Journal: Frontiers in Cell and Developmental Biology
Article Title: The Interaction of TRAF6 With Neuroplastin Promotes Spinogenesis During Early Neuronal Development
doi: 10.3389/fcell.2020.579513
Figure Lengend Snippet: TRAF6 mediates dendritic protrusion formation via neuroplastin. (A,B) The TRAF6 binding motif-deficient Np65Δ-GFP does not rescue dendritic protrusion formation in Nptn –/– neurons. (A) Confocal images of segments of dendrites of Nptn +/+ and Nptn –/– neurons transfected with plasmids encoding GFP, Np65-GFP or Np65Δ-GFP at 7 DIV. At 9 DIV, these neurons were fixed and stained with anti-GFP antibodies followed by an Alexa 488-conjugated antibody (green). (B) Protrusion densities from 2 independent cultures were used to obtain the mean ± SEM as indicated ( Nptn +/+ GFP = 4.12 ± 0.18, n = 34; Nptn –/– GFP = 1.72 ± 0.19, n = 27; Nptn –/– Np65-GFP = 3.67 ± 0.18, n = 33; Nptn –/– Np65Δ-GFP = 1.79 ± 0.16, n = 33). *** p < 0.001 vs. GFP-filled wild type neurons and ### p < 0.001 vs. GFP-filled Nptn –/– neurons using Student‘s t -test. (C,D) TRAF6 knockdown prevents the increase of dendritic protrusions induced by Np65-GFP in hippocampal neurons. Neurons were co-transfected with either control scrambled siRNA or siRNA against TRAF6 mRNA and with GFP-encoding plasmid (6 DIV). Additionally, neurons were co-transfected with siRNA and Np65-GFP or Np65Δ-GFP. After 72 h, neurons were stained with anti-MAP2 and anti-TRAF6 antibodies to control neuronal morphology and TRAF6 KD, respectively. Only neurons with ≥60% reduction in TRAF6 immunoreactivity (arrow heads in C ) were considered for the counting of dendritic protrusions. (D) Transfected neurons from 4 independent cultures were analyzed (sicontrol GFP = 3.73 ± 0.16, n = 59; siTRAF6 GFP = 2.16 ± 0.18, n = 49; siTRAF6 Np65-GFP = 2.09 ± 0.16, n = 22; siTRAF6 Np65Δ-GFP = 1.69 ± 0.17, n = 14). *** p < 0.001 vs. sicontrol GFP using Student‘s t -test. Scale bar = 100 μm. (E–G) Expression of Np65Δ-GFP does not increase the number of dendritic protrusions in 9 DIV-old rat hippocampal neurons. (E) Dendritic segments of neurons expressing GFP or Np65Δ-GFP and stained with antibodies against GFP (white) and Shank2 (red clusters) were photographed using confocal microscopy. Images were processed to identify Shank2 clusters of interest (see section “Materials and Methods”). Scale bar = 10 μm. (F) Quantification of the protrusion densities and (G) the distribution of Shank2-positive and Shank2-negative protrusions from 20-30 neurons per group from 3 independent cultures [Shank2(+): GFP = 0.55 ± 0.06; Np65Δ-GFP = 0.62 ± 0.04]. (H–J) TRAF6 inhibition decreases formation of dendritic protrusions. (H) 7 DIV-old rat neurons were transfected with Np65-GFP, treated with the TRAF6 inhibitor SMI 6860766 (SMI TRAF6, 2 μm) for 48 h, fixed, and stained for GFP (white) and Shank2 (red clusters) at 9 DIV. Scale bar = 10 μm. (I) Protrusion density (DMSO GFP = 3.24 ± 0.118, n = 47; SMI TRAF6 GFP = 2.22 ± 0.23, n = 16; DMSO Np65-GFP = 4.59 ± 0.16, n = 56; SMI TRAF6 Np65-GFP = 3.34 ± 0.16, n = 28) and (J) Distribution of Shank2-positve and Shank2-negative protrusions from transfected neurons per group from 3 independent cultures are displayed. * p < 0.05 or *** p < 0.001 vs. DMSO GFP and ### p < 0.001 vs. SMI TRAF6 GFP using Student’s t -test [Shank2(+): GFP DMSO = 0.58 ± 0.08; SMI TRAF6 = 0.52 ± 0.19; Np65-GFP