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: CNS Neuroscience & Therapeutics

Article Title: Sequential Proteomic Analysis Reveals the Key APOE4 ‐Induced Pathological and Molecular Features at the Presymptomatic Stage in Alzheimer's Disease Mice

doi: 10.1111/cns.70306

Figure Lengend Snippet: Repeated behavioral tests identify the presymptomatic stage of AD in 5xFAD mice expressing human APOE3 or APOE4. (A) Breeding diagram of 5xFAD mice expressing human APOE3 or APOE4 and their littermates (without 5xFAD allele). (B–F) Behavioral tests of mice at 3‐month‐old (3 M) ( n = 8–12 mice/group). (B) Representative images of mouse movement trajectories in the open field test. (C) Total distance of mouse movement (left panel) and the time spent in corner zone (right panel) during the open field test. (D) The number of arm entries (left panel) and the percentage of correct spontaneous alternation in Y maze test (right panel). (E) Representative heatmaps of mouse movement trajectories during novel object recognition test. (F) Recognition time (left panel) and the recognition index (right panel) during the novel object recognition test. (G–L) Behavioral tests of mice at 10‐month‐old (10 M) ( n = 9–14 mice/group). (G) Representative track images of mouse moving during the open field test. (H) Total distance of mouse movement (left panel) and the time spent in corner zone (right panel) during the open field test. (I) The number of arm entries (left panel) and the percentage of correct spontaneous alternation in Y maze test (right panel). (J) Representative track images of mice swimming during the final test in water maze. (K) The latency of mice escaping to the platform during the training process. (L) The speed of mice during the final test in water maze (left panel) and the time spent in the quadrant zone where the platform was located (right panel). Data were analyzed using Kruskal‐Wallis test for (F) left panel, (H) right panel and (L) right panel. The data of latency to platform (K) in water maze test were analyzed using the two‐way repeated measures ANOVA. The other data were analyzed using one‐way ANOVA with post hoc Tukey's test.

Article Snippet: The humanized APOE4 ( APOE4 +/+ , Cat. #NM‐HU‐190002) and APOE3 ( APOE3 +/+ , Cat. #NM‐HU‐190003) knock‐in mice on C57BL/6J background were purchased from Shanghai Model Organisms Center Inc.

Techniques: Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: Sequential Proteomic Analysis Reveals the Key APOE4 ‐Induced Pathological and Molecular Features at the Presymptomatic Stage in Alzheimer's Disease Mice

doi: 10.1111/cns.70306

Figure Lengend Snippet: Human APOE4 causes more Aβ deposition and neuroinflammation than human APOE3 in the brain of 5xFAD mice at the presymptomatic stage. (A, B) Representative images and quantification of immunofluorescence staining of Aβ1‐42 in the cortex and hippocampus of 3‐month‐old (3 M) mice brains. n = 5 mice/group. (C, D) Representative images and quantification of immunofluorescence staining of Aβ1‐42 in the cortex and hippocampus of 10‐month‐old (10 M) mice brains. (E) Representative images of immunofluorescence staining of Iba1 (a marker for microglia) in the cortex and hippocampus of 3‐month‐old (left panel) and 10‐month‐old (right panel) mouse brains. (F‐I) Quantifications of Iba1‐positive cell numbers (F, H) and Iba1‐positive cell area (G, I) in the cortex and hippocampus of 3‐month‐old mouse brains. n = 5 mice/group. (J‐M) Quantifications of Iba1‐positive cell numbers (J, L) and Iba1‐positive cell area (K, M) in the cortex and hippocampus of 10‐month‐old mouse brains. n = 5–7 mice/group. White scale scar: 50 μm, ** p < 0.01, *** p < 0.001. Data were analyzed using one‐way ANOVA with post hoc Tukey's test, except for (H) and (K), which were analyzed using Kruskal‐Wallis test.

Article Snippet: The humanized APOE4 ( APOE4 +/+ , Cat. #NM‐HU‐190002) and APOE3 ( APOE3 +/+ , Cat. #NM‐HU‐190003) knock‐in mice on C57BL/6J background were purchased from Shanghai Model Organisms Center Inc.

