Identification of intellectual disability genes showing circadian clock-dependent expression in the mouse hippocampus
Introduction
Intellectual disability (ID) is defined by an overall intelligence quotient (IQ) lower than 70 and deficits in cognitive abilities with an onset before 18 years of age. This disorder affects 1–3% of the population. The underlying causes of ID are heterogeneous and include genetic and/or environmental factors that influence the development and function of the central nervous system. Genetic factors comprise mutations in genes encoding proteins involved in neurogenesis, neuronal migration, synaptic function, transcription or translation. Many children with ID have problems with memory and learning. Indeed, several memorization processes and associated structures may be impaired in ID, which limits the ability to learn broader cognitive skills. For example, patients with Down’s or Williams syndrome show working memory deficits (Jarrold et al., 1999). Numerous studies report that many children with ID show delayed settling or frequent night waking (Didde and Sigafoos, 2001, Robinson and Richdale, 2004, Sajith and Clarke, 2007).
Sleep is implicated in long-term memory, especially in the reprocessing of recently acquired memory (Gais and Born, 2004, Ji and Wilson, 2007). Indeed, sleep plays a fundamental role in the neurocognitive performance of infants and children (Born and Wilhelm, 2012). More precisely, slow-wave sleep activity helps to consolidate hippocampal-dependent episodic memory. Sleep is controlled by two main regulatory processes: a homeostatic and a circadian one. The homeostatic process tracks the need for sleep whereas the distribution of sleep throughout the day is strongly regulated by circadian rhythm (CR). Circadian clock genes may also be directly involved in sleep homeostasis (Naylor et al., 2000). Sleep/wake-related genes have been reported to be expressed in the cortex, cerebellum, forebrain and hypothalamus in mice and rats (Terao et al., 2003, Terao et al., 2006, Cirelli et al., 2004).
The circadian clock is a fundamental system regulating many aspects of behavior and physiology, including sleep–wake cycles but also blood pressure, body temperature, and metabolism. The suprachiasmatic nucleus (SCN) of the hypothalamus contains the master circadian clock, which is reset daily by light that is detected by retinal ganglion cells and relayed via signaling through the retinohypothalamic tract. The circadian clock consists of a group of highly conserved core clock proteins (e.g. Rev-erbα, Clock, Bmal1, Per1, Per2, Per3), the expression of which is regulated through transcriptional, translational and post-translational feedback loops. These regulatory mechanisms generate and maintain the cyclic expression of clock genes as well as those they regulate (called output genes) over a 24-h period (Bell-Pedersen et al., 2005, Ko and Takahashi, 2006, Dardente and Cermakian, 2007). The SCN coordinates peripheral oscillators like the liver, kidney and cortex, which can be self-sustained, although their correct functioning depends on the other oscillators. Clock mechanisms in the SCN and peripheral oscillators are identical, and involve the 24-h cyclic expression of core clock genes (Reppert and Weaver, 2002, Yoo et al., 2004).
CR has been studied in many non-neuronal tissues, especially in the liver because it regulates metabolism. Several studies report that about 10% of transcripts show circadian clock controlled (CCC) expression in the liver, most of which are involved in metabolic functions (Panda et al., 2002, Storch et al., 2002, Cho et al., 2012). Microarrays and RNA-seq have been used to examine the transcriptome at different Circadian Time (CT) points in several regions of the mouse brain, including the SCN (Panda et al., 2002), prefrontal cortex (Yang et al., 2007), cerebellum, hypothalamus and brain stem (Zhang et al., 2014). The core clock genes are expressed in various brain structures, such as the hippocampus and cortex (Namihira et al., 1999, Reick et al., 2001, Wakamatsu et al., 2001, Shieh, 2003, Yang et al., 2007, Jilg et al., 2010) and studies on mutant mice suggest that the disruption of clock genes alters hippocampal-dependent memory formation (Garcia, 2000, Wang et al., 2009, Jilg et al., 2010). The hippocampus controls memory and learning, processes spatial information and episodic memory, and consolidates information from short-term to long-term memory. These processes are regulated by CR (Chaudhury and Colwell, 2002, Chaudhury et al., 2005, Ruby et al., 2008, Gerstner et al., 2009). For instance, circadian oscillations in hippocampal MAPK activity are implicated in the formation and persistence of memory (Eckel-Mahan et al., 2008, Phan et al., 2011). In addition, adult neurogenesis in the dentate gyrus is also controlled by the circadian molecular clock (Bouchard-Cannon et al., 2013, Schnell et al., 2014).
