Journal: Cell Death & Disease

Article Title: Alpha-synuclein is involved in manganese-induced spatial memory and synaptic plasticity impairments via TrkB/Akt/Fyn-mediated phosphorylation of NMDA receptors

doi: 10.1038/s41419-020-03051-2

Figure Lengend Snippet: a , b After treatment with Mn, western blotting was used to measure the level of α-Syn overexpression in the hippocampus. c – f The levels of phosphorylated NMDA receptors (GluN2B and GluN2A) were measured by western blotting in vivo. n = 6. g , h HT22 cells were transfected with LV-α-Syn shRNA, and the cytotoxicity was measured using the CCK-8 assay and lactate dehydrogenase (LDH) release assay. i – m The levels of phosphorylated NMDA receptors (GluN2B and GluN2A) and α-Syn expression were measured by western blotting in vitro. n = 4. ** P < 0.01 compared to their control counterparts; # # P < 0.01, and # P < 0.05 for comparison between the Mn treatment group.

Article Snippet: These important primary antibodies are listed as follows: PSD95 (1:1000, Cell Signaling Technology, Cat. no. #2507), SynGAP (1:1000, Cell Signaling Technology, Cat. no. #3200), TrkB (1:500, Proteintech, Cat. no. 13129), alpha-Synuclein (1:500, Invitrogen, Cat. no. #MA5-12272), β-actin (1:1000, Cell Signaling Technology, Cat. no. #8457), BDNF (1:1000, Abcam, ab182199), CaMKII-α (1:1000, Cell Signaling Technology, Cat. no. #3357), Phospho-CaMKII (Thr286) (1:1000, Cell Signaling Technology, Cat. no. #12716), NMDA Receptor 2A (GluN2A) (1:1000, Cell Signaling Technology, Cat. no. #4205), NMDA Receptor 2B (GluN2B) (1:1000, Cell Signaling Technology, Cat. no. #4207), AKT (1:500, Proteintech, Cat. no. 10176), AKT-phospho-S473 (1:3000, Proteintech, Cat. no. 66444), Fyn (1:1000, Cell Signaling Technology, Cat. no. #4023), Anti-Fyn (phospho Y530) (1:500, Abcam, ab182661), Phospho-NMDA Receptor 2A (GluN2A) (Tyr1246) (1:1000, Cell Signaling Technology, Cat. no. #4206), and Phospho-NMDA Receptor 2B (GluN2B) (Tyr1472) (1:1000, Cell Signaling Technology, Cat. no. #4208).

Techniques: Western Blot, Over Expression, In Vivo, Transfection, shRNA, CCK-8 Assay, Lactate Dehydrogenase Assay, Expressing, In Vitro, Control, Comparison

Journal: Cell Death & Disease

Article Title: Alpha-synuclein is involved in manganese-induced spatial memory and synaptic plasticity impairments via TrkB/Akt/Fyn-mediated phosphorylation of NMDA receptors

doi: 10.1038/s41419-020-03051-2

Figure Lengend Snippet: a – e After normal or transfected HT22 cells were pretreated with brain-derived neurotrophic factor (BDNF) and Mn, the levels of phospho-Akt and phospho-Fyn, and Akt and Fyn expression were evaluated by western blotting. f – j After normal or transfected HT22 cells were pretreated with BDNF and Mn, the levels of phospho-GluN2B and phospho-GluN2A, and the expression of GluN2B and GluN2A were evaluated by western blotting. n = 4. β-actin was used as a loading control. ** P < 0.01, and * P < 0.05 compared to their control counterparts; ## P < 0.01 compared to their Mn-treated cells; ΔΔ P < 0.01, and Δ P < 0.05 for comparison between their BDNF-pretreated and Mn-treated cells.

