glua2 (n-terminus) alomone agp-073  (Alomone Labs)

Journal: EMBO Reports

Article Title: Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aβ pathology

doi: 10.1038/s44319-024-00090-0

Figure Lengend Snippet: ( A , B ) Immunocytochemistry and quantification showing dendritic marker MAP2, total (t) and surface (s) GluA1 ( A ) and GluA2 ( B ) from WT primary cortical neuron cultures treated with Aβ 1-42 (Aβ42, 1 µM) or scrambled peptide (Scr, 1 µM) for 2 h at DIV 12–14. For GluA1, * P = 0.0192 for surface level, P = 0.42 for total level, and n = 17 and 18 cells from two independent cultures treated with Scr and Aβ42, respectively. For GluA2, ** P = 0.0025 for surface level, P = 0.1584 for total level, and n = 16 and 20 cells from two independent cultures treated with Scr and Aβ42, respectively. ( C , D ) Immunocytochemistry and quantification showing dendritic marker MAP2, total (t) and surface (s) GluA1 ( C ) and GluA2 ( D ) from PSD-95 +/− primary cortical neuron cultures treated with Aβ 1-42 (Aβ42, 1 µM) or scrambled peptide (Scr, 1 µM) for 2 h at DIV 12–14. For GluA1, P = 0.4539 for surface level, P = 0.1311 for total level, and n = 14 and 13 cells from two independent cultures treated with Scr and Aβ42, respectively. For GluA2, P = 0.6643 for surface level, P = 0.659 for total level, and n = 13 and 14 cells from two independent cultures treated with Scr and Aβ42, respectively. Data Information: significance was determined by Student’s t test ( A , C ) or Mann–Whitney U test ( B , D ). Data are represented as mean ± SEM with * P < 0.05, ** P < 0.01, ns non-significant. Scale bar: 5 μm. .

Article Snippet: Mouse anti-GluA1-N-terminus antibody (#MAB2263) and mouse anti-GluA2-N-terminus antibody (#MAB397) are from Millipore.

Techniques: Immunocytochemistry, Marker, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Activation of innate immune receptor TLR9 by mitochondrial DNA plays essential roles in the chemical long-term depression of hippocampal neurons

doi: 10.1016/j.jbc.2024.105744

Figure Lengend Snippet: ODN2088 blocks NMDA-induced internalization of GluA2 containing AMPA receptors. A , immunocytochemical analysis of the effects of ODN2088 on the NMDA-induced reduction of cell surface GluA2. Cultured hippocampal neurons expressing hemagglutinin (HA)-tagged GluA2 were treated with 50 μM NMDA for 10 min without or with ODN2088 (1 μM, 10 min). After fixation, cell surface HA-GluA2 were stained ( red ) and after treatment with Triton X-100, neurons were immunostained for total HA-GluA2 ( blue ). The dendritic regions marked by squares are enlarged in the panels to the right . The scale bar represents 10 μm. Lower graph : quantification of the NMDA-induced reduction in the ratio of surface to total HA-GluA2 fluorescence intensities. Data are represented as the ratio of surface HA-GluA2 immunoreactivity to total HA-GluA2 immunoreactivity. The ratio of control neurons without ODN2088 treatment was defined as 100% (n = 22). Data are presented as mean + SEMs and individual data points. p value by one-way ANOVA, followed by Student-Newman-Keuls post hoc test. B and C , antibody-feeding assay evaluating the effects of ODN2088 on NMDA-induced internalization of cell surface HA-GluA2 ( B ) and endogenous GluA2 ( C ). B , living neurons expressing exogenous HA-GluA2 were labeled with anti-HA antibodies. Neurons were treated with 50 μM NMDA for 10 min with or without ODN2088 treatment. After fixation, cell surface HA antibodies were stained ( red ), and after treatment with Triton X-100, internalized HA antibodies were stained ( green ). The dendritic regions marked by squares are magnified in the panels to the right . The scale bar represents 10 μm. Lower graph : quantification of the NMDA-induced increase in the ratio of internalized to surface HA-antibody fluorescence intensities. The ratio of control neurons without ODN2088 treatment was defined as 100% (n = 14). Data are presented as mean + SEM and individual data points. p value by one-way ANOVA followed by Student−Newman−Keuls post hoc test. C , endogenous GluA2 in living neurons was labeled with antibodies against the extracellular region of GluA2. Neurons were treated with 50 μM NMDA for 10 min with or without ODN2088 treatment. After fixation, cell surface GluA2 antibodies were stained ( red ), and after treatment with Triton X-100, internalized GluA2 antibodies ( green ) and the dendritic marker MAP2 were stained ( blue/white ). The dendritic regions marked by squares are enlarged in the panels to the right . The scale bar represents 10 μm. Lower graph : quantification of the NMDA-induced increase in the ratio of internalized to surface GluA2-antibody fluorescence intensities. The ratio of control neurons without ODN2088 treatment was defined as 100% (n = 11–13). Data are presented as mean + SEM and individual data points. p value by one-way ANOVA followed by Student-Newman-Keuls post hoc test. D , biotinylation assay of endogenous GluA2. Hippocampal cultures were stimulated by NMDA without or with ODN2088. Cell surface proteins were biotinylated and pulled down from the total cell lysates. The amount of GluA2 proteins in the pulled down fraction ( left gel ) and total cell lysate fraction ( right gel ) were analyzed with the immunoblot analysis. NMDA stimulation decreased the amount of the cell surface GluA2, and treatment with ODN2088 blocked the NMDA effect. Right graph : the intensity of the GluA2 band in the biotinylated fraction was normalized to that of the total cell lysate fraction. The ratio of biotinylated/total GluA2 in the control cultures without ODN2088 treatment was arbitrarily set to 100% (n = 4 each). Data are presented as mean + SEM and individual data points. p value by Kruskal–Wallis test and Student-Newman-Keuls post hoc test. AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; NMDA, N-methyl-d-aspartate.

