Apolipoprotein E imbalance in the cerebrospinal fluid of Alzheimer’s disease patients

The CSF samples were obtained by lumbar puncture and centrifuged (2000×g, 10 min) and then immediately aliquoted and stored in ultrafreezers and kept at −80°C until analysis. The time between CSF acquisition and storage was less than 4 h in all cases. The handling of the samples was performed following recommended operating procedures [24]. Freeze-thaw cycles were avoided and new aliquots were used for each independent analysis.

The first cohort was from the longitudinal geriatric population study in Piteå, Sweden [25], the Piteå Dementia Project. The diagnostic evaluation included a clinical examination (detailed medical history and somatic, neuropsychiatric, and neurological status), a neuropsychological test battery, routine blood and CSF tests, and a CT scan to exclude secondary dementias [26]. All clinical diagnoses and evaluations were made without knowledge of the results of the biochemical analyses and vice versa. The cohort consisted of 45 patients with AD (fourteen men and thirty-one women, mean age 77±1 years) and was selected based on the APOE-ε4 status, so that fifteen each had APOE ε3/ε3, APOE ε3/ε4, or APOE ε4/ε4. In addition, fourteen non-AD controls [seven men and seven women, mean age (67 ± 3 years); APOE ε3/ε3: 9, APOE ε3/ε4: 5] were included. APOE genotype was determined by the solid-phase mini-sequencing method as previously described [27]. For this study, patients who were designated as AD or controls also had typical core CSF biomarker levels [Aβ42 and total tau (T-tau)] using cut-offs that are >90% specific for AD [28], but except for CSF Aβ42 and T-tau, all biochemical analyses were made without knowledge of the clinical data. The ethics committees in Umeå University and University of Gothenburg approved the study.

The second cohort was obtained from the Sant Pau Initiative on Neurodegeneration (SPIN cohort) [29] from Hospital Sant Pau (Barcelona, Spain). We included samples from 29 AD patients (thirteen men and sixteen women, mean age 73±1 years; APOE: 10 ε3/ε3, 10 ε3/ε4, 9 ε4/ε4) and ten controls (seven men and three women, mean age 69±2 years; APOE: 5 ε3/ε3, 5 ε3/ε4). Typically, these are patients who present cognitive complaints and are referred to the specialized memory unit from their primary care physician. All patients undergo a full neuropsychological evaluation that demonstrates objective cognitive impairment. Patients were included in the cohort when they presented supportive biomarkers of the AD pathophysiological process. Cognitively normal participants were volunteers without cognitive complaints and normal neuropsychological evaluation. More details about inclusion/exclusion criteria and neuropsychological tests in this cohort are detailed elsewhere [29].

In this cohort, the APOE genotype was determined by direct DNA sequencing and visual analysis of the resulting electropherogram performed to identify the two coding polymorphisms that encode the three possible apoE variants [29].

Each center applied their own internally validated cut-offs, according to their preanalytical and analytical particularities. More details about the cut-offs applied are indicated below. Samples were retrospectively selected from large cohorts to balance age, sex, and APOE status. Most of the selected cases (92%, 43 of 45 from Gothenburg and 25 of 29 from Barcelona) were categorized A+T+ according to [30]; thus, subgrouping by the AT(N) system for analysis was impractical. For full details about the collections, see Table 1.

In the cohort from Gothenburg, the levels of the AD core biomarkers T-tau, P-tau, and Aβ42 were measured in the CSF using INNOTEST ELISAs (Fujirebio-Europe, Gent, Belgium). Patients were designated as AD or controls according to CSF biomarker levels using cut-offs that are >90% specific for AD: Aβ42 <550 pg/mL and total tau (T-tau) >400 pg/mL [20].

For the cohort from Barcelona, cut-offs for AD biomarkers measured in the Lumipulse automated platform (Fujirebio-Europe) were T-tau > 400 pg/mL, P-tau > 63 pg/mL, and 0.062 for the Aβ42/Aβ40 ratio [29].

All samples were analyzed as part of a clinical routine by board-certified laboratory technicians following strict procedures for batch-bridging, analyses, and quality control of individual ELISA plates.

