Journal: Scientific Reports

Article Title: Cortactin regulates endo-lysosomal sorting of AMPARs via direct interaction with GluA2 subunit

doi: 10.1038/s41598-018-22542-z

Figure Lengend Snippet: ΔR156-cortactin expression causes an increase in lysosomal targeting of GluA3 and reduced levels of total GluA3. ( a ) ΔR156-cortactin reduces total levels of endogenous GluA3. Hippocampal neurons transfected with GFP, cortactin shRNA + GFP, cortactin shRNA + sh-resistant GFP-WT-cortactin or cortactin shRNA + sh-resistant GFP-ΔR156-cortactin were permeabilized and stained with GluA3 antibody. Representative 45 μm lengths of dendrites are shown, scale bar = 5 µm. Graph shows fluorescence intensity for total GluA3 staining, values represent mean ± SEM. One-way ANOVA with Bonferroni’s test for multiple comparisons (F(3,66) = 11.94, ***p < 0.0001, n = 23). ( b ) ΔR156-cortactin increases lysosomal targeting of GluA3. GluA3 antibody was applied to live hippocampal neurons transfected with constructs as in a, to label surface receptors. After washing, cultures were returned to the incubator for 60 min prior to fixation and permeabilization. Internalized GluA3 was labelled with AlexaFluor647 - conjugated secondary antibody (cyan), and lysosomes were identified by staining with LAMP1 (magenta). Representative 45 μm lengths of dendrites are shown, scale bar = 5 µm. Graph shows Pearson’s colocalization coefficients for internalized GluA3 and LAMP1, values represent mean ± SEM. One-way ANOVA with Bonferroni’s test for multiple comparisons (F(3,44) = 12.08, ***p < 0.0001, n = 12).

Article Snippet: The antibodies used were as follows: anti-cortactin (clone 4F11, Millipore); anti-GluA1 (Calbiochem); anti-GluA2 for Westerns (Synaptic systems); anti-GluA2 for co-IPs (MAB397, Millipore); anti-GluA2 for immunocytochemistry (BD Pharminogen, mouse); anti-GluA3 (Alomone, rabbit); anti-phosphoY421-cortactin (Sigma); anti-phosphoY466-cortactin (Millipore); anti-phosphoY482-cortactin (Millipore); anti-Myc (Santa Cruz); anti-Flag (Sigma); anti-GST (Cell Signalling Technologies); anti-GFP (Chromtek); anti-LAMP1 (Abcam); anti-PSD-95 (Millipore); control IgG (Thermo).

Techniques: Expressing, Transfection, shRNA, Staining, Fluorescence, Construct

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The STEP 61 interactome reveals subunit-specific AMPA receptor binding and synaptic regulation

doi: 10.1073/pnas.1900878116

Figure Lengend Snippet: AMPARs and NMDARs are differentially regulated in STEP-KO mouse brain. (A) Using adult WT and STEP-KO mouse forebrain, a subcellular fractionation assay was performed as described in Materials and Methods, and lysates were immunoblotted with indicated antibodies. H, homogenate; S1, supernatant; Tx-100 sol., soluble fraction. (B–C) Quantification of blots normalized to β-actin (n = 5 independent experiments). Each band intensity in homogenate (total) using GluA1 (P = 0.066), GluA2 (P = 0.006), GluA3 (P = 0.026), GluN2A (P = 0.041), and GluN2B (P = 0.001) (B), as well as GluA1 (P = 0.339), GluA2 (P = 0.007), GluA3 (P = 0.006), GluN2A (P = 0.148), and GluN2B (P = 0.297) in PSD (synaptic) fraction (C) was measured using ImageJ software. (D) The PSD fraction was isolated from WT and STEP-KO mouse brain, solubilized with 1% SDS lysis buffer, and neutralized with 1% Triton X-100 lysis buffer, and an immunoprecipitation (IP) assay was performed using 4G10 (pan pY) antibody. Proteins were resolved by SDS/PAGE and immunoblotted with GluA2 antibody. Quantification of blots normalized to input GluA2 (n = 3 independent experiments, P = 0.007). Error bars represent +SEM, *P < 0.05, **P < 0.01, P value is compared with WT.

