Circadian Gene Expression Research Articles

Liangxue Jiedu formula improves imiquimod-induced psoriasiform dermatitis with circadian desynchrony by regulating Th17 cell differentiation based on network pharmacological analysis

Ethnopharmacological relevanceLiangxue Jiedu formula (LXJDF) is an effective traditional Chinese medicine (TCM) formula for treating psoriasis of blood-heat syndrome and has been used in clinics for decades. Aim of the studyThis study aimed to discover the mechanism of LXJDF in psoriasis and the circadian clock by network pharmacology and experimental studies. Materials and methodsThe compounds of LXJDF were obtained from the TCMSP and BATMAN-TCM databases. The genes related to psoriasis and circadian rhythm/clock were identified by the OMIM and GeneCards databases. Then, target genes were integrated by Venn and analyzed by the String, CytoNCA, DAVID (GO and KEGG) databases, and the network was constructed using Cytoscape. Mice were raised under light disturbance for fourteen days. On the eighth day, mouse dorsal skin was shaved and smeared with 62.5 mg 5% imiquimod at 8:00 (ZT0) for six successive days. Mice were randomly divided into the model, LXJDF-H (49.2 g/kg·bw), LXJDF-L (24.6 g/kg·bw), and positive drug (dexamethasone) groups. Other mice were smeared with Vaseline under the normal light cycle as the control. The drug of each group was administered at 10:00 (ZT2) and 22:00 (ZT14). The skin lesions were observed, and PASI was scored daily. HE and immunofluorescence were used to measure pathological morphology. Th17 cytokines in serum and skin were measured by flow cytometry and qPCR. Circadian clock gene and protein expression levels were determined by qPCR and Western blotting. ResultsWe found 34 potential targets of LXJDF in the treatment of psoriasis and circadian rhythm and confirmed their importance by topology analysis. KEGG pathway analysis revealed that the two major pathways were Th17 cell differentiation and the HIF-1 signaling pathway. At ZT2 and ZT14, LXJDF improved IMQ-induced light disturbance mouse skin lesions, including alleviating scales, erythema, and infiltration, reducing PASI, and inhibiting keratinocyte hyperproliferation and parakeratosis. LXJDF reduced IL-17A, IL-17F, TNF-α, and IL-6 in serum at ZT2 and increased IL-10 at ZT2 and ZT14. LXJDF downregulated the expression of IL-17A and IL-17F in skin. At ZT2, LXJDF significantly upregulated CLOCK and REV-ERBα expression and downregulated HIF-1α expression. At ZT14, LXJDF decreased HIF-1α and RORγt expression and significantly increased REV-ERBα expression. ConclusionLXJDF improves psoriasis dermatitis with circadian rhythm disorders by regulating Th17 cell differentiation.

Thyroid-stimulating hormone-thyroid hormone signaling contributes to circadian regulation through repressing clock2/npas2 in zebrafish

Thyroid-stimulating hormone (TSH) is important for the thyroid gland, development, growth, and metabolism. Defects in TSH production or the thyrotrope cells within the pituitary gland cause congenital hypothyroidism (CH), resulting in growth retardation and neurocognitive impairment. While human TSH is known to display rhythmicity, the molecular mechanisms underlying the circadian regulation of TSH and the effects of TSH-thyroid hormone (TH) signaling on the circadian clock remain elusive. Here we show that TSH, thyroxine (T4), triiodothyronine (T3), and tshba display rhythmicity in both larval and adult zebrafish and tshba is regulated directly by the circadian clock via both E′-box and D-box. Zebrafish tshba−/− mutants manifest congenital hypothyroidism, with the characteristics of low levels of T4 and T3 and growth retardation. Loss or overexpression of tshba alters the rhythmicity of locomotor activities and expression of core circadian clock genes and hypothalamic-pituitary-thyroid (HPT) axis-related genes. Furthermore, TSH-TH signaling regulates clock2/npas2 via the thyroid response element (TRE) in its promoter, and transcriptome analysis reveals extensive functions of Tshba in zebrafish. Together, our results demonstrate that zebrafish tshba is a direct target of the circadian clock and in turn plays critical roles in circadian regulation along with other functions.