The experiments were carried out using a cohort of 107 rats (53 males and 54 females), including transgenic TgF344-AD rats (n = 52) expressing mutant human APP (APPsw) and presenilin-1 (PS1ΔE9) genes [31] and wild-type Fischer rats (n = 55). Rats were bred in the animal research facilities at the University of Barcelona. Animals were provided with food and water ad libitum and maintained in a temperature-controlled environment in a 12/12-h light-dark cycle. CSF samples (50–100 μL) were collected from ketamine/xylazine-anesthetized animals by cisternal puncture with a glass capillary in the suboccipital region through the atlanto-occipital membrane, with a single incision into the subarachnoid space [32]. CSF aliquots from different time points [4 months: 16 wild-type (8 male, 8 female) and 16 TgF344-AD animals (8 male, 8 female); 10.5 months: 17 wild-type (8 male, 9 female) and 16 TgF344-AD animals (8 male, 8 female); 16.5 months: 22 wild-type (12 male, 10 female) and 20 TgF344-AD animals (9 male, 11 female)] were analyzed. This study was part of a large project assessing various different proteins that included brain analysis at each stage; thus, it was not possible to perform longitudinal measurements in the same animal (repeat sampling) to reduce the number of animals. Animal work was performed in accordance with the local legislation, with the approval of the Experimental Animal Ethical Committee of the University of Barcelona, and in compliance with European legislation.

Following electrophoresis, proteins were blotted onto 0.45-μm nitrocellulose membranes (Bio-Rad Laboratories, GmbH, Munich, Germany). Bands of apoE immunoreactivity were detected using either the antibody AB178479 (goat polyclonal; Merck Millipore) or the antibody AB947 (goat polyclonal; Merck Millipore), both common to all apoE isoforms, or alternatively by an antibody specific to the apoE4 isoform (recognizes an internal domain comprising the Arg112 residue present exclusively in apoE4 species; mouse monoclonal, Novus Biologicals; NBP1-49529). Blots were then probed with the appropriate conjugated secondary antibodies (IRDye secondary antibodies, LI-COR Biosciences, Lincoln, NE, USA) and imaged on an Odyssey CLx Infrared Imaging System (LI-COR Biosciences). Band intensities were analyzed using LI-COR software (ImageStudio Lite). The boxes selected with the ImageQuant Studio software for quantification, as well as the completed blots, are shown as supplementary figures. Recombinant apoE3 (Peprotech, ThermoFisher Scientific# 350-02) was included into each blot to serve as a loading reference and for normalizing the immunoreactivity signal between blots. Specifically, the same amount of recombinant apoE3 was always included, and the immunoreactivity of the apoE bands from each blot was referred to (divided by) the immunoreactivity of recombinant apoE3, thus correcting inter-blot differences and allowing for comparisons across assays.

For blue-native gel electrophoresis, the CSF samples were not heated (native conditions) and were loaded with NuPage LDS 4× Sample Buffer (ThermoFisher Scientific, NP007) into native-PAGE 4–16% gels (ThermoFisher Scientific, BN1002BOX). Buffers were prepared using native-PAGE Running Buffer (ThermoFisher Scientific, BN2001) and native-PAGE Cathode Buffer Additive (ThermoFisher Scientific, BN2002). Immunoreactivity was detected using the AB178479 antibody and HRP anti-goat secondary antibody (ThermoFisher). The signal was visualized by ECL (GE Healthcare Life Science) and analyzed using ImageStudio Lite.

CSF samples (50 μL) were incubated on a roller overnight with 100 μL PureProteome FlexiBind Magnetic Beads (Merck Millipore, LSKMAGN04) coupled with the AB178479 apoE antibody (Merck Millipore). The supernatant was removed, and the beads were washed and then resuspended and boiled at 98 °C for 5 min in SDS-PAGE sample buffer and analyzed by western blot with the AB947 antibody (Merck Millipore) or anti-apoE4 antibody (Novus Biologicals, NBP1-49529). For a control immunoprecipitation, beads were coupled with horse serum and then incubated with CSF samples.

Enzymatic deglycosylation was performed using an Agilent Enzymatic Deglycosylation Kit (Agilent Technologies, GK80110) following the manufacturer’s instructions. Briefly, for each condition, 30 μL of control or AD CSF was mixed with 10-μl incubation buffer and 2.5-μL denaturing buffer and heated at 100 °C for 5 min. The samples were then cooled down to room temperature, and 2.5 μL of detergent (15% NP-40) was added while mixing gently. O- (1 μL sialidase and 1 μL O-glycanase) or N-linked (1 μL N-glycanase) deglycosylating enzymes were then added according to each different condition (O-linked, N-linked, or O- and N-linked deglycosylation) and samples were heated at 37 °C for 3 h. Samples were then analyzed by western blot. As for control of the deglycosylation process, samples exposed to the same heating conditions but without deglycosylating enzymes were included.