Article Snippet: Mouse anti-STEP (cat. no. NB300-202; Novagen), rabbit anti-SynGAP (cat. no. 3200; Cell Signaling), rabbit anti–Kalirin-7 (laboratory made), mouse anti–NLGN-1 (cat. no. 129111; Synaptic Systems), rabbit anti-NBEA (cat. no. 194003; Synaptic Systems), rabbit anti-GluA1 (custom made), rabbit anti-GluA2/3 ( 55 ), mouse anti-GluA2 (clone L21/32; NeuroMab), rabbit anti-GluA2 (cat. 82103; Synaptic Systems), rabbit anti-GluA3 (cat. no. 3437; Cell Signaling), rabbit anti-GluN2A (cat. no. M264; Sigma), mouse anti-GluN2B (clone N59/36; NeuroMab), mouse anti-GluN1 ( 28 ), mouse anti–PSD-95 (clone K28/43; NeuroMab), mouse anti-SAP102 (clone N19/2; NeuroMab), rabbit anti-Fyn (cat. no. 4023; Cell Signaling), rabbit anti-Src (cat. no. 2110; Cell Signaling), mouse anti-GAPDH (cat. no. sc-365062; Santa Cruz), mouse anti-FLAG (cat. no. F1804; Sigma), rabbit anti-FLAG (cat. no. 7425; Sigma), rabbit anti-HA (cat. no. 3724; Cell Signaling), mouse anti-HA (cat. no. 2367; Cell Signaling), rabbit anti-myc (cat. no. 2278; Cell Signaling), rabbit anti-GST (cat. no. A190-122A; Bethyl Laboratories), mouse anti–β-actin (cat. no. G043; abm), and mouse anti-4G10 (cat. no. 05-321; Millipore).

Techniques: Fractionation, Software, Isolation, Lysis, Immunoprecipitation, SDS Page

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The STEP 61 interactome reveals subunit-specific AMPA receptor binding and synaptic regulation

doi: 10.1073/pnas.1900878116

Figure Lengend Snippet: STEP61 overexpression regulates the synaptic expression of AMPARs and NMDARs, and STEP61 regulation of synaptic AMPARs is through the lysosomal degradation pathway. (A–C) Primary cortical neurons were transduced with lentivirus expressing STEP61 at DIV 17, and 7 d later, total protein lysate (n = 5 independent experiments) or PSD fraction (n = 3 independent experiments) was isolated and immunoblotted with GluA1 (P = 0.072), GluA2 (P = 0.227), GluA3 (P = 0.306), GluN2A (P = 0.172), GluN2B (P = 0.479), GluN1 (P = 0.483), and Fyn (P = 0.357) in total lysate (A), as well as with GluA1 (P = 0.421), GluA2 (P = 0.002), GluA3 (P = 3.628e-04), GluN2A (P = 0.009), GluN2B (P = 0.001), GluN1 (P = 3.077e-05), and Fyn (P = 0.173) in PSD fraction (B). (C) Chloroquine (50 μM) or MG-132 (1 μM) was applied for 18 h before protein isolation from the PSD fraction. Immunoblotting was performed (n = 3 independent experiments) with GluA1 (STEP61, P = 0.7360; STEP61 + chloroquine, P = 0.9645; and STEP61 + MG-132, P = 0.9278), GluA2 (STEP61, P = 0.0116; STEP61 + chloroquine, P = 0.9979; and STEP61 + MG-132, P = 0029), and GluA3 (STEP61, P = 0.0001; STEP61 + chloroquine, P = 0.3268; and STEP61 + MG-132, P = 0.0004). All blots were normalized to β-actin. Dunnett’s one-way ANOVA test was performed. Error bars represent +SEM, *P < 0.05, **P < 0.01, ***P < 0.001, P value is compared with control (CTL). (D, Left) Primary hippocampal neurons were transfected with pCAG-IRES-mCherry or pCAG-STEP61-IRES-mCherry at DIV 10 and immunostained at DIV 20. After fixation and permeabilization, total GluA2 was labeled with anti-GluA2 and Alexa 555-conjugated secondary antibody (red), and PSD-95 was visualized with anti-PSD-95 and Alexa 488-conjugated secondary antibody (green). (Scale bar: 5 μm.). Arrowheads indicate GluA2 nonoverlapping with PSD-95 puncta. (D, Right) STEP61 (P = 0.0001), STEP61 + chloroquine (P = 0.9953), and STEP61 + MG-132 (P = 0.0001), Dunnett’s one-way ANOVA test. Error bars represent ±SEM, ***P < 0.001, P value is compared with CTL. ns, not significant.

Article Snippet: Mouse anti-STEP (cat. no. NB300-202; Novagen), rabbit anti-SynGAP (cat. no. 3200; Cell Signaling), rabbit anti–Kalirin-7 (laboratory made), mouse anti–NLGN-1 (cat. no. 129111; Synaptic Systems), rabbit anti-NBEA (cat. no. 194003; Synaptic Systems), rabbit anti-GluA1 (custom made), rabbit anti-GluA2/3 ( 55 ), mouse anti-GluA2 (clone L21/32; NeuroMab), rabbit anti-GluA2 (cat. 82103; Synaptic Systems), rabbit anti-GluA3 (cat. no. 3437; Cell Signaling), rabbit anti-GluN2A (cat. no. M264; Sigma), mouse anti-GluN2B (clone N59/36; NeuroMab), mouse anti-GluN1 ( 28 ), mouse anti–PSD-95 (clone K28/43; NeuroMab), mouse anti-SAP102 (clone N19/2; NeuroMab), rabbit anti-Fyn (cat. no. 4023; Cell Signaling), rabbit anti-Src (cat. no. 2110; Cell Signaling), mouse anti-GAPDH (cat. no. sc-365062; Santa Cruz), mouse anti-FLAG (cat. no. F1804; Sigma), rabbit anti-FLAG (cat. no. 7425; Sigma), rabbit anti-HA (cat. no. 3724; Cell Signaling), mouse anti-HA (cat. no. 2367; Cell Signaling), rabbit anti-myc (cat. no. 2278; Cell Signaling), rabbit anti-GST (cat. no. A190-122A; Bethyl Laboratories), mouse anti–β-actin (cat. no. G043; abm), and mouse anti-4G10 (cat. no. 05-321; Millipore).

