Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: Determination of the interaction affinity between GM1 and APOE. A , schematic illustration of determining the binding affinity between lipid structure and APOE using MST. B , the binding affinity (K D ) between APOE3 or APOE4 and lipid structures containing GM1 or cholesterol (n = 3). C , the binding affinity between APOE3 or APOE4 and lipid structures with different GM1 concentrations. (n = 3). D - H , negative staining images of lipid structures with varying GM1 concentrations. p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). ( A ) is created with BioRender.com . APOE, apolipoprotein E; MST, microscale thermophoresis.

Article Snippet: To determine the binding affinity between APOE-lipoprotein and its receptor LDLR (MedChemExpress), we first labeled APOE3 and APOE4 with RED-NHS 2nd generation, as described earlier.

Techniques: Binding Assay, Negative Staining, Microscale Thermophoresis

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: Analysis of GM1 localization and levels following the cellular uptake of lipid structures. A , schematic illustration of the method used to determine GM1 following the cellular uptake. B , GM1 levels in differentiated PC-12 cells following cellular uptake, determined using the SpectraMax i3 or by quantifying confocal microscopy images. (n ≥ 3) ( C ) representative images showing GM1 and APOE localization in differentiated PC-12 cells following cellular uptake, as determined by confocal microscopy. (n ≥ 6) ( D ) GM1 levels in HEK-293 cells following cellular uptake, determined using the SpectraMax i3 or by quantifying confocal microscopy images. (n ≥ 6) ( E ) (1) cellular uptake of DiD-labeled cholesterol and GM1 lipid structures in U-87 MG cells, and (2) changes in cholesterol levels in U-87 MG cells after cellular uptake of APOE3 and APOE4-enriched cholesterol lipoproteins, as well as a mixture of APOE3 and APOE4-enriched cholesterol and GM1 lipoproteins. (n ≥ 6). p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). ( A ) is created with BioRender.com . Created in BioRender. Dokholyan, N. (2025) https://BioRender.com/g17z389 . APOE, apolipoprotein E; HEK, human embryonic kidney; PC, l -α-phosphatidylcholine.

Article Snippet: To determine the binding affinity between APOE-lipoprotein and its receptor LDLR (MedChemExpress), we first labeled APOE3 and APOE4 with RED-NHS 2nd generation, as described earlier.

Techniques: Confocal Microscopy, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Apolipoprotein E (APOE) regulates the transport of monosialotetrahexosylganglioside (GM1)

doi: 10.1016/j.jbc.2025.110778

Figure Lengend Snippet: The binding affinity of APOE-enriched lipoprotein and APOE receptor LDLR using MST. A and B , the binding affinity between APOE3-enriched lipoprotein with varying GM1 concentration to LDLR. (n = 3) ( C and D ) the binding affinity between APOE4-enriched lipoprotein varying GM1 concentration to LDLR (n = 3). p value: ns (0.05 < p ≤ 1), ∗ (0.01 < p ≤ 0.05, ∗∗ (0.001 < p ≤ 0.01, ∗∗∗ (0.0001 < p ≤ 0.001, ∗∗∗∗ ( p ≤ 0.0001). APOE, apolipoprotein E; LDLR, low-density lipoprotein receptor; MST, microscale thermophoresis.

Article Snippet: To determine the binding affinity between APOE-lipoprotein and its receptor LDLR (MedChemExpress), we first labeled APOE3 and APOE4 with RED-NHS 2nd generation, as described earlier.

Techniques: Binding Assay, Concentration Assay, Microscale Thermophoresis

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: APOE4 triggers dysregulated synaptic vesicle release by disrupting SNARE complex assembly

doi: 10.1007/s00018-025-05787-6

Figure Lengend Snippet: In vitro characterization of the different interactions of APOE3 and APOE4. A Flow chart of the experimental approach to identify APOE genotype-dependent interacting proteins. Apoe -/- mice primary neurons were incubated with either recombinant human APOE3 or APOE4 protein (10 µg/mL for 24 h), then Co-IP and MS analysis were performed to screen the potential APOE trapped proteins. IgG was used as negative control. B Venn-diagram depicting the number of interaction proteins among the APOE3, APOE4, and IgG groups. The top five of the APOE4-specifically enriched proteins were listed according to the abundance in MS data. C GO term enrichment analysis (molecular function) of the APOE3 and APOE4-specifically interacting proteins (top 20). D KEGG pathway analysis of the APOE3 and APOE4-specifically enriched proteins (top 20)

Article Snippet: C57BL/6J background female humanized APOE3- and APOE4 -TR mice were purchased from Cyagen Biosciences.

