Altered sleep homeostasis in rev-erbα knockout mice

Study Objectives: The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep.Methods: EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline.Results: Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1–4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain.Conclusions: Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context

Cortical miR-709 links glutamatergic signaling to NREM sleep EEG slow waves in an activity-dependent manner

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that have been implicated in a plethora of neuronal processes. Nevertheless, their role in regulating brain activity in the context of sleep has so far received little attention. To test their involvement, we deleted mature miRNAs in post-mitotic neurons at two developmental ages, i.e., in early adulthood using conditional Dicer knockout (cKO) mice and in adult mice using an inducible conditional Dicer cKO (icKO) line. In both models, electroencephalographic (EEG) activity was affected and the response to sleep deprivation (SD) altered; while the rapid-eye-movement sleep (REMS) rebound was compromised in both, the increase in EEG delta (1 to 4 Hz) power during non-REMS (NREMS) was smaller in cKO mice and larger in icKO mice compared to controls. We subsequently investigated the effects of SD on the forebrain miRNA transcriptome and found that the expression of 48 miRNAs was affected, and in particular that of the activity-dependent miR-709. In vivo inhibition of miR-709 in the brain increased EEG power during NREMS in the slow-delta (0.75 to 1.75 Hz) range, particularly after periods of prolonged wakefulness. Transcriptome analysis of primary cortical neurons in vitro revealed that miR-709 regulates genes involved in glutamatergic neurotransmission. A subset of these genes was also affected in the cortices of sleep-deprived, miR-709-inhibited mice. Our data implicate miRNAs in the regulation of EEG activity and indicate that miR-709 links neuronal activity during wakefulness to brain synchrony during sleep through the regulation of glutamatergic signaling

Altered Sleep Homeostasis in Rev-erbα Knockout Mice

Abstract Study Objectives: The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. Methods: EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. Results: Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1-4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. Conclusions: Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context. Significance Although circadian clock genes are named for their role in driving circadian rhythms in gene expression, physiology, and behavior, they can fulfill other important functions. The clock gene and transcriptional repressor REV-ERBα plays a role in pathways affecting metabolism and central nervous functioning. Using mice lacking the gene encoding REV-ERBα, Nr1d1, we could extend these finding to include the homeostatic regulation of sleep. Because the activity of REV-ERBα is modulated by cellular redox state, we propose that this molecule can sense and respond to the metabolic imbalance imposed at the neuronal level by periods of extended wakefulness. Recently developed synthetic drugs targeting REV-ERBα could thus be useful in the treatment of both the circadian and homeostatic aspects of sleep-wake related disorders

A Neuron-Specific Deletion of the MicroRNA-Processing Enzyme DICER Induces Severe but Transient Obesity in Mice

<div><p>MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally. MiRNAs are implicated in various biological processes associated with obesity, including adipocyte differentiation and lipid metabolism. We used a neuronal-specific inhibition of miRNA maturation in adult mice to study the consequences of miRNA loss on obesity development. <i>Camk2a-CreERT2</i> (<i>Cre⁺</i>) and floxed <i>Dicer</i> (<i>Dicer^lox/lox</i>) mice were crossed to generate tamoxifen-inducible conditional <i>Dicer</i> knockouts (cKO). Vehicle- and/or tamoxifen-injected <i>Cre⁺;Dicer^lox/lox</i> and <i>Cre⁺;Dicer^+/+</i> served as controls. Four cohorts were used to a) measure body composition, b) follow food intake and body weight dynamics, c) evaluate basal metabolism and effects of food deprivation, and d) assess the brain transcriptome consequences of miRNA loss. cKO mice developed severe obesity and gained 18 g extra weight over the 5 weeks following tamoxifen injection, mainly due to increased fat mass. This phenotype was highly reproducible and observed in all 38 cKO mice recorded and in none of the controls, excluding possible effects of tamoxifen or the non-induced transgene. Development of obesity was concomitant with hyperphagia, increased food efficiency, and decreased activity. Surprisingly, after reaching maximum body weight, obese cKO mice spontaneously started losing weight as rapidly as it was gained. Weight loss was accompanied by lowered O₂-consumption and respiratory-exchange ratio. Brain transcriptome analyses in obese mice identified several obesity-related pathways (e.g. leptin, somatostatin, and nemo-like kinase signaling), as well as genes involved in feeding and appetite (e.g. <i>Pmch, Neurotensin</i>) and in metabolism (e.g. <i>Bmp4</i>, <i>Bmp7</i>, <i>Ptger1</i>, <i>Cox7a1</i>). A gene cluster with anti-correlated expression in the cerebral cortex of post-obese compared to obese mice was enriched for synaptic plasticity pathways. While other studies have identified a role for miRNAs in obesity, we here present a unique model that allows for the study of processes involved in reversing obesity. Moreover, our study identified the cortex as a brain area important for body weight homeostasis.</p></div

Pathway analysis of significant genes at 4 weeks and of the anti-correlated cluster at 8 weeks.

