Circadian Gene Expression Research Articles

Paclitaxel chemotherapy disrupts behavioral and molecular circadian clocks in mice

Cancer patients experience circadian rhythm disruptions in activity cycles and cortisol release that correlate with poor quality of life and decreased long-term survival rates. However, the extent to which chemotherapy contributes to altered circadian rhythms is poorly understood. In the present study, we examined the extent to which paclitaxel, a common chemotherapy drug, altered entrained and free-running circadian rhythms in wheel running behavior, circulating corticosterone, and circadian clock gene expression in the brain and adrenal glands of tumor-free mice. Paclitaxel injections delayed voluntary wheel running activity onset in a light–dark cycle (LD) and lengthened the free-running period of locomotion in constant darkness (DD), indicating an effect on inherent suprachiasmatic nucleus (SCN) pacemaker activity. Paclitaxel attenuated clock gene rhythms in multiple brain regions in LD and DD. Furthermore, paclitaxel disrupted circulating corticosterone rhythms in DD by elevating its levels across a 24-hour cycle, which correlated with blunted amplitudes of Arntl, Nr1d1, Per1, and Star rhythms in the adrenal glands. Paclitaxel also shortened SCN slice rhythms, increased the amplitude of adrenal gland oscillations in PER2::luciferase cultures, and increased the concentration of pro-inflammatory cytokines and chemokines released from the SCN. These findings indicate that paclitaxel disrupts clock genes and behavior driven by the SCN, other brain regions, and adrenal glands, which were associated with chemotherapy-induced inflammation. Together, this preclinical work demonstrates that chemotherapy disrupts both central and peripheral circadian rhythms and supports the possibility that targeted circadian realignment therapies may be a novel and non-invasive way to improve patient outcomes after chemotherapy.

Lectin isolated from Abelmoschus esculentus induces caspase mediated apoptosis in human U87 glioblastoma cell lines and modulates the expression of circadian clock genes

Lectins are a cluster of proteins which are capable of recognizing and binding to glycoconjugates and are extensively found in plants, animals, fungi and bacteria. Plant-derived lectins have been gaining importance over the years due to their innumerable biological activities and also have the added possibility of being compatible to the human system while simultaneously exhibiting properties like antimicrobial and antitumor activities.Abelmoschus esculentus (AE) commonly known as okra is a vegetable with medicinal properties. AE extracts are used to treat disorders such as constipation, microbial infection, urine retention, hypoglycemia and inflammation in humans. Previous studies showed that lectin isolated from AE exhibited anti inflammatory, anti nociceptive, anticancer, antioxidant and hemagglutinating activities. However, the antitumor effect of the lectin derived from this plant against neural cancer cells still remains unexplored.Glioblastoma is a malignant tumor of the nervous system. Treatment options for patients afflicted by glioblastoma is limited to surgical resection, preceded by radiation therapy and followed by chemotherapy. Hence it would be of interest to identify novel bio molecules with ability to selectively target glioblastoma with minimum side effects. In this aspect, lectins from vegetables that are commonly used as food products could offer a promising lead as anticancer molecules.The present study proves the anti-proliferative effect of lectin isolated from AE on human U87 glioma cells. MTT assay showed significant concentration dependent cytotoxic activity and the IC50 value was calculated as 21 μg/ml. Further, annexin V/FITC staining by FACS, the expression of caspase 3 and 7 and the circadian genes clock and Bmal1 using RT-PCR and the generation of intracellular ROS, cell cycle analysis by FACS revealed the ability of AEL to induce effective apoptosis.

Daily coordination of orexinergic gating in the rat superior colliculus-Implications for intrinsic clock activities in the visual system.

The orexinergic system delivers excitation for multiple brain centers to facilitate behavioral arousal, with its malfunction resulting in narcolepsy, somnolence, and notably, visual hallucinations. Since the circadian clock underlies the daily arousal, a timed coordination is expected between the orexin system and its target subcortical visual system, including the superior colliculus (SC). Here, we use a combination of electrophysiological, immunohistochemical, and molecular approaches across 24h, together with the neuronal tract-tracing methods to investigate the daily coordination between the orexin system and the rodent SC. Higher orexinergic input was found to occur nocturnally in the superficial layers of the SC, in time for nocturnal silencing of spontaneous firing in this visual brain area. We identify autonomous daily and circadian expression of clock genes in the SC, which may underlie these day-night changes. Additionally, we establish the lateral hypothalamic origin of the orexin innervation to the SC and that the SC neurons robustly respond to orexin A via OX2 receptor in both excitatory and GABAA receptor-dependent inhibitory manners. Together, our evidence elucidates the combination of intrinsic and extrinsic clock mechanisms that shape the daily function of the visual layers of the SC.

