CLOCKΔ19 mutation modifies the manner of synchrony among oscillation neurons in the suprachiasmatic nucleus.

In mammals, the principal circadian oscillator exists in the hypothalamic suprachiasmatic nucleus (SCN). In the SCN, CLOCK works as an essential component of molecular circadian oscillation, and ClockΔ19 mutant mice show unique characteristics of circadian rhythms such as extended free running periods, amplitude attenuation, and high-magnitude phase-resetting responses. Here we investigated what modifications occur in the spatiotemporal organization of clock gene expression in the SCN of ClockΔ19 mutants. The cultured SCN, sampled from neonatal homozygous ClockΔ19 mice on an ICR strain comprising PERIOD2::LUCIFERASE, demonstrated that the Clock gene mutation not only extends the circadian period, but also affects the spatial phase and period distribution of circadian oscillations in the SCN. In addition, disruption of the synchronization among neurons markedly attenuated the amplitude of the circadian rhythm of individual oscillating neurons in the mutant SCN. Further, with numerical simulations based on the present studies, the findings suggested that, in the SCN of the ClockΔ19 mutant mice, stable oscillation was preserved by the interaction among oscillating neurons, and that the orderly phase and period distribution that makes a phase wave are dependent on the functionality of CLOCK.

Establishment of TSH ß real-time monitoring system in mammalian photoperiodism.

Organisms have seasonal physiological changes in response to day length. Long-day stimulation induces thyroid-stimulating hormone beta subunit (TSHß) in the pars tuberalis (PT), which mediates photoperiodic reactions like day-length measurement and physiological adaptation. However, the mechanism of TSHß induction for day-length measurement is largely unknown. To screen candidate upstream molecules of TSHß, which convey light information to the PT, we generated Luciferase knock-in mice, which quantitatively report the dynamics of TSHß expression. We cultured brain slices containing the PT region from adult and neonatal mice and measured the bioluminescence activities from each slice over several days. A decrease in the bioluminescence activities was observed after melatonin treatment in adult and neonatal slices. These observations indicate that the experimental system possesses responsiveness of the TSHß expression to melatonin. Thus, we concluded that our experimental system monitors TSHß expression dynamics in response to external stimuli.

Variations in PROKR2, but not PROK2, are associated with hypopituitarism and septo-optic dysplasia.

CONTEXT: Loss-of-function mutations in PROK2 and PROKR2 have been implicated in Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia. Recent data suggest overlapping phenotypes/genotypes between KS and congenital hypopituitarism (CH), including septo-optic dysplasia (SOD). OBJECTIVE: We screened a cohort of patients with complex forms of CH (n = 422) for mutations in PROK2 and PROKR2. RESULTS: We detected 5 PROKR2 variants in 11 patients with SOD/CH: novel p.G371R and previously reported p.A51T, p.R85L, p.L173R, and p.R268C-the latter 3 being known functionally deleterious variants. Surprisingly, 1 patient with SOD was heterozygous for the p.L173R variant, whereas his phenotypically unaffected mother was homozygous for the variant. We sought to clarify the role of PROKR2 in hypothalamopituitary development through analysis of Prokr2(-/-) mice. Interestingly, these revealed predominantly normal hypothalamopituitary development and terminal cell differentiation, with the exception of reduced LH; this was inconsistent with patient phenotypes and more analogous to the healthy mother, although she did not have KS, unlike the Prokr2(-/-) mice. CONCLUSIONS: The role of PROKR2 in the etiology of CH, SOD, and KS is uncertain, as demonstrated by no clear phenotype-genotype correlation; loss-of-function variants in heterozygosity or homozygosity can be associated with these disorders. However, we report a phenotypically normal parent, homozygous for p.L173R. Our data suggest that the variants identified herein are unlikely to be implicated in isolation in these disorders; other genetic or environmental modifiers may also impact on the etiology. Given the phenotypic variability, genetic counseling may presently be inappropriate.

Acute induction of Eya3 by late-night light stimulation triggers TSHß expression in photoperiodism.