Techniques: In Vitro, Incubation, Recombinant, Co-Immunoprecipitation Assay, Negative Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: APOE4 triggers dysregulated synaptic vesicle release by disrupting SNARE complex assembly

doi: 10.1007/s00018-025-05787-6

Figure Lengend Snippet: Interaction of APOE with SNARE proteins in an E4 > E3 manner. A , B HA-VAMP2 and Flag-APOE (APOE3 or APOE4) plasmids were transiently co-transfected into HEK-293T cells. Cell lysates were IPed by anti-HA or anti-Flag antibodies and subsequently immunoblotted with the indicated antibodies. C Principle of the BiFC assay. D BIFC analysis of the interaction between VAMP2 and APOE proteins. HEK-293T cells were transiently co-transfected with the BiFC plasmids, VN-VAMP2 and VC-APOE (APOE3 or APOE4), or VAMP2-VN and VC-APOE (APOE3 or APOE4) plasmids. After 48 h of transfection, the cells were imaged by fluorescence microscopy, and the relative fluorescence intensity of Venus was measured using a fluorescence microplate reader. E Endogenous APOE (red) and VAMP2 (green) protein were stained by relative antibodies in the hippocampal tissue from APOE3TR mice. F The lysates of the hippocampal tissue from APOE-TR mice were IPed by anti-VAMP2 or anti-IgG antibodies and subsequently immunoblotted with the indicated antibodies. G Representative images of individual immunofluorescence staining of APOE and VAMP2 interaction in the hippocampus sections of APOE-TR mice tested by Duolink PLA. The red particles (APOE/VAMP2 interaction) represent their interaction. DAPI as a nuclear marker ( n = 6 mice per group, mixed gender). Scale bar: 20 μm. Data information: One-way ANOVA was used for to analyze the BiFC assay, nonpaired Student’s t test was used for other figures. Data are presented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: C57BL/6J background female humanized APOE3- and APOE4 -TR mice were purchased from Cyagen Biosciences.

Techniques: Transfection, Bimolecular Fluorescence Complementation Assay, Fluorescence, Microscopy, Staining, Immunofluorescence, Marker

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: APOE4 triggers dysregulated synaptic vesicle release by disrupting SNARE complex assembly

doi: 10.1007/s00018-025-05787-6

Figure Lengend Snippet: Molecular recognition of the interaction between APOE and VAMP2. A Backbone RMSD of APOE3-VAMP2 and APOE4-VAMP2 complex with time at 300 K. The ordinate represents RMSD (nm), and the abscissa represents time (ns). B The protein structures compactness analysis by Rg of APOE3-VAMP2 and APOE4-VAMP2 complexes during 100 ns of MD simulation. The ordinate represents Rg (nm), and the abscissa represents time (ns). C , D Changes in hydrogen bond number and binding energy between the APOE protein and VAMP2 with MDS time. Color scheme: red indicates APOE3-VAMP2, blue indicates APOE4-VAMP2. E , F Differences in the binding of APOE3 and APOE4 with VAMP2. G The binding patterns of VAMP2 on APOE3 and APOE4 protein surfaces (cartoon model represents VAMP2; red region represents the region forming SNARE complex with SNAP25 and syntaxin-1 proteins; APOE3 and APOE4 are represented by surface model; blue and orange regions on protein surface represent hydrophilic and hydrophobic domains, respectively). H The APOE3 and APOE4 isoforms differ from each other at amino acid residues 112, APOE3 (Cys112) and APOE4 (Arg112). I HA-VAMP2 was co-transfected with Flag-APOE3 (1–136), Flag-APOE4 (1–136), or Flag-APOE (137–299) plasmids into HEK-293T cells. Cell lysates were subjected to immunoprecipitation and subsequent immunoblotting using the indicated antibodies

