Fig 1: Deletion of HBEGF abolishes hUC-MSCs’ ability to improve cognitive dysfunction(A) Schematic of lentiviral vectors used for shRNA-mediated HBEGF knockdown (shHBEGF_1, shHBEGF_2, and shHBEGF_3) and scrambled non-targeting control (Lenti-shNC), driven by a U6 promoter. EGFP was co-expressed under a CMV promoter. Immunoblotting (bottom) assesses knockdown efficiency in hUC-MSCs. (B) Experimental schematic examining HBEGF’s role in cognitive recovery. hUC-MSCs were transduced with Lenti-NC or Lenti-shHBEGF_3 for 72 h. MSCshNC and MSCshHBEGF_3 groups were collected and tested for their ability to improve cognitive function in the anti-NMDAR encephalitis model. (C) Timeline and experimental procedures assessing MSCshNC and MSCshHBEGF_3 effects in the mouse model. (D and E) Quantification of NOL (D) and NOR (E) tests showing that hUC-MSCshHBEGF_3 lost the ability to improve cognitive impairment. n = 8 per group. (F) Representative MWM movement paths for each group and quantification of escape latencies during training and platform crossings in the probe test. n = 8 per group. (G) Representative 3D projections and quantification of PSD95 (red), total cell surface NMDAR clusters (green), and synaptic NMDAR clusters (white). hUC-MSCshHBEGF_3 failed to prevent Pt CSF-induced NMDAR reduction. Scale bar, 2 μm. (H) Immunoblot detection of GluN1 (NMDAR), PSD95, and β-actin, showing that Pt CSF administration reduces total and synaptic NMDAR levels, which cannot prevented by hUC-MSCs with HBEGF knockdown. n = 3 per group. Data in (A) and (D)–(H) are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 2: MAPLEX-mediated exosomes efficiently inhibit NMDAR reduction and improve cognitive dysfunction(A) Schematic of the exosome isolation process. (B) WB analysis of HBEGF expression in HEK293 cells transfected with empty vector or HBEGF plasmids. (C) HBEGF expression in exosomes derived from HEK293 cells transfected with empty vector or HBEGF plasmids. (D) WB of the exosome markers CD163 and CD9 and the exosome-exclusive marker GM130 in exosomes from hUC-MSCs transfected with different plasmids. (E) NTA showing size distribution of exosomes from HEK293 cells transfected with different plasmids. (F) Representative TEM images of exosomes from control or HBEGF plasmid-transfected HEK293 cells. Scale bar, 200 nm. (G) Schematic of MAPLEX-mediated exosome therapy. Top: HBEGF:MAPLEXs were isolated, and HBEGF was separated from the exosomal membrane using 405-nm light. Bottom: HBEGF:MAPLEXs ON was intranasally administered to mice for brain delivery. (H and I) NOL (H) and NOR (I) test quantifications show that intranasal exosomes alleviate cognitive impairment in the anti-NMDAR encephalitis mouse model. (J) Representative MWM movement trajectories and escape latencies and quantification of platform crossings in the probe test for each group. n = 8 per group. (K) Representative 3D projections and quantification of PSD95 (red), total cell surface NMDAR clusters (green), and synaptic NMDAR clusters (white). Exosome treatment effectively prevents Pt CSF-induced NMDAR reduction. Scale bar, 2 μm. All quantitative data are presented as mean ± SEM. Statistical analyses for (H)–(K) were performed using one-way ANOVA with Tukey’s post hoc test. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 3: HBEGF secretion by hUC-MSCs inhibits NMDAR reduction and ameliorates cognitive impairment in anti-NMDAR encephalitis(A) Venn diagram comparing hUC-MSC secretory protein expression via transcriptomic analysis. A total of 101 genes overlap between the GSE165811 database (green) and a secretory protein database (red). Genes from this study are highlighted in blue. (B) Representative confocal images showing PSD95 (red), total cell surface NMDAR clusters (green), and synaptic NMDAR clusters (merge). Quantification in primary mouse hippocampal neurons demonstrates HBEGF to be the most effective treatment to reduce NMDAR reduction compared to CST3, THBS1, CCL2, IGFBP4, and MFGE8. n = 6 per group. Scale bar, 10 μm. (C) Experimental timeline and procedures assessing HBEGF’s role in the anti-NMDAR encephalitis mouse model. (D and E) Quantification of the NOL (D) and NOR (E) tests, showing improved exploratory preference with HBEGF treatment. n = 8 per group. (F) Representative MWM movement paths for each group. In the probe test, Pt CSF-treated mice crossed the target quadrant significantly less often than HBEGF-treated mice. n = 8 per group. (G) 3D projections and quantification of total PSD95 and NMDAR clusters, as well as synaptic NMDAR clusters, from a representative animal in each group. HBEGF treatment inhibits NMDAR reduction. Scale bar, 2 μm. (H) GluN1 protein levels are reduced in the Pt CSF + vehicle group compared to the Pt CSF + HBEGF group. n = 3 per group. Statistical analyses for (B) and (D)–(H) were conducted using one-way ANOVA with Tukey’s post hoc test. Data are presented as mean ± SEM. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 4: The HBEGF receptor and downstream pathway regulating antibody-mediated reduction of NMDAR(A) CoIP assay using GluN1 and EGFR antibodies, with IgG as a negative control, demonstrating GluN1-EGFR interaction. (B) Schematic of lentiviral vectors for shRNA-mediated EGFR knockdown (shEGFR_1, shEGFR_2, and shEGFR_3) and scrambled non-targeting control (Lenti-shNC). (C) shRNA-mediated EGFR knockdown efficiency. n = 3 per group. (D) Experimental timeline and procedures for virus (Lenti-shNC and shEGFR_1) injection. (E) Representative immunofluorescence images showing the co-expression of EGFR with neurons in the hippocampal tissue of mice 1 week after the injection of shEGFR_1. Scale bars, 50 μm. n = 3 animals/group. (F and G) NOL (F) and NOR (G) test quantifications show that shEGFR_1 mice lost the ability to alleviate cognitive impairment in the anti-NMDAR encephalitis model. n = 8 per group. (H) Representative MWM movement paths and quantification of platform crossings and escape latencies during training. n = 8 per group. (I) Immunoblot analysis was performed to examine the expression of hippocampal proteins, including GluN1, PSD95, and β-actin. n = 4 per group. (J) 3D reconstructed micrographs and quantification of PSD95 (red), total membrane-localized NMDAR clusters (green), and synapse-specific NMDAR pools (white). shEGFR_1 failed to prevent Pt CSF-induced NMDAR endocytosis. Scale bar, 2 μm. Data in (C) and (E)–(J) are presented as mean ± SEM. For (E), Student’s t test was used. For (C) and (F)–(J), statistical significance was assessed using one-way ANOVA with Tukey’s post hoc test. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Supplier Page from MedChemExpress for HB-EGF Protein, Mouse