Clock Protein Research Articles

Characterization of clock proteins in the substantia nigra and subthalamic nucleus of the Sapajus apella primate

Clock genes, which are essential for suprachiasmatic nucleus (SCN) function, also play critical roles in other brain regions, and their expression have been the subject of various studies. An increasingly deeper understanding of the expression of these genes in different species contributes to our knowledge of their functions and the factors influencing their expression. Considering that most studies have been conducted in nocturnal rodents, in this study we investigated the presence of Per1, Per2 and Cry1 in neurons of the substantia nigra (SN) and subthalamic nucleus (STN) in a diurnal primate. The immunoreactivity of Per1, Per2, and Cry1 was analyzed using immunohistochemistry, revealing significant Per1-IR, Per2-IR, and Cry1-IR in the SN. While Per1-IR and Per2-IR were also observed in the STN, no Cry1-IR staining was detected in the STN. These results confirm the presence of proteins that regulate circadian rhythms in areas associated with motor behavior.

Exercise improves muscle mitochondrial dysfunction-associated lipid profile under circadian rhythm disturbance.

We investigated whether endurance exercise training (EXT) ameliorates circadian rhythm (CR)-induced risk factors by improving skeletal muscle (SKM) mitochondrial biogenesis, reducing oxidative stress, and modulating apoptotic protein expression. We distinguished between regular and shift workers using the National Health and Nutrition Examination Survey (NHANES) and investigated the health problems caused by shift work (CR disturbance) and the potential therapeutic effects of exercise. In our animal study, 36 rats underwent 12 weeks of CR disturbance, divided into regular and irregular CR groups. These groups were further split into EXT (n = 12) and sedentary (n = 12) for an additional 8 weeks. We analyzed SKM tissue to understand the molecular changes induced by CR and EXT. NHANES data were analyzed using SAS 9.4 and Prism 8 software, while experimental animal data were analyzed using Prism 8 software. The statistical procedures used in each experiment are indicated in the figure legends. Our studies showed that CR disturbance increases dyslipidemia, alters circadian clock proteins (BMAL1, PER2), raises apoptotic protein levels, and reduces mitochondrial biogenesis in SKM. EXT improved LDL-C and HDLC levels without affecting muscle BMAL1 expression. It also enhanced mitochondrial biogenesis (AMPK, PGC-1α, Tfam, NADH-UO, COX-I), antioxidant levels (Catalase, SOD1, SOD2), and apoptotic protein (p53, Bax/Bcl2) expression or activity in SKM. We demonstrated that shift work-induced CR disturbance leads to dyslipidemia, diminished mitochondrial biogenesis, and reduced antioxidant capacity in SKM. However, EXT can counteract dyslipidemia under CR disturbance, potentially lowering the risk of cardiovascular disorders.

Forward genetic approach identifies a phylogenetically conserved serine residue critical for the catalytic activity of UBIQUITIN-SPECIFIC PROTEASE 12 in Arabidopsis

Circadian clocks rely on transcriptional/translational feedback loops involving clock genes and their corresponding proteins. While the primary oscillations originate from gene expression, the precise control of clock protein stability plays a pivotal role in establishing the 24-hour circadian rhythms. Most clock proteins are degraded through the ubiquitin/26S proteasome pathway, yet the enzymes responsible for ubiquitination and deubiquitination remain poorly characterised. We identified a missense allele (ubp12-3, S327F) of the UBP12 gene/protein in Arabidopsis. Despite ubp12-3 exhibited a short period phenotype similar to that of a loss-of-function allele, molecular analysis indicated elevated protease activity in ubp12-3. We demonstrated that early flowering of ubp12 mutants is a result of the shortened circadian period rather than a direct alteration of UBP12 function. Analysis of protease activity of non-phosphorylatable (S327A, S327F) and phosphomimetic (S327D) derivatives in bacteria suggested that phosphorylation of serine 327 inhibits UBP12 enzymatic activity, which could explain the over-functioning of S327F in vivo. We showed that phosphomimetic mutations of the conserved serine in the Neurospora and human orthologues reduced ubiquitin cleavage activity suggesting that not only the primary structures of UBP12-like enzymes are phylogenetically conserved across a wide range of species, but also the molecular mechanisms governing their enzymatic activity.