Article Snippet: C57BL/6J background female humanized APOE3- and APOE4 -TR mice were purchased from Cyagen Biosciences.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: APOE4 triggers dysregulated synaptic vesicle release by disrupting SNARE complex assembly

doi: 10.1007/s00018-025-05787-6

Figure Lengend Snippet: APOE4 inhibits SNARE complex formation in vitro and in vivo. A Flow chart for detecting the effect of different APOE subtypes on SNARE complex assemble in vitro with purified proteins. B A Coomassie-stained gel showing a decrease in the amount of the SNARE complex assemble in the presence of APOE4 in a 20-min reaction. C SNARE complex formation was quantified by western blot following Coomassie-staining, using recombinant human APOE3 or APOE4 protein at doses of 2, 5, and 10 µM. D , E SH-SY5Y cells or Apoe -/- primary neurons were infected with AV-mediated APOE3 and APOE4. SNARE complex levels were determined by western blot. F The illustration of the bilateral hippocampal stereotactic injection and the infection efficiency of AAVs was assessed by immunofluorescence. G Expression levels of the SNARE complex were examined in the hippocampus of 7-month-old AAV (AAV-CMV-EGFP, AAV-APOE3, and AAV-APOE4)- injected female mice ( n = 6–8 mice per group). Data information: Nonpaired Student’s t test was used for B , one-way ANOVA was used for C , D , E and G . Data are presented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001. At least three independent experiments were performed

Article Snippet: C57BL/6J background female humanized APOE3- and APOE4 -TR mice were purchased from Cyagen Biosciences.

Techniques: In Vitro, In Vivo, Purification, Staining, Western Blot, Recombinant, Infection, Injection, Immunofluorescence, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: APOE4 triggers dysregulated synaptic vesicle release by disrupting SNARE complex assembly

doi: 10.1007/s00018-025-05787-6

Figure Lengend Snippet: APOE4 decreases SNARE complex assembly under physiological conditions. A , B Levels of the SNARE complex were examined in the cortex and hippocampus of 6-month-old human APOE3 and APOE4-TR female mice ( n = 6 mice per group). C Relative expression of APOE, VAMP2, SNAP25, and syntaxin-1 in the cortex and hippocampus of 6-month-old human APOE3 and APOE4-TR female mice were determined by immunoblot ( n = 6 mice per group). D , E SNARE-complex assembly in the brain lysates of APOE-TR mice was quantified with Co-IP of SNARE proteins with VAMP2 and SNAP-25 ( n = 3 mice per group). Quantification was performed according to the density of blot bands ratio to GAPDH. Data are presented as mean ± SEM, * p < 0.05. One-way ANOVA was used for A , B and C , nonpaired Student’s t test was used for D and E

Article Snippet: C57BL/6J background female humanized APOE3- and APOE4 -TR mice were purchased from Cyagen Biosciences.

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: APOE4 triggers dysregulated synaptic vesicle release by disrupting SNARE complex assembly

doi: 10.1007/s00018-025-05787-6

Figure Lengend Snippet: APOE4 impedes the phase separation of SNARE complex. A HEK-293T cells were cotransfected with 1 µg/ml EGFP-VAMP2, EGFP-SNAP25, and EGFP- syntaxin1 plasmids for 24 h, then the represent protein condensates were photobleached. The white dashed lines indicate the photobleached area. Scale bar,10 μm. Nine droplets were selected for FRAP analysis. B Fusion and fission (yellow arrowheads) events of droplets formed by VAMP2-mCherry, syntaxin1-ECFP, and syntaxin1-ECFP fusion proteins (1 µg/ml) in conditional solution containing salt ions (1 M NaCl) and crowding reagent (PEG-8000, 10% w/v). Scale bar, 10 μm. C HEK-293T cells were cotransfected with increasing amounts (0, 2.5, or 5.0 µg/ml) of the APOE3 plasmid and the mCherry-VAMP2, EGFP-SNAP25, or syntaxin1-ECFP plasmid (1.0 µg/ml). Representative images were obtained by confocal microscopy 24 h after transfection. D Twelve cells were randomly selected for statistical analysis of droplets size. E The mixture of mCherry-VAMP2, SNAP25-EGFP, and syntaxin1-ECFP fusion proteins were added with different doses of APOE3 or APOE4 recombinant protein (0, 10, 25, and 50 ug/ml) in buffer containing crowding reagent (PEG-8000, 10% w/v) for 5 min. Representative images were obtained by confocal microscopy. F , G Quantification of the droplets number and droplets size. Eighteen fields of view were randomly selected for statistical analysis. Data information: In D , F and G , data are presented as the mean ± SEM. Scale bar: 5 μm. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: C57BL/6J background female humanized APOE3- and APOE4 -TR mice were purchased from Cyagen Biosciences.