Disruptions of Circadian Genes in Cutaneous Melanoma-An In Silico Analysis of Transcriptome Databases.

Circadian genes are a set of genes that regulate the body's internal clock and influence various physiological processes, including sleep-wake cycles, metabolism and immune function. Skin cutaneous melanoma (SKCM) is a type of skin cancer that arises from the pigment-producing cells in the skin and is the most deadly form of skin cancer. This study has investigated the relevance of circadian gene expression and immune infiltrations in the outcomes of cutaneous melanoma patients. In the present study, in silico methods based on the GEPIa, TIMER 2.0 and cBioPortal databases were performed, so as to investigate the transcript level and prognostic value of 24 circadian genes in SKCM and their relationship with the immune infiltration level. The in silico analysis showed that significantly more than half of the investigated circadian genes have an altered transcript pattern in cutaneous melanoma compared to normal skin. The mRNA levels of TIMELES and BHLHE41 were upregulated, whereas those of NFIL3, BMAL1, HLF, TEF, RORA, RORC, NR1D1, PER1, PER2, PER3, CRY2 and BHLHE40 were downregulated. The presented research shows that SKCM patients with at least one alteration of their circadian genes have decreased overall survival. Additionally, majority of the circadian genes are significantly corelated with the immune cells' infiltration level. The strongest correlation was found for neutrophils and was followed by circadian genes: NR1D2 r = 0.52 p < 0.0001, BMAL1 r = 0.509 p < 0.0001; CLOCK r = 0.45 p < 0.0001; CSNKA1A1 r = 0.45 p < 0.0001; RORA r = 0.44 p < 0.0001. The infiltration level of immune cells in skin tumors has been associated with patient prognosis and treatment response. Circadian regulation of immune cell infiltration may further contribute to these prognostic and predictive markers. Examining the correlation between circadian rhythm and immune cell infiltration can provide valuable insights into disease progression and guide personalized treatment decisions.

Comprehensive analysis of the prognosis and biological significance of RORβ in head and neck squamous cell carcinoma.

Recent studies have shown that abnormal expression of the core circadian clock gene, retinoic acid-related orphan receptor β (RORβ ), is closely associated with the occurrence and progression of various malignant tumors. However, the expression and function of RORβ in head and neck squamous cell carcinoma (HNSCC) remains unclear. Here, we comprehensively investigated the altered expression, clinical significance, prognostic value, and biological functions of RORβ in HNSC, as well as its correlation with changes in the tumor immune microenvironment. We found that RORβ expression was decreased in HNSC and 19 other cancers. Low RORβ expression was significantly associated with tumor size, clinical stage, and survival time in HNSC patients, indicating that it may have diagnostic and prognostic value in HNSCC. Epigenetic analysis showed that the promoter methylation level of RORβ was significantly higher in HNSCC compared to adjacent noncancerous tissues. Furthermore, RORβ hypermethylation was significantly associated with low expression levels of RORβ and poor prognosis in HNSCC patients (p < 0.05). Enrichment analysis found that RORβ was involved in immune system regulation and T-cell activation, as well as the PI3K/AKT and ECM receptors interaction pathways. In vitro assays revealed that RORβ regulated the proliferation, migration and invasion ability of HNSCC cells. Additionally, we found that RORβ expression was significantly correlated with changes in the tumor immune microenvironment, suggesting it may affect prognosis by regulating immune infiltration in HNSC patients. Therefore, RORβ may serve as a potential prognostic biomarker and therapeutic target for HNSCC patients.