Circadian Clock Disruption and Growth of Kidney Cysts in Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, and often progresses to kidney failure. ADPKD progression is not uniform among patients, suggesting that factors secondary to the PKD1/2 gene mutation could regulate the rate of disease progression. Here we tested the effect of circadian clock disruption on ADPKD progression. Circadian rhythms are regulated by cell-autonomous circadian clocks composed of clock proteins. BMAL1 is a core constituent of the circadian clock. To disrupt the circadian clock, we deleted Bmal1 gene in the renal collecting ducts of the Pkd1RC/RC (RC/RC) mouse model of ADPKD (RC/RC;Bmal1f/f;Pkhd1cre, called DKO mice), and in Pkd1 knockout mouse inner medullary collecting duct cells (Pkd1Bmal1KO mIMCD3 cells). Only male mice were used. Human nephrectomy ADPKD kidneys showed altered clock gene expression when compared to normal control human kidneys. When compared to RC/RC kidneys, DKO kidneys showed significantly altered clock gene expression, increased cyst growth, cell proliferation, apoptosis and fibrosis. DKO kidneys also showed increased lipogenesis and cholesterol synthesis-related gene expression, and increased tissue triglyceride levels compared to RC/RC kidneys. Similarly, in vitro, Pkd1Bmal1KO cells showed altered clock genes, increased lipogenesis and cholesterol synthesis-related genes, and reduced fatty-acid oxidation-related gene expression compared to Pkd1KO cells. The Pkd1Bmal1KO cells showed increased cell proliferation compared to Pkd1KO cells, which was rescued by pharmacological inhibition of lipogenesis. Renal collecting duct specific Bmal1 gene deletion disrupted the circadian clock and triggered accelerated ADPKD progression by altering lipid metabolism-related gene expression.

O-GlcNAcylation of circadian clock protein Bmal1 impairs cognitive function in diabetic mice.

Neuronal damage in the hippocampus induced by high glucose has been shown to promote the onset and development of cognitive impairment in diabetes, but the underlying molecular mechanism remains unclear. Guided by single-cell RNA sequencing, we here report that high glucose increases O-GlcNAcylation of Bmal1 in hippocampal neurons. This glycosylation promotes the binding of Clock to Bmal1, resulting in the expression of transcription factor Bhlhe41 and its target Dnajb4. Upregulated Dnajb4 in turn leads to ubiquitination and degradation of the mitochondrial Na + /Ca2+ exchanger NCLX, thereby inducing mitochondrial calcium overload that causes neuronal damage and cognitive impairment in mice. Notably, Bhlhe41 downregulation or treatment with a short peptide that specifically blocks O-GlcNAcylation of Bmal1 on Ser424 mitigated these adverse effects in diabetic mouse models. These data highlight the crucial role of O-GlcNAcylation in circadian clock gene expression and may facilitate the design of targeted therapies for diabetes-associated cognitive impairment.

Enhancing Quality of Life in Multiple Sclerosis Patients Through Coadministration of FDA-Approved Immunomodulators and Melatonin

