Fig. 2: Autophagic vesicles are locally formed in dendrites of cultured neurons following LTD. | Nature Communications

Fig. 2: Autophagic vesicles are locally formed in dendrites of cultured neurons following LTD.

From: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

Fig. 2

a Top, representative super-resolution microscopy dSTORM image of a secondary dendrite labeled with an antibody against LC3, 15 min after cLTD. Bottom, magnification of representative U-shaped LC3-positive structures in dendrites, 15 min after NMDA or DHPG pulses. Scale bars: 2 μm and 250 nm, as indicated. (N = 3 independent experiments). b Graph showing the number of LC3-positive U-shaped structures in secondary dendrites visualized in (a), before (control) and 15 min or 60 min after NMDAR- and mGluR-LTD. Bars represent mean values ± SEM. N = 3 independent experiments per condition (n > 9 dendrites per condition). Statistical analysis was performed by one-way ANOVA. For the time point of 15 min F(2, 33) = 17.93, p < 0.0001) (Tukey’s test Pcontrol-NMDA < 0.0001, Pcontrol-DHPG < 0.0001). For the time point of 60 min F(2, 21) = 9.459, p = 0.0012) (Tukey’s test Pcontrol-NMDA = 0.0041, Pcontrol-DHPG = 0.0024). c Graph showing the size distribution (nm) of the dendritic U-shaped LC3-positive structures visualized in a upon NMDAR- and mGluR-LTD. Bars represent mean values ± SEM for each analysed dendrite. N = 3 independent experiments (n > 40 dendrites per condition). d, Confocal images of dendrites immunolabeled with antibodies against WIPI2, LC3, and MAP2 before (control) or after 15 min of NMDAR- and mGluR-LTD. Scale bar: 10 μm. (N = 6 independent experiments). e, Representative confocal images of neurons immunolabeled with antibodies against ULK1, Atg101, Atg13, FIP200 and, along with MAP2 to label dendrites before (control) or 15 min after LTD-inducing pulses. Scale bar: 20 μm. Graphs showing the number of puncta positive for each ULK1-complex component in secondary dendrites, normalized for dendrite length, in every condition, as indicated. Graph bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed using one-way ANOVA. ULK1: F(2,15) = 24.48, P < 0.0001 (Tukey’s multiple comparison test, Pcontrol_NMDAR < 0.0001, Pcontrol_mGluR < 0.0001, PNMDAR_mGluR = 0.8825). Atg101: F(2,15) = 24.31, P < 0.0001 (Tukey’s multiple comparison test, Pcontrol_NMDAR < 0.0001, Pcontrol_mGluR < 0.0001, PNMDAR_mGluR = 0.9329). Atg13: F(2,15) = 8.386, P = 0.0036 (Tukey’s multiple comparison test, Pcontrol_NMDAR = 0.007, Pcontrol_mGluR = 0.0086, PNMDAR_mGluR = 0.9940). FIP200: F(2,15) = 17.66, P = 0.0001 (Tukey’s multiple comparison test, Pcontrol_NMDAR = 0.0002, Pcontrol_mGluR = 0.0009, PNMDAR_mGluR = 0.6440). f Western blot analyses for Atg13, FIP200, ULK1, Atg101, LC3, and β-III tubulin (Tuj1) in neuronal lysates, under control conditions and 15 min after NMDAR- and mGluR-LTD. Graphs showing the normalized protein levels of Atg13, FIP200, ULK1, Atg101 under the aforementioned conditions. Bars represent mean values ± SEM. N = 3 independent experiments for ULK1 complex proteins, N = 5 independent experiments for LC3-II. Statistical analyses were performed using one-way ANOVA. ULK1: F(2,6) = 13.57, P = 0.0059 (Tukey’s multiple comparison test Pcontrol_NMDA = 0.0056, Pcontrol_DHPG = 0.0253). Atg101: F(2,6) = 27.57, P = 0.0009 (Tukey’s multiple comparison test Pcontrol_NMDA = 0.0013, Pcontrol_DHPG = 0.79). Atg13: F(2,6) = 113.1, P < 0.0001 (Tukey’s multiple comparison test Pcontrol_NMDA = 0.0027, Pcontrol_DHPG < 0.0001). FIP200: F(2,6) = 15.14, P = 0.0045 (Tukey’s multiple comparison test Pcontrol_NMDA = 0.0240, Pcontrol_DHPG = 0.041). LC3-II: F (2, 12) = 4,969, P = 0.0268 (Tukey’s multiple comparison test Pcontrol_NMDA = 0.0478, Pcontrol_DHPG = 0.0421).

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