Circadian regulation of response to oxidative stress in Drosophila melanogaster

Circadian rhythms are fundamental biological phenomena generated by molecular genetic mechanisms known as circadian clocks. There is increasing evidence that circadian synchronization of physiological and cellular processes contribute to the wellness of organisms, curbing pathologies such as cancer and premature aging. Therefore, there is a need to understand how circadian clocks orchestrate interactions between the organism’s internal processes and the environment. Here, we explore the nexus between the clock and oxidative stress susceptibility in Drosophila melanogaster. We exposed flies to acute oxidative stress induced by hydrogen peroxide (H₂O₂), and determined that mortality rates were dependent on time at which exposure occurred during the day/night cycle. The daily susceptibility rhythm was abolished in flies with a null mutation in the core clock gene period (per) abrogating clock function. Furthermore, lack of per increased susceptibility to H₂O₂ compared to wild-type flies, coinciding with enhanced generation of mitochondrial H₂O₂ and decreased catalase activity due to oxidative damage. Taken together, our data suggest that the circadian clock gene period is essential for maintaining a robust anti-oxidative defense.     

Effect of hesperidin on the temporal regulation of redox homeostasis in clock mutant (Cryb) of Drosophila melanogaster

Disorganized redox homeostasis is a main factor causing a number of diseases and it is imperative to comprehend the orchestration of circadian clock under oxidative stress in the organism, Drosophila melanogaster. This investigation analyses the influence of hesperidin on the circadian rhythms of lipid peroxidation products and antioxidants during rotenone-stimulated oxidative stress in fruit fly. The characteristics of rhythms of thiobarbituric acid reactive substances (TBARS), antioxidants (superoxide dismutase (SOD) and catalase (CAT)) were noticeably decreased in rotenone administered flies. Supplementation of hesperidin to rotenone-treated flies increased the mesor and modulated the amplitudes of antioxidants and conspicuously decreased the mesor values of TBARS. In addition, delays in acrophase in rotenone-induced flies were reversed by hesperidin treatment. Thus, treatment of hesperidin caused normalization of the altered rhythms. Disorganization of 24 h rhythms in markers of redox homeostasis was observed during rotenone treatment and the impairment is severe in circadian clock mutant (Cryb) flies. Reversibility of rhythms was prominent subsequent to hesperidin treatment in wild-type flies than (Cryb) flies. These observations denote a role of circadian clock in redox homeostasis and the use of Drosophila model in screening putative antioxidative phytomedicines prior to their usage in mammalian systems.     

Modulating effects of hesperidin on circadian pattern indices of rotenone induced redox homeostasis in clock mutant (cryb) of Drosophila melanogaster

The circadian timing system controls drug metabolism and cellular processes over the 24 h period in every cell. Impaired redox homeostasis is a casual factor for a number of diseases and it is desirable to understand the orchestration of circadian clock under oxidative stress in the model organism, Drosophila melanogaster. This study evaluates the effect of hesperidin on the circadian rhythms of lipid peroxidation products and antioxidants during rotenone-induced oxidative stress in fruit fly. The characteristics of temporal rhythms (acrophase, amplitude, and mesor) of glutathione peroxides (GPx), reduced glutathione (GSH)), were markedly declined in rotenone-treated flies when compared to other groups. Treatment of hesperidin to rotenone-treated flies significantly increased the mesor and modified the amplitudes of antioxidants. Further, delays in acrophase in rotenone-induced flies were reversed by hesperidin treatment. Thus, treatment of hesperidin results in normalization of the altered rhythms of these indices plausibly by its cytoprotective and antioxidant effects. Impairment of 24 h rhythms in oxidative stress markers and antioxidants were observed during rotenone treatment and the impairment is severe in circadian clock mutant cry^b flies. A reversibility of rhythms was prominent consequent to hesperidin treatment in wild-type flies than cry^b flies. These findings revealed a role of circadian clock in redox homeostasis and the use of Drosophila model in screening putative antioxidative phytomedicines earlier to their use in mammalian systems.     

