Journal: Brain Communications

Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac081

Figure Lengend Snippet: Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.

Article Snippet: Primary antibodies were as follows: chicken anti-GFP (1:1000; Abcam; AB13970), rabbit anti-NeuN (1:1000; Abcam; AB104225), goat anti-ChAT (1:500; Abcam; AB34419) and guinea pig anti-AMPA receptor 2 subunit (GluA2) (1:500; Alomone Labs; AGP-073).

Techniques: Quantitative RT-PCR, Expressing

Journal: Brain Communications

Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac081

Figure Lengend Snippet: Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.

Article Snippet: Primary antibodies were as follows: chicken anti-GFP (1:1000; Abcam; AB13970), rabbit anti-NeuN (1:1000; Abcam; AB104225), goat anti-ChAT (1:500; Abcam; AB34419) and guinea pig anti-AMPA receptor 2 subunit (GluA2) (1:500; Alomone Labs; AGP-073).

Techniques: Expressing

Journal: Brain Communications

Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac081

Figure Lengend Snippet: Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.

Article Snippet: Primary antibodies were as follows: chicken anti-GFP (1:1000; Abcam; AB13970), rabbit anti-NeuN (1:1000; Abcam; AB104225), goat anti-ChAT (1:500; Abcam; AB34419) and guinea pig anti-AMPA receptor 2 subunit (GluA2) (1:500; Alomone Labs; AGP-073).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

Journal: Brain Communications

Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac081

Figure Lengend Snippet: Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.

Article Snippet: Cells were blocked in 10% (v/v) normal donkey serum with 0.1% (v/v) Triton-X 100 in 0.1 M PBS and incubated overnight at 4°C in the following primary antibodies: goat anti-ChAT (1:500, Millipore; AB144P), chicken anti-β III tubulin (1:1000, Abcam; AB41489) and rabbit anti-GluA2 (1:500; Alomone Labs; AGC-005).

Techniques: Quantitative RT-PCR, Expressing

Journal: Brain Communications

Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac081

Figure Lengend Snippet: Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.

Article Snippet: Cells were blocked in 10% (v/v) normal donkey serum with 0.1% (v/v) Triton-X 100 in 0.1 M PBS and incubated overnight at 4°C in the following primary antibodies: goat anti-ChAT (1:500, Millipore; AB144P), chicken anti-β III tubulin (1:1000, Abcam; AB41489) and rabbit anti-GluA2 (1:500; Alomone Labs; AGC-005).

Techniques: Expressing

Journal: Brain Communications

Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac081

Figure Lengend Snippet: Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.

Article Snippet: Cells were blocked in 10% (v/v) normal donkey serum with 0.1% (v/v) Triton-X 100 in 0.1 M PBS and incubated overnight at 4°C in the following primary antibodies: goat anti-ChAT (1:500, Millipore; AB144P), chicken anti-β III tubulin (1:1000, Abcam; AB41489) and rabbit anti-GluA2 (1:500; Alomone Labs; AGC-005).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

Journal: iScience

Article Title: KIBRA regulates activity-induced AMPA receptor expression and synaptic plasticity in an age-dependent manner

doi: 10.1016/j.isci.2022.105623

Figure Lengend Snippet:

Article Snippet: GluA2, rabbit polyclonal , Alomone Labs , Cat# AGC-005 RRID: AB_2039881.

Techniques: Recombinant, Blocking Assay, Software

Journal: Marine Drugs

Article Title: Astaxanthin Protection against Neuronal Excitotoxicity via Glutamate Receptor Inhibition and Improvement of Mitochondrial Function

doi: 10.3390/md20100645

Figure Lengend Snippet: Astaxanthin inhibits a [Ca 2+ ]i increase in cortical neurons upon ionotropic glutamate receptor activation. ( A – C ) The average [Ca 2+ ]i response in control (black) and AST (red) preincubated neurons stimulated with 50 μM each of NMDA (+ 5 μM glycine), AMPA and KA for 15 min (NMDA: Con n = 23, AST n = 42; AMPA: con n = 27, AST n = 23; KA con n = 30, AST n = 40). ( D ) Dot plot representing the total calcium (area under the curve) after 15 min of NMDA, AMPA and KA stimulation. Arrow heads indicate point of glutamate receptor agonist applications. ( E ) Representative protein expression levels of NMDA (GluN1), AMPA (GluA2) and KA (GluK123) detected by the Western blot analysis with β-actin as the internal reference (individual Western blots figure are provided in ). ( F ) Dot plot indicate the average normalized protein expression for GluN1, GluA2 and GluK123. Data are represented as mean ± SEM from 3–4 different experiments, * p < 0.05. n.s: non-significant.

Article Snippet: Membranes were then probed with specific primary antibodies GluN1 (NMDAR1, 1:1000, Alomone Lab Jerusalem, Israel), GluR2 (GluA2, 1:1000, Alomone Lab), GluK 123 (GluR 567, 1:2000, Santa Cruz, Dallas, TX, USA) and β-Actin, 1:2000, BD).

Techniques: Activation Assay, Expressing, Western Blot