Abstract A048: The importance of time: Circadian rhythm disruption and prostate cancer

Abstract Circadian rhythms are evolutionarily created cycles programmed to integrate human behavior, physiology, and metabolism with the surrounding environment. The core clock machinery is composed of an autoregulatory network. The positive components of the loop are CLOCK and BMAL transcription factors, which heterodimerize to regulate expression of key circadian genes through E-box elements. CLOCK/BMAL regulate expression of cryptochrome (CRY1 and CRY2) and period (PER1, PER2, and PER3) genes, which are the negative components of the circadian loop. CRYs and PERs repress CLOCK/BMAL activity, thus conferring daily rhythmicity of cellular, metabolic, and physiological functions for homeostatic maintenance. Disruption to these rhythms, via nighttime light exposure or other forms of desynchronization, has been linked to poor health outcomes. Specifically, circadian disruption is designated as a probable carcinogen by the World Health Organization. Subsequent epidemiology research identified pilots and other shift workers have an increased risk of prostate cancer (PCa). Additional genomic studies identified multiple core clock genes (CCGs) with associated upregulation and/or downregulation in prostatic tumor tissues compared to benign samples. CCGs have also been found to impact several key oncogenic pathways including cell cycle control, apoptosis, DNA repair, and metabolic regulation. In order to further clarify the risk of an altered circadian genome, we analyzed multiple publicly available human PCa datasets to identify clinical correlates in both primary and metastatic disease. Intriguingly, we found genomic alterations in circadian rhythm were associated with increased Gleason score, cancer staging, and positive node status in primary disease. Furthermore, within a subset of patients with castrate-resistant prostate cancer (CRPC), those with concurrent altered CCGs had a decreased survival compared to patients without altered CCGs. This data further emphasizes the importance of circadian rhythm consideration in the context of malignancy and preventative medicine. With so many unknown questions and a high potential to identify subtypes of tumors with altered CCGs and aggressive disease, future studies are needed to improve therapeutic efficacy. Analysis of circadian genomic alterations with subsequent comparison of demographics including ethnicity, occupation, and sleep habits would expand the understanding of everyday variables on circadian disruption. Additionally, the comparison of short-term versus long-term circadian synchronizations should be analyzed to identify higher-risk populations. Lastly, the long-term goal should be to discover potential circadian targeted treatments or to identify ways of involvement of circadian rhythmicity in cancer treatment, including chronotherapy (i.e., time of day of treatment) to optimize the impact of cancer therapeutics to enhance patient outcome. Citation Format: Sierra T. Pence, Ayesha A. Shafi. The importance of time: Circadian rhythm disruption and prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A048.

Analysis of glucocorticoid responsive and circadian clock molecules in Alzheimer’s disease brain tissues.

AbstractBackgroundUnder basal non‐stressed brain conditions, a control system that includes an inherent molecular clock activation, immunomodulatory role of glucocorticoids and nuclear factor‐kappa B (NF‐κB) signaling is essential to prevent excessive inflammation and to effectively return to homeostasis. This results in a continuous competition between feedback and feedforward control. Chronic stress and dysregulated molecular clock are implicated as significant risk factors for neurodegeneration. In unstimulated mice, the core clock gene BMAL1 and the glucocorticoid induced leucine zipper (GILZ, a glucocorticoid responsive gene) are highly expressed in hippocampal microglia. The expressions of these two genes are positively correlated with circulating corticosterone. Mechanistically, GILZ inhibits nuclear translocation of NF‐κB p65 and functions as an anti‐inflammatory molecule. BMAL1 on the other hand, independent of its circadian partner CLOCK, inhibits the glucocorticoid receptor mediated transactivation of GILZ. Elevated plasma corticosterone and age‐related decrease in BMAL1 are associated with increased activation of NF‐κB p65 and susceptibility to neuropathology. We have observed that the GILZ expression in the hippocampus is inversely related to that of NF‐κB p65 and IBA1 in mouse models of Alzheimer’s disease (AD). Taken together, we hypothesize that the GILZ expression will correlate positively with core clock genes and negatively with markers of gliosis and inflammation in neurodegenerative diseases.MethodWe evaluated the expressions of circadian markers BMAL1 and Period genes (Per1, Per2 and Per3); that of glial cell surface markers Iba1 and GFAP and that of GILZ and NF‐kB as markers of inflammatory system in AD brain tissues by immunohistochemistry and real‐time polymerase chain reaction (RT‐PCR).ResultGILZ expression was reduced in the hippocampus and pre‐frontal cortex of AD tissues, was inversely related to p65, Iba1 and GFAP expressions and directly related to BMAL1 and Per2.ConclusionGILZ could potentially function as a molecular modulator at the intersection of the three systems.

