Coupling-dependent metabolic ultradian rhythms in confluent cells.

Ultradian rhythms in metabolism and physiology have been described previously in mammals. However, the underlying mechanisms for these rhythms are still elusive. Here, we report the discovery of temperature-sensitive ultradian rhythms in mammalian fibroblasts that are independent of both the cell cycle and the circadian clock. The period in each culture is stable over time but varies in different cultures (ranging from 3 to 24 h). We show that transient, single-cell metabolic pulses are synchronized into stable ultradian rhythms across contacting cells in culture by gap junction-mediated coupling. Coordinated rhythms are also apparent for other metabolic and physiological measures, including plasma membrane potential (Δψp), intracellular glutamine, α-ketoglutarate, intracellular adenosine triphosphate (ATP), cytosolic pH, and intracellular calcium. Moreover, these ultradian rhythms require extracellular glutamine, several different ion channels, and the suppression of mitochondrial ATP synthase by α-ketoglutarate, which provides a key feedback mechanism. We hypothesize that cellular coupling and metabolic feedback can be used by cells to balance energy demands for survival.

NPAS4 regulates the transcriptional response of the suprachiasmatic nucleus to light and circadian behavior.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals and is entrained by environmental light. However, the molecular basis of the response of the SCN to light is not fully understood. We used RNA/chromatin immunoprecipitation/single-nucleus sequencing with circadian behavioral assays to identify mouse SCN cell types and explore their responses to light. We identified three peptidergic cell types that responded to light in the SCN: arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), and cholecystokinin (CCK). In each cell type, light-responsive subgroups were enriched for expression of neuronal Per-Arnt-Sim (PAS) domain protein 4 (NPAS4) target genes. Further, mice lacking Npas4 had a longer circadian period under constant conditions, a damped phase response curve to light, and reduced light-induced gene expression in the SCN. Our data indicate that NPAS4 is necessary for normal transcriptional responses to light in the SCN and critical for photic phase-shifting of circadian behavior.

Sleeping Sickness Disrupts the Sleep-Regulating Adenosine System.

Patients with sleeping sickness, caused by the parasite Trypanosoma brucei, have disruptions in both sleep timing and sleep architecture. However, the underlying cause of these sleep disturbances is not well understood. Here, we assessed the sleep architecture of male mice infected with T. brucei and found that infected mice had drastically altered sleep patterns. Interestingly, T. brucei-infected mice also had a reduced homeostatic sleep response to sleep deprivation, a response modulated by the adenosine system. We found that infected mice had a reduced electrophysiological response to an adenosine receptor antagonist and increased adenosine receptor gene expression. Although the mechanism by which T. brucei infection causes these changes remains to be determined, our findings suggest that the symptoms of sleeping sickness may be because of alterations in homeostatic adenosine signaling.SIGNIFICANCE STATEMENT Sleeping sickness is a fatal disease that disrupts the circadian clock, causes disordered temperature regulation, and induces sleep disturbance. To examine the neurologic effects of infection in the absence of other symptoms, in this study, we used a mouse model of sleeping sickness in which the acute infection was treated but brain infection remained. Using this model, we evaluated the effects of the sleeping sickness parasite, Trypanosoma brucei, on sleep patterns in mice, under both normal and sleep-deprived conditions. Our findings suggest that signaling of adenosine, a neuromodulator involved in mediating homeostatic sleep drive, may be reduced in infected mice.

Dual-Color Single-Cell Imaging of the Suprachiasmatic Nucleus Reveals a Circadian Role in Network Synchrony.

The suprachiasmatic nucleus (SCN) acts as a master pacemaker driving circadian behavior and physiology. Although the SCN is small, it is composed of many cell types, making it difficult to study the roles of particular cells. Here we develop bioluminescent circadian reporter mice that are Cre dependent, allowing the circadian properties of genetically defined populations of cells to be studied in real time. Using a Color-Switch PER2::LUCIFERASE reporter that switches from red PER2::LUCIFERASE to green PER2::LUCIFERASE upon Cre recombination, we assess circadian rhythms in two of the major classes of peptidergic neurons in the SCN: AVP (arginine vasopressin) and VIP (vasoactive intestinal polypeptide). Surprisingly, we find that circadian function in AVP neurons, not VIP neurons, is essential for autonomous network synchrony of the SCN and stability of circadian rhythmicity.

Tissue-specific FAH deficiency alters sleep-wake patterns and results in chronic tyrosinemia in mice.

