Tau overexpression impacts a neuroinflammation gene expression network perturbed in Alzheimer's disease.

Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer's disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid

Spatial working memory and object recognition memory in rTg4510 mice.

<p>(A) At 2 and 4 months of age, rTg4510 displayed cognitive deficits in spatial working memory compared to DN (1-way ANOVA followed by Tukey’s post-hoc ***p<0.001; student’s t-test *p<0.05). By 6 months of age, spatial working memory was confounded by stereotypic behavior observed in rTg4510 mice. n = 10–12/group. (B) At 2 months of age, all 3 genotypes displayed a preference for the novel object over the familiar object (2-way ANOVA, Bonferroni’s post-hoc **p<0.01, ***p<0.001). By 4 months of age, there was no significant difference in the amount of time rTg4510 mice explored the novel object compared to the familiar object, and the percent of time that rTg4510 explored the novel object was significantly less than the time spent by DN animals (2-way ANOVA, Bonferroni’s post-hoc *p<0.05, ***p<0.001). By 6 months of age, both rTg4510 and tTA animals explored the novel object less than DN animals (2-way ANOVA, Bonferroni’s post-hoc *p<0.05, ***p<0.001). n = 12–15/group. Error bars indicate SEM.</p

Overview of gene expression changes identified in microdissected hippocampal subfields.

<p>(A) Venn diagram of number of differentially expressed genes in the CA1, CA3 and DG microdissected samples. Most of the gene expression changes were observed in the CA1 region, with only modest overlap with the other two regions. (B) Venn diagram of the number of differentially expressed genes in the hippocampal subfields with the whole hippocampus.</p

Upregulation of the complement system in rTg4510 mice.

<p>The complement cascade, shown here adapted from the IPA library, is the most significant pathway overrepresented by genes with age-dependent changes in expression in rTg4510 hippocampus. All gene expression changes in this pathway were increases in expression. The degree of shading in red corresponds to the amount of upregulation in rTg4510 hippocampus compared to tTA at 6.1 months of age, with the numbers indicating the fold-change.</p

Upregulation of neuroinflammation markers in the rTg4510 brain.

<p>(A) <i>Gfap</i> and <i>Spp1</i> (which encodes for osteopontin) were upregulated at the mRNA level, as measured on Affymetrix microarrays, in rTg4510 hippocampus. <i>Gfap</i> expression increased progressively across the 3 age groups, whereas <i>Spp1</i> increased between 1.9 and 4.7 months of age, but did not differe significantly between 4.7 and 6.1 months of age. Expression levels are normalized to 6.1 month old tTA animals. Significance was determined using a one-way ANOVA followed by the Dunnett multiple comparison test. *p<0.05, ***p<0.001 compared to 1.9 month-old rTg4510. ^###p<0.001 compared to tTA. (B) GFAP and osteopontin protein levels, as measured by ELISA, were significantly highers in the cortex of rTg4510 animals at 4.6 and 6.1 months. Significance was determined using a one-way ANOVA followed by the Dunnett multiple comparison test. ****p<0.0001 compared to tTA of the respective ages. Error bars indicate SEM.</p

Inflammatory genes upregulated in the rTg4510 frontal cortex.

<p><i>C4b</i>, <i>Gfap</i> and <i>Spp1</i> mRNA expression levels were higher in rTg4510 animals compared to all other genotypes in the frontal cortex, as determined by qRT-PCR. mRNA expression levels are all normalized to tTA. **p<0.01, ***p<0.001 compared to tTA using the Dunnett multiple comparison test. Error bars indicate SEM.</p

Heirarchical clustering of age-dependent gene expression changes in rTg4510 mice.

<p>Heirarchical clustering of the 165 probe sets that showed age-dependent changes in rTg4510 mice (A) using all ages and genotyopes analysed, or (B) only 6.1 month old animals, illustrates probe sets that were either downregulated or upregulated with age in rTg4510 animals. Standardization was achieved by shifting the expression values to a mean of zero and scaling to a standard deviation of one.</p

Microglial gene expression network altered in rTg4510 mice.

<p>Most of the genes with age-dependent gene expression changes in rTg4510 mice compared to controls fall into an interconnected network of genes related to microglial function. TYROBP forms a major hub of this network. Colors indicate levels of gene expression changes in rTg4510 mice at 6.1 months of age compared to tTA controls, with red indicating upregulation and green indicating downregulation. The network was generated in Ingenuity Pathway Analysis and additional interactions were added using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106050#pone.0106050-Jensen1" target="_blank">[63]</a>.</p

Pathways affected by genes downregulated in the CA1 region.

<p>624 genes downregulated in the CA1 subregion of rTg4510 were subjected subjected to Ingenuity Pathway Analysis. The top axis represents the –log(p-value) of the probability that the gene set represented the canonical pathway by chance, as determined by the Fisher’s Exact Test, with the red line delineating p = 0.05 threshold, and the bottom axis represents the ratio of the number of genes from the query gene set in the canonical pathway to the total number of genes in the pathway. The top canonical pathways significantly overrepresented by these genes, shown by the blue bars, included pathways implicated in neurodegeneration, neuronal signaling and cell migration. The green bars show that these genes also significantly overlap with synaptic transmission modules affected in AD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106050#pone.0106050-Zhang1" target="_blank">[14]</a>. The query genes in these pathways are shown in Table S8 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106050#pone.0106050.s003" target="_blank">File S1</a>.</p

Gene expression profiling of laser microdissected hippocampal subfields.

<p>(A) Schematic representation of hippocampal circuitry. Regions isolated by laser capture microdissection are outlined in grey. (B) Principle Component Analysis (PCA) of gene expression profiling data of laser microdissected hippocampal subfields from 4.5 month and 6.1 month old rTg4510 animals and controls. The different subfields, designated by colors, separate from each other along the second principle component (z axis), whereas the genotypes, designated by shapes, separate along the first principle component (x axis), particularly at 6.1 months of age. The tables indicate the number of probe sets that meet the false discovery rate cut-off of 0.05 at the two ages.</p