In-gel digestion was performed as previously described [33] in order to investigate the content of western blot immunoreactive bands of interest using an antibody-free method. Briefly, 1 mL of a pool of AD CSF (APOE ε3/ε4 and APOE ε3/ε4 cases) was immunoprecipitated with AB178479 antibody and loaded into SDS-polyacrylamide gel under reducing conditions, as described above. ApoE3 and apoE4 recombinant proteins (Peprotech, ThermoFisher Scientific# 350-02 and 350-04) were also loaded in the gel (10 pmol) and used as a reference for band excising and positive control. Upon electrophoresis, the gel was divided into two parts, one for protein visualization by SimplyBlue^TM SafeStain Coomassie (ThermoFisher Scientific, cat# LC6060) and one for blotting with the AB947 antibody as confirmation of band presence and location. Bands of interest were cut-out from the AD CSF gel lane and recombinant protein lanes and destained using a 1:1 mixture of acetonitrile and 50 mM ammonium bicarbonate solution twice for 15 min. Furthermore, gel pieces were de-hydrated with 100% acetonitrile and dried using a vacuum centrifuge. Samples were subsequently reduced with 10 mM dithiothreitol (DTT) for 1 h at 56 °C and alkylated with 25 mM iodoacetamide (IAA) for 45 min at room temperature in the dark. Gel pieces were further washed with 25mM ammonium bicarbonate, de-hydrated with 100% acetonitrile, and dried using a vacuum centrifuge once more. Samples were digested overnight at 37°C using 100 ng/μL trypsin enzyme (Sequencing Grade Modified Trypsin, #V511A, Promega). The next day, digestion was stopped by the addition of 2% trifluoroacetic acid and 75% acetonitrile solution, and peptides were collected into a new tube (Costar, #3207). Gel pieces were further extracted with the addition of 50% acetonitrile and 0.2% trifluoroacetic acid solution shaking for 30 min. The supernatant containing the peptides was transferred to the collection tube. Pooled extracts for each gel piece were dried through vacuum centrifugation and stored at −80 °C pending mass spectrometry (MS) analysis.

Dried in-gel digested samples were reconstituted in 7 μL 8% acetonitrile/8% formic acid solution and shaken for 30 min. A total of 6 μL of each sample was investigated using mass spectrometry (MS) analysis performed with a Dionex 3000 nanoflow liquid chromatography system coupled to a Q Exactive (both Thermo Fisher Scientific). Briefly, a reversed phase Acclaim PepMap C18 (100 Å pore size, 3 μm particle size, 20 mm length, 75 μm i.d., Thermo Fisher Scientific) trap column was used for online desalting and sample clean-up. Separation was performed with a reversed phase Acclaim PepMap RSLC C18 (100 Å pore size, 2 μm particle size, 75 μm i.d., 150 mm length, Thermo Fisher Scientific) column at a flow rate of 300 nL/min by applying a linear gradient of 0–40% B for 50 min. Mobile phase A was 0.1% formic acid in water (v/v) and mobile phase B was 0.1% formic acid and 84% acetonitrile in water (v/v/v).

Mass spectra were acquired in positive ion mode and in a data-dependent manner with a resolution setting of 70,000 for precursor and 17,500 for fragment ion acquisitions. Fragmentation was obtained by higher energy collision-induced dissociation (HCD) using a normalized collision energy (NCE) setting of 28. Database searches were made using PEAKS Studio XPRO (Bioinformatic Solutions, Inc., Waterloo, Canada).

All the data was analyzed using GraphPad Prism (version 7; GraphPad software, San Diego, CA, USA). The test was used to analyze the distribution of each variable. Firstly, multiple comparisons were performed between groups, ANOVA was used for parametric variables, and the Kruskal-Wallis test for non-parametric variables. A Student’s t-test for parametric variables and a Mann-Whitney U test for non-parametric variables were employed for comparison between two groups and for determining precise p values. For correlations, the Pearson and Spearman tests were used. The results are shown as means ± SEM; the standard deviation (SD) and median values are also displayed as indicated in the figure legends.

