Beyond Conventional Therapies: Molecular Dynamics of Alzheimer's Treatment through CLOCK/BMAL1 Interactions.

BACKGROUND: Alzheimer's Disease (AD) represents a neurodegenerative disorder characterized by cognitive and behavioral impairments significantly hindering social and occupational functioning. Melatonin, a hormone pivotal in regulating the body's intrinsic circadian rhythm, also acts as a catalyst in the breakdown of beta-amyloid deposits, offering a promising therapeutic approach for AD. The upregulation of Brain and Muscle ARNT-Like 1 (Bmal1) gene expression, stimulated by melatonin, emerges as a potential contributor to AD intervention. Current pharmacological interventions, such as FDA-approved cholinesterase inhibitors and the recently authorized monoclonal antibody, Lecanemab, are utilized in AD management. However, the connection between these medications and Bmal1 remains insufficiently explored. OBJECTIVE: This study aims to investigate the molecular effects of FDA-endorsed drugs on the CLOCK: Bmal1 dimer. Furthermore, considering the interactions between melatonin and Bmal1, this research explores the potential synergistic efficacy of combining these pharmaceutical agents with melatonin for AD treatment. METHODS: Using molecular docking and MM/PBSA methodologies, this research determines the binding affinities of drugs within the Bmal1 binding site, constructing interaction profiles. RESULTS: The findings reveal that, among FDA-approved drugs, galanthamine and donepezil demonstrate notably similar binding energy values to melatonin, interacting within the Bmal1 binding site through analogous amino acid residues and functional groups. CONCLUSION: A novel therapeutic approach emerges, suggesting the combination of melatonin with Lecanemab as a monoclonal antibody therapy. Importantly, prior research has not explored the effects of FDA-approved drugs on Bmal1 expression or their potential for synergistic effects.

Circadian regulation of MGMT expression and promoter methylation underlies daily rhythms in TMZ sensitivity in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. METHODS AND RESULTS: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. CONCLUSION: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity and that scoring MGMT methylation status requires controlling for time of day of biopsy.

Hypothalamic astrocytic-BMAL1 regulates energy homeostasis in a sex-dependent manner.

Here, we demonstrate that hypothalamic astrocytic BMAL1 computes cyclic metabolic information to optimize energetic resources in a sexually dimorphic manner. Knockdown of BMAL1 in female astrocytes leads to negative energy balance and alters basal metabolic cycles without affecting circadian locomotor activity. Thus, astrocytic BMAL1 contributes to the control of energy balance through the modulation of the metabolic rate, hepatic and white adipose tissue lipogenesis, and the activity of brown adipose tissue. Importantly, most of these alterations are specific to hypothalamic astrocytic BMAL1. Moreover, female mice with BMAL1 knockdown in astrocytes exhibited a "male-like" metabolic obese phenotype when fed a high-fat diet. Overall, our results suggest a sexually dimorphic effect of astrocytic BMAL1 on the regulation of energy homeostasis, which may be of interest in the physiopathology of obesity and related comorbidities.

The effect of 24-hour sleep deprivation and anaerobic exercise on the expression of BAX, BCL2, BMAL1 and CCAR2 genes in peripheral blood mononuclear cells after L-arginine supplementation.

Sleep deprivation disrupt the circadian clock and exercise performance. Defective oxidative stress caused by sleep deprivation may affect the expression of genes involved in cell apoptosis. Since a number of studies have shown the anti-apoptotic effect of L-arginine, so the aim of this study was to evaluate the effect of eight weeks of L-arginine supplementation on the expression of brain and muscle ARNT-like protein 1 (BMAL1), cell cycle and apoptosis regulator 2 (CCAR2), and BAX and BCL2 genes during sleep deprivation and acute anaerobic exercise. Participants included 20 healthy men age 26-35 years, randomized into the L-arginine intervention group (n = 10) and a placebo control (n = 10). The running-based anaerobic sprint test (RAST) was used for anaerobic exercise. Intervention subjects took one 1000 mg L-arginine tablet daily for 8 weeks. The Real-Time PCR method was used to determine apoptosis gene expression in peripheral blood mononuclear cells (PBMCs). Acute anaerobic exercise and sleep deprivation both increased the expression of BAX and CCAR2 genes, and decreased the expression of BCL2 and BMAL1 genes (p < 0.05 for all). L-arginine supplementation increased the expression of BMAL1 and BCL2 genes and decreased the expression of BAX and CCAR2 genes relative to control (p < 0.05). L-Arginine controlled the increase in expression of BAX and CCAR2 genes and the decrease in expression of BCL2 and BMAL1 genes in response to sleep deprivation and acute anaerobic exercise (p < 0.05). Our results showed that 24-hour sleep deprivation and acute anaerobic exercise increased the expression of pro-apoptotic genes (BAX and CCAR2) and decreased the expression of anti-apoptotic genes (BCL2 and BMAL1), although the effect of sleep deprivation is greater. In this situation, L-arginine supplementation may balance the apoptotic state of peripheral blood mononuclear cells. However, any recommendation about this needs further research.