Techniques: Plasmid Preparation, Confocal Microscopy, Transfection, Recombinant

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: APOE4 triggers dysregulated synaptic vesicle release by disrupting SNARE complex assembly

doi: 10.1007/s00018-025-05787-6

Figure Lengend Snippet: APOE4 decreases synaptic vesicle release. A Experimental protocol for FM4-64-loading and unloading. B Representative fluorescence intensity of neuron cells stained with FM4-64 in each condition. SH-SY5Y cells were infected with AV-mediated APOE3, APOE4, and control adenovirus C . Apoe -/- mice primary neurons were infected with AV-mediated APOE3 and APOE4 for 48 h, then the cells were incubated with FM4-64 dye and treated with 70 mM KCl to label the synaptic vesicles. Next, 70 mM KCl was added to the FM4-64 loaded cells at room temperature to stimulate the exocytosis. D TEM images from the CA1 region of the hippocampus from 7-month-old APOE3-TR and APOE4-TR mice. The vesicles number, vesicles size and docked vesicles were quantified (two synapses were randomly selected from each section, n = 5 mice per group). Docked vesicles were determined as located within 30 nm of the presynaptic active zone. Scale bars: 500 nm. E Representative images shown the docked dense-core vesicles in resting PC12 cells as determined by the counting the neuropeptide Y-td-mOrange2 labeled vesicles. Scale bars, 10 μm. Data information: Data are presented as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001. Two-way ANOVA for B , C . One-way ANOVA for D . Nonpaired Student’s t test was used for E

Article Snippet: C57BL/6J background female humanized APOE3- and APOE4 -TR mice were purchased from Cyagen Biosciences.

Techniques: Fluorescence, Staining, Infection, Control, Incubation, Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: APOE4 triggers dysregulated synaptic vesicle release by disrupting SNARE complex assembly

doi: 10.1007/s00018-025-05787-6

Figure Lengend Snippet: A hypothetical mechanistic model shows that APOE4 bind to individual SNAREs in such a way that impedes SNARE complex assembly, thereby inhibiting pore fusion and SNARE-mediated exocytosis

Article Snippet: C57BL/6J background female humanized APOE3- and APOE4 -TR mice were purchased from Cyagen Biosciences.

Techniques:

Journal: Frontiers in Immunology

Article Title: Single-cell atlas of human skin implicates APOE pro-inflammatory signaling in diabetic foot ulcers

doi: 10.3389/fimmu.2025.1591944

Figure Lengend Snippet: APOE+ fibroblasts promote fibrosis and inflammation in DFU. (A) Representative images showing histological differences between human healthy donor (HD) and diabetic foot ulcer (DFU) skin tissues assessed by HE staining (left) and APOE expression visualized by IHC (right). Insets display magnifications of different regions in the skin. Scale bar in the original images: 500 μm, 2000 μm. Scale bar in the magnified images: 50 μm. Upper: Cellular infiltration was quantified in HE-stained sections by measuring the percentage of infiltrated area. Lower: APOE expression was quantified through positive area percentage analysis of IHC staining. (B) Western blot analysis and quantitative comparison of APOE protein in human HD and DFU skin tissues. (C) RT-qPCR analysis of mRNA levels of α-SMA, COL1A1 and COL3A1 in human HD and DFU skin tissues. (D) Western blot analysis and quantitative comparison of the NF-κB and JAK1/Stat3 signaling pathways in human HD and DFU skin tissues. (E) Western blot analysis and quantitative comparison of the NF-κB and JAK1/Stat3 signaling pathways in human fibroblasts treated with APOE3 at concentrations of 10 ng/mL or 25 ng/mL. (F) Western blot analysis and quantitative comparison of the NF-κB and JAK1/Stat3 signaling pathways in human fibroblasts treated with APOE3 at a concentration of 10 ng/mL for 6 h, 12 h or 24 h. Data are presented as mean ± SD from three independent biological replicates (n = 3). * p < 0.05, *** p < 0.001.

Article Snippet: Cells were treated with human recombinant APOE3 protein (MedChemExpress, China) at 10 ng/mL or 25 ng/mL for 24 hours, or at 10 ng/mL for 6, 12, or 24 hours.

Techniques: Staining, Expressing, Immunohistochemistry, Western Blot, Comparison, Quantitative RT-PCR, Protein-Protein interactions, Concentration Assay