Genetic variability within molecular core clock genes in cluster headache.

Cluster headache is a disorder which has been shown to have both a circadian and a circannual pattern. This review gives an overview of the chronobiology of cluster headache summarizing recent findings on structural variations with a focus on the hypothalamus and the implication of the molecular clock and circadian and circannual variations in gene expression. Recent imaging studies using high-resolution structural and functional MRI have highlighted subtle hypothalamic alterations in cluster headache, specifically at the microstructural and connectivity level rather than clear macrostructural changes. Diffusion-based measures reveal altered fractional anisotropy and diffusivity in the hypothalamus, suggesting modified neuronal connectivity that may relate to attack frequency. In parallel, experimental data suggest significant differences in pain perception between day and night, which correlate to circadian oscillations of gene expression, and several drugs used for cluster headache have been reported to alter the molecular clock. The striking circadian and circannual phenotype of cluster headache opens for the possibility of clock-modulating therapy. The potential to pharmacologically target the molecular clock mechanism is supported by experimental data from mice demonstrating substantial effects of standard cluster headache treatments on the molecular clock.

Background and Purpose: The circadian rhythm, regulated by the peripheral clock, is crucial for lung development. Key molecular clock genes regulate cellular functions and tissue repair, essential for lung maturation. In preterm infants, disruptions in the molecular clock may contribute to bronchopulmonary dysplasia (BPD), characterized by impaired lung development and function. Cellular senescence can further hinder lung maturation. Our studies suggest that disruptions in the molecular clock may accelerate senescence and affect lung function. We hypothesized that molecular clock expression patterns influence cellular senescence. To test this, we used single-nucleus RNA sequencing (snRNA-seq) to analyze developing lungs from preterm to young adult subjects, including BPD patients, and employed CODEX spatial biology to visualize proteins and biomarkers in pediatric tissues. Methods: Pediatric lung tissue samples were obtained from the BRINDL repository. Frozen sections were processed for snRNA-seq using 10X Genomics technology to evaluate circadian and senescence-related gene expression. Samples were grouped into seven categories: Pre-term (31-36 weeks), Full-term (40 weeks), BPD (≤28 weeks), Children (2-8 years), Adolescents (13-16 years), and Young Adults (23-25 years). Bioinformatic analyses, including pseudo-time analysis, identified gene expression patterns. Findings were validated by CODEX staining and multiplex assays (Luminex, NanoString) for protein and gene expression. Results: Distinct circadian gene expression and growth factor levels were observed across developmental stages. In BPD, reduced expression of key clock-related genes (nrd1, arntl, per2, clock, cry1, cry2) in Alveolar Type 2 cells suggests disrupted circadian regulation. Altered growth factors (VEGF, FGF, EGFR, IGF1) indicate impaired lung development pathways. In contrast, pediatric and young adult groups showed lower senescence markers (cdkn1a, cdkn2a) in epithelial cells. CODEX staining revealed increased senescence markers (p16, GLB1) and proteins (COL1A1, α-SMA) in BPD donors, suggesting post-transcriptional modifications. Trajectory analysis revealed the expression patterns of molecular clock and senescence genes during early lung development, with healthier circadian function and reduced senescence markers in early lung development and young adults. Conclusion: This study highlights the role of circadian molecular clock gene expression and growth factors in lung health, particularly in epithelial cells. Disrupted circadian rhythms in BPD may impair alveologenesis, while increased circadian expression supports lung growth. These findings suggest potential therapeutic interventions to enhance circadian function and reduce senescence, improving respiratory outcomes in preterm infants at risk for BPD. Future research should focus on restoring circadian rhythms and supporting growth factor signaling for optimal lung development.

