCI (Albert et al., 2011). Follow-up took place by routine annual visits

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to our memory clinic, in which patient history, cognitive tests and a general physical and neurologic examination were repeated. NINCDS-ADRDA criteria for probable AD were used (McKhann et al., 1984), but all patients with dementia due to AD also fulfilled NIA-AA core clinical criteria for AD (McKhann et al., 2011). Clinical progression was defined as progression to MCI or dementia due to AD in patients with SCD and as progression to dementia due to AD in patients with MCI. If patients with SCD first progressed to MCI and then to AD, the moment of conversion to MCI was taken as time of clinical progression.

Neuropsychological assessment

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ACCEPTED MANUSCRIPT Neuropsychological assessment was described in detail elsewhere (van Harten et al., 2013). Cognitive functions were assessed using a standardized test battery. Part of this battery was used to evaluate associations between ApoE concentration and cognitive performance at baseline and over time. For global cognition we used the mini mental state examination (MMSE) (Folstein et al., 1975)). For memory, we used total immediate recall

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and delayed recall of the Dutch version of the Rey auditory verbal learning task (RAVLT) (Rey, 1964). For attention we used Trail Making Test (TMT) A and to evaluate executive functioning we used TMT B (Reitan, 1958). Selection of these tests was based on an earlier finding that they were sensitive to cognitive decline in preclinical AD (van Harten et al.,

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2013).

APOE genotyping

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APOE genotyping was performed after automated genomic DNA isolation from 7-10 mL EDTA blood. It was subjected to PCR, checked for size and quantity using a QIAxcel DNA Fast Analysis kit (Qiagen, Venlo, The Netherlands) and sequenced using Sanger sequencing on an ABI130XL. Subjects were classified as APOEε4 negative or positive.

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CSF and plasma biomarker measurements

CSF and plasma analyses were performed at the Neurochemistry Laboratory of the Clinical Chemistry department of the VU University Medical Center in Amsterdam. CSF was obtained by lumbar puncture between the L3/L4 or L4/L5 intervertebral space by a 25-

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gauge needle and collected in polypropylene tubes (Sarstedt, Numbrecht, Germany). EDTA plasma and CSF were collected on the same day (BD Vacutainer; BD, Etten Leur, The

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Netherlands). 2.5 mL CSF was used for routine analyses and Aβ42, Tau and pTau measurements using ELISA (Fujirebio, Belgium) as previously described (van Harten et al., 2013). We chose the pTau-181 assay because of its reliability and long time experience in our laboratory with this assay (Mulder et al., 2010). CSF and plasma were centrifuged and 0.5 mL aliquots were stored in polypropylene vials (Sarstedt) at -80°C until ApoE analyses. ApoE concentrations in CSF (mg/L) (n=403) and EDTA plasma (mg/L) (n=412) were measured using a commercial sandwich ELISA, which detects all ApoE isoforms (3712-1H20; Mabtech, Nacka Strand, Sweden). Sample dilutions (1:2000 and 1:20000 for CSF and plasma respectively) and serial dilutions of recombinant ApoE3, used to prepare a standard curve, ranging from 31.6 mg/L to 0.03 mg/L, were made in assay buffer (3652-J2;

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ACCEPTED MANUSCRIPT Mabtech, Sweden) . Surplus routine plasma samples with either high or low ApoE concentrations were pooled, aliquoted, stored at -80 °C and included in each run as a quality control. Intra- assay CV’s were 2.7% for plasma and 3.0% for CSF, inter-assay CV’s tested in 24 ELISA plates were 10.4% and 10.3% for the low and high ApoE plasma

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controls, respectively.

Statistical analyses

Data were analysed using SPSS for Macintosh, version 24. Figures 1 and 3 were created using GraphPad PRISM. Because ApoE, Aβ42, Tau and pTau concentrations were not

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normally distributed, they were log10 transformed and subsequently converted to z-

scores prior to all analyses to allow parametric comparisons and comparison of effect sizes

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on a normalized scale. Groups were compared based on their baseline diagnosis (SCD vs. MCI), outcome (stable vs. clinical progression) using t-tests and chi square tests as appropriate. Differences in ApoE concentration based on APOE genotype and sex were similarly assessed using t-tests. Results of these analyses are presented as median [range] for groups under investigation with p-values corresponding to t-tests after logtransformation and conversion to z-scores. In a post-hoc analysis, we additionally

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stratified the comparison between CSF ApoE concentrations according to baseline diagnosis for APOE genotype.

To investigate whether CSF and plasma ApoE concentration predicted clinical progression

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along the AD continuum, we used Cox proportional hazard models. Clinical progression was taken as outcome measure. Results are presented as Hazard Ratio (HR) (95%CI).

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Because we converted log-transformed ApoE concentration to z-scores prior to analysis, HR’s represent the risk of clinical progression associated with 1 standard deviation increase of the log10-transformed ApoE concentrations. In our first model, the predictor was ApoE concentration in CSF or plasma, adjusted for age, sex, baseline MMSE and baseline diagnosis. Education was not included, because it was neither associated to our predictor of interest, nor to our outcome measure. In the second model, APOE genotype (ε4 positive vs. ε4 negative), and the interaction ApoE concentration*APOE genotype were added. When we found an interaction between ApoE concentration and APOE genotype, results were subsequently stratified for APOE genotype in model 2. When this interaction was absent however, the interaction term was excluded from the model and results were

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ACCEPTED MANUSCRIPT corrected for APOE genotype. In a final Cox proportional hazards model we evaluated whether adding the use of cholesterol lowering drugs changed any of the hazard ratios. This variable was excluded when results remained unchanged. For illustration purposes, ApoE concentrations were additionally divided into tertiles in order to generate Kaplan

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Meier curves (visualization of raw data).

In addition, we investigated the effect of ApoE concentration on baseline cognition and

cognitive decline over time in multiple exploratory analyses using linear mixed models. The models included terms for time, ApoE concentration and ApoE concentration*time

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interaction. Intercept and time were entered as a random effects, all other effects were

fixed. Outcome measures were results of above-mentioned neuropsychological tests. All

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models were adjusted for age and sex (fixed effects). In the first model we investigated the whole group. In a second model we stratified patients based on their APOEε4 status. Because of the exploratory nature of these analyses, they were not corrected for multiple comparisons. Results are presented as betas with p-values.

To investigate associations between ApoE concentration and CSF Aβ42, Tau and pTau, we

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used linear regression models. We included the same two models as we did in the Cox proportional hazard models. CSF Aβ42, Tau and pTau were dependent variables. In the first model the independent variable was ApoE concentration in CSF or plasma, adjusted for age, sex, baseline MMSE and baseline diagnosis. In the second model, APOE genotype

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(ε4 positive vs. ε4 negative), and the interaction ApoE concentration *genotype were added. When we found an interaction between ApoE concentration and APOE genotype,

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results were subsequently stratified for APOE genotype in model 2. When this interaction was absent however, the interaction term was excluded from the model and results were corrected for APOE genotype. Results are given as standardized Beta’s with 95% confidence interval (95% CI).

Subsequently, we repeated all analyses for patients with SCD and MCI separately. A p<0.05 was considered significant for all analyses.

