Journal: bioRxiv

Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic synaptic transmission and plasticity

doi: 10.1101/373597

Figure Lengend Snippet: (a) Representative images of surface GluA1 staining (red) in 14-16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C or shParkin-G430D constructs. Scale bar, 10 μm. (b) Quantification of cell-surface GluA1 intensity expressed as a fraction of shParkin-WT (n ≥70 fields of view per condition with >100 GluA1 puncta per field, results confirmed in 4 independent experiments. *** P <0.001, one-way ANOVA, error bars represent SEM). (c) Representative images of surface GluA1 staining (red) in 14-16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C or shParkin-G430D constructs and non-transduced KO control. Scale bar, 10 μm. (d) Quantification of cell-surface GluA1 intensity expressed as a fraction of Parkin KO (n ≥50 fields of view per condition with >100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P <0.001, one-way ANOVA, error bars represent SEM).

Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), mouse Flag M2 (1:5,000; Sigma).

Techniques: Staining, Expressing, Construct, Control

Journal: bioRxiv

Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic synaptic transmission and plasticity

doi: 10.1101/373597

Figure Lengend Snippet: (a) Representative images of surface GluN1 staining (red) in 14-16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C or shParkin-G430D constructs. Scale bar, 10 μm. (b) Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin-WT (n ≥50 fields of view per condition with >100 GluN1 puncta per field, results confirmed in 4 independent experiments. *** P <0.001, one-way ANOVA, error bars represent SEM). Scale bar, 10 μm. (c) Representative images of surface GluN1 staining (red) in 14-16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C or shParkin-G430D constructs, and non-transduced KO control. Scale bar, 10 μm. (d) Quantification of cell-surface GluN1 intensity expressed as a fraction of Parkin KO (n ≥50 fields of view per condition with >100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P <0.001, one-way ANOVA, error bars represent SEM).

Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), mouse Flag M2 (1:5,000; Sigma).

Techniques: Staining, Expressing, Construct, Control

Journal: bioRxiv

Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic synaptic transmission and plasticity

doi: 10.1101/373597

Figure Lengend Snippet: (a) Representative images of surface GluA1 staining (red) in 14-16 DIV hippocampal neurons expressing shParkin +/- WT, C431S, or W403A Parkin constructs. Scale bar, 10 μm. (b) Same condition as (a), but for surface GluN1 staining (red). Scale bar, 10 μm. (c) Quantification of cell-surface GluA1 intensity expressed as a fraction of shParkin control (n ≥40 fields of view per condition with >100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P <0.001, one-way ANOVA, error bars represent SEM). (d) Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin control (n≥40 fields of view per condition with >100 GluN1 puncta per field, results confirmed in 3 independent experiments. *** P <0.001, one-way ANOVA, error bars represent SEM).

Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), mouse Flag M2 (1:5,000; Sigma).

Techniques: Staining, Expressing, Construct, Control

Journal: bioRxiv

Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic synaptic transmission and plasticity

doi: 10.1101/373597

Figure Lengend Snippet: (a) Representative immunoblots for GFP immunoprecipitation (IP) from HEK293T cell lysates expressing Myc/Myc-Parkin, GFP-GluA1/-GluA2/-GluN1/-GluN2B, and HA-ubiquitin, probed for HA and GFP. Ubiquitin immunoreactivity used for quantification is marked on HA blots. (b) Quantification of GFP-GluA or GluN ubiquitination, expressed as the ratio of marked HA blot intensity (a) with Myc-Parkin (+) to Myc control (-), then normalized to immunoprecipitated GFP, GFP-GluA or GluN (n=3 experiments, * P <0.05; one-way ANOVA, error bars represent SEM). (c) Representative Myc and GFP immunoblots for Myc IP from HEK293T cell lysates expressing Myc-Parkin and GFP-GluA1/-GluA2/-GluN1/-GluN2B. Arrowhead indicates immunoprecipitated Myc-Parkin (just below IgG band). (d) Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/C431S/W403A, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. (e) Quantification of Flag-GluN1 ubiquitination by measurement of marked HA blot intensity (d), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP control (n=3 experiments, ** P <0.01, *** P <0.001, one-way ANOVA, error bars represent SEM). (f) Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/T240M/R275W/R334C/G430D constructs, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. (g) Quantification of Flag-GluN1 ubiquitination by measurement of marked HA intensity (f), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP condition (n=3 experiments, * P <0.05; ** P <0.01, *** P <0.001, one-way ANOVA, error bars represent SEM).

Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), mouse Flag M2 (1:5,000; Sigma).

Techniques: Western Blot, Immunoprecipitation, Expressing, Ubiquitin Proteomics, Control, Construct

Journal: bioRxiv

Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic synaptic transmission and plasticity

doi: 10.1101/373597

Figure Lengend Snippet: (a) Representative images of surface GluA1 staining (red) in 14-16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT, under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. (b) Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. (c) Same as (a), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. (d) Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. (e) Representative images of surface GluA1 staining (red) in 14-16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, under the control condition or after cLTD induction. Scale bar, 10 μm. (f) Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs. For panels (b) and (d), n ≥50 fields of view per condition with >100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P <0.001, unpaired t test. For panel (f), n ≥40 fields of view per condition with >100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P <0.001, one-way ANOVA, error bars represent SEM.

Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), mouse Flag M2 (1:5,000; Sigma).

Techniques: Staining, Expressing, Control, Construct

Journal: bioRxiv

Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic synaptic transmission and plasticity

doi: 10.1101/373597

Figure Lengend Snippet: (a) Representative images of surface GluA1 staining (red) in 14-16 DIV Parkin KO hippocampal neurons expressing shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, and non-transduced KO control under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. (b) Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing the above Parkin constructs. (c) Same as (a), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. (d) Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing above Parkin constructs. For panels (b) and (d), n ≥40 fields of view per condition with >100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P <0.001, one-way ANOVA, error bars represent SEM.

Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), mouse Flag M2 (1:5,000; Sigma).

Techniques: Staining, Expressing, Construct, Control

Journal: bioRxiv

Article Title: The VGCC auxiliary subunit α2δ1 is an extracellular GluA1 interactor and regulates LTP, spatial memory, and seizure susceptibility

doi: 10.1101/2024.12.02.626379

Figure Lengend Snippet: A, Left, purified recombinant HA-GluA1 ATD and HA-GluA2 ATD produced in 293T cells, then used as bait in pull-downs. Center, schematic of pull-down assay after incubation of recombinant ATDs with whole mouse brain lysates. Right, silver stain of proteins eluted after pull-down and PAGE-SDS. B, Subcellular localization of specific and unique GluA1 ATD and GluA2 ATD interactors. C, Partial list of GluA1 ATD (left column, blue) and GluA2 ATD (right column, yellow)-interacting proteins in the mouse brain identified in proteomic screen. D, co-IP analysis of the interaction between recombinant α2δ1 and GluA1 (n≥3). E, co-IP analysis of the interaction between recombinant α2δ1 and GluA2 (n≥3). F, co-IP analysis of the interaction between α2δ1 and HA-GluA1 ATD (n≥3). G, GluA1 / α2δ1 interaction in mouse brain homogenates after pull-down with GluA1 CTD antibody. GluA2 is used as positive control (n=3). H, Left, Molecular model of the ATD of AMPAR (pdb: 6njl) docked to α2δ1 (pdb: 7vfv) in complex with the VGCC allowing a trans interaction obtained in ClusPro docking server. GluA1 subunits are show in light blue, GluA2 subunits are shown in yellow. α2δ1 is shown in dark green, the rest of the VGCC complex in light green. Right, the region boxed in the H is depicted at higher magnification, highlighting some of the residues involved in the interaction between GluA1 ATD and α2δ1. Potential intramolecular H-bonds between the selected residues are indicated in magenta. Rendering of the molecular complexes was performed in ChimeraX.

Article Snippet: After blocking tissue with 5% swine serum (Jackson Immuno Research, # 014-000-121) and 2% BSA (Cell Signaling, #9998S) in permeabilizing conditions (0.1% Triton X-100, Sigma-Aldrich, # T8787), samples were incubated overnight at 4° C with the following primary antibodies: GluA1 (rabbit, Cell signaling, #13185, 1:500 dilution), PSD-95 (mouse, Synaptic Systems, #124 011, 1:500), and VGLUT1 (guinea pig, Synaptic Systems, #135 304, 1:500) followed by incubation with Alexa 488 goat anti-mouse (Life Technologies, #A-11001, 1:500), Alexa 647 goat anti-rabbit (Life Technologies, #A21245, 1:500) and Alexa 568 goat anti-guinea pig (Life Technologies, #A11075, 1:500) secondary antibodies for 1.5 hour at RT.

Techniques: Purification, Recombinant, Produced, Pull Down Assay, Incubation, Silver Staining, Co-Immunoprecipitation Assay, Positive Control

Journal: bioRxiv

Article Title: The VGCC auxiliary subunit α2δ1 is an extracellular GluA1 interactor and regulates LTP, spatial memory, and seizure susceptibility

