Genomic analysis of sleep deprivation reveals translational regulation in the hippocampus

Abstract

Sleep deprivation is a common problem of considerable health and economic impact in today's society. Sleep loss is associated with deleterious effects on cognitive functions such as memory and has a high comorbidity with many neurodegenerative and neuropsychiatric disorders. Therefore, it is crucial to understand the molecular basis of the effect of sleep deprivation in the brain. In this study, we combined genome-wide and traditional molecular biological approaches to determine the cellular and molecular impacts of sleep deprivation in the mouse hippocampus, a brain area crucial for many forms of memory. Microarray analysis examining the effects of 5 h of sleep deprivation on gene expression in the mouse hippocampus found 533 genes with altered expression. Bioinformatic analysis revealed that a prominent effect of sleep deprivation was to downregulate translation, potentially mediated through components of the insulin signaling pathway such as the mammalian target of rapamycin (mTOR), a key regulator of protein synthesis. Consistent with this analysis, sleep deprivation reduced levels of total and phosphorylated mTOR, and levels returned to baseline after 2.5 h of recovery sleep. Our findings represent the first genome-wide analysis of the effects of sleep deprivation on the mouse hippocampus, and they suggest that the detrimental effects of sleep deprivation may be mediated by reductions in protein synthesis via downregulation of mTOR. Because protein synthesis and mTOR activation are required for long-term memory formation, our study improves our understanding of the molecular mechanisms underlying the memory impairments induced by sleep deprivation.

Fig. 1.

Analysis of gene expression following 5 h of sleep deprivation. Microarray experimental design and results. An initial comparison was made between genome-wide mRNA expression patterns in hippocampal tissue taken from young male sleep-deprived (SD) and nonsleep-deprived (NSD) mice, using Mouse 430_2 Affymetrix microarray chips. Following normalization by robust multiarray average (RMA), microarray data were analyzed using the affy and limma packages in R/Bioconductor. BH, Benjamini-Hochberg; DAVID, Database for Annotation Visualization and Integrated Discovery; EASE, Expression Analysis Systematic Explorer.

Fig. 2.

Quantitative RT-PCR validation of genes upregulated or downregulated by sleep deprivation in the hippocampus. Quantitative RT-PCR (dark gray) was used to validate the expression level of genes identified by microarray analysis (light gray) as being changed in the hippocampus by sleep deprivation. For each gene, expression is represented as the fold change in SD mice relative to NSD mice, normalized to the average expression of housekeeping genes Actg, Hprt, and Tuba4a. The fold change values from the microarray for SD/NSD are shown for each gene for comparison. All SD/NSD qPCR comparisons are significant at P < 0.05, except Nr4a1 induction (see Table 2). Bars indicate ± SE.

Fig. 3.

Enriched functions regulated by sleep deprivation. Sleep deprivation downregulates translation and upregulates transcription. Functional annotation terms from the following databases: Gene Ontology (GO) biological process and molecular function, KEGG pathways and protein information resource keywords, were clustered based on similarity using the Database for Annotation Visualization and Integrated Discovery (DAVID). Clusters of functional terms enriched in SD down- or upregulated gene lists compared with the genome as a whole (P value <0.05) are represented as bars. Height of bars represents the enrichment score of each cluster, with the scores of downregulated clusters shown as negative numbers for visualization purposes. Enrichment score is calculated as −log(10) of the geometric mean P value among all clustered terms. Only clusters with enrichment score >1.5 (average P value of functional terms within the cluster <0.05) were considered. Examples of genes found within each cluster are shown, with qPCR-tested genes in boldface. Note that there were significant clusters of ion-binding functional terms found within both the up- and downregulated gene lists. For details of the functional terms included in these clusters, see Supplemental Table S2.

Fig. 4.

The effects of sleep deprivation on the insulin signaling pathway. Genes regulated by sleep deprivation mapped to the insulin signaling pathway (adapted from KEGG and Wikipathways). Upregulated gene names are written in lower case, in bold and italics, and downregulated gene names are written in bold with underlining. Note that genes from several functional clusters and enriched pathways shown in Fig. 3 and Table 2 are contained within this signaling network.

Fig. 5.

Sleep deprivation reduces levels of mTOR and mTOR phosphorylation, and recovery sleep reverses these effects. A: representative Western blots of phosphorylated mTOR (p-mTOR, left) and total mTOR (mTOR, right) from hippocampus homogenates of SD animals (n = 9) and NSD controls (n = 9). β-Tubulin reactivity is shown as a loading control (bottom). Quantitation is shown with protein levels represented as a fold change in SD mice relative to NSD mice, which are normalized to the β-tubulin loading control. B: representative Western blots of p-mTOR (left) and mTOR (right) from hippocampus homogenates of SD and NSD mice that were allowed to sleep for 2.5 h post-SD (SD+R, n = 7). β-Tubulin reactivity is shown as a loading control (bottom). Quantitation is shown with protein levels represented as a fold change in SD+R mice relative to NSD mice, which are normalized to the β-tubulin loading control. Bars represent ± SE. **P < 0.005, *P < 0.01, 2-tailed t-test.

Fig. 6.

qPCR analysis of hippocampal gene expression following 2.5 h of recovery sleep after sleep deprivation. For each gene, expression is represented as the fold change in SD mice allowed to sleep for 2.5 h (SD+R) relative to NSD mice, normalized to the average expression of housekeeping genes Actg, Hprt, and Tuba4a. Black line denotes no change in gene expression between SD and NSD mice (fold change = 1). Bars indicate ± SE. *Significant differences between SD+R relative to SD (P < 0.05).