Objective: The primary goal of this study was to assess the binding characteristics and affinities of melatonin and certain U.S. Food and Drug Administration (FDA)-approved lipophilic drugs used in the treatment of multiple sclerosis (MS) with the brain and muscle Arnt-like 1 (BMAL1) clock protein. Additionally, the study aimed to investigate their potential as modulators of BMAL1 activity for therapeutic applications in MS.Methods: Molecular docking and simulation studies were conducted to investigate the interactions between the BMAL1 protein and the selected agents as ligands. Docking simulations were performed using AutoDock Vina, and key interactions were analyzed with Biovia Discovery Studio Visualizer. A molecular dynamics simulation was conducted using GROMACS 2020.4 and the binding free energies were calculated using the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method. The pharmacokinetic properties of the ligands were predicted using the BOILED-Egg model implemented in the SwissADME web tool.Results: Melatonin demonstrated the highest binding affinity (-8.5 kcal/mol) and most favorable binding free energy (-245.26±1.27 kJ/mol) with BMAL1, suggesting its potential synergistic activity with other FDA-approved drugs in MS therapy. The pharmacokinetic analysis indicated that all three ligands were likely to cross the blood-brain barrier and exhibit high gastrointestinal absorption, with teriflunomide and melatonin potentially achieving a better profile.Conclusion: This study highlights the potential of concomitant use of melatonin to modulate BMAL1 activity, providing a foundation for developing targeted chronotherapeutic strategies in MS treatment.

Cold-induced degradation of core clock proteins implements temperature compensation in the Arabidopsis circadian clock.

The period of circadian clocks is maintained at close to 24 hours over a broad range of physiological temperatures due to temperature compensation of period length. Here, we show that the quantitative control of the core clock proteins TIMING OF CAB EXPRESSION 1 [TOC1; also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)] and PRR5 is crucial for temperature compensation in Arabidopsis thaliana. The prr5 toc1 double mutant has a shortened period at higher temperatures, resulting in weak temperature compensation. Low ambient temperature reduces amounts of PRR5 and TOC1. In low-temperature conditions, PRR5 and TOC1 interact with LOV KELCH PROTEIN 2 (LKP2), a component of the E3 ubiquitin ligase Skp, Cullin, F-box (SCF) complex. The lkp2 mutations attenuate low temperature-induced decrease of PRR5 and TOC1, and the mutants display longer period only at lower temperatures. Our findings reveal that the circadian clock maintains its period length despite ambient temperature fluctuations through temperature- and LKP2-dependent control of PRR5 and TOC1 abundance.

Nuclear interactions between the Pseudo-Response Regulator clock proteins and the Multi-Step Phosphorelay mediator Histidine-containing phosphotransfers in the moss Physcomitrium patens

Pseudo-Response Regulator (PRR) proteins constitute a fundamental set of circadian clock components in plants. PRRs have an amino acid sequence stretch with similarity to the receiver (REC) domain of response regulators (RRs) in the Multi-Step Phosphorelay (MSP). However, it has never been elucidated whether PRRs interact with Histidine-containing Phosphotransfer (HPt) proteins, which transfer a phosphate to RRs. Here, we studied whether PRRs interact with HPts in the moss Physcomitrium patens by the Yeast Two-Hybrid system and Bimolecular Fluorescence Complementation. P. patens PRR1/2/3 interacted with HPt1/2 in the nucleus, but not with HPt3, suggesting that P. patens PRRs function as authentic RRs. We discuss these results in relation to the evolution and diversity of the plant circadian clocks.

Clock protein LHY targets SNAT1 and negatively regulates the biosynthesis of melatonin in Hypericum perforatum.

Hypericum perforatum, also known as "natural fluoxetine," is a commonly used herbal remedy for treating depression. It is unclear whether melatonin in plants regulated by the endogenous circadian clock system is like in vertebrates. In this work, we found that the melatonin signal and melatonin biosynthesis gene, serotonin N-acetyltransferase HpSNAT1, oscillates in a 24-hour cycle in H. perforatum. First, we constructed a yeast complementary DNA library of H. perforatum and found a clock protein HpLHY that can directly bind to the HpSNAT1 promoter. Second, it was confirmed that HpLHY inhibits the expression of HpSNAT1 by targeting the Evening Element. Last, it indicated that HpLHY-overexpressing plants had reduced levels of melatonin in 12-hour light/12-hour dark cycle photoperiod, while loss-of-function mutants exhibited high levels, but this rhythm seems to disappear as well. The results revealed the regulatory role of LHY in melatonin biosynthesis, which may make an important contribution to the field of melatonin synthesis regulation.

