Journal: Brain

Article Title: LGI1 antibodies alter K v 1.1 and AMPA receptors changing synaptic excitability, plasticity and memory

doi: 10.1093/brain/awy253

Figure Lengend Snippet: Patient-derived LG1 IgG causes a decrease of total cell surface and synaptic AMPAR clusters in the hippocampus. (A) 3D projection and analysis of the density of total clusters of GluA1 AMPAR and PSD95, and synaptic clusters of AMPAR (defined as AMPAR clusters that co-localized with PSD95) in one of the CA3 subregions indicated in Fig. 3A, ‘Analysis’. Merged images (A, top and bottom left; GluA1 green, and PSD95 red) were post-processed and used to calculate the density of the indicated clusters (density = spots/μm3). Scale bar = 2 μm. (B–D) Quantification of the density of total (B) and synaptic (C) GluA1 AMPAR clusters, and PSD95 clusters (D) in a pooled analysis of hippocampal subregions (CA1, CA3, dentate gyrus) in animals treated with patient-derived LGI1 IgG (red) or control IgG (black) on the indicated days. Mean density of clusters in control IgG treated animals was defined as 100%. Data are presented as mean ± SEM. For each time point five animals infused with patient-derived IgG and five with control IgG were examined. Significance of treatment effect was assessed by two-way ANOVA with an alpha-error of 0.05 (asterisks) and post hoc testing with Sidak-Holm adjustment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: To determine whether the postsynaptic levels of AMPAR were affected, brain sections were incubated with a polyclonal guinea pig antibody against GluA1 (1:200, #AGP009, Alomone) for 2 h at room temperature, washed in PBS containing 0.3% Triton, and incubated with a rabbit polyclonal antibody against the postsynaptic marker PSD95 (1:250, #18258, Abcam) overnight at 4°C.

Techniques: Derivative Assay, Control

Journal: PLoS ONE

Article Title: Protein Tyrosine Phosphatase PTP1B Is Involved in Hippocampal Synapse Formation and Learning

doi: 10.1371/journal.pone.0041536

Figure Lengend Snippet: Protein extracts from hippocampi of adult WT and KO mice were prepared. (A) Western blots were first probed with a polyclonal antibody specific for ß-catenin-pTyr-654. Subsequently, the membrane was stripped and re-probed with a monoclonal antibody against total ß-catenin. The normalized pY654/ß-catenin signal was calculated from scanned bands. Note that KO mice show a significant increase of the normalized signal compared to the WT mice (KO: 380.5±161.7% vs WT: 100.0±11.6%). (B) N-cadherin was immunoprecipitated using a specific monoclonal antibody. Western blots of N-cadherin immunoprecipitates were first probed to detect total ß-catenin and then re-probed to detect N-cadherin. Experiments from five animals show that normalized ratios of ß-catenin/N-cadherin in KO mice are significantly reduced compared to those in WT mice (KO: 80.8±7% vs WT: 100±3, p<0.05 one-tailed Mann-Whitney test). (C) Amount of GluR1 co-immunoprecipitated with N-cadherin. Experiments from five animals show that normalized ratios of GluR1/N-cadherin are not statistically different between WT and KO mice (KO: 112.1±19.2% vs WT: 100.0±16.8%. Asterisks indicate statistical differences for a p≤0.05, according to the one-tailed Mann-Whitney test.

Article Snippet: For Western blots and immunoprecipitation studies we used the following antibodies: rabbit polyclonal antibody against the phosphorylated tyrosine 654 of ß-catenin (1/100) from AbCam (Cambridge, MA); mouse monoclonal antibody against ß-catenin, clone 14 (1/2000) from BD Biosciences (Franklin Lakes, NJ); mouse monoclonal antibody against N-cadherin, clone GC-4 (1/2500) from Sigma-Aldrich, rabbit polyclonal antibody against glutamate receptor1, clone GluR1 (1/2500) from Millipore (Billerica, MA), rabbit anti-phospho-p44/42 MAPK, clone #9101, rabbit anti-p44/42 MAPK, clone #4695, both from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Immunoprecipitation, One-tailed Test, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Transgenic Mice Overexpressing Human STIM2 and ORAI1 in Neurons Exhibit Changes in Behavior and Calcium Homeostasis but Show No Signs of Neurodegeneration

doi: 10.3390/ijms21030842

Figure Lengend Snippet: Basic electrophysiological properties of hippocampal neurons measured by local field potential recordings from acute brain slices that were isolated from 11-month-old female and male wildtype and STIM2/ORAI1 mice, upper panel: females; lower panel: males. ( A ) Input–output curves reflecting basal synaptic transmission in CA3-CA1 projection recorded from stratum radiatum . ( B ) Input–output curves of population spike responses recorded from stratum pyramidale in CA1 region. ( C ) Paired-pulse ratios of fEPSP slopes measured at different interstimulus intervals. Student’s t-test was used to check statistical significance of the observed differences; p -values are displayed above the respective charts. n = 16 wildtype and n = 19 STIM2/ORAI1 slices (females); n = 9 wildtype and STIM2/ORAI1 slices (males). ( D ) The level of GluR1 phosphorylation was analyzed by Western blot in tissue homogenates from wild type and transgenic mice of both sexes. Three mice per sex and genetic variant were analyzed.

Article Snippet: Polyclonal rabbit monoclonal antibody against phospo-GluR1 Ser845 was from Millipore (catalog no. EPR2148; lot 2377032).

Techniques: Isolation, Transmission Assay, Phospho-proteomics, Western Blot, Transgenic Assay, Variant Assay