Journal: Pharmaceutics

Article Title: Glial Fibrillary Acidic Protein: A Biomarker and Drug Target for Alzheimer’s Disease

doi: 10.3390/pharmaceutics14071354

Figure Lengend Snippet: GFAP is more abundant and more modified in AD aggregates than in AMC. ( a ) Spectral counts for GFAP in sarkosyl-insoluble aggregates isolated by immuno-pulldown (IP) of Aβ or tau, or in total aggregates without IP, from human AD vs. age-matched-control (AMC) hippocampus. AD differs from AMC according to heteroscedastic t -tests: * p < 0.05; ** p < 0.005. ( b ) Western blot of phosphorylated GFAP from AMC(3,3) vs. AD(3,3) or AD(4,4) hippocampal aggregates, detected with antibody to phospho-GFAP (Ser13; ThermoFisher). *** Each AD group differs from AMC according to two-tailed heteroscedastic t -test at p < 0.0001. ( c ) Differential post-translational modifications (PTMs: phosphorylation of S er or T hr residues, or oxidations of M et) observed in GFAP isolated from AMC [ApoE(3,3)], AD [ApoE(3,3)], or AD [ApoE(4,4)] hippocampus, as indicated. Peptide coverage is indicated by yellow highlighting, including PTMs indicated by green highlighting.

Article Snippet: Cells at ~40% confluence were transfected with short interfering RNA (siRNA) constructs targeting GFAP (SAS1_Hs01 00227618), AKT2 (SAS1_Hs01 00035058), ROCK1 (SAS1 Hs 00065571), BARK (SAS1 Hs 00039321), or PKA (SAS1 Hs 00217223), all obtained from Millipore-Sigma (St. Louis, USA).

Techniques: Modification, Isolation, Control, Western Blot, Two Tailed Test, Phospho-proteomics

Journal: Pharmaceutics

Article Title: Glial Fibrillary Acidic Protein: A Biomarker and Drug Target for Alzheimer’s Disease

doi: 10.3390/pharmaceutics14071354

Figure Lengend Snippet: Molecular dynamic analyses of GFAP structure. ( a ) Initial modeled structure of GFAP. Yellow internal cavity is the predicted drug-binding pocket. ( b ) Predicted binding pocket volume for GFAP at 50 ns intervals over a 500 ns span. ( c ) Predicted cavity for ligand binding (yellow) at 200 ns. ( d ) Time course of root mean squared deviation (RMSD) for GFAP structure, comparing 500 ns in silico simulations of AMC (unmodified) GFAP to GFAP with phosphomimetic substitutions to mimic AD(3,3) and AD(4,4). ( e ) Distribution across GFAP (432 residues) of root mean squared fluctuation (RMSF) to illustrate positional variability by residue. ( d , e ) Keys to the right of panels d and e display the color codes for RMSD and RMSF values, respectively.

Article Snippet: Cells at ~40% confluence were transfected with short interfering RNA (siRNA) constructs targeting GFAP (SAS1_Hs01 00227618), AKT2 (SAS1_Hs01 00035058), ROCK1 (SAS1 Hs 00065571), BARK (SAS1 Hs 00039321), or PKA (SAS1 Hs 00217223), all obtained from Millipore-Sigma (St. Louis, USA).

Techniques: Binding Assay, Ligand Binding Assay, In Silico, Residue

Journal: Pharmaceutics

Article Title: Glial Fibrillary Acidic Protein: A Biomarker and Drug Target for Alzheimer’s Disease

doi: 10.3390/pharmaceutics14071354

Figure Lengend Snippet: Effects on aggregation of RNAi knockdowns targeting GFAP or its putative kinases. ( a ) Observed GFAP phosphorylations in human hippocampal aggregates and their putative kinases. ( b ) Fluorescence images of Thioflavin T-stained SH-SY5Y-APP Sw cells, after liposome-mediated transfection with siRNA constructs targeting GFAP or its candidate kinases. ( c ) Histogram showing means ± SEM for Thioflavin T staining of aggregates as in ( b ). ( d ) T98G cells stained with Thioflavin T after transfection by siRNA constructs as shown. ( e ) Histogram of means ± SEM for Thioflavin T staining of aggregates as in panel ( d ). ( c , e ) Significance of differences between treated groups and controls according to heteroscedastic, two-tailed t -tests: *** p ≤ 0.0005; **** p ≤ 0.00005. Experiments were replicated 3–4 times, with consistent results.

Article Snippet: Cells at ~40% confluence were transfected with short interfering RNA (siRNA) constructs targeting GFAP (SAS1_Hs01 00227618), AKT2 (SAS1_Hs01 00035058), ROCK1 (SAS1 Hs 00065571), BARK (SAS1 Hs 00039321), or PKA (SAS1 Hs 00217223), all obtained from Millipore-Sigma (St. Louis, USA).

Techniques: Fluorescence, Staining, Transfection, Construct, Two Tailed Test

Journal: Pharmaceutics

Article Title: Glial Fibrillary Acidic Protein: A Biomarker and Drug Target for Alzheimer’s Disease

doi: 10.3390/pharmaceutics14071354

Figure Lengend Snippet: The drug MSR1 specifically binds GFAP and blocks its role in aggregation . ( a ) Histogram shows stability (Gibbs free energy of binding) of the top three drugs from in silico screening for solvated MM-GBSA docking to GFAP. The ChemBridge structural drug library was screened in three stages of progressively increased stringency, followed by counter-screening to eliminate drugs with tubulin affinity. ( b ) SH-SY5Y-APP Sw cells were stained with Thioflavin T (green fluorescence) and counter-stained with DAPI (not shown). ( c ) Histogram showing 2-fold reduction in amyloid per cell (thioflavin-T fluorescence divided by the count of DAPI + nuclei per field); *** p ≤ 0.0005. ( d ) Total sarkosyl-insoluble aggregate protein, electrophoresed and stained with SYPRO-Ruby after isolation from SY5Y-APP Sw cells. ( e ) The set of proteins totally removed from aggregates by siRNA knockdown of GFAP is nearly identical to the set eliminated by MSR1. The Venn diagram shows proteomic overlap of 251 proteins identified in sarkosyl-insoluble aggregates from untreated SY5Y-APP Sw cells (≥7 hits) but not detected 48 h after transfection with GFAP siRNA or after treatment with 1 µM MSR1. Conversely, four proteins absent from untreated cell aggregates were identified in both treated cell groups. ( f ) Linear regression of log 2 (fold change) of aggregate protein abundance after GFAP siRNA treatment (X-axis) vs. MSR1 treatment (Y-axis). Selected proteins are labeled, including many found by aggregate cross-linking to be immediate neighbors of GFAP (red dots). Dots within the dashed rectangle were shifted <2-fold by either treatment. R = 0.77 for the regression; F -test significance was p < 3 × 10 −280 .

Article Snippet: Cells at ~40% confluence were transfected with short interfering RNA (siRNA) constructs targeting GFAP (SAS1_Hs01 00227618), AKT2 (SAS1_Hs01 00035058), ROCK1 (SAS1 Hs 00065571), BARK (SAS1 Hs 00039321), or PKA (SAS1 Hs 00217223), all obtained from Millipore-Sigma (St. Louis, USA).

Techniques: Binding Assay, In Silico, Drug discovery, Staining, Fluorescence, Isolation, Knockdown, Transfection, Quantitative Proteomics, Labeling