Photomorphogenesis of Myxococcus macrosporus: new insights for light-regulation of cell development

Myxobacteria are non-photosynthetic bacteria distinguished among prokaryotes by a multicellular stage in their life cycle known as fruiting bodies that are formed in response to nutrient deprivation and stimulated by light. Here, we report an entrained, rhythmic pattern of Myxococcus macrosporus fruiting bodies, forming consistently spaced concentric rings when grown in the dark. Light exposure disrupts this rhythmic phenotype, resulting in a sporadic arrangement and reduced fruiting-body count. M. macrosporus genome encodes a red-light photoreceptor, a bacteriophytochrome (BphP), previously shown to affect the fruiting-body formation in the related myxobacterium Stigmatella aurantiaca. Similarly, the formation of M. macrosporus fruiting bodies is also impacted by the exposure to BphP—specific wavelengths of light. RNA-Seq analysis of M. macrosporus revealed constitutive expression of the bphP gene. Phytochromes, as light-regulated enzymes, control many aspects of plant development including photomorphogenesis. They are intrinsically correlated to circadian clock proteins, impacting the overall light-mediated entrainment of the circadian clock. However, this functional relationship remains unexplored in non-photosynthetic prokaryotes. Genomic analysis unveiled the presence of multiple homologs of cyanobacterial core oscillatory gene, kaiC, in various myxobacteria, including M. macrosporus, S. aurantiaca and M. xanthus. RNA-Seq analysis verified the expression of all kaiC homologs in M. macrosporus and the closely related M. xanthus, which lacks bphP genes. Overall, this study unravels the rhythmic growth pattern during M. macrosporus development, governed by environmental factors such as light and nutrients. In addition, myxobacteria may have a time-measuring mechanism resembling the cyanobacterial circadian clock that links the photoreceptor (BphP) function to the observed rhythmic behavior.Graphical abstract

Knockdown of the Clock gene in the liver aggravates MASLD in mice via inhibiting lipophagy.

The increased global prevalence of metabolic dysfunction-associated steatohepatitis (MASLD) has been closely associated with chronic disorders of the circadian clock. Herein, we investigate the role of Clock, a core circadian gene, in the pathogenesis of MASLD. Wild-type (WT) and liver-specific Clock knockdown (Clock-KD) mice were fed a Western diet for 20weeks to induce MASLD. A cellular MASLD model was established by treating AML12 cells with free fatty acids and the effects of Clock knockdown were examined following transfection with Clock siRNA. Increased lipid deposition and more severe steatohepatitis and fibrosis were observed in the livers of Western diet-fed but not normal chow diet-fed Clock-KD mice after 20weeks compared to WT mice. Moreover, the Clock gene was found to be significantly downregulated in WT MASLD mice. The Clock gene was shown to regulate the expression of lipophagy-related proteins (LC3B, P62, RAB7, and PLIN2) in vivo and in vitro. Knockdown of Clock was found to inhibit lipophagy resulting in increased accumulation of lipid droplets in the mouse liver and AML12 cells. Interestingly, the CLOCK protein was shown to interact with P62. However, knockdown of the Clock gene did not promote transcription of the P62 gene but suppressed degradation of the P62 protein during lipophagy in AML12 cells. The hepatic Clock gene regulates lipophagy and affects lipid droplet deposition in liver cells, and thus plays a critical role in the development of MASLD induced by a Western diet.

LIP1 Regulates the Plant Circadian Oscillator by Modulating the Function of the Clock Component GIGANTEA.

