Preserving transcriptional stress responses as an anti-aging strategy.

The progressively increasing frailty, morbidity and mortality of aging organisms coincides with, and may be causally related to, their waning ability to adapt to environmental perturbations. Transcriptional responses to challenges, such as oxidative stress or pathogens, diminish with age. This effect is manifest in the declining function of the stress responsive transcription factor Nrf2. Protective gene expression programs that are controlled by the Drosophila Nrf2 homolog, CncC, support homeostasis and longevity. Age-associated chromatin changes make these genes inaccessible to CncC binding and render them inert to signal-dependent transcriptional activation in old animals. In a previous paper, we have reported that overexpression of the CncC dimerization partner Maf-S counteracts this degenerative effect and preserves organism fitness. Building on this work, we show here that Maf-S overexpression prevents loss of chromatin accessibility and maintains gene responsiveness. Moreover, the same outcome, along with an extension of lifespan, can be achieved by inducing CncC target gene expression pharmacologically throughout adult life. Thus, pharmacological or dietary interventions that can preserve stress responsive gene expression may be feasible anti-aging strategies.

Counting the Minutes.

A newly discovered mechanism that causes the 'Minute' phenotype in fruit flies can explain how organisms are able to eliminate the mutant cells that arise occasionally during development.

SIRT6 Is Responsible for More Efficient DNA Double-Strand Break Repair in Long-Lived Species.

DNA repair has been hypothesized to be a longevity determinant, but the evidence for it is based largely on accelerated aging phenotypes of DNA repair mutants. Here, using a panel of 18 rodent species with diverse lifespans, we show that more robust DNA double-strand break (DSB) repair, but not nucleotide excision repair (NER), coevolves with longevity. Evolution of NER, unlike DSB, is shaped primarily by sunlight exposure. We further show that the capacity of the SIRT6 protein to promote DSB repair accounts for a major part of the variation in DSB repair efficacy between short- and long-lived species. We dissected the molecular differences between a weak (mouse) and a strong (beaver) SIRT6 protein and identified five amino acid residues that are fully responsible for their differential activities. Our findings demonstrate that DSB repair and SIRT6 have been optimized during the evolution of longevity, which provides new targets for anti-aging interventions.

BET-ting on Nrf2: How Nrf2 Signaling can Influence the Therapeutic Activities of BET Protein Inhibitors.

BET proteins such as Brd3 and Brd4 are chromatin-associated factors, which control gene expression programs that promote inflammation and cancer. The Nrf2 transcription factor is a master regulator of genes that protect the organism against xenobiotic attack and oxidative stress. Nrf2 has demonstrated anti-inflammatory activity and can support cancer cell malignancy. This review describes the discovery, mechanism and biomedical implications of the regulatory interplay between Nrf2 and BET proteins. Both Nrf2 and BET proteins are established drug targets. Small molecules that either activate or suppress these proteins are currently tested in clinical trials. The crosstalk between Nrf2 and BET proteins may have important, and until now overlooked, implications for the therapeutic effects of these drugs. Based on the information covered in this review, it should be possible to design combinatorial treatment strategies for cancer and inflammatory diseases, which may improve the efficacy of targeting a Nrf2 or BET proteins individually.

Methylation of the phosphatase-transcription activator EYA1 by protein arginine methyltransferase 1: mechanistic, functional, and structural studies.

The eyes absent (EYA) family proteins are conserved transcriptional coactivators with intrinsic protein phosphatase activity. They play an essential role in the development of various organs in metazoans. These functions are associated with a unique combination of phosphatase and transactivation activities. However, it remains poorly understood how these activities and the consequent biologic functions of EYA are regulated. Here, we demonstrate that 2 conserved arginine residues, R304 and R306, of EYA1 are essential for its in vitro phosphatase activity and in vivo function during Drosophila eye development. EYA1 physically interacts with protein arginine methyltransferase 1, which methylates EYA1 at these residues both in vitro and in cultured mammalian and insect cells. Moreover, we show that wild-type, but not methylation-defective, EYA1 associates with γ-H2A.X in response to ionizing radiation. Taken together, our results identify the conserved arginine residues of EYA1 that play an important role for its activity, thus implicating arginine methylation as a novel regulatory mechanism of EYA function.-Li, X., Eberhardt, A., Hansen, J. N., Bohmann, D., Li, H., Schor, N. F. Methylation of the phosphatase-transcription activator EYA1 by protein arginine methyltransferase 1: mechanistic, functional, and structural studies.

Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein.

Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation.

Cdk12 Is A Gene-Selective RNA Polymerase II Kinase That Regulates a Subset of the Transcriptome, Including Nrf2 Target Genes.