Circadian clock regulation via biomaterials for nucleus pulposus.

Circadian clock disorder during tissue degeneration has been considered the potential pathogenesis for various chronic diseases, such as intervertebral disc degeneration (IVDD). In this study, circadian clock-regulating biomaterials (ClockMPs) that could effectively activate the intrinsic circadian clock of nucleus pulposus cells (NPCs) in IVDD and improve the physiological function of NPCs for disc regeneration were fabricated via air-microfluidic technique and the chemical cross-linking between polyvinyl alcohol and modified-phenylboronic acid. In vitro experiments verified that ClockMPs could scavenge reactive oxygen species to maintain a stable microenvironment for the circadian clock by promoting the binding of BMAL1 and CLOCK proteins. ClockMPs could regulate the expression of core circadian clock genes by activating the PI3K-AKT pathway in NPCs to remodel the intrinsic circadian clock and promote extracellular matrix synthesis. Furthermore, in vivo experiments of IVDD treated with ClockMPs proved that ClockMPs could promote disc regeneration by regulating the circadian clock of NPCs. In conclusion, ClockMPs provided a novel and promising strategy for circadian clock regulation during tissue regeneration. This article is protected by copyright. All rights reserved.

Acute and Repeated Ozone Exposures Differentially Affect Circadian Clock Gene Expression in Mice.

Circadian rhythms have an established role in regulating physiological processes, such as inflammation, immunity, and metabolism. Ozone, a common environmental pollutant with strong oxidative potential, is implicated in lung inflammation/injury in asthmatics. However, whether O3 exposure affects the expression of circadian clock genes in the lungs is not known. In this study, changes in the expression of core clock genes are analyzed in the lungs of adult female and male mice exposed to filtered air (FA) or O3 using qRT-PCR. The findings are confirmed using an existing RNA-sequencing dataset from repeated FA- and O3 -exposed mouse lungs and validated by qRT-PCR. Acute O3 exposure significantly alters the expression of clock genes in the lungs of females (Per1, Cry1, and Rora) and males (Per1). RNA-seq data revealing sex-based differences in clock gene expression in the airway of males (decreased Nr1d1/Rev-erbα) and females (increased Skp1), parenchyma of females and males (decreased Nr1d1 and Fbxl3 and increased Bhlhe40 and Skp1), and alveolar macrophages of males (decreased Arntl/Bmal1, Per1, Per2, Prkab1, and Prkab2) and females (increased Cry2, Per1, Per2, Csnk1d, Csnk1e, Prkab2, and Fbxl3). These findings suggest that lung inflammation caused by O3 exposure affects clock genes which may regulate key signaling pathways.

Epigenetic Activation of Circadian Clock Genes Elicits Inflammation in Experimental Murine Dry Eye

ABSTRACT Purpose To explore whether circadian clock genes contribute to elicit inflammation in experimental dry eye (EDE). Methods RNA sequencing analyzed mRNA expression patterns in EDE model. RT-qPCR and/or Western blot determined the expression of inflammatory factors and circadian genes during EDE. MethylTarget™ assays determined the promoter methylation levels of Per genes in vivo. Per2 or Per3 knockdown assessed their effects on inflammatory factors in vitro. Results We utilized an intelligently controlled environmental system (ICES) to establish a mouse EDE model. The significant upregulated genes were enriched for circadian rhythms. Therein lied oscillatory and time-dependent upregulation of PER2 and PER3, as well as their promoter hypomethylation during EDE. Silencing PER2 or PER3 significantly decreased inflammatory factor expression and also reversed such increased inflammatory response in azacitidine (AZA) treatment in vitro model. Conclusions Our findings suggest that DNA methylation mediated the upregulation of PER2 and PER3, leading to inflammatory response in EDE.