Article Snippet: Commercial antibodies: anti-HA, (Covance 901501), anti-actin (Sigma-Aldrich A3853), anti-GluA2 N terminus (Millipore MAB397), anti-GluA2 C terminus (Millipore AB10529), anti-FLAG (Sigma F7425), anti-caspase-3 (ProteinTech 66470-2-Ig), anti-TLR9 (Abcam ab134368), anti-cytochrome c ProteinTech 10993-1-AP), anti-TOM20 (ProteinTech 11802-1-AP) and anti-DNA (Millipore CBL186) antibodies; Alexa-350 (Thermo Fisher Scientific A-11045), 405 (Thermo Fisher Scientific A-31556), 488 (Thermo Fisher Scientific A-11008), 546 (Thermo Fisher Scientific A-11003), horseradish peroxidase (Rockland 18-8816-33, 18-8817-33)-conjugated secondary antibodies.

Techniques: Cell Culture, Expressing, Staining, Fluorescence, Feeding Assay, Labeling, Marker, Cell Surface Biotinylation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Activation of innate immune receptor TLR9 by mitochondrial DNA plays essential roles in the chemical long-term depression of hippocampal neurons

doi: 10.1016/j.jbc.2024.105744

Figure Lengend Snippet: TLRs function on NMDA-induced reduction of cell surface GluA2. A and B , immunocytochemical analysis of the effects of TLR3 knockdown ( A ) and TLR7 knockdown ( B ) on the NMDA-induced endocytosis of cell surface GluA2. Cultured hippocampal neurons expressing HA-tagged GluA2 with siRNA were treated with 50 μM NMDA for 10 min. Following fixation, the cell surface HA-GluA2 ( red ) and total HA-GluA2 ( blue ) were stained. The dendritic regions marked by squares were enlarged in the panels to the right . The scale bar represents 10 μm. Right graph : quantification of NMDA-induced reduction in the ratio of surface to total HA-GluA2 fluorescence intensity. Data are represented as the ratio of surface HA-GluA2 immunoreactivity normalized by total HA-GluA2 immunoreactivity. The ratio of control neurons was defined as 100% (n = 14). Data are presented as mean + SEM and individual data points. p value by two-tailed Student’s t test. C , immunocytochemical analysis of the effects of TLR9 knockdown on the NMDA-induced endocytosis of cell surface GluA2. Cultured hippocampal neurons expressing HA-tagged GluA2 with scrambled RNA ( top ), siRNA ( middle ), and siRNA with TLR9 resistant -FL ( bottom ) were treated with 50 μM NMDA for 10 min. Following fixation, the cell surface HA-GluA2 ( red ), TLR9 resistant -FL ( green ), and total HA-GluA2 ( blue ) were stained. The dendritic regions marked by squares were enlarged in the panels to the right . The scale bar represents 10 μm. Right graph : quantification of NMDA-induced reduction in the ratio of surface to total GluA2 fluorescence intensity. Data are represented as the ratio of surface HA-GluA2 immunoreactivity normalized by total HA-GluA2 immunoreactivity. The ratio of scramble RNA transfected control neurons was defined as 100% (n = 18–21). Data are presented as mean + SEM and individual data points. p value by Kruskal–Wallis test and Dunn’s post hoc test. HA, hemagglutinin; TLR, toll-like receptor; NMDA, N-methyl-d-aspartate.

