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. 2022 Nov 2;14(1):161.
doi: 10.1186/s13195-022-01108-2.

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

Matthew Paul Lennol  1   2 , Irene Sánchez-Domínguez  3   4 , Inmaculada Cuchillo-Ibañez  1   2   5 , Elena Camporesi  6 , Gunnar Brinkmalm  6 , Daniel Alcolea  2   7 , Juan Fortea  2   7   8 , Alberto Lleó  2   7 , Guadalupe Soria  4   9 , Fernando Aguado  3   4 , Henrik Zetterberg  6   10   11   12   13 , Kaj Blennow  6   10 , Javier Sáez-Valero  14   15   16
Affiliations

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

Matthew Paul Lennol et al. Alzheimers Res Ther. .

Abstract

Objective: The purpose of this study was to examine the levels of cerebrospinal fluid (CSF) apolipoprotein E (apoE) species in Alzheimer's disease (AD) patients.

Methods: We analyzed two CSF cohorts of AD and control individuals expressing different APOE genotypes. Moreover, CSF samples from the TgF344-AD rat model were included. Samples were run in native- and SDS-PAGE under reducing or non-reducing conditions (with or without β-mercaptoethanol). Immunoprecipitation combined with mass spectrometry or western blotting analyses served to assess the identity of apoE complexes.

Results: In TgF344-AD rats expressing a unique apoE variant resembling human apoE4, a ~35-kDa apoE monomer was identified, increasing at 16.5 months compared with wild-types. In humans, apoE isoforms form disulfide-linked dimers in CSF, except apoE4, which lacks a cysteine residue. Thus, controls showed a decrease in the apoE dimer/monomer quotient in the APOE ε3/ε4 group compared with ε3/ε3 by native electrophoresis. A major contribution of dimers was found in APOE ε3/ε4 AD cases, and, unexpectedly, dimers were also found in ε4/ε4 AD cases. Under reducing conditions, two apoE monomeric glycoforms at 36 kDa and at 34 kDa were found in all human samples. In AD patients, the amount of the 34-kDa species increased, while the 36-kDa/34-kDa quotient was lower compared with controls. Interestingly, under reducing conditions, a ~100-kDa apoE complex, the identity of which was confirmed by mass spectrometry, also appeared in human AD individuals across all APOE genotypes, suggesting the occurrence of aberrantly resistant apoE aggregates. A second independent cohort of CSF samples validated these results.

Conclusion: These results indicate that despite the increase in total apoE content the apoE protein is altered in AD CSF, suggesting that function may be compromised.

Keywords: Aberrant complexes; Alzheimer’s disease; Biomarker; Cerebrospinal fluid; Glycoform imbalance; apoE.

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Conflict of interest statement

HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Alector, Annexon, Artery Therapeutics, AZTherapies, CogRx, Denali, Eisai, Nervgen, Novo Nordisk, Pinteon Therapeutics, Red Abbey Labs, Passage Bio, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave; has given lectures in symposia sponsored by Cellectricon, Fujirebio, Alzecure, Biogen, and Roche; and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). KB has served as a consultant, at advisory boards, or at data monitoring committees for Abcam, Axon, Biogen, JOMDD/Shimadzu, Julius Clinical, Lilly, MagQu, Novartis, Prothena, Roche Diagnostics, and Siemens Healthineers and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program, all unrelated to the work presented in this paper. JF has served as a consultant for Novartis and Lundbeck; has received honoraria for lectures from Roche, NovoNordisk, Nestle, Esteve, and Biogen; and served at advisory boards for AC Immune, Zambon, and Lundbeck. D.A. participated in advisory boards from Fujirebio-Europe and Roche Diagnostics and received speaker honoraria from Fujirebio-Europe, Roche Diagnostics, Nutricia, Krka Farmacéutica S.L., Zambon S.A.U., and Esteve Pharmaceuticals S.A. AL has served at scientific advisory boards from Fujirebio-Europe, Nutricia, Roche-Genentech, Biogen, Grifols, and Roche Diagnostics and has filed a patent application of synaptic markers in neurodegenerative diseases.

