Oxidative stress and aberrant signaling in aging and cognitive decline

Brain aging is associated with a progressive imbalance between antioxidant defenses and intracellular concentrations of reactive oxygen species (ROS) as exemplified by increases in products of lipid peroxidation, protein oxidation, and DNA oxidation. Oxidative conditions cause not only structural damage but also changes in the set points of redox-sensitive signaling processes including the insulin receptor signaling pathway. In the absence of insulin, the otherwise low insulin receptor signaling is strongly enhanced by oxidative conditions. Autophagic proteolysis and sirtuin activity, in turn, are downregulated by the insulin signaling pathway, and impaired autophagic activity has been associated with neurodegeneration. In genetic studies, impairment of insulin receptor signaling causes spectacular lifespan extension in nematodes, fruit flies, and mice. The predicted effects of age-related oxidative stress on sirtuins and autophagic activity and the corresponding effects of antioxidants remain to be tested experimentally. However, several correlates of aging have been shown to be ameliorated by antioxidants. Oxidative damage to mitochondrial DNA and the electron transport chain, perturbations in brain iron and calcium homeostasis, and changes in plasma cysteine homeostasis may altogether represent causes and consequences of increased oxidative stress. Aging and cognitive decline thus appear to involve changes at multiple nodes within a complex regulatory network.     

JNK signaling in insulin-producing cells is required for adaptive responses to stress in Drosophila

Adaptation to environmental challenges is critical for the survival of an organism. Repression of Insulin/IGF Signaling (IIS) by stress-responsive Jun-N-terminal Kinase (JNK) signaling is emerging as a conserved mechanism that allows reallocating resources from anabolic to repair processes under stress conditions. JNK activation in Insulin-producing cells (IPCs) is sufficient to repress Insulin and Insulin-like peptide (ILP) expression in rats and flies, but the significance of this interaction for adaptive responses to stress is unclear. In this study, it is shown that JNK activity in IPCs of flies is required for oxidative stress-induced repression of the Drosophila ILP2. It is found that this repression is required for growth adaptation to heat stress as well as adult oxidative stress tolerance, and that induction of stress response genes in the periphery is in part dependent on IPC-specific JNK activity. Endocrine control of IIS by JNK in IPCs is thus critical for systemic adaptation to stress.     

JNK protects Drosophila from oxidative stress by trancriptionally activating autophagy

JNK signaling functions to induce defense mechanisms that protect organisms against acute oxidative and xenobiotic insults. Using Drosophila as a model system, we investigated the role of autophagy as such a JNK-regulated protective mechanism. We show that oxidative stress can induce autophagy in the intestinal epithelium by a mechanism that requires JNK signaling. Consistently, artificial activation of JNK in the gut gives rise to an autophagy phenotype. JNK signaling can induce the expression of several autophagy-related (ATG) genes, and the integrity of these genes is required for the stress protective function of the JNK pathway. In contrast to autophagy induced by oxidative stress, non-stress related autophagy, as it occurs for example in starving adipose or intestinal tissue, or during metamorphosis, proceeds independently of JNK signaling. Autophagy thus emerges as a multifunctional process that organisms employ in a variety of different situations using separate regulatory mechanisms.     

TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development

Transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) is a member of the MAPKKK superfamily and has been characterized as a component of the TGF-beta/bone morphogenetic protein signaling pathway. TAK1 function has been extensively studied in cultured cells, but its in vivo function is not fully understood. In this study, we isolated a Drosophila homolog of TAK1 (dTAK1) which contains an extensively conserved NH(2)-terminal kinase domain and a partially conserved COOH-terminal domain. To learn about possible endogenous roles of TAK1 during animal development, we generated transgenic flies which express dTAK1 or the mouse TAK1 (mTAK1) gene in the fly visual system. Ectopic activation of TAK1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by TAK1 signaling. Expression of a dominant negative form of dTAK during embryonic development resulted in various embryonic cuticle defects including dorsal open phenotypes. Our results strongly suggest that in Drosophila melanogaster, TAK1 functions as a MAPKKK in the JNK signaling pathway and participates in such diverse roles as control of cell shape and regulation of apoptosis.     

