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. 2015 Nov 4;35(44):14717-26.
doi: 10.1523/JNEUROSCI.2053-15.2015.

microRNA-33 Regulates ApoE Lipidation and Amyloid-β Metabolism in the Brain

Jaekwang Kim  1 , Hyejin Yoon  1 , Takahiro Horie  2 , Jack M Burchett  3 , Jessica L Restivo  3 , Noemi Rotllan  4 , Cristina M Ramírez  4 , Philip B Verghese  3 , Masafumi Ihara  5 , Hyang-Sook Hoe  6 , Christine Esau  7 , Carlos Fernández-Hernando  4 , David M Holtzman  3 , John R Cirrito  8 , Koh Ono  9 , Jungsu Kim  10
Affiliations

microRNA-33 Regulates ApoE Lipidation and Amyloid-β Metabolism in the Brain

Jaekwang Kim et al. J Neurosci. .

Abstract

Dysregulation of amyloid-β (Aβ) metabolism is critical for Alzheimer's disease (AD) pathogenesis. Mounting evidence suggests that apolipoprotein E (ApoE) is involved in Aβ metabolism. ATP-binding cassette transporter A1 (ABCA1) is a key regulator of ApoE lipidation, which affects Aβ levels. Therefore, identifying regulatory mechanisms of ABCA1 expression in the brain may provide new therapeutic targets for AD. Here, we demonstrate that microRNA-33 (miR-33) regulates ABCA1 and Aβ levels in the brain. Overexpression of miR-33 impaired cellular cholesterol efflux and dramatically increased extracellular Aβ levels by promoting Aβ secretion and impairing Aβ clearance in neural cells. In contrast, genetic deletion of mir-33 in mice dramatically increased ABCA1 levels and ApoE lipidation, but it decreased endogenous Aβ levels in cortex. Most importantly, pharmacological inhibition of miR-33 via antisense oligonucleotide specifically in the brain markedly decreased Aβ levels in cortex of APP/PS1 mice, representing a potential therapeutic strategy for AD.

Significance statement: Brain lipid metabolism, in particular Apolipoprotein E (ApoE) lipidation, is critical to Aβ metabolism and Alzheimer's disease (AD). Brain lipid metabolism is largely separated from the periphery due to blood-brain barrier and different repertoire of lipoproteins. Therefore, identifying the novel regulatory mechanism of brain lipid metabolism may provide a new therapeutic strategy for AD. Although there have been studies on brain lipid metabolism, its regulation, in particular by microRNAs, is relatively unknown. Here, we demonstrate that inhibition of microRNA-33 increases lipidation of brain ApoE and reduces Aβ levels by inducing ABCA1. We provide a unique approach for AD therapeutics to increase ApoE lipidation and reduce Aβ levels via pharmacological inhibition of microRNA in vivo.

Keywords: ABCA1; Alzheimer's disease; ApoE; abeta; miR-33.

