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 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 P control-NMDAR < 0.0001, P control-mGluR < 0.0001, P NMDAR-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, P control-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 P control/S-NMDA/S = 0.3677, P control/W-NMDA/W = 0.9986, P NMDA/U-NMDA/W < 0.0001, P NMDA/U-NMDA/S < 0.0001, P control/S-DHPG/S = 0.9674, P control/W-DHPG/W = 0.9989, P DHPG/U-DHPG/W < 0.0001, P DHPG/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 P control/scr-control/atg5 = 0.9999, P NMDA/scr-NMDA/atg5 = 0.0025, P DHPG/scr-DHPG/atg5 < 0.0001, P control/scr-NMDA/scr < 0.0001, P control/scr-DGPG/scr < 0.0001, P control/atg5-NMDA/atg5 = 0.8959, P control/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, P NMDA-NMDA+IFE < 0.0001, P DHPG-DHPG+MTEP/JNJ < 0.0001). Scale bars: 10 μm for all panels.

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Cell Culture, Immunolabeling, Labeling, Infection, shRNA

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 Confocal images of dendrites immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction and in the absence or presence of Dynamin-1 inhibitory peptide (50 µM) or SBI-0206965 (500 nM), a selective inhibitor of the ULK1 kinase activity. Inhibitors were applied 25 min before, during and 15 min after the pulses. Scale bar: 10 µm. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 9 independent experiments. Statistical analysis was performed using one-way ANOVA (F (8, 72) = 7.411, P < 0.0001) (Tukey’s test P control-control/D > 0.99, P control-control/S = 0.9971, P NMDA-NMDA/D = 0.0451, P NMDA-NMDA/S = 0.0008, P DHPG-DHPG/D = 0.0017, P DHPG-DHPG/S = 0.0002). b Confocal images of dendrites of neurons expressing 4 scrambled sequences ( sh-scramble ), or 4 sh-RNAs against atg5 ( sh-atg5 ), immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 10 independent experiments. Statistical analysis was performed using one-way ANOVA (F (5, 54) = 30.02, P < 0.0001) (Tukey’s test, P control/scr-control/atg5 = 0.0626, P NMDA/scr-NMDA/atg5 < 0.0001, P DHPG/scr-DHPG/atg5 < 0.0001, P control/atg5-NMDA/atg5 > 0.99, P control/atg5-DHPG/atg5 = 0.8602, P control/scr-NMDA/scr = 0.0008, P control/scr-DHPG/scr < 0.0001). c Representative images of consecutive confocal z-planes of cultured neurons immunostained with antibodies against PSD95, LC3, and MAP2 to label the dendrites, 15 min after cLTD. Note the colocalization of PSD95 and LC3 in dendritic spines (yellow arrows) and in the dendritic shaft (white arrows), in consecutive z-planes. Scale bar: 10 µm. Graph showing the percentage of PSD95 puncta co-localizing with LC3 in consecutive confocal z-planes in dendritic spines and shafts in control neurons or 15 min after chemically induced NMDAR- or mGluR-LTD. Bars represent mean values ± SEM. N = 8 independent experiments. Statistical analysis was performed by one-way ANOVA (F(5,42) = 48.43, P < 0.0001) (Tukey’s test for dendritic shaft, P control-NMDA = 0.0569, P control-DHPG = 0.1948, for dendritic spines, P control-NMDA < 0.0001, P control-DHPG < 0.0001). d Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of Bafilomycin A1 (50 µM) for 15 min before, during, and 15 min after the NMDA and DHPG pulses. e Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of SBI-0206965 (500 nM) for 30 min before, during, and 15 min after the NMDA and DHPG pulses. f Western blot analysis for GluA2 and PSD95 in lysates of cultured shscrambled or sh-atg5 expressing neurons in control conditions or 15 min after NMDAR- and mGluR-LTD. d – f Graphs showing the levels of PSD95 and GluA2 levels in the indicated conditions, normalized to total protein levels. Bars represent mean values ± SEM. Statistical analysis was performed by one-way ANOVA. d (N = 9 independent experiments) PSD95: F(5,48) = 15.08, P < 0.0001 (Tukey’s test P control-control/Baf = 0.7566, P control-NMDA = 0.0016, P control-DHPG = 0.0081, P NMDA-NMDA/Baf < 0.0001, P DHPG-DHPG/Baf = 0.0013. GluA2: F(5,48)=6.627, P < 0.0001 (Tukey’s test P control-control/Baf = 0.9692, P control-NMDA = 0.0014, P control-DHPG = 0.0067, P NMDA-NMDA/Baf = 0.0421, P DHPG-DHPG/Baf = 0.0127. e ( N = 7 independent experiments) PSD95: F(5,36) = 23.80, P < 0.0001. (Tukey’s test P control-control/SBI > 0.99, P NMDA-NMDA/SBI < 0.0001, P DHPG-DHPG/SBI < 0.0001, P control-NMDA < 0.0001, P control-DHPG < 0.0001, P control/SBI-NMDA/SBI = 0.9764, P control/SBI-DHPG/SBI = 0.6286). Panel e, GluA2: F(5,36)=11.73, P < 0.0001. (Tukey’s test P control-control/SBI = 0.9179, P NMDA-NMDA/SBI = 0.0001, P DHPG-DHPG/SBI = 0.0002, P control-NMDA = 0.0099, P control-DHPG = 0.0323, P control/SBI-NMDA/SBI = 0.9959, P control/SBI-DHPG/SBI = 0.9407). f ( N = 7 independent experiments) PSD95: F(5,36) = 10.93, P < 0.0001. (Tukey’s test P control/scr-control/atg5 = 0.7927, P NMDA/scr-NMDA/atg5 = 0.0045, P DHPG/scr-DHPG/atg5 = 0.0003, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0030, P control/atg5-NMDA/atg5 = 0.9488, P control/atg5-DHPG/atg5 = 0.9976). GluA2: F(5,36) = 10.79, P < 0.0001. (Tukey’s test P control/scr-control/atg5 > 0.99, P NMDA/scr-NMDA/atg5 = 0,0001, P DHPG/scr-DHPG/atg5 = 0.0019, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0021, P control/atg5-NMDA/atg5 = 0.5844, P control/atg5-DHPG/atg5 > 0.99).

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Immunolabeling, Activity Assay, Labeling, Expressing, Cell Culture, 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 – 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).

Article Snippet: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

Techniques: Western Blot, Purification, Two Tailed Test

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: GluA2 (N-terminus) , Alomone , AGP-073 , , 1/1000.

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