Article Snippet: Commercial antibodies: anti-HA, (Covance 901501), anti-actin (Sigma-Aldrich A3853), anti-GluA2 N terminus (Millipore MAB397), anti-GluA2 C terminus (Millipore AB10529), anti-FLAG (Sigma F7425), anti-caspase-3 (ProteinTech 66470-2-Ig), anti-TLR9 (Abcam ab134368), anti-cytochrome c ProteinTech 10993-1-AP), anti-TOM20 (ProteinTech 11802-1-AP) and anti-DNA (Millipore CBL186) antibodies; Alexa-350 (Thermo Fisher Scientific A-11045), 405 (Thermo Fisher Scientific A-31556), 488 (Thermo Fisher Scientific A-11008), 546 (Thermo Fisher Scientific A-11003), horseradish peroxidase (Rockland 18-8816-33, 18-8817-33)-conjugated secondary antibodies.

Techniques: Cell Culture, Expressing, Staining, Fluorescence, Two Tailed Test, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Activation of innate immune receptor TLR9 by mitochondrial DNA plays essential roles in the chemical long-term depression of hippocampal neurons

doi: 10.1016/j.jbc.2024.105744

Figure Lengend Snippet: Mitochondrial morphological changes and mitophagy induced by NMDA treatment. A , cultured hippocampal neurons expressing mitochondria-targeted cyan fluorescent protein (mito-CFP) were stimulated with NMDA and observed for up to 7 min. Images of the mitochondria every 1 min after NMDA stimulation from the ODN2088 untreated neuron ( upper panel ) and ODN2088 treated neuron ( lower panel ). The scale bar represents 10 μm. B , quantitative analysis of the length of mitochondria. Cultured hippocampal neurons expressing mito-CFP were stimulated with NMDA for 7 min in the absence or presence of ODN2088. After fixation, the length of mitochondria was quantified. n = 37 to 50 mitochondria from three independent cultures. Data are presented as mean + SEM and individual data points. p value by Kruskal–Wallis test and Dunn’s post hoc test. C , histogram of mitochondrial length in the ODN2088 untreated neurons. White and black bars indicate the frequency of mitochondria without (control) or with the NMDA stimulation, respectively. D , schematic drawing of lipidation and translocation of microtubule-associated protein 1 light chain 3 (LC3) from the cytosol to the isolation membrane upon autophagy induction. Phosphatidylethanolamine is attached to cytosolic LC3 by the Atg16L complex and lipidated LC3 translocates to the isolation membrane of the autophagosome ( , ). E , cultured hippocampal neurons expressing mito-CFP and mCherry-LC3B were stimulated with NMDA and observed for up to 7 min. Images of the neurons before and 7 min after NMDA stimulation are shown. The dendritic regions enclosed by the white squares are magnified in the right panels . The scale bars represent 10 μm in the left panels and 5 μm in the right panels . Arrows indicate the mitochondria surrounded by mCherry-LC3B. Arrowheads indicate mitochondria outside autophagosomes. F , high-resolution images of mCherry-LC3 and mito-CFP from the NMDA-untreated (control) and NMDA-stimulated neurons. The scale bars represent 5 μm. G , line scan of the fluorescence intensities of mCherry-LC3 and mito-CFP. The mCherry-LC3 ( red ) and mito-CFP ( cyan ) fluorescence were quantified along the dendrites indicated by white arrows in (F), indicating that the mito-CFP signal was surrounded by the mCherry-LC3 signal in the NMDA-stimulated neuron, whereas, mCherry-LC3 uniformly distributed along the dendrite in the control neuron. H , quantitative analysis of the number of mitophagy within the 100 μm dendrite. Data are presented as mean + SEM and individual data points. n = 18 from three independent cultures. p value by two-tailed Student’s t test. I , cultured hippocampal neurons expressing HA-GluA2 were pretreated with Mdivi-1 and stimulated with NMDA. The cell surface HA-GluA2 ( red ) and total HA-GluA2 ( blue ) were stained. The dendritic regions marked by squares were enlarged in the panels to the right . The scale bars represent 10 μm. Right graph : quantification of NMDA-induced reduction in the ratio of the surface to total GluA2 fluorescence intensities. Data are represented as the ratio of surface HA-GluA2 immunoreactivity normalized by total HA-GluA2 immunoreactivity. The ratio in Mdivi-1–untreated control neurons was defined as 100% (n = 14–15). Data are presented as mean + SEM and individual data points. ∗ p < 0.05 by one-way ANOVA and Student-Newman-Keuls post hoc test. HA, hemagglutinin; Mdivi, mitochondrial division inhibitor; NMDA, N-methyl-d-aspartate.

