Introduction
Sleep deprivation is a widespread public health problem in the United States and countries around the globe [
1]. In the United States, estimates suggest that nearly 70% of adults and teenagers have insufficient sleep at least one day per month [
2‐
4]. Acute sleep deprivation results in cognitive impairments (reviewed in [
5]), as well as the exacerbation of neuropsychiatric and mood disorders (reviewed in [
6,
7]). The decrements in cognitive function and performance induced by acute sleep deprivation create an economic burden with decreased workplace productivity as well as increased accident risk encumbering public safety [
8‐
11]. Moreover, acute sleep deprivation results in increased levels of amyloid-beta as well as increased levels of tau in cerebral spinal fluid and plasma, which are pathological markers associated with increased risk of Alzheimer’s disease [
12,
13].
The impact of sleep deprivation on long-term memory is phylogenetically conserved as seen in behavioral studies of invertebrates to rodent models to human subjects [
14‐
18]. Moreover, the effects of acute sleep deprivation on memory can extend for days, even with recovery sleep. For example, in the marine mollusk
Aplysia, the effects of acute sleep deprivation persist for at least 48 h inhibiting the formation of long-term memory [
17]. Similarly, in humans, acute deprivation impairs episodic memory and hippocampus dependent memory associations for more than two days, despite recovery sleep [
18]. Long-lasting cellular mechanisms such as changes in gene regulation ostensibly underlie the conserved persistent effects of acute sleep deprivation on memory. The hippocampus is particularly susceptible to the impacts of sleep deprivation with changes apparent in cellular signaling, protein synthesis, and neuronal connectivity following sleep deprivation [
12,
18‐
22], although studies differ as to the effect of acute sleep deprivation on dendritic structure [
13,
22‐
24]. Studies have highlighted the effects of sleep deprivation on gene expression and protein synthesis in the hippocampus [
25‐
27], with more recent analyses conducted to identify gene networks and hubs correlated with sleep deprivation [
28]. Sleep deprivation also induces epigenetic alterations affecting gene expression in animal models and humans ([
29,
30] and reviewed in [
31]). Enhancement of global gene transcription through inhibition of histone deacetylation has been shown to rescue hippocampus-dependent memory and synaptic plasticity in sleep deprived mice [
32]. Thus, our understanding of the extent and specificity of sleep deprivation on gene regulation remains incomplete.
To more fully detail the effects of acute sleep deprivation on transcription, we investigated the effect of 5 h of sleep deprivation on gene expression in the hippocampus using an unbiased deep RNA sequencing (RNA-Seq) approach. We identified 1146 genes differentially regulated after sleep deprivation. Genes significantly upregulated were preferentially associated with the nucleus with functions in RNA binding and processing, whereas genes significantly downregulated after sleep deprivation were associated with cell adhesion, the synapse, dendrites, and postsynaptic membrane. Through comparison with a recently published data set analyzing the effects of acute sleep deprivation on ribosome associated transcripts in excitatory neurons of the hippocampus [
27], we found a considerable difference between the number of genes regulated by sleep deprivation at the total RNA level in the ribosome associated pool of transcripts. Genes regulated by sleep deprivation at both the transcriptional and translational levels showed enrichment in protein kinase and phosphatase activity, as well as potassium and cation channel activity. Functions enriched with genes regulated by sleep deprivation only in the transcriptome included transcription factor binding, histone deacetylase activity, nucleotide binding, nucleotide exchange factor activity and small GTPase regulator activity; whereas genes regulated solely in the translatome displayed network enrichment for the unfolded protein binding pathway, protein binding, peptide binding, protein dimerization and ubiquitin binding. The data set generated with this research highlights the differences in biological function between genes upregulated after sleep deprivation and those downregulated demonstrating the gene specific effects of sleep deprivation and recovery sleep on gene regulation.
