vesicular glutamate transporter 1  (Novus Biologicals)

Journal: bioRxiv

Article Title: Translatome profiling reveals opposing alterations in inhibitory and excitatory neurons of Fragile X mice and identifies EPAC2 as a therapeutic target

doi: 10.1101/2025.04.21.649817

Figure Lengend Snippet: (a) Shared DEGs between Camk2a and Pvalb translatomes, and corresponding GO enrichment terms for biological processes. (b) Heatmap of top 20 DEGs that share the same directionality in both cell types. FMRP target genes indicated in maroon. (c) Positive association between 193 shared DEGs (between Camk2a and Pvalb samples) and both FMRP targets (27 genes, OR=2.2, p=0.005; Fisher’s exact test) and ASD risk genes (12 genes; n.s.) Specific DEGs at major intersections are listed. (d) Rapgef4 is significantly upregulated in both Camk2a and Pvalb samples across both S1 and V1 (FDR=0.05; Pvalb -S1: adj.p=0.048; Pvalb -V1: adj.p=0.01; Camk2a -S1: adj.p=0.002; Camk2a -V1: adj.p=0.008). (e) Example in situ hybridization probing for Rapgef4 , Pvalb and Slc17a7 ( Vglut1 ) a marker of excitatory neurons. (f) Cumulative distribution (Left) and quantification of puncta density (Right) for Rapgef4 + puncta overlapping with Slc17a7 or Pvalb in S1 of WT and Fmr1 KO mice ( Slc17a7 : 0.17±0.002 vs. 0.12±0.001 puncta/pixel 2 , p<0.001; unpaired t-test; n= 2738/2009 neurons from n=3/4 WT and Fmr1 KO mice, respectively; Pvalb : 0.14±0.004 vs. 0.10±0.003 puncta/pixel 2 , p<0.001; n=359/209 neurons from n=3/4 WT and Fmr1 KO mice, respectively). Data represented as median±1.5*IQR. (g) Example immunohistochemistry for EPAC2 (green) and neuronal marker NEUN (blue). (h) Distribution (Left) of the fraction of cell soma area (NEUN+) covered by EPAC2 puncta in WT and Fmr1 KO mice (WT: 0.126±0.004 vs. KO: 0.136±0.006; p=0.03; unpaired t-test; n=2618/2415 neurons from n= 8/6 WT and Fmr1 KO mice, respectively). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The following primary antibodies were used: mouse anti-parvalbumin (1:1,000, SAB4200545, Sigma Aldrich), Anti-EPAC2 (1:250, rabbitAb #43239 and mAb #4156, Cell Signaling), Anti-NEUN (1:1000, MA5-33103, Thermofisher), anti-HA (1:1000, mAb#901513, Covance), anti-GAD (1:1000, Gad6 DSHB), anti-VGLUT1 (1:1000, N28/9 DSHB).

Techniques: In Situ Hybridization, Marker, Immunohistochemistry

Journal: bioRxiv

Article Title: Translatome profiling reveals opposing alterations in inhibitory and excitatory neurons of Fragile X mice and identifies EPAC2 as a therapeutic target

doi: 10.1101/2025.04.21.649817

Figure Lengend Snippet: (a) While most genes in the EPAC2 interactome were upregulated in Camk2a samples from Fmr1 KO mice, their dysregulation was more balanced in Pvalb samples. Note that most of these genes are also FMRP targets, ASD risk genes, or both. (B) Seeded network analysis of Rapgef4 with corresponding pathways of correlated and anticorrelated genes. (C) Representative in situ hybridization image for expression of Rapgef4 , Pvalb , Slc17a7 in S1. (D) Colocalization of GAD65 (top) and VGLUT1 (bottom) (in red) with EPAC2 (green). (E) Colocalization of PSD95 (red) with EPAC2 (green). (F) (Top) Western blot showing enrichment of EPAC2 in synaptoneurosomal preparations. (Bottom) EPAC2 levels are higher in the synaptoneurosomal fraction compared to the total lysate (p=0.0002, t-test). (G) Western blots showing co-immunoprecipitation of EPAC2 with PSD95 (top) and SYT2 (bottom). Note that the intensity of the bands cannot be compared across the two experiments as the quantities loaded differed between IP-PSD95 and pre-IP. (H) Representative image of Epac2 and NEUN (Related to ).

Article Snippet: The following primary antibodies were used: mouse anti-parvalbumin (1:1,000, SAB4200545, Sigma Aldrich), Anti-EPAC2 (1:250, rabbitAb #43239 and mAb #4156, Cell Signaling), Anti-NEUN (1:1000, MA5-33103, Thermofisher), anti-HA (1:1000, mAb#901513, Covance), anti-GAD (1:1000, Gad6 DSHB), anti-VGLUT1 (1:1000, N28/9 DSHB).

Techniques: In Situ Hybridization, Expressing, Western Blot, Immunoprecipitation