DMSO = 0.55 ± 0.06; Np65-GFP SMI TRAF6 = 0.42 ± 0.09]. (K–M) From the experiments in (H–J) we calculated (K) the area of Shank2 clusters (DMSO GFP = 0.105 ± 0.004; DMSO Np65-GFP = 0.137 ± 0.004; DMSO Np65Δ-GFP = 0.106 ± 0.003; SMI TRAF6 GFP = 0.093 ± 0.003; SMI TRAF6 Np65-GFP = 0.094 ± 0.006; SMI TRAF6 Np65Δ-GFP = 0.098 ± 0.005), (L) the fluorescence intensity of the clusters (DMSO GFP = 134.6 ± 1.4; DMSO Np65-GFP = 139.5 ± 1.9; DMSO Np65Δ-GFP = 138.4 ± 2.1; SMI TRAF6 GFP = 133.0 ± 1.7; SMI TRAF6 Np65-GFP = 134.0 ± 1.8; SMI TRAF6 Np65Δ-GFP = 134.3 ± 1.6), and (M) the number of Shank2 clusters per protrusion (DMSO GFP = 1.36 ± 0.17; DMSO Np65-GFP = 1.92 ± 0.14; DMSO Np65Δ-GFP = 1.35 ± 0.14; SMI TRAF6 GFP = 1.39 ± 0.13; SMI TRAF6 Np65-GFP = 1.28 ± 0.09; SMI TRAF6 Np65Δ-GFP = 1.40 ± 0.10). * p < 0.05 between Np65-GFP-expressing and GFP-expressing neurons using Student’s t -test. # p < 0.05 between the treatments for the same transfection. (N) Neuroplastin requires both its TRAF6 binding motif and endogenous TRAF6 activity to promote spinogenic protrusion density. The illustration in the middle shows Np65-GFP-expressing neurons with increased density of Shank2-containing spinogenic protrusions. This phenotype is no longer observed when the TRAF6 binding motif is deleted from the Np65 intracellular tail (Np65Δ-GFP, left). TRAF6 blockage decreases both the density of protrusions and fraction of protrusions with Shank2 clusters (right).
Article Snippet: Other primary antibodies used were: anti-TRAF6 rabbit (Santa Cruz, #sc-7221; 1:100), anti-Synapsin 1 rabbit (Synaptic Systems, #106 103; 1:500), anti-Shank2 guinea pig antibody (Synaptic Systems, #162 204; 1:1,000), anti-Homer1 mouse (Synaptic Systems, #160 011; 1:500); anti-MAP2 guinea pig (Synaptic Systems, #188 004; 1:1,000) primary antibodies for overnight at 4°C.
Techniques: Binding Assay, Transfection, Staining, Knockdown, Control, Plasmid Preparation, Expressing, Confocal Microscopy, Inhibition, Fluorescence, Activity Assay
Synaptic Systems manufactures this product 
![Neuroplastin promotes formation of dendritic protrusions. (A–C) Reduced number of dendritic protrusions in Nptn –/– compared to Nptn +/+ mouse primary hippocampal neurons at 9 DIV. (A) Nptn –/– and Nptn +/+ neurons transfected with GFP-encoding plasmids at 6–7 DIV using Lipofectamine. At 9 DIV, neurons were fixed and stained with anti-GFP antibody followed by an Alexa 488-conjugated antibody to enhance their intrinsic fluorescence (green) and with anti-MAP2 antibodies followed by a proper secondary antibody to detect dendrites (magenta). Images were obtained using a confocal microscope. Scale bar = 100 μm. (B) Protrusion density (number of dendritic protrusions per 10 μm) of GFP-filled Nptn –/– and Nptn +/+ neurons (circles) is expressed as mean ± SEM from three independent cultures. *** p < 0.001 between genotypes using Student‘s t -test ( Nptn +/+ GFP = 4.12 ± 0.18, n = 33; Nptn –/– GFP = 1.72 ± 0.19, n = 36). (C) Protrusion density of GFP-, Np65- GFP-, or Np55-GFP-expressing Nptn –/– neurons from two independent cultures. *** p < 0.001 or ** p < 0.01 vs. Nptn –/– GFP using Student‘s t-test ( Nptn –/– GFP = 1.92 ± 0.22, n = 26; Nptn –/– Np65-GFP = 3.67 ± 0.18, n = 20; Nptn –/– Np55-GFP = 3.77 ± 0.19, n = 26). (D,E) Both neuroplastin isoforms increase dendritic protrusion density in rat neurons at 8 DIV. (D) Confocal