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. 2012 Sep 15;53(6):1371-80.
doi: 10.1016/j.freeradbiomed.2012.07.018. Epub 2012 Aug 3.

Sporadic Alzheimer Disease Fibroblasts Display an Oxidative Stress Phenotype

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Free PMC article

Sporadic Alzheimer Disease Fibroblasts Display an Oxidative Stress Phenotype

Mahesh Ramamoorthy et al. Free Radic Biol Med. .
Free PMC article

Abstract

Alzheimer disease (AD) is a major health problem in the United States, affecting one in eight Americans over the age of 65. The number of elderly suffering from AD is expected to continue to increase over the next decade, as the average age of the U.S. population increases. The risk factors for and etiology of AD are not well understood; however, recent studies suggest that exposure to oxidative stress may be a contributing factor. Here, microarray gene expression signatures were compared in AD-patient-derived fibroblasts and normal fibroblasts exposed to hydrogen peroxide or menadione (to simulate conditions of oxidative stress). Using the 23K Illumina cDNA microarray to screen expression of >14,000 human genes, we identified a total of 1017 genes that are chronically up- or downregulated in AD fibroblasts, 215 of which were also differentially expressed in normal human fibroblasts within 12h after exposure to hydrogen peroxide or menadione. Pathway analysis of these 215 genes and their associated pathways revealed cellular functions that may be critically dysregulated by oxidative stress and play a critical role in the etiology and/or pathology of AD. We then examined the AD fibroblasts for the presence of oxidative DNA damage and found increased accumulation of 8-oxo-guanine. These results indicate the possible role of oxidative stress in the gene expression profile seen in AD.

Conflict of interest statement

Conflict of Interest: The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. The response of normal human fibroblasts to oxidative stress
(A) Bar graphs plotting number of genes up- (up) or down-regulated (down) 0 h after treatment with 100 μM H2O2, or 25 μM menadione (normal fibroblasts) or after mock incubation (AD cells and control cells) (B) Same as (A), except 12 hours after treatment or mock treatment.
Figure 2
Figure 2. Gene expression profiles associated with AD or oxidative stress
(A) Venn diagram showing the number of genes co-regulated in unstressed AD cells (A0C-C0C); H2O2 treated normal fibroblasts (C0H-C0C) or menadione-treated normal fibroblasts (C0M-COC) using cells harvested immediately after the end of treatment or mock treatment. Up-regulated genes are shown in red and down-regulated genes are shown in blue. (B) Same as (A), except using cells harvested 12 h after the end of treatment or mock treatment. (C) Heat map/Cell plot summarizing gene expression ratios immediately after the end of treatment or mock treatment. The color key on the right maps color intensity to the range of positive and negative expression ratios. (D) Same as (C), except using cells harvested 12 h after the end of treatment or mock treatment.
Figure 2
Figure 2. Gene expression profiles associated with AD or oxidative stress
(A) Venn diagram showing the number of genes co-regulated in unstressed AD cells (A0C-C0C); H2O2 treated normal fibroblasts (C0H-C0C) or menadione-treated normal fibroblasts (C0M-COC) using cells harvested immediately after the end of treatment or mock treatment. Up-regulated genes are shown in red and down-regulated genes are shown in blue. (B) Same as (A), except using cells harvested 12 h after the end of treatment or mock treatment. (C) Heat map/Cell plot summarizing gene expression ratios immediately after the end of treatment or mock treatment. The color key on the right maps color intensity to the range of positive and negative expression ratios. (D) Same as (C), except using cells harvested 12 h after the end of treatment or mock treatment.
Figure 3
Figure 3. Oxidative stress triggers overlap of gene ontology changes that resemble Alzheimer's disease expression profile
(A) Heat map summarizing GO terms linked to significant gene expression ratios shown in Figure 2A (see text for details). (B) Venn diagram number of GO-terms/pathways co-regulated in unstressed AD cells (A12C-C12C); H2O2 treated normal fibroblasts (C12H-C2C) or menadione-treated normal fibroblasts (C12M-C2C) using cells harvested 12 h after the end of treatment or mock treatment. The color key on the right maps color intensity to the range of positive and negative expression ratios. (C) Selected prominent GO terms/pathways from (A), with corresponding Z-scores, thought to have potential relevance to AD.
Figure 3
Figure 3. Oxidative stress triggers overlap of gene ontology changes that resemble Alzheimer's disease expression profile
(A) Heat map summarizing GO terms linked to significant gene expression ratios shown in Figure 2A (see text for details). (B) Venn diagram number of GO-terms/pathways co-regulated in unstressed AD cells (A12C-C12C); H2O2 treated normal fibroblasts (C12H-C2C) or menadione-treated normal fibroblasts (C12M-C2C) using cells harvested 12 h after the end of treatment or mock treatment. The color key on the right maps color intensity to the range of positive and negative expression ratios. (C) Selected prominent GO terms/pathways from (A), with corresponding Z-scores, thought to have potential relevance to AD.
Figure 4
Figure 4. Flow cytometric quantitative analysis of 8-OdG in AD fibroblasts grown in culture
(A) Quantified FITC intensity as indicator of 8-OdG in fixed normal fibroblasts (control 1) after exposure to 100 μM H2O2 for 15 min. (B) As in (A), except using untreated normal fibroblasts (control 1). (C) As in (A), except using untreated normal fibroblasts (control 1), untreated normal fibroblasts (control 2), untreated AD fibroblasts (AG07374) and untreated AD fibroblasts (AG05770). (D) Quantification of the data shown in (C) using the mean value across different cell lines. Error bars show ±SEM, n=2 per cell line.
Figure 5
Figure 5. Validation of microarray data
Fold-change in expression of the indicated genes was determined using quantitative PCR. Error bars show ±SD, n=3
Figure 6
Figure 6. Summary of Pubmatrix search
Summary of some of the more interesting genes that we find commonly altered after oxidative stress treatment to normal cells or in untreated AD cells relative to untreated control cells. Pubmatrix [50] was used to identify which of these genes had been previously reported in the literature with AD and those gene products are depicted in red.

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