Fig 1: Decorin knockdown impaired the excitatory synaptogenesis and the insertion of pGluR1-ser831 into the excitatory postsynaptic membranes in vitro. Neurons were infected with lentivirus at DIV 3, and the immunofluorescence assay were performed at DIV 12: (a) Immunocytochemistry of dendrites from neurons in the shctrl and shdecorin groups at DIV 12 colabeled with antibodies against Bassoon (red) and PSD95 (blue) to visualize pre- and postsynaptic levels. Scale bar: 5 µm; (b) Quantification of Bassoon, PSD95 and colocalization puncta in B per 30 µm dendrite length. Values represent the mean ± SEM, n = 30 in three independent experiments. **P < 0.01 compared with the shctrl group; n.s. indicates not significant (P > 0.05); (c) Immunofluorescence images of dendrites in DIV 12 neurons from the shctrl and shdecorin groups colabeled with antibodies against GluR1 (red) and PSD95 (blue). Scale bar: 5 µm; (d) Quantification of GluR1 and colocalization puncta in B per 30 µm dendrite length. Values represent the mean ± SEM, n = 30 in three independent experiments. n.s. indicates not significant (P > 0.05); (e) Representative images of dendrites in DIV12 neurons from the shctrl and shdecorin groups colabeled with antibodies against GluR1 (red) and PSD95 (blue). Scale bar: 5 µm and (f) Quantification of GluR1 and colocalization puncta in D per 30 µm dendrite. Values represent the mean ± SEM, n = 25 in three independent experiments.**P < 0.01 compared with the shctrl group.
Fig 2: Effects of rapamycin and NBQX on the regulation of liraglutide-induced synaptic protein expression in hippocampal cells treated with dexamethasone. Cells were exposed to rapamycin (1 µM) or NBQX (50 µM) for 30 min prior to the addition of distilled water (control) or liraglutide (100 nM) for 4 days with dexamethasone (500 µM). In two different wells per group of each of two independent cultures (total 4 wells), cell lysates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses for each of the primary antibodies. The Western blot and revealed the levels of PSD-95 (A), synapsin I (B), and GluA1 (C). Representative images and quantitative analyses normalized to the a-tubulin band are shown. Values (n = 4) are mean ± standard error of the mean (SEM) expressed as a percentage of the control cell values. *p < 0.05 vs. control cells (no liraglutide and no inhibitors), **p < 0.01 vs. control cells, †p < 0.05 vs. liraglutide-only-treated cells, ††p < 0.01 vs. liraglutide-only-treated cells.
Fig 3: Western blot validations of Syn, PSD95, p-ERK1/2, and GluA1 proteins in the hippocampus. (A) Western blot images. (B) The protein expression levels were quantitatively analyzed with the tubulin level. Data are presented as mean ± SD. Student’s t-test, *p < 0.05, ***p < 0.001 versus the control group. n = 6 for each group.
Fig 4: Importance of CaM acetylation in hippocampal LTP.A, reduced Ac-CaM, p-CaMKIIα, and p-GluR1 in 3KR/3KR hippocampal slices, in response to cLTP stimulation. B–D, quantification of Ac-CaM/CaM (B), p-CaMKIIα/CaMKIIα (C), and p-GluR1/GluR1 (D) in panel A. Data were represented as mean ± SD. ∗∗∗p < 0.0001, two-way ANOVA followed by Tukey’s multiple comparisons test, n = 6, data were normalized to WT slices under control condition. E, diagram showing field EPSP recording at SC-CA1 synapses from WT and 3KR/3KR mice. F, comparable paired pulse facilitation (PPF) at SC-CA1 synapses between WT and 3KR/3KR mice. Data were represented as mean ± SD. F (1,19) = 0.304, p = 0.5876, two-way ANOVA, n = 10 slices from four WT, n = 11 slices from four 3KR/3KR mice. G, normalized fEPSP amplitudes were plotted every 1 min for hippocampal slices from WT and 3KR/3KR mice. H, reduced TBS-induced LTP at SC-CA1 synapses in 3KR/3KR hippocampal slices, compared with WT. Data in panel G were quantified. Data were represented as mean ± SD. ∗∗ p = 0.0021, t test, n = 11 slices from six WT mice, n = 15 slices from eight 3KR/3KR mice. I, Coomassie blue staining of 10 μg GST-tagged WT, 3KR, and 3KQ-CaM proteins purified from bacteria. J, diagram showing whole-cell recording of eEPSC in hippocampal CA1 pyramidal neurons. Recording pipettes were infused with GST-WT, 3KR, or 3KQ-CaM. K, normalized eEPSC amplitudes were plotted every 1 min for CA1 pyramidal neurons infused with 100 nM GST-WT, 3KR, or 3KQ-CaM. L, quantification of LTP in panel K. Data were represented as mean ± SD. NS, not significant, ∗∗∗p = 0.001, one-way ANOVA, n = 9 cells from nine mice for WT and 3KQ-CaM, n = 10 cells from ten mice for 3KR-CaM.
Fig 5: Membrane topology and AMPAR-interacting domains of PRRT1. (A) Confocal images of HEK293 cells stained with anti-HA antibody for surface (top panels) or total (bottom panels) PRRT1. PRRT1-HA showed staining on the surface but not HA-PRRT1 or PRRT1-loop-HA. Note that different coverslips were used for surface and total staining. The calibration bar equals 10 µm. (B) A cartoon showing the topology of PRRT1 based on the results of surface staining in (A). Each of the three places where HA tag is inserted in PRRT1 is depicted with a star. (C) Co-IP experiments were performed with anti-GFP antibody on HEK293 cell lysates expressing Flag-GluA1 and GFP-PRRT1 constructs. Immunoblotting (IB) of immunoprecipitated (IP) samples with anti-Flag antibody (top panel) and of input samples with anti-Flag (middle) and anti-GFP (bottom) antibodies (right). Flag-GluA1 co-immunoprecipitated with GFP-PRRT1 full-length (FL) and N?144 but the co-IP with PRRT1-C?34 and PRRT1-C?60 mutants was weak or absent, respectively. Cartoons of full length and deletion constructs of PRRT1 used in co-IP are shown on the left.
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