Techniques: Over Expression, Expressing, Transduction, Isolation, Western Blot, Transfection, Labeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

doi: 10.3389/fnmol.2018.00199

Figure Lengend Snippet: Expression of the L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits and subunit assemblies in WT and Gria1 R/R mice. (A) Hippocampal expression of GluA1–3, GluN1, αCaMKII and ß-actin in WT and Gria1 R/R mice from P2 till P90. (B) Co-immunoprecipitations (IPs) using polyclonal anti-GluA1 and anti-GluA2/3 antibodies show the presence of GluA1–3 in AMPAR assemblies from hippocampal membrane preparations at P > 60 of WT , Gria1 R/R (R/R) and Gria1 −/− (−/−) mice. (C) Schematic representation of the Gria1 R “knock-in” ( Gria1 tm1Erk ) allele and the Gria1 + allele ( WT ). Below the gene segments, the putative AMPAR subtypes, that can operate at CA3-to-CA1 synapses in Gria1 R/R and WT mice, are schematically depicted (GluA1(R) = GluA1(Q600R)). Large AMPAR symbols for high abundance; small symbols for low abundance; transparent for AMPARs with low single channel conductance. The inset shows the position of the Q600R mutations (R) in two out of the four P-loop segments that form the ion pore of an AMPAR. Exons are in boxes, loxP sites in black triangles and the M1 and P-loop coding sequence in black squares. The position of the mutated codon Q600R codon and codon Q600 in Gria1 tm1Erk and Gria1 are indicated, respectively (see Sprengel et al., ). High resolution images of (A,B) are accessible at https://dx.doi.org/10.17617/3.1i .

Article Snippet: The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000).

Techniques: Expressing, Knock-In, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Nna1 , Essential for Purkinje Cell Survival, Is also Associated with Emotion and Memory

doi: 10.3390/ijms232112961

Figure Lengend Snippet: Immunohistochemical observation in the cerebellum and hippocampus of Nna1 N KO mice. ( a ) Macro image of the brains from Nna1 N KO mice and WT mice in the adult age (n = 3 mice in each genotype). No significant atrophy on the parasagittal image of the cerebellum was observed between Nna1 N KO and WT mice. ( b , c ) Calbindin-D28K IHC on parasagittal sections (n = 3 mice in each genotype). No significant expression difference was observed in the cerebellum between Nna1 N KO and WT mice (n = 3 mice in each genotype). ( d , d′ , d″ ) and ( e , e′ , e″ ) Car8 and VGluT1 double IHC also showed no significant difference between KO and WT mice. ( f , f′ , f″ ) and ( g , g′ , g″ ) Nissl staining on parasagittal sections (n = 3 mice in each genotype). No significant morphological and cell number difference was observed between Nna1 N KO and WT mice. Scale bars: 20 µm in ( d , e , f′ , f″ , g′ , g″ ), Scale bars: 0.5 mm in ( b , c , f , g ), Scale bars: 5 µm in ( d′ , d″ ).

Article Snippet: We used the following primary antibodies: rabbit polyclonal anti-Car-8 (RRID: AB_2571667; 1 μg/mL, Frontier Institute); Calbindin-D28K antibody (RRID: 1:5000; Swant), rabbit polyclonal anti-GLAST (RRID: AB_2571715; 1 μg/mL, Frontier Institute); rabbit polyclonal anti-GluA1 antibody (RRID: AB_2571752; 1 μg/mL, Frontier Institute); rabbit polyclonal anti-GluA2 (RRID: AB_2571754; 1 μg/mL, Frontier Institute), rabbit polyclonal anti-GluA3 (RRID: AB_2571598; 1 μg/mL, Frontier Institute), guinea pig polyclonal anti-GluA4N (RRID: AB_2571756; 1 μg/mL, Frontier Institute) and mixture of Alexa 488-, Cy3-, or Alexa 647-labeled species-specific secondary antibodies for 2h at room temperature at a dilution of 1:200 (Invitrogen; Jackson Immuno Research).

Techniques: Immunohistochemical staining, Expressing, Staining