Techniques: Immunofluorescence, Staining, Marker

Journal: CNS Neuroscience & Therapeutics

Article Title: Sequential Proteomic Analysis Reveals the Key APOE4 ‐Induced Pathological and Molecular Features at the Presymptomatic Stage in Alzheimer's Disease Mice

doi: 10.1111/cns.70306

Figure Lengend Snippet: Sequential proteomic analysis of human APOE4 and APOE3‐induced molecular changes in the hippocampus of 5xFAD mice at the presymptomatic stage. (A, B) Summary of the differentially expressed protein (DEP) numbers identified between different groups in the hippocampus of 3‐month‐old (A) and 10‐month‐old (B) mice, respectively. DEPs were defined as proteins with fold change (FC) > 2 or FC < 0.5, and a p < 0.05. (C, D) Top10 KEGG‐enriched pathways in comparisons between E3‐AD and E3‐WT mice (10‐month), and E4‐AD and E4‐WT mice (10‐month), respectively. (E) Top20 significantly enriched KEGG pathways at level 2 in comparisons between E4‐AD and E3‐AD mice (10‐month). (F, G) The top20 upregulated and downregulated proteins in comparisons between E4‐AD and E3‐AD mice (10‐month). Synapse‐related proteins were marked in green, while mitochondria‐related proteins were marked in purple. (H) Venn diagram showing overlap of upregulated (left panel) and (downregulated) DEPs in comparisons between E4‐AD and E3‐AD mice at the age of 3 months and 10 months, respectively.

Article Snippet: The humanized APOE4 ( APOE4 +/+ , Cat. #NM‐HU‐190002) and APOE3 ( APOE3 +/+ , Cat. #NM‐HU‐190003) knock‐in mice on C57BL/6J background were purchased from Shanghai Model Organisms Center Inc.

Techniques:

Journal: CNS Neuroscience & Therapeutics

Article Title: Sequential Proteomic Analysis Reveals the Key APOE4 ‐Induced Pathological and Molecular Features at the Presymptomatic Stage in Alzheimer's Disease Mice

doi: 10.1111/cns.70306

Figure Lengend Snippet: Sequential phosphoproteomic analysis of human APOE4 and APOE3‐induced molecular changes in the hippocampus of 5xFAD mice at the presymptomatic stage. (A, B) Summary of the differentially expressed phosphopeptides (DEpP) numbers identified between different groups in the hippocampus of 3‐month‐old (A) and 10‐month‐old (B) mice, respectively. DEPs were defined as proteins with fold change (FC) > 2 or FC < 0.5, and a p < 0.05. (C, D) Top10 KEGG‐enriched pathways in comparisons between E3‐AD and E3‐WT mice (10‐month), and E4‐AD and E4‐WT mice (10‐month), respectively. (E) Top20 significantly enriched KEGG pathways at level 2 in comparisons between E4‐AD and E3‐AD mice (10‐month). (F, G) The top20 upregulated and downregulated phosphopeptides in comparisons between E4‐AD and E3‐AD mice (10‐month). Synapse‐related proteins were marked in green, while mitochondria‐related proteins were marked in purple. ANK‐2 was marked with two colors due to its classification as both a synapse‐related protein and a mitochondria‐related protein. A protein name may appear multiple times as different phosphopeptides from the same protein can be detected. (H) Venn diagram showing overlap of upregulated (left panel) and (downregulated) DEpPs in comparisons between E4‐AD and E3‐AD mice at the age of 3 months and 10 months, respectively.

Article Snippet: The humanized APOE4 ( APOE4 +/+ , Cat. #NM‐HU‐190002) and APOE3 ( APOE3 +/+ , Cat. #NM‐HU‐190003) knock‐in mice on C57BL/6J background were purchased from Shanghai Model Organisms Center Inc.