Abstract A027: Defining the role of PER1 and circadian rhythm dysregulation in KRAS/LKB1-mutant lung adenocarcinoma

Abstract KRAS and LKB1 are frequently co-mutated in lung adenocarcinoma and characterize a particularly aggressive, metastatic, and treatment-resistant subtype of this disease. We recently published that the outputs of biological rhythm pathways downstream of the AMPK kinase are altered in KRAS/LKB1-mutant tumors. In a live-cell phenotyping screen we used RNAseq to identify the circadian clock genes PER1 and PER2 as uniquely upregulated in partially transformed and invasive KRAS/LKB1-mutant human bronchial epithelial cells. We have analyzed expression of individual circadian clock genes in KRAS-mutant and KRAS/LKB1-mutant patient tumors and found that expression of the PER genes, particularly PER1, is increased in the KRAS/LKB1 subtype of lung adenocarcinoma. At the protein level, PER1 expression is increased in KRAS/LKB1-mutant HBECs as compared to KRAS-mutant cells. Addback of wild-type LKB1 in A549 cells, which are KRAS/LKB1-mutant, results in a decrease in PER1 protein levels, confirming that LKB1 has a role in controlling PER1 levels. Multiple phosphorylation events lead to PER1 nuclear translocation to control its role as a transcriptional repressor in the core clock. In circadian-synced HBECs, PER1 enters the nucleus in KRAS-mutant cells, but remains cytosolic in KRAS/LKB1-mutant cells. In circadian-synced A549 cells, we also observed a lack of nuclear PER1 localization. Addback of wild-type LKB1 restored nuclear localization of PER1, as did expression of kinase-dead LKB1, suggesting that LKB1’s regulation of PER1 localization is kinase-independent. Our current and future experiments aim to determine how clock-controlled gene expression may be altered in KRAS/LKB1-mutant cells, what the functional impact of altered PER1 expression and localization is on growth of KRAS/LKB1-mutant tumors, and whether pharmacological manipulation of LKB1-related signaling pathways can restore nuclear localization of PER1. Citation Format: Rebecca E. Parker, Junghui Koo, Bhakti Dwivedi, Adam I. Marcus, Melissa Gilbert-Ross. Defining the role of PER1 and circadian rhythm dysregulation in KRAS/LKB1-mutant lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A027.

Correlations between the symptoms of insomnia, depression, sleep quality, antitumor necrosis factor therapy, and disrupted circadian clock gene expression in inflammatory bowel disease.

Inflammatory bowel disease (IBD) might be accompanied by emotional disturbances. Circadian rhythm genes, such as brain and muscle ARNT‑Like 1 (BMAL1), circadian locomotor output cycles kaput (CLOCK), neuronal PAS domain protein 2 (NPAS2), or nuclear receptor subfamily 1 group D member 1 (NR1D1) are related to inflammation and psychiatric symptoms that might modulate their expression. The study aimed to compare the expression of the BMAL1, CLOCK, NPAS2, NR1D1 mRNA in IBD patients and healthy controls (HCs). We evaluated the association between the gene expression and the disease severity, antitumor necrosis factor (TNF) therapy, sleep quality, insomnia, and depression. A total of 81 IBD patients and 44 HCs were recruited and classified according to the disease activity and IBD type (ulcerative colitis [UC] or Crohn disease [CD]). The participants filled out questionnaires assessing their sleep quality, daytime sleepiness, insomnia, and depression. Venous blood samples were collected, and the IBD patients on the anti‑TNF therapy had their blood drawn before and after 14 weeks of the treatment. In comparison with HCs, the IBD group had decreased expression of all studied genes apart from the BMAL1 gene. UC individuals with exacerbation had decreased expression of the CLOCK and the NPAS2 genes, as compared with the remission group. UC severity negatively correlated with the CLOCK, NPAS2, and NR1D1 mRNA levels. The IBD participants with depression symptoms had a decreased expression of the CLOCK and the NR1D1 genes, as compared with those without mood disturbances. Poor sleep quality was associated with a decreased expression of the NR1D1 gene. Biologic treatment decreased the expression of the BMAL1 gene. Disruption of the clock gene expression might constitute a molecular background of sleep disorders and depression in IBD and might contribute to UC exacerbation.