Living organisms detect seasonal changes in day length (photoperiod) [1-3] and alter their physiological functions accordingly to fit seasonal environmental changes. TSHß, induced in the pars tuberalis (PT), plays a key role in the pathway that regulates vertebrate photoperiodism [4, 5]. However, the upstream inducers of TSHß expression remain unknown. Here we performed genome-wide expression analysis of the PT under chronic short-day and long-day conditions in melatonin-proficient CBA/N mice, in which the photoperiodic TSHß expression response is preserved [6]. This analysis identified "short-day" and "long-day" genes, including TSHß, and further predicted the acute induction of long-day genes by late-night light stimulation. We verified this by advancing and extending the light period by 8 hr, which induced TSHß expression within one day. In the following genome-wide expression analysis under this acute long-day condition, we searched for candidate upstream genes by looking for expression that preceded TSHß's, and we identified the Eya3 gene. We demonstrated that Eya3 and its partner Six1 synergistically activate TSHß expression and that this activation is further enhanced by Tef and Hlf. These results elucidate the comprehensive transcriptional photoperiodic response in the PT, revealing the complex regulation of TSHß expression and unexpectedly rapid response to light changes in the mammalian photoperiodic system.

Melanopsin-dependent photo-perturbation reveals desynchronization underlying the singularity of mammalian circadian clocks.

Singularity behaviour in circadian clocks--the loss of robust circadian rhythms following exposure to a stimulus such as a pulse of bright light--is one of the fundamental but mysterious properties of clocks. To quantitatively perturb and accurately measure the dynamics of cellular clocks, we synthetically produced photo-responsiveness within mammalian cells by exogenously introducing the photoreceptor melanopsin and continuously monitoring the effect of photo-perturbation on the state of cellular clocks. Here we report that a critical light pulse drives cellular clocks into singularity behaviour. Our theoretical analysis consistently predicts and subsequent single-cell level observation directly proves that desynchronization of individual cellular clocks underlies singularity behaviour. Our theoretical framework also explains why singularity behaviours have been experimentally observed in various organisms, and it suggests that desynchronization is a plausible mechanism for the observable singularity of circadian clocks. Importantly, these in vitro and in silico findings are further supported by in vivo observations that desynchronization underlies the multicell-level amplitude decrease in the rat suprachiasmatic nucleus induced by critical light pulses.

Distinct localization of prokineticin 2 and prokineticin receptor 2 mRNAs in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian clock that regulates physiological and behavioral circadian rhythms in mammals. Prokineticin 2 (PK2) is highly expressed in the SCN, and its involvement in the generation of circadian locomotor activity has been reported previously. In the present study, using in situ hybridization methods, we investigated the localization of PK2 and prokineticin receptor 2 (PKR2), a specific receptor for PK2, in the rat SCN. In steady light : dark (L : D = 12 : 12 h) and constant dark conditions, rPK2 mRNA displayed a robust circadian oscillation with a peak occurring during the day. Moreover, during peak expression, the rPK2 mRNA-positive neurons were scattered in both the dorsomedial and ventrolateral SCN, which are two functionally and morphologically distinct subregions. Furthermore, double-labeling in situ hybridization experiments revealed that greater than 50% of the rPK2 mRNA-containing neurons co-expressed either vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) or arginine vasopressin (AVP) in the SCN. In contrast, the rPKR2 mRNA levels did not show significant diurnal alterations. rPKR2 mRNA-containing neurons were also clustered in the dorsolateral part of the SCN, which shows negligible labeling of either rAVP, rVIP, rGRP or rPK2 transcripts. In addition, this region exhibited a delayed cycling of the rPer1 gene. These results suggest an intrinsic PK2 neurotransmission and functionally distinct roles for PKR2-expressing neurons in the SCN.

Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2.

Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-)but Pkr2(-/-)mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.

Effect of phosphodiesterase type 4 on circadian clock gene Per1 transcription.

The induction of Per1 gene in the suprachiasmatic nucleus, the center of the circadian clock, is assumed to play significant roles in the adjustment of the internal clock. cAMP is one of the intracellular mediators which activates Per1 transcription. Here, we showed that the amount of the rat Per1 (rPer1) transcript induced by forskolin (FK) was significantly upregulated by the inhibition of phosphodiesterase type 4 (PDE4), a specific phosphodiesterase for cAMP, in rat-1 fibroblasts. Administration of rolipram, a specific inhibitor of PDE4, increased intracellular cAMP concentration, phosphorylation of cAMP response element binding protein (CREB) and enhanced rPer1 induction at their peaks. However, in the falling phase of rPer1 induction, the inhibition of PDE4 hardly affected the profile of rPer1 expression. These findings suggest the involvement of PDE4 for the regulation of rPer1 expression via cAMP metabolism at peak of the induction but little or no participation of PDE4 in the decreasing phase of the gene expression.