Guangrui Yang

Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration

Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator-like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration.

Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1.

Significance The circadian clock allows an organism to anticipate daily changes imposed by the environment. The response to LPS is altered depending on time of day; however, the molecular mechanisms underlying this are unclear. We find that the clock in myeloid cells plays a role in LPS-induced sepsis by altering NF-κB activity and the induction of the microRNA miR-155. LPS causes the repression of BMAL1 via the targeting of miR-155 to two seed sequences in the 3′-untranslated region of Bmal1. Lack of miR-155 has profound effects on circadian function and circadian induction of cytokines by LPS. Thus, the molecular clock is using miR-155 as an important regulatory component to control inflammation variably across the circadian day in myeloid cells. The response to an innate immune challenge is conditioned by the time of day, but the molecular basis for this remains unclear. In myeloid cells, there is a temporal regulation to induction by lipopolysaccharide (LPS) of the proinflammatory microRNA miR-155 that correlates inversely with levels of BMAL1. BMAL1 in the myeloid lineage inhibits activation of NF-κB and miR-155 induction and protects mice from LPS-induced sepsis. Bmal1 has two miR-155–binding sites in its 3′-UTR, and, in response to LPS, miR-155 binds to these two target sites, leading to suppression of Bmal1 mRNA and protein in mice and humans. miR-155 deletion perturbs circadian function, gives rise to a shorter circadian day, and ablates the circadian effect on cytokine responses to LPS. Thus, the molecular clock controls miR-155 induction that can repress BMAL1 directly. This leads to an innate immune response that is variably responsive to challenges across the circadian day.

Timing of expression of the core clock gene Bmal1 influences its effects on aging and survival

Postnatal knockout of a core clock gene in mice prompts reevaluation of the systemic role of the molecular clock in the biology of aging. For clock ticking, timing matters Ironically, antiaging product advertisements often promise to “slow down the clock.” But abolishing the circadian clock—for example, by knocking out Bmal1, a core clock gene—accelerates aging and shortens the life span in mice. As a result, Bmal1 knockout mice often serve as a model system in studies of the role of circadian rhythms in the aging process. Now Yang et al. show that the developmental timing of Bmal1 expression influences the circadian clock’s effects on aging and survival. To assess the role of circadian rhythms in the aging process, the authors made conditional Bmal1 knockout mice that are missing the BMAL1 protein only during adult life. Unlike knockout mice that perpetually lack Bmal1 expression, the new conditional Bmal1 knockout mice displayed loss of circadian rhythm in wheel-running activity, heart rate, and blood pressure, but exhibited normal life spans, fertility, body weight, blood glucose levels, and age-dependent arthropathy; in fact, atherosclerosis and hair growth actually improved, despite obliteration of clock function. Another surprising observation was little changes in overall gene expression in the livers of adult-life Bmal1 knockout mice, even though there’s a quelling of expression of oscillating genes. Both prenatal and postnatal knockout mice displayed similar ocular abnormalities and brain astrogliosis. Taken together, these findings reveal that many phenotypes thought to be caused by circadian rhythm disruption in conventional Bmal1 knockout mice apparently manifest as a result of clock-independent BMAL1 functions. Thus, the systemic role of the molecular clock in the biology of aging requires reinvestigation in order to increase the likelihood of translation for preclinical studies of the aging process. The absence of Bmal1, a core clock gene, results in a loss of circadian rhythms, an acceleration of aging, and a shortened life span in mice. To address the importance of circadian rhythms in the aging process, we generated conditional Bmal1 knockout mice that lacked the BMAL1 protein during adult life and found that wild-type circadian variations in wheel-running activity, heart rate, and blood pressure were abolished. Ocular abnormalities and brain astrogliosis were conserved irrespective of the timing of Bmal1 deletion. However, life span, fertility, body weight, blood glucose levels, and age-dependent arthropathy, which are altered in standard Bmal1 knockout mice, remained unaltered, whereas atherosclerosis and hair growth improved, in the conditional adult-life Bmal1 knockout mice, despite abolition of clock function. Hepatic RNA-Seq revealed that expression of oscillatory genes was dampened in the adult-life Bmal1 knockout mice, whereas overall gene expression was largely unchanged. Thus, many phenotypes in conventional Bmal1 knockout mice, hitherto attributed to disruption of circadian rhythms, reflect the loss of properties of BMAL1 that are independent of its role in the clock. These findings prompt reevaluation of the systemic consequences of disruption of the molecular clock.

