Fig 2: NPTX2-SEP colocalizes with excitatory synapses on PVs, and its exocytosis is activity dependent in vivo.(A and B) AAV-CaMKII-NPTX2-SEP and AAV-Flex-PSD95.FingR-tdTomato were injected into the sensory cortex of PV-Cre mice. Three weeks later, live animals were imaged with two-photon microscopy under isoflurane anesthesia. Three-dimensional and Imaris isosurface images are shown here. Enlarged images (B) from three boxes in (A) show NPTX2-SEP puncta on soma and processes of PVs. Colocalized or associated signals are indicated by white arrows. Yellow arrows indicate PSD95.FingR-tdTomato signal without NPTX2-SEP. MIP, maximum intensity projection. (C) AAV-CaMKII-NPTX2-HA-V5 and AAV-Flex-PSD95.FingR-tdTomato were injected into the sensory cortex of PV-Cre mice. Perfused brain tissue section was stained with anti-HA for confocal microscopy. Maximum intensity projection of z stack was shown (0.38-µm interval for 59 stack). NPTX2 accumulation onto PV-PSD95 is indicated by white arrows. (D) Representative Imaris isosurface images of NPTX2-SEP before and after (15 min, 2, 24, and 48 hours) C1V1 (1040 nm) versus control (800 nm) stimulation in S1 barrel field layer 2/3. Maximum intensity projection of 3D images at 80- to 180-µm depth is shown. Scale bars, 20 µm. 2P, two-photon. (E and F) Quantification of the spot number (E) and total volume (F). N = 5 animals. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with post hoc Tukey’s test compared with “Before.” (G) Representative image showing the expression of C1V1-mCherry in L2/3 PNs. Image was acquired by confocal imaging of frozen tissue section. Scale bar, 20 µm.

Fig 3: EPS15L1 is predominantly expressed in neurons and surviving Eps15L1-KO mice have development and neural deficits.(A) Western blotting of the heart (Ht), ovary (Ov), testis (Te), colon (Co), lung (Lu), liver (Li), spleen (Sp), brain (Br), cerebellum (Ce), and fibroblasts (MEFs); 10 µg of each tissue lysate were loaded and probed for EPS15. Specific bands are indicated by arrows. Various amounts of GST-fusion protein for EPS15 or EPS15L1 were also loaded to allow for a rough estimate of the actual amount of the protein in the cellular lysates. (B) Western blotting as in (A), probed for EPS15L1. Specific bands are indicated by arrows. (C) Immunofluorescence for EPS15 and EPS15L1 expression in the hippocampus of adult mouse brain. Sections were stained for EPS15 or EPS15L1 as indicated (green), synaptophysin (red) and counterstained with DAPI (blue). Bar, 10 µm. (D) Western blotting of adult mouse brain fractions with the indicated antibodies. Equal amounts of homogenate (H), synaptosomal fraction (Syn), and postsynaptic density (PSD) were loaded. Note that, as expected, PSD95 (a post-synaptic marker) was enriched in the PSD, whereas synaptophysin was enriched in the synaptosomal fraction. (E) WT and Eps15L1-KO mice at 4 wk of age. (F) Bodyweight of WT/HET and Eps15L1-KO mice at 4 wk of age. Data from WT and heterozygous animals were pooled as no statistical difference between the two genotypes was detected. The number of mice analyzed is shown in parentheses. (G) Results of a reduced SHIRPA test performed on 4-wk-old WT and Eps15L1-KO mice. **P < 0.01 versus WT; ***P < 0.001 versus WT

Fig 4: PSD-95 restores the decrease in PSD-95 cluster number induced by Pin1. (A) Model showing how post-translational modification regulates PSD-95 entry and stability at the PSD. (B) Confocal images of hippocampal cultured neurons overexpressing PSD-95 and control or Pin1 IRES EGFP. Images show EGFP, PSD-95 staining and the overlay image. Scale bar: 10 µm. (C) wt PSD-95 restores the decrease in cluster number. Normalized number of clusters for dendrites expressing EGFP EGFP 1.00 ± 0.04, n = 49; Pin1 1.21 ± 0.07, n = 53; C113S 1.12 ± 0.07, n = 54, (D) PSD-95 overexpression restores the area of PSD-95 clusters well above the level of non-transfected. EGFP 1.00 ± 0.04, n = 49; Pin1 0.82 ± 0.04, n = 53, C113S 0.85 ± 0.03, n = 55 Kruskal–Wallis chi-square = 13.46, df = 2, p < 0.0012. (E) Similar to wt PSD-95, overexpression of the N3A mutant of PSD-95 also restores to control levels the density of PSD-95 per dendritic area in cells overexpressing Pin1. (F) N3A overexpression restores the cluster of PSD-95 per dendritic area above the level of EGFP expressing cells. EGFP 1.00 ± 0.04, n = 42; Pin1 0.14 ± 0.05, n = 66; C113S 1.32 ± 0.06, n = 51, One-way ANOVA F(2,156) = 7.84, p = 0.0006. (G) N3A overexpression restores the area of PSD-95 clusters well above the level of non-transfected. EGFP 1.00 ± 0.03, n = 42; Pin1 0.86 ± 0.03, n = 66; C113S 1.02 ± 0.04, n = 51 55 Kruskal–Wallis chi-square = 16.21, df = 2, p < 0.0003.

Fig 5: Signaling complexes in WT and AKAP5 mutant hippocampus. A, Immunoprecipitation (IP) of AKAP5, calcineurin (CaN), and RIIa, with immunoblotting (IB) using antibodies against CaN, AKAP5, and RIIa. Input is the equivalent amount of hippocampal extract. B, IP of the dendritic shaft AKAP MAP2, AKAP5, a PSD enriched protein, GluR2/3, and RIIa, with immunoblotting (IB) using antibodies against AKAP5, PSD-95, RIIa, MAP2, and GluR2/3. IP's are 7.5 fold more concentrated relative to the inputs, to facilitate visualization and comparison of the WT vs. KO extracts, as well as to detect weaker interactions.