Kisspeptin Neuron-Specific and Self-Sustained Calcium Oscillation in the Hypothalamic Arcuate Nucleus of Neonatal Mice: Regulatory Factors of its Synchronization.

INTRODUCTION: Synchronous and pulsatile neural activation of kisspeptin neurons in the arcuate nucleus (ARN) are important components of the gonadotropin-releasing hormone pulse generator, the final common pathway for central regulation of mammalian reproduction. However, whether ARN kisspeptin neurons can intrinsically generate self-sustained synchronous oscillations from the early neonatal period and how they are regulated remain unclear. OBJECTIVE: This study aimed to examine the endogenous rhythmicity of ARN kisspeptin neurons and its neural regulation using a neonatal organotypic slice culture model. METHODS: We monitored calcium (Ca2+) dynamics in real-time from individual ARN kisspeptin neurons in neonatal organotypic explant cultures of Kiss1-IRES-Cre mice transduced with genetically encoded Ca2+ indicators. Pharmacological approaches were employed to determine the regulations of kisspeptin neuron-specific Ca2+ oscillations. A chemogenetic approach was utilized to assess the contribution of ARN kisspeptin neurons to the population dynamics. RESULTS: ARN kisspeptin neurons in neonatal organotypic cultures exhibited a robust synchronized Ca2+ oscillation with a period of approximately 3 min. Kisspeptin neuron-specific Ca2+ oscillations were dependent on voltage-gated sodium channels and regulated by endoplasmic reticulum-dependent Ca2+ homeostasis. Chemogenetic inhibition of kisspeptin neurons abolished synchronous Ca2+ oscillations, but the autocrine actions of the neuropeptides were marginally effective. Finally, neonatal ARN kisspeptin neurons were regulated by N-methyl-D-aspartate and gamma-aminobutyric acid receptor-mediated neurotransmission. CONCLUSION: These data demonstrate that ARN kisspeptin neurons in organotypic cultures can generate synchronized and self-sustained Ca2+ oscillations. These oscillations controlled by multiple regulators within the ARN are a novel ultradian rhythm generator that is active during the early neonatal period.

Role of the circadian nuclear receptor REV-ERBα in dorsal raphe serotonin synthesis in mood regulation.

Affective disorders are frequently associated with disrupted circadian rhythms. The existence of rhythmic secretion of central serotonin (5-hydroxytryptamine, 5-HT) pattern has been reported; however, the functional mechanism underlying the circadian control of 5-HTergic mood regulation remains largely unknown. Here, we investigate the role of the circadian nuclear receptor REV-ERBα in regulating tryptophan hydroxylase 2 (Tph2), the rate-limiting enzyme of 5-HT synthesis. We demonstrate that the REV-ERBα expressed in dorsal raphe (DR) 5-HTergic neurons functionally competes with PET-1-a nuclear activator crucial for 5-HTergic neuron development. In mice, genetic ablation of DR 5-HTergic REV-ERBα increases Tph2 expression, leading to elevated DR 5-HT levels and reduced depression-like behaviors at dusk. Further, pharmacological manipulation of the mice DR REV-ERBα activity increases DR 5-HT levels and affects despair-related behaviors. Our findings provide valuable insights into the molecular and cellular link between the circadian rhythm and the mood-controlling DR 5-HTergic systems.

Impact of the circadian nuclear receptor REV-ERBα in dorsal raphe 5-HT neurons on social interaction behavior, especially social preference.

Social interaction among conspecifics is essential for maintaining adaptive, cooperative, and social behaviors, along with survival among mammals. The 5-hydroxytryptamine (5-HT) neuronal system is an important neurotransmitter system for regulating social behaviors; however, the circadian role of 5-HT in social interaction behaviors is unclear. To investigate whether the circadian nuclear receptor REV-ERBα, a transcriptional repressor of the rate-limiting enzyme tryptophan hydroxylase 2 (Tph2) gene in 5-HT biosynthesis, may affect social interaction behaviors, we generated a conditional knockout (cKO) mouse by targeting Rev-Erbα in dorsal raphe (DR) 5-HT neurons (5-HTDR-specific REV-ERBα cKO) using the CRISPR/Cas9 gene editing system and assayed social behaviors, including social preference and social recognition, with a three-chamber social interaction test at two circadian time (CT) points, i.e., at dawn (CT00) and dusk (CT12). The genetic ablation of Rev-Erbα in DR 5-HTergic neurons caused impaired social interaction behaviors, particularly social preference but not social recognition, with no difference between the two CT points. This deficit of social preference induced by Rev-Erbα in 5-HTDR-specific mice is functionally associated with real-time elevated neuron activity and 5-HT levels at dusk, as determined by fiber-photometry imaging sensors. Moreover, optogenetic inhibition of DR to nucleus accumbens (NAc) 5-HTergic circuit restored the impairment of social preference in 5-HTDR-specific REV-ERBα cKO mice. These results suggest the significance of the circadian regulation of 5-HT levels by REV-ERBα in regulating social interaction behaviors.