Fumarylacetoacetate hydrolase (FAH) is the last enzyme in tyrosine catabolism, and mutations in the FAH gene are associated with hereditary tyrosinemia type I (HT1 or TYRSN1) in humans. In a behavioral screen of N-ethyl-N-nitrosourea mutagenized mice we identified a mutant line which we named "swingshift" (swst, MGI:3611216) with a nonsynonymous point mutation (N68S) in Fah that caused age-dependent disruption of sleep-wake patterns. Mice homozygous for the mutation had an earlier onset of activity (several hours before lights off) and a reduction in total activity and body weight when compared with wild-type or heterozygous mice. Despite abnormal behavioral entrainment to light-dark cycles, there were no differences in the period or phase of the central clock in mutant mice, indicating a defect downstream of the suprachiasmatic nucleus. Interestingly, these behavioral phenotypes became milder as the mice grew older and were completely rescued by the administration of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione], an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, which is upstream of FAH. Mechanistically, the swst mutation had no effect on the enzymatic activity of FAH, but rather promoted the degradation of the mutant protein. This led to reduced FAH protein levels and enzymatic activity in the liver and kidney (but not the brain or fibroblasts) of homozygous mice. In addition, plasma tyrosine-but not methionine, phenylalanine, or succinylacetone-increased in homozygous mice, suggesting that swst mutants provide a model of mild, chronic HT1.

Neuronal Myocyte-Specific Enhancer Factor 2D (MEF2D) Is Required for Normal Circadian and Sleep Behavior in Mice.

The transcription factor, myocyte enhancer factor-2 (MEF2), is required for normal circadian behavior in Drosophila; however, its role in the mammalian circadian system has not been established. Of the four mammalian Mef2 genes, Mef2d is highly expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus, a region critical for coordinating peripheral circadian clocks. Using both conventional and brain-specific Mef2d KO (Mef2d-/-) mouse lines, we demonstrate that MEF2D is essential for maintaining the length of the circadian free-running period of locomotor activity and normal sleep patterns in male mice. Crossing Mef2d-/- with Per2::luc reporter mice, we show that these behavioral changes are achieved without altering the endogenous period of the master circadian oscillator in the SCN. Together, our data suggest that alterations in behavior in Mef2d-/- mice may be the result of an effect on SCN output, rather than an effect on timekeeping within the SCN itself. These findings add to the growing body of evidence that MEF2 proteins play important roles in the brain.SIGNIFICANCE STATEMENT These studies are the first to show a role for MEF2 proteins in the brain outside of the hippocampus, and our findings suggest that these proteins may play diverse roles in the CNS. It is important to continue to build on our understanding of the roles of proteins acting in the SCN because SCN dysfunction underlies jet lag in humans and influences the response to shift work schedules, which are now known as risk factors for the development of cancer. Our work on MEF2D could be the basis for opening new lines of research in the development and regulation of circadian rhythms.

Modeling mutant/wild-type interactions to ascertain pathogenicity of PROKR2 missense variants in patients with isolated GnRH deficiency.

A major challenge in human genetics is the validation of pathogenicity of heterozygous missense variants. This problem is well-illustrated by PROKR2 variants associated with Isolated GnRH Deficiency (IGD). Homozygous, loss of function variants in PROKR2 was initially implicated in autosomal recessive IGD; however, most IGD-associated PROKR2 variants are heterozygous. Moreover, while IGD patient cohorts are enriched for PROKR2 missense variants similar rare variants are also found in normal individuals. To elucidate the pathogenic mechanisms distinguishing IGD-associated PROKR2 variants from rare variants in controls, we assessed 59 variants using three approaches: (i) in silico prediction, (ii) traditional in vitro functional assays across three signaling pathways with mutant-alone transfections, and (iii) modified in vitro assays with mutant and wild-type expression constructs co-transfected to model in vivo heterozygosity. We found that neither in silico analyses nor traditional in vitro assessments of mutants transfected alone could distinguish IGD variants from control variants. However, in vitro co-transfections revealed that 15/34 IGD variants caused loss-of-function (LoF), including 3 novel dominant-negatives, while only 4/25 control variants caused LoF. Surprisingly, 19 IGD-associated variants were benign or exhibited LoF that could be rescued by WT co-transfection. Overall, variants that were LoF in ≥ 2 signaling assays under co-transfection conditions were more likely to be disease-associated than benign or 'rescuable' variants. Our findings suggest that in vitro modeling of WT/Mutant interactions increases the resolution for identifying causal variants, uncovers novel dominant negative mutations, and provides new insights into the pathogenic mechanisms underlying heterozygous PROKR2 variants.

X-chromosome dosage affects male sexual behavior.

Sex differences in the brain and behavior are primarily attributed to dichotomous androgen exposure between males and females during neonatal development, as well as adult responses to gonadal hormones. Here we tested an alternative hypothesis and asked if sex chromosome complement influences male copulatory behavior, a standard behavior for studies of sexual differentiation. We used two mouse models with non-canonical associations between chromosomal and gonadal sex. In both models, we found evidence for sex chromosome complement as an important factor regulating sex differences in the expression of masculine sexual behavior. Counter intuitively, males with two X-chromosomes were faster to ejaculate and display more ejaculations than males with a single X. Moreover, mice of both sexes with two X-chromosomes displayed increased frequencies of mounts and thrusts. We speculate that expression levels of a yet to be discovered gene(s) on the X-chromosome may affect sexual behavior in mice and perhaps in other mammals.