Brain Transcriptional and Epigenetic Associations with Autism

<div><h3>Background</h3><p>Autism is a common neurodevelopmental syndrome. Numerous rare genetic etiologies are reported; most cases are idiopathic.</p> <h3>Methodology/Principal Findings</h3><p>To uncover important gene dysregulation in autism we analyzed carefully selected idiopathic autistic and control cerebellar and BA19 (occipital) brain tissues using high resolution whole genome gene expression and whole genome DNA methylation microarrays. No changes in DNA methylation were identified in autistic brain but gene expression abnormalities in two areas of metabolism were apparent: down-regulation of genes of mitochondrial oxidative phosphorylation and of protein translation. We also found associations between specific behavioral domains of autism and specific brain gene expression modules related to myelin/myelination, inflammation/immune response and purinergic signaling.</p> <h3>Conclusions/Significance</h3><p>This work highlights two largely unrecognized molecular pathophysiological themes in autism and suggests differing molecular bases for autism behavioral endophenotypes.</p> </div

Specific domains of the Autism Diagnostic Interview-Revised (ADI-R) are associated with specific gene modules.

<p>(<b>A</b>) Heatmap of module-variable associations for cerebellar hemisphere samples from weighted genome co-expression network analysis, including domain scores for the ADI-R. Cells are color-coded by correlation coefficient and * is placed in cells having associations with p-values ≤0.01. Autism, dichotomous variable autism vs control; PMI, postmortem interval; A, ADI-R Social interaction impairments domain; B, ADI-R Communication and language impairments domain; C, ADI-R Repetitive and stereotyped behaviors domain. (<b>B</b>) Gene ontology enrichment annotation clusters for the three significant gene modules associated with specific ADI-R domain scores. P, p-value; r, Pearson correlation coefficient.</p

No differential methylation of genomic DNA was identified between control and autism cerebellar cortex or BA19 cortex.

<p>(<b>A</b>) Unsupervised correspondence analysis of genome-wide CpG methylation in control and autism cerebellar cortex and BA19 cerebral cortex demonstrates that brain region and age are associated with the largest two sources of variability, respectively, in the data. The first two principal components are listed on the x- and y-axis respectively, with percent of variance explained in parentheses. Bubbles correspond to individual samples with area proportional to age of subject, bubble outline to region, and color to phenotype. (<b>B</b>) Percent of genome-wide methylated probes in cerebellar cortex or BA19 cortex in control and autism samples. Error bars represent standard deviations. There was no significant difference in global methylation between autism vs control cerebellum or BA19 cortex. There was a significant difference between brain regions, as expected (alpha = 0.05; Wilcoxon rank-sum test). (<b>C</b>) Pyrosequencing of bisulfite-treated DNA. Percent methylation at individual CpG dinucleotides is reported in cerebellar cortex samples (n ≥7 per group) for <i>a priori</i> candidate genes suspected to exhibit differential methylation. X-axis represents locus relative to transcription start site. Error bars represent standard deviations.</p

Autistic brain shows transcriptional heterogeneity and differential expression of genes of mitochondrial oxidative phosphorylation and protein translation.

<p>(<b>A</b>) Unsupervised correspondence analysis of whole-genome transcriptome expression data demonstrates that brain region is associated with the greatest source of variability in the data. The second principal component is associated with variability mostly among autistic samples. The first two principal components are listed on the x- and y-axis respectively, with percent of variance explained in parentheses. Bubbles represent individual samples, with area proportional to age, outline to brain region, and color to phenotype. (<b>B</b>) Heatmap of the top 50 differentially expressed probes between autistic and control brain, accounting for brain region, separates most autistic brains from controls (FDR ≤5%). Rows correspond to probes and columns to samples. The dendrogram represents sample similarity on the basis of the top 50 differentially expressed probes. Probes with log₂-fold change >0.7 are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044736#pone.0044736.s007" target="_blank">table S3</a>. (<b>C</b>) The top 300 differentially expressed probes are enriched for gene ontology annotation clusters corresponding to mitochondrial oxidative phosphorylation and protein translation. ADI-R, Autism Diagnostic Interview-Revised; BA19, Brodmann area 19; CER, cerebellum; PMI, postmortem interval.</p

Circulating S100B is taken up by peripheral immune cells.

<p>AlexaFlour 488-tagged S100B protein accumulated in Langerhans cells of the skin (<b>A1, 2</b>), thymic dendritic cells (<b>B1, 2</b>), and in dendritic cells in lymph nodes (a para-aortic lymph node is shown in <b>C1, 2</b>). Note the lack of uptake by non-immune surrounding tissue. Also note the typical appearance of dendritic cells in nodal tissues. Injection consisted of AlexaFlour 488-tagged S100B at a concentration of 0.12 ng/mL introduced via tail vein and allowed to circulate 2–3 hrs. prior to tissue harvesting; DAPI (<i>blue</i>) was added as a nuclear stain. Note the different magnifications among the panels, with scale bar  = 200 µm in B1, 100 µm in A2 and 50 µm in all other panels. Rats were injected with a combination of AlexaFlour 594-tagged S100A1 (<i>red</i>) and AlexaFlour 488-tagged S100B (<i>green</i>). Skin (<b>D</b>) and splenic (<b>E</b>) tissue revealed that Langerhans cells (<i>arrows</i> in D) and splenic cells within the germinal center (<i>GC</i>), mantel zone (<i>MnZ</i>) and marginal zone (<i>MaZ</i>) demonstrate uptake of both S100A1 and S100B; however, the extent and intensity of S100B uptake was much greater as also evident in the merged figures (D3, E3). <b>F</b> shows the preferential segregation of S100B at the membrane of dendritic cells in skin (<b>F1</b> and <b>F2</b>) and thymus (<b>F3</b>); greater detail of a Langerhans cell from D2 (box) is shown in F1 in order to highlight membrane staining. Injection consisted of AlexaFlour 488-tagged S100B at a concentration of 0.10–0.12 ng/mL introduced via tail vein and allowed to circulate 2–3 hrs. prior to tissue harvesting; DAPI (blue) was added as a nuclear stain. <i>GC  =  germinal center, MnZ  =  mantle zone, MaZ  =  marginal zone</i>.</p

Summary of experimental results testing for the presence of S100B at the protein, mRNA levels or after injection of labeled S100B.