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ACCEPTED MANUSCRIPT RESULTS

3.1 baseline characteristics Of the 430 included patients (average age 64.2±9.0 years; 180 (42%) female), 216 (50%) were APOEε4 positive. Average follow-up for the whole group was 2.5±1.5 years. Baseline

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characteristics are given in Table 1. In groups of patients with MCI and SCD, patients who showed clinical progression were older at baseline, more often APOEε4 positive and had CSF Aβ42, Tau and pTau values more consistent with AD (i.e. lower concentrations of CSF Aβ42 and higher concentrations of CSF Tau and pTau). MCI patients who showed clinical

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progression at follow-up also had lower MMSE at baseline, and were more often female, than those who did not show clinical progression. APOEε4 carriers had more abnormal CSF

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Aβ42 (552 [190-1444] vs. 897 [178-1545]), Tau (374 [87-3515] vs. 259 [69-2088]) and pTau values (64 [20-268] vs. 48 [16-190]) than APOEε4 non-carriers (all p<0.001). Eightytwo patients (19%) used cholesterol-lowering drugs. We found no difference in CSF ApoE concentration between those with and without cholesterol lowering drug use (3.2 [1.1-6.3] mg/L vs. 3.4 [0.4-9.3] mg/L, p=0.99). Plasma ApoE concentrations were lower in patients

p=0.004).

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= Insert Table 1 about here =

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who used cholesterol lowering drugs (25.6 [9.2-64.4] mg/L vs. 29.3 [11.2-76.1] mg/L,

3.2 Associations between ApoE levels and known risk factors for AD. We found no correlation between ApoE concentrations in CSF and plasma (r=-0.02.

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p=0.64; Supplementary Figure 1). CSF ApoE concentrations correlated with age (r=0.18, p<0.001, Figure 1), plasma ApoE did not (r=-0.03, p=0.60). Females had slightly higher CSF ApoE concentration than men (3.6 [1.0-9.0] vs. 3.2 [0.4-9.3] mg/L, p<0.05), but we found no sex difference in plasma ApoE (females 28.6 [9.2-57.7], males 27.8 [4.7-76.1] mg/L, p=0.94). APOEε4 carriers had higher CSF ApoE concentrations (3.7 [1.1-9.3] vs. 2.9 [0.47.1] mg/L, p<0.001) and lower plasma ApoE concentrations (25.5 [4.7-73.1] vs. 31 [12.976.1] mg/L, p<0.001) than APOEε4 non-carriers. Finally, patients with a baseline diagnosis of MCI had higher CSF ApoE concentrations than SCD patients at baseline (Table 1, Figure 1). When this analysis was stratified for APOE genotype, the difference between SCD and

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ACCEPTED MANUSCRIPT MCI remained present in APOEε4 carriers only (APOEε4 positive SCD 3.36 [1.30-9.32] and MCI 3.96 [1.06-9.02], p=0.012; APOEε4 negative SCD 2.90 (0.70-6.39] and MCI 3.14 [0.357.08], p=0.64).

3.3 Predictive value of ApoE concentrations for clinical progression

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We used Cox proportional hazard models to evaluate predictive value of CSF and plasma ApoE concentrations for clinical progression over time (Table 2). Adjusted for age, sex,

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baseline MMSE and baseline diagnosis, one SD increase in the log-transformed CSF ApoE concentration (approximately 1.8 mg/L) was associated with 1.3 times increased risk of

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clinical progression at each moment in time (HR [95%CI] 1.3 [1.1-1.6]; p=0.012). In model 2, additionally including APOE genotype, we found an interaction between CSF ApoE concentration and APOE genotype (p=0.03). Subsequent stratification of results showed that in APOEε4 carriers, a 1 SD increase in log-transformed CSF ApoE was associated with a 1.5 times increased risk of clinical progression (HR 1.5 [95% CI 1.1-2.0]; p=0.01). This effect remained unchanged after additional adjustment for CSF Aβ42 (model 3, HR 1.4

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[95% CI 1.1-1.8]; p=0.02), but disappeared after subsequently adding CSF Tau in model 4 (HR 0.8 [95% CI 0.6-1.1]; p=0.26), potentially suggesting mediation between CSF ApoE and pathophysiological processes represented by CSF Tau. In APOEε4 non-carriers, CSF ApoE did not predict clinical progression. When patients with SCD and MCI were assessed

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separately, effect sizes were similar to those in the whole group, although the predictive value of CSF ApoE concentration in APOEε4 carriers did not reach significance and its

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effect size diminished after adjustment for CSF Aβ42 in SCD patients, instead of after adjustment for CSF Aβ42 and Tau. For illustrative purposes, we show Kaplan-Meier curves for survival according to ApoE tertile in APOEε4 carriers and non-carriers separately (Figure 2). Plasma ApoE was not associated with clinical progression (Table 2). Results did not change when the use of cholesterol lowering drugs was added as a covariate (data not shown).

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ACCEPTED MANUSCRIPT 3.4 Association between CSF ApoE and cognition. Next, we evaluated associations between CSF ApoE concentrations and cognitive decline using linear mixed models in a subset of patients with non-missing neuropsychological data (n between 355 and 384 for each cognitive test, Supplementary Table 1). In the whole group, higher CSF ApoE levels were associated with slightly worse baseline performance

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on memory tests (β for immediate recall on the RAVLT: -1.04, p=0.04; delayed recall of the RAVLT: -0.37, p=0.04), but there were no associations with cognitive decline over time.

Subsequent stratification of the analysis based on APOEε4 genotype showed that CSF ApoE was associated with baseline memory in APOEε4 carriers (β for RAVLT immediate recall: -

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1.94, p=0.02; RAVLT delayed recall: -0.67, p=0.03), but not in APOEε4 non-carriers. When SCD and MCI patients were analysed separately, effect sizes in APOEε4 carriers remained

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similar, but results did not remain significant. A trend for decline over time in immediate recall on the RAVLT emerged in SCD patients (β -0.60, p=0.059) and in SCD APOEε4 noncarriers (β -0.85, p=0.08).

3.5 Association between plasma ApoE and cognition.

We found no associations between plasma ApoE and cognition in the whole group

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(Supplementary Table 2). After stratification for APOE genotype APOEε4 carriers showed a trend for an association between high plasma ApoE and low baseline MMSE (β -0.26, p=0.08). After separating groups based on baseline diagnosis, we found a similar trend in

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MCI patients (β -0.29, p=0.07). MCI patients also showed a trend for an association between high plasma ApoE and lower baseline delayed recall (β -0.33, p= 0.07). After

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stratification for APOE genotype, this effect was strongest in APOEε4 non-carriers with MCI (β -0.70, p=0.03). We found no associations between plasma ApoE and cognition at baseline in SCD patients. High plasma ApoE was associated with decline over time in immediate recall (β -0.82, p=0.03) in all SCD patients and APOEε4 carriers with SCD (β 1.00, p=0.03). APOEε4 carriers with SCD also showed an association between plasma ApoE and delayed recall at baseline ((β -0.90, p=0.01).

3.6 Associations between ApoE level and endophenotypical markers of AD We used linear regression models to study relationships between ApoE concentration in CSF or plasma and CSF Aβ42, Tau and pTau as endophenotypical markers of AD (Table 3).

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ACCEPTED MANUSCRIPT Adjusted for age, sex, baseline MMSE and baseline diagnosis, we found moderate positive associations between CSF ApoE and (p)Tau, but no association between CSF ApoE and Aβ42. After additional adjustment for APOE genotype (model 2), we found a borderline significant weakly positive association between CSF ApoE and Aβ42 concentrations (β = 0.09, p=0.055). As interactions between CSF ApoE and APOE genotype were present when

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Tau and pTau were taken as outcome measure (β Tau = 0.23, p=0.005; β pTau = 0.19, p=0.02), we stratified these results based on APOE genotype in model 2. In APOEε4

carriers, we found strong associations between CSF ApoE and Tau and between CSF ApoE and pTau, which indicated that higher CSF ApoE concentrations were related to more AD-

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like (i.e. higher) CSF (p)Tau concentrations(β Tau = 0.51, p<0.0001; β pTau = 0.53,

p<0.001). We also found positive associations between CSF ApoE and CSF (p)Tau in

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APOEε4 non-carriers, but effect sizes were smaller than in APOEε4 carriers (β Tau = 0.29, p<0.001; β pTau = 0.36, p<0.001). See Figure 3 for scatterplots of associations between CSF ApoE and CSF Tau.