Circadian clocks are biochemical timers regulating many physiological and molecular processes according to the day/night cycles. The function of the oscillator relies on negative transcriptional/translational feedback loops operated by the so-called clock genes and the encoded clock proteins. Previously, we identified the small GTPase LIGHT INSENSITIVE PERIOD 1 (LIP1) as a circadian-clock-associated protein that regulates light input to the clock in the model plant Arabidopsis thaliana. We showed that LIP1 is also required for suppressing red and blue light-mediated photomorphogenesis, pavement cell shape determination and tolerance to salt stress. Here, we demonstrate that LIP1 is present in a complex of clock proteins GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB 1 (TOC1). LIP1 participates in this complex via GUANINE EX-CHANGE FACTOR 7. Analysis of genetic interactions proved that LIP1 affects the oscillator via modulating the function of GI. We show that LIP1 and GI independently and additively regulate photomorphogenesis and salt stress responses, whereas controlling cell shape and photoperiodic flowering are not shared functions of LIP1 and GI. Collectively, our results suggest that LIP1 affects a specific function of GI, possibly by altering binding of GI to downstream signalling components.

Differential serum levels of CACNA1C, circadian rhythm and stress response molecules in subjects with bipolar disorder: Associations with genetic and clinical factors

BackgroundMany patients with bipolar disorder (BD) do not respond to or have difficulties tolerating lithium and/or other mood stabilizing agents. There is a need for personalized treatments based on biomarkers in guiding treatment options. The calcium voltage-gated channel CACNA1C is a promising candidate for developing personalized treatments. CACNA1C is implicated in BD by genome-wide association studies and several lines of evidence suggest that targeting L-type calcium channels could be an effective treatment strategy. However, before such individualized treatments can be pursued, biomarkers predicting treatment response need to be developed. MethodsAs a first step in testing the hypothesis that CACNA1C genotype is associated with serum levels of CACNA1C, we conducted ELISA measures on serum samples from 100 subjects with BD and 100 control subjects. ResultsWe observed significantly higher CACNA1C (p < 0.01) protein levels in subjects with BD. The risk single nucleotide polymorpshism (SNP) (rs11062170) showed functional significance as subjects homozygous for the risk allele (CC) had significantly greater CACNA1C protein levels compared to subjects with one (p = 0.013) or no copies (p = 0.009). We observed higher somatostatin (SST) (p < 0.003) protein levels and lower levels of the clock protein aryl hydrocarbon receptor nuclear translocator-like (ARTNL) (p < 0.03) and stress signaling factor corticotrophin releasing hormone (CRH) (p < 0.001) in BD. SST and period 2 (PER2) protein levels were associated with both alcohol dependence and lithium response. ConclusionsOur findings represent the first evidence for increased serum levels of CACNA1C in BD. Along with altered levels of SST, ARNTL, and CRH our findings suggest CACNA1C is associated with circadian rhythm and stress response disturbances in BD.

The Biological Clock of Liver Metabolism in Metabolic Dysfunction-Associated Steatohepatitis Progression to Hepatocellular Carcinoma.

Circadian rhythms are endogenous behavioral or physiological cycles that are driven by a daily biological clock that persists in the absence of geophysical or environmental temporal cues. Circadian rhythm-related genes code for clock proteins that rise and fall in rhythmic patterns driving biochemical signals of biological processes from metabolism to physiology and behavior. Clock proteins have a pivotal role in liver metabolism and homeostasis, and their disturbances are implicated in various liver disease processes. Encoded genes play critical roles in the initiation and progression of metabolic dysfunction-associated steatohepatitis (MASH) to hepatocellular carcinoma (HCC) and their proteins may become diagnostic markers as well as therapeutic targets. Understanding molecular and metabolic mechanisms underlying circadian rhythms will aid in therapeutic interventions and may have broader clinical applications. The present review provides an overview of the role of the liver's circadian rhythm in metabolic processes in health and disease, emphasizing MASH progression and the oncogenic associations that lead to HCC.

Transcriptome Analysis Revealed ZmPTOX1 Is Required for Seedling Development and Stress Tolerance in Maize.

