Histone H3 Ser57 and Thr58 phosphorylation in the brain of 5XFAD mice

Abstract

Alzheimer's disease has been shown to have a global reduction in gene expression, called an epigenetic blockade, which may be regulated by histone post-translational modifications. Histone H3 has been shown to be highly regulated by phosphorylation. We, therefore, chose H3 for investigation of phosphorylation of the core sites serine-57 (S57) and threonine-58 (T58). Hemispheres of brains from a mouse model of rapid amyloid deposition (5XFAD) were used for measurement of S57 and T58 phosphorylation. Multiple reaction monitoring (MRM) was used to measure the level of phosphorylation, which was normalized to a non-modified "housekeeping" peptide of H3. S57 phosphorylation was decreased by 40%, T58 phosphorylation was decreased by 45%, and doubly phosphorylated S57pT58p was decreased by 30% in 5XFAD brain in comparison to C57BL/6J age- and sex-matched wild type controls. Amyloid-β (Aβ) and amyloid precursor protein were also measured to confirm that 5XFAD mice produced high levels of Aβ. Decreased phosphorylation of these sites in close proximity to DNA may lead to stabilization of DNA-histone interactions and a condensed chromatin state, consistent with the epigenetic blockade associated with AD. Our findings of H3 sites S57 and T58 exhibiting lower levels of phosphorylation in 5XFAD model compared to wild type control implicate these sites in the epigenetic blockade in neurodegeneration pathology.

Keywords: 5XFAD; APP, amyloid precursor protein; Alzheimer’s disease; Aβ, amyloid-β; Histone; MRM, multiple reaction monitoring; Multiple reaction monitoring; PTM, post-translational modification; Phosphorylation; S57, serine-57; T58, threonine-58.

Graphical abstract

Fig. 1

Peptide identification by MRM and selection of transitions for quantification. (A, D and F) Numerous transitions were monitored to determine retention time and confirm identity of each peptide. (B, E and H) Spectra of ions used to confirm identity and site of phosphorylation. (C, F and I) A single transition was selected from each peptide for quantification. ^*Transition 425.72 → 409.21 had high signal intensity and was removed from chromatogram (G) and reduced in spectrum (H) to better illustrate other monitored transitions.

Fig. 2

Quantification of APP and Aβ in 5XFAD and control. (A) Amyloid precursor protein, APP, (gray) is a large transmembrane protein that contains the amino acid sequence for Aβ (red). Aβ is released as a fragment peptide during processing of APP by β- and γ-secretases. Therefore, measurements for Aβ include the peptide present in unprocessed APP and free Aβ. Total APP peptides AVIQHFQEK and VESLEQEAANER and Aβ peptide LVFFAEDVGSNK were measured in whole hemisphere homogenates from (B) wild type mice (n = 3) and (C) 5XFAD mice (n = 3). Measurements represent mean ± standard deviation of biological replicates (n = 3) using three transitions for AVIQHFQEK, four transitions for VESLEQEAANER, and four transitions for LVFFAEDVGSNK for absolute quantification using QconCAT as internal standard. ^***p < 0.001.

Fig. 3

Effect of 5XFAD model on S57 and T58 phosphorylation. Phosphorylation was measured in whole hemisphere homogenates from mixed wild type mice (n = 3) and 5XFAD mice (n = 3). Measurements represent mean ± standard deviation of biological and analytical replicates (n = 9) normalized to respective phosphorylation site in mixed wild type control. ^**p < 0.01; ^***p < 0.001.

Fig. 4

Proximity of H3 S57 and T58 to DNA. (A) S57 is less than 1.1 nm from DNA and (B) T58 is less than 0.9 nm from DNA. DNA is shown in gray, H3 in blue, and S57 and T58 sites in red. Distance measurements were performed in PyMOL Molecular Graphics System Version 1.7.2 using the crystal structure of the human nucleosome (PDB ID: 2CV5) . (C) Surface view of phosphorylated S57 and T58 on histone core (green) in close proximity to the interface of the positively charged phosphate backbone (red) of DNA (gray). All modeling was performed in PyMOL and insertion of phosphate groups was performed using a PyMOL plugin called PyTMs .

Fig. 5

Location of H3 residues S57 and T58 within nucleosome. (A) Core histones (green), with flexible histone tails truncated in structure, is shown wrapped in DNA (gray). The S57T58 regions (spheres with oxygen in red and nitrogen in blue) are in the turn of a helix-turn-helix motif that brings the residues in close proximity to DNA and are easily accessible to kinases and phosphatases. (B) Phosphorylated S57 and T58 create a repulsive charge–charge interaction with negatively charged DNA, weakening the affinity of the nucleosome. (C) Dephosphorylation of S57 and T58 greatly reduces negative charge on the histone interface, strengthening the affinity of the DNA–histone interaction. Modeling was performed using PyMOL Molecular Graphics System Version 1.7.2 and the crystal structure of the human nucleosome (PDB: 2CV5) . Insertion of phosphate groups was performed using the PyTMs plugin .