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: Neural Plasticity

Article Title: Acute Footshock Stress Induces Time-Dependent Modifications of AMPA/NMDA Protein Expression and AMPA Phosphorylation

doi: 10.1155/2016/7267865

Figure Lengend Snippet: Time-dependent changes of protein expression levels of GluA1 (a), GluA2 (b), GluA1 phospho-Ser 831 (c), GluA1 phospho-Ser 845 (d), and GluA2 phospho-Ser 880 (e) in PFC/FC total homogenate of rats subjected to FS-stress and sacrificed immediately after stress and 2 h and 24 h from stress beginning. Data are represented as percentage of controls at each time point, as means ± SEM ( n = 8). Statistics: Generalized Linear Models (GLM) and Bonferroni Post Hoc Test (see for details). ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: AGC004, Alomone Labs, Jerusalem, Israel) and GluA2 (1 : 2500, cod.

Techniques: Expressing

Journal: Neural Plasticity

Article Title: Acute Footshock Stress Induces Time-Dependent Modifications of AMPA/NMDA Protein Expression and AMPA Phosphorylation

doi: 10.1155/2016/7267865

Figure Lengend Snippet: Time-dependent changes of protein expression levels of GluA1 (a), GluA2 (b), GluA1 phospho-Ser 831 (c), GluA1 phospho-Ser 845 (d), and GluA2 phospho-Ser 880 (e) in PFC/FC postsynaptic spine membranes of rats subjected to FS-stress and sacrificed immediately after stress and 2 h and 24 h from stress beginning. Data are represented as percentage of controls at each time point, as means ± SEM ( n = 8). Statistics: Generalized Linear Models (GLM) and Bonferroni Post Hoc Test (see for details). ∗∗ p < 0.01.

Article Snippet: AGC004, Alomone Labs, Jerusalem, Israel) and GluA2 (1 : 2500, cod.

Techniques: Expressing

Journal: Journal of Neurodegenerative Diseases

Article Title: Differential Changes in Postsynaptic Density Proteins in Postmortem Huntington's Disease and Parkinson's Disease Human Brains

doi: 10.1155/2014/938530

Figure Lengend Snippet: GluA2 expression levels in human postmortem HD and PD hippocampus and striatum. (a) No significant changes in GluA2 expression in HD or PD hippocampal regions. (b) GluA2 specific changes occur in the putamen (Put) of HD and PD tissue. * P < 0.05, ** P < 0.005. (c) Representative images are shown for GluA2 in the dentate gyrus, CA3 and CA1 regions of the hippocampus. (d) Representative images of GluA2 immunostaining in the caudate nucleus and putamen of the striatum. Scale bar for (c) and (d) is 25 μ m. (e) High power example image of GluA2 immunolabelling, showing the somatic and dendritic localisations.

Article Snippet: Sections were then incubated for 72 hours (4°C) in primary antibodies against mouse GluA2 (Alomone) 1 : 200 (WT n = 6, YAC n = 5), rabbit PSD-95 (Sigma) 1 : 200 (WT n = 6, YAC n = 5), rabbit SAP97 (ABR) 1 : 1000 (WT n = 6, YAC n = 5), and mouse GluN1 (Neuromab) 1 : 300 (WT n = 5, YAC n = 6).

Techniques: Expressing, Immunostaining

Journal: Journal of Neurodegenerative Diseases

Article Title: Differential Changes in Postsynaptic Density Proteins in Postmortem Huntington's Disease and Parkinson's Disease Human Brains

doi: 10.1155/2014/938530

Figure Lengend Snippet: Quantitative immunohistochemistry of SAP97, PSD-95, GluN1, and GluA2 expression in YAC128 hippocampal sections. Sections were prepared from symptomatic 1-year-old YAC128 mice to provide a comparison to the end stage of human HD. (a)–(d). Top: Quantification of (a) SAP97, (b) PSD-95, (c) GluA2, and (d) GluN1 levels in dentate gyrus (DG), area CA3, and area CA1. Below: Example immunohistochemical staining for each glutamatergic synaptic protein in the hippocampal CA1 region in control (wildtype) and YAC128 mice. (e) Immunohistochemical quantification of DARPP-32 expression in wild-type and YAC128 striatum (caudate putamen, CPu). * P < 0.05.

Article Snippet: Sections were then incubated for 72 hours (4°C) in primary antibodies against mouse GluA2 (Alomone) 1 : 200 (WT n = 6, YAC n = 5), rabbit PSD-95 (Sigma) 1 : 200 (WT n = 6, YAC n = 5), rabbit SAP97 (ABR) 1 : 1000 (WT n = 6, YAC n = 5), and mouse GluN1 (Neuromab) 1 : 300 (WT n = 5, YAC n = 6).

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining, Staining

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