Journal: British journal of pharmacology

Article Title: Memantine-induced functional rewiring of the glutamate synapse in the striatum of dopamine transporter knockout rats.

doi: 10.1111/bph.17403

Figure Lengend Snippet: FIGURE 1 Presynaptic changes in the striatum of DAT+/+ and DAT/ rats. Protein levels of (a) VGLUT1, (b) VGLUT2, the ratio of (d) pSynapsinSer603/Synapsin1, (e) GLT-1 and (f) xCT were measured in the whole homogenate of the striatum of DAT+/+ and DAT/ rats. The ratio (c) pαCaMKIIThr286/αCaMKII was investigated in the postsynaptic density. (g) Representative immunoblots are shown for VGLUT1, VGLUT2, pαCaMKIIThr286, αCaMKII, pSynapsinSer603, Synapsin1, GLT-1, xCT and β-actin. Results are expressed as mean percentage ± mean standard error from five independent determinations for each experimental group. Unpaired Student's t test *P < 0.05 versus DAT+/+ rats.

Article Snippet: The conditions of the primary antibodies were the following:- anti-GLT-1 (1:2000, Abcam, Cat# Ab41621, RRID:AB_ 941782), anti-vGluT1 (1:1000, Cell Signalling Technology, Cat# 12331, RRID:AB_2797887), anti vGluT2 (1:1000, Cell Signalling Technology, Cat# 71555, RRID:AB_2799805), anti xCT [SLC7A11] (1:1000, Abcam, Cat# ab216876), anti phospho-Synapsin1 Ser603 (1:1000, Cell Signalling Technology, Cat# 88246, RRID:AB_2800119), anti Synapsin1 (1:2000, Santa Cruz Biotechnology, Cat# sc-8295, RRID:AB_677472), anti phospho-αCaMKII Thr286 (1:1000, Thermo Fisher Scientific, Cat# MA1047, RRID:AB_325402), anti αCaMKII (1:2000, Millipore, Cat# 05-532, RRID:AB_309787), anti GluN1 (1:1000, Cell Signalling Technology, Cat# 5704, RRID:AB_1904067), anti GluN2A (1:1000, Cell Signalling Technology, Cat# 4205, RRID: nloaded from https://bpspubs.onlinelibrary.w iley.com /doi/10.1111/bph.17403 by IN A SP - N E PA L , W iley O nline L ibrary on [13/12/2024].

Techniques: Western Blot

Journal: Frontiers in Neuroscience

Article Title: PKR downregulation prevents copper-induced synaptic dysfunction and cognitive impairment in a murine model of Wilson’s disease

doi: 10.3389/fnins.2024.1447304

Figure Lengend Snippet: Evidence of synaptic dysfunction in animal model of Wilson’s disease (WD). (A) Total dendritic lengths in wild type (WT) and toxic milk (TX) mice ( n = 3). (B) Number of dendrites in TX mice compared to the number in control mice ( n = 3). (C) Images of hippocampal neurons of TX and WT mice. Scale bar, 10 μm. (D,E) Microplate reader results showing hippocampal GDH and GOGAT activities ( n = 3). (F) LC/MS quantitative results for glutamate/GABA metabolic loop-related indicators in WT and TX mice ( n = 3). (G) RT-qPCR results for mRNA expressions of Synapsin-1, Syn and Vamp2 in presynaptic membranes, and PSD93 and PSD95 in postsynaptic membranes of WT and TX mice ( n = 3). (H) Levels of synapsin1, synaptophysin, and VAMP2 in presynaptic membranes, and of PSD93 and PSD95 in postsynaptic membranes of WT and TX mice ( n = 3), as quantified using western blots (we normalized the relative protein levels against those of the tubulin loading control and present them as relative protein levels compared to the control). Numeric values represent means ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: We incubated the membranes with different antibodies, namely rabbit anti-PKR antibody (1:2000 dilution, ab184257, Abcam), rabbit anti-Phospho-PKR (Thr451) antibody (1:1000 dilution, 44-668G, Invitrogen), rabbit anti-eIF2α antibody (1:1000 dilution, 5324, Cell Signaling Technology), rabbit anti-Phospho-eIF2α (Ser51) antibody (1:1000 dilution, 3398, Cell Signaling Technology), rabbit anti-ATF-4 antibody (1:1000 dilution, 11815, Cell Signaling Technology), rabbit anti-CREB antibody (1:1000 dilution, 9197, Cell Signaling Technology), rabbit anti-Phospho-CREB (Ser133) antibody (1:1000 dilution, 9198, Cell Signaling Technology), rabbit anti-CHOP antibody (1:1000 dilution, 5554, Cell Signaling Technology), rabbit anti-Synapsin1 antibody (1:1000 dilution, 5297, Cell Signaling Technology), rabbit anti-Synaptophysin antibody (1:1000 dilution, 36406, Cell Signaling Technology), rabbit anti-PSD-93 antibody (1:1000 dilution, 19046, Cell Signaling Technology), rabbit anti-PSD-95 antibody (1:1000 dilution, 3450, Cell Signaling Technology), and rabbit anti-VAMP2 antibody (1:1000 dilution, GB11451, Service bio) at 4°C overnight.