Article Snippet: Commercial antibodies: anti-HA, (Covance 901501), anti-actin (Sigma-Aldrich A3853), anti-GluA2 N terminus (Millipore MAB397), anti-GluA2 C terminus (Millipore AB10529), anti-FLAG (Sigma F7425), anti-caspase-3 (ProteinTech 66470-2-Ig), anti-TLR9 (Abcam ab134368), anti-cytochrome c ProteinTech 10993-1-AP), anti-TOM20 (ProteinTech 11802-1-AP) and anti-DNA (Millipore CBL186) antibodies; Alexa-350 (Thermo Fisher Scientific A-11045), 405 (Thermo Fisher Scientific A-31556), 488 (Thermo Fisher Scientific A-11008), 546 (Thermo Fisher Scientific A-11003), horseradish peroxidase (Rockland 18-8816-33, 18-8817-33)-conjugated secondary antibodies.

Techniques: Cell Culture, Expressing, Translocation Assay, Isolation, Membrane, Fluorescence, Two Tailed Test, Staining

Journal: The Journal of Biological Chemistry

Article Title: Activation of innate immune receptor TLR9 by mitochondrial DNA plays essential roles in the chemical long-term depression of hippocampal neurons

doi: 10.1016/j.jbc.2024.105744

Figure Lengend Snippet: Mitochondrial DNA amount affected the NMDA-induced internalization of AMPA receptors. A , dideoxycytidine (ddC) reduced the amount of mitochondrial DNA (mtDNA). Cultured hippocampal neurons were treated with ddC for 96 h and stained by the antiMAP2 and DNA antibodies. The fluorescence intensities of DNA staining within the dendrites were quantified. The average fluorescence intensities of ddC untreated (control) neurons were defined as 100% (n = 16). Data are presented as mean + SEM and individual data points. p value by two-tailed Student’s t test. The scale bars represent 10 μm. B , cultured hippocampal neurons expressing HA-GluA2 were pretreated with ddC and stimulated with NMDA. The cell surface HA-GluA2 ( red ) and total HA-GluA2 ( blue ) were stained. The dendritic regions marked by squares were enlarged in the panels to the right . The scale bars represent 10 μm. Lower graph : quantification of NMDA-induced reduction in the ratio of the surface to total GluA2 fluorescence intensities. Data are represented as the ratio of surface HA-GluA2 immunoreactivity normalized by total HA-GluA2 immunoreactivity. The ratio in ddC-untreated control neurons was defined as 100% (n = 15). Data are presented as mean + SEM and individual data points. p value by Kruskal–Wallis test and Student-Newman-Keuls post hoc test. C , ddC effects on the NMDA-induced AMPA receptor endocytosis in the TLR9 knocked down neurons. The ratio in ddC-untreated control neurons was defined as 100% (n = 16). Data are presented as mean + SEM and individual data points. No significant difference was detected by one-way ANOVA. The scale bars represent 10 μm. AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; HA, hemagglutinin; NMDA, N-methyl-d-aspartate; TLR, toll-like receptor.