Prostanoids and inflammatory pain.

Prostanoids play pivotal roles in inflammation and pain. Cyclooxygenase (COX) inhibitors, the nonsteroidal anti-inflammatory drugs (NSAIDs), depress prostanoid formation and are widely used to treat inflammatory pain. However, their therapeutic benefit is offset by serious side-effects, primarily gastrointestinal and cardiovascular complications. Pathway elements downstream of the COX enzymes, particularly the terminal synthases and receptors of prostaglandin E2, have been proposed as alternative targets for the development of novel NSAID like drugs. Here, we summarize the current knowledge on the roles of individual prostanoids in modulating inflammatory pain.

Cell selective cardiovascular biology of microsomal prostaglandin E synthase-1.

Background— Global deletion of microsomal prostaglandin E synthase 1 (mPGES-1) in mice attenuates the response to vascular injury without a predisposition to thrombogenesis or hypertension. However, enzyme deletion results in cell-specific differential use by prostaglandin synthases of the accumulated prostaglandin H2 substrate. Here, we generated mice deficient in mPGES-1 in vascular smooth muscle cells, endothelial cells, and myeloid cells further to elucidate the cardiovascular function of this enzyme. Methods and Results— Vascular smooth muscle cell and endothelial cell mPGES-1 deletion did not alter blood pressure at baseline or in response to a high-salt diet. The propensity to evoked macrovascular and microvascular thrombogenesis was also unaltered. However, both vascular smooth muscle cell and endothelial cell mPGES-1–deficient mice exhibited a markedly exaggerated neointimal hyperplastic response to wire injury of the femoral artery in comparison to their littermate controls. The hyperplasia was associated with increased proliferating cell nuclear antigen and tenascin-C expression. In contrast, the response to injury was markedly suppressed by myeloid cell depletion of mPGES-1 with decreased hyperplasia, leukocyte infiltration, and expression of proliferating cell nuclear antigen and tenascin-C. Conditioned medium derived from mPGES-1–deficient macrophages less potently induced vascular smooth muscle cell proliferation and migration than that from wild-type macrophages. Conclusions— Deletion of mPGES-1 in the vasculature and myeloid cells differentially modulates the response to vascular injury, implicating macrophage mPGES-1 as a cardiovascular drug target.

Knitting Up the Raveled Sleave of Care

Recent advances in our understanding of molecular clocks highlight their relevance to human physiology and disease. Recent advances in our understanding of molecular clocks highlight their relevance to human physiology and disease. This Review is based on the Franklin Epstein Lecture delivered at Beth Israel Deaconess Hospital on 25 April 2013. We discuss recent advances in our understanding of molecular clocks and highlight their relevance to human physiology and disease.