Watching the clock in glioblastoma.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor, accounting for 14.2% of all diagnosed tumors and 50.1% of all malignant tumors, and the median survival time is approximately 8 months irrespective of whether a patient receives treatment without significant improvement despite expansive research (Ostrom QT, Price M, Neff C, et al. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2015-2019. Neurooncology. 2022; 24(suppl 5):v1-v95.). Recently, important roles for the circadian clock in GBM tumorigenesis have been reported. Positive regulators of circadian-controlled transcription, brain and muscle ARNT-like 1 (BMAL1), and circadian locomotor output cycles kaput (CLOCK), are highly expressed also in GBM and correlated with poor patient prognosis. BMAL1 and CLOCK promote the maintenance of GBM stem cells (GSCs) and the establishment of a pro-tumorigenic tumor microenvironment (TME), suggesting that targeting the core clock proteins may augment GBM treatment. Here, we review findings that highlight the critical role the circadian clock plays in GBM biology and the strategies by which the circadian clock can be leveraged for GBM treatment in the clinic moving forward.

Grape Seed Proanthocyanidins Modulate the Hepatic Molecular Clock via MicroRNAs.

SCOPE: Circadian rhythm is an endogenous and self-sustained timing system, responsible for the coordination of daily processes in 24-h timescale. It is regulated by an endogenous molecular clock, which is sensitive to external cues as light and food. This study has previously shown that grape seed proanthocyanidins extract (GSPE) regulates the hepatic molecular clock. Moreover, GSPE is known to interact with some microRNAs (miRNAs). Therefore, the aim of this study is to evaluate if the activity of GSPE as modulator of hepatic clock genes can be mediated by miRNAs. METHODS AND RESULTS: 250 mg kg-1 of GSPE is administered to Wistar rats before a 6-h jet lag and sacrificed at different time points. GSPE modulated both expression of Bmal1 and miR-27b-3p in the liver. Cosinor-based analysis reveals that both Bmal1 and miR-27b-3p expression follow a circadian rhythm, a negative interaction between them, and the role of GSPE adjusting the hepatic peripheral clock via miRNA. Additionally, in vitro studies show that Bmal1 is sensitive to GSPE (25 mg L-1 ). However, this effect is independent of miR-27b-3p. CONCLUSION: miRNA regulation of peripheral clocks via GSPE may be part of a complex mechanism that involves the crosstalk with the central system rather than a direct effect.

Sleep deprivation affects sex hormones secretion by regulating the expression of the circadian clock gene in the hypothalamus and pituitary via the PI3K/Akt signaling pathway in pregnant rats.