Article Snippet: These important primary antibodies are listed as follows: PSD95 (1:1000, Cell Signaling Technology, Cat. no. #2507), SynGAP (1:1000, Cell Signaling Technology, Cat. no. #3200), TrkB (1:500, Proteintech, Cat. no. 13129), alpha-Synuclein (1:500, Invitrogen, Cat. no. #MA5-12272), β-actin (1:1000, Cell Signaling Technology, Cat. no. #8457), BDNF (1:1000, Abcam, ab182199), CaMKII-α (1:1000, Cell Signaling Technology, Cat. no. #3357), Phospho-CaMKII (Thr286) (1:1000, Cell Signaling Technology, Cat. no. #12716), NMDA Receptor 2A (GluN2A) (1:1000, Cell Signaling Technology, Cat. no. #4205), NMDA Receptor 2B (GluN2B) (1:1000, Cell Signaling Technology, Cat. no. #4207), AKT (1:500, Proteintech, Cat. no. 10176), AKT-phospho-S473 (1:3000, Proteintech, Cat. no. 66444), Fyn (1:1000, Cell Signaling Technology, Cat. no. #4023), Anti-Fyn (phospho Y530) (1:500, Abcam, ab182661), Phospho-NMDA Receptor 2A (GluN2A) (Tyr1246) (1:1000, Cell Signaling Technology, Cat. no. #4206), and Phospho-NMDA Receptor 2B (GluN2B) (Tyr1472) (1:1000, Cell Signaling Technology, Cat. no. #4208).

Techniques: Transfection, Derivative Assay, Expressing, Western Blot, Control, Comparison

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: NR2A and NR2B antagonists prevented and reversed mechanical allodynia in rats with SNI. a The threshold force of SNI-induced mechanical allodynia on the ipsilateral hind paw was significantly decreased on day 7 and continued until day 14. Intrathecal treatment with the NR2A-selective antagonist NVP-AAM077 (4 nmol) and the NR2B-selective antagonist Ro25-6981 (20 nmol) once daily for 14 days prevented the development of mechanical allodynia on the hind paw ipsilateral to SNI on days 3, 5, 7, 10, and 14. c A single intrathecal administration of NVP-AAM077 (4 nmol) and Ro25-6981 (20 nmol) on day 14 attenuated mechanical allodynia at 30 min after the treatment, and the effect of NVP-AAM077 was maintained for 24 h. b , d NVP-AAM077 and Ro25-6981 did not change the mechanical nociceptive threshold of the contralateral hind paw in the same rat. e A single intrathecal administration of 10 nmol MK-801 on day 14 reversed SNI-induced mechanical allodynia 30 min after the treatment. NVP, NVP-AAM077; Ro25, Ro25-6981; MK, MK-801. Data are shown as the means ± SE. * P < 0.05, ** P < 0.01 versus vehicle. # P < 0.05, ## P < 0.01 versus before the single intrathecal treatment

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques:

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: NR2A and NR2B antagonists prevented exogenous leptin-induced mechanical allodynia. a Intrathecal leptin (50 μg) treatment in naïve rats, given once daily for 7 days, induced mechanical allodynia on day 7. Coadministration of leptin with 4 nmol NVP-AAM077 or 20 nmol Ro25-6981 attenuated the behavioral changes ( n = 5). b NVP-AAM077 and Ro25-6981 alone did not change the baseline nociceptive threshold ( n = 6). lep, leptin; NVP, NVP-AAM077; Ro25, Ro25-6981. Data are shown as the means ± SE. ** P < 0.01 versus day 0

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques:

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: Leptin enhancement of NR2B- but not NR2A-mediated currents in dissociated lamina II neurons in naïve rats. a Treatment with the NR2A-selective antagonist NVP-AAM077 (0.4 μM) plus the NR2B-selective antagonist Ro25-6981 (1 μM) blocked NMDAR-mediated currents ( n = 8). b Exposure to leptin (100 nM) for 5 min did not change NMDAR-mediated currents after blockade with Ro25-6981 (1 μM) ( n = 10). c Exposure to leptin (100 nM) for 5 min enhanced NMDAR-mediated currents after inhibition by 0.4 μM NVP-AAM077 ( n = 9). d Histograms showing the effect of leptin on NMDAR-mediated currents after inhibition by NVP-AAM077 or Ro25-6981. Data are shown as the means ± SE. lep, leptin; NVP, NVP-AAM077; Ro, Ro25-6981. * P < 0.05, ** P < 0.01 vs. vehicle; # P < 0.05 vs NVP