Pharmacological Rescue with SR8278, a Circadian Nuclear Receptor REV-ERBα Antagonist as a Therapy for Mood Disorders in Parkinson's Disease.

Parkinson's disease is a neurodegenerative disease characterized by progressive dopaminergic neuronal loss. Motor deficits experienced by patients with Parkinson's disease are well documented, but non-motor symptoms, including mood disorders associated with circadian disturbances, are also frequent features. One common phenomenon is "sundowning syndrome," which is characterized by the occurrence of neuropsychiatric symptoms at a specific time (dusk), causing severe quality of life challenges. This study aimed to elucidate the underlying mechanisms of sundowning syndrome in Parkinson's disease and their molecular links with the circadian clock. We demonstrated that 6-hydroxydopamine (6-OHDA)-lesioned mice, as Parkinson's disease mouse model, exhibit increased depression- and anxiety-like behaviors only at dawn (the equivalent of dusk in human). Administration of REV-ERBα antagonist, SR8278, exerted antidepressant and anxiolytic effects in a circadian time-dependent manner in 6-OHDA-lesioned mice and restored the circadian rhythm of mood-related behaviors. 6-OHDA-lesion altered DAergic-specific Rev-erbα and Nurr1 transcription, and atypical binding activities of REV-ERBα and NURR1, which are upstream nuclear receptors for the discrete tyrosine hydroxylase promoter region. SR8278 treatment restored the binding activities of REV-ERBα and NURR1 to the tyrosine hydroxylase promoter and the induction of enrichment of the R/N motif, recognized by REV-ERBα and NURR1, as revealed by ATAC-sequencing; therefore, tyrosine hydroxylase expression was elevated in the ventral tegmental area of 6-OHDA-injected mice, especially at dawn. These results indicate that REV-ERBα is a potential therapeutic target, and its antagonist, SR8278, is a potential drug for mood disorders related to circadian disturbances, namely sundowning syndrome, in Parkinson's disease.

microRNA-25 as a novel modulator of circadian Period2 gene oscillation.

Circadian clock controls an organism's biological rhythm and regulates its physiological processes in response to external time cues. Most living organisms have their own time-keeping mechanism that is maintained by transcriptional-translational autoregulatory feedback loops involving several core clock genes, such as Period. Recent studies have found the relevance between the modulation of circadian oscillation and posttranscriptional modifications by microRNAs (miRNAs). However, there are limited studies on candidate miRNAs that regulate circadian oscillation. Here, we characterize the functions of novel miRNA-25 regulating circadian Period2 (Per2) expression. Using several in silico algorithms, we identified novel miR-25-3p that, together with miR-24-3p, targets the Per2 gene. Luciferase reporter assays validated that miR-25-3p and miR-24-3p repressed Per2 expression and confirmed their predicted binding sites in the 3'-untranslated region (UTR) of Per2 mRNA. Real-time bioluminescence analyses using Per2::Luc mouse embryonic fibroblasts confirmed that PER2 protein oscillation patterns were responsive to miR-25-3p and miR-24-3. The overexpression of miR-25-3p or miR-24-3p resulted in the dampening and period shortening of the PER2::LUC oscillation, while inhibition of either miRNA increased the relative amplitude of the PER2::LUC oscillation. Notably, endogenous miR-25-3p expression in the suprachiasmatic nucleus (SCN) showed no circadian rhythmicity, but the expression levels differed in various brain regions and peripheral tissues. These results suggest that the posttranscriptional regulation of miR-25-3p and miR-24-3p may differ according to Per2 gene expression in different tissue regions. In summary, we found that novel miR-25-3p was involved in fine-tuning circadian rhythmicity by regulating Per2 oscillation at the posttranscriptional level and that it functioned synergistically with miR-24-3p to affect Per2 oscillation.

Gut Microbial Metabolites Induce Changes in Circadian Oscillation of Clock Gene Expression in the Mouse Embryonic Fibroblasts.

