The Polymethoxyflavone Sudachitin Modulates the Circadian Clock and Improves Liver Physiology.

SCOPE: Polymethoxylated flavones (PMFs) are a group of natural compounds known to display a wide array of beneficial effects to promote physiological fitness. Recent studies reveal circadian clocks as an important cellular mechanism mediating preventive efficacy of the major PMF Nobiletin against metabolic disorders. Sudachitin is a PMF enriched in Citrus sudachi, and its functions and mechanism of action are poorly understood. METHODS AND RESULTS: Using circadian reporter cells, it shows that Sudachitin modulates circadian amplitude and period of Bmal1 promoter-driven reporter rhythms, and real-time qPCR analysis shows that Sudachitin alters expression of core clock genes, notably Bmal1, at both transcript and protein levels. Mass-spec analysis reveals systemic exposure in vivo. In mice fed with high-fat diet with or without Sudachitin, it observes increased nighttime activity and daytime sleep, accompanied by significant metabolic improvements in a circadian time-dependent manner, including respiratory quotient, blood lipid and glucose profiles, and liver physiology. Focusing on liver, RNA-sequencing and metabolomic analyses reveal prevalent diurnal alteration in both gene expression and metabolite accumulation. CONCLUSION: This study elucidates Sudachitin as a new clock-modulating PMF with beneficial effects to improve diurnal metabolic homeostasis and liver physiology, suggesting the circadian clock as a fundamental mechanism to safeguard physiological well-being.

The circadian E3 ligase FBXL21 regulates myoblast differentiation and sarcomere architecture via MYOZ1 ubiquitination and NFAT signaling.

Numerous molecular and physiological processes in the skeletal muscle undergo circadian time-dependent oscillations in accordance with daily activity/rest cycles. The circadian regulatory mechanisms underlying these cyclic processes, especially at the post-transcriptional level, are not well defined. Previously, we reported that the circadian E3 ligase FBXL21 mediates rhythmic degradation of the sarcomere protein TCAP in conjunction with GSK-3ß, and Psttm mice harboring an Fbxl21 hypomorph allele show reduced muscle fiber diameter and impaired muscle function. To further elucidate the regulatory function of FBXL21 in skeletal muscle, we investigated another sarcomere protein, Myozenin1 (MYOZ1), that we identified as an FBXL21-binding protein from yeast 2-hybrid screening. We show that FBXL21 binding to MYOZ1 led to ubiquitination-mediated proteasomal degradation. GSK-3ß co-expression and inhibition were found to accelerate and decelerate FBXL21-mediated MYOZ1 degradation, respectively. Previously, MYOZ1 has been shown to inhibit calcineurin/NFAT signaling important for muscle differentiation. In accordance, Fbxl21 KO and MyoZ1 KO in C2C12 cells impaired and enhanced myogenic differentiation respectively compared with control C2C12 cells, concomitant with distinct effects on NFAT nuclear localization and NFAT target gene expression. Importantly, in Psttm mice, both the levels and diurnal rhythm of NFAT2 nuclear localization were significantly diminished relative to wild-type mice, and circadian expression of NFAT target genes associated with muscle differentiation was also markedly dampened. Furthermore, Psttm mice exhibited significant disruption of sarcomere structure with a considerable excess of MYOZ1 accumulation in the Z-line. Taken together, our study illustrates a pivotal role of FBXL21 in sarcomere structure and muscle differentiation by regulating MYOZ1 degradation and NFAT2 signaling.

ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by poor response to standard therapies and therefore unfavorable clinical outcomes. Better understanding of TNBC and new therapeutic strategies are urgently needed. ROR nuclear receptors are multifunctional transcription factors with important roles in circadian pathways and other processes including immunity and tumorigenesis. Nobiletin (NOB) is a natural compound known to display anticancer effects, and our previous studies showed that NOB activates RORs to enhance circadian rhythms and promote physiological fitness in mice. Here, we identified several TNBC cell lines being sensitive to NOB, by itself or in combination. Cell and xenograft experiments showed that NOB significantly inhibited TNBC cell proliferation and motility in vitro and in vivo. ROR loss- and gain-of-function studies showed concordant effects of the NOB-ROR axis on MDA-MB-231 cell growth. Mechanistically, we found that NOB activates ROR binding to the ROR response elements (RRE) of the IκBα promoter, and NOB strongly inhibited p65 nuclear translocation. Consistent with transcriptomic analysis indicating cancer and NF-κB signaling as major pathways altered by NOB, p65-inducible expression abolished NOB effects, illustrating a requisite role of NF-κB suppression mediating the anti-TNBC effect of NOB. Finally, in vivo mouse xenograft studies showed that NOB enhanced the antitumor efficacy in mammary fat pad implanted TNBC, as a single agent or in combination with the chemotherapy agent Docetaxel. Together, our study highlights an anti-TNBC mechanism of ROR-NOB via suppression of NF-κB signaling, suggesting novel preventive and chemotherapeutic strategies against this devastating disease.