Reversible dysregulation of renal circadian rhythm in lupus nephritis

BackgroundWe have found disruption of expression of major transcriptional regulators of circadian rhythm in the kidneys of several mouse models of lupus nephritis. Here we define the consequence of this disturbance with respect to circadian gene expression and renal homeostatic function in a mouse model of lupus nephritis.MethodsMolecular profiling of kidneys from 47 young and 41 nephritic female NZB/W F1 mice was performed at 4 hourly intervals over a 24 h period. Disruption of major circadian transcriptional regulators was confirmed by qPCR. Molecular data was normalized and analyzed for rhythmicity using RAIN analysis. Serum aldosterone and glucose and urine sodium and potassium were measured at 4 hourly intervals in pre-nephritic and nephritic mice and blood pressure was measured every 4 h. Analyses were repeated after induction of complete remission of nephritis using combination cyclophosphamide and costimulatory blockade.ResultsWe show a profound alteration of renal circadian rhythms in mice with lupus nephritis affecting multiple renal pathways. Using Cosinor analysis we identified consequent alterations of renal homeostasis and metabolism as well as blood pressure dipper status. This circadian dysregulation was partially reversed by remission induction therapy.ConclusionsOur studies indicate the role of inflammation in causing the circadian disruption and suggest that screening for loss of normal blood pressure dipping should be incorporated into LN management. The data also suggest a potential role for circadian agonists in the treatment of lupus nephritis.

The potential remedy of melatonin on osteoarthritis.

Osteoarthritis (OA), the most common arthritis worldwide, is a degenerative joint disease characterized by progressive cartilage breakdown, subchondral remodeling, and synovial inflammation. Although conventional pharmaceutical therapies aimed to prevent further cartilage loss and joint dysfunction, there are no ideal strategies that target the pathogenesis of OA. Melatonin exhibits a variety of regulatory properties by binding to specific receptors and downstream molecules and exerts a myriad of receptor-independent actions via intracellular targets as a chondrocyte protector, an anti-inflammation modulator, and a free radical scavenger. Melatonin also modulates cartilage regeneration and degradation by directly/indirectly regulating the expression of main circadian clock genes, such as transcriptional activators [brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal) and circadian locomotor output cycles kaput (Clock)], transcriptional repressors [period circadian regulator (Per)1/2, cryptochrome (Cry)1/2, and Dec2], and nuclear hormone receptors [Rev-Erbs and retinoid acid-related orphan receptors (Rors)]. Owing to its effects on cartilage homeostasis, we propose a potential role for melatonin in the prevention and therapy of OA via the modulation of circadian clock genes, mitigation of chondrocyte apoptosis, anti-inflammatory activity, and scavenging of free radicals.

Period 2: A Regulator of Multiple Tissue-Specific Circadian Functions.

The zebrafish represents a powerful model for exploring how light regulates the circadian clock due to the direct light sensitivity of its peripheral clocks, a property that is retained even in organ cultures as well as zebrafish-derived cell lines. Light-inducible expression of the per2 clock gene has been predicted to play a vital function in relaying light information to the core circadian clock mechanism in many organisms, including zebrafish. To directly test the contribution of per2 to circadian clock function in zebrafish, we have generated a loss-of-function per2 gene mutation. Our results reveal a tissue-specific role for the per2 gene in maintaining rhythmic expression of circadian clock genes, as well as clock-controlled genes, and an impact on the rhythmic behavior of intact zebrafish larvae. Furthermore, we demonstrate that disruption of the per2 gene impacts on the circadian regulation of the cell cycle in vivo. Based on these results, we hypothesize that in addition to serving as a central element of the light input pathway to the circadian clock, per2 acts as circadian regulator of tissue-specific physiological functions in zebrafish.

Chronic stress induces NPD-like behavior in APPPS1 and WT mice with subtle differences in gene expression.

