Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE4 alters autophagic flux in in HEK293 cells stably expressing fluorescently tagged APOE. (A,C) HEK293 cells expressing APOE3–mCh or APOE4–mCh or mCh vector were analyzed by western blotting. (B) HEK293 cells stably expressing APOE3–mCh, APOE4–mCh, or mCh vector were treated with Baf and analyzed as for A and C (50 nM 4 h), Rap (10 nM 4 h) or both. Quantitative results are mean±s.e.m. Revert, protein stain; NT, no treatment. * P <0.05, ** P <0.01, **** P <0.0001 (one-way ANOVA with multiple comparisons correction).

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Staining

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE is turned over by autophagy in HEK293 cells with stable APOE expression. Representative images and fluorescence intensity of APOE3–mCh, APOE4–mCh, or mCh cells treated with Baf (50 nM 4 h), Epox (100 nM), or MG132 (50 µM). Quantitative results are mean±s.e.m. Bars over graphs indicate time points at which P <0.05 on two-way ANOVA with post-hoc Dunnett test.

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Expressing, Fluorescence

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE4 colocalizes with enlarged lysosomes. (A) HEK293 cells stably expressing APOE3– or APOE4–mCh (red) and transfected with CellLight Lamp1–GFP (green), and stained with DAPI (blue). (B) HEK293 cells stably expressing APOE3– or APOE4–mCh–SepH. SepH shown in green. (C) HEK293 cells expressing APOE2, APOE3, APOE4 or vector were treated with oleic acid (OA, overnight) and Baf (4 h). NT, no treatment. Quantitative results are mean±s.e.m. In A–C, three wells were imaged per APOE isoform (three images per well) by confocal microscopy. Images were analyzed using Imaris software. Quantitative results are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 [Student's two-tailed unpaired t -test (A,B) or one-way ANOVA with a post-hoc Tukey–Kramer test (C)].

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Stable Transfection, Expressing, Transfection, Staining, Plasmid Preparation, Confocal Microscopy, Software, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE transiently overexpressed in ST14A cells is degraded by LAMP2A-dependent autophagy. (A) Schematic of dual-tag fluorescent APOE with quenching of green SepHluorin in lysosomes. (B) APOE3–mCh–SepH and mCh–SepH tag fluorescence intensity in ST14A cells with Baf (50 nM, 4 h). (C) APOE3 mRNA in ST14A cells expressing APOE3–Myc–flag with Baf treatment. (D) APOE3–Myc–Flag abundance in ST14A cells following Baf treatment (4 h 50 nM). (E) APOE3–mCh–SepH fluorescence intensity following BFA (5 μg/ml) treatment. (F) ST14A cells expressing APOE3-myc-flag and treated with BFA (5 μg/ml, 4 h) were analyzed by western blotting. Quantitative results are mean±s.e.m. Revert, protein stain; NT, no treatment. * P <0.05, *** P <0.001 [Student's unpaired two-tailed t -test was used (D); bars above graphs indicate time points at which FDR<0.05 by two-way ANOVA with post-hoc Tukey–Kramer test (B,E)].

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Fluorescence, Expressing, Western Blot, Staining, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: LAMP2A is required for autophagy of APOE3 in ST14A cells. (A,B) ST14A cells were co-transfected with shRNA targeting LAMP2A and (A) APOE3–Myc–Flag or (B) APOE3–mCh, and APOE3 levels were assessed by western blot or fluorescence intensity. shCtrl, control shRNA. Bars above graph in B indicates time points at which FDR<0.05 by two-way ANOVA. (C) APOE levels in mouse brain tissue from 2-year-old wild-type and LAMP2 knockout mice. Quantitative results are mean±s.e.m. * P <0.05, ** P <0.01, **** P <0.0001 [Student's two-tailed unpaired t -test (A,C)].