Sleep deprivation (SD) has many deleterious health effects and occurs in more than 70% of pregnant women. However, the changes in sex hormones and relevant mechanisms after SD have not been well clarified. The aim of the present study was to explore the effects of SD on the secretion of sex hormones and the underlying mechanisms. Twelve pregnant Wistar rats were divided into control (CON, n = 6) and SD (n = 6) groups. Pregnant rats in the SD group were deprived of sleep for 18 h, and allowed free rest for 6 h, and then the above procedures were repeated until delivery. The CON group lived in a 12 h light/dark light cycle environment. Estradiol (E2) and progesterone (P4) levels were detected by enzyme-linked immunosorbent assay (ELISA), and the expression of circadian clock genes, Bmal1, Clock and Per2, in hypothalamus and pituitary gland tissues were evaluated by immunohistochemistry (IHC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The PI3K and Akt phosphorylation levels in the hypothalamic and pituitary tissues were determined by Western blot. The results showed that, compared with the CON group, the SD group exhibited significantly reduced serum E2 and P4 levels, down-regulated Bmal1, Clock and Per2 expression, as well as decreased phosphorylation levels of PI3K and Akt. But there was no significant difference of the total PI3K and Akt protein expression levels between the two groups. These results suggest that SD might affect the expression of the circadian clock genes in the hypothalamus and pituitary via PI3K/Akt pathway, and subsequently regulate the secretion of sex hormones in the pregnant rats, which hints the important roles of SD-induced changes of serum sex hormone levels in the pregnant rats.

Drugging the circadian clock feedback cycle to ameliorate cartilage degeneration.

Dampened peripheral clocks have been linked to osteoarthritis (OA), yet it is unclear whether drugging the clock can ameliorate OA. Given that RORs and REV-ERBs mediate respectively, positive and negative transcriptional feedback of the master clock gene BMAL1, we investigate whether RORs agonist Nobiletin (NOB) and SR1078, and REV-ERBs antagonist SR8278 can enhance BMAL1 expression and attenuate cartilage degeneration. NOB and SR8278 promoted BMAL1 expression and elicited mitigating effects against IL-1ß-induced degeneration of cartilage explants, as evidenced by increased cellular density and collagen synthesis along with alleviated catabolism and collagen denaturation. Despite promoted BMAL1 expression, SR1078 concomitantly suppressed chondrocyte anabolism and catabolism. Consistent with these findings, NOB and SR8278 treatment, but not SR1078, effectively attenuated structural destruction of articular cartilage in surgery-induced OA mouse models. Notably, the beneficial effects of NOB and SR8278 were evidently observed in IL-1ß-induced degeneration of human cartilage explants and immortalized human chondrocytes. Moreover, BMAL1 knockdown assays indicated that NOB and SR8278 enhanced clock function and concordantly rendered protection against altered anabolism and catabolism in a BMAL1-dependent regime. Collectively, our study suggests that targeting RORs and REV-ERBs to promote the dampened peripheral clocks could be a route taken to apply chronotherapy within the context of OA.

Efficacy of Kushen decoction on high-fat-diet-induced hyperlipidemia in rats.

OBJECTIVE: To investigate the efficacy and underlying mechanisms of action of Kushen decoction on high-fat-diet-induced hyperlipidemia in rats using RNA-seq technology. METHODS: The efficacy of a Kushen decoction, at a concentration of 1 mL/g of crude medicine prepared according to the method commonly used in clinical practice, was investigated on 24 specific pathogen-free male Sprague-Dawley rats. Liver tissues were compared using RNA-Seq technology. The differentially expressed genes were further investigated by real-time fluorescent quantitative polymerase chain reaction (qPCR and Western blot (WB). RESULTS: Serum triglycerides (TG), liver low-density lipoprotein cholesterol (LDL-C), body weight, body length, and Lee's index were significantly increased in the untreated hyperlipidemia-induced group (model) compared with the control group, whereas liver high-density lipoprotein cholesterol (HDL-C) was significantly decreased. Serum TG, liver LDL-C, bodyweight, and Lee's index were decreased in the high-dose Kushen decoction group (HDKS) compared with the model group, whereas liver HDL-C was significantly increased. Similarly, liver TG tended to decline in the HDKS group. Comparison of the gene expression profiles in the livers from different groups indicated that the Kushen decoction significantly affected metabolic pathways, PPAR signalling pathway, and circadian rhythm ( ≤ 0.05), with the genes ARNTL, PER3, and CLOCK being differentially expressed. qPCR and WB analysis confirmed the differential expression of the genes discovered by transcriptomics analysis. CONCLUSION: The Kushen decoction may achieve a lipid-lowering effect on hyperlipidemic rats by regulating metabolic pathways and the circadian rhythm pathway and in particular, their related genes ARNTL, PER3, and CLOCK.