Unique self-sustaining circadian oscillators within the brain of Drosophila melanogaster

In Drosophila circadian rhythms persist in constant darkness (DD). The small ventral Lateral Neurons (s-LN_v) mainly control the behavioral circadian rhythm in consortium with the large ventral Lateral Neurons (l-LN_v) and dorsal Lateral Neurons (LN_d). It is believed that the molecular oscillations of clock genes are the source of this persistent behavior. Indeed the s-LN_v, LN_d, Dorsal Neurons (DN)-DN₂ and DN₃ displayed self-sustained molecular oscillations in DD both at RNA and protein levels, except the DN₂ oscillates in anti-phase. In contrast, the l-LN_v and DN₁ displayed self-sustained oscillations at the RNA level, but protein oscillations quickly dampened. Having self-sustained and dampened molecular oscillators together in the DN groups suggested that they play different roles. However, all DN groups seemed to contribute together to the light-dark (LD) behavioral rhythm. The LD entrainment of LN oscillators is achieved through Rhodopsin (RH) and Cryptochrome (CRY). CRY's expression in all DN groups implicates also its role in LD entrainment of DN, like in DN₁. However, mutations in cry and glass that did not inflict LD synchronization of the DN₂, DN₃ oscillator implicate the existence of a novel photoreceptor at least in DN₃.     

Linear motifs in the C-terminus of D. melanogaster cryptochrome

The C-terminus of cryptochrome (CRY) regulates light responses in Drosophila. These include the light-dependent binding of Drosophila dCRY to the clock proteins PERIOD and TIMELESS in a yeast two-hybrid system, which we proved to be a convenient and reliable readout of the behavior of dCRY in vivo. In this study, we present a combination of in silico analysis and experimental validation in yeast, to identify novel functional motifs in the C-terminal region of dCRY. Our results suggest that linear motifs are present in this small region, which is a likely hotspot for molecular interactions.     

High sucrose consumption promotes obesity whereas its low consumption induces oxidative stress in Drosophila melanogaster

The effects of sucrose in varied concentrations (0.25–20%) with constant amount of yeasts in larval diet on development and metabolic parameters of adult fruit fly Drosophila melanogaster were studied. Larvae consumed more food at low sucrose diet, overeating with yeast. On high sucrose diet, larvae ingested more carbohydrates, despite consuming less food and obtaining less protein derived from yeast. High sucrose diet slowed down pupation and increased pupa mortality, enhanced levels of lipids and glycogen, increased dry body mass, decreased water content, i.e. resulted in obese phenotype. Furthermore, it suppressed reactive oxygen species-induced oxidation of lipids and proteins as well as the activity of superoxide dismutase. The activity of catalase was gender-related. In males, at all sucrose concentrations used catalase activity was higher than at its concentration of 0.25%, whereas in females sucrose concentration virtually did not influence the activity. High sucrose diet increased content of protein thiols and the activity of glucose-6-phosphate dehydrogenase. The increase in sucrose concentration also enhanced uric acid level in females, but caused opposite effects in males. Development on high sucrose diets was accompanied by elevated steady-state insulin-like peptide 3 mRNA level. Finally, carbohydrate starvation at yeast overfeeding on low sucrose diets resulted in oxidative stress reflected by higher levels of oxidized lipids and proteins accompanied by increased superoxide dismutase activity. Potential mechanisms involved in regulation of redox processes by carbohydrates are discussed.     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

Role of oxidative stress in epileptic seizures

Oxidative stress resulting from excessive free-radical release is likely implicated in the initiation and progression of epilepsy. Therefore, antioxidant therapies aimed at reducing oxidative stress have received considerable attention in epilepsy treatment. However, much evidence suggests that oxidative stress does not always have the same pattern in all seizures models. Thus, this review provides an overview aimed at achieving a better understanding of this issue. We summarize work regarding seizure models (i.e., genetic rat models, kainic acid, pilocarpine, pentylenetetrazol, and trimethyltin), oxidative stress as an etiologic factor in epileptic seizures (i.e., impairment of antioxidant systems, mitochondrial dysfunction, involvement of redox-active metals, arachidonic acid pathway activation, and aging), and antioxidant strategies for seizure treatment. Combined, this review highlights pharmacological mechanisms associated with oxidative stress in epileptic seizures and the potential for neuroprotection in epilepsy that targets oxidative stress and is supported by effective antioxidant treatment.     

Genome-wide association analysis of oxidative stress resistance in Drosophila melanogaster

Background

Aerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress.

Methods and Findings

We used 167 wild-derived inbred lines of the Drosophila Genetic Reference Panel for a genome-wide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67–79% and 56–66% of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis.

Conclusions

We identified novel candidate genes associated with variation in resistance to oxidative stress that have context-dependent effects. These results form the basis for future translational studies to identify oxidative stress susceptibility/resistance genes that are evolutionary conserved and might play a role in human disease.     

