Journal: Cell Death & Disease

Article Title: The amino acid transporter LAT1 coordinates proper motor function at the perinatal stage

doi: 10.1038/s41419-026-08663-8

Figure Lengend Snippet: A Schematic diagram of generation of neuron-specific Slc7a5 knockout mice. B Kaplan–Meier survival analysis of Slc7a5 fl/fl ( n = 8) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 12). C Schematic diagram of behavioral analyses in Slc7a5 fl/fl and Syn1-Cre;Slc7a5 fl/fl mice. D Body weight of Slc7a5 fl/fl (1 week: n = 20; 2 week: n = 22) and Syn1-Cre;Slc7a5 fl/fl mice (1 week: n = 7; 2 week: n = 10). E – G Quantitative data of hindlimb suspension test; ( E ) latency to fall (seconds), ( F ) number of pulls, and ( G ) hindlimb score of Slc7a5 fl/fl (1 week and 2 week: n = 20) and Syn1-Cre;Slc7a5 fl/fl mice (1 week and 2 week: n = 7). Solid bars represent the results of the first trial, while dashed bars represent the results of the second trial. H Quantitative data of negative geotaxis test; latency to upward (seconds) of Slc7a5 fl/fl (1 week: n = 20; 2 week: n = 25) and Syn1-Cre;Slc7a5 fl/fl mice (1 week: n = 7; 2 week: n = 9). I – K Representative pictures and quantitative data of tail suspension test (self-clasping test); ( J ) clasping time (seconds), and ( K ) clasping score of Slc7a5 fl/fl (1 week: n = 11; 2 week: n = 21) and Syn1-Cre;Slc7a5 fl/fl mice (1 week: n = 7; 2 week: n = 8). L Quantitative data of righting reflex test; latency to right (seconds) of Slc7a5 fl/fl (1 week: n = 20; 2 week: n = 25) and Syn1-Cre;Slc7a5 fl/fl mice (1 week: n = 7; 2 week: n = 9). NS not significant. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Syn1-Cre mice (#003966) were obtained from Jackson Laboratory.

Techniques: Knock-Out, Suspension

Journal: Cell Death & Disease

Article Title: The amino acid transporter LAT1 coordinates proper motor function at the perinatal stage

doi: 10.1038/s41419-026-08663-8

Figure Lengend Snippet: A Schematic diagram of histological analyses in Slc7a5 fl/fl and Syn1-Cre;Slc7a5 fl/fl mice. B – E Immunofluorescent images and quantitative data of ChAT/DAPI staining in lumbar spinal cord; ( C ) the number of ChAT positive cell, and ( D , E ) the distribution of cell body area (μm 2 ) of Slc7a5 fl/fl (1 week and 2 week: n = 6) and Syn1-Cre;Slc7a5 fl/fl mice (1 week and 2 week: n = 6). Arrowheads indicate representative ChAT positive cells. F – H Immunofluorescent images and quantitative data of NeuN staining in lumbar spinal cord; ( G ) the number of NeuN positive cell (/mm 2 ), and ( H ) NeuN immunointensity of Slc7a5 fl/fl ( n = 3) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 3) at 2 weeks of age. Arrowheads indicate representative NeuN-positive cells. I , J Immunofluorescent images and quantitative data of cleaved caspase 3/ChAT/DAPI staining in lumbar spinal cord; ( J ) the number of cleaved caspase 3 and ChAT double positive cells of Slc7a5 fl/fl ( n = 6) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 6) at 1 week of age. Arrowheads indicate representative cleaved caspase 3, ChAT, and double-positive cells. K , L Immunofluorescent images and quantitative data of LC3B/ChAT/DAPI staining in lumbar spinal cord; ( L ) the number of LC3B and ChAT double positive cells of Slc7a5 fl/fl ( n = 3) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 3) at 1 week of age. Arrowheads indicate representative LC3B, ChAT, and double positive cells. NS not significant, VH ventral horn, DH dorsal horn. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Syn1-Cre mice (#003966) were obtained from Jackson Laboratory.

Techniques: Staining

Journal: Cell Death & Disease

Article Title: The amino acid transporter LAT1 coordinates proper motor function at the perinatal stage

doi: 10.1038/s41419-026-08663-8

Figure Lengend Snippet: A – C Immunofluorescent images and quantitative data of GFAP staining in lumbar spinal cord; ( B ) the number of GFAP positive cell (/mm 2 ), and ( C ) GFAP immunointensity of Slc7a5 fl/fl ( n = 3) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 3) at 2 weeks of age. Arrowheads indicate representative GFAP-positive cells. D – F Immunofluorescent images and quantitative data of MAG staining in lumbar spinal cord; ( E ) the number of MAG positive cell (/mm 2 ), and ( F ) MAG immunointensity of Slc7a5 fl/fl ( n = 3) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 3) at 2 weeks of age. G – I Immunofluorescent images and quantitative data of Iba1 staining in lumbar spinal cord; ( H ) the number of Iba1 positive cell (/mm 2 ), and (I) Iba1 immunointensity of Slc7a5 fl/fl ( n = 3) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 3) at 2 weeks of age. Arrowheads indicate representative Iba1 positive cells. NS not significant, GM gray matter, WH white matter. * P < 0.05 and ** P < 0.01.