Neuropsychiatric disturbances (NPDs) are considered hallmarks of Alzheimer's disease (AD). Nevertheless, treatment of these symptoms has proven difficult and development of safe and effective treatment options is hampered by the limited understanding of the underlying pathophysiology. Thus, robust preclinical models are needed to increase knowledge of NPDs in AD and develop testable hypotheses and novel treatment options. Abnormal activity of the hypothalamic–pituitary–adrenal (HPA) axis is implicated in many psychiatric symptoms and might contribute to both AD and NPDs development and progression. We aimed to establish a mechanistic preclinical model of NPD‐like behavior in the APPPS1 mouse model of AD and wildtype (WT) littermates. In APPPS1 and WT mice, we found that chronic stress increased anxiety‐like behavior and altered diurnal locomotor activity suggestive of sleep disturbances. Also, chronic stress activated the HPA axis, which, in WT mice, remained heightened for additional 3 weeks. Chronic stress caused irregular expression of circadian regulatory clock genes (BMAL1, PER2, CRY1 and CRY2) in both APPPS1 and WT mice. Interestingly, APPPS1 and WT mice responded differently to chronic stress in terms of expression of serotonergic markers (5‐HT1A receptor and MAOA) and inflammatory genes (IL‐6, STAT3 and ADMA17). These findings indicate that, although the behavioral response to chronic stress might be similar, the neurobiochemical response was different in APPPS1 mice, which is an important insight in the efforts to develop safe and effective treatments options for NPDs in AD patients. Further work is needed to substantiate these findings.

The Histone Variant MacroH2A1 Impacts Circadian Gene Expression and Cell Phenotype in an In Vitro Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. A foremost risk factor for HCC is obesity/metabolic syndrome-related non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), which is prompted by remarkable changes in transcription patterns of genes enriching metabolic, immune/inflammatory, and circadian pathways. Epigenetic mechanisms play a role in NAFLD-associated HCC, and macroH2A1, a variant of histone H2A, is involved in the pathogenesis modulating the expression of oncogenes and/or tumor suppressor genes and interacting with SIRT1, which crucially impacts the circadian clock circuitry. Hence, we aimed to appraise if and how macroH2A1 regulated the expression patterns of circadian genes in the setting of NAFLD-associated HCC. We took advantage of an in vitro model of liver cancer represented by HepG2 (human hepatocarcinoma) cells stably knocked down for macroH2A1 and conducted whole transcriptome profiling and deep phenotyping analysis. We found up-regulation of PER1 along with several deregulated circadian genes, enriching several important pathways and functions related to cancer onset and progression, such as epithelial-to-mesenchymal transition, cell cycle deregulation, and DNA damage. PER1 silencing partially mitigated the malignant phenotype induced by the loss of macroH2A1 in HCC cells. In conclusion, our findings suggest a modulatory role for the core circadian protein PER1 in liver carcinogenesis in the context of a lack of the macroH2A1 epigenetic and transcriptional landscape.

Comparative Transcriptome Analysis Reveals bmo-miR-6497-3p Regulate Circadian Clock Genes during the Embryonic Diapause Induction Process in Bivoltine Silkworm.

Simple SummaryDiapause is a type of seasonal polyphenism and is induced by multiple cues, such as light and temperature. However, the molecular mechanisms of how the environment induces diapause remains unclear. We are interested in the epigenetic regulation, especially the non-coding RNA, involved in environmentally induced diapause. The progeny eggs of bivoltine silkworm strains undergoing diapause or not depends on the incubation temperature during the maternal embryonic stage. In this research, we compared the differentially expressed microRNAs in embryos incubated under diapause-inducing temperature (25 °C) and non-diapause-inducing temperature (15 °C) in silkworm. Our results indicate that miRNAs respond to diapause-inducing temperature and regulate the expression of circadian clock genes. Our research not only provides reference for the study of the diapause induction process, but also gives insights into the role of epigenetic modification in diapause.Diapause is one of the survival strategies of insects for confronting adverse environmental conditions. Bombyx mori displays typical embryonic diapause, and offspring diapause depends on the incubation environment of the maternal embryo in the bivoltine strains of the silkworm. However, the molecular mechanisms of the diapause induction process are still poorly understood. In this study, we compared the differentially expressed miRNAs (DEmiRs) in bivoltine silkworm embryos incubated at diapause- (25 °C) and non-diapause (15 °C)-inducing temperatures during the blastokinesis (BK) and head pigmentation (HP) phases using transcriptome sequencing. There were 411 known miRNAs and 71 novel miRNAs identified during the two phases. Among those miRNAs, there were 108 and 74 DEmiRs in the BK and HP groups, respectively. By the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the predicted target genes of the DEmiRs, we found that aside from metabolism, the targets were also enriched in phototransduction-fly and insect hormone biosynthesis in the BK group and the HP group, respectively. Dual luciferase reporter assay illustrated that bmo-miR-6497-3p directly regulated Bmcycle and subsequently regulated the expression of circadian genes. These results imply that microRNAs, as vitally important regulators, respond to different temperatures and participate in the diapause induction process across species.