Myeloid cell microsomal prostaglandin E synthase-1 fosters atherogenesis in mice

Significance Inhibitors of microsomal prostaglandin E synthase-1 (mPGES-1) are being developed as analgesics. Although global depletion of mPGES-1 suggests they will be less likely to confer a cardiovascular hazard than NSAIDs selective for inhibition of COX-2, mPGES-1–derived PGE2 may have contrasting effects on discrete cellular components of the vasculature. mPGES-1 inhibition may result in substrate rediversion to other PG synthases, the products of which differ between cell types and exert contrasting biologies. Here, myeloid cell mPGES-1, reflective of the macrophage enzyme, promotes atherogenesis, fostering inflammation and oxidative stress. By contrast, mPGES-1 in endothelial and vascular smooth muscle cells has no discernable effect. Selective targeting of macrophage mPGES-1 may conserve cardiovascular benefit while avoiding adverse effects related to enzyme blockade in other tissues. Microsomal prostaglandin E synthase-1 (mPGES-1) in myeloid and vascular cells differentially regulates the response to vascular injury, reflecting distinct effects of mPGES-1–derived PGE2 in these cell types on discrete cellular components of the vasculature. The cell selective roles of mPGES-1 in atherogenesis are unknown. Mice lacking mPGES-1 conditionally in myeloid cells (Mac-mPGES-1-KOs), vascular smooth muscle cells (VSMC-mPGES-1-KOs), or endothelial cells (EC-mPGES-1-KOs) were crossed into hyperlipidemic low-density lipoprotein receptor-deficient animals. En face aortic lesion analysis revealed markedly reduced atherogenesis in Mac-mPGES-1-KOs, which was concomitant with a reduction in oxidative stress, reflective of reduced macrophage infiltration, less lesional expression of inducible nitric oxide synthase (iNOS), and lower aortic expression of NADPH oxidases and proinflammatory cytokines. Reduced oxidative stress was reflected systemically by a decline in urinary 8,12-iso-iPF2α-VI. In contrast to exaggeration of the response to vascular injury, deletion of mPGES-1 in VSMCs, ECs, or both had no detectable phenotypic impact on atherogenesis. Macrophage foam cell formation and cholesterol efflux, together with plasma cholesterol and triglycerides, were unchanged as a function of genotype. In conclusion, myeloid cell mPGES-1 promotes atherogenesis in hyperlipidemic mice, coincident with iNOS-mediated oxidative stress. By contrast, mPGES-1 in vascular cells does not detectably influence atherogenesis in mice. This strengthens the therapeutic rationale for targeting macrophage mPGES-1 in inflammatory cardiovascular diseases.