doi: 10.1101/2024.12.02.626379

Figure Lengend Snippet: A, Schematic of the presynaptic α2δ1 deletion at CA3➔CA1 synapses, comparing α2δ1 f/f (left) with α2δ1 ΔCA3 (right) mice. B, α2δ1 mRNA ISH in the hippocampus of α2δ1 f/f (top) and α2δ1 ΔCA3 (bottom) mice, showing low magnification (left); CA3 (center) and CA1 (right) photomicrographs. Asterisks identify putative interneurons preserving α2δ1 expression in field CA3 in α2δ1 ΔCA3 mice. C, Representative immunostaining of GluA1 (red) and PSD-95 (green) and VGLUT1 (blue) in hippocampal field CA1 in α2δ1 f/f (top) and α2δ1 ΔCA3 mice (bottom) samples. Scale bar, 25 µm (10 µm insets). D, Representative Structured Illumination Microscopy (SIM) images of GluA1 (red), PSD-95 (green) and VGLUT1 (blue) in hippocampal area CA1 SR in α2δ1 f/f (left) and α2δ1 ΔCA3 (right) samples. Scale bar, 1 µm. E, F, average density of VGLUT1 and PSD-95 positive puncta, respectively, in CA1 SR. G, Proportion of PSD-95 colocalizing with VGLUT1. H, average density of GluA1-positive puncta. I, J, Proportion of GluA1 colocalizing with VGLUT1 and PSD-95, respectively. K, Representative mEPSC traces for α2δ1 f/f (top) and α2δ1 ΔCA3 (bottom) CA1 PNs. L,M, mEPSC amplitude and frequency, respectively, in α2δ1 f/f and α2δ1 ΔCA3 PNs. N, Representative individual mEPSC traces. O, P, mEPSC 10-90% rise time and decay tau, respectively, in α2δ1 f/f and α2δ1 ΔCA3 CA1 PNs. Q, Schematic of the preparation used for evoked EPSC recordings in R-T. R, S, Paired-pulse ratios (PPR) and AMPAR/NMDAR EPSC ratios in α2δ1 f/f and α2δ1 ΔCA3 CA1 PNs, respectively. T, AMPAR EPSC normalized to the mean AMPAR EPSC amplitude before LTP induction (arrow). AMPAR EPSC current traces from α2δ1 f/f (black) and α2δ1 ΔCA3 (teal) neurons shown to the right of R-T. n=3-8 mice/genotype (C-J), n=5-14 cells/genotype (K-T). Scale bars: 5 pA, 200 ms (K), 2 pA, 50 ms (N), 50 pA, 50 ms (R-T). *, p≤0.05; n.s., not statistically significant, unpaired t-test (E-J), Mann-Whitney U test (L-T). SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Article Snippet: After blocking tissue with 5% swine serum (Jackson Immuno Research, # 014-000-121) and 2% BSA (Cell Signaling, #9998S) in permeabilizing conditions (0.1% Triton X-100, Sigma-Aldrich, # T8787), samples were incubated overnight at 4° C with the following primary antibodies: GluA1 (rabbit, Cell signaling, #13185, 1:500 dilution), PSD-95 (mouse, Synaptic Systems, #124 011, 1:500), and VGLUT1 (guinea pig, Synaptic Systems, #135 304, 1:500) followed by incubation with Alexa 488 goat anti-mouse (Life Technologies, #A-11001, 1:500), Alexa 647 goat anti-rabbit (Life Technologies, #A21245, 1:500) and Alexa 568 goat anti-guinea pig (Life Technologies, #A11075, 1:500) secondary antibodies for 1.5 hour at RT.

Techniques: Preserving, Expressing, Immunostaining, Microscopy, MANN-WHITNEY

Journal: Molecular medicine reports

Article Title: Naringin ameliorates memory deficits and exerts neuroprotective effects in a mouse model of Alzheimer's disease by regulating multiple metabolic pathways.

doi: 10.3892/mmr.2021.11971

Figure Lengend Snippet: Figure 8. Naringin exerts neuroprotective effects by regulating the glutamate receptor system and apoptosis. (A) Protein levels of NMDAR1, GluR2 and CAMKII. (B) Protein levels of Bad, Bcl‑2 and cleaved‑caspase‑3. **P<0.01 vs. sham group; #P<0.05 and ##P<0.01 vs. model group; &P<0.05, &&P<0.01 vs. naringin group. NMDAR1, glutamate receptor subunit 1; GluR2, glutamate receptor 2; CAMKII, calcium/calmodulin‑dependent protein kinase type II.

Article Snippet: After blocking with 5% non‐fat dried milk for 2.5 h at 37 ̊C, the PVDF membrane was incubated with primary antibodies against: APP (1:1,000; cat. no. bs‐12503R; BIOSS), BACE1 (1:1,000; cat. no. 5606T; Cell Signaling Technology, Inc.), CDK5 (1:1,000; cat. no. bs‐10258Rm; BIOSS), p‐Tau396 (1:1,000; cat. no. bs‐3446R; BIOSS), glutamate receptor subunit 1 (NMDAR1) (1:1,000; cat. no. bs‐23343R; BIOSS), glutamate receptor 2 (GluR2; 1:1,000; cat. no. bs‐13385R; BIOSS), calcium/calmod‐ ulin‐dependent protein kinase type II (CAMKII; 1:1,000; Boster), Bad (1:500; cat. no. A00183; Boster), caspase‐3 (1:500; cat. no. bs‐0081R; BIOSS), Bcl‐2 (1:500; cat. no. bs‐0032R; BIOSS), ERβ (1:1,000; cat. no. kl437Hu22; KALANG; https://www.biomart.cn/infosupply/31407572.htm), p‐P38 (1:1,000; cat. no. bs‐2210R; BIOSS) and β‐actin (1:1,000; cat. no. bs‐0061R; BIOSS) overnight.

Techniques: