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Reduced Levels of ABCA1 Transporter Are Responsible for the Cholesterol Efflux Impairment in β-Amyloid-Induced Reactive Astrocytes: Potential Rescue from Biomimetic HDLs

Sierri, Giulia, Dal Magro, Roberta, Vergani, Barbara, Leone, Biagio Eugenio, Formicola, Beatrice, Taiarol, Lorenzo, Fagioli, Stefano, Kravicz, Marcelo, Tremolizzo, Lucio, Calabresi, Laura, Re, Francesca·2021·International Journal of Molecular Sciences
DOI 10.3390/ijms23010102PMC PMC8745016
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Abstract

The cerebral synthesis of cholesterol is mainly handled by astrocytes, which are also responsible for apoproteins’ synthesis and lipoproteins’ assembly required for the cholesterol transport in the brain parenchyma. In Alzheimer disease (AD), these processes are impaired, likely because of the astrogliosis, a process characterized by morphological and functional changes in astrocytes. Several ATP-binding cassette transporters expressed by brain cells are involved in the formation of nascent discoidal lipoproteins, but the effect of beta-amyloid (Aβ) assemblies on this process is not fully understood. In this study, we investigated how of Aβ1-42-induced astrogliosis affects the metabolism of cholesterol in vitro. We detected an impairment in the cholesterol efflux of reactive astrocytes attributable to reduced levels of ABCA1 transporters that could explain the decreased lipoproteins’ levels detected in AD patients. To approach this issue, we designed biomimetic HDLs and evaluated their performance as cholesterol acceptors. The results demonstrated the ability of apoA-I nanodiscs to cross the blood–brain barrier in vitro and to promote the cholesterol efflux from astrocytes, making them suitable as a potential supportive treatment for AD to compensate the depletion of cerebral HDLs.