<p><b>A.</b> Top 10 enriched pathways in cKO mice at 4 weeks. The % genes affected within each pathway are represented. The bar colors indicate categories to which the pathway belongs. <b>B.</b> Top 10 enriched cellular processes (left) and pathways (right) found in the cluster of anti-correlated genes (i.e., the green cluster in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116760#pone.0116760.g004" target="_blank">Fig. 4B</a>). </p

Gene expression of <i>Dicer</i> cKO and control mice in the cortex.

<p>A.P-value histograms for [cKO-control] comparisons in the cortex at 4 and 8 weeks. The horizontal line represents the number of P values expected by chance. The continuous decrease in P values from 0 to 0.4 suggests that a majority of genes are differentially expressed at 4 weeks. At 8 weeks none of the transcripts were differentially expressed. <b>B.</b> Scatter plot and distributions of the moderated t-statistic at 4 weeks and 8 weeks for 14′713 genes in the cortex. X- and Y-axis: moderated t from the cKO-control contrast at 4 and 8 weeks, respectively. At 4 weeks, the t-statistic distribution is skewed with a majority of genes having positive values. At 8 weeks, the distribution is symmetrical with a modal value close to zero. The three clusters (brown, green, blue) were defined by fitting a two-component Gaussian mixture model on each of the two marginal distributions (“mclust” R). Brown: data belonging to component 1 in both distributions. Green: data belonging to component 1 in the 4 weeks distribution and to component 2 in the 8 weeks distribution. Blue: data belonging to the 4 weeks component 1 which did not separate into two distinct clusters at 8 weeks. <b>C</b>. Mean (±1 SEM) expression of genes related to food intake, thermogenesis, and lipid metabolism (n = 3/condition). Blue bars represent the cKO mice and black bars the controls. Bars are grouped by 4-week (4 w, left pair of bars) and 8-week (8 w, right bar pair) values. </p

<i>Dicer</i> cKO mice show transient obesity.

<p>A.Photographs of two representative <i>Cre⁺;Dicer^lox/lox</i> mice injected either with tamoxifen (cKO, 55.2 gr) or vehicle (27.2 gr) taken 49 days after injection. The same two mice are represented from a dorsal (left picture) and frontal (right picture) view. <b>B.</b><i>Upper panel.</i> Body weight of cKO and control mice at day 49 (n = 3/group). <i>Lower panel.</i> Fat and lean body mass of cKO and control mice at day 65 (n = 3/group). Note that at this time, body weight in cKO mice already decreased. <b>C.</b> Body weight and food intake in cKO mice and controls (n = 4/group) over a 17-week period. Dashed grey panel highlights the 5 days of tamoxifen treatment. cKO mice increased their body weight concomitant with increasing food intake until reaching a maximum at day 38. Values reverted to control levels within 3 weeks. Food efficiency was calculated per week. Data expressed as mean (±1 SEM). cKO and control mice are represented in blue and black, respectively. Stars mark significant differences (t-tests p<0.05).</p

<i>Dicer</i> cKO mice show reduced basal metabolism and activity, and an altered response to fasting.

<p>Metabolic parameters and locomotor activity were measured during an <i>ad lib</i> fed state (72h baseline) followed by a 15h overnight fasting period and subsequent 24h refeeding. Data are shown as mean (±1 SEM) and statistical differences are indicated by stars above graphs (t-tests, p<0.05). Values of the three control groups (see text) were averaged. <b>A and B</b>. Compared to control mice (black; n = 18), obese cKO mice (blue; n = 6) showed reduced respiratory exchange ratio both under baseline and in response to fasting, mostly due to a lower VO₂. Pre-obese mice show only a reduction in VO₂ during fasting. <b>C.</b> Both pre-obese and obese cKO mice fail to enhance locomotor activity and showed a larger and smaller drop in heat production, respectively. <b>D</b>. Obese cKO mice have an overall reduction of locomotor activity, both in baseline and in response to fasting. </p