Journal: Cells

Article Title: JRM-28, a Novel HDAC2 Inhibitor, Upregulates Plasticity-Associated Proteins in Hippocampal Neurons and Enhances Morphological Plasticity via Activation of CREB: Implications for Alzheimer’s Disease

doi: 10.3390/cells13231964

Figure Lengend Snippet: HDAC proteins, antibodies, and application.

Article Snippet: GluR1 Monoclonal Antibody , Invitrogen , MA5-32344 , Rabbit , WB, IF , 1:500 (WB) 1:250 (IF).

Techniques: Binding Assay

Journal: Cells

Article Title: JRM-28, a Novel HDAC2 Inhibitor, Upregulates Plasticity-Associated Proteins in Hippocampal Neurons and Enhances Morphological Plasticity via Activation of CREB: Implications for Alzheimer’s Disease

doi: 10.3390/cells13231964

Figure Lengend Snippet: List of antibodies used for western blot (WB) and immunofluorescence (IF).

Article Snippet: GluR1 Monoclonal Antibody , Invitrogen , MA5-32344 , Rabbit , WB, IF , 1:500 (WB) 1:250 (IF).

Techniques: Western Blot, Immunofluorescence

Journal: Cells

Article Title: JRM-28, a Novel HDAC2 Inhibitor, Upregulates Plasticity-Associated Proteins in Hippocampal Neurons and Enhances Morphological Plasticity via Activation of CREB: Implications for Alzheimer’s Disease

doi: 10.3390/cells13231964

Figure Lengend Snippet: JRM-28 enhances post-synaptic calcium influx in E18 mouse primary hippocampal neurons. Mouse primary hippocampal neurons were generated from E18 fetal brains as described in the Methods section. After 18 days of differentiation, these neurons were treated with 5 µM of JRM-28 for 48 h and then analyzed for IF and calcium influx assay. The representative images demonstrate dual IF analyses of ( A ) MAP2 (green) plus NMDA receptor subunit NR2A (red); and ( B ) MAP2 (green) plus AMPA receptor subunit GluR1 (red). Nuclei were stained with DAPI (blue). ( C ) MFIs (mean fluorescence intensities) of NR2A and ( D ) GluR1 were calculated in ImageJ software in 50 different NR2A-ir and GluR1-ir receptors (as shown in red-colored dense bodies) per group and then plotted as dotted histograms. An unpaired t -test was adopted to test the significance of the means between groups, resulting in **** p < 0.0001 versus control ( t = 5.334; df = 98) in NR2A analysis and **** p < 0.0001 versus control ( t = 9.608; df = 98) for GluR1. ( E ) Ionotropic calcium assay was performed in hippocampal neurons with increasing doses of JRM-28. Recording started 0.1 s after the addition of 20 μM NMDA via a dual pump autoinjector, and the kinetic plot was recorded at Ex: Em 485:535 nm filter set at 0.1 s interval for 100 readings. The result was normalized with baseline and plotted as a linear scale. ( F ) AMPA-driven calcium influx assay in hippocampal neurons treated with 2, 5, and 10 μM of JRM-28. The recording was performed at a 0.1 s time interval for 100 repeats following the addition of 20 μM AMPA. ( G ) Hippocampal neurons were transfected with creb siRNA for 24 h, followed by treatment with different doses of JRM-28 for another 24 h. After that, an NMDA-sensitive calcium influx assay was performed. ( H ) AMPA-sensitive calcium entry assay was measured in creb siRNA-transfected hippocampal neurons after treatment with increasing doses of JRM-28. ( I ) Pre-synaptic protein synaptotagmin was dual labeled with NR2A. The enhanced interaction between these two proteins may represent augmented synaptic transmission. (a) In control cells, synaptotagmin-ir endocytic membrane fold (circular; green arrow) is found to be present near the post-synaptic NMDA receptor (red dot; NR2A; red arrow). (b) Synaptotagmin-ir endocytic membrane complex (green arrow) was found to be latched onto NR2A-ir (red arrow) NMDA receptor in JRM-28- (5 µM) treated neuron. Scale bar = 5 µm. Results are confirmed after three independent experiments.

Article Snippet: GluR1 Monoclonal Antibody , Invitrogen , MA5-32344 , Rabbit , WB, IF , 1:500 (WB) 1:250 (IF).