Although the link between cognitive impairment, memory and sleep as well as that between CR and sleep are well established, little is known about the relationship between the circadian clock and ID. The enrichment of CCC genes among the known ID genes would favor the hypothesis that the circadian clock-mediated deregulation of memory processes is involved in the pathogenesis of ID.
In this context, we analyzed genome-wide rhythmic gene expression in the hippocampus, focusing on circadian regulation. Using the JTK cycle algorithm, we demonstrate here that about 3.5% of transcripts show circadian-regulated expression in the mouse hippocampus. We divided these genes into four categories based on their temporal expression, and show that each category is characterized by specific molecular and cellular signatures. The category containing genes that were strongly expressed at the transition phase between light and dark was highly enriched in sleep/wake-related genes.
Furthermore, from a list of 673 ID-associated genes, we identified 30 genes (4.4%) showing circadian-dependent expression in the hippocampus. This proportion was not significantly higher than that expected by chance (3.5%), showing that the pathogenesis of ID is probably not related to disturbances in the circadian clock in the hippocampus.
Section snippets
Animals
Animal experiments were performed in accordance with the European Communities Council Directive (86/809/EEC) on the care and use of animals and were approved by the local ethics committee. All animals were housed singly in cages in a room maintained at 23 ± 1 °C under a 12/12 light–dark cycle (bright light, 200 lx). Nine-week-old male C57/BL6 mice were killed in the dark and hippocampi were quickly excised under a red-light lamp and snap-frozen in liquid nitrogen. The dark/dark period started at 7
CCC genes in the hippocampus
To identify CCC genes in the mouse hippocampus, we conducted our study in dark–dark conditions to overcome synchronizing effect of the SCN in response to light. We previously reported that the expression of some clock genes (Rev-erbα and Arntl) oscillates in the hippocampus in such conditions (Valnegri et al., 2011). To identify systematically genes with a CCC pattern of expression, we used Agilent microarray technology to examine the transcriptome of the hippocampus of three mice at four CT
Discussion
Circadian clocks drive the CR of biological processes in various tissues. This system is organized into a master circadian clock, the SCN that synchronizes the peripheral oscillators. Instead of a single cerebral clock, the brain contains a system of multiple local oscillators exhibiting CR in different structures (Guilding and Piggins, 2007). Indeed, the expression of Per1 and Per2 clock genes only partially overlaps in various brain regions and their promoters are activated in a cell
Conclusions
Our analysis demonstrates that about 3.5% of genes expressed in the mouse hippocampus show a CCC pattern of expression and that ID genes are not enriched among these CCC genes. In addition, we identify a list of 30 ID genes showing CCC expression. Mutations in these genes may lead to defects in the circadian regulation of hippocampal-dependent memory consolidation and/or in sleep/wake disturbances observed in patients with ID.
Acknowledgments
We thank Cochin Institute genomic facility for technical assistance. We thank Dr. Etienne Challet (from INCI, Strasbourg, France) for his expertise on CR. This work was supported by grants from Inserm, Agence Nationale de la Recherche (ANR-2010-BLANC-1434-03), Europe (Euro FP7, Gencodys, 241995) and Roche (Agreement, N°121531A10). J.R. received a postdoctoral fellowship from Roche.
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These authors contributed equally to this work.