Article Snippet: Commercial antibodies: anti-HA, (Covance 901501), anti-actin (Sigma-Aldrich A3853), anti-GluA2 N terminus (Millipore MAB397), anti-GluA2 C terminus (Millipore AB10529), anti-FLAG (Sigma F7425), anti-caspase-3 (ProteinTech 66470-2-Ig), anti-TLR9 (Abcam ab134368), anti-cytochrome c ProteinTech 10993-1-AP), anti-TOM20 (ProteinTech 11802-1-AP) and anti-DNA (Millipore CBL186) antibodies; Alexa-350 (Thermo Fisher Scientific A-11045), 405 (Thermo Fisher Scientific A-31556), 488 (Thermo Fisher Scientific A-11008), 546 (Thermo Fisher Scientific A-11003), horseradish peroxidase (Rockland 18-8816-33, 18-8817-33)-conjugated secondary antibodies.

Techniques: Cell Culture, Staining, Fluorescence, Two Tailed Test, Expressing

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

Journal: Cell Death & Disease

Article Title: SIVA-1 regulates apoptosis and synaptic function by modulating XIAP interaction with the death receptor antagonist FAIM-L

doi: 10.1038/s41419-020-2282-x

Figure Lengend Snippet: a Representative confocal images of hippocampal neurons. At 7 days in vitro, immunofluorescence was performed by staining with anti-SIVA-1 (green), Hoechst (nucleus marker, blue). MitoTracker (mitochondria marker), anti-Rab5 (early endosome marker), anti-calnexin (ER marker), anti-β III Tubulin (cytoskeleton marker), and anti-TrkA (cytosolic membrane marker) were stained in red. Scale bar 10 µm. b Graph represents Mander’s coefficients M1 corresponding to the channel 1 (SIVA-1) signal that co-located with the channel 2 (cellular markers) signal. Only values higher than 0.5 were considered to indicate co-localization. c Subcellular protein fractionation assay of adult mouse brain. Histone 3 antibody was used as a nuclear marker, GluA2 as a plasma membrane marker, anti-calnexin as reticular membrane protein, cytochrome C as a mitochondrial protein, and GAPDH as a cytosolic protein. N nuclear fraction, HM heavy membrane fraction, LM light membrane fraction, C cytosolic fraction. d Representative confocal images of hippocampal neurites. At 14 days in vitro, immunofluorescence was performed by staining with anti-SIVA-1 (green), presynaptic markers synapsin II (red) postsynaptic marker PSD95 (blue). The graph represents Mander’s coefficient corresponding to co-localization between SIVA-1 and the synaptic markers. SIVA-1 was found to locate in some of the synapses (arrows and asterisks). Scale bar 2 µm.

Article Snippet: Hippocampal neurons at 12–14 DIV were incubated with antibodies against the N-terminus of GluA2 (2 μg/ml, mouse monoclonal, clone 6C4, Millipore Cat# MAB397) for 60 min at 20 °C.

Techniques: In Vitro, Immunofluorescence, Staining, Marker, Membrane, Fractionation, Clinical Proteomics

Journal: Cell Death & Disease

Article Title: SIVA-1 regulates apoptosis and synaptic function by modulating XIAP interaction with the death receptor antagonist FAIM-L

doi: 10.1038/s41419-020-2282-x

Figure Lengend Snippet: a Representative confocal images of neurons transfected with SIVA-1, FAIM-L, or empty vector. GluA2 internalization assay was performed in neurons treated with NMDA to stimulate LTD and in untreated neurons. Only GFP-positive cells (first column) were considered for quantification. Internalized GluA2 (second column, red in merge) and surface GluA2 (third column, green in merge) were measured. Scale bar 10 µm. b Results were normalized to empty vector, untreated cells. Induction of chemical LTD induced GluA2 internalization in empty vector condition and SIVA-1 transfected cells ( F (1, 632) = 15.85). Non-stimulated cells overexpressing SIVA-1 showed an increase in GluA2 internalization. FAIM-L overexpression blocked LTD induction ( F (3, 632) = 15.17), and its overexpression with SIVA-1 in untreated cells restored basal levels of receptor internalization ( F (3, 632) = 15.85). Each point represents an independent experimental repeat in which 15–20 cells were analyzed. UT untreated, NMDA N-methyl-D-aspartate. c GluA2 surface receptor was isolated with the Biotin surface assay in hippocampal cells transfected with SIVA-1 or empty vector as a control. Lower panel shows quantification of GluA2 surface receptor ( t (2) = 3,060). Two-way ANOVA * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.0001.