Techniques:

Journal: CNS Neuroscience & Therapeutics

Article Title: Sequential Proteomic Analysis Reveals the Key APOE4 ‐Induced Pathological and Molecular Features at the Presymptomatic Stage in Alzheimer's Disease Mice

doi: 10.1111/cns.70306

Figure Lengend Snippet: Human APOE4 results in more pronounced synaptic degeneration than human APOE3 in the brain of 5xFAD mice at the presymptomatic stage. (A) Representative images of Golgi staining of apical dendrites (highlighted by arrows) of pyramidal neuron in the hippocampal CA1 region of 10‐month‐old mice. (B) Quantification of spines on the apical dendrites of pyramidal neuron stained in (A) Each point represents a neuron, 20 neurons per mouse, n = 5 mice/group. ** p < 0.01, *** p < 0.001, Kruskal‐Wallis test. White scale scar: 10 μm. (C, D) Western blotting measurement of PSD95, Synaptophysin, and BIG2 expression levels. n = 6–7 mice/group, * p < 0.05, one‐way ANOVA with post hoc Tukey's test. (E, F) All the significantly upregulated and downregulated synapse‐related proteins in comparison between E4‐AD and E3‐AD mice (10‐month). (G, H) All the significantly upregulated and downregulated synapse‐related phosphopeptides in comparison between E4‐AD and E3‐AD mice (10‐month). A protein name may appear multiple times as different phosphopeptides from the same protein can be detected. (I) Protein–Protein Interaction (PPI) network map of all differentially expressed synapse‐related proteins and phosphopeptides in (E‐H). The hub proteins (connecting with 3 or more other proteins) identified with PPI network were marked with red (upregulated) or blue (downregulated) in the PPI network and boxed in (E‐H).

Article Snippet: The humanized APOE4 ( APOE4 +/+ , Cat. #NM‐HU‐190002) and APOE3 ( APOE3 +/+ , Cat. #NM‐HU‐190003) knock‐in mice on C57BL/6J background were purchased from Shanghai Model Organisms Center Inc.

Techniques: Staining, Western Blot, Expressing, Comparison

Journal: CNS Neuroscience & Therapeutics

Article Title: Sequential Proteomic Analysis Reveals the Key APOE4 ‐Induced Pathological and Molecular Features at the Presymptomatic Stage in Alzheimer's Disease Mice

doi: 10.1111/cns.70306

Figure Lengend Snippet: Human APOE4 impairs neuronal mitochondrial function in the brain of 5xFAD mice at the presymptomatic stage. (A, B) Western blotting measurement of mitochondrial function‐related protein expressions, including TOM20, MFN1, DRP1, Parkin, in mouse hippocampal neuronal cell line HT22 cells expressing human APOE3 or APOE4 fused to HA tag. GAPDH served as an internal control. (C) HT22 cells incubated with Mito‐GFP were analyzed by flow cytometry to determine mitochondrial content. (D, E) Representative images and quantification of mitochondrial membrane potential with TMRM. (F) ATP content measured by firefly luciferase. (G) HT22 cells incubated with DCFH‐DA were analyzed by flow cytometry to determine ROS levels. n = 3–5 biological replicates per treatment. * p < 0.05, ** p < 0.01, Student's t ‐test. (H, I) Significantly upregulated and downregulated mitochondria‐related proteins in comparison between E4‐AD and E3‐AD mice (10‐month). (J, K) Significantly upregulated and downregulated mitochondria‐related phosphopeptides in comparison between E4‐AD and E3‐AD mice (10‐month). A protein name may appear multiple times as different phosphopeptides from the same protein can be detected. (L) Protein–Protein Interaction (PPI) network map of all differentially expressed mitochondria‐related proteins and phosphopeptides in (H‐K). The hub proteins (connecting with 3 or more other proteins) identified with PPI network were marked with red (upregulated) or blue (downregulated) in the PPI network and boxed in (H‐K). (M) PPI network map of all differentially expressed proteins and phosphopeptides associated with mitochondria and synapse. Proteins in the key nodes were determined with having direct connection to synaptic proteins PPI network or mitochondrial proteins PPI network rather than having connection to a single protein. The gene names in the key nodes marked with red or blue means upregulated or downregulated in protein or phosphopeptides.

Article Snippet: The humanized APOE4 ( APOE4 +/+ , Cat. #NM‐HU‐190002) and APOE3 ( APOE3 +/+ , Cat. #NM‐HU‐190003) knock‐in mice on C57BL/6J background were purchased from Shanghai Model Organisms Center Inc.

Techniques: Western Blot, Expressing, Control, Incubation, Flow Cytometry, Membrane, Luciferase, Comparison