The Nrf2 transcription factor is well conserved throughout metazoan evolution and serves as a central regulator of adaptive cellular responses to oxidative stress. We carried out an RNAi screen in Drosophila S2 cells to better understand the regulatory mechanisms governing Nrf2 target gene expression. This paper describes the identification and characterization of the RNA polymerase II (Pol II) kinase Cdk12 as a factor that is required for Nrf2 target gene expression in cell culture and in vivo. Cdk12 is, however, not essential for bulk mRNA transcription and cells lacking CDK12 function are viable and able to proliferate. Consistent with previous findings on the DNA damage and heat shock responses, it emerges that Cdk12 may be specifically required for stress activated gene expression. Transcriptome analysis revealed that antioxidant gene expression is compromised in flies with reduced Cdk12 function, which makes them oxidative stress sensitive. In addition to supporting Reactive Oxygen Species (ROS) induced gene activation, Cdk12 suppresses genes that support metabolic functions in stressed conditions. We suggest that Cdk12 acts as a gene-selective Pol II kinase that engages a global shift in gene expression to switch cells from a metabolically active state to "stress-defence mode" when challenged by external stress.

Mechanisms and functions of Nrf2 signaling in Drosophila.

The Nrf2 transcription factor belongs to the Cap'n'collar family, named after the founding member of this group, the product of the Drosophila Cap'n'collar gene. The encoded protein, Cap'n'collar, abbreviated Cnc, offers a convenient and accessible model to study the structure, function, and biology of Nrf2 transcription factors at the organismic, tissular, cellular, and molecular levels, using the powerful genetic, genomic, and biochemical tools available in Drosophila. In this review we provide an account of the original identification of Cnc as a regulator of embryonic development. We then describe the discovery of Nrf2-like functions of Cnc and its role in acute stress signaling and aging. The establishment of Drosophila as a model organism in which the mechanisms and functions of Nrf2 signaling can be studied has led to several discoveries: the regulation of stem cell activity by an Nrf2-mediated redox mechanism, the interaction of Nrf2 with p62 and Myc in the control of tissue growth and the unfolded protein response, and more. Several of these more recent lines of investigation are highlighted. Model organisms such as the fly and the worm remain powerful experimental platforms that can help to unravel the many remaining puzzles regarding the role of Nrf2 and its relatives in controlling the physiology and maintaining the health of multicellular organisms.

Proteasome dysfunction in Drosophila signals to an Nrf2-dependent regulatory circuit aiming to restore proteostasis and prevent premature aging.

The ubiquitin-proteasome system is central to the regulation of cellular proteostasis. Nevertheless, the impact of in vivo proteasome dysfunction on the proteostasis networks and the aging processes remains poorly understood. We found that RNAi-mediated knockdown of 20S proteasome subunits in Drosophila melanogaster resulted in larval lethality. We therefore studied the molecular effects of proteasome dysfunction in adult flies by developing a model of dose-dependent pharmacological proteasome inhibition. Impaired proteasome function promoted several 'old-age' phenotypes and markedly reduced flies' lifespan. In young somatic tissues and in gonads of all ages, loss of proteasome activity induced higher expression levels and assembly rates of proteasome subunits. Proteasome dysfunction was signaled to the proteostasis network by reactive oxygen species that originated from malfunctioning mitochondria and triggered an Nrf2-dependent upregulation of the proteasome subunits. RNAi-mediated Nrf2 knockdown reduced proteasome activities, flies' resistance to stress, as well as longevity. Conversely, inducible activation of Nrf2 in transgenic flies upregulated basal proteasome expression and activity independently of age and conferred resistance to proteotoxic stress. Interestingly, prolonged Nrf2 overexpression reduced longevity, indicating that excessive activation of the proteostasis pathways can be detrimental. Our in vivo studies add new knowledge on the proteotoxic stress-related regulation of the proteostasis networks in higher metazoans. Proteasome dysfunction triggers the activation of an Nrf2-dependent tissue- and age-specific regulatory circuit aiming to adjust the cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis circuit accelerates aging.

Assessing neurodegenerative phenotypes in Drosophila dopaminergic neurons by climbing assays and whole brain immunostaining.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's (5). Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human α-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function (2). Accordingly, this model has been successfully used to identify potential therapeutic targets in PD (8). Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line (3, 10).

Differential regulation of proteasome functionality in reproductive vs. somatic tissues of Drosophila during aging or oxidative stress.