Discussion
Numerous behavioral and molecular studies have demonstrated the requirement of sleep for memory and neural plasticity (reviewed in [
55,
56]). Previous research has shown that the hippocampus is highly susceptible to the effects of acute sleep deprivation inhibiting long-term memory with changes apparent in neuronal connectivity and morphology [
14,
20,
22,
57,
58]. Although studies using mice and rats have shown that acute sleep deprivation affects gene expression in the hippocampus and the forebrain [
25,
50,
54,
59‐
64], much of the previous research has focused on specific gene sets or used microarray analysis, rendering an incomplete picture of the effects of sleep deprivation on gene expression. Thus, we conducted an unbiased investigation of the effects of sleep deprivation on hippocampal gene expression using RNA-Seq. As predicted, the RNA-Seq experiments provided a more in-depth investigation into the effects of sleep deprivation on the transcriptome with more genes analyzed than previous microarray experiments. We found that five hours of sleep deprivation upregulated or downregulated gene expression dependent upon the biological functions and cellular components associated with the genes. The RNA-Seq results were validated through independent sleep deprivation and recovery experiments followed by RT-qPCR for genes of interest.
Analysis of genes upregulated by sleep deprivation revealed associations with nuclear functions including genes involved in RNA binding, processing, and splicing potentially increasing RNA splicing misregulation, nonsense mediated decay and RNA degradation. For example, the
Upf2 gene, a mediator of nonsense mediated decay, was upregulated by sleep deprivation consistent with the hypothesis that sleep deprivation could result in changes in RNA splicing that lead to increased RNA degradation. Misregulation of RNA splicing affects neural plasticity and function (reviewed in [
50]). Dysregulation of RNA binding proteins and splicing has been associated with aberrant neural function and neurodegenerative diseases including Alzheimer’s disease (reviewed in [
65,
66]). Thus, acute sleep deprivation has the potential to induce widespread changes in neuronal and synaptic plasticity through changes in RNA processing.
In the present study, we found that significant downregulation of genes by sleep deprivation was associated with cell adhesion and synaptic protein functions including
Nlgn1,
Nlgn3,
Ncam1,
Nectin3 and
Nectin4. Cell adhesion molecules, such as the post-synaptic adhesion protein Neuroligin-1 has been previously associated with sleep regulation ([
64] and reviewed in [
67]). Sleep deprivation downregulated metalloproteases such as
Adam23, involved in cell–cell interactions. Although multiple cellular components were significantly enriched for genes downregulated by sleep deprivation, the top three cellular locations were the dendrite, postsynaptic membrane, and the synapse. Postsynaptic density scaffolding proteins, such as members of the Disc large associated protein family
Dlgap1 and
Dlgap3, were significantly downregulated by sleep deprivation. Thus, the probable outcomes of the downregulation of genes by sleep deprivation are weakened synaptic plasticity and cell–cell interactions. Our results are consistent with previously observed decreases in hippocampal plasticity seen following brief periods of sleep deprivation [
68‐
71]
. Sleep deprivation appears to have some of the largest cellular impacts at the synapse as recent studies on whole forebrain tissue found that acute sleep deprivation reduced the rhythms in protein phosphorylation of synaptic proteins [
72,
73]. Although differences in the effect of sleep deprivation on transcription and translation are apparent between brain regions ([
50,
53] and reviewed in [
5]), acute sleep deprivation also affects synaptic proteins in the cortex [
74].
Three hours of recovery sleep following acute sleep deprivation normalized gene expression for most genes we investigated, similar to what has been observed for many genes in the hippocampus and the cortex [
25,
54]. However, we did find that the transcription factor
Nr4a1 remained upregulated after recovery sleep, albeit to a smaller extent. The
Nr4a family of transcription factors are ligand-independent nuclear receptors regulated by CREB that function in long-term hippocampus dependent memory [
75‐
78]. Inhibition of Nr4a transcriptional activity impairs long-term memory, although short-term memory is not affected [
77].