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When we repeated the linear regression analyses for each diagnostic group separately, we found an additional interaction between APOE genotype and CSF ApoE concentration (β = 0.30, p=0.02) when CSF Aβ42 was taken as outcome measure in patients with SCD

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(Supplementary Table 3A and B). Effect sizes indicated higher CSF ApoE was associated to slightly higher (less AD-like) Aβ42 in APOEε4 non-carriers with SCD, while it was associated to lower (more AD-like) Aβ42 in APOEε4 carriers. Neither individual effect was

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significant, however. Other results were similar to those in the whole group. The association between CSF ApoE and CSF Tau/pTau did not differ between SCD and MCI patients.

Plasma ApoE was weakly associated to CSF Aβ42 and pTau prior to correction for APOE genotype (Model 1; Table 3)(β Aβ42 = 0.14, p=0.002; β pTau = -0.12, p=0.005). After additional correction for APOE genotype, only the association between plasma ApoE and pTau remained significant (β = -0.09, p=0.048).

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ACCEPTED MANUSCRIPT DISCUSSION

In this study, we showed that high CSF ApoE concentration is associated with clinical and pathophysiological manifestation of AD in non-demented APOEε4 carriers. Most importantly, CSF ApoE predicted clinical progression in APOEε4 carriers, but not in

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APOEε4 non-carriers. The predictive value of CSF ApoE remained unchanged after

adjustment for Aβ42, but its effect lost significance after additional adjustment for CSF Tau. CSF ApoE and CSF Tau/pTau were strongly correlated in APOEε4 carriers, but less so in APOEε4 non-carriers. The cross-sectional nature of our biomarker data precludes

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conclusions about causality and directionality of effects. Still, if one hypothesizes CSF ApoE could be an intermediate between APOE genotype and AD pathophysiology; our findings

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may indicate that ApoE concentration exerts its effect on clinical progression through the pathophysiological processes represented by CSF Tau/pTau in APOEε4 carriers. When looking at cognitive performance, the difference in clinical progression was mainly due to worse baseline memory performance associated with higher CSF ApoE in both diagnostic groups, without significant differences in rate of cognitive decline over time, although trends for increased decline in immediate recall did emerge in APOEε4 carriers with SCD.

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Contrary to CSF ApoE, plasma ApoE did not predict clinical progression.

The finding that high CSF ApoE concentration is associated with worse prognosis was

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further substantiated by several positive correlations between CSF ApoE and known risk factors for AD. CSF ApoE concentration was higher in APOEε4 carriers with MCI than in those with SCD. It was higher in females than in men and correlated positively with age.

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These findings partly confirm earlier findings in patients along the clinical continuum of AD (Baker-Nigh et al., 2016; Cruchaga et al., 2012; Darreh-Shori et al., 2011; Schmidt et al., 2014; Toledo et al., 2014), although Toledo et al found females had lower CSF ApoE concentrations than men (Toledo et al., 2014). In line with our other findings, we found APOEε4 carriers had higher CSF ApoE concentrations than APOEε4 non-carriers. This corroborates findings from 2 prior studies in patients with probable AD or Lewy-body disease (Darreh-Shori et al., 2011; Vijayaraghavan et al., 2014), but stands in contrast to several other studies in AD (Bekris et al., 2008; Cruchaga et al., 2012; Khan et al., 2015; Martínez-Morillo et al., 2014; Schmidt et al., 2014).

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ACCEPTED MANUSCRIPT Because we found CSF ApoE predicted clinical progression in APOEε4 carriers only, our results may support the existence of a functional difference between different isoforms of ApoE. Several studies have shown that the ApoE4 protein is the predominant isoform produced in the brains of APOEε4 carriers, whether they are heterozygous or homozygous

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carriers and that this imbalance is reflected in CSF (Baker-Nigh et al., 2016; Fukumoto et al., 2003). If this is the case, functional differences between ApoE isoforms, mainly ApoE3 and ApoE4, may explain why CSF ApoE predicts clinical progression in APOEε4 carriers, but not in APOEε4 non-carriers. Based on our findings, we would hypothesize that high

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ApoE4 predicts clinical progression along the AD continuum (in APOEε4 carriers), while high ApoE3 (or ApoE2) does not (in APOEε4 non-carriers). A functional difference between

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ApoE4 and other isoforms may also explain why the association between CSF ApoE and Tau is stronger in APOEε4 carriers than in non-carriers. A wealth of in vitro and animal model evidence supports the existence of functional differences between different isoforms of ApoE, [for a review see (Yu et al., 2014)] but to our knowledge, we are the first to confirm the clinical relevance of this difference in the central nervous system in vivo.

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In view of these findings, and in view of our finding that CSF ApoE is no longer predictive of clinical progression after adjustment for Tau, the most important consequence of our study may be pathophysiological instead of clinical. In literature, multiple different ways in

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which APOEε4 genotype increases risk of dementia due to AD have been proposed. Within the brain a distinction can be made between amyloid dependent and amyloid independent pathways (Yu et al., 2014; Liu et al., 2013). Cross-sectionally we found strong positive

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associations between CSF ApoE and CSF Tau/pTau in APOEε4 carriers, but no association between CSF ApoE and CSF Aβ42. Based on our current data we cannot conclude that this cross-sectional association represents a causal relationship between CSF ApoE and CSF Tau. If it does, our results may indicate that amyloid independent pathways are more influenced by ApoE concentration in APOEε4 carriers than amyloid dependent pathways. Interestingly, several in vitro studies have suggested ApoE4 causes hyperphosphorylation of Tau and tangle formation, while ApoE3 does not (Andrews-Zwilling et al., 2010; Harris et al., 2003; Strittmatter et al., 1994). Also, AD transgenic mice that express human ApoE4 (3xTG-ApoE4 mice) had more total tau accumulation than AD transgenic mice with other

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ACCEPTED MANUSCRIPT APOE genotypes, while there was no difference in amyloid deposition (Bennett et al., 2013).

Another on-going debate concerns the question whether the predominant effect of ApoE4 is a gain of toxic functions or a loss of protective functions compared to ApoE3 (Liu et al.,

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2015; Liu et al., 2013). The answer to this question has important therapeutic consequences: a gain of toxic function would imply lowering ApoE in the central nervous system (CNS) is the best therapeutic approach, while a loss of protective function would advocate increasing CNS ApoE concentrations (Cramer et al., 2012; Kim et al., 2011; Liao et

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al., 2014; Ramanan et al., 2014; Sen et al., 2012). The detrimental effect of high ApoE in

APOEε4 carriers in our study, combined with the above-mentioned evidence from mouse

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models and the fact that absence of ApoE in APP/PSdE9 mice ameliorates AD pathology (Fitz et al., 2015), implies a gain of toxic function may be the predominant effect in the predementia stage of AD in APOEε4 carriers. Speculatively, these subjects may therefore benefit most from a therapeutic approach that lowers CNS ApoE (Bien-Ly et al., 2012; Pankiewicz et al., 2014). It is important to note, however, that we cannot conclude from our findings that all ApoE is detrimental and ApoE4 is just more detrimental than the other

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isoforms. If all ApoE had been detrimental, high CSF ApoE concentrations would have predicted clinical progression in APOEε4 non-carriers as well, but to a lesser degree than in APOEε4 carriers. This was not the case, although we did find a small association between CSF ApoE and CSF Tau/pTau and a trend for more decline in delayed recall over

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time in APOEε4 non-carriers with SCD. This association may not have reached significance

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due to insufficient statistical power in this relatively small subgroup.

One prior study including ADNI participants with a research question similar to our own reached a notably different conclusion (Toledo et al., 2014). They found positive associations between CSF ApoE and Tau and between CSF ApoE and age. In initial models high CSF ApoE predicted clinical progression from MCI to dementia due to AD, but after additional adjustment for CSF tau, relatively low CSF ApoE concentrations were associated with clinical progression, which led to the conclusion that low CSF ApoE may indicate a failed protective response in early AD. No real differences were found between APOEε4 carriers and non-carriers. Various (pre-analytical, analytical and study-group) variables may account for the difference between these findings in ADNI and the current study. For 15

ACCEPTED MANUSCRIPT example, the ADNI sample was about 10 years older than our study sample and associations between ApoE concentration and AD may change as people grow older or reach a different disease stage. Also, ApoE was measured using a multi-analyte panel in ADNI, while we used an extensively validated ELISA method. From a broader perspective, similar between-study differences may explain much of the differences between existing

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case-control studies regarding ApoE concentration and AD (Talwar et al., 2016; Wang et al., 2014). Standardization of the analytical process may lead to more consistent results in the future.

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In addition to mechanisms directly related to pathophysiological processes in the brain, the APOEε4 genotype could also increase risk of dementia due to AD by influencing

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cardiovascular risk (Caselli e Reiman, 2013; Holtzman et al., 2012; Kester et al., 2010; Mahley e Rall, 2000). If this is the case, plasma ApoE may be a better reflection of this mechanism in vivo than CSF ApoE, because the two are not correlated and it is therefore likely that plasma ApoE is derived from a peripheral source (the liver), while CSF ApoE is derived from the brain (Baker-Nigh et al., 2016; Carlsson et al., 1991; Cruchaga et al., 2012; Fagan et al., 2000). Our finding that plasma ApoE did not predict clinical progression while

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CSF ApoE did, could imply that the activity of ApoE in the brain determines the risk of developing dementia due to AD to a larger extent than its cardiovascular effects. This is in line with results of the Rotterdam study in which APOE genotype and atherosclerosis were found to be independent risk factors for dementia due to AD (Slooter et al., 1999). In

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seeming contrast to these early findings, low plasma ApoE predicted incident dementia due to AD in a later publication from the Rotterdam study and a population based study

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from Denmark (Rasmussen et al., 2015; Wolters et al., 2016). Adjustment for cardiovascular risk did not reduce hazard ratios, which was taken to mean that a different mechanism could be at play (Wolters et al., 2016). In general, our results did not support any role for plasma ApoE in clinical progression. As such, our results are more in line with three other longitudinal studies: in spite of promising cross-sectional results, plasma ApoE did not predict clinical progression in MCI patients from ADNI and a multi-centre study combining 3 cohorts, nor did it predict conversion from CDR 0 to a higher CDR score in the Sydney Memory and Aging study (Hye et al., 2014; Song et al., 2012; Toledo et al., 2014). Still, our use of linear mixed models with cognitive performance as outcome measure adds to existing evidence by revealing that effects of plasma ApoE may differ according to

16

ACCEPTED MANUSCRIPT clinical disease stage and APOEε4 carrier status. In APOEε4 carriers with SCD high plasma ApoE was associated to worse delayed recall at baseline and steeper decline in immediate recall over time, while high plasma ApoE concentration was associated to worse baseline delayed recall in APOEε4 non-carriers with MCI. If anything, these results further support the thought that high ApoE may be detrimental. These results warrant replication in a

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larger population, however. Increasingly smaller sample size due to stratification based on baseline diagnosis and APOE genotype combined with multiple comparisons may have led to false-positive results due to multiple testing and false-negative results due to

diminished statistical power. Overall results of the analyses with cognitive decline as

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outcome measure do seem plausible, though. We mainly observed associations between ApoE concentrations and memory and this domain has previously been proven to be the

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first domain to decline in early AD (van Harten et al., 2013).

Strengths of the current study include that we used a highly standardized protocol for CSF and plasma collection, storage and ApoE determination. We used a commercially available test that was validated (plasma and CSF) before use in the study. Also, we investigated a large group of non-demented subjects with substantial longitudinal follow-up and included

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interactions between ApoE concentration and APOE genotype in our analyses. In retrospect, the study may have been improved by using isoform-specific tests. Also, we might have included a larger population to be able to present our results in an APOEε4 gene-dose manner, but with the current sample size this would not have produced

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sufficiently robust results. In the current set-up of the study some of the associations we found may have been rendered non-significant due to lack of statistical power in

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increasingly small subgroups based on stratification for baseline diagnosis and APOE genotype. We included patients with SCD and MCI. This could be seen as a limit, because ApoE could have different effects along the AD continuum. In fact, we have several indications that this may be the case, because we found an interaction between CSF ApoE concentration and APOE genotype in patients with SCD when CSF Aβ42 was taken as dependent variable, while we did not find this in MCI and we found several differential effects regarding plasma ApoE when cognitive performance was taken as outcome measure. Main results regarding the predictive value of ApoE concentrations were very similar however.

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ACCEPTED MANUSCRIPT Our results suggest CSF ApoE is an important mediator of APOEε4 associated risk of dementia due to AD. High CSF ApoE concentrations may be especially detrimental because of their association with (p)Tau. Whether this is indeed a causal relationship will have to be evaluated in studies using longitudinal CSF sampling. Our results obtained in APOEε4 non-carriers underscore the importance to further elucidate disease mechanisms

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unrelated to ApoE.

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ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS

We would like to thank Hans Heijst and Mariska van der Wal for their excellent technical support. Research of the VUmc Alzheimer center is part of the neurodegeneration research program of Amsterdam Neuroscience. The VUmc Alzheimer Center is supported by Stichting

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Alzheimer Nederland and Stichting VUmc fonds. The clinical database structure was developed with funding from Stichting Dioraphte. Willem Meindert de Hoop stichting financially supported the measurement of ApoE biomarkers. Wiesje van der Flier received a grant to study SCD from

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Gieskes Strijbis fonds.

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ACCEPTED MANUSCRIPT REFERENCES Albert MS, DeKosky ST, Dickson D, Dubois B, Feldman HH, Fox NC, Gamst A, Holtzman DM, Jagust WJ, Petersen RC, Snyder PJ, Carrillo MC, Thies B, Phelps CH. The diagnosis of mild cognitive impairment due to alzheimer's disease: Recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease. Alzheimers dement 2011;7:270-79.

RI PT

Andrews-Zwilling Y, Bien-Ly N, Xu Q, Li G, Bernardo A, Yoon SY, Zwilling D, Yan TX, Chen L, Huang Y. Apolipoprotein E4 causes age- and tau-dependent impairment of gabaergic interneurons, leading to learning and memory deficits in mice. J neurosci 2010;30:13707-717. Baker-Nigh AT, Mawuenyega KG, Bollinger JG, Ovod V, Kasten T, Franklin EE, Liao F, Jiang H, Holtzman D, Cairns NJ, Morris JC, Bateman RJ. Human central nervous system (CNS) apoe isoforms are increased by age, differentially altered by amyloidosis, and relative amounts reversed in the CNS compared to plasma. J biol chem 2016;291(53):27204-18.

M AN U

SC

Bekris LM, Millard SP, Galloway NM, Vuletic S, Albers JJ, Li G, Galasko DR, DeCarli C, Farlow MR, Clark CM, Quinn JF, Kaye JA, Schellenberg GD, Tsuang D, Peskind ER, Yu CE. Multiple snps within and surrounding the apolipoprotein E gene influence cerebrospinal fluid apolipoprotein E protein levels. J alzheimers dis 2008;13:25566. Bennett RE, Esparza TJ, Lewis HA, Kim E, Mac Donald CL, Sullivan PM, Brody DL. Human apolipoprotein E4 worsens acute axonal pathology but not amyloid-β immunoreactivity after traumatic brain injury in 3xtg-AD mice. J neuropathol exp neurol 2013;72:396-403. Bertram L, McQueen MB, Mullin K, Blacker D, Tanzi RE. Systematic meta-analyses of alzheimer disease genetic association studies: The alzgene database. Nature genetics 2007;39:17-23.

