Prenatal age and early infancy are associated with transition of H3K4me3 landscapes in prefrontal cortex.

<p>A. Correlation of H3K4me3 occupancy in RefSeq promoters between all pairs of samples. Samples are sorted by age. The ages of the samples are listed on the x-axis in years (except where gw = week of gestation), with males in blue and females in pink. Note that the six youngest brains (prenatal and infant brains <1 year of age) are less strongly correlated with older brains (Pearson correlation coefficient R = 0.91–0.97), while brains ranging from 1.3 to 81 years of age mostly show very high (R = 0.94–0.99) between-sample correlations. B. Principal Component Analysis (PCA) analysis of differential H3K4me3 peaks for discovering similarity between samples. Prenatal samples are in green, infant samples in blue, and the remaining samples in red. Ages for prenatal samples are in gw and for all other samples in years. Each sample was compared with every other sample to calculate the number of peaks that are different, and the resulting 31 by 31 matrix was used as the input for the PCA analysis. The first principal component clearly separates prenatal samples from the other samples and the second component shows lower values for both prenatal and infant samples than for older samples. C. The same as A but for the five NeuN– samples included in this study.</p

Average RNA expression levels.

<p>Average RNA expression levels for the genes near the up H3K4me3 peaks (A) and for the genes near the down H3K4me3 peaks (B). Expression data were taken from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003433#pgen.1003433-Colantuoni1" target="_blank">[5]</a>. Each dot indicates one sample, for which the expression levels of the genes near the up (or down) H3K4me3 peaks were averaged. Color scheme is the same as for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003433#pgen-1003433-g002" target="_blank">Figure 2</a>.</p

Averaged age profiles.

<p>Average age profiles for H3K4me3 level for the up (A) and down (B) peaks. Green corresponds to prenatal samples, blue for infant samples, and red for the remaining samples.</p

Samples used in this study and sequencing statistics.

<p>Brain Banks: HBTRC - Harvard Brain Tissue Resource Center (Dr. Francine Benes); UM-BTB - University of Maryland Brain and Tissue Bank for Developmental Disorders (Dr. Ron Zielke); MPRC - Maryland Psychiatric Research Center (Dr. Rosalinda Roberts and Dr. Andree Lessard); UCI/UCD - University of California Irvine/Davis (Dr. Ted Jones and William E. Bunney Jr). AGP = Shulha et al. Archives of General Psychiatry 69:314–324 (2012); PNAS = Cheung et al., Proceedings National Acad Sci USA 107:8824–8829 (2010). gw = gestational week.</p

Behavioral summary in group housed versus single housed (3 months of social isolation) <i>L3mbtl1</i> null mutant and wildtype mice.

<p>The figure summarizes the following tests in group and single housed mice, as indicated (<b>A</b>) General activity/locomotion. (<b>B</b>) Depression/ forced swim. (<b>C</b>) Anxiety/open field test. (N = 8 group housed <i>L3mbtl1-/-</i>, N = 10 group housed <i>L3mbtl1+/+</i>, N = 10 single housed <i>L3mbtl1-/-</i>, N = 10 single housed <i>L3mbtl1+/+</i>). (<b>D</b>) Cognition/radial arm maze (N = 8 group housed <i>L3mbtl1-/-</i>, N = 11 group housed <i>L3mbtl1+/+</i>, N = 11 single housed <i>L3mbtl1-/-</i>, N = 10 single housed <i>L3mbtl1+/+</i>). Behavioral assays were conducted in the order from the least to the most stressful, and thus (i) activity/locomotion, followed by (ii) radial arm maze, followed by (iii) anxiety/open field, and (iv) depression / forced swim as the final test. All data shown as mean ± S.E.M., * P <0.05; independent-samples t-test after two-way ANOVA: for (A) locomotion: F_(1,34) = 3.807, P = 0.05, Bonferroni post test P <0.05 for single housed <i>L3mbtl1-/-</i> vs. <i>L3mbtl1+/</i> +); for (B) forced swim: F_(1,34) = 10.93, P = 0.022, Bonferroni post test P <0.01 for single vs. group housed; for (C), (D) no significance. All data shown in Fig 2 are from batches of mice different from those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121252#pone.0121252.g001" target="_blank">Fig 1</a>.</p

Locomotor effects of D1 agonist SKF81297.