Circadian regulation of apolipoprotein gene expression affects testosterone production in mouse testis

The circadian clock system plays an important role in regulating testosterone synthesis in mammals. Male Bmal1−/− mice are infertile with low serum testosterone levels and decreased expression of testicular steroidogenic genes, suggesting that circadian clock genes regulate testosterone biosynthesis by activating steroidogenic gene transcription. However, whether the circadian clock regulates testosterone production via other genes remains unknown. Using Bmal1−/− mice and their wild-type (WT) siblings, we aimed to identify additional genes by which the circadian clock regulates testosterone synthesis. WT and Bmal1−/− mouse testes sections had similar normal morphologies, although there was a decrease in testicular spermatozoa in the Bmal1−/− mice. Low serum testosterone levels were detected in the Bmal1−/− mice. RNA sequencing identified 37 and 48 genes that were differentially expressed between WT and Bmal1−/− mouse testes at circadian time (CT2 and CT14), respectively. The cholesterol metabolism pathway was significantly enriched in the KEGG pathway analysis, and there was lower expression of three apolipoprotein genes (Apoa1, Apoa2, and Apoc3) at CT2 in the testes of Bmal1−/− mice than in those of WT mice. These decreases in Apoa1, Apoa2, and Apoc3 expression were verified by quantitative polymerase chain reaction analysis, which also revealed downregulation of the expression of the circadian clock (Per2, Dbp, and Nr1d1) and steroidogenic (StAR, Cyp11a1, and Hsd17b3) genes. The expression of circadian clock genes was relatively stable in WT mice over a 20-h period, whereas there was clear circadian rhythmic expression of Apoa1, Apoa2, Apoc3, StAR, Cyp11a1, Hsd3b2, and Hsd17b3. Bmal1−/− mice showed severely reduced expression of testicular circadian clock genes at three time points (CT4, CT12, and CT20), and a reduction in mRNA expression levels of Apo (Apoa1, Apoa2, and Apoc3) and steroidogenic (StAR, Cyp11a1, Hsd3b2, and Hsd17b3) genes. Oil Red O staining showed decreased lipid aggregation in the Leydig cells of Bmal1−/− mouse testes. Considering the vital role of Apo genes in high-density lipoprotein formation and cholesterol transport, the present data suggest that the circadian clock system regulates testosterone production by orchestrating the rhythmic expression of Apo genes. These data extend our understanding of the role of the circadian clock in regulating testosterone production in mammals.

Interpreting machine learning models to investigate circadian regulation and facilitate exploration of clock function

The circadian clock is an important adaptation to life on Earth. Here, we use machine learning to predict complex, temporal, and circadian gene expression patterns in Arabidopsis Most significantly, we classify circadian genes using DNA sequence features generated de novo from public, genomic resources, facilitating downstream application of our methods with no experimental work or prior knowledge needed. We use local model explanation that is transcript specific to rank DNA sequence features, providing a detailed profile of the potential circadian regulatory mechanisms for each transcript. Furthermore, we can discriminate the temporal phase of transcript expression using the local, explanation-derived, and ranked DNA sequence features, revealing hidden subclasses within the circadian class. Model interpretation/explanation provides the backbone of our methodological advances, giving insight into biological processes and experimental design. Next, we use model interpretation to optimize sampling strategies when we predict circadian transcripts using reduced numbers of transcriptomic timepoints. Finally, we predict the circadian time from a single, transcriptomic timepoint, deriving marker transcripts that are most impactful for accurate prediction; this could facilitate the identification of altered clock function from existing datasets.

Large-scale analysis reveals spatiotemporal circadian patterns of cilia transcriptomes in the primate brain.

