Journal: The Journal of Neuroscience

Article Title: Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aβ-Bearing Mice

doi: 10.1523/JNEUROSCI.0877-17.2017

Figure Lengend Snippet: Astrocyte activation in 5xFAD mice is associated with increased CN/NFAT signaling. A, Representative Western blots for CN, GFAP, and GAPDH loading control in three WT and three 5xFAD mice. Full-length CN (CN-FL) is an ∼60 kDa band, and the hyperactive ΔCN fragment falls at ∼48 kDa (arrow). B–D, Mean ± SEM hippocampal protein levels [in arbitrary units (A.U.)] for CN-FL, ΔCN, and GFAP in WT (n = 15) and 5xFAD (n = 16) mice. E, Scatter plot showing the correlation between ΔCN and GFAP levels in 5xFAD mice. F, G, Confocal micrographs showing immunolabeling of GFAP and ΔCN in the dentate gyrus (F) and CA1 (G) of WT and 5xFAD mice. H, Confocal micrographs showing immunolabeling of GFAP and NFAT4 in CA1 of WT and 5xFAD mice. DAPI labeling of cell nuclei is in blue. I, J, Mean ± SEM NFAT4 labeling intensity in astrocytes (I, total) and astrocyte nuclei (J) in WT mice (n = 8) and 5xFAD mice (n = 8). NFAT4 labeling was compared across genotypes using Student's t test.

Article Snippet: Nuclear NFAT4 localization images were obtained with a 100× objective in a Z-series (0.5 μm steps), and image analyses were performed using NIS Element software (Nikon; RRID: SCR_014329) and 3D reconstructions performed using Imaris 8.1.2 software (RRID: SCR_007370).

Techniques: Activation Assay, Western Blot, Control, Immunolabeling, Labeling

Journal: The Journal of Neuroscience

Article Title: Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aβ-Bearing Mice

doi: 10.1523/JNEUROSCI.0877-17.2017

Figure Lengend Snippet: AAV-Gfa2-VIVIT reduces astrocytic NFAT activation and improves cognition in 5xFAD mice. A, B, Experimental treatments and timeline. Mice received intrahippocampal injections of AAV-Gfa2-EGFP control or AAV-Gfa2-VIVIT-EGFP at 1.5–2 months of age. Behavioral, glutamate signaling, and/or electrophysiology endpoint measures were collected at 6–8 months of age. C, Transverse hippocampal section from an AAV-Gfa2-EGFP-treated mouse at 8 months of age showing extensive EGFP labeling. D, Confocal micrographs of EGFP (green), NFAT4 (red), and DAPI in 5xFAD mice treated with AAV-Gfa2-EGFP or AAV-Gfa2-VIVIT-EGFP. Left panels are 2-D confocal micrographs, and right panels are 3-D reconstructed images of ∼15 μm z-stacks (0.5 μm sections). E, Mean ± SEM NFAT4 activity levels (ratio of nuclear NFAT4 to total NFAT4 labeling) in EGFP-positive astrocytes of 5xFAD mice treated with AAV-Gfa2-EGFP (n = 6) or AAV-Gfa2-VIVIT-EGFP (n = 6). Nuclear NFAT4 levels were compared across AAV treatment groups using Student's t test. F, G, Mean ± SEM error rate (percentage of total errors on block 1) on the 2 d RAWM task in WT mice (F) and 5xFAD mice (G) treated with AAV-Gfa2-EGFP (CT) or AAV-Gfa2-VIVIT-EGFP. Genotype and AAV treatment effects on learning rates were determined with ANOVA and Fisher's LSD test. H, Mean ± SEM total errors committed on days 1 and 2 of the RAWM task. WT-CT group, 28 mice/group; WT-VIVIT group, 15 mice/group; 5xFAD-CT group, 22 mice/group; 5xFAD-VIVIT group, 10 mice/group. Genotype, AAV, and training day effects (along with significant interactions) were determined with a three-way rmANOVA and follow-up one-way rmANOVAs within each experimental group.

Article Snippet: Nuclear NFAT4 localization images were obtained with a 100× objective in a Z-series (0.5 μm steps), and image analyses were performed using NIS Element software (Nikon; RRID: SCR_014329) and 3D reconstructions performed using Imaris 8.1.2 software (RRID: SCR_007370).

Techniques: Activation Assay, Control, Labeling, Activity Assay, Blocking Assay