<p>The figure summarizes SKF81297-induced locomotion at (<b>A</b>) 0.5mg/kg and (<b>B</b>) 1mg/kg i.p., administered 60 min after placing mice into the open field apparatus and 30 min after a saline control injection. Assays were conducted in group and single housed animals, as indicated (N = 5 group housed <i>L3mbtl1-/-</i>, N = 9 group housed <i>L3mbtl1+/+)</i> and single-housed animals N = 4 single housed <i>L3mbtl1-/-</i>, N = 5 single housed <i>L3mbtl1+/+</i>). All data are shown as mean ± S.E.M. (B) repeated measures ANOVA F_(1,14) = 3.203 with time point T _{60 min} Bonferroni post test P <0.05, other time timepoints *, P <0.05 by independent sample t-test. All data in Fig 3 are from experimentally naive mice.</p

Behavioral summary of <i>L3mbtl1</i> null mutant and control mice at baseline.

<p>(<b>A</b>) General activity, (a) body weight and (b) locomotion. (<b>B</b>) Depression, (a) forced swim and (b) tail suspension (N = 25-28/genotype and test). (<b>C</b>) Anxiety, (a) elevated plus maze, (b) light-dark box, (c) open-field test (N = 17–21 genotype/test). (<b>D</b>) cognition, assessed by contextual fear conditioning (N = 12 <i>L3mbtl1-/-</i> and N = 17 <i>L3mbtl1+/+</i>). Animals used for general activity and depression assays were from a different batch than the animals used for anxiety assays. A third batch of animals was used for the fear conditioning assays. All animals are kept under baseline conditions (group housing). All data are shown as mean ± S.E.M., * P <0.05; independent-samples t test and, for fear conditioning assay, repeated measures ANOVA.</p

TDP-43 aggregation in NSC34 cells.

<p>Values were averages of 4 or more experiments. Distribution of soluble TDP-43 fusion proteins was determined in cells that did not contain aggregates. All distribution was determined based on GFP signal except construct 1, which was determined by staining for TDP-43 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015878#pone-0015878-g003" target="_blank">Fig. 3</a> and the text). Nuc means nucleus; Cyto means cytoplasm; % transfected means the percentage of the transfected cells detected by visualizing GFP; % nucleus means percentage of aggregate-positive cells where the aggregates were located in the nucleus; % cytoplasm means percent of aggregate-positive cells where the aggregates were located in the cytoplasm; SE, standard error.</p

Coaggregation of wild type TDP-43 with its fragments.

<p>(<b>A</b>) The full-length TDP-43-tdTomato construct expressed the fusion protein. Proteins were extracted 48 hrs after the transfection of NSC-34 cells. Fifteen µg of the protein was resolved by SDS-PAGE and blotted. TDP-43-tdTomato was detected using a TDP43 antibody or an HA-tag antibody. (<b>B</b>) The EGFP-tagged, aggregation-prone TDP-43 fragments (<b>2</b>, <b>3</b>, <b>4</b>, <b>6</b>, <b>7</b> and <b>8</b>) were cotransfected with the full-length TDP-43-tdTomato construct. The full-length TDP-43 was colocalized with all of these mutants in aggregates. Two mutants (<b>5</b> and <b>11</b>) that do not form aggregates were also cotransfected with the full-length TDP-43. In these cells, the mutants (green) were diffusely distributed throughout the cells and the full-length wild type (red) was predominantly in the nucleus.</p

Expression of TDP-43 fragments inhibits neurite growth in primary rat forebrain neurons.

<p>(<b>A</b>) The rat forebrain neural precursors were transfected with EGFP and each of the TDP-43 constructs as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015878#pone-0015878-g001" target="_blank">Fig. 1</a>. The cells were also transfected with tdTomato for visualizing neurites. Twenty-four hours after the transfection, the cells were differentiated to neurons by withdrawing basic fibroblast growth factor 2 from the medium and cultured for another 72 hours before being fixed and photographed. (<b>B</b>) Quantification of neurite-bearing cells as shown in (A). Values are averages of four independent experiments. Error bars represent standard error.</p