The clock modulator Nobiletin mitigates astrogliosis-associated neuroinflammation and disease hallmarks in an Alzheimer's disease model.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, and there is a pressing need to identify disease-modifying factors and devise interventional strategies. The circadian clock, our intrinsic biological timer, orchestrates various cellular and physiological processes including gene expression, sleep, and neuroinflammation; conversely, circadian dysfunctions are closely associated with and/or contribute to AD hallmarks. We previously reported that the natural compound Nobiletin (NOB) is a clock-enhancing modulator that promotes physiological health and healthy aging. In the current study, we treated the double transgenic AD model mice, APP/PS1, with NOB-containing diets. NOB significantly alleviated ß-amyloid burden in both the hippocampus and the cortex, and exhibited a trend to improve cognitive function in these mice. While several systemic parameters for circadian wheel-running activity, sleep, and metabolism were unchanged, NOB treatment showed a marked effect on the expression of clock and clock-controlled AD gene expression in the cortex. In accordance, cortical proteomic profiling demonstrated circadian time-dependent restoration of the protein landscape in APP/PS1 mice treated with NOB. More importantly, we found a potent efficacy of NOB to inhibit proinflammatory cytokine gene expression and inflammasome formation in the cortex, and immunostaining further revealed a specific effect to diminish astrogliosis, but not microgliosis, by NOB in APP/PS1 mice. Together, these results underscore beneficial effects of a clock modulator to mitigate pathological and cognitive hallmarks of AD, and suggest a possible mechanism via suppressing astrogliosis-associated neuroinflammation.

Effects of the Clock Modulator Nobiletin on Circadian Rhythms and Pathophysiology in Female Mice of an Alzheimer's Disease Model.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder and the most common cause of dementia. Various pathogenic mechanisms have been proposed to contribute to disease progression, and recent research provided evidence linking dysregulated circadian rhythms/sleep and energy metabolism with AD. Previously, we found that the natural compound Nobiletin (NOB) can directly activate circadian cellular oscillators to promote metabolic health in disease models and healthy aging in naturally aged mice. In the current study, using the amyloid-ß AD model APP/PS1, we investigated circadian, metabolic and amyloid characteristics of female mice and the effects of NOB. Female APP/PS1 mice showed reduced sleep bout duration, and NOB treatment exhibited a trend to improve it. While glucose tolerance was unchanged, female APP/PS1 mice displayed exaggerated oxygen consumption and CO2 production, which was mitigated by NOB. Likewise, cold tolerance in APP/PS1 was impaired relative to WT, and interestingly was markedly enhanced in NOB-treated APP/PS1 mice. Although circadian behavioral rhythms were largely unchanged, real-time qPCR analysis revealed altered expression of several core clock genes by NOB in the cerebral cortex, notably Bmal1, Npas2, and Rora. Moreover, NOB was also able to activate various clock-controlled metabolic genes involved in insulin signaling and mitochondrial function, including Igf1, Glut1, Insr, Irs1, Ucp2, and Ucp4. Finally, we observed that NOB attenuated the expression of several AD related genes including App, Bace1, and ApoE, reduced APP protein levels, and strongly ameliorated Aß pathology in the cortex. Collectively, these results reveal novel genotype differences and importantly beneficial effects of a natural clock-enhancing compound in biological rhythms and related pathophysiology, suggesting the circadian clock as a modifiable target for AD.

Clock-Modulating Activities of the Anti-Arrhythmic Drug Moricizine.

Dysregulated circadian functions contribute to various diseases, including cardiovascular disease. Much progress has been made on chronotherapeutic applications of drugs against cardiovascular disease (CVD); however, the direct effects of various medications on the circadian system are not well characterized. We previously conducted high-throughput chemical screening for clock modulators and identified an off-patent anti-arrhythmic drug, moricizine, as a clock-period lengthening compound. In Per2:LucSV reporter fibroblast cells, we showed that under both dexamethasone and forskolin synchronization, moricizine was able to increase the circadian period length, with greater effects seen with the former. Titration studies revealed a dose-dependent effect of moricizine to lengthen the period. In contrast, flecainide, another Class I anti-arrhythmic, showed no effects on circadian reporter rhythms. Real-time qPCR analysis in fibroblast cells treated with moricizine revealed significant circadian time- and/or treatment-dependent expression changes in core clock genes, consistent with the above period-lengthening effects. Several clock-controlled cardiac channel genes also displayed altered expression patterns. Using tissue explant culture, we showed that moricizine was able to significantly prolong the period length of circadian reporter rhythms in atrial ex vivo cultures. Using wild-type C57BL/6J mice, moricizine treatment was found to promote sleep, alter circadian gene expression in the heart, and show a slight trend of increasing free-running periods. Together, these observations demonstrate novel clock-modulating activities of moricizine, particularly the period-lengthening effects on cellular oscillators, which may have clinical relevance against heart diseases.

