1. AβOs bind to and impact hypothalamic neurons Representative immunocytochemistry images of mature hypothalamic neurons in culture exposed to vehicle (Veh) or AβOs (500 nM) for 3 h.
AβOs bind to and impact hypothalamic neurons Representative immunocytochemistry images of mature hypothalamic neurons in culture exposed to vehicle (Veh) or AβOs (500 nM) for 3 h. Binding of AβOs was detected using anti‐oligomer monoclonal antibody NU4 (red). Neurons were double‐labeled using MAP‐2 antibody (green). Images represent typical results from experiments with three independent hypothalamic cultures (three coverslips/experimental condition per independent experiment). Scale bar = 30 and 10 μm for main panels and insets, respectively. Representative immunocytochemistry image of mature hypothalamic culture exposed to AβOs (500 nM) and immunolabeled with anti‐GFAP (green) and NU4 (red) antibodies. Insets show AβOs binding to neuronal dendrites, whereas no binding was detected to GFAP‐positive cells. Representative DCF fluorescence images from hypothalamic neuronal cultures exposed to vehicle or AβOs (500 nM, 4 h). Insets show optical zoom images of the indicated areas. Scale bars = 100 and 50 μm for main panels and insets, respectively. Graph shows integrated DCF fluorescence intensities (relative units; see Materials and Methods) (n = 3 independent hypothalamic cultures; three wells/experimental condition per experiment; three images acquired per well). Bars represent means ± SEM. #P = 0.0604, one‐sample t‐test compared with a fixed value of 100 RUs. LDH activity (IU/l) in culture media of hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h). Representative immunofluorescence images of eIF2α‐P in hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Scale bar = 30 μm. Graph represents integrated immunofluorescence intensities of eIF2α‐P levels from three independent hypothalamic cultures (three coverslips/experimental condition per experiment, 20 images per coverslip). Bars represent means ± SEM. *P = 0.0489, one‐way ANOVA followed by Bonferroni post hoc test comparing AβO‐treated versus vehicle‐treated cultures. Representative images of hypothalamic cultures exposed to AβOs (500 nM, 3 h) and double‐labeled with NU4 (oligomer‐sensitive) and eIF2α‐P antibodies. Arrow points to a neuron presenting high levels of eIF2α‐P in the absence of AβO binding. Nuclear staining (DAPI) is shown in blue. Scale bar = 30 μm. Representative images of hypothalamic neurons labeled with NU4 antibody exposed to AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Similar patterns of AβO binding were observed in both conditions. Scale bar = 20 μm.
Julia R Clarke et al. EMBO Mol Med. 2015;7:
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