Proteasome is central to proteostasis maintenance, as it degrades both normal and damaged proteins. Herein, we undertook a detailed analysis of proteasome regulation in the in vivo setting of Drosophila melanogaster. We report that a major hallmark of somatic tissues of aging flies is the gradual accumulation of ubiquitinated and carbonylated proteins; these effects correlated with a ~50% reduction of proteasome expression and catalytic activities. In contrast, gonads of aging flies were relatively free of proteome oxidative damage and maintained substantial proteasome expression levels and highly active proteasomes. Moreover, gonads of young flies were found to possess more abundant and more active proteasomes than somatic tissues. Exposure of flies to oxidants induced higher proteasome activities specifically in the gonads, which were, independently of age, more resistant than soma to oxidative challenge and, as analyses in reporter transgenic flies showed, retained functional antioxidant responses. Finally, inducible Nrf2 activation in transgenic flies promoted youthful proteasome expression levels in the aged soma, suggesting that age-dependent Nrf2 dysfunction is causative of decreasing somatic proteasome expression during aging. The higher investment in proteostasis maintenance in the gonads plausibly facilitates proteome stability across generations; it also provides evidence in support of the trade-off theories of aging.

Declining signal dependence of Nrf2-MafS-regulated gene expression correlates with aging phenotypes.

Aging is a degenerative process characterized by declining molecular, cell and organ functions, and accompanied by the progressive accumulation of oxidatively damaged macromolecules. This increased oxidative damage may be causally related to an age-associated dysfunction of defense mechanisms, which effectively protect young individuals from oxidative insults. Consistently, older organisms are more sensitive to acute oxidative stress exposures than young ones. In studies on the Drosophila Nrf2 transcription factor CncC, we have investigated possible causes for this loss of stress resistance and its connection to the aging process. Nrf2 is a master regulator of antioxidant and stress defense gene expression with established functions in the control of longevity. Here, we show that the expression of protective Nrf2/CncC target genes in unstressed conditions does not generally decrease in older flies. However, aging flies progressively lose the ability to activate Nrf2 targets in response to acute stress exposure. We propose that the resulting inability to dynamically adjust the expression of Nrf2 target genes to the organism's internal and external conditions contributes to age-related loss of homeostasis and fitness. In support of this hypothesis, we find the Drosophila small Maf protein, MafS, an Nrf2 dimerization partner, to be critical to maintain responsiveness of the Nrf2 system: overexpression of MafS in older flies preserves Nrf2/CncC signaling competence and antagonizes age-associated functional decline. The maintenance of acute stress resistance, motor function, and heart performance in aging flies overexpressing MafS supports a critical role for signal responsiveness of Nrf2 function in promoting youthful phenotypes.

A versatile ΦC31 based reporter system for measuring AP-1 and Nrf2 signaling in Drosophila and in tissue culture.

This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ΦC31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined integration sites furthermore enable the experimenter to compare the activity of different reporters in one organism. The reporter constructs have a modular design to facilitate the combination of promoter elements (synthetic transcription factor binding sites or natural regulatory sequences), reporter genes (eGFP, or DsRed.T4), and genomic integration sites. The system was used to analyze and compare the activity and signal response profiles of two stress inducible transcription factors, AP-1 and Nrf2. To complement the transgenic reporter fly lines, tissue culture assays were developed in which the same synthetic ARE and TRE elements control the expression of firefly luciferase.

Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Oxidative stress has been associated with the etiology of both sporadic and monogenic forms of PD. The transcription factor Nrf2, a conserved global regulator of cellular antioxidant responses, has been implicated in neuroprotection against PD pathology. However, direct evidence that upregulation of the Nrf2 pathway is sufficient to confer neuroprotection in genetic models of PD is lacking. Expression of the PD-linked gene encoding α-synuclein in dopaminergic neurons of Drosophila results in decreased locomotor activity and selective neuron loss in a progressive age-dependent manner, providing a genetically accessible model of PD. Here we show that upregulation of the Nrf2 pathway by overexpressing Nrf2 or its DNA-binding dimerization partner, Maf-S, restores the locomotor activity of α-synuclein-expressing flies. Similar benefits are observed upon RNA-interference-mediated downregulation of the prime Nrf2 inhibitor, Keap1, as well as in conditions of keap1 heterozygosity. Consistently, the α-synuclein-induced dopaminergic neuron loss is suppressed by Maf-S overexpression or keap1 heterozygosity. Our data validate the sustained upregulation of the Nrf2 pathway as a neuroprotective strategy against PD. This model provides a genetically accessible in vivo system in which to evaluate the potential of additional Nrf2 pathway components and regulators as therapeutic targets.

c-Jun localizes to the nucleus independent of its phosphorylation by and interaction with JNK and vice versa promotes nuclear accumulation of JNK.