Plant seedling morphogenesis is considerably related to photosynthesis, pigment synthesis, and circadian periodicity during seedling development. We identified and cloned a maize zebra or crossbanding leaves mutant wk3735, which produces pale white kernels and was identified and plays a role in the equilibrium of the Redox state the in/out of ETC by active oxygen scavenging. Interestingly, it produces the zebra leaves during the production of the first seven leaves, which is apparently different from the mutation of homologs AtPTOX in Arabidopsis. It is intriguing to investigate how and why yellow crossbands (zebra leaf phenotype) emerge on leaves. As expected, chlorophyll concentration and photosynthetic efficiency both significantly declined in the yellow sector of wk3735 leaves. Meanwhile, we observed the circadian expression pattern of ZmPTOX1, which was further validated by protein interaction assays of the circadian clock protein TIM1 and ZmPTOX1. The transcriptome data of yellow (muW) and green (muG) sectors of knock-out lines and normal leaves of overexpression lines (OE) at the 5th-leaf seedling stage were analyzed. Zebra leaf etiolated sections exhibit a marked defect in the expression of genes involved in the circadian rhythm and rhythmic stress (light and cold stress) responses than green sections. According to the analysis of co-DEGs of muW vs. OE and muG vs. OE, terms linked to cell repair function were upregulated while those linked to environmental adaptability and stress response were downregulated due to the mutation of ZmPTOX1. Further gene expression level analyses of reactive oxygen species (ROS) scavenging enzymes and detection of ROS deposition indicated that ZmPTOX1 played an essential role in plant stress resistance and ROS homeostasis. The pleiotropic roles of ZmPTOX1 in plant ROS homeostasis maintenance, stress response, and circadian rhythm character may collectively explain the phenotype of zebra leaves during wk3735 seedling development.

Circadian clock disruption and growth of kidney cysts in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, and often progresses to kidney failure. ADPKD progression is not uniform among patients, suggesting that factors secondary to the PKD1/2 gene mutation could regulate the rate of disease progression. Here we tested the effect of circadian clock disruption on ADPKD progression. Circadian rhythms are regulated by cell-autonomous circadian clocks composed of clock proteins. BMAL1 is a core constituent of the circadian clock. To disrupt the circadian clock, we deleted Bmal1 gene in the renal collecting ducts of the Pkd1 RC/RC (RC/RC) mouse model of ADPKD (RC/RC; Bmal1 f/f ; Pkhd1 cre , called DKO mice), and in Pkd1 knockout mouse inner medullary collecting duct cells ( Pkd1Bmal1 KO mIMCD3 cells). Only male mice were used. Human nephrectomy ADPKD kidneys and Pkd1 KO mIMCD3 cells showed reduced Bmal1 gene expression compared to normal controls. When compared to RC/RC kidneys, DKO kidneys showed significantly altered clock gene expression, increased cyst growth, cell proliferation, apoptosis and fibrosis. DKO kidneys also showed increased lipogenesis and cholesterol synthesis-related gene expression, and increased tissue triglyceride levels compared to RC/RC kidneys. Similarly, in vitro, Pkd1Bmal1 KO cells showed altered clock genes, increased lipogenesis and cholesterol synthesis-related genes, and reduced fatty-acid oxidation-related gene expression compared to Pkd1KO cells. The Pkd1Bmal1 KO cells showed increased cell proliferation compared to Pkd1KO cells, which was rescued by pharmacological inhibition of lipogenesis. Renal collecting duct specific Bmal1 gene deletion disrupts the circadian clock and triggers accelerated ADPKD progression by altering lipid metabolism-related gene expression. Lack of BMAL1, a circadian clock protein in renal collecting ducts disrupted the clock and increased cyst growth and fibrosis in an ADPKD mouse model.BMAL1 gene deletion increased cell proliferation by increasing lipogenesis in kidney cells.Thus, circadian clock disruption could be a risk factor for accelerated disease progression in patients with ADPKD.

