Fig 2: Sec13 regulates the OL secretome.(A) Electron microscopic analysis of the ER structure in spinal cords from control and Sec13-cKO mice at P7. Scale bars: 1 µm. (B) Quantification of ER thickness in spinal cords from control and Sec13-cKO mice at P7 (n = 3 independent experiments). (C and D) Immunoblotting for the indicated proteins in primary control and Sec13-cKO OPCs. (E) Immunoblotting for the indicated proteins in primary rat OPCs or differentiating immature OLs following treatment with scrambled (siSCR) or Sec13 siRNAs. (F) Real-time PCR analysis of myelination-associated genes in primary rat OPCs under differentiation conditions following treatment with DMEM/F-12 or CM (n = 3 independent experiments). (G) Volcano plot showing the differential protein abundance (highlighted in color; fold change >2; P < 0.05) in the CM of primary rat differentiating immature OLs relative to that in OPCs. (H) Volcano plot showing the differential protein abundance (highlighted in color; fold change >2; P < 0.05) in the CM of primary rat differentiating immature OLs treated with Sec13 siRNA relative to scrambled siRNA. (I) Venn diagram showing the overlap between differentially secreted factors upon OPC differentiation and knockdown of Sec13 in differentiating immature OLs. (J) GO functional category analysis of the proteins with reduced secretion after knockdown of Sec13. Data represent the mean ± SD. *P < 0.05 and ***P < 0.001, by 2-tailed, unpaired Student’s t test. Down, downregulated; Up, upregulated.

Fig 3: Morphological reorganization of ER, ERGIC and Golgi apparatus in aberrant AT-1 models. (a) Representative ER morphology in primary-cultured MEFs using ER3-mCherry (scale bar, 3 µm). White asterisk (*) indicates an enlarged cisternae within the perinuclear rough ER. High-magnification areas for WT and AT-1 sTg are shown (scale bar, 2 µm). (b–f) Quantification of puncta revealed using SIM microscopy in primary-cultured MEFs in (b) Sec13 puncta (n = 14 WT; n = 13 AT-1 sTg; n = 10 AT-1S113R/+), (c) Sec31 puncta (n = 25 WT; n = 20 AT-1 sTg; n = 21 AT-1S113R/+), (d) Sec23 puncta (n = 4 WT; n = 7 AT-1 sTg; n = 5 AT-1S113R/+), (e) Sec24 puncta (n = 12 WT; n = 15 AT-1 sTg; n = 14 AT-1S113R/+), (f) Sec16 puncta (n = 19 WT; n = 15 AT-1 sTg; n = 14 AT-1S113R/+). (g–j) Pearson’s coefficient of puncta revealed using SIM microscopy in primary-cultured MEFs in (g) Sec31 and Sec13 puncta (n = 8 WT; n = 7 AT-1 sTg; n = 5 AT-1S113R/+), (h) Sec23 and Sec24 puncta (n = 4 WT; n = 7 AT-1 sTg; n = 5 AT-1S113R/+), (i) Sec31 and Sec24 puncta (n = 8 WT; n = 8 AT-1 sTg; n = 9 AT-1S113R/+), (j) Sec13 and Sec16 puncta (n = 6 WT; n = 6 AT-1 sTg; n = 5 AT-1S113R/+). (k) ERGIC morphology in primary-cultured MEFs using ERGIC-53 antibody (scale bar, 3 µm) and quantified using Imaris reconstruction of volume and number of puncta (n = 7 WT; n = 7 AT-1 sTg; n = 7 AT-1S113R/+). (l) Golgi apparatus morphology in primary-cultured MEFs using GM130 antibody (scale bar, 3 µm) and quantified using Imaris reconstruction of volume and area (n = 11 WT; n = 12 AT-1 sTg; n = 12 AT-1S113R/+). (m) Representative electron microscopy of MEFs from WT, AT-1 sTg, and AT-1S113R/+. Yellow outlines Golgi structures and orange outlines disorganized secretory structures and vesicles. (scale bar, 500 nm). MEFs from multiple cells from 3 biologically independent animals for each genotype for (b–l). One-way ANOVA. *P < 0.05; **P < 0.005.

Fig 4: PTN promotes OL differentiation via autocrine signaling.(A) Real-time PCR analysis of myelination-associated genes in primary rat OPCs under differentiation conditions without T3, but in the presence or absence of the indicated recombinant factors (PTN, 100 nM; LIF, 50 ng/μL; APOE, 10 μg/μL; MDK, 100 nM; CSAD, 1.3 ng/μL). n = 3 independent experiments. (B) Immunolabeling for CNP in primary rat OPCs under differentiation conditions for 72 hours following treatment with scrambled or PTN siRNAs. Scale bars: 100 μm. (C) Immunoblotting for transfected PTN-FLAG in CM and cellular whole-cell lysate (WCL) from Oli-neu cells transduced with scrambled or Sec13 shRNA. (D and E) Immunostaining (D) and quantification (E) of CNP signals in primary rat OPCs under differentiation conditions following PTN or PTN-mutant (L18&20R) overexpression (n = 3 independent experiments). Scale bar: 50 μm. (F and G) Immunostaining (F) and quantification (G) of MBP signals in primary rat OPCs under differentiation conditions following treatment with scrambled or Sec13 siRNAs and recombinant PTN protein, respectively (n = 3 independent experiments). Scale bar: 50 μm. (H) Co-IP of the indicated endogenous factors with PTN in primary rat differentiating immature OLs. (I) Immunoblotting for the indicated proteins in primary rat differentiating immature OLs following treatment with recombinant PTN for 1 hour. (J) Real-time PCR analysis of myelination-associated genes in primary rat OPCs under differentiation conditions following treatment with scrambled or p190 siRNAs and recombinant PTN protein, respectively (n = 3 independent experiments). Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Tukey’s correction for multiple comparisons.

Fig 5: The COPII complex is implicated in remyelination after demyelination.(A) Immunostaining for Sec13, Olig2, and MBP at 7 dpl in nonlesion white matter control, PBS-injected, and LPC-injured spinal cords from 8-week-old mice. Arrows indicate Sec13+ and Olig2+ cells. Scale bars: 20 µm. (B) Immunostaining for Sec31A, Olig2, and MBP at 7 dpl in nonlesion white matter control, PBS-injected, and LPC-injured spinal cords from 8-week-old mice. Arrows indicate Sec31A+ and Olig2+ cells. Scale bars: 20 µm. (C and D) Quantification of Sec13A+Olig2+ cells among Olig2+ cells (C) and Sec31A+Olig2+ cells among Olig2+ cells (D) in LPC lesion sites (n = 3 animals/treatment). Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Tukey’s correction for multiple comparisons. (E) Immunoblotting of the indicated proteins in cultured rat OPCs and differentiating OLs after T3 treatment. Ctrl, control.