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques: Inhibition

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: Leptin enhancement of NR2B, but not NR2A, expression in cultured DRG neurons. a Immunohistochemistry results showed that administration of leptin in culture medium for 72 h upregulated NR2B expression in a dose-dependent manner (2 ng/ml leptin had the maximal enhancement effect), and cotreatment with 1 μM Ro25-6981 diminished the upregulation. b Leptin at 2 ng/ml slightly enhanced NR2A expression, which was attenuated by 0.4 μM NVP-AAM077. c – f Western blot results showed that administration of leptin (2 ng/ml) to culture medium for 72 h significantly upregulated NR2B expression ( c , d ) but not NR2A expression ( e , f ) in cultured DRG neurons. The NR2B upregulation was blocked by 1 μM Ro25-6981 ( c , d ). Neither 1 μM Ro25-6981 nor 0.4 μM NVP-AAM077 alone changed the baseline expression of NR2B or NR2A. lep, leptin; NVP, NVP-AAM077; Ro, Ro25-6981. n = 3. Scale bar, 50 μm. * P < 0.05 vs vehicle

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques: Expressing, Cell Culture, Immunohistochemistry, Western Blot

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: Leptin-mediated enhancement of nNOS expression was blocked by an NR2B antagonist. Immunohistochemistry ( a ) and Western blot ( b and c ) results showed that administration of leptin (2 ng/ml) to culture medium for 72 h significantly upregulated nNOS expression in cultured DRG neurons. The upregulation of nNOS expression by leptin was significantly prevented by coapplication of the NR2B antagonist Ro25-6981 (1 μM) and slightly attenuated by the NR2A antagonist NVP-AAM077 (0.4 μM). Ro25-6981 (1 μM) and NVP-AAM077 (0.4 μM) alone did not change baseline nNOS expression. Lep, leptin; NVP, NVP-AAM077; Ro, Ro25-6981. n = 3. Scale bar, 50 μm. ** P < 0.01 vs vehicle; # P < 0.05 vs leptin

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques: Expressing, Immunohistochemistry, Western Blot, Cell Culture

Journal: Cell reports

Article Title: GluN3A subunit tunes NMDA receptor synaptic trafficking and content during postnatal brain development

doi: 10.1016/j.celrep.2023.112477

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-GluN2A , Merck Millipore , Cat# M264.

Techniques: Cell Culture, Recombinant, Software

Journal: Scientific Reports

Article Title: New Alzheimer’s disease model mouse specialized for analyzing the function and toxicity of intraneuronal Amyloid β oligomers

doi: 10.1038/s41598-019-53415-8

Figure Lengend Snippet: Immunoblot analysis of expression ratios of the pre and postsynaptic proteins in Aβ-GFP Tg and non-Tg mice synapse. ( A,B ) Synaptosome fractions from hippocampus (20 μg/lane) were immunoblotted against presynaptic markers ( A ; synaptophysin, VAMP-2, syntaxin-1, and Munc13–1) and postsynaptic markers ( B ; PSD-95, GluN1, GluN2A, GluN2B, GluA2, and neuroligin). We prepared 4 sets of Aβ-GFP Tg/non-Tg samples. Band intensities of Aβ-GFP Tg mice were normalized by those of actin and the expression were calculated ratios with the values of non-Tg mice as 1. There was no difference in expression ratios of the presynaptic proteins we examined. However, expression of the postsynaptic proteins GluN2B, Glu2A, and neuroligin were significantly decreased in Aβ-GFP Tg mice at 3-month-old (*p < 0.05 by Mann-Whitney’s U test, n = 4 that included hippocampi from 3 animals each, data are presented as means ± SEM). ( C ) The expression ratios of GluN2B in homogenate. There was no significant difference in the expression ratios of GluN2B in homogenates of Aβ-GFP Tg and non-Tg mice (Mann-Whitney’s U test, n = 5 animals each, data are presented as means ± SEM).

Article Snippet: VAMP-2 (1:5000, Abcam, Cambridge, UK), Munc13-1(1:1000, Synaptic Systems, Gottingen, Germany), syntaxin-1 (1:1000, Merck), synaptophysin (1:2000, Sigma-Aldrich), PSD-95 (1:200, Merck), GluN1 (1:200, Merck), GluN2A (1:500, Merck), GluN2B (1:200, Merck), GluA2 (1:200, Merck), neuroligin 1(1:500, Synaptic Systems), and actin (1:2000, Merck) antibodies were used to probe the corresponding proteins and ECL was used for detection (Immunostar Zeta and Immunostar LD, FUJIFILM Wako Pure Chemical Corporation).

Techniques: Western Blot, Expressing