TE D

Bien-Ly N, Gillespie K, Walker D, Yoon Y, Huang Y. Reducing human apolipoprotein E levels attenuates agedependent Amyloid-beta accumulation in mutant human amyloid precursor protein transgenic mice. Journal of neuroscience 2012;32:4803-11. Blair CK, Folsom AR, Knopman DD, Bray MS, Mosley TH, Boerwinkle E. APOE genotype and cognitive decline in a middle-aged cohort. Neurology 2005;64:268-76.

EP

Boyle PA, Buchman AS, Wilson RS, Kelly JF, Bennett DA. The APOE ε4 allele is associated with incident mild cognitive impairment among community-dwelling older persons. Neuroepidemiology 2010;34:43-9.

AC C

Carlsson J, Armstrong VW, Reiber H, Felgenhauer K, Seidel D. Clinical relevance of the quantification of apolipoprotein E in cerebrospinal fluid.. Clin chim acta 1991; 196: 167-176. Caselli RJ, Reiman EM. Characterizing the preclinical stages of alzheimer's disease and the prospect of presymptomatic intervention.. J alzheimers dis 2013;33 Suppl 1:S405-416. Cramer PE, Cirrito JR, Wesson DW, Lee CY, Karlo JC, Zinn AE, Casali BT, Restivo JL, Goebel WD, James MJ, Brunden KR, Wilson DA, Landreth GE. ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models. Science 2012;335:1503-06. Cruchaga C, Kauwe JS, Nowotny P, Bales K, Pickering EH, Mayo K, Bertelsen S, Hinrichs A, Fagan AM, Holtzman DM, Morris JC, Goate AM. Cerebrospinal fluid APOE levels: An endophenotype for genetic studies for alzheimer's disease. Hum mol genet 2012;21:4558-71. Darreh-Shori T, Modiri N, Blennow K, Baza S, Kamil C, Ahmed H, Andreasen N, Nordberg A.The apolipoprotein E ε4 allele plays pathological roles in AD through high protein expression and interaction with butyrylcholinesterase. Neurobiology of aging 2011;32:1236-48.

20

ACCEPTED MANUSCRIPT Elias-Sonnenschein S, Viechtbauer W, Ramakers IHGB, Verhey FRJ, Visser J. Predictive value of APOE- 4 allele for progression from MCI to ad-type dementia: A meta-analysis. Journal of neurology, neurosurgery & psychiatry 2011;82:1149-56. Fagan AM, Younkin LH, Morris JC, Fryer JD, Cole TG, Younkin SG, Holtzman DM. Differences in the abeta40/abeta42 ratio associated with cerebrospinal fluid lipoproteins as a function of apolipoprotein E genotype. Ann neurol 2000;48:201-10.

RI PT

Fitz NF, Tapias V, Cronican AA, Castranio EL, Saleem M, Carter AY, Lefterova M, Lefterov I, Koldamova R. Opposing effects of apoe/apoa1 double deletion on amyloid-β pathology and cognitive performance in APP mice. Brain 2015;138:3699-715. Folstein MF, Folstein SE, McHugh PR. "Mini-mental state," A practical method for grading the cognitive state of patients for the clinician. J psychiatr res 1975;12:189-98.

SC

Fukumoto H, Ingelsson M, Gårevik N, Wahlund LO, Nukina N, Yaguchi Y, Shibata M, Hyman BT, Rebeck GW, Irizarry MC. APOE epsilon 3/ epsilon 4 heterozygotes have an elevated proportion of apolipoprotein E4 in cerebrospinal fluid relative to plasma, independent of alzheimer's disease diagnosis. Exp neurol 2003;183:249-53.

M AN U

Harris FM, Brecht WJ, Xu Q, Tesseur I, Kekonius L, Wyss-Coray T, Fish JD, Masliah E, Hopkins PC, ScearceLevie K, Weisgraber KH, Mucke L, Mahley RW, Huang Y. Carboxyl-terminal-truncated apolipoprotein E4 causes alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice. Proc natl acad sci U S A 2003;100:10966-71. Holtzman DM, Herz J, Bu G. Apolipoprotein E and apolipoprotein E receptors: Normal biology and roles in alzheimer disease. Cold spring harb perspect med 2012;2:a006312.

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Hye A, Riddoch-Contreras J, Baird AL, Ashton NJ, Bazenet C, Leung R, Westman E, Simmons A, Dobson R, Sattlecker M, Lupton M, Lunnon K, Keohane A, Ward M, Pike I, Zucht HD, Pepin D, Zheng W, Tunnicliffe A, Richardson J, Gauthier S, Soininen H, Kłoszewska I, Mecocci P, Tsolaki M, Vellas B, Lovestone S. Plasma proteins predict conversion to dementia from prodromal disease. Alzheimers dement 2014;10:799-807.e2. Kester MI, van der Flier WM, Mandic G, Blankenstein MA, Scheltens P, Muller M. Joint effect of hypertension and APOE genotype on CSF biomarkers for alzheimer’s disease. Journal of alzheimer's disease 2010;20:1083-90.

EP

Khan W, Aguilar C, Kiddle SJ, Doyle O, Thambisetty M, Muehlboeck S, Sattlecker M, Newhouse S, Lovestone S, Dobson R, Giampietro V, Westman E, Simmons A, Alzheimer’s Disease Neuroimaging Initiative. A subset of cerebrospinal fluid proteins from a multi-analyte panel associated with brain atrophy, disease classification and prediction in alzheimer's disease. PLoS One 2015;10:e0134368.

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Kim J, Jiang H, Park S, Eltorai AE, Stewart FR, Yoon H, Basak JM, Finn MB, Holtzman DM. Haploinsufficiency of human APOE reduces amyloid deposition in a mouse model of amyloid-β amyloidosis. J neurosci 2011;31:18007-12. Liao F, Hori Y, Hudry E, Bauer AQ, Jiang H, Mahan TE, Lefton KB, Zhang TJ, Dearborn JT, Kim J, Culver JP, Betensky R, Wozniak DF, Hyman BT, Holtzman DM. Anti-ApoE antibody given after plaque onset decreases aβ accumulation and improves brain function in a mouse model of aβ amyloidosis. J neurosci 2014;34:7281-92. Liu CC, Kanekiyo T, Xu H, Bu G. Apolipoprotein E and alzheimer disease: Risk, mechanisms and therapy. Nature reviews neurology 2013;9:106-18. Liu DS, Pan XD, Zhang J, Shen H, Collins NC, Cole AM, Koster KP, Ben Aissa M, Dai XM, Zhou M, Tai LM, Zhu YG, LaDu M, Chen XC. APOE4 enhances age-dependent decline in cognitive function by down-regulating an NMDA receptor pathway in efad-tg mice. Mol neurodegener 2015;10:7. Łojkowska W, Witkowski G, Bednarska-Makaruk M, Wehr H, Sienkiewicz-Jarosz H, Graban A, Bochyńska A, Wiśniewska A, Gugała M, Sławińska K, Sawicka B, Poniatowska R, Ryglewicz D. Correlations between cerebellar

21

ACCEPTED MANUSCRIPT and brain volumes, cognitive impairments, apoe levels, and APOE genotypes in patients with AD and MCI. Curr alzheimer res 2013;10: 964-72. Mahley RW, Rall SC. Apolipoprotein E: Far more than a lipid transport protein. Annu Rev Genomics Hum Genet 2000;1:507-37.

