Examination of Target Genes in Late-Onset AD Brains.

<p><i>A–B</i>. qPCR of NLRP2 (<i>A</i>) and <i>ASB9</i> (<i>B</i>) from mRNA from Brodmann's area (BA38) from control and AD brains. Black bars (1–5) are controls and red bars represent AD patients (6–16), which are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s005" target="_blank">Fig S5</a>. qPCR data was normalized internally to <i>GAPDH</i> expression and also to the average of 5 control lines. Statistical significance was determined by Student's t-Test and error bars reflect SEM. For control vs. AD, n = 5 for control, n = 11 for AD, p = 0.005. <i>C</i>. List of <i>PSEN1</i> NPC target genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s008" target="_blank">Table S2</a>) that have differential expression in independent microarray data of laser captured microdissected (LCM) cortical neurons from one of three brain areas (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s005" target="_blank">Fig S5</a>). All comparisons are either non-demented AD pathology (NDAD) or AD versus control samples. HIP refers to hippocampus, EC for entorhinal cortex, and MTG for middle temporal gyrus. Fold change and significance (FDR: false discover rate) reflect values for LCM neuron arrays. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s004" target="_blank">Figure S4</a>.</p

Characterization and Molecular Profiling of <i>PSEN1</i> Familial Alzheimer's Disease iPSC-Derived Neural Progenitors

<div><p>Presenilin 1 (<i>PSEN1</i>) encodes the catalytic subunit of γ-secretase, and <i>PSEN1</i> mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of <i>PSEN1</i>, we characterized neural progenitor cells (NPCs) derived from FAD mutant <i>PSEN1</i> subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying <i>PSEN1</i> mutations. <i>PSEN1</i> mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in <i>PSEN1</i> NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in <i>PSEN1</i> NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our <i>PSEN1</i> iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.</p></div

Validation of Target Genes in <i>PSEN1</i> NPCs.

<p>All qPCR data was normalized internally to <i>GAPDH</i> expression and also to cell line 7889O. Statistical significance was determined by Student's t-Test and error bars reflect SEM. <i>A</i>–<i>B</i>. <i>NLRP2</i> mRNA expression was assessed in undifferentiated iPSCs (<i>control vs. PSEN1</i>, n = 4 for each genotype, <i>p = 0.016</i>) and NPCs (<i>control vs.PSEN1, n = 4 for each genotype, p = 0.03</i>). <i>C</i>. Western blot analysis of NLRP2 protein expression in NPCs. α-Tubulin was used as a loading control <i>D</i>. Representative experiment showing <i>ASB9</i> mRNA expression in NPCs. For control vs. PSEN1, n = 4 for each genotype, p = 0.03. <i>E</i>. Representative experiment showing <i>NDP</i> mRNA expression in NPCs. For control vs. PSEN1, n = 4 for each genotype, p = 0.005. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s008" target="_blank">Table S2</a>.</p

Aβ42/Aβ40 Ratio is Elevated in <i>PSEN1</i> Cells.

<p>All assays detected Aβ1-40 and 1-42 using ELISA (Wako) on conditioned media from the cell type indicated. Ratios were normalized against the first control line listed on each panel. Statistical significance was determined via Student's t-Test, error bars reflect SEM. Each n equals an individual cell line (averaged biological triplicates) in 1 independent experiment. <i>A</i>. Aβ42/Aβ40 ratio is increased in day 14 differentiated NPCs/early neurons. Control and <i>PSEN1</i> NPCs were generated from the core set of iPSC lines, and one of three independent experiments is shown. For control compared to PSEN1 NPCs (n = 4 for each genotype), p = 0.003. <i>B–C.</i> Aggregate data is shown from 3 independent fibroblast and 3 independent NPCs/early neuron experiments. N = 7 for each fibroblast genotype data point, and n = 12 for each NPC/early neuron genotype data point. <i>B</i>. Aβ 42/40 ratios are shown for both control and PSEN1 fibroblasts and NPCs. For control fibroblasts vs. <i>PSEN</i>1 fibroblast, p = 0.001; for control NPCs vs. <i>PSEN1</i> NPCs, p = 0.000005; for <i>PSEN1</i> fibroblasts vs. <i>PSEN1</i> NPCs, p = 0.036. <i>C</i>. Total Aβlevels (Aβ40 + Aβ42) are statistically similar between control and <i>PSEN1</i> fibroblasts and NPCs/early neurons. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s003" target="_blank">Figure S3</a>.</p

Gene Expression Profiling of Control vs. PS1 NPCs/Early Neurons.

<p>All 8 core iPSC lines were differentiated for 14 days in triplicate wells, lysed for RNA, amplified to generate cRNA, and ran on the Illumina HumanHT-12-14 BeadChip platform. <i>A</i>. Clustering of 8 core lines by correlation. UR stands for unrelated control. <i>B</i>. Scatter plot (log scale) of the correlation of gene expression between 4 control lines and 4 PSEN1 lines. The red lines indicate a 3-fold expression difference. <i>C</i>. Chart indicating the number of upregulated (shown in blue) and downregulated (shown in yellow) genes for each threshold of analysis. “DiffScore” refers to genes with a Diff Score of >13 (upregulated) or <13 (downregulated), which indicate a change in expression with a pValue of p≤0.05, without regard to the relative fold change. Criteria for fold change categories include the listed fold change as well as statistical significance. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s007" target="_blank">Table S1</a>.</p

Core set of iPSC Lines.

<p>See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084547#pone.0084547.s002" target="_blank">Fig S2</a>.</p