Journal: Experimental neurology

Article Title: A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

doi: 10.1016/j.expneurol.2019.112973

Figure Lengend Snippet: (A) Schematic representation of GAT-1 protein topology and locations of GAT-1 variants identified in patients associated with a spectrum of epilepsy syndromes. It is predicted that GAT-1 contains 12 transmembrane domains. G234S is located at the junction of the second intracellular loop and the 5th transmembrane domain of the GAT-1 protein. The positions of variants are based on the published LeuT crystal structure. (B) Amino acid sequence homology shows that glycine (G) at residue 234 is highly conserved in SCL6A1 in human (Accession NO. NP_003033.3) and across species. (C) Tertiary structures of both the wildtype and G234S mutant protein GAT-1 are predicted by I-TASSER. Residue 234 is highlighted as red and Glycine is mutated to Serine. (D) From interatomic interactions predictions by DynaMut, wild-type (upper) and G234S mutation (bottom) residues are colored in light-green and are also represented as sticks alongside with the surrounding residues. Halogen bonds are depicted in blue. Hydrogen bonds are colored in red. Machine learning methods as Supplementary Table 1 predicted this mutation destabilized the global conformation of the GAT-1 protein.

Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone, 1:200) or mouse monoclonal GFP (Millipore, Billerica, MA, 1:200).

Techniques: Sequencing, Mutagenesis

Journal: Experimental neurology

Article Title: A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

doi: 10.1016/j.expneurol.2019.112973

Figure Lengend Snippet: A-B. HEK293T cells were transfected with GAT-1YFP (3μg) for 48 hrs. (A) Total lysates were analyzed by SDS-PAGE and western blot. The membranes were blotted with mouse anti-GFP antibody. (B) Rat cortical neurons were transfected with the wildtype or the mutant GAT-1(G234S)YFP cDNAs at day 7 days old in cultured dish. The total lysates were harvested from rat cortical neurons expressing the wildtype GAT-1YFP (wt) or mutant GAT-1(G234S)YFP (G234S) transporters after 8 days of transfection. The total lysates were then analyzed by SDS-PAGE. In HEK 293T cells (A), three protein bands were detected in both the wildtype and the mutant conditions. In rat cortical neurons (B), only a single strong band was detected in both the wildtype and the mutant conditions. In A and B, 1:500 means the ratio of the GFP antibody in buffer (1μg of GFP in 500 μl 1XPBS). (C, D). The total protein integrated density values (IDVs) were measured. The abundance of the mutant (GAT-1(G234S) transporter was normalized to the wildtype condition. In C, the total protein abundance was measured by adding up all the three bands run between 90–110 KDa. In both C and D, the total protein IDVs of either the wildtype or the mutant was normalized to its loading control. The abundance of the mutant transporter was then normalized to the wildtype. (***p < 0.001 vs. wt, n=4 different transfections).

Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone, 1:200) or mouse monoclonal GFP (Millipore, Billerica, MA, 1:200).

Techniques: Transfection, SDS Page, Western Blot, Mutagenesis, Cell Culture, Expressing

Journal: Experimental neurology

Article Title: A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

doi: 10.1016/j.expneurol.2019.112973

Figure Lengend Snippet: (A) The flow cytometry histograms depict surface expression levels of GAT-1 from HEK293T cells were transfected with wildtype GAT-1YFP (wt), or the mutant GAT-1(G234S)YFP cDNAs or control (insert). Cell surface wild type and mutant GAT-1 stained with polyclonal anti-GAT-1 antibody that was fluorescently conjugated with Alexa Fluor-555. (B) Surface protein was isolated by biotinylating live cells expressing wildtype GAT-1YFP (wt), or the mutant GAT-1(G234S)YFP and analyzed by SDS-PAGE. The membranes were visualized by polyclonal anti-GAT-1 protein. (C) The normalized relative fluorescence intensity (FI) (C) or the surface protein intensity values (IDVs) (D) were normalized to those obtained with wildtype GAT-1 while the FI or protein IDVs in the wildtype was arbitrarily taken as 1 in each experiment (***p < 0.001 vs. wt, n=4 different transfections). (E). The GABA uptake assay was carried out with 3H radioactive GABA transport in HeLa cells transiently expressing the wildtype or the mutant GAT-1YFP for 48 hrs. GABA flux was measured after 15 min transport at room temperature (**p < 0.01 vs. wt, n=6 for wildtype and 7 for the mutant which represents number of transfections).

Article Snippet: Membranes were incubated with primary rabbit polyclonal antibodies against GAT-1 (Alomone, 1:200) or mouse monoclonal GFP (Millipore, Billerica, MA, 1:200).

Techniques: Flow Cytometry, Expressing, Transfection, Mutagenesis, Staining, Isolation, SDS Page, Fluorescence

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