Journal: Neuropsychopharmacology

Article Title: The Membrane Proximal Region of AMPA Receptors in Lateral Amygdala is Essential for Fear Memory Formation

doi: 10.1038/npp.2015.121

Figure Lengend Snippet: TAT-MPR(AA) in LA impairs long-term but not short-term fear memory formation. (a) The MPR(AA) peptide is derived from the MPR region of GluA4. The GluA4 MPR is different from GluA1 MPR region at positions 816 and 818 where the GluA4 contains alanines (highlighted by squares). (b) Microinjection of biotin-labeled TAT-conjugated MPR(AA) into the LA (50 μg/μl, 0.5 μl per LA) led to its insertion into cells when tested 30 min later as detected by the Alexa Fluor 568-streptavidin (red) labeling (blue—DAPI staining). (c) Rat were microinjected with fluorescent (FITC)-labeled TAT-MPR(AA) into LA 30 min before fear conditioning. Two hours after training, surface expression of the GluA4 (upper panels) or GluA2 (lower panels) receptors was monitored. We used antibodies against the extracellular portion of the GluA4 or GluA2 to detect surface-expressing receptors. As seen, the surface expression of GluA4 (open arrows) 2 h after fear conditioning training is reduced in neurons that contain the TAT-MPR(AA) peptide (closed arrows) compared with neurons that do not contain the peptide. The GluA2 (open arrow) is expressed at the surface of neurons that contain the TAT-MPR(AA) peptide (closed arrow), indicating that the peptide is not effective in these neurons. (d) Freezing throughout the tone presentations during training of the same animals as in panel (e) was analyzed. There is no significant difference between animals injected with TAT-MPR(AA) peptide and rats injected with saline (F(4)=0.649, p>0.6). (e) Microinjection of the TAT-MPR(AA) peptide (50 μg/μl, 0.5 μl per LA n=10) into LA 30 min before fear conditioning significantly impaired long-term fear memory formation tested 24 h after training when compared with saline-injected rats (0.5 μl saline per LA n=9) (F(1)=9.128, p<0.009). (f) Freezing throughout the tone presentations during training of the same animals as in panel (g) was analyzed. There is no significant difference between animals injected with TAT-MPR(AA) peptide and rats injected with saline (F(2.535)=1.228, p>0.3). (g) Microinjection of the TAT-MPR(AA) peptide (50 μg/μl, 0.5 μl per LA n=11) into LA 30 min before fear conditioning had no significant effect on short-term fear memory formation tested 2 h after training when compared with saline-injected rats (0.5 μl saline per LA n=8) (F(1)=0.679. p>0.4). □, Saline; ▴, TAT-MPR(AA).

Article Snippet: Sections were then incubated overnight at 4 °C with the anti-GluA1, anti-GluA2, or anti-GluA4 primary antibodies that recognize the extracellular portion of the receptors (1:200; Alomone Labs, Israel) in 0.01 M PBS containing 1% BSA.

Techniques: Derivative Assay, Labeling, Staining, Expressing, Injection

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Effects of PDI knockdown on PP2A bindings to PDI and GluA2 in the hippocampus following KA injection. KA increases PDI:PP2A and GluA2:PP2A bindings in control siRNA-infused animals, but not in PDI siRNA-infused animals. ( a ) Representative Western blot images for the PDI:PP2A and GluA2:PP2A bindings. ( b – c ) Quantitative analyses of the effects of PDI siRNA on PP2A level ( b ) and PDI:PP2A binding ( c ) following KA injection (*, # p < 0.05 vs. control siRNA vs. saline; n = 7, respectively; Kruskal–Wallis test followed by Tukey post-hoc test).

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques: Knockdown, Injection, Control, Western Blot, Binding Assay, Saline

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Effects of PDI knockdown on PICK1 bindings to GluA2 in the hippocampus following KA injection. PDI siRNA does not influence PICK1 level in both saline- and KA-treated groups. As compared to control siRNA, PDI siRNA increases GluA2A:PICK1 binding in saline-treated group. Although KA does not affect GluA2:PICK1 binding in control siRNA-infused group, it increases it in PDI siRNA-infused group. ( a ) Representative Western blot images for the GluA2:PICK1 binding. ( b – c ) Quantitative analyses of the effects of PDI siRNA on PICK1 level ( b ) and GluA2:PICK1 binding ( c ) following KA injection (*, # p < 0.05 vs. control siRNA vs. saline; n = 7, respectively; Kruskal–Wallis test followed by Tukey post-hoc test). ( d ) Scheme of the role of PDI in AMPAR internalization. PDI may reduce lead to reduction-induced PP2A activation, which would abolish PICK1-mediated AMPAR internalization by dephosphorylating GluA2 S880 and CaMKII T286 sites.

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques: Knockdown, Injection, Saline, Control, Binding Assay, Western Blot, Activation Assay

Journal: Scientific Reports

Article Title: PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus

doi: 10.1038/s41598-023-41014-7

Figure Lengend Snippet: Primary antibodies used in the present study.

Article Snippet: p-GRIA2 Y869/873/876 , Rabbit , Cell signaling (#3921) , 1:1,000 (WB).

Techniques:

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