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Figure 1 Assessment of Aβ aggregation. (a) Oligomers’ and fibrils’ preparations of Aβ1-42 were characterized by SDS-PAGE/WB. Representative Western blot of Aβ1-42 probed with anti-Aβ 6E10 and visualized by ECL is shown. (b) Quantification of the optical density (O.D.) of bands, expressed as %. (c) Turbidity at 330 nm. (d) Thioflavin T (ThT) fluorescence. Experiments were performed in triplicate and data are presented as mean ± standard deviation.
Figure 1 Assessment of Aβ aggregation. (a) Oligomers’ and fibrils’ preparations of Aβ1-42 were characterized by SDS-PAGE/WB. Representative Western blot of Aβ1-42 probed with anti-Aβ 6E10 and visualized by ECL is shown. (b) Quantification of the optical density (O.D.) of bands, expressed as %. (c) Turbidity at 330 nm. (d) Thioflavin T (ThT) fluorescence. Experiments were performed in triplicate and data are presented as mean ± standard deviation.CC BY · PubMed Central
Figure 2 Cell viability after exposure to Aβ. NHA cells were incubated with different doses of Aβ oligomers (a) or fibrils (b) ranging from 1 to 20 μM, for 24 or 48 h. The cytotoxicity of treatments was assessed by MTT assay; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p< 0.0001 (two-way ANOVA).
Figure 2 Cell viability after exposure to Aβ. NHA cells were incubated with different doses of Aβ oligomers (a) or fibrils (b) ranging from 1 to 20 μM, for 24 or 48 h. The cytotoxicity of treatments was assessed by MTT assay; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p< 0.0001 (two-way ANOVA).CC BY · PubMed Central
Figure 3 Quantification of GFAP expression levels in NHA after exposure to Aβ1-42 oligomers via SDS-PAGE/WB. NHA cells were treated with a preparation of Aβ1-42, enriched in oligomers, at increasing concentrations (1 μM, 10 μM, and 20 μM) for 24 (a) and 48 (b) h. The data are expressed as the mean of the samples in triplicate ± SEM; * p <0.05 (one-way ANOVA).
Figure 3 Quantification of GFAP expression levels in NHA after exposure to Aβ1-42 oligomers via SDS-PAGE/WB. NHA cells were treated with a preparation of Aβ1-42, enriched in oligomers, at increasing concentrations (1 μM, 10 μM, and 20 μM) for 24 (a) and 48 (b) h. The data are expressed as the mean of the samples in triplicate ± SEM; * p <0.05 (one-way ANOVA).CC BY · PubMed Central
Figure 4 Quantification of GFAP expression levels in NHA after exposure to Aβ1-42 fibrils via SDS-PAGE/WB. NHA cells were treated with a preparation of Aβ1-42, enriched in fibrils, at increasing concentrations (1 μM, 10 μM, and 20 μM) for 24 (a) and 48 (b) h. The data are expressed as the mean of the samples in triplicate ± SEM; **** p < 0.0001 (one-way ANOVA).
Figure 4 Quantification of GFAP expression levels in NHA after exposure to Aβ1-42 fibrils via SDS-PAGE/WB. NHA cells were treated with a preparation of Aβ1-42, enriched in fibrils, at increasing concentrations (1 μM, 10 μM, and 20 μM) for 24 (a) and 48 (b) h. The data are expressed as the mean of the samples in triplicate ± SEM; **** p < 0.0001 (one-way ANOVA).CC BY · PubMed Central
Figure 5 Evaluation of cholesterol efflux from astrocytes in AD-like conditions, in the presence of ACM as acceptor of cholesterol. The cholesterol efflux was measured using the Cholesterol Efflux Assay kit, after treatment with Aβ1-42 oligomers (1 μM or 10 μM) for 24 (a) or 48 (b) h. The fluorescence of supernatant and of cellular lysates was measured by spectrofluorometer. Data are expressed as the mean of the samples in triplicate ± SEM; * p < 0.05; **** p < 0.0001 (2way ANOVA).
Figure 5 Evaluation of cholesterol efflux from astrocytes in AD-like conditions, in the presence of ACM as acceptor of cholesterol. The cholesterol efflux was measured using the Cholesterol Efflux Assay kit, after treatment with Aβ1-42 oligomers (1 μM or 10 μM) for 24 (a) or 48 (b) h. The fluorescence of supernatant and of cellular lysates was measured by spectrofluorometer. Data are expressed as the mean of the samples in triplicate ± SEM; * p < 0.05; **** p < 0.0001 (2way ANOVA).CC BY · PubMed Central
Figure 6 Quantification of ABCA1/ABCG1 expression levels in NHA after exposure to Aβ1-42 oligomers via SDS-PAGE/WB. NHA cells were treated with increasing concentrations (1 μM, 10 μM) of oligomers-enriched Aβ1-42 samples for 24, 48, or 72 h, and ABCA1 (a) and ABCG1 (b) levels were evaluated. The data are expressed as the mean of the samples in triplicate ± SEM; * p < 0.05; ** p < 0.01; **** p < 0.0001 (two-way ANOVA).