RI PT

Martínez-Morillo E, Hansson O, Atagi Y, Bu G, Minthon L, Diamandis EP, Nielsen HM. Total apolipoprotein E levels and specific isoform composition in cerebrospinal fluid and plasma from alzheimer's disease patients and controls. Acta neuropathol 2014;127:633-43. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of alzheimer's disease: Report of the NINCDS-ADRDA work group under the auspices of department of health and human services task force on alzheimer's disease. Neurology 1984;34:939-44.

SC

McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR, Kawas CH, Klunk WE, Koroshetz WJ, Manly JJ, Mayeux R, Mohs RC, Morris JC, Rossor MN, Scheltens P, Carrillo MC, Thies B, Weintraub S, Phelps CH. The diagnosis of dementia due to alzheimer's disease: Recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease. Alzheimers dement 2011;7:263-9.

M AN U

Mulder C, Verwey A, van der Flier M, Bouwman H, Kok A, van Elk J, Scheltens P, Blankenstein A. Amyloid- (142), total tau, and phosphorylated tau as cerebrospinal fluid biomarkers for the diagnosis of alzheimer disease. Clinical chemistry 2010;56:248-53. Pankiewicz JE, Guridi M, Kim J, Asuni AA, Sanchez S, Sullivan PM, Holtzman DM, Sadowski MJ. Blocking the apoe/Aβ interaction ameliorates Aβ-related pathology in APOE ε2 and ε4 targeted replacement Alzheimer model mice. Acta neuropathol commun 2014;2:75. Petersen RC, Smith GE, Waring SC, Ivnik RJ, Tangalos EG, Kokmen E. Mild cognitive impairment: Clinical characterization and outcome. Arch neurol 1999;56:303-8.

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Petersen RC. Mild cognitive impairment as a diagnostic entity. J intern med 2004;256:183-194.

EP

Ramanan VK, Risacher SL, Nho K, Kim S, Swaminathan S, Shen L, Foroud TM, Hakonarson H, Huentelman MJ, Aisen PS, Petersen RC, Green RC, Jack CR, Koeppe RA, Jagust WJ, Weiner MW, Saykin AJ, Alzheimer’s Disease Neuroimaging Initiative. APOE and BCHE as modulators of cerebral amyloid deposition: A florbetapir PET genome-wide association study. Mol psychiatry 2014;19 351-7. Rasmussen KL, Tybjaerg-Hansen A, Nordestgaard BG, Frikke-Schmidt R. Plasma levels of apolipoprotein E and risk of dementia in the general population. Ann neurol 2015;77:301-311.

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Reitan RM. Validity of the trail making test as indicator of organic brain damage. Percept mot skills 1958;8:271-6. Rey R. L'examen clinique en psychologie. Paris. Presse Universitaire de France 1964. Scarabino D, Broggio E, Gambina G, Maida C, Gaudio MR, Corbo RM. Apolipoprotein E genotypes and plasma levels in mild cognitive impairment conversion to alzheimer's disease: A follow-up study. Am J med genet B neuropsychiatr genet 2016;171(8):1131-8. Schmidt C, Becker H, Zerr I. Cerebrospinal fluid apolipoprotein E concentration and severity of cognitive impairment in patients with newly diagnosed alzheimer's disease..Am J alzheimers dis other demen 2014;29:54-60. Sen A, Alkon DL, Nelson TJ. Apolipoprotein E3 (apoe3) but not apoe4 protects against synaptic loss through increased expression of protein kinase C epsilon. J biol chem 2012;287:15947-58. Simon R, Girod M, Fonbonne C, Salvador A, Clement Y, Lanteri P, Amouyel P, Lambert C, Lemoine J. Total apoe and apoe4 isoform assays in an alzheimer's disease case-control study by targeted mass spectrometry (n = 669): A pilot assay for methionine-containing proteotypic peptides. Molecular & cellular proteomics 2012;11:1389-1403.

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ACCEPTED MANUSCRIPT Slooter AJ, Cruts M, Ott A, Bots ML, Witteman JC, Hofman A, Van Broeckhoven C, Breteler MM, van Duijn CM. The effect of APOE on dementia is not through atherosclerosis: The rotterdam study. Neurology 1999;53:1593-5. Song F, Poljak A, Crawford J, Kochan NA, Wen W, Cameron B, Lux O, Brodaty H, Mather K, Smythe GA, Sachdev PS. Plasma apolipoprotein levels are associated with cognitive status and decline in a community cohort of older individuals.. PLoS one 2012;7:e34078.

RI PT

Strittmatter WJ, Saunders AM, Goedert M, Weisgraber KH, Dong LM, Jakes R, Huang DY, Pericak-Vance M, Schmechel D, Roses AD. Isoform-specific interactions of apolipoprotein E with microtubule-associated protein tau: Implications for alzheimer disease. Proc natl acad sci U S A 1994;91:11183-6. Talwar P, Sinha J, Grover S, Agarwal R, Kushwaha S, Srivastava MV, Kukreti R. Meta-analysis of apolipoprotein E levels in the cerebrospinal fluid of patients with alzheimer's disease. J neurol sci 2016;360:179-187.

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Toledo JB, Da X, Weiner MW, Wolk DA, Xie SX, Arnold SE, Davatzikos C, Shaw LM, Trojanowski JQ, Alzheimer’s Disease Neuroimaging Initiative. CSF apo-e levels associate with cognitive decline and MRI changes.. Acta neuropathol 2014;127:621-632.

M AN U

van Harten AC, Smits LL, Teunissen CE, Visser PJ, Koene T, Blankenstein MA, Scheltens P, van der Flier WM. Preclinical AD predicts decline in memory and executive functions in subjective complaints. Neurology 2013;81:1409-16. Vijayaraghavan S, Maetzler W, Reimold M, Lithner CU, Liepelt-Scarfone I, Berg D, Darreh-Shori T. High apolipoprotein E in cerebrospinal fluid of patients with lewy body disorders is associated with dementia. Alzheimers dement 2014;10:530-540.e1. Wang C, Yu JT, Wang HF, Jiang T, Tan CC, Meng XF, Soares HD, Tan L. Meta-analysis of peripheral blood apolipoprotein E levels in alzheimer's disease. PLoS one 2014;9:e89041.

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Wolters FJ, Koudstaal PJ, Hofman A, van Duijn CM, Ikram MA. Serum apolipoprotein E is associated with longterm risk of alzheimer's disease: The rotterdam study. Neurosci lett 2016;617:139-142. Yasuno F, Tanimukai S, Sasaki M, Ikejima C, Yamashita F, Kodama C, Hidaka S, Mizukami K, Asada T. Effect of plasma lipids, hypertension and APOE genotype on cognitive decline. Neurobiol aging 2012;33:2633-40.

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Yu JT, Tan L, Hardy J.Apolipoprotein E in alzheimer's disease: An update. Annu rev neurosci 2014;37:79-100.

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SCD

MCI

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Table 1: baseline characteristics according to baseline and follow-up diagnosis.