BackgroundCluster headache is a severe primary headache disorder commonly featuring a strikingly distinct circadian attack pattern. Therefore, the circadian system has been suggested to play a crucial role in the pathophysiology of cluster headache. Cryptochromes are key components of the molecular clock generating circadian rhythms and have previously been shown to be associated with several psychiatric disorders, including seasonal affective disorder, bipolar disorder, and depression.MethodsIn this case-control study, we investigated the role of cryptochrome (CRY) genes in cluster headache by screening 628 cluster headache patients and 681 controls from Sweden for four known genetic variants in the CRY1 (rs2287161 and rs8192440) and CRY2 (rs10838524 and rs1554338) genes. In addition, we analyzed CRY1 gene expression in primary fibroblast cell lines from eleven patients and ten controls.ResultsThe exonic CRY1 variant rs8192440 was associated with cluster headache on allelic level (p=0.02) and this association was even more pronounced in a subgroup of patients with reported diurnal rhythmicity of attacks (p=0.002). We found a small significant difference in CRY1 gene expression between cluster headache patients and control individuals (p=0.04), but we could not identify an effect of the associated variant rs8192440 on CRY1 expression.ConclusionsWe discovered a disease-associated variant in the CRY1 gene and slightly increased CRY1 gene expression in tissue from cluster headache patients, strengthening the hypothesis of circadian dysregulation in cluster headache. How this gene variant may contribute to the pathophysiology of the disease remains subject to further studies.

The circadian rhythm regulates the 24-hour physiological and behavioral cycles through endogenous molecular clocks governed by core clock genes via the transcription-translation feedback loop (TTFL). In mammals, the suprachiasmatic nucleus (SCN) serves as the central pacemaker, coordinating the timing of physiological processes throughout the body by regulating clock genes such as CLOCK, BMAL1, PER, and CRY. The molecular clocks of peripheral tissues and cells are synchronized by the SCN through TTFLs to regulate metabolism, immunity, and energy homeostasis. Numerous studies indicate that circadian rhythm disruption is closely related to obesity, type 2 diabetes, metabolic syndrome and other diseases, and the mechanism involves the dysregulation of glucose and lipid metabolism, abnormal insulin signaling and low-grade inflammation. In recent years, small-molecule compounds targeting the core clock components such as CRY, REV-ERB, and ROR have been identified and shown potential to modulate metabolic diseases by stabilizing or inhibiting the activity of key clock proteins. This review summarizes the mechanisms and advances in these compounds, and explores the challenges and future directions for their clinical translation, providing insights for chronotherapy-based metabolic disease interventions.

The peripheral tissue pacemaker is responsive to light and other zeitgebers, especially food availability. Generally, the pacemaker can be reset and entrained independently of the central circadian structures. Studies involving clock-gene expressional patterns in fish peripheral tissues have attracted considerable attention. However, the rhythmic expression of clock genes in skeletal muscle has only scarcely been investigated. The present study was designed to investigate the core clock and functional gene expression rhythms in crucian carp. Meanwhile, the synchronized effect of food restrictions (short-term fasting) on these rhythms in skeletal muscle was carefully examined. In fed crucian carp, three core clock genes (Clock, Bmal1a, and Per1) and five functional genes (Epo, Fas, IGF1R2, Jnk1, and MyoG) showed circadian rhythms. By comparison, four core clock genes (Clock, Bmal1a, Cry3, and Per2) and six functional genes (Epo, GH, IGF2, Mstn, Pnp5a, and Ucp1) showed circadian rhythms in crucian carp muscle after 7-day fasting. In addition, three core clock genes (Clock, Per1, and Per3) and six functional genes (Ampk1a, Lpl, MyoG, Pnp5a, PPARα, and Ucp1) showed circadian rhythms in crucian carp muscle after 15-day fasting. However, all gene rhythmic expression patterns differed from each other. Furthermore, it was found that the circadian genes could be altered by feed deprivation in crucian carp muscle through the rhythms correlation analysis of the circadian genes and functional genes. Hence, food-anticipatory activity of fish could be adjusted through the food delivery restriction under a light–dark cycle. These results provide a potential application in promoting fish growth by adjusting feeding conditions and nutritional state.

BackgroundUnder 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.

Although the intracellular molecular clocks that regulate circadian (~24 h) behavioral rhythms are well understood, it remains unclear how molecular clock information is transduced into rhythmic neuronal activity that in turn drives behavioral rhythms. To identify potential clock outputs, the authors generated expression profiles from a homogeneous population of purified pacemaker neurons (LN(v)s) from wild-type and clock mutant Drosophila. They identified a group of genes with enriched expression in LN(v)s and a second group of genes rhythmically expressed in LN(v)s in a clock-dependent manner. Only 10 genes fell into both groups: 4 core clock genes, including period (per) and timeless (tim), and 6 genes previously unstudied in circadian rhythms. The authors focused on one of these 6 genes, Ir, which encodes an inward rectifier K(+) channel likely to regulate resting membrane potential, whose expression peaks around dusk. Reducing Ir expression in LN(v)s increased larval light avoidance and lengthened the period of adult locomotor rhythms, consistent with increased LN(v) excitability. In contrast, increased Ir expression made many adult flies arrhythmic and dampened PER protein oscillations. The authors propose that rhythmic Ir expression contributes to daily rhythms in LN(v) neuronal activity, which in turn feed back to regulate molecular clock oscillations.