Article Snippet: Hippocampal neurons at 12–14 DIV were incubated with antibodies against the N-terminus of GluA2 (2 μg/ml, mouse monoclonal, clone 6C4, Millipore Cat# MAB397) for 60 min at 20 °C.

Techniques: Transfection, Plasmid Preparation, Over Expression, Isolation, Control

Journal: Cell Death & Disease

Article Title: SIVA-1 regulates apoptosis and synaptic function by modulating XIAP interaction with the death receptor antagonist FAIM-L

doi: 10.1038/s41419-020-2282-x

Figure Lengend Snippet: a Representative confocal images of neurons transfected with SIVA-1 or empty vector and treated with 10 μM of Q-VD (caspase inhibitor). Only GFP-positive cells (first column) were considered for quantification (left panel). Scale bar 10 µm. b The results of the quantification of internalized GluA2 vs. surface GluA2 were normalized to empty vector, untreated cells (right panel). Each point represents an independent experimental repeat in which 15–20 cells were analyzed. SIVA-1-transfected cells showed greater internalization of GluA2 receptor ( F (1, 97) = 3.840), Q-VD treatment restored internalization to basal levels ( F (1, 97) = 7.345). Two-way ANOVA *** p ≤ 0.001. c Representative confocal images of neurons transfected with vectors carrying shSIVA-1 or shScramble as control. The GluA2 internalization assay was performed in neurons treated with NMDA to stimulate LTD and in untreated neurons. Only GFP-positive cells (first column) were considered for quantification. Internalized GluA2 (second column, red in merge) and surface GluA2 (third column, green in merge) were measured. Scale bar 10 µm. d The results of the quantification of internalized GluA2 vs. surface GluA2 were normalized to shScrambled, untreated cells. Each point represents an independent experimental repeat in which 15–20 cells were analyzed. NMDA treatment induced GluA2 internalization in both shScrambled and shSIVA-1 cells ( F (1, 84) = 167.1). However, shSIVA-1 cells treated with NMDA showed a significant decrease in receptor internalization ( F (1, 84) = 33.5). Two-way ANOVA ** p ≤ 0.01; *** p ≤ 0.001. shScr shScrambled.

Article Snippet: Hippocampal neurons at 12–14 DIV were incubated with antibodies against the N-terminus of GluA2 (2 μg/ml, mouse monoclonal, clone 6C4, Millipore Cat# MAB397) for 60 min at 20 °C.

Techniques: Transfection, Plasmid Preparation, Control

Journal: Cell Death & Disease

Article Title: SIVA-1 regulates apoptosis and synaptic function by modulating XIAP interaction with the death receptor antagonist FAIM-L

doi: 10.1038/s41419-020-2282-x

Figure Lengend Snippet: a Primary neuronal cells were treated with 50 μM of NMDA at different time points (0–30 min) and protein expression of SIVA-1 was analyzed by SDS-PAGE. Anti-tubulin was used as a loading control. b SIVA-1 expression increased with 5 min, and 15 min treatment of NMDA. t test ( t (15) = 2427; t (15) = 2755). * p ≤ 0.05. c qPCR revealed no change in SIVA-1 mRNA expression after treatment. d The increase in SIVA-1 levels (two-way ANOVA F (2, 6) = 1712) was blocked by pretreatments with 50 µM of BAPTA-AM for 30 min or 1 µg/ml of cycloheximide for 1 h (two-way ANOVA ( F (3, 9) = 1326)). * p ≤ 0.05 for comparison between time points in the same treatment, # p ≤ 0.05 for comparison between treatments. e Schematic representation of SIVA-1 function. In neurons, SIVA-1 was increased after chemical LTD induction. SIVA-1 destabilized XIAP by increasing its ubiquitination and impairing its interaction with FAIM-L. We propose that this is one of the mechanisms through which SIVA-1 triggers caspase-3 activation in neurons and consequent apoptosis and caspase-3-dependent GluA2 internalization. CHX cycloheximide, BAPTA-AM 1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis(acetoxymethyl ester), chLTD chemical long-term depression.

Article Snippet: Hippocampal neurons at 12–14 DIV were incubated with antibodies against the N-terminus of GluA2 (2 μg/ml, mouse monoclonal, clone 6C4, Millipore Cat# MAB397) for 60 min at 20 °C.