Cilia are dynamic subcellular systems, with core structural and functional components operating in a highly coordinated manner. Since many environmental stimuli sensed by cilia are circadian in nature, it is reasonable to speculate that genes encoding cilia structural and functional components follow rhythmic circadian patterns of expression. Using computational methods and the largest spatiotemporal gene expression atlas of primates, we identified and analyzed the circadian rhythmic expression of cilia genes across 22 primate brain areas. We found that around 73% of cilia transcripts exhibited circadian rhythmicity across at least one of 22 brain regions. In 12 brain regions, cilia transcriptomes were significantly enriched with circadian oscillating transcripts, as compared to the rest of the transcriptome. The phase of the cilia circadian transcripts deviated from the phase of the majority of the background circadian transcripts, and transcripts coding for cilia basal body components accounted for the majority of cilia circadian transcripts. In addition, adjacent or functionally connected brain nuclei had large overlapping complements of circadian cilia genes. Most remarkably, cilia circadian transcripts shared across the basal ganglia nuclei and the prefrontal cortex peaked in these structures in sequential fashion that is similar to the sequential order of activation of the basal ganglia-cortical circuitry in connection with movement coordination, albeit on completely different timescales. These findings support a role for the circadian spatiotemporal orchestration of cilia gene expression in the normal physiology of the basal ganglia-cortical circuit and motor control. Studying orchestrated cilia rhythmicity in the basal ganglia-cortical circuits and other brain circuits may help develop better functional models, and shed light on the causal effects cilia functions have on these circuits and on the regulation of movement and other behaviors.

What if melatonin could help patients with severe COVID-19?

What if melatonin could help patients with severe COVID-19?

The Vascular Circadian Clock in Chronic Kidney Disease.

Chronic kidney disease is associated with extremely high cardiovascular mortality. The circadian rhythms (CR) have an impact on vascular function. The disruption of CR causes serious health problems and contributes to the development of cardiovascular diseases. Uremia may affect the master pacemaker of CR in the hypothalamus. A molecular circadian clock is also expressed in peripheral tissues, including the vasculature, where it regulates the different aspects of both vascular physiology and pathophysiology. Here, we address the impact of CKD on the intrinsic circadian clock in the vasculature. The expression of the core circadian clock genes in the aorta is disrupted in CKD. We propose a novel concept of the disruption of the circadian clock system in the vasculature of importance for the pathology of the uremic vasculopathy.

MicroRNA-21 Controls Circadian Regulation of Apoptosis in Atherosclerotic Lesions.

The necrotic core partly formed by ineffective efferocytosis increases the risk of an atherosclerotic plaque rupture. Microribonucleic acids contribute to necrotic core formation by regulating efferocytosis and macrophage apoptosis. Atherosclerotic plaque rupture occurs at increased frequency in the early morning, indicating diurnal changes in plaque vulnerability. Although circadian rhythms play a role in atherosclerosis, the molecular clock output pathways that control plaque composition and rupture susceptibility are unclear. Circadian gene expression, necrotic core size, apoptosis, and efferocytosis in aortic lesions were investigated at different times of the day in Apoe-/-Mir21+/+ mice and Apoe-/-Mir21-/- mice after consumption of a high-fat diet for 12 weeks. Genome-wide gene expression and lesion formation were analyzed in bone marrow-transplanted mice. Diurnal changes in apoptosis and clock gene expression were determined in human atherosclerotic lesions. The expression of molecular clock genes, lesional apoptosis, and necrotic core size were diurnally regulated in Apoe-/- mice. Efferocytosis did not match the diurnal increase in apoptosis at the beginning of the active phase. However, in parallel with apoptosis, expression levels of oscillating Mir21 strands decreased in the mouse atherosclerotic aorta. Mir21 knockout abolished circadian regulation of apoptosis and reduced necrotic core size but did not affect core clock gene expression. Further, Mir21 knockout upregulated expression of proapoptotic Xaf1 (XIAP-associated factor 1) in the atherosclerotic aorta, which abolished circadian expression of Xaf1. The antiapoptotic effect of Mir21 was mediated by noncanonical targeting of Xaf1 through both Mir21 strands. Mir21 knockout in bone marrow cells also reduced atherosclerosis and necrotic core size. Circadian regulation of clock gene expression was confirmed in human atherosclerotic lesions. Apoptosis oscillated diurnally in phase with XAF1 expression, demonstrating an early morning peak antiphase to that of the Mir21 strands. Our findings suggest that the molecular clock in atherosclerotic lesions induces a diurnal rhythm of apoptosis regulated by circadian Mir21 expression in macrophages that is not matched by efferocytosis, thus increasing the size of the necrotic core.

