Machine Learning and In-Vivo Expression Analyses Reveal Circadian Clock Features Predictive of Anxiety in Humans

Mood disorders, including anxiety, are associated with disruptions in circadian rhythms and are linked to polymorphisms in circadian clock genes. Molecular mechanisms underlying these connections may be direct—via transcriptional activity of clock genes on downstream mood pathways in the brain, or indirect—via clock gene influences on the phase and amplitude of circadian rhythms which, in turn, modulate physiological processes influencing mood. Employing machine learning combined with statistical approaches, we explored clock genotype combinations that predict risk for anxiety symptoms in a deeply phenotyped population. We identified multiple novel circadian genotypes predictive of anxiety, with the PER3B-AG/CRY1-CG genotype being the strongest predictor of anxiety risk in males. Molecular chronotyping, using clock gene expression oscillations, revealed that advanced circadian phase and robust circadian amplitudes are associated with high levels of anxiety symptoms. Further analyses revealed that individuals with advanced phases and pronounced circadian misalignment were at higher risk for severe anxiety symptoms. Our results support both direct and indirect influences of clock gene variants on mood: while sex-specific clock genotype combinations predictive of anxiety symptoms suggest direct effects on mood pathways, the mediation of PER3B effects on anxiety via diurnal preference measures and the association of circadian phase with anxiety symptoms provide evidence for indirect effects of the molecular clockwork on mood. Unraveling the complex molecular mechanisms underlying the links between circadian physiology and mood is essential to identifying the core clock genes to target in future functional studies, thereby advancing the development of non-invasive treatments for anxiety-related disorders.

The Association between Circadian Clock Gene Polymorphisms and Metabolic Syndrome: A Systematic Review and Meta-Analysis

Metabolic syndrome (MetS) is a combination of cardiovascular risk factors associated with type 2 diabetes, obesity, and cardiovascular diseases. The circadian clock gene polymorphisms are very likely to participate in metabolic syndrome genesis and development. However, research findings of the association between circadian rhythm gene polymorphisms and MetS and its comorbidities are not consistent. In this study, a review of the association of circadian clock gene polymorphisms with overall MetS risk was performed. In addition, a meta-analysis was performed to clarify the association between circadian clock gene polymorphisms and MetS susceptibility based on available data. The PubMed and Scopus databases were searched for studies reporting the association between circadian rhythm gene polymorphisms (ARNTL, BMAL1, CLOCK, CRY, PER, NPAS2, REV-ERBα, REV-ERBβ, and RORα) and MetS, and its comorbidities diabetes, obesity, and hypertension. Thirteen independent studies were analyzed with 17,381 subjects in total. The results revealed that the BMAL1 rs7950226 polymorphism was associated with an increased risk of MetS in the overall population. In contrast, the CLOCK rs1801260 and rs6850524 polymorphisms were not associated with MetS. This study suggests that some circadian rhythm gene polymorphisms might be associated with MetS in different populations and potentially used as predictive biomarkers for MetS.

Expression Patterns of Clock Gene mRNAs and Clock Proteins in Human Psoriatic Skin Samples

Psoriasis is a systemic inflammatory skin disorder that can be associated with sleep disturbance and negatively influence the daily rhythm. The link between the pathomechanism of psoriasis and the circadian rhythm has been suggested by several previous studies. However, there are insufficient data on altered clock mechanisms in psoriasis to prove these theories. Therefore, we investigated the expression of the core clock genes in human psoriatic lesional and non-lesional skin and in human adult low calcium temperature (HaCaT) keratinocytes after stimulation with pro-inflammatory cytokines. Furthermore, we examined the clock proteins in skin biopsies from psoriatic patients by immunohistochemistry. We found that the clock gene transcripts were elevated in psoriatic lesions, especially in non-lesional psoriatic areas, except for rev-erbα, which was consistently downregulated in the psoriatic samples. In addition, the REV-ERBα protein showed a different epidermal distribution in non-lesional skin than in healthy skin. In cytokine-treated HaCaT cells, changes in the amplitude of the bmal1, cry1, rev-erbα and per1 mRNA oscillation were observed, especially after TNFα stimulation. In conclusion, in our study a perturbation of clock gene transcripts was observed in uninvolved and lesional psoriatic areas compared to healthy skin. These alterations may serve as therapeutic targets and facilitate the development of chronotherapeutic strategies in the future.

Depriving mice of sleep also deprives of food

Both sleep-wake behavior and circadian rhythms are tightly coupled to energy metabolism and food intake. Altered feeding times in mice are known to entrain clock-gene rhythms in brain and liver and sleep-deprived humans tend to eat more and gain weight. Previous observations in mice showing that sleep deprivation (SD) changes clock-gene expression might thus relate to altered food intake and not to the loss of sleep per se. Whether SD affects food intake in the mouse and how this might affect clock-gene expression is, however, unknown. We therefore quantified i) the cortical expression of the clock genes Per1, Per2, Dbp, and Cry1 in mice that had access to food or not during a 6h SD, and ii) food intake during baseline, SD, and recovery sleep. We found that food deprivation did not modify the SD-incurred clock-gene changes in the cortex. Moreover, we discovered that although food intake during SD did not differ from baseline, mice lost weight and increased food intake during subsequent recovery. We conclude that SD is associated with food deprivation and that the resulting energy deficit might contribute to the effects of SD that are commonly interpreted as a response to sleep loss.

