Fig 2: Schematic representations of the potential mechanisms of Sec22b and Ykt6 in autophagosome retrograde trafficking and autophagic flux in neurons exposed to I/R. The special neurite structure of neurons leads to unique localizations of lysosomes and autophagosomes. Lysosomes are mainly located in the cell soma, while autophagosomes are preferentially generated at the neurite tip. The balance of Sec22b and Ykt6 expression is essential for autophagosome retrograde trafficking and autophagic flux. The reperfusion-induced increase in Sec22b and decrease in Ykt6 disrupt autophagosome transport and lead to damaged autophagic flux. Autophagy inducer pretreatment could rescue reperfusion-induced disordered expression of Sec22b and Ykt6, maintain autophagic flux, and finally alleviate I/R-induced neuronal injury. In contrast, once reperfusion-induced autophagic flux damage occurs, autophagy inducer posttreatment leads to excessive autophagosome accumulation in neurite terminals and even neuronal injury.

Fig 3: Sec22b knock-down and Ykt6 overexpression induce neuroprotection in MCAO/R mice via autophagy. A, Neurologic scoring of male and female mice at 2 d after MCAO/R. Data are presented as the mean ± SD. Male: **p = 0.0032 for F/+:Nes:MCAO/R + vec versus MCAO + Sec22b-sh group; *p = 0.0111 for MCAO/R + vec versus MCAO + Ykt6-OE group; Female: ***p = 0.0002 for F/+:Nes:MCAO/R + vec versus MCAO + Sec22b-sh group; *p = 0.0111 for MCAO/R + vec versus MCAO + Ykt6-OE group. N.S.: not significant (F(3,132) = 40.53, p < 0.0001, two-way ANOVA). N = 9 mice per group. B, Statistical analysis of the infarct volume of male and female mice assessed by TTC staining at 2 d after MCAO/R. Data are presented as the mean ± SD. Male: ***p < 0.0001 for F/+:Nes:MCAO/R + vec versus MCAO + Sec22b-sh group; ***p < 0.0001 for MCAO/R + vec versus MCAO + Ykt6-OE group; female: ***p < 0.0001 for F/+:Nes:MCAO/R + vec versus MCAO + Sec22b-sh group; ***p < 0.0001 for MCAO/R + vec versus MCAO + Ykt6-OE group. N.S.: not significant (F(3,96) = 78.80, p < 0.0001, two-way ANOVA). N = 9 mice per group. C, Representative TTC staining images of male and female mice at 2 d after MCAO/R. D, E, Adhesive removal test. The time for the mice to touch and remove the stickers was recorded and statistically analyzed. Data are presented as the mean ± SD. ***p < 0.001, ###p < 0.001, &&&p < 0.001, N.S.: not significant by two-way ANOVA with Tukey's multiple comparisons test, n = 12. F/+:Nes: Atg7flox/+; Nestin-Cre; F/F:Nes: Atg7flox/flox; Nestin-Cre.

Fig 4: Sec22b and Ykt6 play key roles in autophagosome retrograde trafficking in neurons exposed to I/R. A, Western blot analysis of Sec22b in penumbra tissue of male and female mice 6 h after MCAO/R. B, The relative protein level of Sec22b was quantified. Data are presented as the mean ± SD. ***p < 0.0001 for sham versus MCAO/R in males; ###p < 0.0001 for sham versus MCAO/R in females (F(1,20) = 0.2836, p = 0.6002, one-way ANOVA). N = 6 mice per group. C, Immunofluorescence analysis was performed in the penumbra tissue of mice at 6 h after MCAO/R. Sec22b antibody (red), NeuN antibody (white) and GFAP antibody (green) were used, and nuclei were fluorescently labeled with DAPI (blue). Scale bar: 50 µm. D, Double immunofluorescence analysis was performed with antibodies against Sec22b (green) and LC3 (red) in neurons at 6 h after OGD/R. Nuclei were fluorescently labeled with DAPI (blue). Representative images of reproducible triplicate experiments. Arrows indicate the colocalization of Sec22b and LC3 in neurites. Scale bar: 20 µm. E, Western blot analysis of Ykt6 in penumbra tissue of male and female mice 6 h after MCAO/R. F, The relative protein level of Ykt6 was quantified. Data are presented as the mean ± SD. *p = 0.0165 for sham versus MCAO/R in males; ##p = 0.0039 for sham versus MCAO/R in females (F(1,20) = 0.06,209, p = 0.8058, one-way ANOVA). N = 6 mice per group. G, Western blot assay of Ykt6 in cultured neurons 6 h after OGD/R. H, The relative protein level of Ykt6 was quantified. Data are presented as the mean ± SD. **p = 0.0073 for Nor versus OGD/R group by unpaired t test. N = 3 independent replicates. I, Immunofluorescence analysis was performed with an antibody against LC3 (red) in neurons overexpressing GFP-Ykt6 at 6 h after OGD/R. Nuclei were fluorescently labeled with DAPI (blue). Representative images of reproducible triplicate experiments. Arrows indicate the colocalization of GFP-Ykt6 and LC3 in the proximal neurite. Scale bar: 20 µm. J, Western blotting was performed to detect the response of Sec22b knock-down and Ykt6 overexpression to baf A1 in cultured neurons under non-OGD conditions. Rapamycin treatment was used as a positive control. K, The LC3-II/I ratio was calculated. Data are presented as the mean ± SD. **p = 0.0062 for baf A1– versus baf+ in the Rapa group (F(1,20) = 13.97, p = 0.0013, two-way ANOVA). N = 3 independent replicates. L, Western blot analysis of LC3 in neurons exposed to the indicated treatments 6 h after OGD/R. M, The LC3-II/LC3-I ratio was calculated. **p = 0.0039 for baf A1– versus baf A1+ in Sec22b-si group; Data are presented as the mean ± SD. **p = 0.0054 for baf A1– versus baf A1+ in Ykt6-OE group (F(1,12) = 23.73, p = 0.0004, two-way ANOVA). N = 3 independent replicates. Original images of Western blottings for this figure are shown in Extended Data Figure 3-1.

Fig 5: Sec22b knock-down and Ykt6 overexpression improved the outcomes of rapamycin posttreatment in MCAO/R mice. A, Western blot analysis of Sec22b in the penumbra brain tissue 6 h after MCAO/R. B, The relative protein level of Sec22b was quantified. Data are presented as the mean ± SD. ***p < 0.0001 for MCAO/R versus sham group, ##p = 0.0038 for MCAO/R + Rapa-pre versus MCAO/R group, N.S.: not significant (F(3,20) = 17.50, p < 0.0001, one-way ANOVA). N = 6 mice per group. C, Western blot analysis of Ykt6 in the penumbra brain tissue 6 h after MCAO/R. D, The relative protein level of Ykt6 was quantified. Data are presented as the mean ± SD. ***p = 0.0005 for MCAO/R versus sham group, #p = 0.0109 for MCAO/R + Rapa-pre versus MCAO/R group, N.S.: not significant (F(3,20) = 20.12, p < 0.0001, one-way ANOVA). N = 6 mice per group. E, Representative TTC staining images of mice at 2 d after MCAO/R. F, Statistical analysis of infarct volume. Data are presented as the mean ± SD. **p = 0.0072, &p = 0.0162, &&p = 0.003 versus MCAO/R group, N.S.: not significant versus MCAO/R + Rapa-post group, ###p < 0.0001 (F(4,25) = 22.73, p < 0.0001, one-way ANOVA). N = 6 mice per group. Original images of Western blottings for this figure are shown in Extended Data Figure 6-1.