Involvement of oxidative stress in 4-vinylcyclohexene-induced toxicity in Drosophila melanogaster

4-Vinylcyclohexene (VCH) is a dimer of 1,3-butadiene produced as a by-product of pesticides, plastic, rubber, flame retardants, and tire production. Although, several studies have reported the ovotoxicity of VCH, information on a possible involvement of oxidative stress in the toxicity of this occupational chemical is scarce. Hence, this study was carried out to investigate further possible mechanisms of toxicity of VCH with a specific emphasis on oxidative stress using a Drosophila melanogaster model. D. melanogaster (both genders) of 1 to 3 days old were exposed to different concentrations of VCH (10 µM–1 mM) in the diet for 5 days. Subsequently, the survival and negative geotaxis assays and the quantification of reactive oxygen species (ROS) generation were determined. In addition, we evaluated RT-PCR expressions of selected oxidative stress and antioxidant mRNA genes (HSP27, 70, and 83, SOD, Nrf-2, MAPK2, and catalase). Furthermore, catalase, glutathione-S-transferase (GST), delta aminolevulinic acid dehydratase (δ-ALA-D), and acetylcholinesterase (AChE) activities were determined. VCH exposure impaired negative geotaxic behavior and induced the mRNA of SOD, Nrf-2, and MAPK2 genes expressions. There were increases in catalase and ROS production, as well as inhibitions of GST, δ-ALA-D, and AChE activities (P<0.05). Our results suggest that the VCH mechanism of toxicity is associated with oxidative damage, as evidenced by the alteration in the oxidative stress-antioxidant balance, and possible neurotoxic consequences due to decreased AChE activity, and impairments in negative geotaxic behavior. Thus, we conclude that D. melanogaster is a useful model for investigating the toxicity of VCH exposure, and here, we have provided further insights on the mechanism of VCH-induced toxicity.     

Role of oxidative stress in Drosophila aging.

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.     

Hesperidin modulates the rhythmic proteomic profiling in Drosophila melanogaster under oxidative stress

The circadian clock regulates vital aspects of physiology including protein synthesis and oxidative stress response. In this investigation, we performed a proteome-wide scrutiny of rhythmic protein accrual in Drosophila melanogaster on exposure to rotenone, rotenone + hesperidin and hesperidin in D. melanogaster. Total protein from fly samples collected at 6 h intervals over the 24 h period was subjected to two-dimensional gel electrophoresis and mass spectrometry. Bioinformatics tool, Protein ANalysis THrough Evolutionary Relationships classification system was used to the determine the biological processes of the proteins of altered abundance. Conspicuous variations in the proteome (151 proteins) of the flies exposed to oxidative stress (by rotenone treatment) and after alleviating oxidative stress (by hesperidin treatment) were observed during the 24 h cycle. Significantly altered levels of abundance of a wide variety of proteins under oxidative stress (rotenone treatment) and under alleviation of oxidative stress (rotenone + hesperidin treatment) and hesperidin (alone) treatment were observed. These proteins are involved in metabolism, muscle activity, heat shock response, redox homeostasis, protein synthesis/folding/degradation, development, ion-channel/cellular transport, and gustatory and olfactory function of the flies. Our data indicates that numerous cellular processes are involved in the temporal regulation of proteins and widespread modulations happen under rotenone treatment and, action of hesperidin could also be seen on these categories of proteins.     

The biological clock: the bodyguard of temporal homeostasis

In order for any organism to function properly, it is crucial that it be table to control the timing of its biological functions. An internal biological clock, located, in mammals, in the suprachiasmatic nucleus of the hypothalamus (SCN), therefore carefully guards this temporal homeostasis by delivering its message of time throughout the body. In view of the large variety of body functions (behavioral, physiological, and endocrine) as well as the large variety in their preferred time of main activity along the light:dark cycle, it seems logical to envision different means of time distribution by the SCN. In the present review, we propose that even though it presents a unimodal circadian rhythm of general electrical and metabolic activity, the SCN seems to use several sorts of output connections that are active at different times along the light: dark cycle to control the rhythmic expression of different body functions. Although the SCN is suggested to use diffusion of synchronizing factors in the rhythmic control of behavioral functions, it also needs neuronal connections for the control of endocrine functions. The distribution of the time-of-day message to neuroendocrine systems is either directly onto endocrine neurons or via intermediate neurons located in specific SCN targets. In addition, the SCN uses its connections with the autonomic nervous system for spreading its time-of-day message, either by setting the sensitivity of endocrine glands (i.e., thyroid, adrenal, ovary) or by directly controlling an endocrine output (i.e., melatonin synthesis). Moreover, the SCN seems to use different neurotransmitters released at different times along the light: dark cycle for each of the different connection types presented. Clearly, the temporal homeostasis of endocrine functions results from a diverse set of biological clock outputs.     

