In mammals, circadian control of physiology and behavior is driven by a master pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. We have used gene expression profiling to identify cycling transcripts in the SCN and in the liver. Our analysis revealed approximately 650 cycling transcripts and showed that the majority of these were specific to either the SCN or the liver. Genetic and genomic analysis suggests that a relatively small number of output genes are directly regulated by core oscillator components. Major processes regulated by the SCN and liver were found to be under circadian regulation. Importantly, rate-limiting steps in these various pathways were key sites of circadian control, highlighting the fundamental role that circadian clocks play in cellular and organismal physiology.

SUMMARY Mutations that cause Intellectual Disability (ID) and Autism Spectrum Disorder (ASD) are commonly found in genes that encode for synaptic proteins. However, it remains unclear how mutations that disrupt synapse function impact intellectual ability. In the SYNGAP1 mouse model of ID/ASD, we found that dendritic spine synapses develop prematurely during the early postnatal period. Premature spine maturation dramatically enhanced excitability in the developing hippocampus, which corresponded with the emergence of behavioral abnormalities. Inducing SYNGAP1 mutations after critical developmental windows closed had minimal impact on spine synapse function, while repairing these pathogenic mutations in adulthood did not improve behavior and cognition. These data demonstrate that SynGAP protein acts as a critical developmental repressor of neural excitability that promotes the development of life-long cognitive abilities. We propose that the pace of dendritic spine synapse maturation in early life is a critical determinant of normal intellectual development.

Circadian rhythms of cell and organismal physiology are controlled by an autoregulatory transcription-translation feedback loop that regulates the expression of rhythmic genes in a tissue-specific manner. Recent studies have suggested that components of the circadian pacemaker, such as the Clock and Per2 gene products, regulate a wide variety of processes, including obesity, sensitization to cocaine, cancer susceptibility, and morbidity to chemotherapeutic agents. To identify a more complete cohort of genes that are transcriptionally regulated by CLOCK and/or circadian rhythms, we used a DNA array interrogating the mouse protein-encoding transcriptome to measure gene expression in liver and skeletal muscle from WT and Clock mutant mice. In WT tissue, we found that a large percentage of expressed genes were transcription factors that were rhythmic in either muscle or liver, but not in both, suggesting that tissue-specific output of the pacemaker is regulated in part by a transcriptional cascade. In comparing tissues from WT and Clock mutant mice, we found that the Clock mutation affects the expression of many genes that are rhythmic in WT tissue, but also profoundly affects many nonrhythmic genes. In both liver and skeletal muscle, a significant number of CLOCKregulated genes were associated with the cell cycle and cell proliferation. To determine whether the observed patterns in cell-cycle gene expression in Clock mutants resulted in functional dysregulation, we compared proliferation rates of fibroblasts derived from WT or Clock mutant embryos and found that the Clock mutation significantly inhibits cell growth and proliferation.cell cycle ͉ circadian rhythms ͉ Clock mutation ͉ gene expression ͉ protein-encoding transcriptome M any organisms have Ϸ24-h rhythms in metabolism, physiology, and behavior that are driven by cell autonomous circadian pacemakers (1). These circadian rhythms allow organisms to coordinate a myriad of physiological processes with the changing environment. In mammals, the circadian pacemaker is composed of interlocked transcription-translation feedback loops: the primary loop is composed of the basic helix-loophelix transcription factors CLOCK and BMAL1, which drive transcription of the Period (Per1, Per2) and Cryptochrome (Cry1, Cry2) genes (1, 2). PER and CRY proteins form the negative limb of the feedback loop by inhibiting their own CLOCK: BMAL1-induced transcription; turnover of PER and CRY allows the cycle to begin anew. The interlocked loop consists of REV-ERB-␣ and ROR␣, which repress and activate the Bmal1 gene, thereby modulating its function (3, 4). Mutation or deletion of Clock (5), Bmal1 (6), Per1/2 genes (7, 8), or Cry1/2 (9, 10) genes results in behavioral arrhythmicity and disruption of the autoregulatory loop, whereas disruption of components of the secondary loop results in short period-length phenotypes (3, 4).The molecular components of the circadian clock are present in the majority of neurons in the suprachiasmatic nucleus (SCN), a bilateral body in the anterior hypot...