Article Snippet: Syn1-Cre mice (#003966) were obtained from Jackson Laboratory.

Techniques: Staining

Journal: Cell Death & Disease

Article Title: The amino acid transporter LAT1 coordinates proper motor function at the perinatal stage

doi: 10.1038/s41419-026-08663-8

Figure Lengend Snippet: A – E H&E and Nissl staining images and quantitative data of the area of ( B ) Layer V and ( C ) Layer II/III of cerebral cortex (×10 5 μm 2 ), ( D ) the neuronal density in Layer V of cerebral cortex (N cells ×10 4 /mm 3 ), and ( E ) the distribution of somatic area in Layer V of cerebral cortex (μm 2 ) of Slc7a5 fl/fl ( n = 3) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 3) at 2 weeks of age. F – J Immunofluorescent images and quantitative data of GFAP/DAPI staining in cerebral cortex and hippocampus; the % of GFAP positive cells in ( G ) cerebral cortex and ( H ) hippocampus, and the GFAP immunointensity in ( I ) cerebral cortex and ( J ) hippocampus of Slc7a5 fl/fl ( n = 3) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 3) at 2 weeks of age. K-N . H&E staining images and quantitative data of the thickness of (L) molecular layer and (M) granular layer of cerebellum (μm), and (N) the number of Purkinje cells in cerebellum (/mm) of Slc7a5 fl/fl ( n = 3) and Syn1-Cre;Slc7a5 fl/fl mice ( n = 3) at 2 weeks of age. NS not significant.

Article Snippet: Syn1-Cre mice (#003966) were obtained from Jackson Laboratory.

Techniques: Staining

Journal: Cell Death & Disease

Article Title: The amino acid transporter LAT1 coordinates proper motor function at the perinatal stage

doi: 10.1038/s41419-026-08663-8

Figure Lengend Snippet: A – E H&E staining images and quantitative data of the distribution of fiber area (μm 2 ) of ( B , D ) quadriceps and ( C , E ) gastrocnemius of Slc7a5 fl/fl (1 week and 2 week: n = 5, 6) and Syn1-Cre;Slc7a5 fl/fl mice (1 week and 2 week: n = 5). F – L Immunofluorescent images of synaptophysin, neurofilament, and BTX staining and quantitative data of NMJ in EDL muscle; ( G , J ) NMJ area (μm 2 ), ( H , K ) NMJ maturity (%), and ( I , L ) NMJ innervation (denervation, partial innervation, and full innervation) (%) of Slc7a5 fl/fl (1 week and 2 week: n = 6) and Syn1-Cre;Slc7a5 fl/fl mice (1 week and 2 week: n = 6). NS not significant. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Syn1-Cre mice (#003966) were obtained from Jackson Laboratory.

Techniques: Staining

Journal: Cell Death & Disease

Article Title: The amino acid transporter LAT1 coordinates proper motor function at the perinatal stage

doi: 10.1038/s41419-026-08663-8

Figure Lengend Snippet: A Schematic diagram of calpeptin administration schedule followed by histological analyses in Slc7a5 fl/fl and Syn1-Cre;Slc7a5 fl/fl mice. B Kaplan–Meier survival analysis of Slc7a5 fl/fl mice and Syn1-Cre;Slc7a5 fl/fl mice treated with vehicle or calpeptin ( Slc7a5 fl/fl mice + vehicle, n = 9; Slc7a5 fl/fl mice + calpeptin, n = 7; Syn1-Cre;Slc7a5 fl/fl mice + vehicle, n = 5; Syn1-Cre;Slc7a5 fl/fl mice + calpeptin, n = 7). C – E Immunofluorescent images and quantitative data of cleaved caspase 3/ChAT/DAPI staining in lumbar spinal cord; ( D ) the number of ChAT positive cells, and ( E ) the number of cleaved caspase 3 and ChAT double positive cells of Slc7a5 fl/fl mice and Syn1-Cre;Slc7a5 fl/fl mice treated with vehicle or calpeptin ( Slc7a5 fl/fl mice + vehicle, n = 5; Slc7a5 fl/fl mice + calpeptin, n = 5; Syn1-Cre;Slc7a5 fl/fl mice + vehicle, n = 5; Syn1-Cre;Slc7a5 fl/fl mice + calpeptin, n = 5). Arrowheads indicate representative cleaved caspase 3, ChAT, and double positive cells. F – I Immunofluorescent images of synaptophysin/neurofilament/BTX staining and quantitative data of NMJ in EDL muscle; ( G ) NMJ area (μm 2 ), ( H ) NMJ maturity (%), and ( I ) NMJ innervation (%) of Slc7a5 fl/fl mice and Syn1-Cre;Slc7a5 fl/fl mice treated with vehicle or calpeptin ( Slc7a5 fl/fl mice + vehicle, n = 5; Slc7a5 fl/fl mice + calpeptin, n = 5; Syn1-Cre;Slc7a5 fl/fl mice + vehicle, n = 5; Syn1-Cre;Slc7a5 fl/fl mice + calpeptin, n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001, and # P < 0.05.

Article Snippet: Syn1-Cre mice (#003966) were obtained from Jackson Laboratory.

Techniques: Staining

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