Research ArticleAlzheimer’s Disease

The E3 ubiquitin ligase Idol controls brain LDL receptor expression, ApoE clearance, and Aβ amyloidosis

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Science Translational Medicine  18 Nov 2015:
Vol. 7, Issue 314, pp. 314ra184
DOI: 10.1126/scitranslmed.aad1904
  • Fig. 1. The LXR-Idol axis regulates LDLR abundance in the brain.

    (A) Real-time polymerase chain reaction (PCR) analysis of Idol mRNA expression in different mouse brain cell types, normalized to microglial expression. MG, microglia; Astr, astrocytes; Hip N, hippocampal neurons; Cort N, cortical neurons. Error bars represent SEM. (B) Representative micrographs showing immunofluorescence staining of brain sections from the frontal cortex of Idol+/− mice. Images in top row show Idol expression in neurons. Images in bottom row show Idol expression in microglia. Green, NeuN (neuron) and Iba1 (microglia); red, Idol; blue, 4′,6-diamidino-2-phenylindole (DAPI; nucleus). Scale bars, 10 μm. (C) Real-time PCR analysis of LXR target gene expression in primary microglia treated with dimethyl sulfoxide (DMSO) or 1 μM GW3965 for 6 hours, normalized to vehicle (DMSO) control. Error bars represent SEM. (D) Immunoblot analysis of whole-cell lysates from primary microglia treated with DMSO or 1 μM GW3965 for 6 hours. Lanes represent samples from individual cultures. (E) Immunoblot analysis of whole-cell lysates from primary microglia isolated from Idol+/+ or Idol−/− mice. Lanes represent samples from individual animals. WT, wild type. (F) Immunoblot analysis of whole-cell lysates from the frontal cortex of Idol+/+, Idol+/−, or Idol−/− mice. Lanes represent samples from individual animals. Immunoblot signals are quantified on the right. Error bars represent SEM. *P < 0.05, **P < 0.01 by Student’s t test. Each experiment was performed at least twice.

  • Fig. 2. Loss of Idol expression inhibits plaque formation in a mouse model of Aβ amyloidosis.

    (A) Quantification of Aβ40 and Aβ42 peptides in the insoluble guanidine fraction and the soluble RIPA fraction from the frontal cortex of APP/PS1;Idol+/+, APP/PS1;Idol+/− (for Aβ42), and APP/PS1;Idol−/− mice. Error bars represent SEM. *P < 0.05, **P < 0.01 by Student’s t test. n = 5 to 7. (B) Immunoblot analysis of brain RIPA fractions from APP/PS1;Idol+/+ and APP/PS1;Idol−/− mice. APP was detected with 4G8 antibody. Aβ and β-CTF were detected with 82E1 antibody. (C) Immunoblot analysis of total brain lysates from APP/PS1;Idol+/+ and APP/PS1;Idol−/− mice. (D) Representative micrographs showing immunohistochemical staining of brain sections from APP/PS1;Idol+/+, APP/PS1;Idol+/−, and APP/PS1;Idol−/− mice with Aβ-specific 82E1B antibody. Black stain indicates Aβ plaques. Scale bar, 250 μm. (E) Quantification of Aβ plaque load in the frontal cortex and hippocampus of APP/PS1;Idol+/+, APP/PS1;Idol+/−, and APP/PS1;Idol−/− mice by 82E1B antibody staining for Aβ. Error bars represent SEM. *P < 0.05, **P < 0.01 by Student’s t test. n = 6.

  • Fig. 3. Reducing Idol expression attenuates the deposition of fibrillar amyloid.

    (A) Representative micrographs showing thioflavin S staining of brain sections from APP/PS1;Idol+/+, APP/PS1;Idol+/−, or APP/PS1;Idol−/− mice. White color indicates thioflavin S fluorescence. Scale bar, 250 μm. (B) Quantification of thioflavin S (ThioS)–stained Aβ plaque load in the frontal cortex and hippocampus of male or female APP/PS1;Idol+/+, APP/PS1;Idol+/−, or APP/PS1;Idol−/− mice. Error bars represent SEM. *P < 0.05, **P < 0.01 by Student’s t test. n = 6.

  • Fig. 4. Reducing Idol expression attenuates microgliosis in a mouse model of Aβ amyloidosis.

    (A) Representative micrographs showing immunohistochemical staining of brain sections from APP/PS1;Idol+/+, APP/PS1;Idol+/−, or APP/PS1;Idol−/− mice with CD45 antibody. Black color indicates CD45-positive lesions. Scale bar, 200 μm. (B) Quantification of CD45 staining load in the frontal cortex or hippocampus of APP/PS1;Idol+/+, APP/PS1;Idol+/−, or APP/PS1;Idol−/− mice. Error bars represent SEM. *P < 0.05, **P < 0.01 by Student’s t test. n = 6. (C) Representative micrographs showing immunohistochemical staining of the hippocampal CA1 regions of APP/PS1;Idol+/+, APP/PS1;Idol+/−, or APP/PS1;Idol−/− mice with Iba1 antibody. Black color indicates Iba1+ cells. Scale bar, 50 μm. (D) Quantification of the number of Iba1+ cells in the frontal cortex or hippocampus of APP/PS1;Idol+/+, APP/PS1;Idol+/−, or APP/PS1;Idol−/− mice. Error bars represent SEM. *P < 0.05, **P < 0.01 by Student’s t test. n = 6.

  • Fig. 5. Idol regulates ApoE in the brain.