The first-line cluster headache medication verapamil alters the circadian period and elicits sex-specific sleep changes in mice.

Verapamil is the first-line preventive medication for cluster headache, an excruciating disorder with strong circadian features. Whereas second- and third-line preventives include known circadian modulators, such as melatonin, corticosteroids, and lithium, the circadian effects of verapamil are poorly understood. Here, we characterize the circadian features of verapamil using both in vitro and in vivo models. In Per2::LucSV reporter fibroblasts, treatment with verapamil (0.03-10 µM) showed a dose-dependent period shortening of the reporter rhythm which reached a nadir at 1 µM, and altered core clock gene expression at 10 µM. Mouse wheel-running activity with verapamil (1 mg/mL added to the drinking water) also resulted in significant period shortening and activity reduction in both male and female free-running wild-type C57BL6/J mice. The temporal patterns of activity reduction, however, differ between the two sexes. Importantly, piezo sleep recording revealed sexual dimorphism in the effects of verapamil on sleep timing and bout duration, with more pronounced adverse effects in female mice. We also found altered circadian clock gene expression in the cerebellum, hypothalamus, and trigeminal ganglion of verapamil-treated mice. Verapamil did not affect reporter rhythms in ex vivo suprachiasmatic nucleus (SCN) slices from Per2:Luc reporter mice, perhaps due to the exceptionally tight coupling in the SCN. Thus, verapamil affects both peripheral (trigeminal ganglion) and central (hypothalamus and cerebellum) nervous system structures involved in cluster headache pathophysiology, possibly with network effects instead of isolated SCN effects. These studies suggest that verapamil is a circadian modulator in laboratory models at both molecular and behavioral levels, and sex is an important biological variable for cluster headache medications. These observations highlight the circadian system as a potential convergent target for cluster headache medications with different primary mechanisms of action.

The GSK-3ß-FBXL21 Axis Contributes to Circadian TCAP Degradation and Skeletal Muscle Function.

FBXL21 is a clock-controlled E3 ligase modulating circadian periodicity via subcellular-specific CRYPTOCHROME degradation. How FBXL21 regulates tissue-specific circadian physiology and what mechanism operates upstream is poorly understood. Here we report the sarcomere component TCAP as a cytoplasmic substrate of FBXL21. FBXL21 interacts with TCAP in a circadian manner antiphasic to TCAP accumulation in skeletal muscle, and circadian TCAP oscillation is disrupted in Psttm mice with an Fbxl21 hypomorph mutation. GSK-3ß phosphorylates FBXL21 and TCAP to activate FBXL21-mediated, phosphodegron-dependent TCAP degradation. GSK-3ß inhibition or knockdown diminishes FBXL21-Cul1 complex formation and delays FBXL21-mediated TCAP degradation. Finally, Psttm mice show significant skeletal muscle defects, including impaired fiber size, exercise tolerance, grip strength, and response to glucocorticoid-induced atrophy, in conjunction with cardiac dysfunction. These data highlight a circadian regulatory pathway where a GSK-3ß-FBXL21 functional axis controls TCAP degradation via SCF complex formation and regulates skeletal muscle function.

The Daam2-VHL-Nedd4 axis governs developmental and regenerative oligodendrocyte differentiation.

Dysregulation of the ubiquitin-proteasomal system (UPS) enables pathogenic accumulation of disease-driving proteins in neurons across a host of neurological disorders. However, whether and how the UPS contributes to oligodendrocyte dysfunction and repair after white matter injury (WMI) remains undefined. Here we show that the E3 ligase VHL interacts with Daam2 and their mutual antagonism regulates oligodendrocyte differentiation during development. Using proteomic analysis of the Daam2-VHL complex coupled with conditional genetic knockout mouse models, we further discovered that the E3 ubiquitin ligase Nedd4 is required for developmental myelination through stabilization of VHL via K63-linked ubiquitination. Furthermore, studies in mouse demyelination models and white matter lesions from patients with multiple sclerosis corroborate the function of this pathway during remyelination after WMI. Overall, these studies provide evidence that a signaling axis involving key UPS components contributes to oligodendrocyte development and repair and reveal a new role for Nedd4 in glial biology.

