Altered response to social defeat in F2 MSUS males.

<p><b>a,</b> Sucrose consumption in F2 MSUS and control males subjected to SD and non-SD. <b>b</b>, Images of the SD test arena showing representative tracking of position and movements in defeated (SD, right) and non-defeated (non-SD, left) animals. Interaction and corner zones are outlined with blue boxes. Trace from a control male, black. Trace from a MSUS male, red. <b>c,</b> Time in the interaction zone, and (<b>d</b>) time in corners in F2 MSUS and control males subjected to SD and non-SD. <b>e,</b> Number of daily attacks over the 2-weeks of SD in F2 control and MSUS mice. # p<0.05, SD versus non-defeated (Non-SD) F2 MSUS or control males.* p<0.05, F2 MSUS versus F2 control within SD treatment, # p<0.05, ## p<0.001 SD versus non-SD F2 MSUS or F2 control animals.</p

5HT1AR binding and serotonin level in the brain of MSUS offspring.

<p><b>a–e,</b> 5HT1AR binding in (<b>a</b>) PAG, (<b>b</b>) DR, (<b>c</b>) MR, (<b>d</b>) hippocampus, (<b>e</b>) thalamus, (<b>f</b>) hypothalamus, (<b>g</b>) frontal cortex, and (<b>h</b>) striatum (lateral PAG: (t(8) = 2.79, p<0.05; DR: t(8) = 2.72, p<0.05; CA1: t(8) = 29.34, p<0.05; DG: t(8) = 2.26, p = 0.05); posterior thalamus: t(8) = 2.36, p<0.05; medial thalamus: t(8) = 3.67, p<0.01; anterior thalamus: t(8) = 3.81, p<0.01). Serotonin levels in (<b>i</b>) frontal cortex (t(16) = −2.54, p<0.05) and (<b>j</b>) dorsal hippocampus (t(16) = .43, ns) in MSUS and control males. (<b>k</b>) Effect of 8-OH-DPAT treatment on social exploration in MSUS and control males (F(1, 22) = 11.47, p<0.01). Abbreviations: Anterior hypothalamic area, AHC; anterior thalamus, ant. thal.; caudate putamen, CPu; cingulate cortex, Cg1/2; dentate gyrus, DG; dorsal raphe, DR; motor cortex, M1/M2; medial thalamus, med. thal.; median raphe, MR; nucleus accumbens, NAcc; paraventricular nucleus of the hypothalamus, PVN. periacqueductal grey, PAG; posterior thalamus, posterior thal. +, p = 0.05, *, p<0.05, **, p<0.01 as indicated by unpaired t-test, or Fisher's PLSD post-hoc when appropriate.</p

Abnormal sociability in F2 and F3 MSUS males.

<p><b>a–c</b> Level of investigation of a same-sex unfamiliar conspecific in (<b>a</b>) F1 MSUS and control males, and (<b>b),</b> F2 and (<b>c</b>) F3 MSUS and control males and females (F1: ns; F2 male: t(13) = 2.38, p<0.05; F2 female: ns; F3 male: t(21) = 3.73, p<0.01; F3 female: ns).</p

Abnormal social memory in F1, F2 and F3 MSUS mice.

<p><b>a-c</b>, Social recognition discrimination ratio in (<b>a</b>) F1, (<b>b</b>) F2, and (<b>c</b>) F3 control and MSUS mice (F1: t(14) = 2.224, p<0.05; F2: t(14) = 3.43, p<0.01; F3: t(13) = 3.52, p<0.01). <b>d–f</b>, Olfactory memory discrimination ratio in (<b>d</b>) F1 MSUS and control males, and (<b>e</b>) F2, and (<b>f</b>) F3 MSUS and control females. One-sample t-test demonstrates significant decrease from expected value (0.5) in F1, F2, and F3 MSUS and control mice in the olfactory recognition test. *, p<0.05, **, p<0.01 as indicated by unpaired t-test, or Fisher's PLSD post-hoc where appropriate.</p

Blockade of Ca²⁺ rise and delayed cell death induced by OGD by ifenprodil and PP1α in CA1 pyramidal cells.

<p>(<b>a</b>) OGD-induced increase in ΔF/F (% relative to basal level) in control and NVP-AAM077-treated slices (control, n = 7; NVP-AAM077, n = 7). This increase is prevented by ifenprodil perfusion (n = 8) or by PP1α expression (n = 7). Schematic representation of a hippocampal slice showing the patch-clamp electrode filled with OGB-1 (left inset). Image of a CA1 pyramidal neuron loaded with OGB-1 with outlined region of interest (cell soma) used to calculate fluorescence (middle inset). Relative fluorescence (ΔF/F) traces of individually recorded CA1 pyramidal neurons in control slices, PP1α expressing slices, and slices treated with NVP-AAM077 or ifenprodil subjected to OGD (right inset). (<b>b</b>) Quantitative histogram illustrating the area under the curve used as a measure of intracellular Ca²⁺ overload during 4 min OGD, given by the calculation of ΔF/F integral normalized to control. *p<0.05. (<b>c</b>) Propidium iodide labeling of hippocampal slices showing massive cell death in the CA1 pyramidal cell layer (red staining) 48 h after OGD (control OGD, n = 7). Ifenprodil treatment (n = 6) and PP1α expression (n = 5) significantly reduced cell death as shown by an almost absent PI labeling after OGD. No cell death was observed in slices maintained in ACSF (control ACSF, n = 8; ifenprodil ACSF, n = 8; PP1α ACSF, n = 7). Scale bar: 400 µm. (<b>d</b>) Quantitative histogram with normalized labeling intensity. ***p<0.001.</p

PP1α reverses CaMKIIα Thr286 phosphorylation and mediates the dephosphorylation of NR2B Ser1303 upon OGD.