<p>+ indicates presence, ++ presence at levels significantly greater than in tissues labeled with +. ND =  Not determined, - indicates absence of measurable signal.</p

Circulating S100B fails to invade barrier organs; however S100B gene transcription and protein synthesis occurs in both brain and testis.

<p>Injection (exogenous) strategies demonstrate privilege of barrier organs to transendothelial diffusion of S100B while immunodetection of endogenous S100B demonstrates brain- and testis specific S100B protein by astrocytes and Sertoli cells. mRNA detection in the same barrier organs confirms S100B expression. <b>(A1)</b> shows the lack of fluorescent signal (Alexa Fluor (AF) 488, in <i>green</i>) in brain regions where endogenous S100B was readily detected <b>(A2)</b>. The section used for immunohistochemistry contained the CA2 sector of the hippocampus. Note that, as expected, S100B was present in glial cells but not in neurons; neuronal cell bodies in hippocampal CA2 region are seen as unstained ghosts. Testicular tissue yielded similar results albeit in testis the barrier is established by Sertoli and not endothelial cells <b>(B1 and B2)</b>. Note that intravascular S100B was restricted to the stroma of the seminiferous tubules in the testis where endogenous S100B was not present. S100B+ cells in the stroma (<i>arrowheads</i>) are CD4+ dendritic cells (<i>insert</i> in <b>B2</b>). DAPI (<i>blue</i>) was added as a nuclear stain <b>(B1)</b>. AlexaFlour 488-tagged S100B was injected to mimic blood-brain barrier disruption (e.g., ref. 13). The labeled protein was allowed to circulate 3 hrs. prior to tissue harvesting. The mRNA bands shown reflect levels of expression by brain and testis. <i>Sem. T.  =  seminiferous tubule</i>.</p

Comparison of S100B and S100A1 sequences to underscore similarities (normal character) and differences (in bold).

<p>There is 94% (4 aa are different) similarity between bovine S100B used for these experiments and rat S100B. Thus, the uptake by immune cells is not due to foreign antigen sequence. Same considerations can be made for human S100A1 used as a tracer, since the human protein used shared 96% identity with rat protein.</p

Schematic representation of the events that follow BBBD.

<p>See text for details.</p

Splenic S100B-positive cells change in both number and morphology following pilocarpine-induced seizure.

<p>S100B positive cells can be viewed within splenic follicles both in an unmanipulated animal (<b>A</b>), as well as following simulation of BBBD via IV injection of Alexa Fluor 488-labeled S100B (<b>B</b>) and BBBD from pilocarpine administration (<b>C</b>). The pattern of staining is preserved in all 3 conditions, however, staining is clearly augmented in BBBD simulation and to a much greater degree in actual induced BBBD. S100B-labeled lymphocytes and dendritic cells can be observed in all regions of the splenic follicle. Note that the morphology of labeled cells also appears to change with induction of BBBD in (<b>C</b>), where cells appeared to have a more dendritic and interconnected staining pattern than in other conditions. The identity of these cells was verified via the immune cell markers, CD4 and CD8 immunostaining (<b>D1–D2</b>); S100B+ and CD4+/CD8+ double positive cells are again found throughout the follicle, with emphasized staining in the marginal zone vs other areas. Injection consisted of AlexaFlour 488-tagged S100B at a concentration of 0.10–0.12 ng/mL introduced via tail vein and allowed to circulate 2–3 hrs prior to tissue harvesting. For induced BBBD, the animal was treated with pilocarpine and spleen was removed prior to onset of status epilepticus. Sections in <b>A</b> and <b>C</b> were treated with mouse anti-S100B antibody (Ab) and donkey anti-mouse 2° Ab conjugated to FITC (Jackson). In <b>D1–D2</b>, sections were treated with rabbit anti-S100B Ab and donkey rabbit 2° Ab conjugated to Texas Red (Jackson) and rat anti-CD4 or CD8 antibody and mouse anti-rat 2° Ab conjugated to FITC (Jackson). DAPI was added as a nuclear stain. In <b>E–F</b>, sections shows rats injected with S100B tracer in control (<b>E1–E3</b>) and pilocarpine administrated rats (<b>F1–F3</b>). Co-localization of S100B+CD86 (F1) and high individual staining of CD86+ (<b>F2</b>) in pilocarpine compared to controls <b>E1</b> and <b>E2</b> indicates that activated immune cells capture S100B. The dendritic cell nature of these cells was further demonstrated by their CD86+ staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101477#pone.0101477.s003" target="_blank">Figure S3</a>). Scale bar in A = 100 µm for all images. <i>GC  =  germinal center, MnZ  =  mantle zone, MaZ  =  marginal zone, WP  =  white pulp, RP  =  red pulp</i>.</p