Journal: Nature Communications

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

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: 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 P control-NMDA < 0.0001, P control-DHPG < 0.0001). For the time point of 60 min F(2, 21) = 9.459, p = 0.0012) (Tukey’s test P control-NMDA = 0.0041, P control-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, P control_NMDAR < 0.0001, P control_mGluR < 0.0001, P NMDAR_mGluR = 0.8825). Atg101: F(2,15) = 24.31, P < 0.0001 (Tukey’s multiple comparison test, P control_NMDAR < 0.0001, P control_mGluR < 0.0001, P NMDAR_mGluR = 0.9329). Atg13: F(2,15) = 8.386, P = 0.0036 (Tukey’s multiple comparison test, P control_NMDAR = 0.007, P control_mGluR = 0.0086, P NMDAR_mGluR = 0.9940). FIP200: F(2,15) = 17.66, P = 0.0001 (Tukey’s multiple comparison test, P control_NMDAR = 0.0002, P control_mGluR = 0.0009, P NMDAR_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 P control_NMDA = 0.0056, P control_DHPG = 0.0253). Atg101: F(2,6) = 27.57, P = 0.0009 (Tukey’s multiple comparison test P control_NMDA = 0.0013, P control_DHPG = 0.79). Atg13: F(2,6) = 113.1, P < 0.0001 (Tukey’s multiple comparison test P control_NMDA = 0.0027, P control_DHPG < 0.0001). FIP200: F(2,6) = 15.14, P = 0.0045 (Tukey’s multiple comparison test P control_NMDA = 0.0240, P control_DHPG = 0.041). LC3-II: F (2, 12) = 4,969, P = 0.0268 (Tukey’s multiple comparison test P control_NMDA = 0.0478, P control_DHPG = 0.0421).

Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

Techniques: Super-Resolution Microscopy, Labeling, Control, Immunolabeling, Comparison, Western Blot

Journal: Nature Communications

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

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: a – e Representative confocal images of the CA1 stratum radiatum area of wild-type P22-P28 hippocampal slices (as shown in the schematic representation), immunolabeled with antibodies against a Arc, b ULK1, c Atg101, d Atg13, and e FIP200 along with MAP2. Immuno-labeling was performed in slices under control conditions or 15 min after chemical NMDAR- or mGluR-LTD. Scale bars: 50 μm. Graphs showing the number of puncta positive for each ULK1-complex component, normalized to MAP2. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed using one-way ANOVA. ULK1: F(2,15) = 32.10, P < 0.0001 (Tukey’s multiple comparison test P control_NMDAR < 0.0001, P control_mGluR < 0.0001, P NMDAR_mGluR = 0.9906). Atg101: F(2,15) = 32.92, P < 0.0001 (P control_NMDAR < 0.0001, P control_mGluR < 0.0001, P NMDAR_mGluR = 0.8920). Atg13: F(2,15) = 17.88, P = 0.0001 (P control_NMDAR = 0.0002, P control_mGluR = 0.0006, P NMDAR_mGluR = 0.8267). FIP200: F(2,15) = 16.91, P = 0.0001 (P control_NMDAR = 0.0003, P control_mGluR = 0.0006, P NMDAR_mGluR = 0.9247). f Representative electron micrographs of neurobiotin-labeled dendrites of pyramidal neurons in the CA1 stratum radiatum area in hippocampal slices under control conditions and 15 min or 1 h after LTD induction by LFS. Black arrows indicate the labeled dendritic spines. Note the presence of multiple autophagic vesicles in the dendritic shaft near the neck of the spines (asterisks). Bottom panels show characteristic double-membrane-bound autophagic vesicles at higher magnification and the presence of vesicle-like structures inside dendritic spines. Scale bars: 500 nm (white bars), 200 nm (black bars). ( N = 6 animals per condition). g Graph showing the percentage of labeled postsynaptic dendrites containing phagophores in control condition or 15 min after LFS and in the presence or absence of Ciliobrevin-D (25 μM), bath applied for 2 h prior, during and after LFS. Bars represent mean values ± SEM. N = 6 animals per condition, n = 48 dendrites per condition. Statistical analyses were performed by one-way ANOVA, F(2,15) = 41.99, P < 0.0001 (Tukey’s multiple comparison test P control_LFS < 0.0001, P LFS_LFS/CBD = 0.9574, P control_LFS/CBD < 0.0001). h Graph showing the density of autophagic vesicles in postsynaptic labeled dendrites under control conditions and 1 h after LFS-induced LTD with or without bath application of Ciliobrevin-D. Graph bars represent mean values ± SEM. n = 34 dendrites per condition from N = 6 animals. Statistical analyses were performed using Mann–Whitney U test, comparing the LFS condition with or without bath application of Ciliobrevin-D to control (P control_LTD < 0.0001; P control_LTD/CBD < 0.0001; P LTD_LTD/CBD = 0.0209). i Graph showing the size distribution of autophagic vesicles in postsynaptic labeled dendrites 1 h after LFS-induced LTD with or without bath application of Ciliobrevin-D. Bars represent mean values ± SEM. N = 89 autophagic vesicles. Statistical analyses were performed using Mann–Whitney U test ( P = 0.2648).

Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

Techniques: Immunolabeling, Control, Comparison, Labeling, Membrane, MANN-WHITNEY

Journal: Nature Communications

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

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

Techniques: Concentration Assay, Activity Assay, Plasmid Preparation, Avidin-Biotin Assay, Control, Virus, Infection, In Vivo

Journal: Nature Communications

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

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: 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 P control-NMDA < 0.0001, P control-DHPG < 0.0001). For the time point of 60 min F(2, 21) = 9.459, p = 0.0012) (Tukey’s test P control-NMDA = 0.0041, P control-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, P control_NMDAR < 0.0001, P control_mGluR < 0.0001, P NMDAR_mGluR = 0.8825). Atg101: F(2,15) = 24.31, P < 0.0001 (Tukey’s multiple comparison test, P control_NMDAR < 0.0001, P control_mGluR < 0.0001, P NMDAR_mGluR = 0.9329). Atg13: F(2,15) = 8.386, P = 0.0036 (Tukey’s multiple comparison test, P control_NMDAR = 0.007, P control_mGluR = 0.0086, P NMDAR_mGluR = 0.9940). FIP200: F(2,15) = 17.66, P = 0.0001 (Tukey’s multiple comparison test, P control_NMDAR = 0.0002, P control_mGluR = 0.0009, P NMDAR_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 P control_NMDA = 0.0056, P control_DHPG = 0.0253). Atg101: F(2,6) = 27.57, P = 0.0009 (Tukey’s multiple comparison test P control_NMDA = 0.0013, P control_DHPG = 0.79). Atg13: F(2,6) = 113.1, P < 0.0001 (Tukey’s multiple comparison test P control_NMDA = 0.0027, P control_DHPG < 0.0001). FIP200: F(2,6) = 15.14, P = 0.0045 (Tukey’s multiple comparison test P control_NMDA = 0.0240, P control_DHPG = 0.041). LC3-II: F (2, 12) = 4,969, P = 0.0268 (Tukey’s multiple comparison test P control_NMDA = 0.0478, P control_DHPG = 0.0421).

Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

Techniques: Super-Resolution Microscopy, Labeling, Control, Immunolabeling, Comparison, Western Blot

Journal: Nature Communications

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

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet: a – e Representative confocal images of the CA1 stratum radiatum area of wild-type P22-P28 hippocampal slices (as shown in the schematic representation), immunolabeled with antibodies against a Arc, b ULK1, c Atg101, d Atg13, and e FIP200 along with MAP2. Immuno-labeling was performed in slices under control conditions or 15 min after chemical NMDAR- or mGluR-LTD. Scale bars: 50 μm. Graphs showing the number of puncta positive for each ULK1-complex component, normalized to MAP2. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed using one-way ANOVA. ULK1: F(2,15) = 32.10, P < 0.0001 (Tukey’s multiple comparison test P control_NMDAR < 0.0001, P control_mGluR < 0.0001, P NMDAR_mGluR = 0.9906). Atg101: F(2,15) = 32.92, P < 0.0001 (P control_NMDAR < 0.0001, P control_mGluR < 0.0001, P NMDAR_mGluR = 0.8920). Atg13: F(2,15) = 17.88, P = 0.0001 (P control_NMDAR = 0.0002, P control_mGluR = 0.0006, P NMDAR_mGluR = 0.8267). FIP200: F(2,15) = 16.91, P = 0.0001 (P control_NMDAR = 0.0003, P control_mGluR = 0.0006, P NMDAR_mGluR = 0.9247). f Representative electron micrographs of neurobiotin-labeled dendrites of pyramidal neurons in the CA1 stratum radiatum area in hippocampal slices under control conditions and 15 min or 1 h after LTD induction by LFS. Black arrows indicate the labeled dendritic spines. Note the presence of multiple autophagic vesicles in the dendritic shaft near the neck of the spines (asterisks). Bottom panels show characteristic double-membrane-bound autophagic vesicles at higher magnification and the presence of vesicle-like structures inside dendritic spines. Scale bars: 500 nm (white bars), 200 nm (black bars). ( N = 6 animals per condition). g Graph showing the percentage of labeled postsynaptic dendrites containing phagophores in control condition or 15 min after LFS and in the presence or absence of Ciliobrevin-D (25 μM), bath applied for 2 h prior, during and after LFS. Bars represent mean values ± SEM. N = 6 animals per condition, n = 48 dendrites per condition. Statistical analyses were performed by one-way ANOVA, F(2,15) = 41.99, P < 0.0001 (Tukey’s multiple comparison test P control_LFS < 0.0001, P LFS_LFS/CBD = 0.9574, P control_LFS/CBD < 0.0001). h Graph showing the density of autophagic vesicles in postsynaptic labeled dendrites under control conditions and 1 h after LFS-induced LTD with or without bath application of Ciliobrevin-D. Graph bars represent mean values ± SEM. n = 34 dendrites per condition from N = 6 animals. Statistical analyses were performed using Mann–Whitney U test, comparing the LFS condition with or without bath application of Ciliobrevin-D to control (P control_LTD < 0.0001; P control_LTD/CBD < 0.0001; P LTD_LTD/CBD = 0.0209). i Graph showing the size distribution of autophagic vesicles in postsynaptic labeled dendrites 1 h after LFS-induced LTD with or without bath application of Ciliobrevin-D. Bars represent mean values ± SEM. N = 89 autophagic vesicles. Statistical analyses were performed using Mann–Whitney U test ( P = 0.2648).

Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

Techniques: Immunolabeling, Control, Comparison, Labeling, Membrane, MANN-WHITNEY

Journal: Nature Communications

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

doi: 10.1038/s41467-022-28301-z

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

Techniques: Concentration Assay, Activity Assay, Plasmid Preparation, Avidin-Biotin Assay, Control, Virus, Infection, In Vivo