Fig 2: Knockdown of Netrin-1 (NTN-1) in hippocampal CA1 regions decreased not only visceral sensitivity but also the expression levels of mRNA and proteins of Netrin-1, DCC, GluA1, and PSD95. (A) The representative external abdominal oblique muscle EMG recordings. (B) VMR amplitudes to 40 and 60 mmHg CRD were significantly reduced in rats expressing Lenti-shNTN-1 (MS + shNTN-1) than in rats injected Lenti-Scramble (MS + Scramble). MS + Scramble: n = 12. MS + shNTN-1: n = 12. All data were given as mean ± SEM. ***P < 0.001, two-tailed unpaired Student’s t-test. (C) The mRNA and (E) protein expressions of Netrin-1, DCC, GluA1, and PSD95 in the hippocampus were significantly reduced in the MS + shNTN-1 group. (D) Representative immunoblots showed the expression of Netrin-1, DCC, GluA1, NR2A, NR2B, PSD95, and ß-Tubulin. (F) A representative fluorescence image confirmed the successful GFP expression in shNTN-1 infected cells in the hippocampus. Scale bar, 500 µm. MS + Scramble: n = 4. MS + shNTN1: n = 4. All data were given as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t-test.

Fig 3: Netrin-1 and GluA1 protein were upregulated in the hippocampus in rats of three MS groups. Representative immunoblots showed the expression of Netrin-1, DCC, Neo-1, GluA1, NR1, NR2A, NR2B, PSD95, and ß-Tubulin on PND21 (A), PND35 (C), and PND70 (E). (B) Western blot analysis showed increased expression of Netrin-1, GluA1, NR2A, NR2B, and PSD95 in the hippocampus in MS rats on PND21, (D) PND35, and (F) PND70. Furthermore, the protein expression level of DCC was upregulated on PND35. On PND21: NC, n = 8; MS, n = 8. On PND35: NC, n = 8; MS, n = 8. On PND70: NC, n = 8; MS, n = 8. All data were given as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t-test.

Fig 4: RNA-seq results of the hippocampus in MS and NC rats of three different age periods (n = 4 per group). (A–C) The differentially expressed genes (DEGs) on PND21 (A), PND35 (B), and PND70 (C) were significantly enriched in various KEGG signaling pathways. The top-ranked pathways were visualized with bubble plots. (D) Venn diagram of enriched pathways showed that axon guidance pathway was the only shared enriched pathway which ranked among top five of all age periods. (E) Venn diagram of DEGs in the hippocampus showed that 54 DEGs were shared in different age periods. (F) A total of 54 shared DEGs were significantly enriched in various pathways, including the axon guidance pathway. (G) A simplified schematic of the axon guidance pathway. Netrin-1, its specific receptor deleted in colorectal cancer (DCC), as well as other two molecules marked in red were upregulated among MS rats of three periods. Netrin-1 and DCC were specific ligands or receptors for each other.

Fig 5: Gai1 and Gai3 associate with Netrin-1-stimualted CD146, required for CD146 internalization. Expression of listed signaling proteins of the Netrin-1 cascade in WT and Gai1/3 DKO MEFs was shown (A). WT MEFs were treated with Netrin-1 (25 ng/mL) for 5 min, CD146-Gai1/3-Gab1 association (“IP”) and expression (“Input”) was shown (B). DCC, Neogenin and UNC5B did not associate with Gai1/3 in Netrin-1-treated WT MEFs (B). Puromycin-selected stable WT MEFs with CD146 shRNA (“shCD146”) or the scramble control shRNA (“shC”) were treated with Netrin-1 (25 ng/mL) for 15 min, expression of listed proteins was shown (C). WT MEFs were treated with Netrin-1 (25 ng/mL) for 1-5 min, expression of listed protein in membrane fraction lysates, cytosol fraction lysates and total cell lysates was tested (D). WT or Gai1/3 DKO MEFs were treated with Netrin-1 (25 ng/mL) for 5 min, expression of listed protein in membrane fraction lysates and total cell lysates was tested (E). Quantifications were from five biological replicates (n = 5). *P < 0.05 versus “shC” or “0 min” (D). #P < 0.05.