Lifespan extension by preserving proliferative homeostasis in Drosophila

Regenerative processes are critical to maintain tissue homeostasis in high-turnover tissues. At the same time, proliferation of stem and progenitor cells has to be carefully controlled to prevent hyper-proliferative diseases. Mechanisms that ensure this balance, thus promoting proliferative homeostasis, are expected to be critical for longevity in metazoans. The intestinal epithelium of Drosophila provides an accessible model in which to test this prediction. In aging flies, the intestinal epithelium degenerates due to over-proliferation of intestinal stem cells (ISCs) and mis-differentiation of ISC daughter cells, resulting in intestinal dysplasia. Here we show that conditions that impair tissue renewal lead to lifespan shortening, whereas genetic manipulations that improve proliferative homeostasis extend lifespan. These include reduced Insulin/IGF or Jun-N-terminal Kinase (JNK) signaling activities, as well as over-expression of stress-protective genes in somatic stem cell lineages. Interestingly, proliferative activity in aging intestinal epithelia correlates with longevity over a range of genotypes, with maximal lifespan when intestinal proliferation is reduced but not completely inhibited. Our results highlight the importance of the balance between regenerative processes and strategies to prevent hyperproliferative disorders and demonstrate that promoting proliferative homeostasis in aging metazoans is a viable strategy to extend lifespan.     

Involvement of oxidative stress and caspase 2-mediated intrinsic pathway signaling in age-related increase in muscle cell apoptosis in mice

Apoptosis has been implicated as a mechanism of loss of muscle cells in normal aging and plays an important role in age-related sarcopenia. To test the hypothesis that caspase 2 and c-Jun NH₂-terminal kinase (JNK)-mediated intrinsic pathway signaling contribute to skeletal muscle cell apoptosis in aging, we compared activation of caspase 2 and JNK and the in vivo expression of 4-hydroxynonenal protein adducts (4-HNE), inducible nitric oxide synthase (iNOS), glucose-6-phosphate dehydrogenase (G6PDH), B-cell lymphoma-2 (BCL-2), BAX, and phospho-BCL-2 in gastrocnemius muscles of young (5 months old) and old (25 months old) mice. A distinct age-related increase in 4-HNE and iNOS expression was readily detected in mice. Increased oxidative stress and iNOS induction were further accompanied by a decrease in G6PDH expression, activation of caspase 2 and JNK, and inactivation of BCL-2 through phosphorylation at serine 70, and caspase 9 activation. Regression analysis further revealed that increased muscle cell death in aging was significantly correlated with changes in the levels of these molecules. Taken together, our data indicate that caspase 2 and JNK-mediated intrinsic pathway signaling is one of the mechanisms involved in age-related increase in muscle cell apoptosis.     

Cytochrome P4502E1, oxidative stress, JNK, and autophagy in acute alcohol-induced fatty liver

Binge alcohol drinking induces hepatic steatosis. Recent studies showed that chronic ethanol-induced fatty liver was, at least in part, CYP2E1 dependent. The mechanism of acute alcohol-induced steatosis and whether CYP2E1 plays any role are still unclear. Increasing oxidative stress by alcohol can activate the JNK MAP kinase signaling pathway, suggesting that JNK might be a target for prevention of alcohol-induced steatosis. We used CYP2E1 knockout (KO) mice, a JNK inhibitor, and JNK1 or JNK2 knockout mice to test the role of CYP2E1, JNK, and the individual role of JNK1 and JNK2 in acute alcohol-induced steatosis. In wild-type (WT) mice, acute alcohol activates CYP2E1 and increases oxidative stress, which reciprocally increases activation of the JNK signaling pathway. Acute alcohol-induced fatty liver and oxidative stress were blunted in CYP2E1 KO mice and by the JNK inhibitor in WT mice. The antioxidant N-acetylcysteine decreased the acute alcohol-induced oxidative stress, the activation of JNK, and the steatosis but not the activation of CYP2E1. Acute alcohol decreased autophagy and increased expression of SREBP, effects blocked by the JNK inhibitor. Acute alcohol-induced fatty liver was the same in JNK1 and JNK2 KO mice as in WT mice; thus either JNK1 or JNK2 per se is sufficient for induction of steatosis by acute alcohol. The results show that acute alcohol elevation of CYP2E1, oxidative stress, and activation of JNK interact to lower autophagy and increase lipogenic SREBP resulting in fatty liver.     

Bendless modulates JNK-mediated cell death and migration in Drosophila

The TNF–JNK pathway is a highly conserved signaling pathway that regulates a wide spectrum of biological processes including cell death and migration. To further delineate this pathway, we carried out a genetic screen for dominant modifiers of the cell death phenotype triggered by ectopic expression of Eiger (Egr), the Drosophila TNF ortholog. Here we show that Bendless (Ben), an E2 ubiquitin-conjugating enzyme, modulates Egr-induced JNK activation and cell death through dTRAF2. Furthermore, Ben physically interacts with dTRAF2 and regulates Egr-induced dTRAF2 polyubiquitination. Finally, Ben is required for JNK-dependent tumor progression, cell migration, oxidative stress resistance and longevity. Our results indicate that Ben constitutes an essential component of the evolutionarily conserved TNF–JNK pathway that modulates cell death and invasion, tumor progression, stress response and lifespan in metazoans.     