To determine whether altered CSF apoE levels could be indicative of pathology-associated changes, we initially examined them in a rat transgenic AD model. The TgF344-AD rat expresses human APP with the Swedish mutation and human PSEN1 with the Δ exon 9 mutation. As mentioned above, while humans have three versions of the APOE gene, other mammals such as rats only have one isoform of the apoE protein, which presents Arg at position 112 (https://​web.​expasy.​org/​variant_​pages/​VAR_​000652.​html) and thus shares the inability to form disulfide-linked dimers with human apoE4. We examined apoE levels in the CSF of 4-, 10.5-, and 16.5-month-old transgenic rats and wild-type littermates by SDS-PAGE using the AB178479 antibody. In all the animals and at all ages, apoE appeared as a single ~35-kDa band (Fig. 1A) and no differences were found between males and females (p> 0.05 for the comparison at every age). We did not find different glycoforms. Although no differences were found at 4 and 10.5 months between wild-type and TgF344-AD rats, a trend of apoE increment was observed. At 16.5 months of age, apoE levels were 50% higher in TgF344-AD animals than in wild-types (p = 0.003, Fig. 1B). The significant differences for CSF apoE levels detected between TgF344-AD and controls at 16.5 months of age were maintained when the animals were subgrouped by gender (male: control vs TgF344: p = 0.019; female: control vs TgF344: p = 0.048).

We examined the presence of apoE species in CSF samples by SDS-PAGE and western blot under reducing conditions (in presence of the reducing agent β-mercaptoethanol that breaks disulfide bonds) from a cohort of control and AD patients from Gothenburg (Sweden; see Table 1) expressing different APOE genotypes.

In all CSF samples, using the AB178479 antibody, apoE appeared as two distinct immunoreactive bands of ~34 and ~36 kDa (Fig. 2A). Immunoprecipitation with this antibody and subsequent immunoblotting with an alternative apoE antibody, AB947, confirmed the characterization of both apoE monomeric species in APOE ε3/ε3 samples (Fig. 2B). The same occurred when using an anti-apoE4 antibody in APOE ε3/ε4 samples (Fig. 2C). An apoE band of ~100 kDa was also observed, almost exclusively in the AD CSF samples, and this band was immunoprecipitated similarly to monomers (Fig. 2A–C). ApoE3 and apoE2 isoforms form disulfide-linked dimers in CSF, but these dimers should be sensitive to the reducing agent β-mercaptoethanol. Additional bands between the monomers and the 100-kDa bands did not appear to follow any specific pattern related with the pathology condition or the APOE genotype and were not consistently represented in the immunoprecipitated fraction; therefore, they were not considered for further investigations. Immunoprecipitated complexes of 100 kDa, using the AB178479 antibody, were dissected after electrophoresis and examined by MS analysis identifying 14 tryptic peptides spanning throughout the sequence of human apoE (Uniprot entry P02649_HUMAN), and both apoE3 and apoE4 isoforms were detected. Matching sequences are displayed in Table 2.

We first aimed to understand why the monomers appeared as two distinct bands with different molecular masses. We hypothesized that the bands likely represent different glycoforms of the protein and, thus, an enzymatic deglycosylation assay was performed. While, as expected, N-deglycosylation did not alter the apoE band pattern, O-deglycosylation simplified the apoE pattern to a single immunoreactive band, suggesting that O-glycosylation could account for the differences in the molecular mass between the 36- and 34-kDa apoE species (Fig. 2D). The small differences in the electrophoretic mobility between glycosylated and deglycosylated apoE monomers are probably related to the slight carbohydrate mass associated to O-glycosylation, but also to changes in protein shape that affect their electrophoretic migration. Glycosylated proteins are normally more globular than non-glycosylated proteins, because the carbohydrate chains are not linear, even in reducing conditions. The apparent levels of the 100-kDa apoE band were not significantly modified following enzymatic deglycosylation, suggesting that these species are resistant to enzymatic deglycosylation (Fig. 2D). We observed the occurrence of apoE dimers in the recombinant protein (a non-glycosylated species since it is produced in bacteria), suggesting that sugar residues are not relevant epitopes for disulfide-linked dimer formation.