PPARs and Metabolic Syndrome

Peroxisome proliferator-activated receptors (PPARs) exert versatile biological effects, notably in energy metabolism. During the last two decades, numerous studies have demonstrated that PPARs act as pivotal regulators of metabolic syndrome, a series of disorders in energy utilization and storage that are implicated with type 2 diabetes, diabetic nephropathy, and cardiovascular diseases, to mention a few. PPARα and PPARγ are the molecular targets of a number of marketed drugs for the treatment of these diseases, and accumulating evidence suggested PPARβ/δ as a potential therapeutic drug target as well. Although energy metabolism and metabolic syndrome are the most intensively studied domain of PPARs, it has not been addressed specifically in any issue of PPAR Research ever since its launch. Here, we gathered 3 reviews and 5 research articles that encompass metabolic syndrome and its complications. M. Aprile et al. tackled the subject of PPARγ and human adipogenesis in their research article. Rather than focusing on canonical PPARγ transcripts, authors largely emphasized on the critical contribution of PPARγ dominant negative isoforms to adipogenesis and their implied potential role in pathological conditions. In addition, a novel of PPARγ dominant negative transcript, γ1ORF4, was first identified in this study. In regard to nonalcoholic fatty liver diseases, the hepatic expression of the metabolic syndrome, M. Sharif et al. conducted a thorough analysis of previously published data about the steatogenic role of PPARγ and summarized two probable PPARγ ligand-dependent toxicological modes of action: (i) activation of PPARγ in hepatocytes and (ii) inhibition in adipocytes. Two papers, one review and a research article, by Z. Jia and Y. Sun et al., appraised the role of PPARγ in diabetic nephropathy (DN). Their comprehensive review summarized the limitations of traditional PPARγ agonists, addressed the advantages of newly developed PPARγ agonists, and rendered new insights into the therapeutic potential of PPARγ agonists in the treatment of DN, while the research article suggested that a combination of PPARγ agonists with COX-2/PGE2 inhibitors may be an alternative way of dealing with DN. In another research article, J. Jin et al. analyzed the correlation between PPAR gene polymorphisms and pediatric primary nephrotic syndrome (PNS) by comparing children with PNS against healthy subjects. They found that PPARγ (Pro12Ala) and PGC-1α (Gly482Ser) polymorphisms are associated with abnormal insulin and triglyceride metabolism in pediatric PNS patients, suggesting that these polymorphisms may be relevant to the prognosis of this chronic disease. The knowledge of the role of PPARα in metabolic disorder-associated cardiovascular diseases was well recognized in this special issue. Z. Jia et al.s research article asserted the involvement of HMGB1 (high mobility group box 1) in the protective effect of PPARα in cardiac hypertrophy and provided a novel approach to study the pathogenesis of cardiac hypertrophy. Although most studies showed that PPARα activation confers protection against atherogenesis, the intriguing possibility that PPARα might foster atherogenesis is also considered. In this current issue, M. Vechoropoulos et al. found that PPARα mediates the proatherogenic effect of chronic nitric oxide synthesis inhibition and this effect is independent of blood pressure and serum lipids alterations. These data further shaped the view that the role of PPARα in atherosclerosis needs to be reevaluated. Lastly, in the review article “PPARs Integrate the Mammalian Clock and Energy Metabolism,” we collected recent findings about the role of PPARs in biological clocks. This brand new function of PPARs bridges energy metabolism with circadian rhythm whose relationship has been known for long time, but not well understood. We summarized the circadian function of three PPAR subtypes one by one and concluded that the abnormality of PPARs and circadian rhythm could impinge on each other and thus leads to metabolic disorders. Further investigation of PPARs in this field will give us a new perspective on the therapeutic advances in the treatment of metabolic ailments. In conclusion, this special issue is packed with intriguing novel breakthroughs and insights into PPARs and metabolic syndrome. We hope that these advances will generate more interest from the scientific community in better understanding of the role of PPARs in metabolic syndrome and associated complications. Lihong  Chen Zhanjun  Jia Guangrui  Yang

A Pilot Characterization of the Human Chronobiome

Physiological function, disease expression and drug effects vary by time-of-day. Clock disruption in mice results in cardio-metabolic, immunological and neurological dysfunction; circadian misalignment using forced desynchrony increases cardiovascular risk factors in humans. Here we integrated data from remote sensors, physiological and multi-omics analyses to assess the feasibility of detecting time dependent signals - the chronobiome – despite the “noise” attributable to the behavioral differences of free-living human volunteers. The majority (62%) of sensor readouts showed time-specific variability including the expected variation in blood pressure, heart rate, and cortisol. While variance in the multi-omics is dominated by inter-individual differences, temporal patterns are evident in the metabolome (5.4% in plasma, 5.6% in saliva) and in several genera of the oral microbiome. This demonstrates, despite a small sample size and limited sampling, the feasibility of characterizing at scale the human chronobiome “in the wild”. Such reference data at scale are a prerequisite to detect and mechanistically interpret discordant data derived from patients with temporal patterns of disease expression, to develop time-specific therapeutic strategies and to refine existing treatments.

Molecular clocks and the human condition: approaching their characterization in human physiology and disease.

Molecular clockworks knit together diverse biological networks and compelling evidence from model systems infers their importance in metabolism, immunological and cardiovascular function. Despite this and the diurnal variation in many aspects of human physiology and the phenotypic expression of disease, our understanding of the role and importance of clock function and dysfunction in humans is modest. There are tantalizing hints of connection across the translational divide and some correlative evidence of gene variation and human disease but most of what we know derives from forced desynchrony protocols in controlled environments. We now have the ability to monitor quantitatively ex vivo or in vivo the genome, metabolome, proteome and microbiome of humans in the wild. Combining this capability, with the power of mobile telephony and the evolution of remote sensing, affords a new opportunity for deep phenotyping, including the characterization of diurnal behaviour and the assessment of the impact of the clock on approved drug function.