Figure 6 Quantification of ABCA1/ABCG1 expression levels in NHA after exposure to Aβ1-42 oligomers via SDS-PAGE/WB. NHA cells were treated with increasing concentrations (1 μM, 10 μM) of oligomers-enriched Aβ1-42 samples for 24, 48, or 72 h, and ABCA1 (a) and ABCG1 (b) levels were evaluated. The data are expressed as the mean of the samples in triplicate ± SEM; * p < 0.05; ** p < 0.01; **** p < 0.0001 (two-way ANOVA).CC BY · PubMed Central
Figure 7 TEM image of the nanodiscs. Characteristic negative stain electron microscopic image of nascent HDL, along with two expanded views of the indicated insets. The scale bar corresponds to 100 or 50 nm. (a) Nanodiscs viewed laterally are indicated by yellow arrows. (b) Enlargement of a nanodisc decorated with apoA-I.
Figure 7 TEM image of the nanodiscs. Characteristic negative stain electron microscopic image of nascent HDL, along with two expanded views of the indicated insets. The scale bar corresponds to 100 or 50 nm. (a) Nanodiscs viewed laterally are indicated by yellow arrows. (b) Enlargement of a nanodisc decorated with apoA-I.CC BY · PubMed Central
Figure 8 Evaluation of the ability of apoA-1 nanodiscs as cholesterol acceptors. The cholesterol efflux was measured using the Cholesterol Efflux Assay kit. NHA cells were treated with different cholesterol acceptors: free apoA-I, nanodiscs, apoA-I nanodiscs, and ACM. The fluorescence of supernatant and of cell lysates was measured by spectrofluorometer; * p < 0.05; *** p < 0.001 (one-way ANOVA).
Figure 8 Evaluation of the ability of apoA-1 nanodiscs as cholesterol acceptors. The cholesterol efflux was measured using the Cholesterol Efflux Assay kit. NHA cells were treated with different cholesterol acceptors: free apoA-I, nanodiscs, apoA-I nanodiscs, and ACM. The fluorescence of supernatant and of cell lysates was measured by spectrofluorometer; * p < 0.05; *** p < 0.001 (one-way ANOVA).CC BY · PubMed Central
Figure 9 Evaluation of apoA-I nanodiscs efficacy in AD-like condition. (a) The cholesterol efflux was measured using the Cholesterol Efflux Assay kit. After treating the cells with Aβ1-42 oligomers (1 μM or 10 μM) for 24 h, two different cholesterol acceptors, ACM or apoA-I nanodiscs (40 μg/mL), were added. The fluorescence of supernatant and cell lysates was measured by spectrofluorometer. (b) Detection of cell viability, GFAP, and ABCA1 levels to confirm the apoA-I efficacy in restoring some cellular processes related to AD that were compromised by Aβ oligomers; * p <0.05; ** p ≤ 0.01; *** p ≤ 0.001 (one-way ANOVA).
Figure 9 Evaluation of apoA-I nanodiscs efficacy in AD-like condition. (a) The cholesterol efflux was measured using the Cholesterol Efflux Assay kit. After treating the cells with Aβ1-42 oligomers (1 μM or 10 μM) for 24 h, two different cholesterol acceptors, ACM or apoA-I nanodiscs (40 μg/mL), were added. The fluorescence of supernatant and cell lysates was measured by spectrofluorometer. (b) Detection of cell viability, GFAP, and ABCA1 levels to confirm the apoA-I efficacy in restoring some cellular processes related to AD that were compromised by Aβ oligomers; * p <0.05; ** p ≤ 0.01; *** p ≤ 0.001 (one-way ANOVA).CC BY · PubMed Central
Figure 10 Characterization of the BBB in vitro model and evaluation of the passage of nanodiscs. (a) TEER values of the cell monolayer from day 3 to day 7 of growth on transwell reaching a maximum value of 44 Ω × cm2 on the seventh day of growth. (b) Evaluation of the passage of fluorescently labelled nanodiscs and apoA-I nanodiscs; * p <0.05 (Student’s t test).
Figure 10 Characterization of the BBB in vitro model and evaluation of the passage of nanodiscs. (a) TEER values of the cell monolayer from day 3 to day 7 of growth on transwell reaching a maximum value of 44 Ω × cm2 on the seventh day of growth. (b) Evaluation of the passage of fluorescently labelled nanodiscs and apoA-I nanodiscs; * p <0.05 (Student’s t test).CC BY · PubMed Central

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Authors
Sierri, G., Dal Magro, R., Vergani, B., Leone, B. E., Formicola, B., Taiarol, L., Fagioli, S., Kravicz, M., Tremolizzo, L., Calabresi, L., & Re, F.
Affiliations
  • BioNanoMedicine Center NANOMIB, School of Medicine and Surgery, University of Milano-Bicocca, 20900 Monza, Italy
  • School of Medicine and Surgery, University of Milano-Bicocca, 20900 Monza, Italy
  • Department of Pharmacological and Biomolecular Science, Centro Grossi Paoletti, University of Milan, 20133 Milan, Italy
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2021-12-22
Volume
23
Issue
1
Pages
102
ISSN
1422-0067
DOI
10.3390/ijms23010102
PMC
PMC8745016
Cited by
16
References
2
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
PRIN Research Italy (2017PFYK27)

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