A ketogenic diet containing medium-chain triglycerides reduces REM sleep duration without significant influence on mouse circadian phenotypes

Ketogenic diets (KDs) affect the circadian rhythms of behavior and clock gene expression in experimental animals. However, these diets were designed to simulate a fasting state; thus, whether these effects are caused by diet-induced ketogenesis or persistent starvation is difficult to distinguish. The present study aimed to define the effects of a KD containing medium-chain triglycerides (MCT-KD) that increase blood ketone levels without inducing carbohydrate starvation, on circadian rhythms and sleep regulation. Mice were fed with a normal diet (CTRL) or MCT-KD for 2 weeks. Blood β-hydroxybutyrate levels were significantly increased up to 2 mM by the MCT-KD, whereas body weight gain and blood glucose levels were identical between the groups, suggesting that ketosis accumulated without carbohydrate starvation in the MCT-KD mice. Circadian rhythms of wheel-running activity and core body temperature were almost identical, although wheel-running was slightly reduced in the MCT-KD mice. The circadian expression of the core clock genes, Per1, Per2, Bmal1, and Dbp in the hypothalamus, heart, liver, epididymal adipose tissues, and skeletal muscle were almost identical between the CTRL and MCT-KD mice, whereas the amplitude of hepatic Per2 and adipose Per1 expression was increased in MCT-KD mice. The MCT-KD reduced the duration of rapid-eye-movement (REM) sleep without affecting the duration of non-REM sleep and the duration of wakefulness. These findings suggested that the impact of ketone bodies on circadian systems are limited, although they might reduce locomotor activity and REM sleep duration.

Integrative analysis of the association between circadian rhythm and lupus nephritis.

Lupus nephritis (LN) is an autoimmune nephropathy associated with systemic lupus erythematosus. Circadian rhythms are involved in the development of several diseases, especially inflammation-related diseases, but their relationship with LN is unclear. This was an integrative bioinformatics study. The expression profile from glomeruli, tubular interstitium and renal whole tissue samples was used to assess the expression levels and relevance of circadian rhythm-related genes. To screen for circadian rhythm-related signatures, we employed the LASSO and SVM-RFE algorithms. A consensus clustering algorithm was used to classify LN patients into two circadian rhythm patterns (cluster A and cluster B). We made immune cell infiltration analysis. We used the weighted gene co-expression network analysis (WGCNA) algorithm to identify cluster-specific differentially expressed genes. Nephroseq data were used to observe the relationship between genes and renal function. We found more significant differences in circadian rhythm-related gene expression in LN glomeruli compared with tubulointerstitial and whole-kidney tissues. We established a circadian rhythm-related signature consisting of eight genes that can easily distinguish LN from healthy individuals. Patients in cluster A were associated with B-cell-dominated immunity, whereas patients in cluster B were associated with T-cell-dominated immunity. As most of the patients with proliferative LN combined with membranous LN belonged to cluster B, patients in cluster B may have more severe renal pathology compared with patients in cluster A. Fifteen circadian rhythm-related genes associated with LN and LN typing were screened using the WGCNA algorithm, with COL1A2 and DOCK2 associated with renal prognosis. This study found that circadian rhythms are associated with the occurrence of LN, providing new ideas for the development of new LN treatment options from the perspective of circadian rhythms.

Association of Circadian Clock Gene Expression with Pediatric/Adolescent Asthma and Its Comorbidities.