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Figures

Figure 1.
Figure 1.
miR-33 is enriched in the brain. A, Relative expression levels of miR-33 in different mouse tissues. Endogenous levels of miR-33 were analyzed by qRT-PCR in different mouse tissues from 3.5-month-old C57B6 mice (n = 3). Each level was represented as a percentage of liver. B, Relative expression of miR-33 in the mouse brain. Mature miR-33 levels were analyzed by qRT-PCR and were normalized with corresponding U6 levels (n = 2). Data are shown as a percentage of hippocampus. C, Relative expression levels of miR-33 in primary cells isolated from mice brains. miR-33 levels were analyzed in mouse primary neurons, astrocytes, and microglia by qRT-PCR. Data are shown as a percentage of neurons (n = 3). Values are mean ± SEM.
Figure 2.
Figure 2.
miR-33 suppresses ABCA1 expression in the brain. A, Schematic diagram of the conserved target sites of miR-33 in the 3′UTR of ABCA1 mRNA. The mir-33 gene is located within the intron of Sterol regulatory element binding transcription factor 2 (SREBF2) gene. The genomic sequences around mir-33 are highly conserved in mammals. The mature sequence of miR-33 is shown in red within pre-mir-33. miR-33 has three potential targeting sites in the ABCA1. Blue underlines indicate 3′UTR. *Conserved nucleotides. B, miR-33 directly targets the 3′UTR of ABCA1 mRNA. (n = 6 per group). Luciferase reporter assays were performed with the reporter construct containing the full-length 3′UTR of hABCA1 mRNA downstream of Renilla luciferase. Renilla luciferase activity was normalized with the corresponding firefly luciferase activity. C, D, miR-33 decreased ABCA1 levels in N2a cells (n = 6) and mouse primary astrocytes (n = 5). Cells were transfected with miR-33 or scrambled negative control (Ctl). At 48 h after transfection, cells were harvested for Western blot analyses. ABCA1 protein levels were normalized by actin levels and quantified as a percentage of control. E, miR-33 is selectively deleted in cortex of miR-33 (−/−) mice without altering the expression of its host gene, SREBF2, in cortex. GAPDH levels were used for normalization. Data are shown as a percentage of WT. F, G, ABCA1 levels were increased in cortex of mir-33 knock-out mice (miR-33 (−/−), n = 5), compared with WT mice (miR-33 (+/+), n = 5). ABCA1 levels were analyzed by Western blot at 4 months of age. Actin levels were used for normalization. Data are shown as a percentage of control, and values are mean ± SEM. **p < 0.01 (t test). ***p < 0.001 (t test).
Figure 3.
Figure 3.
miR-33 regulates lipidation and level of ApoE in the brain. A, miR-33 impaired cellular cholesterol efflux in N2a and astrocytes. N2a and astrocytes were transfected with miR-33 or scrambled negative control for 24 h and then incubated the cells with radioactively labeled 3H-cholesterol. After 24 h, cells were incubated with human ApoE3 as a cholesterol acceptor. Data are shown as a percentage of total cellular 3H-cholesterol content (total effluxed 3H-cholesterol + cell-associated 3H-cholesterol). Values are mean ± SEM. **p < 0.01 (t test). ***p < 0.001 (t test). n = 3. B, mir-33 deletion increased highly lipidated ApoE-containing particles. PBS-soluble fractions were analyzed by native PAGE and ApoE-specific Western blot. Total ApoE levels applied for each lane were shown below. C, D, PBS-soluble apoE levels were decreased in mir-33-deficient mice, compared with WT. Extracellular protein-enriched PBS-soluble fraction from cortex of mir-33-deficient (n = 6) and WT mice (n = 8) were analyzed by Western blots. Total protein levels (Ponceau staining) were used for normalization. Data are shown as a percentage of control, and values are mean ± SEM. ***p < 0.001 (t test).
Figure 4.
Figure 4.
Overexpression of miR-33 increases Aβ secretion by suppressing ABCA1 in neural cells. Mouse N2a (A, B) and human H4 cells (C, D) expressing APPsw were transfected with miR-33 or scrambled control. At 48 h after transfection, media were changed to fresh serum-free media. At 6 h after media change, cells and media were collected for analyses. Intracellular ABCA1, APP, and CTFβ levels and the secreted Aβ levels in the media were measured by Western blots (n = 6 per group). Actin levels were used for normalization. E, F, Overexpression of Abca1 ORF restored miR-33-mediated alteration of secreted Aβ levels. Abca1 ORF plasmids were contransfected with miR-33 or scrambled control to N2a-expressing APPsw, and the secreted Aβ levels in the media were measured by Western blots (n = 5 per group). Actin levels were used for normalization. Data are shown as a percentage of control, and values are mean ± SEM. *p < 0.05 (t test). ***p < 0.001 (t test).
Figure 5.
Figure 5.
Overexpression of miR-33 impairs Aβ clearance in neural cells. A, B, Overexpression of miR-33 decreased Aβ clearance in neural cells. N2a, H4, and mouse primary astrocytes were transfected with miR-33 or scrambled control. At 48 h after transfection, cells were incubated with synthetic Aβ40 monomers for 24 h. The remaining Aβ40 levels in the media were analyzed by Western blots (n = 5 per group). C, D, Overexpression of Abca1 ORF restored the reduction of Aβ clearance. Abca1 ORF plasmids were contransfected with miR-33 or scrambled control to mouse primary astrocytes. At 48 h after transfection, cells were incubated with synthetic Aβ40 monomers for 24 h. The remaining Aβ40 levels in the media were analyzed by Western blots (n = 4 per group). Data are shown as a percentage of control, and values are mean ± SEM. **p < 0.01 (t test). ***p < 0.001 (t test).
Figure 6.
Figure 6.
miR-33 regulates extracellular Aβ degradation. A, B, Overexpression of miR-33 impaired extracellular Aβ degradation in the conditioned media from astrocytes. Primary astrocytes were transfected with miR-33 or scrambled control. At 48 h after transfection, conditioned media were collected and were incubated with 200 nm of synthetic Aβ40. After 24 h of incubation, the levels of Aβ40 remaining in the media were analyzed by Western blots (n = 3 per group). C, D, Deletion of miR-33 facilitated extracellular Aβ degradation in PBS-soluble fractions of cortices. PBS-soluble fraction of each mouse cortex was adjusted to the same amount of total proteins and then incubated with 200 nm of synthetic Aβ40 for the indicated time (n = 6 per group). Data are shown as a percentage of control, and values are mean ± SEM. ***p < 0.001 (two-way ANOVA).
Figure 7.
Figure 7.
mir-33 deletion decreases mouse endogenous Aβ levels in cortex. Compared with WT mice (n = 8), ABCA1 levels were dramatically increased in cortex of mir-33 knock-out mice (n = 7) at 4 months of age, without altering APP levels (A, B). The levels of Aβ40 (C) and Aβ42 (D) are significantly decreased in cortex of mir-33 knock-out mice (n = 7), compared with WT (n = 9). Actin levels were used for normalization of Western blots. Aβ levels were measured by Aβ-specific ELISA and normalized by total protein levels. Data are shown as a percentage of control, and values are mean ± SEM. ***p < 0.001 (t test).
Figure 8.
Figure 8.
Brain-specific miR-33 inhibition decreases Aβ levels in cortex of APP/PS1 mice. Two-month-old APP/PS1 mice were treated with anti-miR-33 or the mismatch negative control for 4 weeks via intracerebroventricular infusion. Anti-miR-33 markedly increased ABCA1 levels in cortex of APP/PS1 mice (n = 9), compared with the mismatch control (n = 6) (A, B). In the anti-miR-33 treatment group (n = 9), Aβ40 levels were significantly decreased (p = 0.0286) (C) and Aβ42 levels showed a trend toward decrease (p = 0.0954) (D), compared with the mismatch control (n = 6). Actin levels were used for normalization of Western blots. Aβ levels were measured by Aβ-specific ELISA and normalized by total protein levels. Data are shown as a percentage of control, and values are mean ± SEM. *p < 0.05 (t test). ***p < 0.001 (t test).
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