Techniques: Animal Model, Control, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Neuroscience

Article Title: PKR downregulation prevents copper-induced synaptic dysfunction and cognitive impairment in a murine model of Wilson’s disease

doi: 10.3389/fnins.2024.1447304

Figure Lengend Snippet: C16, a PKR inhibitor, attenuates the synaptic dysfunction of Wilson’s disease (WD) in murine model. (A) Ultrastructural organization of hippocampal organotypic slices in saline- or C16-treated toxic milk (TX) and wild type (WT) mice by transmission electron microscope. The right-side images are enlarged versions of the black boxes on the left images, and the arrow points to synaptic vesicles and the pre- and post-synaptic membranes. Scale bars on the left represent 1 μm, and those on the right represent 500 nm. (B) Schematic photomicrographs of neurons with dendrites between repeated 20 mm-spaced concentric rings in the hippocampus of untreated or C16-treated TX and WT mice quantified using Golgi-Cox staining. (C) Total dendritic length and number of dendrites in the hippocampus of untreated or C16-treated TX and WT mice quantified using Golgi-Cox staining. (D,E) Levels of synapsin1, synaptophysin, VAMP2, PSD93, and PSD95 in the hippocampus of saline- or C16-treated TX and WT mice quantified using western blotting ( n = 3) (we normalized the relative protein levels against those of the tubulin loading control and present them as relative protein levels compared to the control). (F,G) RT-qPCR results for mRNA expressions of Synapsin-1 , Syn and Vamp 2 in presynaptic membranes, and PSD93 and PSD95 in postsynaptic membranes of saline- or C16-treated TX and WT mice ( n = 3). Numeric values represent means ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: We incubated the membranes with different antibodies, namely rabbit anti-PKR antibody (1:2000 dilution, ab184257, Abcam), rabbit anti-Phospho-PKR (Thr451) antibody (1:1000 dilution, 44-668G, Invitrogen), rabbit anti-eIF2α antibody (1:1000 dilution, 5324, Cell Signaling Technology), rabbit anti-Phospho-eIF2α (Ser51) antibody (1:1000 dilution, 3398, Cell Signaling Technology), rabbit anti-ATF-4 antibody (1:1000 dilution, 11815, Cell Signaling Technology), rabbit anti-CREB antibody (1:1000 dilution, 9197, Cell Signaling Technology), rabbit anti-Phospho-CREB (Ser133) antibody (1:1000 dilution, 9198, Cell Signaling Technology), rabbit anti-CHOP antibody (1:1000 dilution, 5554, Cell Signaling Technology), rabbit anti-Synapsin1 antibody (1:1000 dilution, 5297, Cell Signaling Technology), rabbit anti-Synaptophysin antibody (1:1000 dilution, 36406, Cell Signaling Technology), rabbit anti-PSD-93 antibody (1:1000 dilution, 19046, Cell Signaling Technology), rabbit anti-PSD-95 antibody (1:1000 dilution, 3450, Cell Signaling Technology), and rabbit anti-VAMP2 antibody (1:1000 dilution, GB11451, Service bio) at 4°C overnight.

Techniques: Saline, Transmission Assay, Microscopy, Staining, Western Blot, Control, Quantitative RT-PCR

Journal: Science Advances

Article Title: Monoallelic de novo AJAP1 loss-of-function variants disrupt trans-synaptic control of neurotransmitter release

doi: 10.1126/sciadv.adk5462

Figure Lengend Snippet: ( A ) Transcellular recruitment of neuronal GBRs to HEK293T cells expressing AJAP1 constructs. HEK293T cells expressing AJAP1-mCherry, AJAP1-SDBM-mCherry or AJAP1-ΔCTD-mCherry were cocultured for 36 hours with hippocampal neurons (DIV12). Immunolabeling for GB2 shows a strong transcellular recruitment of GBRs to the soma of HEK293T cells expressing AJAP1-mCherry and AJAP1-ΔCTD-mCherry but not AJAP1-SDBM-mCherry. AJAP1-expressing cells did not recruit Synapsin1/2. β-TubulinIII was used as a marker for neurites. ( B ) GB2 and Synapsin1/2 immunofluorescence at the soma of HEK293T cells expressing AJAP1 constructs. The background fluorescence in areas devoid of transfected HEK293T cells was subtracted. n.s., P > 0.05, **** P < 0.0001, Welch’s ANOVA, Dunnett’s T3 multiple comparisons test (GB2), Kruskal-Wallis test and Dunn’s multiple comparisons test (Synapsin1/2), n = 33 to 35 cells per condition. ( C ) Transcellular recruitment of neuronal GBRs to HEK293T cells expressing AJAP1-mCherry and the variants AJAP1-W183C-mCherry, AJAP1-P244S-mCherry, and AJAP1-Δ273–412-mCherry. ( D ) GB2 immunofluorescence at the soma of HEK293T cells expressing AJAP1 variants. n.s., P > 0.05, **** P < 0.0001, Kruskal-Wallis test and Dunn’s multiple comparisons test, n = 27 to 28 cells per condition.

Article Snippet: The following primary antibodies were used: Sheep anti-AJAP1 (AF7970, R&D Systems), rabbit anti-GluA2 (AB1768-I, Merck), mouse anti-Bassoon (VAPS003, Stressgen), rabbit anti-Bassoon (141 003, Synaptic Systems), chicken anti-MAP2 (ab5392, Abcam), mouse anti–Ankyrin-G (75–147, Neuromab), mouse anti–PSD-95 (124 011, Synaptic Systems), rabbit anti-VGluT1 (135 303, Synaptic Systems), mouse anti-gephyrin (147 021, Synaptic Systems), rabbit anti-VGAT (131 003, Synaptic Systems), guinea pig anti-GB2 (322 205, Synaptic Systems), mouse anti-GB1a , rabbit anti-Synapsin1/2 (106 002, Synaptic Systems), mouse anti-β-TubulinIII (T8660, Sigma-Aldrich), mouse anti-FLAG (F1804, Sigma-Aldrich), mouse anti-Calbindin1 (300, Swant).

Techniques: Expressing, Construct, Immunolabeling, Marker, Immunofluorescence, Fluorescence, Transfection