Wavelength-specific artificial light disrupts molecular clock in avian species: A power-calibrated statistical approach

The MYC oncoprotein and its family members N-MYC and L-MYC are known to drive a wide variety of human cancers. Emerging evidence suggests that MYC has a bi-directional relationship with the molecular clock in cancer. The molecular clock is responsible for circadian (~24 h) rhythms in most eukaryotic cells and organisms, as a mechanism to adapt to light/dark cycles. Disruption of human circadian rhythms, such as through shift work, may serve as a risk factor for cancer, but connections with oncogenic drivers such as MYC were previously not well understood. In this review, we examine recent evidence that MYC in cancer cells can disrupt the molecular clock; and conversely, that molecular clock disruption in cancer can deregulate and elevate MYC. Since MYC and the molecular clock control many of the same processes, we then consider competition between MYC and the molecular clock in several select aspects of tumor biology, including chromatin state, global transcriptional profile, metabolic rewiring, and immune infiltrate in the tumor. Finally, we discuss how the molecular clock can be monitored or diagnosed in human tumors, and how MYC inhibition could potentially restore molecular clock function. Further study of the relationship between the molecular clock and MYC in cancer may reveal previously unsuspected vulnerabilities which could lead to new treatment strategies.

Circadian rhythm dysfunction occurs in both common and rare neurodegenerative diseases. This dysfunction manifests as sleep cycle mistiming, alterations in body temperature rhythms, and an increase in symptomatology during the early evening hours known as Sundown Syndrome. Disruption of circadian rhythm homeostasis has also been implicated in the etiology of neurodegenerative disease. Indeed, individuals exposed to a shifting schedule of sleep and activity, such as health care workers, are at a higher risk. Thus, a bidirectional relationship exists between the circadian system and neurodegeneration. At the heart of this crosstalk is the molecular circadian clock, which functions to regulate circadian rhythm homeostasis. Over the past decade, this connection has become a focal point of investigation as the molecular clock offers an attractive target to combat both neurodegenerative disease pathogenesis and circadian rhythm dysfunction, and a pivotal role for neuroinflammation and stress has been established. This review summarizes the contributions of molecular clock dysfunction to neurodegenerative disease etiology, as well as the mechanisms by which neurodegenerative diseases affect the molecular clock.

The endogenous molecular clock in skeletal muscle is necessary for maintenance of phenotype and function. Loss of Bmal1 solely from adult skeletal muscle (iMSBmal1(-/-) ) results in reductions in specific tension, increased oxidative fibre type and increased muscle fibrosis with no change in feeding or activity. Disruption of the molecular clock in adult skeletal muscle is sufficient to induce changes in skeletal muscle similar to those seen in the Bmal1 knockout mouse (Bmal1(-/-) ), a model of advanced ageing. iMSBmal1(-/-) mice develop increased bone calcification and decreased joint collagen, which in combination with the functional changes in skeletal muscle results in altered gait. This study uncovers a fundamental role for the skeletal muscle clock in musculoskeletal homeostasis with potential implications for ageing. Disruption of circadian rhythms in humans and rodents has implicated a fundamental role for circadian rhythms in ageing and the development of many chronic diseases including diabetes, cardiovascular disease, depression and cancer. The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop with Bmal1 encoding a core molecular clock transcription factor. Germline Bmal1 knockout (Bmal1 KO) mice have a shortened lifespan, show features of advanced ageing and exhibit significant weakness with decreased maximum specific tension at the whole muscle and single fibre levels. We tested the role of the molecular clock in adult skeletal muscle by generating mice that allow for the inducible skeletal muscle-specific deletion of Bmal1 (iMSBmal1). Here we show that disruption of the molecular clock, specifically in adult skeletal muscle, is associated with a muscle phenotype including reductions in specific tension, increased oxidative fibre type, and increased muscle fibrosis similar to that seen in the Bmal1 KO mouse. Remarkably, the phenotype observed in the iMSBmal1(-/-) mice was not limited to changes in muscle. Similar to the germline Bmal1 KO mice, we observed significant bone and cartilage changes throughout the body suggesting a role for the skeletal muscle molecular clock in both the skeletal muscle niche and the systemic milieu. This emerging area of circadian rhythms and the molecular clock in skeletal muscle holds the potential to provide significant insight into intrinsic mechanisms of the maintenance of muscle quality and function as well as identifying a novel crosstalk between skeletal muscle, cartilage and bone.