Nr4a1 is highly expressed in the hippocampus and is significantly upregulated during learning acquisition and memory consolidation for contextual fear conditioning and spatial object location training [
75,
77]. Thus, the aberrant regulation of
Nr4a1 after sleep deprivation and recovery sleep suggests a possible mechanism for the interference with long-term memory consolidation observed with acute sleep deprivation [
15]. In addition, the RNA splicing factor
Srsf7 reversed direction showing a significant decrease in expression after recovery sleep. Recently, Srsf7 was shown to affect polyadenylation of the 3’UTR with increased Srsf7 resulting in shorter polyA tails [
79]. Polyadenylation and the length of the 3’UTR affects mRNA stability, nuclear export, and translation efficiency. Dysfunctional regulation of polyadenylation has been implicated in multiple diseases including neurological diseases [
80]. Our results indicate that recovery from sleep deprivation is gene specific potentially extending the impacts of acute sleep deprivation, rather than a universal return of gene expression to baseline levels.
Previously, expression of the transcription factor
Elk1 in the hippocampus was shown to remain high after 2.5 h of recover sleep [
25]. Studies in the cortex found that some genes normalize expression levels quickly with recovery sleep, while other genes require up to 6 h of recovery sleep to return to baseline levels [
54]. Of note, genes that responded less quickly to recovery sleep included genes involved in RNA splicing and RNA binding proteins [
54]. Thus, in addition to the more immediate effects of acute sleep deprivation on gene expression and the subsequent impact on synaptic plasticity and cellular signaling, acute sleep deprivation may also exert longer lasting effects on gene regulation through the continued dysregulation of transcription factors and genes related to RNA processing. Further cell-specific research needs to be completed to fully investigate the persistent effects of acute sleep deprivation on RNA splicing and processing.
The canonical view of gene regulation and the central dogma of molecular biology suggest that RNA and protein abundance are highly correlated. However, as understanding of RNA processing increased, it has become apparent that gene regulation occurs at multiple levels (reviewed in [
81])
. Acute sleep deprivation, in particular, appears to distinctly impact transcription and translation as we found when we compared the results of the current RNA-Seq data set with the translatome of excitatory neurons in the hippocampus after sleep deprivation. Although differences arose between these data sets due to cell type differences, genes impacted similarly by sleep deprivation in the transcriptome and translatome encompassed genes involved in kinase and phosphatase signaling pathways, as well as cation and potassium channels. Changes in the expression of membrane channels and cellular signaling pathways have the potential to rapidly impact synaptic strength and plasticity following sleep deprivation. A large number of genes, more than 1,000, were upregulated in the transcriptome, but not in the translatome. Genes regulated only at the level of the transcriptome included genes involved in RNA processing, nucleotide binding and small GTPase signaling. Potentially genes upregulated in the transcriptome, but not the translatome, may reflect transcripts with alternative splicing that are not translated efficiently or that undergo degradation. Alternatively, genes upregulated in the transcriptome by sleep deprivation may also be sequestered in dynamic RNA granules that may then be released when conditions normalize (reviewed in [
82‐
84])
. Notably, there are also genes downregulated in the transcriptome but not in the translatome. Potentially, the translatome reflects isoform specific transcript association with the ribosome, while our RNA-Seq data set reflects total RNA abundance. A comparatively small number of genes, approximately 150 genes, showed separate regulation by sleep deprivation in the translatome of excitatory neurons but not in the overall transcriptome including genes associated with the unfolded protein response and ubiquitination suggesting that additional regulation of protein degradation may occur with sleep deprivation. The analyses presented provide further insight into the nuanced effects of sleep deprivation on gene regulation at multiple levels.
The results presented here provide an unbiased in-depth perspective of the effects of acute sleep deprivation on gene expression in the hippocampus. Notably, our results clearly demonstrate that sleep deprivation differentially upregulates or downregulates genes dependent upon biological function, instead of a more general mechanism resulting in global changes in gene expression. Moreover, our analyses provided new insight into the effects of sleep deprivation revealing the strong association of genes upregulated by sleep deprivation with nuclear functions. In contrast, genes downregulated by sleep deprivation were associated with multiple cellular components, in particular, dendrites and synapses. These distinctions highlight the need for future research investigating the effects of sleep deprivation on the hippocampus taking advantage of technological advances in single cell and spatial transcriptomics. Although the results presented here establish a strong foundation for comparison with data from other brain regions to more precisely understand brain region specific impacts of sleep deprivation, further research is needed to understand the persistent effects of acute sleep deprivation on long-term memory, as well as to identify the effects of chronic sleep restriction on the hippocampus.
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