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

The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia.

The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1-10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1-10 mg/kg, sc) and set shift performance in rats (1-10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1-3 mg/kg, po). BMS-933043 also improved auditory gating (0.56-3 mg/kg, sc) and mismatch negativity (0.03-3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans

Suppression of tau expression leads to reversal of inflammation.

<p>(A) Administration of doxycycline (Dox) to rTg4510 mice resulted in a decrease in total human tau (HT7) in the cortex and hippocampus. The most dramatic reduction was observed in cortical layer IV (arrowhead). Doxycycline administration also resulted in a decrease of Iba-1 and GFAP staining in the cortex. Scale bar, 200 µm. (B) Doxycycline resulted in a decrease in human tau and <i>Gfap</i> gene expression measured by qRT-PCR after 1 week of treatment. Further reductions in <i>Gfap</i> gene expression were observed with progressively longer treatments prior to 5.3 months of age. (C) Quantitation of GFAP levels by ELISA reveal a 3-fold reduction in rTg4510 mice with 6 weeks of doxycycline treatment, but no change in tTA mice. (D) Plasma levels of the monocyte marker, CD40, were higher in rTg4510 animals than tTA controls, and were suppressed to tTA levels after 6 weeks of doxycycline administration. **p<0.01, ***p<0.001 using the Dunnett multiple comparison test. 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

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

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

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

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