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: Alzheimer's Research & Therapy

Article Title: CI-994 is a dual modulator of class I HDACs and Wnt/β-catenin signaling for the treatment of Alzheimer's disease

doi: 10.1186/s13195-026-01982-0

Figure Lengend Snippet: CI-994 suppresses tau phosphorylation in patient-specific iPSC-derived cerebral organoids. Related to Fig. , the human cerebral organoids carrying APOE ε4/ε4 ( A - G ) or MAPT pP301L mutation ( H - N ) at 3 months of age cultured in 6-well plates (3 organoids per well) were treated in triplicate with CI-994 at indicated concentrations for 72 h. Western blotting analyzed the levels of phosphor-tau detected by AT8 or AT180 antibodies, total cellular tau by HT7 antibody and neuronal marker detected by β3-tubulin antibody, and normalized with β-actin levels. All the values are mean ± SEM. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test

Article Snippet: The primary antibodies and their dilutions used in this study are as follows: LRP6 (Cell Signaling Technology, 3395S, 1:1000), anti-ADAM10 (Santa Cruz Biotechnology, 3395S, 1:1000), β-catenin (BD Biosciences, 610,154, 1:5000), acetyl-histone H3 (Cell Signaling Technology, 9649S, 1:1000), histone H3 (Cell Signaling Technology, 4499S, 1:1000), phospho-tau (AT8) (Fisher Healthcare, clone AT8, ENMN1020, 1:500), phospho-tau (AT180) (Fisher Healthcare, clone AT180, ENMN1040, 1:500), tau (Fisher Healthcare, clone HT7, clone HT7, ENMN1000, 1:1000), synaptophysin (Cell Signaling Technology, 36406S, 1:1000), synapsin-1 (Cell Signaling Technology, 5297S, 1:1000), β3-tubulin (Cell Signaling Technology, 5568S, 1:5000), β-actin (Cell Signaling Technology, 3700S and 4970S, 1:5000), and α-tubulin (Cell Signaling Technology, 2125S and 3873S, 1:5000).

Techniques: Phospho-proteomics, Derivative Assay, Mutagenesis, Cell Culture, Western Blot, Marker, Comparison

Journal: Alzheimer's Research & Therapy

Article Title: CI-994 is a dual modulator of class I HDACs and Wnt/β-catenin signaling for the treatment of Alzheimer's disease

doi: 10.1186/s13195-026-01982-0

Figure Lengend Snippet: Activation of Wnt/β-catenin signaling is associated with CI-994-mediated tau phosphorylation inhibition in patient-specific iPSC-derived cerebral organoids. A Wnt reporter assay in Wnt3A-expressing HEK293 cells with the treatment of CI-994 and LGK974 at indicated concentrations for 24 h. B - H The human cerebral organoids carrying APOE ε4/ε4 at 2 months of age cultured in 6-well plates (3 organoids per well) were treated in triplicate with CI-994 (8 µM) and LGK974 (200 nM) for 72 h. B - D The levels of acetyl histone H3K9, total histone H3, total β-catenin, β-actin, and α-tubulin were examined by Western blotting and quantified. E – H The levels of tau phosphorylation (AT8), total tau (HT7), β3-tubulin, and β-actin were examined by Western blotting and quantified. All the values are mean ± SEM. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test

Article Snippet: The primary antibodies and their dilutions used in this study are as follows: LRP6 (Cell Signaling Technology, 3395S, 1:1000), anti-ADAM10 (Santa Cruz Biotechnology, 3395S, 1:1000), β-catenin (BD Biosciences, 610,154, 1:5000), acetyl-histone H3 (Cell Signaling Technology, 9649S, 1:1000), histone H3 (Cell Signaling Technology, 4499S, 1:1000), phospho-tau (AT8) (Fisher Healthcare, clone AT8, ENMN1020, 1:500), phospho-tau (AT180) (Fisher Healthcare, clone AT180, ENMN1040, 1:500), tau (Fisher Healthcare, clone HT7, clone HT7, ENMN1000, 1:1000), synaptophysin (Cell Signaling Technology, 36406S, 1:1000), synapsin-1 (Cell Signaling Technology, 5297S, 1:1000), β3-tubulin (Cell Signaling Technology, 5568S, 1:5000), β-actin (Cell Signaling Technology, 3700S and 4970S, 1:5000), and α-tubulin (Cell Signaling Technology, 2125S and 3873S, 1:5000).