Circadian rhythm is an endogenous oscillation of about 24-h period in many physiological processes and behaviors. This daily oscillation is maintained by the molecular clock machinery with transcriptional-translational feedback loops mediated by clock genes including Period2 (Per2) and Bmal1. Recently, it was revealed that gut microbiome exerts a significant impact on the circadian physiology and behavior of its host; however, the mechanism through which it regulates the molecular clock has remained elusive. 3-(4-hydroxyphenyl)propionic acid (4-OH-PPA) and 3-phenylpropionic acid (PPA) are major metabolites exclusively produced by Clostridium sporogenes and may function as unique chemical messengers communicating with its host. In the present study, we examined if two C. sporogenes-derived metabolites can modulate the oscillation of mammalian molecular clock. Interestingly, 4-OH-PPA and PPA increased the amplitude of both PER2 and Bmal1 oscillation in a dosedependent manner following their administration immediately after the nadir or the peak of their rhythm. The phase of PER2 oscillation responded differently depending on the mode of administration of the metabolites. In addition, using an organotypic slice culture ex vivo, treatment with 4-OH-PPA increased the amplitude and lengthened the period of PER2 oscillation in the suprachiasmatic nucleus and other tissues. In summary, two C. sporogenes-derived metabolites are involved in the regulation of circadian oscillation of Per2 and Bmal1 clock genes in the host's peripheral and central clock machineries.

Abrogation of the Circadian Nuclear Receptor REV-ERBα Exacerbates 6-Hydroxydopamine-Induced Dopaminergic Neurodegeneration.

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive degeneration of dopaminergic (DAergic) neurons, particularly in the substantia nigra (SN). Although circadian dysfunction has been suggested as one of the pathophysiological risk factors for PD, the exact molecular link between the circadian clock and PD remains largely unclear. We have recently demonstrated that REV-ERBα, a circadian nuclear receptor, serves as a key molecular link between the circadian and DAergic systems. It competitively cooperates with NURR1, another nuclear receptor required for the optimal development and function of DA neurons, to control DAergic gene transcription. Considering our previous findings, we hypothesize that REV-ERBα may have a role in the onset and/or progression of PD. In the present study, we therefore aimed to elucidate whether genetic abrogation of REV-ERBα affects PD-related phenotypes in a mouse model of PD produced by a unilateral injection of 6-hydroxydopamine (6-OHDA) into the dorsal striatum. REV-ERBα deficiency significantly exacerbated 6-OHDA-induced motor deficits as well as DAergic neuronal loss in the vertebral midbrain including the SN and the ventral tegmental area. The exacerbated DAergic degeneration likely involves neuroinflammation-mediated neurotoxicity. The Rev-erbα knockout mice showed prolonged microglial activation in the SN along with the overproduction of interleukin 1ß, a pro-inflammatory cytokine, in response to 6-OHDA. In conclusion, the present study demonstrates for the first time that genetic abrogation of REV-ERBα can increase vulnerability of DAergic neurons to neurotoxic insults, such as 6-OHDA, thereby implying that its normal function may be beneficial for maintaining DAergic neuron populations during PD progression.

Implications of Circadian Rhythm in Dopamine and Mood Regulation.

Mammalian physiology and behavior are regulated by an internal time-keeping system, referred to as circadian rhythm. The circadian timing system has a hierarchical organization composed of the master clock in the suprachiasmatic nucleus (SCN) and local clocks in extra-SCN brain regions and peripheral organs. The circadian clock molecular mechanism involves a network of transcription-translation feedback loops. In addition to the clinical association between circadian rhythm disruption and mood disorders, recent studies have suggested a molecular link between mood regulation and circadian rhythm. Specifically, genetic deletion of the circadian nuclear receptor Rev-erbα induces mania-like behavior caused by increased midbrain dopaminergic (DAergic) tone at dusk. The association between circadian rhythm and emotion-related behaviors can be applied to pathological conditions, including neurodegenerative diseases. In Parkinson's disease (PD), DAergic neurons in the substantia nigra pars compacta progressively degenerate leading to motor dysfunction. Patients with PD also exhibit non-motor symptoms, including sleep disorder and neuropsychiatric disorders. Thus, it is important to understand the mechanisms that link the molecular circadian clock and brain machinery in the regulation of emotional behaviors and related midbrain DAergic neuronal circuits in healthy and pathological states. This review summarizes the current literature regarding the association between circadian rhythm and mood regulation from a chronobiological perspective, and may provide insight into therapeutic approaches to target psychiatric symptoms in neurodegenerative diseases involving circadian rhythm dysfunction.