Journal: bioRxiv

Article Title: Splicing of the SynGAP Carboxyl-Terminus Enables Isoform-Specific Tuning of NMDA Receptor Signaling Linked to Cognitive Function

doi: 10.1101/2020.01.18.911487

Figure Lengend Snippet: (A) Schematic diagram for exon map and alternative use of exon 21 in the Syngap1 gene. Exon 21b encodes for α1 isoform. exon 21a encodes for α2 isoform. Point mutations indicated in red alter exon 21b coding sequence without influencing exon21a open reading frame. (B) Schematics of SynGAP-α1 and PSD95 domain structure and the location of point mutations. (C) Illustrations of constructs expressed in HeLa cells to study PDZ-dependent interaction between SynGAP and PSD95. EGFP-CC constructs are homologous to SynGAP-α1 C-terminus. (D) Co-localization of EGFP-CCα1 and PSD95-tRFP in HeLa Cells. Representative images showing subcellular localizations of WT (α1) or PDZ-binding mutant (PBM) EGFP-CCα1 and PSD95-tRFP in HeLa cells when expressed individually or together. (E) Quantification of D. ANOVA with Tukey’s multiple comparisons test, F(3, 96) = 531.4, p<0.0001, n=23-25 cells per condition. (F) Schematics of the targeting strategy. The targeting vector was spanning Exon20 & 21. The vector included point mutations in Exon21, a neomycin resistance selection cassette flanked by Cre recombination sites and diphtheria toxin selection cassette (DTA). (G) Southern blot analysis showing the genomic DNA of the tested heterozygous mice compared to C57BL/6J wild-type DNA following AflII digestion and hybridization with external 5’ probe spanning exon 19. (H) PCR based genotyping strategy. Primers flanking leftover LoxP site yields 61bp product in WT and 120bp product in mutated allele. (I) Representative western blots showing expression levels of total SynGAP and individual isoforms in forebrain lysates. (J) Quantification of I. Relative intensity of bands normalized to total protein signal. ANOVA with Tukey’s multiple comparisons test. SynGAP-α1: F(2, 14) = 24.86, p<0.0001; +/+ vs +/PBM: p=0.0009; +/+ vs PBM/PBM: p<0.0001. n=5 per genotype.

Article Snippet: The lysate was then centrifuged at 35,000 RPM for 30 minutes at 4°C and lysate containing 1 mg of protein was incubated with 2 μg Psd95 antibody (Neuromab, catalog # 75-048) at 4°C overnight with rotation.

Techniques: Sequencing, Construct, Binding Assay, Mutagenesis, Plasmid Preparation, Selection, Southern Blot, Hybridization, Western Blot, Expressing

Journal: bioRxiv

Article Title: Splicing of the SynGAP Carboxyl-Terminus Enables Isoform-Specific Tuning of NMDA Receptor Signaling Linked to Cognitive Function

doi: 10.1101/2020.01.18.911487

Figure Lengend Snippet: (A) Dendritic spine size and surface GluA1 expression levels in steady state primary forebrain cultures at DIV18. Average dendritic spine diameter. Unpaired t-test t(36)=2.508 p=0.0168. Average surface GluA1 expression in dendritic spines. t(36)=4.443 p<0.0001. n=19, each n representing an average of 25-30 spines fromge a dendritic segment belonging to distinct neuron. Scale bar 2 μm. (B) Hippocampal input-output relationships at PND 14-16 in dentate gyrus (DG). Linear regression comparing slopes and intercepts. Slope F(1, 6) = 13.36, p=0.0106, n=3 each n representing an individual mice (3-4 slices were recorded from each). (C) Seizure threshold measured as the time taken to reach the 1st clonus (event onset) during the procedure. Unpaired t-test. t(25)=3.420, p=0.0022, n=13-14 mice per genotype. (D) Western blots showing relative distribution of SynGAP in PSD and Syn/Tx fractions from adult hippocampi. (E-F) Quantification of western blots probing total SynGAP, Synaptophysin and PSD95. For PSD fractions PSD95 and for Syn/Tx fractions Synaptophysin (Syp) were used as loading control. PSD fractions: t(22)=3.733, p=0.0011 n=12 (3 technical replicates for each sample), Syn/TX fractions: t(6)=3.049, p=0.0226, n=4. Each sample represents hippocampi pooled from 2 mice. (G) Western blots showing relative enrichment of SynGAP and PSD95 in PSD fractions isolated from DIV18-21 cultures (left), phospho- and total-ERK1/2 levels in whole cell lysates in steady state (right). (H-I) Quantification of (G). Synaptic enrichment of SynGAP in steady state. Unpaired t-test: t(6)=2.961, p=0.0253, n=4. ERK1/2 phosphorylation is calculated as ratio of phospho-ERK1/2 to total-ERK1/2 in homogenates. t(12)=3.035, p=0.0103, n=6-8. (J) Western blots showing relative enrichment of SynGAP and PSD95 in PSD fractions (right), phospho and total-ERK1/2 levels in whole cell lysates in inactivated state (left). (K-L) Quantification of J. (K) Synaptic enrichment of SynGAP in inactivated state: t(6)=0.5385, p=0.6069, n=4. (L) ERK1/2 phosphorylation in inactivated state: t(4)=1.144, p=0.3163, n=3.

