Cytoplasmic polyadenylation element-binding protein regulates neurotrophin-3-dependent beta-catenin mRNA translation in developing hippocampal neurons - PubMed (original) (raw)

NT3-mediated increase in β-catenin protein in growth cones is protein synthesis dependent. A, Cultured hippocampal neurons (1 DIV) were stimulated with 50 ng/ml NT3 for 15 min and processed for ICC with antibodies directed against β-catenin and CPEB1. NT3 induces a 42% increase in β-catenin within growth cones, while CPEB1 levels remain unchanged (n = 69 growth cones, 4 experiments). The increase in β-catenin is completely blocked by the protein synthesis inhibitor cycloheximide (CHX, 100 μ

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, n = 51 growth cones, 3 experiments) but not by actinomycin D, which still showed a 35% increase in β-catenin (ActD, 5 μ

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, n = 66 growth cones, 3 experiments). Bright-field, phase-contrast (Phase) images of the growth cones are also shown and were used to quantify immunofluorescence in the growth cone (see Materials and Methods). Scale bar: 5 μm. B, NT3 stimulation as in A in the presence of a polyadenylation inhibitor cordycepin (CDY, 20 μ

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) also abolishes the increase in β-catenin in growth cones (n = 44 growth cones, 3 experiments). Scale bar: 5 μm. C, Quantification of results in A and B along with the adenosine (Ade, 20 μ

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) control data (left graph), with β-catenin (solid bars) and CPEB1 (clear bars); ***p < 0.001. In a subset of neurons, fluorescence was quantified in the cell body, all processes, and growth cones of axons and dendrites. Data represent abundance of β-catenin following NT3 compared with the fluorescence intensity in unstimulated cells. In individual cells, an increase in β-catenin is only detected within the growth cones; n = 32 cells from 3 experiments for cell body and processes, 24 axon growth cones, and 59 dendritic growth cones; **p < 0.01, ***p < 0.001.