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Transfection, shRNA, Western Blot, Fluorescence, Knock-Out, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: Fluorescently tagged APOE is endocytosed in an isoform-dependent manner and alters endosomal morphology. (A,B) APOE–mCh-conditioned medium was collected from HEK293T cells and applied to (A) HepG2 and (B) ST14A cells. Cells were imaged and red fluorescence/phase area calculated. Bars represent times when FDR<0.05 between APOE isoforms by two-way ANOVA. (C) ST14A cells were treated for 24 h with conditioned medium with APOE3–mCh or APOE4–mCh (red), and immunocytochemistry for EEA1 or Rab7 (green) was performed, and cells stained with DAPI (blue). Cells were imaged by confocal microscopy and analyzed using Imaris image. Magnified view indicates enlarged images from white boxes in merge panel. (D) HepG2 cells were treated for 24 h with APOE3–mCh or APOE4–mCh conditioned medium, lysosomes were immunoprecipitated and proteomic contents analyzed by mass spectrometry. Proteins reduced in APOE4 lysosomes that were not reduced in mCh-treated cells included mitochondrial proteins such as prohibitin. Western blot analysis for prohibitin was performed on lyso-depleted flow-through. Revert, protein stain. HepG2 cells treated with APOE-mCh conditioned medium were also analyzed by qPCR. Quantitative results are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001; ns, not significant (one-way ANOVA with Tukey–Kramer test).

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Fluorescence, Immunocytochemistry, Staining, Confocal Microscopy, Immunoprecipitation, Mass Spectrometry, Western Blot

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: Knockdown of Rubicon or ATG7 reduces APOE internalization. (A,B) ATG7 and Rubicon were knocked down using siRNA in HepG2 cells, and (A) conditioned medium with APOE3–mCh or (B) medium containing LDL-pHrodo was applied. siCtrl, control siRNA. (C,D) HepG2 cells with siRNA knockdown of (C) ATG7 or (D) Rubicon were analyzed by qPCR. (E) HepG2 cells were treated with inhibitors and APOE3–mCh conditioned medium. Concentrations of inhibitors used were: 50 nM Bafilomycin A1, 20 mM ammonium chloride. NT, no treatment. Cells were imaged and fluorescence quantified by incucyte. Imaging and fluorescence quantification by incucyte. Quantitative results are mean±s.e.m. **** P <0.0001 [Student's two-tailed unpaired t -test (C,D); bars represent times when FDR<0.05 between APOE isoforms by two-way ANOVA with a post-hoc Dunnett's test (A,B,E)]

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Fluorescence, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: FSH and ApoE4 contribute to Alzheimer’s disease-like pathogenesis via C/EBPβ/δ-secretase in female mice

doi: 10.1038/s41467-023-42282-7

Figure Lengend Snippet: Four months old female ApoE4 TR mice were subjected to sham or ovariectomy (OVX) operation, some of the mice after OVX were consecutively treated with anti-FSH antibody (FSH-Ab) (200 μg per day, i.p.) 4 days after OVX for 8 weeks (OVX + FSH Ab group). A , K Representative images of western blot showed the expression of C/EBPβ, AEP, cleaved APP, Tau and p-Tau, GAD67, VGLUT1, PSD95, synapsin, and synaptophysin in the hippocampus. B , K Quantification of protein expression. Data represent mean ± SEM ( n = 4 mice per group, Two-way ANOVA for all the protein quantification except APP N585, one-way ANOVA followed by Tukey’s multiple comparisons test for APP N585). C – H AEP enzymatic activity ( C ), Aβ40 and Aβ42 concentration ( D ), AT8 (red)/Tau N368 (green) immune-reactivity ( E , F ), proteinaceous deposits ( E ) and GFAP (red) or IBA1 (green) positive cells ( G , H ) in the hippocampus were assessed. (scale bar = 50 μm ( E ), or 100 μm ( G )). Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SEM. ( n = 4 mice per group, one-way ANOVA followed by Tukey’s multiple comparisons test). I In order to show the aggregated Tau pathology, total homogenates (TH-S), Sarkosyl-soluble (S1) and Sarkosyl-insoluble (P2) fractions were blotted with antibody against AT8, or T22 for Tau oligomers, respectively. AD human cortex was a positive control. J Golgi staining showed the dendritic spine density. (scale bar = 10 μm). Data represent mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SEM ( n = 4 mice per group, one-way ANOVA followed by Tukey’s multiple comparisons test). L Morris water maze analysis of cognitive functions of the ApoE4-TR mice after sham or OVX operation with or without FSH-Ab treatment. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SEM ( n = 7 mice per group, two-way ANOVA for latency, and speed analyze, one-way ANOVA for AUC latency, Time in target quadrant).