Unravelling the neuroendocrine system of nocturnal spawning regulated by circadian clock in the razor clam, Sinonovacula constricta

The razor clam (Sinonovacula constricta), as one of important cultivated molluscs, holds high economic importance. Unlike other bivalve species, S. constricta exclusively spawns at night, so the endogenous regulatory mechanisms driving spawning were preliminarily investigated. To identify crucial genes/proteins that may be involved in regulating nocturnal spawning, ovarian transcriptomes and phosphoproteomes were analysed at four diurnal time points (00:00/ZT16, 06:00/ZT22, 12:00/ZT28, 18:00/ZT34) in S. constricta. Transcriptome analysis revealed 503 and 25 differentially expressed genes (DEGs) when comparing ZT16 vs ZT28 and ZT22 vs ZT34, respectively. DEGs associated with energy and substance metabolism, were predominantly overexpressed in ZT16. Notably, genes related to the circadian clock, the neuro-endocrine system, and reproduction, such as clock, timeless homolog (htim), estradiol 17-beta-dehydrogenase (hsd17b11), 5-hydroxytryptamine receptor (htr), and forkhead box protein O (foxo), exhibited significant changes between the light and dark cycles (P < 0.05). Phosphoproteome analysis identified 177 differentially phosphorylated proteins at ZT16 vs ZT28, which were mainly involved in energy and substance metabolism. The association analysis showed that reproduction related proteins including FoxO were heightened phosphorylation at ZT28. Based on our findings, the circadian clock (clock, htim, etc.), neuro-endocrine system (hsd17bs, htr, gpcr, etc.), and reproduction (foxo and rptor) genes and protein phosphorylation all exhibited circadian rhythm fluctuations, suggesting that they may all be involved in nocturnal spawning of razor clams. These findings establish a foundation for understanding the molecular mechanisms underlying spawning and offer valuable insights into the artificial breeding of molluscs.

Abstract Tu132: Estrogen modulation of ethanol-evoked cardiac oxidative stress and dysfunction: Role of circadian clock period-2 and ferroptosis in estrogen deficient rats

Background: Alcohol triggers exacerbated cardiovascular effects in females, compared to males, in estrogen (E2) dependent manner. E2 provides cardiac protection in premenopausal women and models of menopause, yet the interplay between the circadian clock proteins and E2 in the female heart, particularly in the presence of ethanol (EtOH) remains unclear. Aim: This study aims to determine if the suppression of the circadian clock protein Period 2 (Per2) and associated redox enzymes contribute to E2-mediated exacerbation of the EtOH-induced cardiac ferroptosis, and dysfunction. Methods: Two groups of female Sprague-Dawley rats (n=6-8) were subjected to bilateral ovariectomy (OVX) and fed with EtOH (5% in liquid diet) in the presence of E2 or its vehicle for 8 weeks. Measurements of cardiac function using radiotelemetry and echocardiography along with ex vivo biochemical/molecular studies. Results: Compared to vehicle treated OVX+EtOH group, the E2-treated OVX+EtOH group exhibited: (1) reduced (p&lt;0.05) body weight gain and fat mass; (2) higher (p&lt;0.05) telemetry-measured blood pressure and heart rate; (3) reductions (p&lt;0.05) in echocardiography derived contractility indices, ejection fraction and fractional shortening. In the same group, ex vivo molecular analysis demonstrated: (1) lower cardiac Per2 and suppressed (p&lt;0.05) redox enzymes; (2) increased (p&lt;0.05) ROS level and glutathione depletion (p&lt;0.05), leading to the degradation of glutathione peroxidase 4 (GPX4); (3) heme oxygenase-1 upregulation leading to iron overload (p&lt;0.05). These ferroptosis-related effects resulted in increased lipid peroxidation (p&lt;0.05) compared to findings in OVX+EtOH rats in the absence of E2. Conclusion: This study is the first to suggest that abrogation of the E2-mediated Per2 upregulation unraveled cardiac ferroptosis and contributes to the detrimental cardiac effects of ethanol in E2-replete rats. These novel findings underscore the potential risk for myocardial ferroptosis and dysfunction in menopausal women who are consuming ethanol while on E2 replacement therapy.