Stable

Clinical progression All

stable

N

207

181

26

223

132

Age, years

61.0±8.8

60.3±8.8

66.0±7.6**

67.1±8.2***

65.4±8.0

69.5±7.9***

Sex, female

86 (42%)

77 (43%)

9 (35%)

94 (42%)

45 (34%)

49 (54%)**

Baseline MMSE

28.3±1.6

28.3±1.6

27.9±1.7

26.6±2.4***

26.9±2.2

26.1±2.6*

Follow-up duration, years

2.8±1.7

2.8±1.7

2.5±2.1

2.3±1.3

2.4±1.4

2.2±1.3

Aβ42, ng/L

840 (232-1370)

888 (2931363)

547 (232-1370)***

552 (178-1545)*** 680 (255-1545) 473 (178-1012)***

Tau, ng/L

248 (69-2088)

243 (69-2088) 390 (116-993)***

PTau, ng/L

46 (18-190)

44 (18-190)

58 (20-125)**

APOEε4 carrier

86 (42%)

70 (39%)

16 (62%)*

ApoE CSF, mg/L†

3.1 (0.7-9.3)

3.0 (0.7-7.9)

3.2 (1.5-9.3)

ApoE plasma, mg/L‡

28.6 (9.2-76.1)

28.6 (9.2-76.1

28.8 (16.4-58.7)

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91

310 (80-1650)

673 (165-3515)***

69(16-268)***

54 (16-199)

87 (27-268)***

130 (58%)**

67 (51%)

63 (69%)**

3.6 (0.4-9.0)**

3.5 (0.4-7.1)

3.8 (1.0-9.0)*

27.8 (4.7-73.1)

27.4 (4.7-73.1)

28.8 (10.5-57.4)

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423 (80-3515)***

Clinical progression

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All

Data are presented as mean±SD, no (%) or median (range). †n=403. ‡n=412. CSF and plasma biomarker concentrations were logtransformed and converted to z-scores prior to analyses. All MCI patients were compared to all patients with SCD and within each diagnostic group patients who showed clinical progression were compared to patients who remained stable. *p<0.05; **p<0.01; ***p<0.001.

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Figure 1: Association between CSF ApoE concentration and known risk factors for dementia due to AD.

A: correlation between age and CSF ApoE (r=0.18, p<0.001). B: Association between gender and CSF ApoE (3.6 [1.0-9.0] vs. 3.2 [0.4-9.3] mg/L, p<0.05). C: Association between baseline diagnosis and CSF ApoE stratified for APOEε4 genotype (APOEε4 non-carriers carriers vs. carriers in SCD 2.9 [0.7-6.4] vs. 3.4 [1.3-9.3] mg/L, p=0.001; APOE ε4 non-carriers carriers vs. carriers in MCI 2.8 [0.4-7.1] vs. 4.1 [1.1-9.0] mg/L, p<0.001. CSF ApoE in APOEε4 carriers with SCD vs. those with MCI 3.4 [1.3-9.3] vs. 4.0 [1.1-9.0] mg/L, p=0.012). 25

ACCEPTED MANUSCRIPT Table 2: Predictive value of CSF and plasma ApoE for clinical progression along the AD continuum over time.

CSF ApoE

1 2

3 4

Plasma ApoE

APOEε4 non-carriers APOEε4 carriers

Subjects All 1.3 (1.1-1.6) 1.0 (0.8-1.4) 1.5 (1.1-2.0)

SCD 1.2 (0.8-2.0) 0.9 (0.5-1.7) 1.5 (0.8-2.9)

APOEε4 non-carriers APOEε4 carriers APOEε4 non-carriers APOEε4 carriers

1.2 (0.9-1.5) 1.4 (1.1-1.8) 1.0 (0.8-1.4) 0.8 (0.6-1.1)

0.8 (0.4-1.5) 0.9 (0.5-1.7) 0.7 (0.4-1.3) 0.7 (0.4-2.2)

1.2 (0.9-1.7) 1.5 (1.1-2.2) 1.1 (0.8-1.2) 0.9 (0.6-1.3)

1.0 (0.8-1.2)

1.0 (0.6-1.6)

1.0 (0.8-1.2)

1 2

MCI 1.3 (1.1-1.7) 1.1 (0.8-1.5) 1.5 (1.1-2.1)

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APOEε4 non-carriers 1.1 (0.9-1.3) 1.1 (0.7-1.8) 1.1 (0.8-1.3) APOEε4 carriers Data are presented as HR (95% CI). CSF ApoE: n=403. Plasma ApoE: n=412. ApoE concentrations were log-transformed and transformed to z-scores prior to analysis. A HR of 1.5 therefore means 1 SD increase in log-transformed ApoE concentration increases the risk of clinical progression 1.5 times at each moment in time: 2 SD increase in log-transformed ApoE concentration increases risk of clinical progression 3 times; 3 SD increase, 4.5 times etc. Model 1: Cox regression model adjusted for age, sex, baseline MMSE and baseline diagnosis. P-values for significant analysis in model 1 with CSF ApoE as predictor: all subjects: p=0.012; MCI: p=0.016. Model 2: Model 1 with additional inclusion of APOE genotype. When we found an interaction (p<0.05) between ApoE concentration and APOE genotype, results were stratified for dichotomized APOE genotype. P-values for significant analyses in model 2 with CSF ApoE as predictor: all APOEε4 carriers p=0.01; APOEε4 carriers with MCI p=0.021. Model 3: model 2 with additional adjustment for baseline CSF Aβ42. P-values for significant analysis in model 3: all APOEε4 carriers p=0.023; all APOEε4 carriers with MCI p=0.012. Model 4: model 3 with additional adjustment for baseline CSF Tau.

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Figure 2: Kaplan Meier curves according to CSF ApoE tertiles. B: APOEε4 carriers

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A: APOEε4 non-carriers

Unadjusted Kaplan-Meier curves for clinical progression with separate lines for CSF ApoE

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tertiles: green=tertile 1, blue=tertile 2, purple=tertile 3. Numbers at baseline and entering the second year interval are depicted below each figure. Censored cases are indicated as vertical

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lines. Kaplan-Meier curves depict the number of subjects that still have their baseline diagnoses at a certain follow-up with 1.0 indicating 100% and 0.6 indicating 60%.

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Model 1

CSF ApoE

Aβ42 Tau pTau

-0.02 (-0.11: 0.08) 0.39 (0.31: 0.47) 0.43 (0.36: 0.51)

Model 2 APOEe4 nonAPOEe4 carriers carriers 0.09 (0.00: 0.18) 0.29 (0.19: 0.39) 0.51 (0.38: 0.65) 0.36 (0.25: 0.46) 0.53 (0.40: 0.66)

Plasma ApoE

Aβ42 Tau pTau

0.14 (0.05: 0.22) -0.07 (-0.15: 0.02) -0.12 (-0.21: -0.04)

0.05 (-0.04: 0.13) -0.03 (-0.12: 0.05) -0.09 (-0.17: -0.00)

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Data are depicted as standardized Beta (95% CI). CSF: n=403, plasma: n=412. Model 1: linear

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regression model adjusted for age, sex, and baseline MMSE and baseline diagnosis. Model 2: Model 1 with additional inclusion of APOE genotype. As we found APOE genotype*ApoE

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concentration interactions in CSF (p=0.006 for Tau; p=0.02 for pTau), we stratified results for

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Tau and pTau in model 2.

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carriers.

A: Association between CSF ApoE (mg/L) and CSF Tau (ng/L) in APOEε4 non-carriers. B: Association between CSF ApoE and CSF Tau in APOEε4 carriers. Figure B does not show an outlier (Tau 3515 ng/L and ApoE 4,58 mg/L). For corresponding linear regression models (after log-transformation and transformation to z-scores of the data) see Table 3.

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Supplementary figure 1: correlations between CSF ApoE concentration and plasma ApoE concentrations, separated by APOE genotype.

A: Association between CSF ApoE concentration and plasma ApoE concentration in APOEε4 non-carriers (correlation after log-transformation: r=0.03, p=0.68). B: Association between CSF ApoE concentration and plasma ApoE concentration in APOEε4 carriers (correlation after log-transformation: r=0.12, p=0.09).