images show rat neurons transfected with plasmids encoding GFP, Np65-GFP or Np55-GFP at 7 DIV. At 8 DIV, neurons were fixed and stained with anti-GFP antibody followed by an Alexa 488-conjugated antibody (white). Scale bar = 10 μm (E) Protrusion densities of 40–50 neurons per group (circles) from 3 to 4 independent cultures. *** p < 0.001 vs. GFP transfected cells using Student‘s t -test (GFP = 1.95 ± 0.19, n = 39; Np65-GFP = 3.23 ± 0.14, n = 56; Np55-GFP = 3.58 ± 0.16, n = 38). (F–H) Overexpression of Np65-GFP increases the number of newly formed <t>Shank2-containing</t> dendritic protrusions. (F) Cropped confocal images of dendritic segments of rat neurons transfected with GFP or Np65-GFP at 7 DIV. At 9 DIV, neurons were fixed and stained with primary antibodies against GFP (white) and Shank2 (red). Arrow heads point to Shank2 spots in dendritic protrusions. Scale bar = 10 μm. (G) Protrusion density (GFP = 3.151 ± 0.182, n = 48; Np65-GFP = 4.642 ± 0.145, n = 54) and (H) Distribution of Shank2-positive and Shank2-negative protrusions were calculated as a fraction from n = 40–50 neurons per group from N = 3 independent experiments. Plots display mean ± SEM as indicated. ** p < 0.01 for Np65-GFP vs. GFP using Student‘s t -test [Shank2(+): GFP = 0.54 ± 0.07; Np65-GFP = 0.60 ± 0.06]. (I–K) (I) Size of puncta (area; GFP = 0.10 ± 0.01, n = 747; Np65-GFP = 0.11 ± 0.01, n = 738), (J ) Fluorescence intensity (GFP = 127.6 ± 2.1; Np65-GFP = 131.5 ± 1.9) and (K) Number of Shank2 clusters/protrusion in neurons (GFP = 1.46 ± 0.17, n = 43; Np65-GFP = 1.91 ± 0.18, n = 49) of the experiments displayed in (F) . * p < 0.05 between Np65-GFP-expressing and GFP-expressing neurons using Student‘s t -test. (L) The upper sketch on the left illustrates dendritic protrusions enriched on Shank2 in control GFP-filled hippocampal neurons at 9 DIV. Np65-GFP-expressing neurons (lower sketch) display more spinogenic protrusions with Shank2 clusters.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5605/pmc07755605/pmc07755605__fcell-08-579513-g003.jpg)
![TRAF6 mediates dendritic protrusion formation via neuroplastin. (A,B) The TRAF6 binding motif-deficient Np65Δ-GFP does not rescue dendritic protrusion formation in Nptn –/– neurons. (A) Confocal images of segments of dendrites of Nptn +/+ and Nptn –/– neurons transfected with plasmids encoding GFP, Np65-GFP or Np65Δ-GFP at 7 DIV. At 9 DIV, these neurons were fixed and stained with anti-GFP antibodies followed by an Alexa 488-conjugated antibody (green). (B) Protrusion densities from 2 independent cultures were used to obtain the mean ± SEM as indicated ( Nptn +/+ GFP = 4.12 ± 0.18, n = 34; Nptn –/– GFP = 1.72 ± 0.19, n = 27; Nptn –/– Np65-GFP = 3.67 ± 0.18, n = 33; Nptn –/– Np65Δ-GFP = 1.79 ± 0.16, n = 33). *** p < 0.001 vs. GFP-filled wild type neurons and ### p < 0.001 vs. GFP-filled Nptn –/– neurons using Student‘s t -test. (C,D) TRAF6 knockdown prevents the increase of dendritic protrusions induced by Np65-GFP in hippocampal neurons. Neurons were co-transfected with either control scrambled siRNA or siRNA against TRAF6 mRNA and with GFP-encoding plasmid (6 DIV). Additionally, neurons were co-transfected with siRNA and Np65-GFP or Np65Δ-GFP. After 72 h, neurons were stained with anti-MAP2 and anti-TRAF6 antibodies to control neuronal morphology and TRAF6 KD, respectively. Only neurons with ≥60% reduction in TRAF6 