The Circadian Clock of Müller Glia Is Necessary for Retinal Homeostasis and Neuronal Survival

Biological processes throughout the body are orchestrated in time through the regulation of local circadian clocks. The retina is among the most metabolically active tissues, with demands depending greatly on the light/dark cycle. Most cell types within the retina are known to express the circadian clock in rodents; however, retinal clock expression in the human has not previously been localized. Moreover, the effect of local circadian clock dysfunction on retinal homeostasis is incompletely understood. We demonstrate an age-dependent decline in circadian clock gene and protein expression in the human retina. Using an animal model of targeted Bmal1 deficiency, we identify the circadian clock of the retinal Müller glia as essential for neuronal survival, vascular integrity, and retinal function. These results suggest a potential role for the local retinal circadian clock within the Müller glia in age-related retinal disease and retinal degeneration.

Molecular circadian clock disruption in the leukocytes of individuals with type 2 diabetes and overweight, and its relationship with leukocyte–endothelial interactions

Aims/hypothesisAlterations in circadian rhythms increase the likelihood of developing type 2 diabetes and CVD. Circadian rhythms are controlled by several core clock genes, which are expressed in nearly every cell, including immune cells. Immune cells are key players in the pathophysiology of type 2 diabetes, and participate in the atherosclerotic process that underlies cardiovascular risk in these patients. The role of the core clock in the leukocytes of people with type 2 diabetes and the inflammatory process associated with it are unknown. We aimed to evaluate whether the molecular clock system is impaired in the leukocytes of type 2 diabetes patients and to explore the mechanism by which this alteration leads to an increased cardiovascular risk in this population.MethodsThis is an observational cross-sectional study performed in 25 participants with type 2 diabetes and 28 healthy control participants. Clinical and biochemical parameters were obtained. Peripheral blood leukocytes were isolated using magnetic bead technology. RNA and protein lysates were obtained to assess clock-related gene transcript and protein levels using real-time PCR and western blot, respectively. Luminex XMAP technology was used to assess levels of inflammatory markers. Leukocyte–endothelial interaction assays were performed by perfusing participants’ leukocytes or THP-1 cells (with/without CLK8) over a HUVEC monolayer in a parallel flow chamber using a dynamic adhesion system.ResultsParticipants with type 2 diabetes showed increased BMAL1 and NR1D1 mRNA levels and decreased protein levels of circadian locomotor output cycles kaput (CLOCK), cryptochrome 1 (CRY1), phosphorylated basic helix-loop-helix ARNT like 1 (p-BMAL1) and period circadian protein homologue 2 (PER2). Correlation studies revealed that these alterations in clock proteins were negatively associated with glucose, HbA1c, insulin and HOMA-IR levels and leukocyte cell counts. The leukocyte rolling velocity was reduced and rolling flux and adhesion were enhanced in individuals with type 2 diabetes compared with healthy participants. Interestingly, inhibition of CLOCK/BMAL1 activity in leukocytes using the CLOCK inhibitor CLK8 mimicked the effects of type 2 diabetes on leukocyte–endothelial interactions.Conclusions/interpretationOur study demonstrates alterations in the molecular clock system in leukocytes of individuals with type 2 diabetes, manifested in increased mRNA levels and decreased protein levels of the core clock machinery. These alterations correlated with the impaired metabolic and proinflammatory profile of the participants with type 2 diabetes. Our findings support a causal role for decreased CLOCK/BMAL1 activity in the increased level of leukocyte–endothelial interactions. Overall, our data suggest that alterations in core clock proteins accelerate the inflammatory process, which may ultimately precipitate the onset of CVD in patients with type 2 diabetes.Graphical