Techniques: Generated, Staining, Fluorescence, Software, Control, Calcium Assay, Transfection, Labeling, Transmission Assay, Membrane

Journal: Cells

Article Title: JRM-28, a Novel HDAC2 Inhibitor, Upregulates Plasticity-Associated Proteins in Hippocampal Neurons and Enhances Morphological Plasticity via Activation of CREB: Implications for Alzheimer’s Disease

doi: 10.3390/cells13231964

Figure Lengend Snippet: The effect of JRM-28-mediated activation of CREB on the upregulation of NR2A, GluR1, and induction of ionotropic calcium entry in AD neurons. AD neurons were differentiated from iPSC-derived neural stem cells (NSCs) as described under the Methods section, followed by treatment with 5 µM of JRM-28 for 48 h, and then analyzed for IF and calcium influx assay. ( A ) The representative image exhibits a dual IF analysis of NR2A (green) and CREB (red). Nuclei were stained with DAPI (blue). ( B ) A non-parametric Spearman correlation analysis of mean fluorescence intensities (MFIs) was performed between CREB and NR2A. The resultant scatter plot displays a strong positive correlation (*** p < 0.005). A total of 30 random neurons were selected from six different images (three images per group) in this analysis. MFI was calculated in ImageJ software, the raw values were recorded, and the derived dataset was analyzed for the normality test in GraphPad Prism 8 software. Spearman correlation analysis was decided once the dataset failed to pass the D’Agostino–Pearson normality test. MFI measurement was restricted to cell bodies only. ( C ) A representative dual IF image of GluR1 (green) and CREB (red). Nuclei were labeled with DAPI (blue). ( D ) A parametric Pearson correlation statistic of MFIs between CREB and GluR1 was performed after the normality test was successfully executed in GraphPad Prism 8 software. A total of 30 neurons from six independent images (three images/group) were selected in this analysis. Results are confirmed after three different experiments. ( E ) Ionotropic calcium assay was performed in AD neurons with increasing doses of JRM-28. Recording started 0.1 s after the addition of 20 μM NMDA via a dual pump autoinjector, and the kinetic plot was recorded at Ex: Em 485:535 nm filter set at 0.1 s interval for 100 readings. The result was normalized with baseline and plotted. * p < 0.05 and ** p < 0.01 versus maximum fluorescence value of control reading. ( F ) AMPA-driven calcium influx assay in AD neurons treated with 2, 5, and 10 μM of JRM-28. The recording was performed at a 0.1 sec time interval for 100 repeats following the addition of 20 μM AMPA. ( G ) AD neurons were transfected with the creb siRNA for 24 h, followed by treatment with different doses of JRM-28 for another 24 h. After that, an NMDA-sensitive calcium influx assay was performed. ( H ) AMPA-sensitive calcium entry assay was measured in creb siRNA-transfected AD neurons after the treatment with increasing doses of JRM-28. Results are confirmed after three independent experiments.

Article Snippet: GluR1 Monoclonal Antibody , Invitrogen , MA5-32344 , Rabbit , WB, IF , 1:500 (WB) 1:250 (IF).

Techniques: Activation Assay, Derivative Assay, Staining, Fluorescence, Software, Labeling, Calcium Assay, Control, Transfection

Journal: eLife

Article Title: Retinoic acid-gated BDNF synthesis in neuronal dendrites drives presynaptic homeostatic plasticity

doi: 10.7554/eLife.79863

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Glur1-NT (N-terminus) (Mouse monoclonal) , Millipore , MAB2263; Clone RH95 , WB (1:2000).

Techniques: Recombinant, Expressing, Plasmid Preparation, Sequencing, Protease Inhibitor, Software, In Vitro, RNA Binding Assay

Journal: Frontiers in Neuroscience

Article Title: Injection of Anti-proBDNF Attenuates Hippocampal-Dependent Learning and Memory Dysfunction in Mice With Sepsis-Associated Encephalopathy

doi: 10.3389/fnins.2021.665757

Figure Lengend Snippet: MAb-proB upregulated the expression of neuronal cells and synapse-associated proteins in sepsis-associated encephalopathy mice. (A–C) Representative images and quantitation of Nissl bodies and NeuN-positive neuronal cells (scale bar = 50 μm, n = 4/group). (D,E) Immunoblot analysis of GluR1, GluR4, NR1, NR-2A, NR-2B, and PTSD95 ( n = 5/group). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001, LPS + IgG vs. LPS + mAb-proB.

Article Snippet: After blocking with 1% gelatin in Tris-buffered saline plus Tween-20 (TBST) for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: BDNF antibody, 1:1,000, catalog no. ab108319, Abcam; p75 antibody, 1:1,000, catalog no. ab8874, Abcam; sortilin antibody, 1:2,000, catalog no. ab16640, Abcam; tropomyosin receptor kinase B (TrkB) antibody, 1:1,000, catalog no. 13129-1-AP, Proteintech; β-actin antibody, 1:5,000, catalog no. 66009-1-Ig, Proteintech; GluR1 antibody, 1:1,000, catalog no. ab183797, Abcam; GluR4 antibody, 1:1,000, catalog no. SAB4501296, Sigma; NR1 antibody, 1:1,000, catalog no. ab174309, Abcam; NR2A antibody, 1:1,000, catalog no. 19953-1-AP, Proteintech; NR2B antibody, 1:1,000, catalog no. ab254356, Abcam; and PSD95 antibody, 1:1,000, catalog no. 2507, Cell Signaling Technology.

Techniques: Expressing, Quantitation Assay, Western Blot