Fig 2: NMDAR- and mGluR-LTD trigger the rapid appearance of autophagic structures in dendrites of cultured neurons.a Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled for surface GluA2 (under non-permeabilizing conditions). Graph showing the number of surface GluA2 labeling, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA, F (2, 15) = 38.28) (Tukey’s test Pcontrol-NMDAR < 0.0001, Pcontrol-mGluR < 0.0001, PNMDAR-mGluR = 0.8438). b Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled with an antibody against endogenous LC3 (autophagic structures) and MAP2 (dendrites). Graph showing the number of dendritic LC3-positive puncta in secondary dendrites, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F2,24) = 15.11, P < 0.0001) (Tukey’s test Pcontrol-NMDA = 0.0005, Pcontrol-mGluR = 0.0001). c Same as in b, but neurons were pretreated for 1 h before, during and after the pulse with wortmannin (500 nM) or SBI-0206965 (500 nM). Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions (U: untreated, W: wortmannin, S: SBI-0206965). Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(8,45) = 33.83, P < 0.0001) (Tukey’s test Pcontrol/S-NMDA/S = 0.3677, Pcontrol/W-NMDA/W = 0.9986, PNMDA/U-NMDA/W < 0.0001, PNMDA/U-NMDA/S < 0.0001, Pcontrol/S-DHPG/S = 0.9674, Pcontrol/W-DHPG/W = 0.9989, PDHPG/U-DHPG/W < 0.0001, PDHPG/U-DHPG/S < 0.0001). d Same as in b with neurons that were infected with AAV plasmids carrying 4 shRNA sequences against atg5 (sh-atg5) or scrambled control (sh-scramble), under the CamK2a promoter. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(5,30) = 16.94, P < 0.0001) (Tukey’s test Pcontrol/scr-control/atg5 = 0.9999, PNMDA/scr-NMDA/atg5 = 0.0025, PDHPG/scr-DHPG/atg5 < 0.0001, Pcontrol/scr-NMDA/scr < 0.0001, Pcontrol/scr-DGPG/scr < 0.0001, Pcontrol/atg5-NMDA/atg5 = 0.8959, Pcontrol/atg5-DHPG/atg5 = 0.9637). e Same as in b, but neurons were immunolabeled 15 min after NMDAR- and mGluR-LTD and treated for 1 h before, during and after the pulse with Ifenprodil (10 µM) or MTEP (10 µM) and JNJ16259685 (10 µM) to pharmacologically inhibit NR2B and mGluR1/5 receptors, respectively. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F (3,32) = 74.46, P < 0.0001) (Tukey’s test, PNMDA-NMDA+IFE < 0.0001, PDHPG-DHPG+MTEP/JNJ < 0.0001). Scale bars: 10 µm for all panels.

Fig 3: Proteomic profiling of the autophagic cargo during NMDAR-LTD.a–d Western blot analyses of different fractions along the autophagic vesicle purification procedure, using antibodies against a autophagosomal markers (LC3B, p62, Atg16L1, and Atg9A), b ER-Golgi markers (TGN, LMAN1, SAR1a), c endosomal markers (Rab11b, EEA1), and d markers of the plasma-membrane (Stx4), extracellular vesicles (Alix) and nuclear extracts (TBP). N = 3 independent experiments. e Graph showing the cell component analysis, as false discovery rate (FDR)-corrected p-values, of the dynamic cargo (total of 393 proteins) that is enriched (up) or less abundant (down) in AVs after LTD, compared to control. f Graphical representation of proteins enriched in AVs upon LTD, with relation to the synapse. g Western blot analysis of PK-treated control and LTD-AVs, validating the presence of the proteins identified by the proteomic analyses in the autophagic vesicles. Postsynaptic density (PSD) fraction was used as reference. Graph showing the fold change of the normalized levels of the proteins validated by western blot, as a ratio of LTD to control. Cargo proteins were normalized to the levels of p62, which remains unaffected at the early phase of LTD. N = 3 independent AV preparations. Bars represent mean values ± SEM. Statistical analysis was performed using paired, two-tailed Student’s t-test (GluA1,N = 6, P = 0.0002; GluA2, N = 6, P = 0.0039; Pick1, N = 5, P = 0.011; SAP97, N = 5, P = 0.0179; FYN, N = 8, P < 0.0001; CamKIIa, N = 8, P < 0.0001; IL1RAPL1, N = 8, P = 0.0004; Adam22, N = 4, P = 0.0018; INA, N = 3, P = 0.0287; MYH10, N = 8, P < 0.0001; ITPKA, N = 6, P = 0.0006; KCC2, N = 4, P = 0. 0352; cofilin-1, N = 6, P = 0.005; dynamin, N = 6, P = 0.0005; p62, N = 6, P = 0.9809). All indicated molecular weights in a–d and g are in kDaltons (kD).