Serum from type 2 diabetes patients consuming a three-meal diet resets circadian rhythms in cultured hepatocytes

AimsFeeding regimens alter circadian rhythms in peripheral tissues, but the mechanism is not understood. We aimed to study whether soluble factors, rather than neuronal-based communication, directly influence circadian rhythms in the liver, in response to a nutritional treatment in type 2 diabetes (T2D) patients. MethodsCultured hepatocytes were treated with serum of insulin-treated T2D patients following either a three-meal diet (3Mdiet) or six-meal diet (6Mdiet) and the circadian expression of clock and metabolic genes was measured. ResultsSerum of the 3Mdiet group led to increased amplitudes and daily mRNA levels of the positive limb of the circadian clock (Clock, Bmal1, Rorα). In parallel, serum of the 3Mdiet group led to the downregulation of the negative limb of the circadian clock (Cry1 and Per1), compared to both baseline and 6Mdiet. In contrast, serum of the 6Mdiet group led to a more distorted expression pattern. The catabolic genes Sirt1 and Ampk were significantly upregulated only by serum of the 3Mdiet group. ConclusionsOur results show that serum of type 2 diabetes patients consuming the 3Mdiet contains soluble factors that reset circadian rhythms leading to an expression pattern similar to that of healthy people. This clock pattern contributes to improved glucose metabolism.

Identification of a circadian gene signature that predicts overall survival in lung adenocarcinoma.

BackgroundLung adenocarcinoma (LUAD) is one of the most common subtypes of lung cancer which is the leading cause of death in cancer patients. Circadian clock disruption has been listed as a likely carcinogen. However, whether the expression of circadian genes affects overall survival (OS) in LUAD patients remains unknown. In this article, we identified a circadian gene signature to predict overall survival in LUAD.MethodsRNA sequencing (HTSeq-FPKM) data and clinical characteristics were obtained for a cohort of LUAD patients from The Cancer Genome Atlas (TCGA). A multigene signature based on differentially expressed circadian clock-related genes was generated for the prediction of OS using Least Absolute Shrinkage and Selection Operator (LASSO)-penalized Cox regression analysis, and externally validated using the GSE72094 dataset from the GEO database.ResultsFive differentially expressed genes (DEGs) were identified to be significantly associated with OS using univariate Cox proportional regression analysis (P < 0.05). Patients classified as high risk based on these five DEGs had significantly lower OS than those classified as low risk in both the TGCA cohort and GSE72094 dataset (P < 0.001). Multivariate Cox regression analysis revealed that the five-gene-signature based risk score was an independent predictor of OS (hazard ratio > 1, P < 0.001). Receiver operating characteristic (ROC) curves confirmed its prognostic value. Gene set enrichment analysis (GSEA) showed that Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to cell proliferation, gene damage repair, proteasomes, and immune and autoimmune diseases were significantly enriched.ConclusionA novel circadian gene signature for OS in LUAD was found to be predictive in both the derivation and validation cohorts. Targeting circadian genes is a potential therapeutic option in LUAD.

Systems and Circuits Linking Chronic Pain and Circadian Rhythms.

Research over the last 20 years regarding the link between circadian rhythms and chronic pain pathology has suggested interconnected mechanisms that are not fully understood. Strong evidence for a bidirectional relationship between circadian function and pain has been revealed through inflammatory and immune studies as well as neuropathic ones. However, one limitation of many of these studies is a focus on only a few molecules or cell types, often within only one region of the brain or spinal cord, rather than systems-level interactions. To address this, our review will examine the circadian system as a whole, from the intracellular genetic machinery that controls its timing mechanism to its input and output circuits, and how chronic pain, whether inflammatory or neuropathic, may mediate or be driven by changes in these processes. We will investigate how rhythms of circadian clock gene expression and behavior, immune cells, cytokines, chemokines, intracellular signaling, and glial cells affect and are affected by chronic pain in animal models and human pathologies. We will also discuss key areas in both circadian rhythms and chronic pain that are sexually dimorphic. Understanding the overlapping mechanisms and complex interplay between pain and circadian mediators, the various nuclei they affect, and how they differ between sexes, will be crucial to move forward in developing treatments for chronic pain and for determining how and when they will achieve their maximum efficacy.

