Figure 3A. Eigen value of the 3-microRNA signature measured in the mice that performed longitudinal water maze training. Note the significantly increased expression of the signature already at 15 months of age, suggesting that increased expression levels precede detectable cognitive impairment. Number of biological replicates = 10/group, unpaired two-sided Wilcox rank test. B. Cognitive score measured in the same mice reveals cognitive decline between 15 and 16,5 months of age (p value 0.0079). Number of biological replicates = 10/group, unpaired two-sided Wilcox rank test. C. Eigen value showing the expression of the 3-microRNA signature in the hippocampus of 3 and cognitively impaired 16,5 months old mice. Number of biological replicates (3 months = 8, 16.5 months = 9), unpaired two-sided Wilcox rank test. D. (Top) Relative enrichment of the three microRNAs across different cell types. Quantitative expression of microRNAs in primary hippocampal neurons, primary astrocytes and primary microglia. miR-146a-5p is significantly enriched in microglia, while miR-148a-3p and miR-181a-5p are significantly enriched in neurons. N = 5/group, Two-way ANOVA, Tukey's multiple comparisons test, *P<0.05, **P<0.01. Bars and error bar indicates mean ± sem. (Bottom) miRNA expression in different cell types of mouse brain stem. The data was retrieved from Hoye et al. 2017. E. Overexpression of microRNAs in relevant cell types for 48 hours followed by genome wide RNA-seq analysis. miR-181a-5p was overexpressed in primary hippocampal neurons, while miR-146a-5p was overexpressed in microglia culture. Immortalized microglial cell line was used for this purpose. Given that miR-148a-3p was highly enriched in neurons (D), primary hippocampal neurons were treated with the corresponding miR-148a-3p mimic. PCA plot shows that the mimic and control treated samples cluster distinguishingly separate from one another. Volcano plot displays the genes significantly deregulated in mimic treated samples compared to control samples (FDR <0.05). Red color indicates the up-regulated genes while the blue color represents the genes those were down-regulated. F. Gene ontology analyses for up- and down-regulated genes. Panel F summarizes top significant up- and down- regulated biological processes corresponding to each microRNA and comparison among them. Over-expression of miR-181a-5p and miR-148a-3p led to down-regulation of genes related to cognition and synaptic functions, while downregulated genes due to increased expression of miR-146a-5p represent ncRNA processing, defense response and protein folding mechanisms. The up-regulated genes due to overexpression of these microRNAs represent several processes including extracellular matrix, endoplasmic reticulum stress. G. Comparison of increased stress and inflammatory responses related significant biological processes among microRNAs. Interestingly, miR-146a-5p overexpression in microglia led to increased expression of inflammatory related genes. Overexpression of both miR-146a-5p and miR-148a-3p can increase stress related responses. Size of the dot represents the number of genes belonging to the given process and the color represent the p value after multiple corrections. H. qPCR analysis confirms the over-expression of pro-inflammation related genes (IL-1ß, IL-6, TNF-alpha) due to overexpression of miR-146a-5p. Expression of anti-inflammatory gene, IL-10 was downregulated in mimic treated cells compared to the controls. Unpaired t-tests, two-tailed, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Bars and error bar indicates mean ± sem. Number of biological replicates: 5-6/group. I. Hypergeometric overlap of the up- and down-regulated genes (E) with gene-sets from different datasets. We calculated enrichment of the deregulated genes relative to those gene-sets and used a Fisher's exact test p value after multiple adjustments to estimate the significance of the overlap. Immune related genes based on expression quantitative loci (e-QTL) were retrieved from a previous study. RNAseq data from CKp25 mice at 2 and 6 weeks after induction were retrieved from GSE65159 and up- and downregulated genes compared to littermate controls were determined after differential expression (significant genes; adjusted p value<0.05). Up- and downregulated transcripts in human AD patients compared to control subjects were determined by analysis of the available data (GSE44770). Proteins those are over- and reduced-expressed in AD patients compared to controls were retrieved from a previous study. Overlap analysis for the up- and down-regulated genes due to over-expression of microRNAs were performed to those separately from the disease conditions. Human orthologs of the mouse deregulated genes were used to perform the overlap analysis in human datasets. Color code represents fold enrichment. *FDR<0.05, **FDR<0.01, ***FDR<0.001