Techniques: Activation Assay, Phospho-proteomics, Inhibition, Derivative Assay, Reporter Assay, Expressing, Cell Culture, Western Blot, Comparison

Journal: Alzheimer's Research & Therapy

Article Title: CI-994 is a dual modulator of class I HDACs and Wnt/β-catenin signaling for the treatment of Alzheimer's disease

doi: 10.1186/s13195-026-01982-0

Figure Lengend Snippet: CI-994 enhances spontaneous electrical activity and synaptic network formation in patient-specific iPSC-derived neurons carrying APOE ε4/ε4. A Spontaneous firing patterns in neurons derived from patient-specific iPSCs carrying APOE ε4/ε4 were measured by MED64 Presto for 8 weeks after differentiation from NPCs with the treatment of CI-994 at 4 µM. Each spike was indicated with a red arrow. P value was calculated by two-tailed, unpaired t-test at week 8. B The frequency of spontaneous firing was monitored in the iPSC-derived neurons for 8 weeks after differentiation and CI-994 treatment ( n = 8 technical replicates). C Extracellular recordings of spontaneous firing including burst firing (red brackets) in the iPSC-derived neurons were measured by MED64 Presto 8 weeks after differentiation and CI-994 treatment. D The incidence of burst firing was monitored in the iPSC neurons for 8 weeks after differentiation and CI-994 treatment ( n = 8 technical replicates). Data represents mean ± SEM. P value was calculated by two-tailed, unpaired t-test at week 8. E – G After the final MEA measurement, the neurons in each well were harvested, and the levels of synapsin-1, synaptophysin, β3-tubulin and β-actin in RIPA lysates were examined by Western blotting. Data represent mean ± SEM, n = 8 technical replicates. P values were calculated using two-tailed, unpaired t test

Article Snippet: The primary antibodies and their dilutions used in this study are as follows: LRP6 (Cell Signaling Technology, 3395S, 1:1000), anti-ADAM10 (Santa Cruz Biotechnology, 3395S, 1:1000), β-catenin (BD Biosciences, 610,154, 1:5000), acetyl-histone H3 (Cell Signaling Technology, 9649S, 1:1000), histone H3 (Cell Signaling Technology, 4499S, 1:1000), phospho-tau (AT8) (Fisher Healthcare, clone AT8, ENMN1020, 1:500), phospho-tau (AT180) (Fisher Healthcare, clone AT180, ENMN1040, 1:500), tau (Fisher Healthcare, clone HT7, clone HT7, ENMN1000, 1:1000), synaptophysin (Cell Signaling Technology, 36406S, 1:1000), synapsin-1 (Cell Signaling Technology, 5297S, 1:1000), β3-tubulin (Cell Signaling Technology, 5568S, 1:5000), β-actin (Cell Signaling Technology, 3700S and 4970S, 1:5000), and α-tubulin (Cell Signaling Technology, 2125S and 3873S, 1:5000).

Techniques: Activity Assay, Derivative Assay, Two Tailed Test, Western Blot

Journal: Alzheimer's Research & Therapy

Article Title: CI-994 is a dual modulator of class I HDACs and Wnt/β-catenin signaling for the treatment of Alzheimer's disease

doi: 10.1186/s13195-026-01982-0

Figure Lengend Snippet: CI-994 enhances histone H3 acetylation and β-catenin levels and suppresses tau phosphorylation in patient-specific iPSC-derived cerebral organoids carrying APOE ε4/ε4. Related to Fig. , patient-specific iPSCs carrying APOE ε4/ε4 were measured by MED64 Presto for 8 weeks after differentiation from NPCs with the treatment of CI-994 at 4 µM. After the final MEA measurement, the neurons in each well were harvested, and RIPA lysates were prepared. A - C The levels of acetyl histone H3, total histone H3 and total β-catenin were examined by Western blotting. D - F The levels of tau phosphorylation, total tau and β3-tubulin and actin were examined by Western blotting. Data represent mean ± SEM, n = 8 technical replicates. P values were calculated using two-tailed, unpaired t test

Article Snippet: The primary antibodies and their dilutions used in this study are as follows: LRP6 (Cell Signaling Technology, 3395S, 1:1000), anti-ADAM10 (Santa Cruz Biotechnology, 3395S, 1:1000), β-catenin (BD Biosciences, 610,154, 1:5000), acetyl-histone H3 (Cell Signaling Technology, 9649S, 1:1000), histone H3 (Cell Signaling Technology, 4499S, 1:1000), phospho-tau (AT8) (Fisher Healthcare, clone AT8, ENMN1020, 1:500), phospho-tau (AT180) (Fisher Healthcare, clone AT180, ENMN1040, 1:500), tau (Fisher Healthcare, clone HT7, clone HT7, ENMN1000, 1:1000), synaptophysin (Cell Signaling Technology, 36406S, 1:1000), synapsin-1 (Cell Signaling Technology, 5297S, 1:1000), β3-tubulin (Cell Signaling Technology, 5568S, 1:5000), β-actin (Cell Signaling Technology, 3700S and 4970S, 1:5000), and α-tubulin (Cell Signaling Technology, 2125S and 3873S, 1:5000).

Techniques: Phospho-proteomics, Derivative Assay, Western Blot, Two Tailed Test