As previously mentioned, the ~100-kDa apoE band was observed almost exclusively in AD CSF samples, including APOE ε4/ε4 samples (Fig. 2A). To further characterize this apoE species, we performed SDS-PAGE studies in non-reducing conditions to preserve the disulfide bonds (absence of β-mercaptoethanol). The 100-kDa apoE band in AD samples appeared to be indistinguishable from the one observed in reducing conditions, and remarkably, this band appeared in the control samples in non-reducing conditions, probably representing apoE dimers linked by disulfide bonds (Fig. 3A). When a specific antibody for apoE4 was used, NBP1-49529, the 100-kDa immunoreactivity was also detected in samples from APOE ε3/ε4 AD subjects under both reducing and non-reducing conditions, while no immunoreactivity was detected in APOE ε3/ε4 control samples under non-reducing conditions (Fig. 3B). This could corroborate that apoE4 in AD samples participates in complexes to form 100-kDa stable species, in both apoE3/4 or apoE4/4 subjects, that do not rely on disulfide bonds, due to its lack of Cys112, thus representing aberrant/anomalous apoE aggregates. In APOE ε3/ε4 control subjects, however, apoE4 does not participate in the 100-kDa species observed under non-reducing conditions, given its complete reliance on disulfide bonds to form functional dimers.

To determine the different contributions of the apoE 100-kDa species in AD and control cases, we first estimated the apoE dimer/monomer balance by native-PAGE electrophoresis. We included a CSF control sample under fully reducing and denaturing conditions, as well as an apoE3 recombinant protein under native conditions, which served to identify the monomeric and dimeric apoE bands. Two immunoreactive apoE bands were observed, likely representing apoE monomers and dimers (Fig. 3C). An immunoreactive band compatible with dimeric complexes was detected in CSF from AD APOE ε4/ε4 cases, whose lack of Cys112 should eliminate their ability to form disulfide-bond-dependent complexes. Given the difficulty of finding age-matched APOE ε4/ε4 control subjects (low prevalence of this genotype in the general and healthy population), we cannot compare AD APOE ε4/ε4 with control ε4/ε4 cases. We compared the apoE dimer/monomer quotient (ratio D/M) between AD CSF samples and controls, subgrouping the samples by APOE genotype (Fig. 3D). In the control group, the dimer/monomer quotient was significantly lower in the APOE ε3/ε4 group (ratio D/M = 0.38) compared to that of the ε3/ε3 group (ratio D/M = 2.20; p= 0.006), associated to the inability of the apoE4 isoform to form disulfide-linked dimers. The same situation was found in the AD group, where the dimer/monomer ratio decreased as the ε4 allele was present (ε3/ε3: ratio D/M = 2.27; ε3/ε4: ratio D/M = 1.04; ε4/ε4: ratio D/M = 0.24). For APOE ε3/ε3 subjects, the dimer/monomer ratio in controls was not significantly different to that found in AD subjects; however, for APOE ε3/ε4 subjects, the quotient was higher in the AD group compared with controls (p = 0.02; Fig. 3D). This may be reflecting the accumulation of aberrant aggregates in the AD samples expressing apoE4, in addition to the physiological disulfide-bound dimers present in controls.

Given the differences in CSF apoE aggregates found under native conditions between AD and controls, we evaluated the levels of the 34-kDa, 36-kDa, and 100-kDa apoE species by SDS-PAGE in reducing conditions and western blotting, using the AB178479 antibody (Fig. 4A). The 34-kDa apoE band was significantly increased in AD compared with controls (p = 0.003; Fig. 4B). When discriminating by APOE genotype, only the APOE ε3/ε3 genotype was significantly elevated in AD compared with control samples (p = 0.02; Fig. 4C). The same analysis within the APOE ε3/ε4 genotype exhibited less statistical power because the size of the control group is small; nonetheless, we observed a trend of 34-kDa apoE increment in ε3/ε4 genotype AD samples with respect to controls (p = 0.09; Fig. 4C). The 36-kDa apoE appeared significantly increased in AD compared with controls overall (p = 0.002; Fig. 4D), but not among APOE genotypes (Fig. 4E).

