Journal: Frontiers in Aging Neuroscience

Article Title: Detrimental effects of soluble α-synuclein oligomers at excitatory glutamatergic synapses

doi: 10.3389/fnagi.2023.1152065

Figure Lengend Snippet: Effects of in vivo striatal injection of αSyn soluble oligomers in mice on ERK and CREB signaling. (A) Levels of pERK were evaluated by Western blot in striatal homogenates of sOligo- and PBS-injected mice 84 dpi. pERK level was normalized on total ERK expression and reported as OD% of PBS-mice. n = 9–10 mice. (B) Levels of pCREB were evaluated by Western blot in striatal homogenates of sOligo- and PBS-injected mice 84 dpi. pCREB level was normalized on total CREB expression and reported as OD% of PBS-mice. n = 10–11 mice. Data are represented as mean ± SEM. * P < 0.05 (Student’s t -test; data with non-normal distribution were tested with Mann–Whitney test).

Article Snippet: The primary antibodies used were: mouse anti-tubulin (1:30,000, #T9026, Sigma), rabbit anti-GAPDH (1:5,000, #sc-25778, Santa Cruz), rabbit anti-GluN2A (1:1,000, #M264, Sigma), rabbit anti-GluN2B (1:1,000, #718600, Invitrogen), mouse anti-GluN2D (1:1,000, #MAB5578, Millipore), rabbit anti-GluA1 (1:1,000, #13185, Cell Signaling), mouse anti-GluA2 (1:1,000, #75–002, Neuromab), mouse anti-GluA3 (1:1,000, #MAB5416, Millipore), polyclonal anti-Rph3A (1:2,000, Protein Tech, #11396-1-AP), mouse anti-PSD-95 (1:1,000, #K28/43, Neuromab), rabbit anti-tyrosine hydroxylase (1:10,000, #AB152, Millipore), rabbit anti-phospho-extracellular signal-regulated kinase (ERK) 44/42 (1:1,000, Cell Signaling, #9101), rabbit anti-ERK 44/42 (1:1,000, #9102, Cell Signaling), rabbit anti-phospho-cAMP responsive element binding protein (CREB) (1:1,000, #9198, Cell Signaling), and rabbit anti-CREB (1:1,000, #9197, Cell Signaling).

Techniques: In Vivo, Injection, Western Blot, Expressing, MANN-WHITNEY

Journal: Frontiers in Aging Neuroscience

Article Title: Detrimental effects of soluble α-synuclein oligomers at excitatory glutamatergic synapses

doi: 10.3389/fnagi.2023.1152065

Figure Lengend Snippet: Molecular effects induced by αsyn sOligo at the hippocampal synapse in vitro . Primary cultures of rat hippocampal neurons were treated at DIV9 with either 2 μg/μl sOligo or PBS and analyses were performed at DIV16. Post-synaptic levels of (A) AMPAR (GluA1, GluA2, and GluA3) and NMDAR (GluN2A and GluN2B) subunits and (B) the scaffolding protein Rph3A and PSD-95 were evaluated by Western blot in TIF from sOligo- or PBS-neurons. Protein levels normalized on tubulin were reported as OD% of PBS-neurons. n = 6 independent cultures. (C) Representative confocal images and quantification of spine morphology analyses (spine density, spine length and spine width) of sOligo- and PBS-neurons at DIV16. Scale bar: 3 μm. n = 23–24 neurons from 3 independent cultures. (D) Levels of pERK were evaluated by Western blot in homogenates of sOligo- and PBS-neurons at DIV16. pERK level was normalized on total ERK expression and reported as OD% of PBS-neurons. n = 12–13 independent cultures. (E) Levels of pCREB were evaluated by Western blot in homogenates of sOligo- and PBS-neurons at DIV16. pCREB level was normalized on total CREB expression and reported as OD% of PBS-neurons. n = 14 independent cultures. Data are represented as mean ± SEM. * P < 0.05 (Student’s t -test; data with non-normal distribution were tested with Mann–Whitney test).

Article Snippet: The primary antibodies used were: mouse anti-tubulin (1:30,000, #T9026, Sigma), rabbit anti-GAPDH (1:5,000, #sc-25778, Santa Cruz), rabbit anti-GluN2A (1:1,000, #M264, Sigma), rabbit anti-GluN2B (1:1,000, #718600, Invitrogen), mouse anti-GluN2D (1:1,000, #MAB5578, Millipore), rabbit anti-GluA1 (1:1,000, #13185, Cell Signaling), mouse anti-GluA2 (1:1,000, #75–002, Neuromab), mouse anti-GluA3 (1:1,000, #MAB5416, Millipore), polyclonal anti-Rph3A (1:2,000, Protein Tech, #11396-1-AP), mouse anti-PSD-95 (1:1,000, #K28/43, Neuromab), rabbit anti-tyrosine hydroxylase (1:10,000, #AB152, Millipore), rabbit anti-phospho-extracellular signal-regulated kinase (ERK) 44/42 (1:1,000, Cell Signaling, #9101), rabbit anti-ERK 44/42 (1:1,000, #9102, Cell Signaling), rabbit anti-phospho-cAMP responsive element binding protein (CREB) (1:1,000, #9198, Cell Signaling), and rabbit anti-CREB (1:1,000, #9197, Cell Signaling).

