Journal: Nature medicine

Article Title: Exercise-linked FNDC5/irisin rescues synaptic plasticity and memory defects in Alzheimer’s models

doi: 10.1038/s41591-018-0275-4

Figure Lengend Snippet: (a) Effect of irisin on AβO-induced increases in eIF2α-P (green) and upregulation of nuclear ATF4 (red). Nuclei were counterstained in blue (DAPI). Scale bar = 5 mm. (b,c) Summary quantification of immunocytochemistry experiments (N = 4 experiments with independent neuronal cultures and AβO preparations). *p<0.05; two-way ANOVA with Holm-Sidak correction; two-sided. Data are represented by mean ± SEM. (d,e) Summary quantification of of protein synthesis in hippocampal neurons, as measured by non-radioactive puromycin incorporation (SUnSET) normalized by β-actin levels (N = 4 experiments with independent hippocampal cultures and AβO preparations). *p<0.05; two-way ANOVA with Holm-Sidak correction; two-sided. Data are represented by mean ± SEM. (f,g) Representative images of dendritic spines in hippocampal neurons, as measured by F-actin labeling with Alexa-conjugated phalloidin (N = 5 experiments with independent neuronal cultures and AβO preparations). Scale bar = 20 mm. *p<0.05, two-way ANOVA. Data are shown as mean ± SEM. At least 30 neurons were analyzed per condition per experiment in immunocytochemistry experiments. (h,i) AβO binding to cultured hippocampal neurons, as detected by AβO-sensitive antibody NU4 (red), after treatment with recombinant irisin (25 nM). Scale bar = 10 mm. The experiments were repeated 5 times with similar results. (i) Summary quantification of 5 experiments with independent neuronal cultures and AβO preparations. Data are shown as mean ± SEM. *p<0.05; paired one-way ANOVA; two-sided. (j) AβO interaction with different proteins in a plate-binding assay. BSA was used as a negative control, while neuroligin-1 was used as a positive control (N = 3 experiments with independent AβO preparations) . Representative dots were cropped from the same film. See Source Data 7 for original data. (k) Double immunocytochemistry co-localization between AβOs (red) and surface FNDC5 (green) in primary cultured hippocampal neurons (3 experiments with independent neuronal cultures and AβO preparations, with 20-25 images (from 2-3 coverslips) per experiment). Scale bar = 5 mm. The experiments were repeated 3 times with similar results. (l-o) Levels of soluble (l,n) and insoluble Aβ 42 (m,o) in and hippocampus (N = 3 for AdGFP, 5 for AdFNDC5) and cortex (N = 3 for AdGFP, 4 for AdFNDC5) of APP/PS1 M146L mice. (m,o) Levels of insoluble Aβ 42 in the hippocampus (N = 3 for AdGFP, 4 for AdFNDC5) (l) and cortex (N = 3 per group) (o) of APP/PS1 M146L mice. Data are shown as mean ± SEM. *p<0.05; Student’s t-test; two-sided.

Article Snippet: Anti-eIF2α−P (phosphoS51) (#BML-SA405-100), anti-eIF2α (total) (#ADI-KAP-CP130), and PKA activity kits were from Enzo Life Sciences (Farmingdale, NY).

Techniques: Immunocytochemistry, Labeling, Binding Assay, Cell Culture, Recombinant, Negative Control, Positive Control