Light sets the brain’s daily clock by regional quickening and slowing of the molecular clockworks at dawn and dusk

How daily clocks in the brain are set by light to local environmental time and encode the seasons is not fully understood. The suprachiasmatic nucleus (SCN) is a central circadian clock in mammals that orchestrates physiology and behavior in tune with daily and seasonal light cycles. Here, we have found that optogenetically simulated light input to explanted mouse SCN changes the waveform of the molecular clockworks from sinusoids in free-running conditions to highly asymmetrical shapes with accelerated synthetic (rising) phases and extended degradative (falling) phases marking clock advances and delays at simulated dawn and dusk. Daily waveform changes arise under ex vivo entrainment to simulated winter and summer photoperiods, and to non-24 hr periods. Ex vivo SCN imaging further suggests that acute waveform shifts are greatest in the ventrolateral SCN, while period effects are greatest in the dorsomedial SCN. Thus, circadian entrainment is encoded by SCN clock gene waveform changes that arise from spatiotemporally distinct intrinsic responses within the SCN neural network.

Expression of CRY2 Gene in the Brain Is Related to Human Navigation

Navigation is a complex cognitive process. CRY2 gene has been proposed to play an important role in navigation behaviors in various non-human animal species. Utilizing a recently developed neuroimaging-transcriptomics approach, the present study reported a tentative link between the CRY2 gene and human navigation. Specifically, we showed a significant pattern similarity between CRY2 gene expression in the human brain and navigation-related neural activation in functional magnetic resonance imaging. To further illuminate the functionality of CRY2 in human navigation, we examined the correlation between CRY2 expression and various cognitive processes underlying navigation, and found high correlation of CRY2 expression with neural activity of multiple cognitive domains, particularly object and shape perception and spatial memory. Further analyses on the relation between the neural activity of human navigation and the expression maps of genes of two CRY2-related pathways, i.e., the magnetoreceptive and circadian-related functions, found a trend of correlation for the CLOCK gene, a core circadian regulator gene, suggesting that CRY2 may modulate human navigation through its role in circadian rhythm. This observation was further confirmed by a behavioral study where individuals with better circadian regularity in daily life showed better sense of direction. Taken together, our study presents the first neural evidence that links CRY2 with human navigation, possibly through the modulation of circadian rhythm.

Circadian Clock Genes Modulate Immune, Cell Cycle and Apoptosis in the Diagnosis and Prognosis of Pan-Renal Cell Carcinoma

Background: Pan-renal cell carcinoma (pan-RCC) is mainly divided into renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), and chromophobe cell carcinoma (KICH). Pan-RCC is a common malignant neoplasm with a high incidence and poor prognosis. Several studies have demonstrated a close association between cancer development and circadian rhythms; however, the clinical significance and molecular mechanism of the clock gene remain unclear in pan-RCC.Methods: In this study, we systematically characterized the alterations of 15 well-known clock genes of three types of kidney cancer. Bioinformatics methods, including differential expression analysis, survival analysis, signing pathway analysis, co-expression network analysis, and drug sensitivity analysis were used to study the diagnosis, prognostic role, and mechanism of clock genes.Results: Thirteen rhythmic genes fluctuated in circadian rhythm in the kidney tissue of mice, and the opposite trend of these rhythm phases was also found in baboons. There are twelve clock genes that were differentially expressed in at least two types of RCC, of which NR1D1, DBP, BHLHE40, CRY1, and CLOCK had the same trend in RCC. Changes in clock control genes may be regulated through methylation, copy number, and mutations. Five rhythmic genes, including PER2, DBP, PER3, CRY2, and RORA, have significant prognostic role in patient survival in at least two types of kidney cancer. Immune infiltration analysis showed that the expression of these rhythmic genes related to prognosis was positively correlated with the infiltration levels of CD4 and CD8 T cells. Pathway analysis suggests that the clock genes is widely related to cancer-related signaling pathways, such as apoptosis, cell cycle, and other pathways. The PPI network showed that circadian genes are closely linked to cancer-related genes such as HIF-1A, TP53, and ERBB2. Moreover, clock gene expression is correlated with the sensitivity of anticancer drugs such as bleomycin and methotrexate in pan-RCC.Conclusion: Taken together, the abnormal expression of biological clock genes plays an important role in the clinical prognosis of RCC through immunity, cell cycle, and apoptosis. These findings provide a reliable basis for the diagnosis, prognosis, and drug guidance for RCC.

Circadian Clock Component BMAL1 in the Paraventricular Nucleus Regulates Glucose Metabolism

It is suggested that clock genes link the circadian rhythm to glucose and lipid metabolism. In this study, we explored the role of the clock gene Bmal1 in the hypothalamic paraventricular nucleus (PVN) in glucose metabolism. The Sim1-Cre-mediated deletion of Bmal1 markedly reduced insulin secretion, resulting in impaired glucose tolerance. The pancreatic islets’ responses to glucose, sulfonylureas (SUs) and arginine vasopressin (AVP) were well maintained. To specify the PVN neuron subpopulation targeted by Bmal1, the expression of neuropeptides was examined. In these knockout (KO) mice, the mRNA expression of Avp in the PVN was selectively decreased, and the plasma AVP concentration was also decreased. However, fasting suppressed Avp expression in both KO and Cre mice. These results demonstrate that PVN BMAL1 maintains Avp expression in the PVN and release to the circulation, possibly providing islet b-cells with more AVP. This action helps enhance insulin release and, consequently, glucose tolerance. In contrast, the circadian variation of Avp expression is regulated by feeding, but not by PVN BMAL1.