Physiological Temperature Changes Fine-Tune ß 2- Adrenergic Receptor-Induced Cytosolic cAMP Accumulation.

Norepinephrine (NE) controls many vital body functions by activating adrenergic receptors (ARs). Average core body temperature (CBT) in mice is 37°C. Of note, CBT fluctuates between 36 and 38°C within 24 hours, but little is known about the effects of CBT changes on the pharmacodynamics of NE. Here, we used Peltier element-controlled incubators and challenged murine hypothalamic mHypoA -2/10 cells with temperature changes of ±1°C. We observed enhanced NE-induced activation of a cAMP-dependent luciferase reporter at 36 compared with 38°C. mRNA analysis and subtype specific antagonists revealed that NE activates ß 2- and ß 3-AR in mHypoA-2/10 cells. Agonist binding to the ß 2-AR was temperature insensitive, but measurements of cytosolic cAMP accumulation revealed an increase in efficacy of 45% ± 27% for NE and of 62% ± 33% for the ß 2-AR-selective agonist salmeterol at 36°C. When monitoring NE-promoted cAMP efflux, we observed an increase in the absolute efflux at 36°C. However, the ratio of exported to cytosolic accumulated cAMP is higher at 38°C. We also stimulated cells with NE at 37°C and measured cAMP degradation at 36 and 38°C afterward. We observed increased cAMP degradation at 38°C, indicating enhanced phosphodiesterase activity at higher temperatures. In line with these data, NE-induced activation of the thyreoliberin promoter was found to be enhanced at 36°C. Overall, we show that physiologic temperature changes fine-tune NE-induced cAMP signaling in hypothalamic cells via ß 2-AR by modulating cAMP degradation and the ratio of intra- and extracellular cAMP. SIGNIFICANCE STATEMENT: Increasing cytosolic cAMP levels by activation of G protein-coupled receptors (GPCR) such as the ß 2-adrenergic receptor (AR) is essential for many body functions. Changes in core body temperature are fundamental and universal factors of mammalian life. This study provides the first data linking physiologically relevant temperature fluctuations to ß 2-AR-induced cAMP signaling, highlighting a so far unappreciated role of body temperature as a modulator of the prototypic class A GPCR.

Withanolide Derivative 2,3-Dihydro-3ß-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3ß-methoxy withaferin-A (3ßmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3ßmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3ßmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 µM and that 3ßmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 µM). We propose that 3ßmWi-A is a novel modulator of circadian rhythms.

Efficient induction of proximity-dependent labelling by biotin feeding in BMAL1-BioID knock-in mice.

Proximity-dependent biotin identification (BioID) is a useful method to identify unknown protein-protein interactions. Few reports have described genetically engineered knock-in mouse models for in vivo BioID. Thus, little is known about the proper method for biotin administration and which tissues are applicable. Here, we established a BioID knock-in mouse model of Brain and Muscle ARNT-Like 1 (BMAL1) and the BirA biotin ligase with R118G mutation (BirA*). The BMAL1-BioID mouse model was used to investigate the effect of biotin diet feeding on protein biotinylation in several tissues. The BMAL1-BirA* fusion protein-retained proper intracellular localization of BMAL1 and binding to CLOCK protein in HEK293T cells. A biotin labelling assay in mouse embryonic fibroblasts revealed the protein biotinylation activity of BMAL1-BirA* expressed in knock-in mouse cells depending on biotin supplementation. Lastly, feeding a 0.5% biotin diet for 7 days induced protein biotinylation in the brain, heart, testis and liver of BMAL1-BioID mice without adverse effects on spermatogenesis. In the kidney, the biotin diet increased biotinylated protein levels in BMAL1-BioID and control mice, suggesting the existence of endogenous biotinylation activity. These results provide valuable information to optimize the in vivo BioID procedure.

Obese Neotomodon alstoni mice exhibit sexual dimorphism in the daily profile of circulating melatonin and clock proteins PER1 and BMAL1 in the hypothalamus and peripheral oscillators.