Circadian rhythms are approximate 24-h behavioral and physiological cycles that function to prepare an organism for daily environmental changes. The basic clock mechanism is a network of transcriptional-translational feedback loops that drive rhythmic expression of genes over a 24-h period. The objectives of this study were to identify transcripts with a circadian pattern of expression in adult skeletal muscle and to determine the effect of the Clock mutation on gene expression. Expression profiling on muscle samples collected every 4 h for 48 h was performed. Using COSOPT, we identified a total of 215 transcripts as having a circadian pattern of expression. Real-time PCR results verified the circadian expression of the core clock genes, Bmal1, Per2, and Cry2. Annotation revealed cycling genes were involved in a range of biological processes including transcription, lipid metabolism, protein degradation, ion transport, and vesicular trafficking. The tissue specificity of the skeletal muscle circadian transcriptome was highlighted by the presence of known muscle-specific genes such as Myod1, Ucp3, Atrogin1 (Fbxo32), and Myh1 (myosin heavy chain IIX). Expression profiling was also performed on muscle from the Clock mutant mouse and sarcomeric genes such as actin and titin, and many mitochondrial genes were significantly downregulated in the muscle of Clock mutant mice. Defining the circadian transcriptome in adult skeletal muscle and identifying the significant alterations in gene expression that occur in muscle of the Clock mutant mouse provide the basis for understanding the role of circadian rhythms in the daily maintenance of skeletal muscle.

Spontaneous action potentials in the suprachiasmatic nucleus (SCN) are necessary for normal circadian timing of behavior in mammals. The SCN exhibits a daily oscillation in spontaneous firing rate (SFR), but the ionic conductances controlling SFR and the relationship of SFR to subsequent circadian behavioral rhythms are not understood. We show that daily expression of the large conductance Ca(2+)-activated K(+) channel (BK) in the SCN is controlled by the intrinsic circadian clock. BK channel-null mice (Kcnma1(-/-)) have increased SFRs in SCN neurons selectively at night and weak circadian amplitudes in multiple behaviors timed by the SCN. Kcnma1(-/-) mice show normal expression of clock genes such as Arntl (Bmal1), indicating a role for BK channels in SCN pacemaker output, rather than in intrinsic time-keeping. Our findings implicate BK channels as important regulators of the SFR and suggest that the SCN pacemaker governs the expression of circadian behavioral rhythms through SFR modulation.

Classic experiments have shown that ovulation and estrous cyclicity are under circadian control and that surgical ablation of the suprachiasmatic nuclei (SCN) results in estrous acyclicity in rats. Here, we characterized reproductive function in the circadian Clock mutant mouse and found that the circadian Clock mutation both disrupts estrous cyclicity and interferes with the maintenance of pregnancy. Clock mutant females have extended, irregular estrous cycles, lack a coordinated luteinizing hormone (LH) surge on the day of proestrus, exhibit increased fetal reabsorption during pregnancy, and have a high rate of full-term pregnancy failure. Clock mutants also show an unexpected decline in progesterone levels at midpregnancy and a shortened duration of pseudopregnancy, suggesting that maternal prolactin release may be abnormal. In a second set of experiments, we interrogated the function of each level of the hypothalamic-pituitary-gonadal (HPG) axis in order to determine how the Clock mutation disrupts estrous cyclicity. We report that Clock mutants fail to show an LH surge following estradiol priming in spite of the fact that hypothalamic levels of gonadotropin-releasing hormone (GnRH), pituitary release of LH, and serum levels of estradiol and progesterone are all normal in Clock/Clock females. These data suggest that Clock mutants lack an appropriate circadian daily-timing signal required to coordinate hypothalamic hormone secretion. Defining the mechanisms by which the Clock mutation disrupts reproductive function offers a model for understanding how circadian genes affect complex physiological systems.

Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes up-regulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A , GATA2 , and DPYSL3 , are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.

MicroRNAs (miRNAs) are small regulatory RNAs that individually regulate up to several hundred genes, and collectively may regulate as much as two-thirds of the transcriptome. Recent evidence supports a role for miRNA dysregulation in psychiatric and neurological disorders, including schizophrenia, bipolar disorder, and autism. Small changes in miRNA expression can fine-tune the expression of multiple genes within a biological network, suggesting that miRNA dysregulation may underlie many of the molecular changes observed in psychiatric disease, and that therapeutic regulation of miRNA levels may represent a novel treatment option.