    (A) Immunoblot analysis of total brain lysates from the frontal cortex of APP/PS1;Idol+/+ or APP/PS1;Idol−/− mice. Lanes represent samples from individual mice. (B to D) Immunoblot analysis of ApoE and tubulin protein in the (B) PBS-soluble fraction, (C) RIPA fraction, and (D) insoluble (guanidine) fraction from the frontal cortex of APP/PS1;Idol+/+ or APP/PS1;Idol−/− mice. Lanes represent samples from individual mice. Quantification of the immunoblot signals is presented on the right. Error bars represent SEM. *P < 0.05, **P < 0.01 by Student’s t test. Each experiment was performed at least twice.

  • Fig. 6. Idol modulates cellular Aβ uptake and Aβ clearance in brain-derived cell lines.

    (A) Immunoblot analysis of LDLR protein in Neuro2a (N2a) cells, hApoE3 cells, and BV2 cells transfected with control or Idol-specific siRNA. Quantification of the immunoblot signals is presented on the right. Error bars represent SEM. **P < 0.01 by Student’s t test. (B) Immunoblot analysis of intracellular Aβ protein in Neuro2a cells, hApoE3 cells, and BV2 cells transfected with control or Idol-specific siRNA and then incubated for 3 hours with recombinant Aβ. Quantification of the immunoblot signals is presented on the right. Error bars represent SEM. **P < 0.01 by Student’s t test. (C) Immunoblot analysis of extracellular Aβ protein in the media of cultures of Neuro2a cells, hApoE3 cells, and BV2 cells transfected with control or Idol-specific siRNA and then incubated for 24 hours with recombinant Aβ. Quantification of the immunoblot signals is presented on the right. Error bars represent SEM. **P < 0.01 by Student’s t test. (D) Representative micrographs showing uptake of fluorescently labeled aggregated HiLyte555-Aβ42 after a 4-hour incubation in primary hippocampal neurons from Idolflox/flox mice treated in vitro with control green fluorescent protein (GFP)–expressing or GFP/Cre (fusion protein with nuclear localization signal)–expressing AAV vector. Red, Aβ42; green, GFP; blue, DAPI. Scale bar, 5 μm. GFP or GFP-Cre expression was driven from a neuron-specific calcium/calmodulin-dependent protein kinase IIa (CaMKIIa) promoter. Quantification of the fluorescence intensity is presented on the right. Error bars represent SEM. n ≥ 40. Each experiment was performed at least twice.

  • Fig. 7. Idol modulates cellular Aβ and ApoE uptake by microglia.

    (A) Representative micrographs showing uptake of fluorescently labeled aggregated HiLyte488-Aβ42 in primary microglia from WT and Idol−/− mice. Blue, DAPI; green, Aβ42. Arrowheads denote Aβ42-positive cells. Arrows denote representative cells shown in the magnified field. Scale bar, 20 μm. (B) Representative micrographs showing uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)–labeled reconstituted discoidal HDL particles containing ApoE by primary microglia from WT and Idol−/− mice. Blue, DAPI; red, DiI rHDL-ApoE. Arrows denote representative cells shown in the magnified field. Scale bar, 20 μm. (C) Quantification of the results shown in (A). Error bars represent SEM. **P < 0.01 by Student’s t test. (D) Quantification of the results shown in (B). Error bars represent SEM. *P < 0.05, **P < 0.01 by Student’s t test. (E) Representative micrographs showing uptake of fluorescently labeled aggregated HiLyte555-Aβ42 after a 30-min incubation in primary microglia from Idolflox/flox mice infected with AAV5/CMV-GFP or AAV5/CMV-Cre/GFP and treated with Accell siRNA nontargeting control (siNT) or siRNA against mouse LDLR (siLDLR). Arrows denote representative cells shown in the magnified field. Scale bar, 20 μm. (F) Quantification of the results shown in (E). Error bars represent SEM. **P < 0.01 by Student’s t test. Each experiment was performed at least twice.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/314/314ra184/DC1

    Fig. S1. Idol decreases LDLR protein and promotes LDLR ubiquitination in mouse brain cells.

    Fig. S2. Loss of Idol does not directly affect inflammatory responses.

    Fig. S3. Idol inhibition promotes the clearance of Aβ by microglia.

    Table S1. Data for Aβ40 shown in Fig. 2A.

    Table S2. Data for Aβ42 shown in Fig. 2A.

    Table S3. Quantification of Aβ plaque load in Fig. 2E.

    Table S4. Quantification of thioflavin S plaque load in Fig. 3B.

    Table S5. Quantification of CD45 load in Fig. 4B.

    Table S6. Quantification of Iba+ cells in Fig. 4D.

  • Supplementary Material for:

    The E3 ubiquitin ligase Idol controls brain LDL receptor expression, ApoE clearance, and Aβ amyloidosis

    Jinkuk Choi, Jie Gao, Jaekwang Kim, Cynthia Hong, Jungsu Kim,* Peter Tontonoz*

    *Corresponding author. E-mail: ptontonoz{at}mednet.ucla.edu (P.T.); kim.jungsu{at}mayo.edu (J.K.)

    Published 18 November 2015, Sci. Transl. Med. 7, 314ra184 (2015)
    DOI: 10.1126/scitranslmed.aad1904

    This PDF file includes:

    • Fig. S1. Idol decreases LDLR protein and promotes LDLR ubiquitination in mouse brain cells.
    • Fig. S2. Loss of Idol does not directly affect inflammatory responses.
    • Fig. S3. Idol inhibition promotes the clearance of Aβby microglia.
    • Table S1. Data for Aβ40 shown in Fig. 2A.
    • Table S2. Data for Aβ42 shown in Fig. 2A.
    • Table S3. Quantification of Aβ plaque load in Fig. 2E.
    • Table S4. Quantification of thioflavin S plaque load in Fig. 3B.
    • Table S5. Quantification of CD45 load in Fig. 4B.
    • Table S6. Quantification of Iba+ cells in Fig. 4D.

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