Techniques: Expressing, SDS Page, Control, Comparison, Ubiquitin Proteomics, Activation Assay

Journal: Scientific Reports

Article Title: FAIM-L regulation of XIAP degradation modulates Synaptic Long-Term Depression and Axon Degeneration

doi: 10.1038/srep35775

Figure Lengend Snippet: Antibody-feeding internalization assay for endogenous GluA2 in hippocampal neurons stimulated with NMDA (50 μM for 15 min, ( B , D , F , H ). Neurons were previously infected with lentivirus containing with the shRNA vectors (scrambled – A , B , C , D – or XIAP – E , F , G , H –) and after 48 h were re-infected with either EMPTY-EGFP ( A , B , E , F ) or FAIM-L-EGFP ( C , D , G , H ) vectors, as indicated. The figure shows triple-label immunostaining for infected GFP-positive neurons (first and second column), surface-remaining GluA2 (third column, green in merge), internalized GluA2 (fourth column, red in merge), and merge (fifth column). Individual channels are shown in gray scale. Images in columns 2, 3, 4 and 5 represent magnifications from selected areas of the first columns. The scale bar represents 20 μm. ( I ) Shows quantitation of internalization index for the experiment represented in ( A–H ) integrated fluorescence intensity of internalized GluA2/integrated fluorescence intensity of surface-remaining GluA2. Results were not normalized to untreated cells (−NMDA) . As a consequence, A.U have no dimensions. N = 55 to 67 neurons from 3 independent experiments, for each group. Data represent mean ± SEM and and One-way ANOVA test followed by Tukey’s multiple comparison post-hoc test was used to calculate significant levels between the indicated groups. *p < 0.05; ***P < 0.001; ****p < 0.0001. ( J ) control experiment to demonstrate that the used XIAP-shRNA efficiently down-regulates the expression of its target protein. Hippocampal neurons were infected with lentivirus containing the scramble-shRNA vectors (upper panels) or XIAP-shRNA vectors (lower panels) and 72 h later they were fixed and immune-stained against XIAP. In lower panels, it can be clearly appreciated that only neurons infected with XIAP-shRNA showed a clear reduction of XIAP protein levels. However, the XIAP protein levels are not affected in non-infected neurons or neurons infected with scramble-shRNA.

Article Snippet: Briefly, hippocampal neurons at 15–18 days DIV were incubated with antibodies against the N-terminus of GluA2 (2 μg/ml, mouse monoclonal, clone 6C4, Millipore) for 30–60 min at 18–20 °C.

Techniques: Infection, shRNA, Immunostaining, Quantitation Assay, Fluorescence, Comparison, Control, Expressing, Staining

Journal: Scientific Reports

Article Title: FAIM-L regulation of XIAP degradation modulates Synaptic Long-Term Depression and Axon Degeneration

doi: 10.1038/srep35775

Figure Lengend Snippet: ( A – D ) antibody-feeding internalization assay for endogenous GluA2 in hippocampal neurons stimulated with NMDA (50 μM for 15 min). Neurons were infected with lentiviruses containing the shRNA constructs (scrambled or FAIM-L). The figure shows triple-label immunostaining for infected GFP-positive neurons (first and second column), surface-remaining GluA2 (third column, green in merge), internalized GluA2 (fourth column, red in merge), and merge (fifth column). Individual channels are shown in grayscale. Images in columns 2, 3, 4 and 5 represent magnifications from selected areas of the first columns. The scale bar represents 20 μm. ( E ) quantification of GluA2 internalization in the indicated conditions calculated as in . Results were not normalized to untreated cells (−NMDA). As a consequence, A.U have no dimensions. N = 48 to 53 neurons from 3 independent experiments, for each group. Data represent means ± SEM and were analyzed by the One-way ANOVA test followed by Newman-Keuls multiple comparison post-hoc test, ***p < 0.001, *p < 0.05.

Article Snippet: Briefly, hippocampal neurons at 15–18 days DIV were incubated with antibodies against the N-terminus of GluA2 (2 μg/ml, mouse monoclonal, clone 6C4, Millipore) for 30–60 min at 18–20 °C.

Techniques: Infection, shRNA, Construct, Immunostaining, Comparison