Article Snippet: Antibody to C/EBPβ (HT-7) (catalog#: sc-7962, 1:200 for immunofluorescence and 1:1000 dilution for Western blot) was from Santa Cruz; anti-AEP (6E3) was a gift from Dr. Colin Watts (1:1000 dilution for Western blot), Professor of Immunobiology, Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK; antibodies to phosphor-Tau (Ser202-Thr205) (AT8, catalog#: MN1020, 1:300 for immunofluorescence and 1:1000 dilution for Western blot), phosphor-Tau (Thr212, Ser214) (AT100, catalog#: MN1020, 1:200 for immunohistochemistry) and IBA1 (catalog#: PA5-18039, 1:400 dilution for immunofluorescence) were from Thermo Fisher Scientific; antibodies to AEP (D6S4H) (catalog#: 93627, 1:500 for immunofluorescence and 1:2000 dilution for Western blot), PSD95 (catalog#: 2507, 1:1000 dilution for Western blot) and synapsin (catalog#: 5297, 1:1000 dilution for Western blot)were obtained from Cell Signaling Technology; antibody to Aβ (4G8) (catalog#: 800701, 1:200 dilution for immunofluorescence) was obtained from Biolegend; antibody to Tau 5 (catalog#: MAB361, 1:2000 dilution for Western blot), β-actin (catalog#: A5316, 1:3000 dilution for Western blot) and GFAP (catalog#: MAB360, 1:400 dilution for immunofluorescence)were from Sigma-Aldrich; antibody to ApoE (catalog#: AB947, 1:1000 dilution for Western blot), T22 (catalog#: ABN454, 1:700 dilution for immunofluorescence) and GAD67 (catalog#: MAB5406, 1:2000 dilution for Western blot and 1:500 dilution for immunofluorescence) was bought from Millipore Sigma; antibody to VGLUT1 (catalog#: 135311, 1:500 dilution for immunofluorescence) were from Synaptic Systems; antibody to synaptophysin (catalog#: ab32127, 1:1000 dilution for Western blot) were from Abcam; antibodies to Tau N368 (1:3000 dilution for Western blot and 1:700 for immunofluorescence) and APP N585 (1:1000 dilution for western blot) were produced in the Ye lab. Human ApoE3 (catalog#: 350-02) and ApoE4 (catalog#: 350-04) recombinant protein were purchased from ThermoFisher Scientific.

Techniques: Western Blot, Expressing, Activity Assay, Concentration Assay, Positive Control, Staining

Journal: Nature Communications

Article Title: FSH and ApoE4 contribute to Alzheimer’s disease-like pathogenesis via C/EBPβ/δ-secretase in female mice