Figures

Fig. 1
Fig. 1
Analysis of CSF apoE in the TgF344-AD rats. A Representative blot of CSF obtained from wild-type (Wt) or transgenic (Tg) rats at 4, 10.5, and 16.5 months. The 100-kDa section of the blot presents enhanced contrast. B Quantification of apoE values obtained from western blots. Data is shown as a percentage with respect to the Wt values obtained at each age. The graphs represent mean ± SEM, and the numbers below represent median ± SD. A significant p value is indicated
Fig. 2
Fig. 2
Characterization of apoE protein in human control (Ct) and AD CSF samples. A Representative immunoblot of CSF samples immunoblotted with apoE antibody (AB178479). CSF samples immunoprecipitated with the apoE AB178479 antibody (originated in goat) and immunoblotted with B apoE antibody AB947 (originated in goat) common to all apoE variants or C NBP1-49529 (originated in mouse), an antibody specific to the apoE4 isoform. Arrowheads: non-specific immunoglobulins (detected also in beads coupled with AB178479 in absence of human CSF; not shown). The bands detected between 34- and 36-kDa monomers and 100-kDa dimers were not consistently immunoprecipitated across trials. Total, CSF before IP; IP, immunoprecipitated protein; IPc, control immunoprecipitation. D CSF samples immunoblotted with apoE antibody (Ab178479) following O-linked, N-linked, or O- and N-linked deglycosylation. As a control of the deglycosylation process, samples under standard conditions and samples heated to 37 °C in the absence of deglycosylating enzymes were also included. Representative blots of three independent immunoprecipitation or deglycosylation experiments are shown
Fig. 3
Fig. 3
Characterization of CSF apoE species under reducing, non-reducing, and native conditions. A, B Control (Ct) and AD CSF samples immunoblotted under reducing (R) or non-reducing (NR) SDS-PAGE with A apoE antibody (Ab178479) or B apoE4-specific antibody (NBP1-49529). Note that apoE4 is identified as part of the 100-kDa bands despite the inability to form disulfide-linked dimers. C Representative blot of native-PAGE studies, showing the apoE monomers and dimers. The discrepancy in molecular weight is likely due to the differences between native-PAGE and SDS-PAGE electrophoretic conditions which affect the migration of proteins. Rec, recombinant apoE3; Den CSF, denatured CSF. D Quantification of the ratio of apoE dimers compared to monomers across the different APOE genotypes, estimated by native-PAGE (see C). Scatter plots of apoE levels are represented. The graphs represent mean ± SEM, and the numbers below represent median ± SD. Significant p values are indicated
Fig. 4
Fig. 4
Analysis of CSF apoE species from the Gothenburg cohort. Control (Ct) and AD CSF samples analyzed by SDS-PAGE. Each individual band was quantified and normalized to the reference value (recombinant apoE). A Representative immunoblot of CSF samples with apoE antibody and legend for graphs. The 100-kDa section of the blot presents enhanced contrast. B, C Statistical analysis of the 34-kDa apoE immunoreactive band in B control and AD and by CAPOE genotype. D, E Statistical analysis of the 36-kDa apoE immunoreactive band in D control and AD and by EAPOE genotype. F, G Statistical analysis of the ratio of 36-kDa/34-kDa immunoreactive bands in F control and AD and by GAPOE genotype. H, I Statistical analysis of the 100-kDa apoE immunoreactive band in H control and AD and by IAPOE genotype. The graphs represent mean ± SEM, and the numbers below represent median ± SD. Significant p values are indicated
Fig. 5
Fig. 5
Analysis of CSF apoE species from the Barcelona cohort. Control (Ct) and AD CSF samples analyzed by SDS-PAGE. Each individual band was quantified and normalized to the reference value (recombinant apoE). A Representative immunoblot of CSF samples with apoE antibody and legend for graphs. The 100-kDa section of the blot presents enhanced contrast. B, C Statistical analysis of the 34-kDa apoE immunoreactive band in B control and AD and by CAPOE genotype. D, E Statistical analysis of the 36-kDa apoE immunoreactive band in D control and AD and by EAPOE genotype. F, G Statistical analysis of the ratio of 36-kDa/34-kDa immunoreactive bands in F control and AD and by GAPOE genotype. H, I Statistical analysis of the 100-kDa apoE immunoreactive band in H control and AD and by IAPOE genotype. The graphs represent mean ± SEM, and the numbers below represent median ± SD. Significant p values are indicated
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