<p>(<b>a</b>) Representative Western blots and corresponding quantitative analysis of CaMKIIα Thr286 phosphorylation. CaMKIIα phosphorylation significantly increases 1 min after OGD (control 1 min post-OGD, n = 8) with no significant change at 16 min (control 16 min post-OGD, n = 6) in control slices. PP1α slices do not display any change in CaMKIIα phosphorylation either 1 min (PP1α 1 min post-OGD, n = 6) or 16 min post-OGD (PP1α 16 min post-OGD, n = 7) compared to control. Phospho-protein levels were normalized to non-phosphorylated protein levels and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05. (<b>b</b>) Representative Western blots and corresponding quantitative analysis of NR2B Ser1303 phosphorylation. Increased level of phospho-NR2B in control slices 1 min after OGD (control 1 min post-OGD, n = 9), but not 16 min after OGD (control 16 min post-OGD, n = 8). PP1α expression or KN-93 treatment blocks this increase (PP1α 1 min post-OGD, n = 6; KN-93 1 min post-OGD, n = 6), but has no effect 16 min post-OGD (PP1α 16 min post-OGD, n = 8; KN-93 16 min post-OGD, n = 5). Phospho-protein levels were normalized to non-phosphorylated protein levels, and β-actin was used as a loading control. Quantitative data for each condition were normalized to levels of non-OGD condition (control non-OGD, n = 14) from the same blot and exposure. *p<0.05.</p

NR2B Ser1303 phosphorylation is necessary and sufficient to drive Ca²⁺ overload.

<p>(<b>a</b>) Expression of wild-type NR2B (NR2B S1303 WT, n = 6) increased OGD-induced ΔF/F (% relative to basal level), while expression of NR2B mutated on Ser1303 (NR2B S1303A, n = 5) was not changed compared to control slices (control, n = 6). C-terminus amino-acid sequence of NR2B S1303 WT and NR2B S1303A mutated version with substitution of serine with alanine on residue 1303 in shaded grey (left inset). Relative fluorescence (ΔF/F) traces of individually recorded CA1 pyramidal neurons in control, NR2B S1303A, and NR2B S1303 WT slices subjected to OGD (middle inset). Image of a CA1 pyramidal neuron expressing the calcium sensitive dye TN-XXL with outlined region of interest (cell soma) used to assess fluorescence and [Ca²⁺]_i (right inset). (<b>b</b>) Quantitative histogram illustrating the area under the curve used as a measure of total intracellular Ca²⁺ load, given by the calculation of ΔF/F integral normalized to control. *p<0.05. (<b>c</b>) Representative Western blot showing co-immunoprecipitation of NR2B and NR1 with PP1α using a PP1α antibody in mouse hippocampus lysates. No protein interaction was observed with non-specific IgG. PP1α and IgG immunoprecipitation was performed using a comparable amount of antibodies, and samples were loaded on the same blot (same exposure). (<b>d</b>) PP1 directly dephosphorylates Ser1303 <i>in vitro</i>. Immunoblot analyses showing the time course of PP1-mediated dephosphorylation on Ser1303 from a GST-fused NR2B C-terminal peptide. GST-NR2B (aa 1221–1482) was incubated with active PP1 following Ser1303 phosphorylation by CaMKII. NR2B was used as loading control.</p

PP1α expression and CaMIIα inhibition normalize NR2B-containing NMDAR-mediated currents after excitotoxicity.

<p>(<b>a</b>) Significant enhancement of NMDAR currents following 4 min OGD in control slices (control, n = 6). Expression of PP1α (n = 5), ifenprodil treatment (n = 8), and KN-93 treatment (n = 6) led to a full recovery of NMDAR currents 6 min after the end of OGD. NVP-AAM077 (n = 5) has no effect on NMDAR currents that remain potentiated throughout recording. Top left, Schematic representation of an organotypic hippocampal slice showing the positioning of the recording patch-clamp electrode and the puffing pipette. Top right, Individual responses from single CA1 pyramidal neurons before (1), during (2) and 10 min after (3) OGD. (<b>b</b>) Time course of OGD mean effect on NMDAR currents. ⁺⁺⁺p<0.001, ***p<0.001.</p

Summary of overrepresented motifs at PTM sites.

<p>A total of ten motifs were detected, flanking either acetylation, methylation or phosphorylation sites. For each motif, bibliographic references for the same or similar motifs are listed, as well as whether it is known to function as a binding motif. Unknown motifs were novel at the time of writing. Many of the identified motifs are novel and distinct from human motifs in the human protein reference database (HPRD). In support of our dataset many of the sites also matched known motifs for the enzymes that catalyse these PTMs, and/or known binding motifs that require modified residues.</p

A,B) Mass spectra of identified endogenous peptides with lysine propionylation and butyrylation and their synthetic counterparts.

<p>Major peaks are labelled in the mass spectra and the fragment ions indicated in the peptide sequence using standard Mascot nomenclature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036980#pone.0036980-Roepstorff1" target="_blank">[56]</a>. <b>A</b>) A novel site of lysine butyrylation on residue K95 of H2A. <b>B</b>) A novel site of lysine propionylation on residue K95 of H2A. <b>C</b>) Highly modified peptides that were detected using ETD-MS/MS included the N-terminal peptide from H4 ac-SGRGKacGGKacGLGKacGGAKacRHRKme2VLR, which contains 5 sites of acetylation and 1 site of dimethylation <b>E</b>) A novel site of lysine crotonylation on residue K108 of H2B.</p