The pathology of asthma is characterized by marked day-night variation, which is likely controlled by circadian clock activity. This study aimed to clarify the association of core circadian clock gene expression with clinical features of asthma. For this purpose, we accessed the National Center for Biotechnology Information database and analyzed transcriptomes of peripheral blood mononuclear cells and clinical characteristics of 134 pediatric/adolescent patients with asthma. Based on the expression patterns of seven core circadian clock genes (CLOCK, BMAL1, PER1-3, CRY1-2), we identified three circadian clusters (CCs) with distinct comorbidities and transcriptomic expressions. In the three CC subtypes, allergic rhinitis, and atopic dermatitis, both asthma comorbidities occurred in different proportions: CC1 had a high proportion of allergic rhinitis and atopic dermatitis; CC2 had a high proportion of atopic dermatitis but a low proportion of allergic rhinitis; and CC3 had a high proportion of allergic rhinitis but a low proportion of atopic dermatitis. This might be associated with the low activity of the FcεRI signaling pathway in CC2 and the cytokine-cytokine receptor interaction pathways in CC3. This is the first report to consider circadian clock gene expression in subcategories of patients with asthma and to explore their contribution to pathophysiology and comorbidity.

1058 Examination of circadian clock gene expression in human skin explants and pharmacological modulation with small molecule compounds

1058 Examination of circadian clock gene expression in human skin explants and pharmacological modulation with small molecule compounds

Clock-regulated coactivators selectively control gene expression in response to different temperature stress conditions in Arabidopsis

Plants respond to severe temperature changes by inducing the expression of numerous genes whose products enhance stress tolerance and responses. Dehydration-responsive element (DRE)-binding protein 1/C-repeat binding factor (DREB1/CBF) transcription factors act as master switches in cold-inducible gene expression. Since DREB1 genes are rapidly and strongly induced by cold stress, the elucidation of the molecular mechanisms of DREB1 expression is vital for the recognition of the initial responses to cold stress in plants. A previous study indicated that the circadian clock-related MYB-like transcription factors REVEILLE4/LHY-CCA1-Like1 (RVE4/LCL1) and RVE8/LCL5 directly activate DREB1 expression under cold stress conditions. These RVEs function in the regulation of circadian clock-related gene expression under normal temperature conditions. They also activate the expression of HSF-independent heat-inducible genes under high-temperature conditions. Thus, there are thought to be specific regulatory mechanisms whereby the target genes of these transcription factors are switched when temperature changes are sensed. We revealed that NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK) proteins act as coactivators of RVEs in cold and heat stress responses in addition to regulating circadian-regulated genes at normal temperatures. We found that among the four Arabidopsis LNKs, LNK1 and LNK2 function under normal and high-temperature conditions, and LNK3 and LNK4 function under cold conditions. Thus, these LNK proteins play important roles in inducing specific genes under different temperature conditions. Furthermore, LNK3 and LNK4 are specifically phosphorylated under cold conditions, suggesting that phosphorylation is involved in their activation.

Disturbed circadian rhythm and retinal degeneration in a mouse model of Alzheimer’s disease

The circadian clock is synchronized to the 24 h day by environmental light which is transmitted from the retina to the suprachiasmatic nucleus (SCN) primarily via the retinohypothalamic tract (RHT). Circadian rhythm abnormalities have been reported in neurodegenerative disorders such as Alzheimer's disease (AD). Whether these AD-related changes are a result of the altered clock gene expression, retina degeneration, including the dysfunction in RHT transmission, loss of retinal ganglion cells and its electrophysiological capabilities, or a combination of all of these pathological mechanisms, is not known. Here, we evaluated transgenic APP/PS1 mouse model of AD and wild-type mice at 6- and 12-month-old, as early and late pathological stage, respectively. We noticed the alteration of circadian clock gene expression not only in the hypothalamus but also in two extra-hypothalamic brain regions, cerebral cortex and hippocampus, in APP/PS1 mice. These alterations were observed in 6-month-old transgenic mice and were exacerbated at 12 months of age. This could be explained by the reduced RHT projections in the SCN of APP/PS1 mice, correlating with downregulation of hypothalamic GABAergic response in APP/PS1 mice in advanced stage of pathology. Importantly, we also report retinal degeneration in APP/PS1 mice, including Aβ deposits and reduced choline acetyltransferase levels, loss of melanopsin retinal ganglion cells and functional integrity mainly of inner retina layers. Our findings support the theory that retinal degeneration constitutes an early pathological event that directly affects the control of circadian rhythm in AD.