Dopamine adjusts the circadian gene expression of Per2 and Per3 in human dermal fibroblasts from ADHD patients

A link between dopamine levels, circadian gene expression, and attention deficit hyperactivity disorder (ADHD) has already been demonstrated. The aim of this study was to investigate the extent of these relationships by measuring circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after dopamine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different dopamine concentrations in human dermal fibroblast (HDF) cultures, the rhythmicity of circadian gene expression (Clock, Bmal1, Per1-3, Cry1) was analyzed via qRT-PCR. We found no statistical significant effect in the actigraphy of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, wake after sleep onset, and total number of wake bouts. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. Dopamine has no effect on Per3 expression in healthy controls, but produces a significant difference in the ADHD group at ZT24 and ZT28. In the ADHD group, incubation with dopamine, either 1 µM or 10 µM, resulted in an adjustment of Per3 expression to control levels. A similar effect also was found in the expression of Per2. Statistical significant differences in the expression of Per2 (ZT4) in the control group compared to the ADHD group were found, following incubation with dopamine. The present study illustrates that dopamine impacts on circadian function. The results lead to the suggestion that dopamine may improve the sleep quality as well as ADHD symptoms by adjustment of the circadian gene expression, especially for Per2 and Per3.

Abstract 863: Circadian gene expression in metastatic sites and association with survival in metastatic castration-resistant prostate cancer

Abstract Introduction: In experimental and epidemiological studies, alterations in several core circadian genes at the germline and tumor level have been associated with prostate cancer. The aim of this study was to investigate mRNA expression of circadian related genes in men with metastatic castration-resistant prostate cancer (mCRPC), and the association with survival. Methods: We assessed whole exome and RNA sequencing data from 317 mCRPC patients from the Stand Up to Cancer-Prostate Cancer Foundation (SU2C-PCF) database. Data were obtained from six sites: metastasis to bone (n=107; n=65 deaths), lymph node (n=129; n=88 deaths), liver (n=42; n=35 deaths), lung (n=6; n=3 deaths), and other soft tissue (n=26; n=20 deaths), as well as primary prostate (n=7; n=5 deaths) over a median follow-up of 71.8 months. We evaluated expression of twelve core circadian genes (ARNTL, CLOCK, CRY1, CRY2, CSNK1E, NR1D1, NPAS2, PER1, PER2, PER3, RORA, TIMELESS) as transcripts per million (TPM). We used the correlation of correlations method to estimate inter-gene correlations between tissue. Unpaired Wilcoxon rank sum tests compared circadian expression differences with tumor mutations in AR and p53, two of the most common genomic alterations in mCRPC. We conducted multivariable Cox regression, overall and stratified by tissue type, to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs) for expression (modeled continuously) and overall survival, adjusted for age and PSA at diagnosis, Gleason, treatment, and histology. Results: Many genes showed low or negative correlation across tissues, with the greatest discordance in CSNK1E (μICC=0.10), and greatest concordance in TIMELESS (μICC=0.55). Lower expression of ARNTL was found in patients with alterations to both p53 and AR. Similarly, higher expression of PER2 and RORA was found in AR-/p53+, compared to those AR+ and AR-/p53-. Higher expression of TIMELESS was associated with risk of death overall and across all tissue sites (HRoverall: 1.02, 95% CI: 1.01-1.03). In liver, higher expression of CLOCK (HR: 0.22, 95% CI: 0.07 - 0.71) and CSNK1E (HR: 0.87, 95% CI: 0.76 - 1.00), and lower expression of CRY1 (HR: 1.62, 95% CI: 1.16 - 2.26) was associated with a lower risk of death. Higher expression of CRY2 (HR: 1.25, 95% CI: 1.02 - 1.53) in liver, but lower expression in bone (HR: 0.95, 95% CI: 0.90 - 1.00) was associated with an increased risk of death. We found no association between ARNTL, NR1D1, NPAS2, PER3, or RORA and survival in any metastatic site. Conclusion: Our results show that circadian gene expression is altered in tissue from mCRPC patients, with substantial heterogeneity in circadian related expression patterns between metastatic tissue sites. These results support prior research on the role of circadian gene expression, particularly CRY1 and CLOCK, and outcomes in localized prostate cancer. Citation Format: Benjamin D. Booker, Konrad H. Stopsack, Travis A. Gerke, Kathryn Penny, Philip W. Kantoff, Lorelei A. Mucci, Sarah C. Markt, PCF/SU2C International Prostate Cancer Dream Team. Circadian gene expression in metastatic sites and association with survival in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 863.