Fig 2: Generation and basic characterization of Pv-Cre;Shank2fl/fl mice. (A) Breeding scheme for the production of WT and Pv-Cre;Shank2fl/fl conditional KO (cKO) mice. Pv-Cre indicates mice heterozygous for the modified Pv allele, Pv-IRES-Cre; Pv-wild indicates normal WT mice without a Cre gene insert. (B) Genotyping of WT and Pv-Cre;Shank2fl/fl mice, by PCRs on WT and cKO cerebellar samples (4 months). Note that only Pv-Cre;Shank2fl/fl mice are positive for both the Pv-Cre allele and the Shank2 KO allele. (C) Levels of Shank2 protein in different brain regions of WT and Pv-Cre;Shank2fl/fl mice (4 months). Ctx, cortex; Str, striatum; Hp, hippocampus; Cb, cerebellum. (n = 4 mice for WT and cKO, *P < 0.05, ns, not significant, Mann-Whitney U test). (D) Pv-Cre;Shank2fl/fl mice (16 weeks) show normal numbers of PV-positive neurons and normal intensities of PV protein signals, as shown by the ratio of PV-positive cells and NeuN (neuronal marker)-positive cells and the intensity of PV protein signals normalized to cell area in Cg1 and M2 cortical regions. Note that a 200 × 200 μm block (middle) rather than the whole area (800 × 1000 μm; left) is shown to better visualize individual cells (n = 8 mice for WT and 7 mice for cKO, ns, not significant, Student’s t-test) The images from the Allen Mouse Brain Atlas was used in (D) (brain diagrams on the left; Lein et al., 2007). Scale bar, 50 μm.

Fig 3: TRAF6 mediates dendritic protrusion formation via neuroplastin. (A,B) The TRAF6 binding motif-deficient Np65?-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 Np65?-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–/– Np65?-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 Np65?-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 Np65?-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 Np65?-GFP does not increase the number of dendritic protrusions in 9 DIV-old rat hippocampal neurons. (E) Dendritic segments of neurons expressing GFP or Np65?-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; Np65?-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 Np65?-GFP = 0.106 ± 0.003; SMI TRAF6 GFP = 0.093 ± 0.003; SMI TRAF6 Np65-GFP = 0.094 ± 0.006; SMI TRAF6 Np65?-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 Np65?-GFP = 138.4 ± 2.1; SMI TRAF6 GFP = 133.0 ± 1.7; SMI TRAF6 Np65-GFP = 134.0 ± 1.8; SMI TRAF6 Np65?-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 Np65?-GFP = 1.35 ± 0.14; SMI TRAF6 GFP = 1.39 ± 0.13; SMI TRAF6 Np65-GFP = 1.28 ± 0.09; SMI TRAF6 Np65?-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 (Np65?-GFP, left). TRAF6 blockage decreases both the density of protrusions and fraction of protrusions with Shank2 clusters (right).

Fig 4: Multicolor imaging of sparsely labeled pyramidal neurons in CA1 imaged by LSFEM. (a) Maximum intensity projection of an expanded mouse brain slice expressing EGFP. The endogenous EGFP fluorescence was enhanced by antibody staining against EGFP, the secondary antibody was conjugated to Alexa 488. The total axial extension was 390 μm. (b) Magnification of the ROI marked in (a). Dendrites were stained with Alexa488 (blue), postsynaptic proteins (shank2) stained with Alexa568 (green), presynaptic proteins (bassoon) stained with Alexa647 (red). The shown images were deconvolved. (c) Magnification a region indicated in (b) (white arrow), lateral field size 17.3×17.3 μm2, indicating synaptic connectivity. The right panel in (c) shows the intensity profiles along the dotted line. (d) 3-D surface rendering of the synapse region shown in (c).

Fig 5: A model for Shank mediation of zinc-dependent subunit switch of AMPAR in developing synapses. A glutamatergic synapse of a wild-type (WT) young neuron is shown with low levels of Shank3 (green) and GluA2 (teal) and high levels of Shank2 (blue) and GluA1 (orange). The synaptic complement changes with zinc-sensitive recruitment of synaptic GluA2 via exocytosis and lateral diffusion (blue arrows), leading to potentiation of AMPAR-mediated synaptic activity. The zinc-recruitment of GluA2 correlated with the level of synaptic Shank3. Zinc addition also led to reduction of synaptic GluA1, which is most likely driven by Shank2. Hence, loss of either Shank2 or Shank3 abolished the zinc-sensitive potentiation of AMPAR-mediated synaptic transmission.