Balance of Activity between LNvs and Glutamatergic Dorsal Clock Neurons Promotes Robust Circadian Rhythms in Drosophila

SCA1+ Cells from the Heart Possess a Molecular Circadian Clock and Display Circadian Oscillations in Cellular Functions

Introduction: Ferroptosis, an iron mediated regulated form of cell death has been implicated in myocardial ischemia-reperfusion (IR) injury among other cardiac conditions. Cellular lipid metabolism and lipid oxidation are key processes in ferroptosis. Circadian clocks, transcriptional feedback loops present in all tissues, regulate diurnal variations in various cardiac physiologic processes including the response to IR injury. The degree to which the clock machinery regulates ferroptosis is unknown. We hypothesize that ferroptosis pathways are induced differently in response to ischemia reperfusion injury at different times of day. Methods: We housed wild-type C57BL6/J mice at a regular 12:12 light dark cycle. We performed a closed chest ischemia reperfusion (IR) procedure to the left anterior descending coronary artery either 2 hours post lights on (Zeitgeber Time 2) or 2 hours post lights off (ZT 14). We sacrificed the mice 24 hours post reperfusion and collected hearts for molecular clock and ferroptosis gene expression quantification by RT-qPCR and to measure a large panel of oxidized lipids and metabolites of oxidation pathways. Because the surgical mice were sacrificed at opposing times of day, we accounted for normal day to night variations by reporting all measurements as the number of standard deviations away from the measurement mean of non-surgical mice sacrificed at the same time of day. Results: The expression of the anti-ferroptosis molecular clock gene Bmal1 was decreased beyond its normal diurnal variations in the mice with the IR procedure at ZT2 compared to those at ZT14. Furthermore, the expression levels of ferroptosis genes Alox15 and Acsl4 were higher in the ZT2 group compared to ZT14 indicating higher induction of ferroptosis in the ZT2 group beyond that of normal diurnal variation. From our metabolomics panel, we find a higher ratio of NADP+ to NADPH in the ZT 2 group indicating a higher redox burden. Also, acyl carnitines of differing chain lengths showed a consistent decrease in the ZT 2 group. Conclusions: In response to ischemia reperfusion injury, ferroptosis is induced in a diurnal manner reflective of alterations in the core clock gene Bmal1, lipid metabolism and oxidation pathways.

Cluster headache (CH) is a debilitating condition with severe and recurrent headaches characterized by circannual and circadian rhythms. A genetic contingent was suggested, and several loci were described in large cohorts. However, no variant associated with CH for multiplex families has been described. The purpose of our study was to examine candidate genes and new genetic variants in a multigenerational family of cluster headaches in which two members have original chronobiological characteristics that we have called the phenomenon of "family periodicity". We performed a whole genome sequencing in four patients in a large multigenerational family of cluster headache to identify additional loci associated with CH. This allowed us to replicate the genomic association of HCRTR2 and CLOCK as candidate genes. In two family members with the same phenotypic circadian pattern (familial periodicity) the association of polymorphism NM_001526.4:c.922G > A was shown in the HCRTR2 gene, and NM_004898.4:c.213T > C in the CLOCK gene. This whole genome sequencing reproduced two genetic risk loci for CH already involved in its pathogenicity. This is the first time that the combination of HCRTR2 and CLOCK gene variants is identified in a multigenerational family of CH with striking periodicity characteristics. Our study supports the hypothesis that the combination of HCRTR2 and CLOCK gene variants can contribute to the risk of cluster headache and offer the prospect of a new area of research on the molecular circadian clock.