Obesity is a global health threat and a risk factor for several metabolic conditions. Though circadian dysfunction has been considered among the multiple causes of obesity, little work has been done to explore the relationship between obesity, circadian dysfunction, and sexual dimorphism. The Neotomodon alstoni mouse is a suitable model for such research. This study employed N. alstoni mice in a chronobiological analysis to determine whether there is circadian desynchronization of relative PER1 and BMAL1 protein levels in the hypothalamus, liver, visceral white adipose tissue, kidney, and heart. It also compared differences between sexes and lean and obese N. alstoni adult mice, by recording behavior and daily circulating serum melatonin as markers of circadian output. We found that obese mice display reduced locomotor activity. Additionally, Cosinor analyses of the relative expression of PER1 and BMAL1 show differences between lean and obese mice in a sex-linked manner. The PER1 24 h rhythm was absent in all tissues of obese males and significant in the tissues of obese females. The BMAL1 24 h rhythm also was significant in most of the tissues tested in lean males, whereas it was significant and shifted the acrophase (peak time of rhythm) in most of the tissues in obese females. Both lean male and female mice showed a rhythmic 24 h pattern of circulating serum melatonin. This daily profile was not only absent in obese mice of both sexes but showed sexual dimorphism. Obese male mice showed lower circulating levels of melatonin compared to lean male mice, but they were higher in obese females compared to lean females. Our results suggest that obesity in N. alstoni is associated with an internal circadian desynchronization in a sex-dependent manner. Overall, this study reinforces the need for further research on the neuroendocrinology of obesity and circadian rhythms using this biological model.

Bromide impairs the circadian clock and glycolytic homeostasis via disruption of autophagy in rat H9C2 cardiomyocytes.

BACKGROUND: Trace elements function as essential cofactors that are involved in various biochemical processes in mammals. Autophagy is vital for nutrient supplement, which is an important Zeitegber for the circadian homeostasis in heart. Here, we considered the possibility that autophagy, as well as the cardiomyocyte clock and glycolysis are interlinked. Detrimental effects were observed when cardiac system is exposed to bromine containing drugs. This study investigated the effects and mechanisms of bromide on the circadian clock and glycolytic metabolism of H9C2 cardiomyocytes. RESULTS: In the present study, bromide does not affect cell viability and apoptosis of H9C2 cardiomyocytes. Bromide dampens the clock and glycolytic (Hk2 and Pkm2) gene expression rhythmicity in a dose-dependent manner. Additionally, bromide inhibits autophagic process in H9C2 cardiomyocytes. In contrast, rapamycin (an autophagy inducer) dramatically restores the inhibitory effect of NaBr on the mRNA expression levels of clock genes (Bmal1, Cry1 and Rorα) and glycolytic genes (Hk2 and Pkm2). CONCLUSIONS: Our results reveal that bromide represses the clock and glycolytic gene expression patterns, partially through inhibition of autophagy.

The circadian clock gene Bmal1 facilitates cisplatin-induced renal injury and hepatization.

Cisplatin is one of the most potent chemotherapy drugs to treat cancers, but its clinical application remains limited due to severe nephrotoxicity. Several approaches have been developed to minimize such side effects, notably including chronotherapy, a well-known strategy based on the circadian clock. However, the component of the circadian clock machinery that particularly responses to the cisplatin stimulation remains unknown, including its functions in cisplatin-induced renal injury. In our present study, we demonstrated that Bmal1, as a key clock gene, was induced by the cisplatin stimulation in the mouse kidney and cultured human HK-2 renal cells. Gain- and loss-of-function studies indicated that Bmal1 facilitated cisplatin-induced renal injury both in vivo and in vitro, by aggravating the cell apoptotic process. More importantly, RNA-seq analysis revealed that Bmal1 triggered the expression of hallmark genes involved in renal hepatization, a critical event accompanied by the injury. At the molecular level, Bmal1 activated the transcription of hepatization-associated genes through direct recruitment to the E-box motifs of their promoters. Our findings suggest that Bmal1, a pivotal mediator induced renal injury in response to cisplatin treatment, and the therapeutic intervention targeting Bmal1 in the kidney may be a promising strategy to minimize the toxic side-effects of cisplatin in its clinical applications.

Circadian control of interferon-sensitive gene expression in murine skin.