doi: 10.1038/s41467-023-42282-7

Figure Lengend Snippet: At 4 months of age, female ApoE4-TR mice received sham or ovariectomy. After ovariectomy, some of the mice were subcutaneously embedded with a 90–day–release pellets (E2, 0.36 mg) of 17β-estradiol to render them biochemically eugonadal, and then with or without FSH treatment. The mice were randomly divided into four groups: sham, OVX, OVX + E2 and OVX + E2 + FSH. A , I Representative images of western blot showed the expression of C/EBPβ, AEP, cleaved APP, Tau, p-Tau, cleaved Tau, GAD67, VGLUT1, PSD95, synapsin and synaptophysin in the hippocampus. B Quantification of the protein expression. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SEM. ( n = 4 mice per group, two-way ANOVA or one-way ANOVA followed by Tukey’s multiple comparisons test). C , D AEP enzymatic activity ( C ), Aβ40, and Aβ42 concentration ( D ) in the hippocampus were examined. Data are presented as mean ± SEM. ( n = 4 mice per group, one-way ANOVA followed by Tukey’s multiple comparisons test). E – G Immunofluorescent (IF) staining showed AT8 (red)/Tau N368 (green) ( E ), AT8 (red)/T22 (green) immuno-reactivity ( F ), and GFAP (red) or IBA1 (green) positive cells ( G ) in the hippocampus. Silver Staining showed the proteinaceous deposits in the CA1 regions ( F ). (scale bar = 50 μm ( E , F ), 100 μm ( G )). Data are shown as mean ± SEM ( n = 4 mice per group, one-way ANOVA followed by Tukey’s multiple comparisons test)). H Golgi staining showed the dendritic spine density in CA1. (scale bar, 10 μm). Data represent mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SEM ( n = 4 mice per group, one-way ANOVA followed by Tukey’s multiple comparisons test). J Morris water maze analysis of cognitive functions showed E2 supplement alleviated OVX-induced learning and memory impairments, but FSH injection after E2 supplement augmented learning and memory impairments. Data shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SEM ( n = 7 mice per group, two-way ANOVA for latency, and speed analyze, one-way ANOVA for AUC latency, time in target quadrant).

Article Snippet: Antibody to C/EBPβ (HT-7) (catalog#: sc-7962, 1:200 for immunofluorescence and 1:1000 dilution for Western blot) was from Santa Cruz; anti-AEP (6E3) was a gift from Dr. Colin Watts (1:1000 dilution for Western blot), Professor of Immunobiology, Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee, Dundee, UK; antibodies to phosphor-Tau (Ser202-Thr205) (AT8, catalog#: MN1020, 1:300 for immunofluorescence and 1:1000 dilution for Western blot), phosphor-Tau (Thr212, Ser214) (AT100, catalog#: MN1020, 1:200 for immunohistochemistry) and IBA1 (catalog#: PA5-18039, 1:400 dilution for immunofluorescence) were from Thermo Fisher Scientific; antibodies to AEP (D6S4H) (catalog#: 93627, 1:500 for immunofluorescence and 1:2000 dilution for Western blot), PSD95 (catalog#: 2507, 1:1000 dilution for Western blot) and synapsin (catalog#: 5297, 1:1000 dilution for Western blot)were obtained from Cell Signaling Technology; antibody to Aβ (4G8) (catalog#: 800701, 1:200 dilution for immunofluorescence) was obtained from Biolegend; antibody to Tau 5 (catalog#: MAB361, 1:2000 dilution for Western blot), β-actin (catalog#: A5316, 1:3000 dilution for Western blot) and GFAP (catalog#: MAB360, 1:400 dilution for immunofluorescence)were from Sigma-Aldrich; antibody to ApoE (catalog#: AB947, 1:1000 dilution for Western blot), T22 (catalog#: ABN454, 1:700 dilution for immunofluorescence) and GAD67 (catalog#: MAB5406, 1:2000 dilution for Western blot and 1:500 dilution for immunofluorescence) was bought from Millipore Sigma; antibody to VGLUT1 (catalog#: 135311, 1:500 dilution for immunofluorescence) were from Synaptic Systems; antibody to synaptophysin (catalog#: ab32127, 1:1000 dilution for Western blot) were from Abcam; antibodies to Tau N368 (1:3000 dilution for Western blot and 1:700 for immunofluorescence) and APP N585 (1:1000 dilution for western blot) were produced in the Ye lab. Human ApoE3 (catalog#: 350-02) and ApoE4 (catalog#: 350-04) recombinant protein were purchased from ThermoFisher Scientific.

Techniques: Western Blot, Expressing, Activity Assay, Concentration Assay, Staining, Silver Staining, Injection