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Supplementary Table 1: Association between CSF ApoE and cognitive performance. APOE genotype

TMT A

TMT B

Estimated effect on

Estimated effect

Estimated effect on

Estimated effect

baseline scores

over time

baseline scores

over time

baseline scores

over time

All

-0.02

-0.10

0.09

-0.09

0.03

-0.09

APOEε4 carriers

-0.09

-0.11

0.22

0.02

-0.41

-0.15

APOEε4 non-carriers

0.01

0.05

-0.00

-0.09

0.07

0.14

All

-1.04*

-0.19

-0.31

-0.60

-0.62

0.15

APOEε4 carriers

-1.94*

0.03

-1.41

-0.34

-0.88

0.20

APOEε4 non-carriers

-0.42

-0.25

-0.21

-0.85

-0.13

0.26

All

-0.37*

-0.04

-0.10

-0.12

-0.19

0.02

APOEε4 carriers

-0.67*

0.01

-0.44

-0.09

-0.21

0.06

APOEε4 non-carriers

-0.05

-0.03

-0.06

-0.13

0.10

0.04

All

-1.23

0.37

-0.83

-0.45

-1.94

0.15

APOEε4 carriers

-1.71

0.10

-3.71

0.48

-0.77

-0.28

APOEε4 non-carriers

0.28

0.57

0.26

-0.21

-0.30

All

-3.53

0.31

-1.27

0.81

-9.59

0.48

APOEε4 carriers

3.30

0.10

-4.76

1.54

7.28

-0.06

APOEε4 non-carriers

-5.49

-0.45

0.82

-0.37

-16.37

0.48

-0.19

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Estimated effect

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MCI

Estimated effect on

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RAVLT total

SCD

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MMSE

All subjects

MMSE: n= 376 RAVLT: n= 355, TMTA: n= 371, TMTB: n= 362. Data are presented as beta’s with p-values. *p<0.05. Analyses were adjusted for age and sex. MMSE = mini mental status examination; RAVLT = the Dutch version of the Rey Auditory Verbal Learning Task (total immediate recall and delayed recall), a 1 point increase means 1 more word recalled; TMT A = part A of the trail making test as measured in 31

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seconds. TMT B = part B of the trail making test as measured in seconds. Please note that a higher score on the MMSE, RAVLT total and RAVLT delayed indicates better performance, while a higher score on the TMT A and TMT B indicates worse performance. Additional associations that reached trend-level, (p<0.1) but not significance were: the association between CSF ApoE and decline over time in RAVLT

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total performance in all SCD patients and in APOEε4 non carriers with SCD (beta -0.60 and -0.85 respectively).

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Supplementary table 2: Association between plasma ApoE and cognitive performance.

RAVLT delayed

TMT A

TMT B

MCI

Estimated effect

Estimated effect

Estimated effect

Estimated effect

on baseline scores

effect over time

on baseline scores

over time

on baseline scores

over time

All

-0.18

0.04

-0.17

-0.03

-0.29

0.03

APOEε4 carriers

-0.26

-0.00

-0.26

-0.17

-0.30

-0.01

APOEε4 non-carriers

-0.09

-0.02

-0.06

0.06

-0.19

-0.03

All

-0.07

-0.15

-0.34

-0.82*

-0.45

0.02

APOEε4 carriers

-0.59

-0.25

-1.49

-1.00*

-0.65

-0.11

APOEε4 non-carriers

0.27

-0.11

0.79

-0.38

-0.74

0.15

All

-0.14

0.05

-0.28

-0.02

-0.33

0.04

APOEε4 carriers

-0.37

-0.01

-0.90*

-0.07

-0.35

-0.01

APOEε4 non-carriers

-0.11

0.09

0.24

0.10

-0.70*

0.10

All

-0.59

-0.37

-0.17

-0.11

-0.61

APOEε4 carriers

-1.87

-0.89

-1.59

-0.05

-1.56

-1.02

APOEε4 non-carriers

-0.70

0.19

0.32

-0.40

-1.29

0.41

All

-0.89

-1.53

-050

0.59

2.28

-1.67

APOEε4 carriers

-3.67

-1.09

-1.70

1.27

-2.59

-1.47

APOEε4 non-carriers

-0.99

-0.50

-0.30

0.66

-0.18

-1.50

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SCD

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MMSE

All subjects

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MMSE: n = 384, RAVLT: n=358, TMTA n = 379, TMTB n = 370. Data are presented as beta’s with p-values. *p<0.05. Analyses were adjusted for age and sex. MMSE = mini mental status examination; RAVLT = the Dutch version of the Rey Auditory Verbal Learning Task (total immediate recall and delayed recall), a 1 point increase means 1 more word recalled; TMT A = part A of the trail making test as measured in seconds. TMT

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B = part B of the trail making test as measured in seconds. Please note that a higher score on the MMSE, RAVLT total and RAVLT delayed indicates better performance, while a higher score on the TMT A and TMT B indicates worse performance. Additional associations that reached trend level (p<0.1), but not significance were: the association between plasma APOE and baseline MMSE in all APOEε4 carriers (beta -0.26), the

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association between plasma ApoE and baseline MMSE in MCI patients (beta -0.29) and the association between plasma ApoE and baseline

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RAVLT delayed in all MCI patients (beta -0.33).

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Supplementary table 3A: linear regression models for the association between ApoE concentrations in CSF/plasma and classical CSF biomarkers for patients with SCD. Outcome

Model 1

CSF ApoE

Aβ42 Tau pTau

-0.02 (-0.14: 0.11) 0.34 (0.25: 0.44) 0.38 (0.28: 0.48)

Plasma ApoE

Aβ42 Tau pTau

0.11 (-0.003:0.23) -0.02 (-0.13: 0.09) -0.05 (-0.16: 0.06)

Model 2 APOEe4 non-carriers 0.10 (-0.04: 0.23) 0.32 (0.20: 0.44) 0.37 (0.25: 0.48)

APOEe4 carriers -0.15 (-0.38: 0.08) 0.43 (0.28: 0.59) 0.44 (0.26: 0.63)

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0.06 (-0.06: 0.17) 0.00 (-0.11: 0.11) -0.04 (-0.15: 0.08)

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Data are presented as standardized Beta (95% CI). CSF: n=191. Plasma: n=202. Model 1: linear regression model corrected for age, sex and baseline MMSE. Model 2: Model 1 with additional inclusion of APOE genotype. As in the models taking all non-demented patients into account, we stratified for APOE genotype in model 2 when CSF ApoE was the predictor. In patients with SCD, the interaction between CSF ApoE and APOE genotype was significant when CSF Aβ42 was the outcome measure (p<0.05), but not when Tau or pTau were taken as outcome measures.

Supplementary table 3B: linear regression models for the association between ApoE concentrations in CSF and plasma and classical CSF biomarkers for patients with MCI. Outcome

Model 1

CSF ApoE

Aβ42 Tau pTau

-0.02 (-0.16: 0.12) 0.43 (0.30: 0.55) 0.48 (0.37: 0.60)

Aβ42 Tau pTau

0.17 (0.03: 0.29) -0.12 (-0.25: 0.01) -0.20 (-0.32: -0.07)

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Plasma ApoE

Model 2 APOEe4 non-carriers APOEe4 carriers 0.15 (0.02: 0.28) 0.25 (0.08: 0.43) 0.54 (0.35: 0.74) 0.34 (0.16: 0.53) 0.57 (0.39: 0.75)

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Predictor

0.03 (-0.10: 0.15) -0.07 (-0.20: 0.06) -0.14 (-0.27: -0.01)

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Data are presented as standardized Beta (95% CI). CSF: n=212. Plasma: n=210. Model 1: linear regression model corrected for age, sex and baseline MMSE. Model 2: Model 1 with additional inclusion of APOE genotype. As in the models taking all non-demented patients into account, we stratified for APOE genotype in model 2 when CSF ApoE was the predictor. In patients with MCI, the interaction between CSF ApoE and APOE genotype was significant when CSF Tau (p<0.05) was taken as outcome measure. We found a trend for an interaction when CSF pTau was taken as outcome measure (p<0.10).

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