Antimicrobial peptides increase tolerance to oxidant stress in Drosophila melanogaster

It is well appreciated that reactive oxygen species (ROS) are deleterious to mammals, including humans, especially when generated in abnormally large quantities from cellular metabolism. Whereas the mechanisms leading to the production of ROS are rather well delineated, the mechanisms underlying tissue susceptibility or tolerance to oxidant stress remain elusive. Through an experimental selection over many generations, we have previously generated Drosophila melanogaster flies that tolerate tremendous oxidant stress and have shown that the family of antimicrobial peptides (AMPs) is over-represented in these tolerant flies. Furthermore, we have also demonstrated that overexpression of even one AMP at a time (e.g. Diptericin) allows wild-type flies to survive much better in hyperoxia. In this study, we used a number of experimental approaches to investigate the potential mechanisms underlying hyperoxia tolerance in flies with AMP overexpression. We demonstrate that flies with Diptericin overexpression resist oxidative stress by increasing antioxidant enzyme activities and preventing an increase in ROS levels after hyperoxia. Depleting the GSH pool using buthionine sulfoximine limits fly survival, thus confirming that enhanced survival observed in these flies is related to improved redox homeostasis. We conclude that 1) AMPs play an important role in tolerance to oxidant stress, 2) overexpression of Diptericin changes the cellular redox balance between oxidant and antioxidant, and 3) this change in redox balance plays an important role in survival in hyperoxia.     

Presence of robust circadian clock oscillation under constitutive over-expression of mCry1 in rat-1 fibroblasts

In mammals, mCRY proteins are essential and are major negative elements in circadian feedback loops. In this study, robust circadian clock oscillation was present even under conditions with constitutive over-expression of mCry1 in rat-1 cells. Rat-1 cells were produced to stably express mPer2 promoter-driven luciferase reporter, in which mCry1 was overexpressed under a tetracycline-dependent gene expression (Tet-On) system. Using these cells, we show that circadian clock oscillations in rat-1 fibroblasts persist when the mCRY1 protein constitutively accumulates in the nuclei.     

Oogenesis in the female sterile (1)42 mutant of Drosophila melanogaster.

The sex-linked mutation fs(1)42 was induced by ethyl methane sulfonate. It has no effect on either the external morphology or longevity of adult hemizygotes or homozygotes. Heterozygotes and hemizygotes are fertile, but homozygotes are sterile. Egg chamber development proceeds through stages 8, and thereafter chambers degenerate. Dying follicle cells are seen in chambers at all positions in the ovarioles. Profollicle cells also die within germaria, and clusters of sister cystocytes take longer than normal to receive their coverings of follicle cells. Egg chambers in the vitellarium contain only about 60% the normal number of follicle cells, these generally have greater lateral dimensions, and their nuclei and nucleoli are also larger than normal. The follicular envelope of mutant chambers often contains gaps through which cystocytes send cytoplasmic projections. Abnormalities seen in development of the fs(1)42 oocyte are likely to be due to its envelope of defective follicle cells.     

Control of oxidative stress resistance by IP3 kinase in Drosophila melanogaster

Oxidative damage is thought to be a major causal factor of aging, and is implicated in several human pathologies such as Alzheimer's and Parkinson's diseases. Nevertheless the genetical determinants of in vivo oxidative stress response are still poorly understood. To identify cellular components whose deregulation leads to oxidative stress resistance, we performed a genetic screen in Drosophila melanogaster. We thus identified in this screen Drosophila Inositol 1,4,5-triphosphate kinase I (D-IP3K1), a Drosophila gene homologous to mammalian IP3Ks. In vertebrates, IP3Ks phosphorylate the second messenger Inositol 1,4,5-triphosphate (IP3) to produce Inositol 1,3,4,5 tetrakiphosphate (IP4). IP3 binding to its receptor (IP3R) triggers Ca(2+) release from the endoplasmic reticulum (ER) to the cytosol, whereas IP4 physiological role remains elusive. We show here that ubiquitous overexpression of D-IP3K1 confers resistance of flies to H(2)O(2)- but not to paraquat-induced oxidative stress. Additional genetic analysis with other members of IP3 and IP4 signaling pathways led us to propose that the D-IP3K1 protective effect is mainly mediated through the reduction of IP3 level (which probably results in reduced Ca(2+) release from internal stores), rather than through the rise of IP4 level.