Fig 2: NRP2 regulates exocytosis in the NE-like PCa cells by controlling the vesicular fusion pathway(A) VAMP2-positive vesicle localization (pink) in C4–2 cells under +/- NRP2 condition.(B) VAMP2-positive vesicle localization (pink) in DKD cells under +/- NRP2 condition.(C) Quantitation of (A) and (B).(D) Cell viability of DKD cells co-cultured with VAMP2 +/- DKD cells (PI, red).(E) Quantitation of (D).(F) Quantitation of pAKT (Ser 473) (green) co-localization with plasma membrane in DKD cells co-cultured with VAMP2 +/- DKD cells.(G) Differential gene expression in NE-like PCa patients compared with adenocarcinoma (GSE126078).(H) Differential gene expression in NE-like PCa patients compared with adenocarcinoma (SU2C/PCF).(I) Differential gene expression in DKD cells compared with C4–2.(J) Real-time PCR of a set of genes identified in gene set enrichment analysis (GSEA).(K) Western blot of Syn1 upon NRP2 knockdown. Data represented as mean ± SEM (n = 3).

Fig 3: Blockade of clathrin-mediated endocytic pathway via Fc II/III receptors inhibits SynI antibody uptake and restores synaptic transmission.a Representative images of SynI-mAb (1.5 µg/mL)-treated hippocampal neurons incubated either with sodium azide (NaN3; 500 nM), PitStop 2 (30 µM) or a Fc?R II/III antibody at the indicated concentrations for 72 h. The control condition indicates cells exposed to SynI-mAb in the presence of the NaN3/PitStop2 solvent. After treatments, cells were fixed, permeabilized and incubated with anti-mouse fluorescent secondary antibodies (green). Double staining for ßIII-tubulin (blue) was performed after fixation. Scale bars, 10 µm. b Quantification of the immunoreactivity intensity of internalized SynI-mAb. Graph shows mean ± SEM, single neuron data points independent preparations; **p < 0.01, ***p < 0.001, one-way ANOVA/Dunnett’s tests versus control. c Mean frequency and amplitude of mEPSCs (upper panels) and mIPSCs (lower panels) in hippocampal neurons treated with either SynI-mAb (1.5 µg/mL) or vehicle for 72 h in the presence or absence of 500 nM NaN3. d Mean frequency and amplitude of mEPSCs (upper panels) and mIPSCs (lower panels) in hippocampal neurons treated with either control CSF or patient #1 CSF (1.5 µg/mL) for 72 h in the presence or absence of 500 nM NaN3. Graphs show means ± SEM, single neuron data points from three independent preparations; *p <0.05, **p < 0.01, two-way ANOVA/Bonferroni’s tests. e Expression of Fc?II/III receptors in WT and SynI KO hippocampal neurons. Representative images of primary WT and SynI KO hippocampal neurons at 14.

Fig 4: The synaptic effects of patient-derived SynI autoantibodies are mimicked by affinity-purified anti-SynI antibodies.a, b Mean frequency and amplitude of mEPSCs (a) and mIPSCs (b) in neurons exposed for 72 h to affinity-purified anti-SynI monoclonal antibodies (Synaptic System #106011; SynI-mAb, 1.5 µg/mL, red bar), as compared to vehicle (white bar). Graphs show means ± SEM, single neuron data points from three independent preparations; *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney test.

Fig 5: Patient autoantibodies and affinity-purified SynI antibodies have a similar distribution in primary neurons and their intracellular accumulation requires SynI.Representative images of WT (a) or SynI KO (b) neurons incubated for 72 h with either LE-CSF, SynI-mAb or equal volumes of the respective vehicles. Neurons were fixed and primary antibodies were visualized with anti-human and anti-mouse fluorescently conjugated secondary antibodies (green), respectively. Double staining for ßIII tubulin (blue) was performed after fixation. Scale bars, 10 and 1 µm for low and high magnification, respectively. c Quantification of the intensity of primary antibody immunoreactivity. The graph shows means ± SEM, single neuron data points from 4 independent preparations; ***p < 0.001, two-way ANOVA/Bonferroni’s tests. d Representative TEM images of nerve terminals from cultured hippocampal neurons incubated with either control CSF or LE-CSF and processed for immunogold against human IgG antibody. Arrow indicates gold particles after gold enhancement. Scale bars, 200 nm. e Histograms representing the percentage of gold-positive synapses. Graphs show means ± SEM, n = 4–5 synapses from two independent preparations; *p < 0.05, two-tailed Student’s t-test.