Journal: iScience

Article Title: GPR3 is an immediate-early gene-like GPCR regulating CREB-dependent neuronal differentiation

doi: 10.1016/j.isci.2026.114944

Figure Lengend Snippet: Gpr3 mRNA expressions after NGF-, Fsk-, and KCl-mediated neuronal differentiation in PC12 cells (A and B) PC12 cells were induced to differentiate using serum deprivation (0.5% FBS) and stimulation with NGF (50 ng/mL), Fsk (20 μM), or KCl (50 mM). (A) Representative images from PC12 cells stimulated with NGF, Fsk, or KCl at 8, 12, 24, and 48 h after induced differentiation. Scale bars are 50 μm. (B) To evaluate neuronal differentiation induced by various stimuli in PC12 cells, the expression of βIII-tubulin ( Tub-βIII ) and synapsin I ( Syn1 ) was examined. Representative immunofluorescence images of PC12 cells cultured in normal serum-containing medium (15% FBS) or stimulated with NGF, Fsk, or KCl for 48 h are shown. Cells were double-stained with anti-Tub-βIII (red) and anti-SYN1 (green) antibodies. Scale bars are 50 μm. (C) Representative immunoblots showing Tub-βIII and SYN1 protein expression in PC12 cells at 0, 12, 24, and 48 h after stimulation with NGF, Fsk, or KCl. GAPDH is shown as an endogenous loading control. Right margin indicates molecular mass standards (in kilodaltons, kDa). (D and E) Densitometric quantification of time-dependent changes in Tub-βIII (D) and SYN1 (E) protein expression. Data were normalized to GAPDH and are expressed relative to the 0 h value. Data are presented as the mean ± SEM from five independent cultures (biological replicates). ∗∗∗, p < 0.001; ∗∗, p < 0.01 vs. 0 h for each condition. (F) The expression of Gpr3 mRNA was evaluated at 0, 2, 4, 6, 12, 24, 48, and 96 h after induced differentiation, using RT-qPCR. Data represent relative Gpr3 expression (fold change vs. β-actin ) and are presented as the mean ± SEM from 4–6 independent cultures (biological replicates). ∗∗∗ p < 0.001, ∗∗ p < 0.01 and ∗ p < 0.05 vs. 0 h. NGF, neuronal growth factor; Fsk, forskolin; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Source data for this figure are provided in .

Article Snippet: After washing, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% normal goat serum in PBS for 1 h. Cells were then incubated overnight at 4°C with rabbit monoclonal anti-SYN1 antibody (1:200; Cell Signaling Technology, #5297) and/or mouse monoclonal anti-Bassoon antibody (1:200; Enzo, clone SAP7F407).

Techniques: Expressing, Immunofluorescence, Cell Culture, Staining, Western Blot, Control, Quantitative RT-PCR

Journal: iScience

Article Title: GPR3 is an immediate-early gene-like GPCR regulating CREB-dependent neuronal differentiation

doi: 10.1016/j.isci.2026.114944

Figure Lengend Snippet: Induced Gpr3 expression during neuronal differentiation modulates Syn1–3 expression in PC12 cells (A–C) Effects of the suppression of Gpr3 expression on Syn mRNA expression. PC12 cells were transfected with control or Gpr3 siRNA. Twenty-four hours after transfection, differentiation was induced using serum deprivation (0.5% FBS) and NGF (50 ng/mL). Syn1 (A), Syn2 (B), and Syn3 (C) mRNA expression was evaluated using RT-qPCR at 0, 24, 48, and 96 h after inducing differentiation. Data represent relative mRNA expression normalized to β-actin and are presented as the mean ± SEM for each condition (three independent replicates). ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 vs. control at each time point. (D) Effects of the suppression of Nr4a expression on GPR3-mediated Syn1 mRNA expression. PC12 cells were transfected with Nr4a1 siRNA, Nr4a2 siRNA, or Nr4a3 siRNA together with pc-GPR3mAGFL plasmids. For the control, cells were co-transfected with control siRNA and pc-mAGFL (mock). Syn1 mRNA expression was evaluated using RT-qPCR 24 h after transfection. Data represent relative mRNA expression normalized to β-actin and are presented as the mean ± SEM for each condition (three biological replicates). ∗∗∗ p < 0.001 for each indicated group. (E) Primary cerebellar granule neurons (CGNs) were transfected with pc-GPR3mAGFL together with control siRNA, Nr4a1 siRNA, or Nr4a2/3 siRNAs. Syn1 mRNA expression was evaluated using RT-qPCR 24 h after transfection. Gpr3 upregulation significantly increased Syn1 expression, and this effect was abolished by Nr4a1 siRNA, whereas Nr4a2/3 siRNAs showed no significant suppression. Data represent relative mRNA expression normalized to β-actin and are presented as the mean ± SEM for each condition (three biological replicates). ∗∗ p < 0.01 and ∗ p < 0.05 for each indicated group.

