Fig 1: Experimental timeline. The mice were administered Netrin-1 (45 μg/kg) or vehicle control (sterile PBS) at 1 h after surgery (Day 0). The mice in Cohort 1 were used to detect behavior 6 h after surgery. Once the behavior test was completed, they were sacrificed immediately for immunofluorescence and ELISA. The mice in Cohort 2 were used to detect behavior at 9 h after surgery. Once the behavior test was completed, they were sacrificed immediately for WB. The mice in Cohort 3 were used to detect behavior 24 h after surgery. Twenty mice in Cohort 4 were injected intravenously with 100 μl 10-kDa dextran at 6 h after surgery. Fifteen minutes after injection, each mouse was prepared for immunofluorescence. Another 20 mice in Cohort 4 were injected intravenously with 100 μl 10-kDa dextran at 24 h after surgery. Fifteen minutes after injection, each mouse was prepared for spectrophotometric quantification of 10-kDa dextran.
Fig 2: Exogenous recombinant human Netrin-1 decreased the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokines in the sera, prefrontal cortex, and hippocampus of surgical mice. (A–C) Expression levels of IL-6, IL-10, and HMGB-1 in serum at 6 h after surgery/anesthesia. (D–F) ELISA was used to detect the expression of IL-6, IL-10, and Netrin-1 in the hippocampus at 6 h postoperatively. (G–I) Altered expression of IL-6, IL-10, and Netrin-1 in the prefrontal cortex at 6 h postoperatively. The data are presented as mean ± standard error of the mean for each group (n = 5 per cohort). *p < 0.05 vs. the control group, #p < 0.05 vs. the surgery group.
Fig 3: Earlier alterations of NTN‐1 and DCC in nigral dopaminergic neurons after MPTP intoxication. (A) Experimental design in a subacute MPTP mouse model. Brains were collected from mice at 6 h after 1 (MPTP‐1'd), 3 (MPTP‐3'd), or 5 (MPTP‐5'd) doses of MPTP injection and on day 7 (MPTP+7d) after the last injection. (B) Representative images of NTN‐1 (Green) in dopaminergic neurons (TH‐positive, Red) of the midbrain at 6 h after 1, 3, or 5 doses of MPTP injection and on day 7 after the last injection. Scale bar, 50 μm. (C) Representative images of DCC (Green) in dopaminergic neurons (TH‐positive, Red) of the midbrain at 6 h after 1, 3, or 5 doses of MPTP injection and on day 7 after the last injection. Scale bar, 50 μm. MPTP: 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine; NTN‐1, netrin‐1; DCC: deleted in colorectal carcinoma; TH: tyrosine hydroxylase.
Fig 4: Dose‐ and time‐dependent alterations of NTN‐1‐DCC signaling after MPP+ stimulation in SH‐SY5Y cells. (A) Western blotting analysis of p‐PAK and p‐Src at 24 h after different doses of MPP+ stimulation in SH‐SY5Y cells. Data were presented as mean ± SEM for four independent experiments in each group and analyzed by one‐way ANOVA followed by Tukey post hoc test. ***p < 0.001, **p < 0.01 versus Con group. (B) Western blotting analysis of Bax, Bcl‐2, cleaved caspase‐3, and caspase‐3 at 24 h after different doses of MPP+ stimulation in SH‐SY5Y cells. Data were presented as mean ± SEM for four independent experiments in each group and analyzed by one‐way ANOVA followed by Tukey post hoc test. ***p < 0.001, **p < 0.01, *p < 0.05 versus Con group. (C) Western blotting analysis of p‐PAK and p‐Src at different times after 100 μM MPP+ stimulation in SH‐SY5Y cells. Data were presented as mean ± SEM for four independent experiments in each group and analyzed by one‐way ANOVA followed by Tukey post hoc test. **p < 0.001, **p < 0.01, *p < 0.05 versus Con group. (D) Western blotting analysis of Bax, Bcl‐2, cleaved caspase‐3, and caspase‐3 at different times after 100 μM MPP+ stimulation in SH‐SY5Y cells. Data were presented as mean ± SEM for four independent experiments in each group and analyzed by one‐way ANOVA followed by Tukey post hoc test. ***p < 0.001, **p < 0.01, *p < 0.05 versus Con group. Con, control; DCC, deleted in colorectal carcinoma; MPP+: 1‐methyl‐4‐phenylpyridinium iodide; NTN‐1, netrin‐1.
Fig 5: Plasma NTN‐1 level and its correlation with UPDRS score in PD patients. (A) Scatter diagram of plasma NTN‐1 levels between the HC and PD groups. Data were presented as dot plots in each group and analyzed by Mann–Whitney test. (B) ROC curves of plasma NTN‐1 levels for distinguishing PD patients from HC. The whiskers in the box plots indicate the values from minimum to maximum, and the center line indicates the median. (C) Relationship between plasma NTN‐1 levels and UPDRS I among PD patients. (D) Relationship between plasma NTN‐1 levels and UPDRS II among PD patients. (E) Relationship between plasma NTN‐1 levels and UPDRS III among PD patients. AUC, area under the ROC curve; HC, healthy control; NTN‐1, netrin‐1; PD, Parkinson's disease; UPDRS, Unified Parkinson disease rating scale.
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