Senescence-induced alteration of circadian phagocytic activity of retinal pigment epithelium cell line ARPE-19

Renewal of retinal photoreceptor outer segments is conducted through daily shedding of distal photoreceptor outer segment tips and subsequent their phagocytosis by the adjacent retinal pigment epithelium (RPE) monolayer. Dysregulation of the diurnal clearance of photoreceptor outer segment tips has been implicated in age-related retinal degeneration, but it remains to be clarified how the circadian phagocytic activity of RPE cells is modulated by senescence. In this study, we used the human RPE cell line ARPE-19 to investigate whether hydrogen peroxide (H2O2)-induced senescence in ARPE-19 cells alters the circadian rhythm of their phagocytic activity. After synchronization of the cellular circadian clock by dexamethasone treatment, the phagocytic activity of normal ARPE-19 cells exhibited significant 24-h oscillation, but this oscillation was modulated by senescence. The phagocytic activity of senescent ARPE-19 cells increased constantly throughout the 24-h period, which still exhibited blunted circadian oscillation, accompanied by an alteration in the rhythmic expression of circadian clock genes and clock-controlled phagocytosis-related genes. The expression levels of REV-ERBα, a molecular component of the circadian clock, were constitutively increased in senescent ARPE-19 cells. Furthermore, pharmacological activation of REV-ERBα by its agonist SR9009 enhanced the phagocytic activity of normal ARPE-19 cells and increased the expression of clock-controlled phagocytosis-related genes. Our present findings extend to understand the role of circadian clock in the alteration of phagocytic activity in RPE during aging. Constitutive enhancement of phagocytic activity of senescent RPE may contribute to age-related retinal degeneration.

Circadian profile, daytime activity, and the Parkinson's phenotype: A motion sensor pilot study with neurobiological underpinnings

Circadian rhythm impairment may play a role in Parkinson's disease (PD) pathophysiology. Recent literature associated circadian rhythm features to the risk of developing Parkinson and to its progression through stages. The association between the chronotype and the phenotype should be verified on a clinical and biological point of view. Herein we investigate the chronotype of a sample of 50 PD patients with the Morningness Eveningness Questionnaire and monitor their daily activity with a motion sensor embedded in a smartphone. Fibroblasts were collected from PD patients (n = 5) and from sex/age matched controls (n = 3) and tested for the circadian expression of clock genes (CLOCK, BMAL1, PER1, CRY1), and for cell morphology, proliferation, and death. Our results show an association between the chronotype and the PD phenotype. The most representative clinical chronotypes were “moderate morning” (56%), the “intermediate” (24%) and, in a minor part, the “definite morning” (16%). They differed for axial motor impairment, presence of motor fluctuations and quality of life (p < 0.05). Patients with visuospatial dysfunction and patients with a higher PIGD score had a blunted motor daily activity (p = 0.006 and p = 0.001, respectively), independently by the influence of age and other motor scores. Fibroblasts obtained by PD patients (n = 5) had an impaired BMAL1 cycle compared to controls (n = 3, p = 0.01). Moreover, a PD flat BMAL1 profile was associated with the lowest cell proliferation and the largest cell morphology. This study contributes to the growing literature on CR abnormalities in the pathophysiology of Parkinson's disease providing a link between the clinical and biological patient chronotype and the disease phenomenology.

Immunotherapeutic approach to reduce senescent cells and alleviate senescence-associated secretory phenotype in mice.

Accumulation of senescent cells (SNCs) with a senescence-associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age-related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF-β and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet β cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro-inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single-nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell-cycle, and senescence-associated pathways in naturally aged mice. Long-term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.