Figure 1: Early AP-2 depletion has defect in axon formation in hippocampal neurons.
From: Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons
(a–c) Representative images of axon extension in hippocampal neurons (cultured for 7 days) expressing GFP-Tau taken from: (a) Control cell (expressing GFP-Tau alone), (b) AP-2KD cell (expressing GFP-Tau + shRNA targeting μ2 subunit of AP-2), and (c) Rescue cell (Expressing GFP-Tau, shRNA targeting μ2 subunit of AP-2, and shRNA-μ2 resistant μ2 plasmid). Knockdown shRNA was added at 36–48 hours after plating. (a–c) (Inset) magnified cell body of neurons that were immunolabeled with left to right: anti-GFP (green) and anti-α-adaptin AP-2 subunit antibody (α subunit of AP-2 complex), (red) and merged (overlay of red and green imaged). (d) Total axon length of neurons from all three conditions (Control, AP-2KD, and Rescue). Total length of axon including primary and all sub-branched axons were measured: Total length of axon; GFP-Tau con = 258.3 ± 27.5 μm (n = 12), GFP-Tau AP-2KD = 56.8 ± 15.2 μm (n = 18), GFP-Tau Rescue = 255.39 ± 33.4 μm (n = 12), (e–g) Total –axon length of neurons from all three conditions (Control, AP-2KD, and Rescue) using exogenously expressed cytosolic marker (e; EGFP only) or axonal markers (f; GFP-VAMP2, (g) GFP-Synapsin I respectively) with the same method as (d). (e) EGFP con = 186.9 ± 34.5 μm (n = 12), EGFPAP-2KD = 37.4 ± 13.0 μm (n = 14), EGFP Rescue = 169.3 ± 21.6 μm (n = 8). (f) GFP-VAMP2 con = 198.8 ± 29.3 μm (n = 16), GFP-VAMP2 AP-2KD = 21.1 ± 9.5 μm (n = 9), GFP-VAMP2Rescue = 191.9 ± 27.4 μm (n = 10). (g) GFP-Synapsin I con = 240.1 ± 35.2 μm (n = 10), GFP-Synapsin I AP-2KD = 21.4 ± 9.0 μm (n = 13), GFP-Synapsin I Rescue = 168.1 ± 33.4 μm (n = 5). Scale bar represents 10 μm. ***p < 0.001. One-way ANOVA.