Article Snippet: The lysate was then centrifuged at 35,000 RPM for 30 minutes at 4°C and lysate containing 1 mg of protein was incubated with 2 μg Psd95 antibody (Neuromab, catalog # 75-048) at 4°C overnight with rotation.

Techniques: Expressing, Western Blot, Control, Isolation, Phospho-proteomics

Journal: bioRxiv

Article Title: Splicing of the SynGAP Carboxyl-Terminus Enables Isoform-Specific Tuning of NMDA Receptor Signaling Linked to Cognitive Function

doi: 10.1101/2020.01.18.911487

Figure Lengend Snippet: (A) Volcano plot demonstrating the label-free quantitative mass-spectrometry profile of the logarithmic difference in protein levels in the immunoprecipitated PSD95 complexes derived from WT and PBM/PBM cultures in inactivated state. Only Gpm6a (shown in black) was significantly altered beyond the predetermined threshold (p<0.01 and ± 0.5 Log2 cutoff). Blue dots represent proteins with type 1 PDZ-ligands. Green dots represent DLG family proteins. P values were calculated via t-test for each protein. Samples were derived from individual cultures (4 per genotype) that were immunoprecipitated separately. (B) List of proteins differentially expressed beyond p>0.05 cutoff. Note that Iqseq2 and Dlgap3 are PDZ-binding proteins. (C) Blots showing distribution of SynGAP in PSD and Syn/Tx fractions upon chemical LTP stimulus (200μM Glycine, no Mg +2 ) in +/+ neurons. PSD: t(2)=5.696, p=0.0295. Syn/Tx: t(2)=7.511, p=0.0173. n=2, each n represents samples from separate cultures that are fractionated separately. (D) Representative blots from showing relative levels of SynGAP and PSD95 in PSD fractions or GluA1 Ser845 phosphorylation in basal state or weak chemical LTP (wcLTP: 10μM Glycine, no Mg +2 ) (E-F) Quantification of D. (E) Bar graphs represents the level of total-SynGAP/PSD95 ratio relative to the baseline of each genotype. +/+: t(4)=0.8354 p=0.4505, PBM: t(4)=17.52, p<0.0001, n=4 (F) GluA1 Ser845 phosphorylation. +/+: t(6)=0.4921 p=0.64, PBM: t(6)=3.152 p=0.0198 (G) Blots showing isoform enrichment in PSD fractions following wcLTP. (H-J) Quantification of G. (H) For SynGAP-α1 +/+: t(6)=0.8266 p=0.4401, PBM: t(6)=16.74 p<0.0001. (I) For SynGAP-α2, +/+: t(6)=5.706 p=0.0013, PBM: t(6)=7.263 p<0.0003. (J) For SynGAP-β, +/+: t(6)=5.894 p=0.0011, PBM: t(6)=10.52 p<0.0001, n=4. Note that α1 isoform is dispersed upon wcLTP only in PDZ-binding mutants.

Article Snippet: The lysate was then centrifuged at 35,000 RPM for 30 minutes at 4°C and lysate containing 1 mg of protein was incubated with 2 μg Psd95 antibody (Neuromab, catalog # 75-048) at 4°C overnight with rotation.

Techniques: Mass Spectrometry, Immunoprecipitation, Derivative Assay, Binding Assay, Phospho-proteomics

Journal:

Article Title: Profilin2 contributes to synaptic vesicle exocytosis, neuronal excitability, and novelty-seeking behavior

doi: 10.1038/sj.emboj.7601737

Figure Lengend Snippet: Lack of actin polymerization in pfn2−/− synaptosomes correlates with increased vesicle docking/priming. Specific binding of the WAVE-complex to profilin2. (A) Synaptic vesicle priming based on vSNARE/tSNARE ratios after immunoprecipitation of tSNARE (syntaxin1) from pfn2−/− synaptosomes is increased by 30%. vSNARE/tSNARE ratios from control mice (n=5) were set to 100% and the mutant ratios (n=4) expressed accordingly. (B) Synaptosomes from control and pfn2−/− mice were analyzed by Western blot using a panel of antibodies detecting different markers of the vesicle release machinery. Two different mutant mice are shown to account for sample variability. (C) Synaptic F/G-actin ratios were not significantly different in resting synaptosomes (0 s), after 60 s stimulation with 20 mM K+ the F-actin/G-actin ratio raised in the control, while no significant increase was seen in the mutants (n=3 for mutant and control; ANOVA, interaction genotype x KCl stimulation: F[1,8]=5.365, P=0.049). (D) Distribution of WAVE-complex components in synaptosomal fractions. WAVE1, Sra1/Cyfip, and Nap1 were found enriched in the presynaptic matrix and the PSD. (E) In synaptosomal lysate, profilin2 co-immunoprecipitated with WAVE1 (left panel) and WAVE1 co-immunoprecipitated with profilin2 (right panel). (F) Specific binding of WAVE1-complex to profilin2. Pull down of WAVE1 from cortical protein extracts using profilin1- and profilin2-coupled beads shows specificity for profilin2.

Article Snippet: Antibodies for WAVE1 (BD Transduction Lab.) and Nap1 (Upstate) were purchased, antibodies for profilin2 and profilin1 were described before ( Witke et al , 1998 ), monoclonal antibodies for Cyfip were raised against recombinant protein (mab5C9).

Techniques: Binding Assay, Immunoprecipitation, Mutagenesis, Western Blot