The circadian clock coordinates a variety of immune responses with signals from the external environment to promote survival. We investigated the potential reciprocal relationship between the circadian clock and skin inflammation. We treated mice topically with the Toll-like receptor 7 (TLR7) agonist imiquimod (IMQ) to activate IFN-sensitive gene (ISG) pathways and induce psoriasiform inflammation. IMQ transiently altered core clock gene expression, an effect mirrored in human patient psoriatic lesions. In mouse skin 1 d after IMQ treatment, ISGs, including the key ISG transcription factor IFN regulatory factor 7 (Irf7), were more highly induced after treatment during the day than the night. Nuclear localization of phosphorylated-IRF7 was most prominently time-of-day dependent in epidermal leukocytes, suggesting that these cell types play an important role in the diurnal ISG response to IMQ. Mice lacking Bmal1 systemically had exacerbated and arrhythmic ISG/Irf7 expression after IMQ. Furthermore, daytime-restricted feeding, which affects the phase of the skin circadian clock, reverses the diurnal rhythm of IMQ-induced ISG expression in the skin. These results suggest a role for the circadian clock, driven by BMAL1, as a negative regulator of the ISG response, and highlight the finding that feeding time can modulate the skin immune response. Since the IFN response is essential for the antiviral and antitumor effects of TLR activation, these findings are consistent with the time-of-day-dependent variability in the ability to fight microbial pathogens and tumor initiation and offer support for the use of chronotherapy for their treatment.

Circadian Regulation of IOP Rhythm by Dual Pathways of Glucocorticoids and the Sympathetic Nervous System.

Purpose: Elevated IOP can cause the development of glaucoma. The circadian rhythm of IOP depends on the dynamics of the aqueous humor and is synchronized with the circadian rhythm pacemaker, that is, the suprachiasmatic nucleus. The suprachiasmatic nucleus resets peripheral clocks via sympathetic nerves or adrenal glucocorticoids. However, the detailed mechanisms underlying IOP rhythmicity remain unclear. The purpose of this study was to verify this regulatory pathway. Methods: Adrenalectomy and/or superior cervical ganglionectomy were performed in C57BL/6J mice. Their IOP rhythms were measured under light/dark cycle and constant dark conditions. Ocular administration of corticosterone or norepinephrine was also performed. Localization of adrenergic receptors, glucocorticoid receptors, and clock proteins Bmal1 and Per1 were analyzed using immunohistochemistry. Period2::luciferase rhythms in the cultured iris/ciliary bodies of adrenalectomized and/or superior cervical ganglionectomized mice were monitored to evaluate the effect of the procedures on the local clock. The IOP rhythm of retina and ciliary epithelium-specific Bmal1 knockout mice were measured to determine the significance of the local clock. Results: Adrenalectomy and superior cervical ganglionectomy disrupted IOP rhythms and the circadian clock in the iris/ciliary body cultures. Instillation of corticosterone and norepinephrine restored the IOP rhythm. ß2-Adrenergic receptors, glucocorticoid receptors, and clock proteins were strongly expressed within the nonpigmented epithelia of the ciliary body. However, tissue-specific Bmal1 knock-out mice maintained their IOP rhythm. Conclusions: These findings suggest direct driving of the IOP rhythm by the suprachiasmatic nucleus, via the dual corticosterone and norepinephrine pathway, but not the ciliary clock, which may be useful for chronotherapy of glaucoma.

Effect of Electroacupuncture on Neurological Deficit and Activity of Clock and Bmal1 in Cerebral Ischemic Rats.