Techniques: In Vitro, Scaffolding, Western Blot, Expressing, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Nonionotropic Action of Endothelial NMDA Receptors on Blood–Brain Barrier Permeability via Rho/ROCK-Mediated Phosphorylation of Myosin

doi: 10.1523/JNEUROSCI.0969-19.2019

Figure Lengend Snippet: CNS endothelial NMDARs display the regulatory GluN2B and GluN3A subunits. Confocal photomicrographs represent coimmunostaining for the GluN1 subunit (anti-N-terminal domain; red) with the following regulatory NMDAR subunit (green): (A) Glun2B, GluN3A (corresponding 3D reconstruction in B), (C) GluN2A, GluN2C, GluN2D, and GluN3B in ECs of the mouse spinal cord. Yellow represents colocalizations. Scale bar, 10 μm.

Article Snippet: To analyze NMDAR subunits and tight junction proteins, the spinal cord tissue sample sections were incubated with the following primary antibodies: mouse monoclonal anti-GluN1 (Glunomab, 1:500) ( Lesept et al., 2016 ; Macrez et al., 2016 ), goat polyclonal anti-GluN2A (#1468, Santa Cruz Biotechnology, 1:200), rabbit polyclonal anti-GluN2B (#9057, Santa Cruz Biotechnology, 1:200), goat polyclonal anti-GluN2C (#1470, Santa Cruz Biotechnology, 1:200), goat polyclonal anti-GluN2D (#31551, Santa Cruz Biotechnology, 1:200), rabbit polyclonal anti-GluN3A (#07-356, Merck Millipore, 1:200), rabbit polyclonal anti-GluN3B (#50474, Santa Cruz Biotechnology, 1:200), rabbit monoclonal anti-claudin-5 (#131259, Abcam, 1:1000), rabbit monoclonal anti-occludin (#167161, Abcam, 1:1000), rabbit polyclonal anti-ZO-1 (zonula occludens -1 #59720, Abcam, 1:1000), rabbit polyclonal anti-VE-Cadherin (vascular endothelial cadherin #33168, Abcam, 1:1000), and goat anti-collagen IV antibody (#1340, Southern Biotechnology, 1:1000).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Nonionotropic Action of Endothelial NMDA Receptors on Blood–Brain Barrier Permeability via Rho/ROCK-Mediated Phosphorylation of Myosin

doi: 10.1523/JNEUROSCI.0969-19.2019

Figure Lengend Snippet: Inflammation-like conditions modulate the expression of NMDAR in hCMEC/D3 cells. A, Photomicrographs of hCMEC/D3 cells represent immunostaining for the NMDAR subunits GluN1, GluN2B, and GluN3A in control conditions or after TNF treatment (1 ng/ml). Scale bar, 20 μm. B, Corresponding quantification. Histograms represent mean ± SEM immunofluorescence. ****p < 0.0001. C, Immunoblotting against GluN1, GluN2B, and GluN3A of proteins extracted from hCMEC/D3 cells in control conditions or after TNF treatment and corresponding quantification. Histograms represent mean ± SEM (n = 3). *p < 0.05. **p < 0.01.

Article Snippet: To analyze NMDAR subunits and tight junction proteins, the spinal cord tissue sample sections were incubated with the following primary antibodies: mouse monoclonal anti-GluN1 (Glunomab, 1:500) ( Lesept et al., 2016 ; Macrez et al., 2016 ), goat polyclonal anti-GluN2A (#1468, Santa Cruz Biotechnology, 1:200), rabbit polyclonal anti-GluN2B (#9057, Santa Cruz Biotechnology, 1:200), goat polyclonal anti-GluN2C (#1470, Santa Cruz Biotechnology, 1:200), goat polyclonal anti-GluN2D (#31551, Santa Cruz Biotechnology, 1:200), rabbit polyclonal anti-GluN3A (#07-356, Merck Millipore, 1:200), rabbit polyclonal anti-GluN3B (#50474, Santa Cruz Biotechnology, 1:200), rabbit monoclonal anti-claudin-5 (#131259, Abcam, 1:1000), rabbit monoclonal anti-occludin (#167161, Abcam, 1:1000), rabbit polyclonal anti-ZO-1 (zonula occludens -1 #59720, Abcam, 1:1000), rabbit polyclonal anti-VE-Cadherin (vascular endothelial cadherin #33168, Abcam, 1:1000), and goat anti-collagen IV antibody (#1340, Southern Biotechnology, 1:1000).

Techniques: Expressing, Immunostaining, Immunofluorescence, Western Blot