Brooke H. Miller

Coordinated Transcription of Key Pathways in the Mouse by the Circadian Clock

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.

Circadian and CLOCK-controlled regulation of the mouse transcriptome and cell proliferation

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 CLOCK-regulated 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.

Circadian Clock Mutation Disrupts Estrous Cyclicity and Maintenance of Pregnancy

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.

MicroRNA-132 dysregulation in schizophrenia has implications for both neurodevelopment and adult brain function

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.

BK calcium-activated potassium channels regulate circadian behavioral rhythms and pacemaker output

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 Ca2+-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.

Pathogenic SYNGAP1 mutations impair cognitive development by disrupting maturation of dendritic spine synapses.

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, whereas 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.

MicroRNA dysregulation in psychiatric disease

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.

Vasopressin regulation of the proestrous luteinizing hormone surge in wild-type and Clock mutant mice.

Abstract In the female mouse, ovulation and estrous cyclicity are under both hormonal and circadian control. We have shown that mice with a mutation in the core circadian gene Clock have abnormal estrous cycles and do not have a luteinizing hormone (LH) surge on the afternoon of proestrus due to a defect at the hypothalamic level. In the present study, we tested the hypotheses that vasopressin (AVP) can act as a circadian signal to regulate the proestrous release of LH, and that this signal is deficient in the Clock mutant. We found that Avp expression in the suprachiasmatic nucleus (SCN) and AVP 1a receptor (Avpr1a) expression in the hypothalamus is reduced in Clock mutant mice compared to wild-type mice. Intracerebroventricular (i.c.v.) injection of AVP on the afternoon of proestrus is sufficient to induce LH secretion, which reaches surge levels in 50% of Clock mutant mice. The effect of AVP on the Clock mutant LH surge is mediated by AVPR1A, as co-infusion of AVP and an AVPR1A-specific antagonist prevents AVP induction of LH release, although infusion of an AVPR1A antagonist into wild-type mice failed to prevent a proestrous LH surge. These results suggest that reduced hypothalamic AVP signaling plays a role in the absence of the proestrous LH surge in Clock mutant mice. The results also support the hypothesis that AVP produced by the SCN may be a circadian signal that regulates LH release.

Genetic regulation of behavioral and neuronal responses to fluoxetine.

Despite widespread use of antidepressants, the factors underlying the behavioral response to antidepressants are unknown. It has been shown that antidepressant treatment promotes the proliferation and survival of neurons in the adult hippocampus via enhanced serotonergic signaling, but it is unclear whether hippocampal neurogenesis is responsible for the behavioral response to antidepressants. Furthermore, a large subpopulation of patients fails to respond to antidepressant treatment due to presumed underlying genetic factors. In the present study, we have used the phenotypic and genotypic variability of inbred mouse strains to show that there is a genetic component to both the behavioral and neuronal effects of chronic fluoxetine treatment, and that this antidepressant induces an increase in hippocampal cell proliferation only in the strains that also show a positive behavioral response to treatment. Furthermore, the behavioral and neuronal responses are associated with an upregulation of genes known to promote neuronal proliferation and survival. These results suggest that inherent genetic predisposition to increased serotonin-induced neurogenesis may be a determinant of antidepressant efficacy.

Phenotypic Characterization of a Genetically Diverse Panel of Mice for Behavioral Despair and Anxiety

Background Animal models of human behavioral endophenotypes, such as the Tail Suspension Test (TST) and the Open Field assay (OF), have proven to be essential tools in revealing the genetics and mechanisms of psychiatric diseases. As in the human disorders they model, the measurements generated in these behavioral assays are significantly impacted by the genetic background of the animals tested. In order to better understand the strain-dependent phenotypic variability endemic to this type of work, and better inform future studies that rely on the data generated by these models, we phenotyped 33 inbred mouse strains for immobility in the TST, a mouse model of behavioral despair, and for activity in the OF, a model of general anxiety and locomotor activity. Results We identified significant strain-dependent differences in TST immobility, and in thigmotaxis and distance traveled in the OF. These results were replicable over multiple testing sessions and exhibited high heritability. We exploited the heritability of these behavioral traits by using in silico haplotype-based association mapping to identify candidate genes for regulating TST behavior. Two significant loci (-logp >7.0, gFWER adjusted p value <0.05) of approximately 300 kb each on MMU9 and MMU10 were identified. The MMU10 locus is syntenic to a major human depressive disorder QTL on human chromosome 12 and contains several genes that are expressed in brain regions associated with behavioral despair. Conclusions We report the results of phenotyping a large panel of inbred mouse strains for depression and anxiety-associated behaviors. These results show significant, heritable strain-specific differences in behavior, and should prove to be a valuable resource for the behavioral and genetics communities. Additionally, we used haplotype mapping to identify several loci that may contain genes that regulate behavioral despair.