As expected, when we considered the sum of the apoE immunoreactivity for the 34- and 36-kDa bands, increased levels were seen in AD patients (27 ± 5%), as compared with controls (p = 0.005). Despite the fact that these results indicate a net increase of CSF apoE in AD samples, when defining a quotient between the apoE monomeric glycoforms (ratio 36 kDa/34 kDa) we detected an imbalance in the AD samples, which displayed a decreased 36-kDa/34-kDa ratio compared with controls (p = 0.007; Fig. 4F). These differences were maintained when the samples were separated by APOE genotype (ε3/ε3 control: ratio 36 kDa/34 kDa = 1.61 vs ε3/ε3 AD: ratio 36 kDa/34 kDa = 1.46, p = 0.01; ε3/ε4 control: ratio 36 kDa/34 kDa = 1.67 vs ε3/ε4 AD: ratio = 1.38, p = 0.001; Fig. 4G). Within the AD group, we also observed significant differences between the genotypes, as the 36-kDa/34-kDa ratio was significantly lower in APOE ε3/ε3 (p < 0.0001) and ε3/ε4 (p < 0.0001) samples when compared with ε4/ε4 AD samples (ratio 36 kDa/34 kDa = 1.64). In each group, CSF apoE levels appeared unaltered when subgrouping between males and females (p > 0.05 for all the subgroups). There were no clear correlations between the level of the 34- or 36-kDa apoE or the 36-kDa/34-kDa ratio with the age of the subjects, in either of the groups considered individually.

The immunoreactivity of the 100-kDa apoE species was quite faint in control samples, and accordingly, substantial differences were found between AD samples and controls (p < 0.0001; Fig. 4H, I). In the AD group, the 100-kDa apoE species levels were significantly lower in the APOE ε4/ε4 group when compared to both ε3/ε3 (p < 0.0001) and ε3/ε4 (p < 0.0001) groups. Interestingly, in the APOE ε4/ε4 AD cases, the samples with the lowest 100-kDa apoE immunoreactivity belonged to the youngest subjects (n = 5, 68±2 years), as compared to the other cases (n= 10, 76±1 years; p=0.002). Within the AD APOE ε4/ε4 subgroup, we also observed a statistically significant positive correlation between the age of participants and the immunoreactivity of the 100-kDa apoE band (r = 0.622, p = 0.013). For the rest of the groups, we failed to determine a correlation between the 100-kDa band and age.

Interestingly, the quotient of 36 kDa/34 kDa monomeric glycoforms (r = 0.40, p = 0.007), as well as the levels of the 34-kDa species (r = 0.32, p = 0.034), correlated with Aβ42 in AD individuals. Meanwhile, the levels of the 100-kDa apoE complexes correlated with T-tau levels (r = 0.33, p = 0.028), yet failed to achieve significance with the Aβ42 levels (r = 0.27, p = 0.070).

We attempted to validate our results in a second independent cohort of CSF samples from Barcelona (see Table 1, Fig. 5A). As in the first cohort, the analyses were performed by SDS-PAGE under reducing conditions. In this cohort, the 34-kDa apoE levels were significantly higher in AD compared with controls (p=0.001), and specifically only for those with APOE ε3/ε3 genotype (p = 0.01) (Fig. 5B, C). In contrast to the first cohort, no differences in the 36-kDa species were detected (Fig. 5D, E). The 36-kDa/34-kDa ratio was again lower in AD compared with control samples (p < 0.001, Fig. 5F), and this difference was maintained when the samples were separated by genotype (AD vs controls for APOE ε3/ε3, p=0.02, and for APOE ε3/ε4, p=0.03) (Fig. 5G). ApoE levels once again appeared to be unaltered when subgrouping between males and females (p> 0.05 for all comparisons). In this cohort, we also failed to correlate 34- or 36-kDa levels or the 36-kDa/34-kDa ratio with the age of the subjects.

As in the first cohort, the 100-kDa apoE levels were higher in AD than in controls (p = 0.005; Fig. 5H). When the samples were stratified by APOE genotype (Fig. 5I), the significant differences between AD and controls were maintained in the ε3/ε3 group (p = 0.01) and were in the limit of statistical significance in the APOE ε3/ε4 group (p = 0.050) despite the small number of controls. Within the AD group, the APOE ε4/ε4 samples once again displayed significantly lower 100-kDa apoE levels compared with ε3/ε3 cases (p = 0.02). The results from this cohort corroborate that 100-kDa apoE is associated to AD and that apoE4 has a reduced capacity to form these complexes.

In this cohort, and exclusively in the AD group overall, we detected a significant correlation between the age of the subjects and the 100-kDa apoE species (r =0.645, p= 0.0002), indicating that the appearance of the aberrant apoE aggregates may be related to aging in AD. This association was maintained within the APOE ε3/ε3 (r = 0.813, p= 0.004) and the ε3/ε4 (r = 0.799, p= 0.006) AD groups.

In this cohort, only the levels of the 100-kDa apoE species correlated with Aβ42 levels (r= 0.41, p= 0.027). Thus, despite finding some correlations, none of these significant correlations resulted in consistency between the two cohorts.