In order to activate gene expression, transcription factors such as c-Jun have to reside in the nucleus. The abundance of c-Jun in the nucleus correlates with the activity of its target genes. As a consequence of excessive c-Jun activation, cells undergo apoptosis or changes in differentiation whereas decreased c-Jun function can reduce proliferation. In the present study we addressed how nuclear accumulation of the transcription factor c-Jun is regulated. First, we analyzed which functions of c-Jun are required for efficient nuclear accumulation. Mutants of c-Jun deficient in dimerization or DNA-binding show no defect in nuclear transport. Furthermore, c-Jun import into the nucleus of living cells occurred when the c-Jun phosphorylation sites were mutated as well in cells that lack the major c-Jun kinase, JNK, suggesting that c-Jun transport into the nucleus does not require JNK signaling. Conversely, however, binding of c-Jun seemed to enhance nuclear accumulation of JNK. In order to identify proteins that might be relevant for the nuclear translocation of c-Jun we searched for novel binding partners by a proteomic approach. In addition to the heat shock protein HSP70 and the DNA damage repair factors Ku70 and 80, we isolated human importin 8 as a novel interactor of c-Jun. Interaction of Imp 8 with c-Jun in human cells was confirmed by co-immunoprecipitation experiments. Nuclear accumulation of c-Jun does not require its functions as a transcription factor or the interaction with its kinase JNK. Interestingly, nuclear accumulation of JNK is regulated by interaction with c-Jun. Unraveling the mechanisms of c-Jun and JNK transport to the nucleus and its regulation will improve our understanding of their role in biological and pathophysiological processes.

Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila.

In Drosophila, intestinal stem cells (ISCs) respond to oxidative challenges and inflammation by increasing proliferation rates. This phenotype is part of a regenerative response, but can lead to hyperproliferation and epithelial degeneration in the aging animal. Here we show that Nrf2, a master regulator of the cellular redox state, specifically controls the proliferative activity of ISCs, promoting intestinal homeostasis. We find that Nrf2 is constitutively active in ISCs and that repression of Nrf2 by its negative regulator Keap1 is required for ISC proliferation. We further show that Nrf2 and Keap1 exert this function in ISCs by regulating the intracellular redox balance. Accordingly, loss of Nrf2 in ISCs causes accumulation of reactive oxygen species and accelerates age-related degeneration of the intestinal epithelium. Our findings establish Keap1 and Nrf2 as a critical redox management system that regulates stem cell function in high-turnover tissues.

Computational systems biology of aging.

Computational systems biology is expected to make major contributions to unravel the complex molecular mechanisms underlying the progression of aging in cells, tissues, and organisms. The development of computational approaches is, however, challenged by a wide spectrum of aging mechanisms participating on different levels of biological organization. The tight connectivity between the molecular constituents, functions, and cell states requires frameworks and strategies that extend beyond current practice to model, simulate, and predict the progression of aging and the emerging aging phenotypes. We provide a general overview of the specific computational tasks and opportunities in aging research, and discuss some illustrative systems level concepts in more detail. One example provided here is the assembly of a conceptual whole cell model that considers the temporal dynamics of the aging process grounded on molecular mechanisms. Another application is the assembly of interactomes, such as protein networks that allow us to analyze changes in network topology and interaction of proteins that have been implicated in aging with other cellular constituents and processes. We introduce the necessary key steps to build these applications and discuss their merits and future extensions for aging research. WIREs Syst Biol Med 2011 3 414-428 DOI: 10.1002/wsbm.126

The role of the antioxidant and longevity-promoting Nrf2 pathway in metabolic regulation.

PURPOSE OF REVIEW: The vertebrate cap'n'collar family transcription factor Nrf2 and its invertebrate homologues SKN-1 (in worms) and CncC (in flies) function as master mediators of antioxidant and detoxification responses and regulators of the cellular redox state. Nrf2 controls gene expression programs that defend various tissues against diverse electrophilic stressors and oxidative insults, thus protecting the organism from disorders that are caused or exacerbated by such stresses. Moreover, studies on model organisms implicate the Nrf2 pathway in the prevention of aging-related diseases and suggest that SKN-1-regulated and CncC-regulated gene expression can promote longevity. These facets of Nrf2 signaling have been thoroughly reviewed. This article discusses another aspect of the Nrf2 pathway's function that has not yet received the same degree of attention, but emerges as a topic of increasing interest and potential clinical impact: its role in metabolic regulation and its interaction with central signaling systems that respond to nutritional inputs. RECENT FINDINGS: Recent evidence identifies Nrf2 signaling as a mediator of the salutary effects of caloric restriction. Nrf2 signaling also crosstalks with metabolic signaling systems such as the insulin/Akt pathway as well as with the metabolism of lipids. Moreover, Nrf2 has a protective role in models of diabetic nephropathy. SUMMARY: The emerging role of Nrf2 as an effector of metabolic and longevity signals offers new therapeutic perspectives. The potential impact of pharmacological manipulation of Nrf2 signaling as a strategy for the prevention and treatment of metabolic disease can be envisioned.