Acute focal cerebral ischemic stroke (IS) is a leading cause of morbidity and mortality worldwide. Acupuncture is an emerging alternative therapy that has been beneficial to acute brain ischemia. However, the underlying protective mechanism of its neuroprotective effect remains unclear. Human original circadian rhythm will be lost after IS, which seriously affects the quality of life and functional recovery of stroke patients. We hypothesize that acupuncture treats IS by regulating the balance of Clock and Bmal1. This study aims to explore the effect of acupuncture at acupoints GV20 and BL23 on neuroprotection and anti-apoptosis in middle cerebral artery occlusion (MCAO) rats and expression of apoptosis and circadian rhythm related proteins. Male Sprague-Dawley (SD) rats were randomly divided into five groups: normal group (Normal), sham model group (Sham MCAO), MCAO model group (MCAO), sham electroacupuncture group (Sham EA) and electroacupuncture group (EA). The MCAO model was prepared by electrocoagulation. The first acupuncture treatment was performed within 2 h after surgery, and then acupuncture therapy was performed on 1st day, 2nd day and 3rd day respectively. After their neurological examination at 72 h of ischemia, the rats from each group were sacrificed. Triphenyltetrazolium chloride (TTC) staining was used to evaluate the brain infarct size. Ultrastructural observation on cerebral ischemic cortex and serum inflammatory cytokines were evaluated. TUNEL staining was used to detect cell apoptosis of brain tissue. The expression levels of proteins Bax, bcl-2, caspase-3, Clock and Bmal1 in the cerebral ischemic region were detected by immunofluorescence staining. Here, we presented evidence that EA at GV20 and BL23 could significantly improve the neurological deficit score and infarct size, and alleviate the cell apoptosis of brain tissue. Moreover, acupuncture treatment upregulated the anti-apoptotic Bcl-2/Bax ratio and reversed the upregulation of caspase-3 following 72-h cerebral ischemia. In addition, the expression levels of circadian proteins Clock and Bmal1 were upregulated in EA group while compared with MCAO group. Our study demonstrated that acupuncture exerted neuroprotective effect against neuronal apoptosis after stroke and the mechanism might be related with regulation of circadian rhythm proteins Clock and Bmal1.

Non-obesogenic doses of palmitate disrupt circadian metabolism in adipocytes.

Saturated fatty acids, such as palmitate, lead to circadian disruption. We aimed at studying the effect of low doses of palmitate on circadian metabolism and to decipher the mechanism by which fatty acids convey their effect in adipocytes. Mice were fed non-obesogenic doses of palm or olive oil and adipocytes were treated with palmitate and oleate. Cultured adipocytes treated with oleate showed increased AMPK activity and induced the expression of mitochondrial genes indicating increased fatty acid oxidation, while palmitate increased ACC activity and induced the expression of lipogenic genes, indicating increased fatty acid synthesis. Low doses of palmitate were sufficient to alter circadian rhythms, due to changes in the expression and/or activity of key metabolic proteins including GSK3ß and AKT. Palmitate-induced AKT and GSK3ß activation led to the phosphorylation of BMAL1 that resulted in low levels as well as high amplitude of circadian clock expression. In adipocytes, the detrimental metabolic alteration of palmitate manifests itself early on even at non-obesogenic levels. This is accompanied by modulating BMAL1 expression and phosphorylation levels, which lead to dampened clock gene expression.

Impact of chronically alternating light-dark cycles on circadian clock mediated expression of cancer (glioma)-related genes in the brain.

Disruption of the circadian rhythm is a risk factor for cancer, while glioma is a leading contributor to mortality worldwide. Substantial efforts are being undertaken to decrypt underlying molecular pathways. Our understanding of the mechanisms through which disrupted circadian rhythm induces glioma development and progression is incomplete. We, therefore, examined changes in the expression of glioma-related genes in the mouse brain after chronic jetlag (CJL) exposure. A total of 22 candidate tumor suppressor (n= 14) and oncogenes (n= 8) were identified and analyzed for their interaction with clock genes. Both the control and CJL groups were investigated for the expression of candidate genes in the nucleus accumbens, hippocampus, prefrontal cortex, hypothalamus, and striatum of wild type, Bmal1-/- and Cry1/2 double knockout male mice. We found significant variations in the expression of candidate tumor suppressor and oncogenes in the brain tissues after CJL treatment in the wild type, Bmal1-/- and Cry1/2 double knockout mice. In response to CJL treatment, some of the genes were regulated in the wild type, Bmal1-/- and Cry1/2 similarly. However, the expression of some of the genes indicated their association with the functional clock. Overall, our result suggests a link between CJL and gliomas risk at least partially dependent on the circadian clock. However, further studies are needed to investigate the molecular mechanism associated with CJL and gliomas.