The soma-germline communication: implications for somatic and reproductive aging

Aging is characterized by a functional decline in most physiological processes, including alterations in cellular metabolism and defense mechanisms. Increasing evidence suggests that caloric restriction extends longevity and retards age-related diseases at least in part by reducing metabolic rate and oxidative stress in a variety of species, including yeast, worms, flies, and mice. Moreover, recent studies in invertebrates – worms and flies, highlight the intricate interrelation between reproductive longevity and somatic aging (known as disposable soma theory of aging), which appears to be conserved in vertebrates. This review is specifically focused on how the reproductive system modulates somatic aging and vice versa in genetic model systems. Since many signaling pathways governing the aging process are evolutionarily conserved, similar mechanisms may be involved in controlling soma and reproductive aging in vertebrates.     

Hedgehog does not guide migrating Drosophila germ cells

In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies.     

Modulation of methuselah expression targeted to Drosophila insulin‐producing cells extends life and enhances oxidative stress resistance

Ubiquitously reduced signaling via Methuselah (MTH), a G‐protein‐coupled receptor (GPCR) required for neurosecretion, has previously been reported to extend life and enhance stress resistance in flies. Whether these effects are due to reduced MTH signalling in specific tissues remains unknown. We determined that reduced expression of mth targeted to the insulin‐producing cells (IPCs) of the fly brain was sufficient to extend life and enhance oxidative stress resistance. Paradoxically, we discovered that overexpression of mth targeted to the same cells has similar phenotypic effects to reduced expression due to MTH's interaction with β‐arrestin, which uncouples GPCRs from their G‐proteins. We confirmed the functional relationship between MTH and β‐arrestin by finding that IPC‐targeted overexpression of β‐arrestin alone mimics the longevity phenotype of reduced MTH signaling. As reduced MTH signaling also inhibits insulin secretion from the IPCs, the most parsimonious mechanistic explanation of its longevity and stress‐resistance enhancement might be through reduced insulin/IGF signaling (IIS). However, examination of phenotypic features of long‐lived IPC‐mth modulated flies as well as several downstream IIS targets implicates enhanced activity of the JNK stress‐resistance pathway more directly than insulin signaling in the longevity and stress‐resistance phenotypes.     

Activation of senescence and aging characteristics by mitochondrially generated ROS: How are they linked?

This article discusses the molecular mechanism(s) that link oxidative stress (ROS) due to mitochondrial dysfunction to the activation of the ROS-sensitive signaling pathways whose levels of activity promote the development of senescence, aging, longevity, and resistance to oxidative stress. Most significantly, our discussion links ROS generated by mitochondrial dysfunction (ROS) to the activation of the ASK1-signalosome - p38 pathway. Our hypothesis argues that this is a major pathway that promotes physiological senescence, aging and age-associated diseases. We thus conclude that the ASK1-signalosome serves as an ROS-sensory system that regulates the levels of ROS-responsive p38 MAPK signals and serves as a signaling center that mediates the physiological consequences of mitochondrial dysfunction.     

c-Jun N-terminal kinases (JNK) antagonize cardiac growth through cross-talk with calcineurin-NFAT signaling

The c-Jun N-terminal kinase (JNK) branch of the mitogen-activated protein kinase (MAPK) signaling pathway regulates cellular differentiation, stress responsiveness and apoptosis in multicellular eukaryotic organisms. Here we investigated the functional importance of JNK signaling in regulating differentiated cellular growth in the post-mitotic myocardium. JNK1/2 gene-targeted mice and transgenic mice expressing dominant negative JNK1/2 were determined to have enhanced myocardial growth following stress stimulation or with normal aging. A mechanism underlying this effect was suggested by the observation that JNK directly regulated nuclear factor of activated T-cell (NFAT) activation in culture and in transgenic mice containing an NFAT-dependent luciferase reporter. Moreover, calcineurin Abeta gene targeting abrogated the pro-growth effects associated with JNK inhibition in the heart, while expression of an MKK7-JNK1 fusion protein in the heart partially reduced calcineurin-mediated cardiac hypertrophy. Collectively, these results indicate that JNK signaling antagonizes the differentiated growth response of the myocardium through direct cross-talk with the calcineurin-NFAT pathway. These results also suggest that myocardial JNK activation is primarily dedicated to modulating calcineurin-NFAT signaling in the regulation of differentiated heart growth.