Fig 2: haFGF14-154 inhibits AEP activity via miR-206-3p/BDNF pathway in the brain of APP/PS1 mice. (A-C) miR-206-3p level in the PFC and hippocampal DG region of APP/PS1 mice with intranasal administration of 600 µg/kg haFGF14-154-loaded cationic liposomes (L+H600) or 0.9% saline (vehicle), measured by fluorescence in situ hybridization (FISH), n = 4/5. (D-F) The expression of BDNF and AEP (full-length and cleaved AEP) in the PFC of APP/PS1 mice with intranasal administration of L+H600 or vehicle, n = 6. The plasma levels of miR-206-3p (G) and BDNF (H) in patients with Alzheimer's disease (AD), PD and Schizophrienia detected by qPCR. P values are calculated by age-adjusted ANCOVA model with Tukey's post-hoc test. n = 25/16, 18/18, 15/13 and 12/8 for normal control and patients with AD, PD and Schizophrienia in the experiments of qPCR and ELISA, respectively. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. P values are calculated by one-way ANOVA with Tukey's post-hoc test and student's t-test.

Fig 3: Overexpression of the synapsin I fragmentation induces synaptic dysfunction. (a) Schematic diagram of synapsin I domain and its cleavage by AEP. The binding sites of synapsin Ⅰ to SVs and F‐actin are shown. (b) Fluorescent micrographs showing the distribution of full‐length synapsin Ⅰ and synapsin Ⅰ C83 fragment. Scale bar, 20 μm. (c) Immunostaining showing the colocalization of full‐length and synapsin Ⅰ C83 fragment with synaptophysin. Scale bar, 20 μm. (d, e) FM4‐64 uptake assay. The uptake of FM4‐64 is decreased in neurons expressing synapsin Ⅰ C83 fragment when compared with neurons expressing full‐length synapsin Ⅰ. Scale bar, 20 μm. (f) FM4‐64 releasing assay. The release of FM4‐64 was decreased in neurons expressing synapsin Ⅰ C83 fragment when compared with neurons expressing full‐length synapsin Ⅰ. Scale bar, 20 μm. (g) Dil staining of dendritic spines. The density of dendritic spines in neurons expressing synapsin Ⅰ C83 is lower than that in neurons expressing full‐length synapsin Ⅰ. Scale bar, 10 μm. Bar graphs are the quantification of the dendritic densities (n = 10). (h) Representative traces of mEACs recorded from isolated hippocampal neurons expression EGFP‐vector, EGFP‐synapsin I, and EGFP‐synapsin Ⅰ C83. (i) Cumulative plots and mean values (inset) of mEACs amplitude in isolated hippocampal neurons expression vector, synapsin Ⅰ, and synapsin Ⅰ C83. (j) Cumulative plots of mEACs inter‐event interval and mean values (inset) of mEACs frequency in isolated hippocampal neurons in various conditions. Kolmogorov‐Smirnov test in cumulative plots (n = 4–6 per group). Data are mean ± SEM; one‐way ANOVA; * p < 0.005, ** p < 0.001, *** p < 0.001, **** p < 0.0001

Fig 4: MO decreases production and increases clearance of Aβ in APP/PS1 mice. (A) The expression level of BACE1, PS1, AEP, IDE, NEP, and LRP1 from hippocampal lysates of WT mice and APP/PS1 mice with or without MO treatment was evaluated by Western blotting. (B–G) The quantification of the Western blot bands from panel A. β-actin serves as the loading control. The data are presented as Mean ± SEM, (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001 vs. WT or vs. APP/PS1 mice groups.

Fig 5: Netrin‐1 reduction in human PD brains, associated with AEP activation and UNC5C cleavage. A) Netrin‐1 expression profiling by array of PD substantia nigra from dataset GDS3129 (reference series: GSE8397). Means + s.e.m. are shown (18 Control, 29 PD cases; unpaired t‐test). B) Western blot (WB) showing that netrin‐1 reduction accompanied by AEP and caspase‐3 activation in substantia nigra (SN) samples from subjects with PD. Actin, loading control; MW, molecular weight; ◄, full length; ←, cleaved. The data of WB quantitative analysis are presented as means ± s.e.m.(n = 3; 2‐tailed t‐test). C) AEP activity assay of the human SN samples from subjects with PD and age‐matched healthy controls. RFU, relative fluorescence units. Means + s.e.m. are shown (n = 5; 2‐tailed t‐test). D) Immunohistochemistry of UNC5C N467 fragment and TH in SN sections from people with PD and age‐matched healthy controls. Scale bar, 100 µm. Means + s.e.m. are shown (n = 5; 2‐tailed t‐test). E–G) Immunofluorescence co‐localization analysis was used to detect the expression of netrin‐1, TH, N467, and total AEP. Images are representative of nine sections from three subjects with PD and age‐matched healthy controls. Scale bar, 30 µm. Bar graph shows data as mean ± s.e.m.; 2‐tailed t‐test.