Journal: Nanomaterials

Article Title: Raman Spectroscopy Reveals Photobiomodulation-Induced α-Helix to β-Sheet Transition in Tubulins: Potential Implications for Alzheimer’s and Other Neurodegenerative Diseases

doi: 10.3390/nano14131093

Figure Lengend Snippet: Characteristic parameters of the intranasal LED applicator of the Vielight Neuro Alpha brain PBM device.

Article Snippet: The exposure of reconstituted tubulin samples was performed with the intranasal LED applicator of the Vielight Neuro Alpha transcranial–intranasal brain PBM device; its parameters are reported in .

Techniques:

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: Here, we demonstrate that a critical function of PBM binding is to limit the mobility of SynGAP-α1 in response to synaptic NMDAR activation.

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) 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: Here, we demonstrate that a critical function of PBM binding is to limit the mobility of SynGAP-α1 in response to synaptic NMDAR activation.

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