Article Snippet: After washing, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% normal goat serum in PBS for 1 h. Cells were then incubated overnight at 4°C with rabbit monoclonal anti-SYN1 antibody (1:200; Cell Signaling Technology, #5297) and/or mouse monoclonal anti-Bassoon antibody (1:200; Enzo, clone SAP7F407).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: iScience

Article Title: GPR3 is an immediate-early gene-like GPCR regulating CREB-dependent neuronal differentiation

doi: 10.1016/j.isci.2026.114944

Figure Lengend Snippet: Developmental changes in Gpr3 , Syn1 , and Nr4a family gene expression in primary cortical neurons from wild-type and Gpr3 −/− mice (A–E) Primary cortical neurons were isolated from postnatal day 0–1 (P0–P1) wild-type (WT), and Gpr3 knockout ( Gpr3 −/− ) mice and cultured in vitro for up to 14 days (DIV0–DIV14). mRNA expression levels of Gpr3 (A), Syn1 (B), Nr4a1 (C), Nr4a2 (D), and Nr4a3 (E) were measured by RT-qPCR at DIV0, DIV0.7 (16 h), DIV1, DIV2, DIV4, DIV7, and DIV14. Data represent relative expression normalized to β-actin and are presented as the mean ± SEM for each condition (three biological replicates). ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 vs. WT neurons at each time point.

Article Snippet: After washing, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% normal goat serum in PBS for 1 h. Cells were then incubated overnight at 4°C with rabbit monoclonal anti-SYN1 antibody (1:200; Cell Signaling Technology, #5297) and/or mouse monoclonal anti-Bassoon antibody (1:200; Enzo, clone SAP7F407).

Techniques: Gene Expression, Isolation, Knock-Out, Cell Culture, In Vitro, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: GPR3 is an immediate-early gene-like GPCR regulating CREB-dependent neuronal differentiation

doi: 10.1016/j.isci.2026.114944

Figure Lengend Snippet: Reduced SYN1-positive puncta in cortical neurons from Gpr3 −/− mice in vitro (A) Staining for SYN1 and Bassoon in cortical neurons obtained from wild-type and Gpr3 −/− mice. Primary neurons were isolated from postnatal day 0–1 (P0–P1) and cultured for 14 days in vitro (DIV14). Neurons were fixed and immunostained with anti-SYN1 (green) and anti-Bassoon (red) antibodies. Representative images are shown. (B) For quantification, neurons were stained with anti-SYN1 (red) and DAPI (blue) at DIV7 and DIV14; representative images are shown. (C) SYN1-positive puncta were quantified within a region 10–25 μm from the nucleus. Quantification was performed using neurons from wild-type ( n = 6) and Gpr3 −/− ( n = 9) mice at DIV7 (left), and from wild-type ( n = 11) and Gpr3 −/− ( n = 9) mice at DIV14 (right) (C). Data are presented as the mean ± SEM for each condition. ∗ p < 0.05 for each indicated group.

Article Snippet: After washing, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% normal goat serum in PBS for 1 h. Cells were then incubated overnight at 4°C with rabbit monoclonal anti-SYN1 antibody (1:200; Cell Signaling Technology, #5297) and/or mouse monoclonal anti-Bassoon antibody (1:200; Enzo, clone SAP7F407).

Techniques: In Vitro, Staining, Isolation, Cell Culture

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