immunoreactivity (arrow heads in C ) were considered for the counting of dendritic protrusions. (D) Transfected neurons from 4 independent cultures were analyzed (sicontrol GFP = 3.73 ± 0.16, n = 59; siTRAF6 GFP = 2.16 ± 0.18, n = 49; siTRAF6 Np65-GFP = 2.09 ± 0.16, n = 22; siTRAF6 Np65Δ-GFP = 1.69 ± 0.17, n = 14). *** p < 0.001 vs. sicontrol GFP using Student‘s t -test. Scale bar = 100 μm. (E–G) Expression of Np65Δ-GFP does not increase the number of dendritic protrusions in 9 DIV-old rat hippocampal neurons. (E) Dendritic segments of neurons expressing GFP or Np65Δ-GFP and stained with antibodies against GFP (white) and Shank2 (red clusters) were photographed using confocal microscopy. Images were processed to identify Shank2 clusters of interest (see section “Materials and Methods”). Scale bar = 10 μm. (F) Quantification of the protrusion densities and (G) the distribution of Shank2-positive and Shank2-negative protrusions from 20-30 neurons per group from 3 independent cultures [Shank2(+): GFP = 0.55 ± 0.06; Np65Δ-GFP = 0.62 ± 0.04]. (H–J) TRAF6 inhibition decreases formation of dendritic protrusions. (H) 7 DIV-old rat neurons were transfected with Np65-GFP, treated with the TRAF6 inhibitor SMI 6860766 (SMI TRAF6, 2 μm) for 48 h, fixed, and stained for GFP (white) and Shank2 (red clusters) at 9 DIV. Scale bar = 10 μm. (I) Protrusion density (DMSO GFP = 3.24 ± 0.118, n = 47; SMI TRAF6 GFP = 2.22 ± 0.23, n = 16; DMSO Np65-GFP = 4.59 ± 0.16, n = 56; SMI TRAF6 Np65-GFP = 3.34 ± 0.16, n = 28) and (J) Distribution of Shank2-positve and Shank2-negative protrusions from transfected neurons per group from 3 independent cultures are displayed. * p < 0.05 or *** p < 0.001 vs. DMSO GFP and ### p < 0.001 vs. SMI TRAF6 GFP using Student’s t -test [Shank2(+): GFP DMSO = 0.58 ± 0.08; SMI TRAF6 = 0.52 ± 0.19; Np65-GFP DMSO = 0.55 ± 0.06; Np65-GFP SMI TRAF6 = 0.42 ± 0.09]. (K–M) From the experiments in (H–J) we calculated (K) the area of Shank2 clusters (DMSO GFP = 0.105 ± 0.004; DMSO Np65-GFP = 0.137 ± 0.004; DMSO Np65Δ-GFP = 0.106 ± 0.003; SMI TRAF6 GFP = 0.093 ± 0.003; SMI TRAF6 Np65-GFP = 0.094 ± 0.006; SMI TRAF6 Np65Δ-GFP = 0.098 ± 0.005), (L) the fluorescence intensity of the clusters (DMSO GFP = 134.6 ± 1.4; DMSO Np65-GFP = 139.5 ± 1.9; DMSO Np65Δ-GFP = 138.4 ± 2.1; SMI TRAF6 GFP = 133.0 ± 1.7; SMI TRAF6 Np65-GFP = 134.0 ± 1.8; SMI TRAF6 Np65Δ-GFP = 134.3 ± 1.6), and (M) the number of Shank2 clusters per protrusion (DMSO GFP = 1.36 ± 0.17; DMSO Np65-GFP = 1.92 ± 0.14; DMSO Np65Δ-GFP = 1.35 ± 0.14; SMI TRAF6 GFP = 1.39 ± 0.13; SMI TRAF6 Np65-GFP = 1.28 ± 0.09; SMI TRAF6 Np65Δ-GFP = 1.40 ± 0.10). * p < 0.05 between Np65-GFP-expressing and GFP-expressing neurons using Student’s t -test. # p < 0.05 between the treatments for the same transfection. (N) Neuroplastin requires both its TRAF6 binding motif and endogenous TRAF6 activity to promote spinogenic protrusion density. The illustration in the middle shows Np65-GFP-expressing neurons with increased density of Shank2-containing spinogenic protrusions. This phenotype is no longer observed when the TRAF6 binding motif is deleted from the Np65 intracellular tail (Np65Δ-GFP, left). TRAF6 blockage decreases both the density of protrusions and fraction of protrusions with Shank2 clusters (right).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5605/pmc07755605/pmc07755605__fcell-08-579513-g005.jpg)