Circadian regulation of chemotherapy-induced peripheral neuropathic pain and the underlying transcriptomic landscape.

Growing evidence demonstrates circadian rhythms of pain hypersensitivity in various chronic disorders. In chemotherapy-induced peripheral neuropathy (CIPN), agents such as paclitaxel are known to elicit chronic neuropathic pain in cancer patients and seriously compromise their quality of life. Here, we report that the mechanical threshold for allodynia in paclitaxel-treated rats exhibited a robust circadian oscillation, reaching the nadir during the daytime (inactive phase). Using Per2::LucSV circadian reporter mice expressing a PER2::LUC fusion protein, we isolated dorsal root ganglia (DRG), the primary sensory cell body for peripheral nerve injury generated hypersensitivity, and monitored ex vivo reporter bioluminescence. We observed strong circadian reporter rhythms in DRG neurons which are highly entrainable by external cues. Paclitaxel treatment significantly lengthened DRG circadian periods, with little effects on the amplitude of oscillation. We further observed the core protein BMAL1 and PER2 in DRG neurons and satellite cells. Using DRG and dorsal horn (DH; another key structure for CIPN pain response) tissues from vehicle and paclitaxel treated rats, we performed RNA-sequencing and identified diurnal expression of core clock genes as well as clock-controlled genes in both sites. Interestingly, 20.1% and 30.4% of diurnal differentially expressed genes (DEGs) overlapped with paclitaxel-induced DEGs in the DRG and the DH respectively. In contrast, paclitaxel-induced DEGs displayed only a modest overlap between daytime and nighttime (Zeitgeber Time 8 and 20). Furthermore, paclitaxel treatment induced de novo diurnal DEGs, suggesting reciprocal interaction of circadian rhythms and chemotherapy. Our study therefore demonstrates a circadian oscillation of CIPN and its underlying transcriptomic landscape.

Cardiolipin Synthesis in Skeletal Muscle Is Rhythmic and Modifiable by Age and Diet.

Circadian clocks regulate metabolic processes in a tissue-specific manner, which deteriorates during aging. Skeletal muscle is the largest metabolic organ in our body, and our previous studies highlight a key role of circadian regulation of skeletal muscle mitochondria in healthy aging. However, a possible circadian regulation of cardiolipin (CL), the signature lipid class in the mitochondrial inner membrane, remains largely unclear. Here, we show that CL levels oscillate during the diurnal cycle in C2C12 myotubes. Disruption of the Ror genes, encoding the ROR nuclear receptors in the secondary loop of the circadian oscillator, in C2C12 cells was found to dampen core circadian gene expression. Importantly, several genes involved in CL synthesis, including Taz and Ptpmt1, displayed rhythmic expression which was disrupted or diminished in Ror-deficient C2C12 cells. In vivo studies using skeletal muscle tissues collected from young and aged mice showed diverse effects of the clock and aging on the oscillatory expression of CL genes, and CL levels in skeletal muscle were enhanced in aged mice relative to young mice. Finally, consistent with a regulatory role of RORs, Nobiletin, a natural agonist of RORs, was found to partially restore transcripts levels of CL synthesis genes in aged muscle under a dietary challenge condition. Together, these observations highlight a rhythmic CL synthesis in skeletal muscle that is dependent on RORs and modifiable by age and diet.

Nobiletin fortifies mitochondrial respiration in skeletal muscle to promote healthy aging against metabolic challenge.

Circadian disruption aggravates age-related decline and mortality. However, it remains unclear whether circadian enhancement can retard aging in mammals. We previously reported that the small molecule Nobiletin (NOB) activates ROR (retinoid acid receptor-related orphan receptor) nuclear receptors to potentiate circadian oscillation and protect against metabolic dysfunctions. Here we show that NOB significantly improves metabolic fitness in naturally aged mice fed with a regular diet (RD). Furthermore, NOB enhances healthy aging in mice fed with a high-fat diet (HF). In HF skeletal muscle, the NOB-ROR axis broadly activates genes for mitochondrial respiratory chain complexes (MRCs) and fortifies MRC activity and architecture, including Complex II activation and supercomplex formation. These mechanisms coordinately lead to a dichotomous mitochondrial optimization, namely increased ATP production and reduced ROS levels. Together, our study illustrates a focal mechanism by a clock-targeting pharmacological agent to optimize skeletal muscle mitochondrial respiration and promote healthy aging in metabolically stressed mammals.