Joanna Kotwica-Rolinska

Circadian Regulation of Glutathione Levels and Biosynthesis in Drosophila melanogaster

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.

Relationships between the circadian system and Alzheimer's disease-like symptoms in Drosophila

Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimers disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, we utilized transgenic Drosophila expressing various Amyloid-β (Aβ) peptides, which are prone to form aggregates characteristic of AD pathology in humans. We compared development of AD-like symptoms in adult flies expressing Aβ peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per01). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Aβ peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. We report that rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Aβ peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Aβ peptides causes rhythm degradation downstream from the central clock mechanism.

Manipulations of Amyloid Precursor Protein Cleavage Disrupt the Circadian Clock in Aging Drosophila

Alzheimers disease (AD) is a neurodegenerative disease characterized by severe cognitive deterioration. While causes of AD pathology are debated, a large body of evidence suggests that increased cleavage of Amyloid Precursor Protein (APP) producing the neurotoxic Amyloid-β (Aβ) peptide plays a fundamental role in AD pathogenesis. One of the detrimental behavioral symptoms commonly associated with AD is the fragmentation of sleep-activity cycles with increased nighttime activity and daytime naps in humans. Sleep-activity cycles, as well as physiological and cellular rhythms, which may be important for neuronal homeostasis, are generated by a molecular system known as the circadian clock. Links between AD and the circadian system are increasingly evident but not well understood. Here we examined whether genetic manipulations of APP-like (APPL) protein cleavage in Drosophila melanogaster affect rest-activity rhythms and core circadian clock function in this model organism. We show that the increased β-cleavage of endogenous APPL by the β-secretase (dBACE) severely disrupts circadian behavior and leads to reduced expression of clock protein PER in central clock neurons of aging flies. Our data suggest that behavioral rhythm disruption is not a product of APPL-derived Aβ production but rather may be caused by a mechanism common to both α and β-cleavage pathways. Specifically, we show that increased production of the endogenous Drosophila Amyloid Intracellular Domain (dAICD) caused disruption of circadian rest-activity rhythms, while flies overexpressing endogenous APPL maintained stronger circadian rhythms during aging. In summary, our study offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimers disease.

Aging alters circadian regulation of redox in Drosophila.

Circadian coordination of metabolism, physiology, and neural functions contributes to healthy aging and disease prevention. Clock genes govern the daily rhythmic expression of target genes whose activities underlie such broad physiological parameters as maintenance of redox homeostasis. Previously, we reported that glutathione (GSH) biosynthesis is controlled by the circadian system via effects of the clock genes on expression of the catalytic (Gclc) and modulatory (Gclm) subunits comprising the glutamate cysteine ligase (GCL) holoenzyme. The objective of this study was to determine whether and how aging, which leads to weakened circadian oscillations, affects the daily profiles of redox-active biomolecules. We found that fly aging is associated with altered profiles of Gclc and Gclm expression at both the mRNA and protein levels. Analysis of free aminothiols and GCL activity revealed that aging abolishes daily oscillations in GSH levels and alters the activity of glutathione biosynthetic pathways. Unlike GSH, its precursors and products of catabolism, methionine, cysteine and cysteinyl-glycine, were not rhythmic in young or old flies, while rhythms of the glutathione oxidation product, GSSG, were detectable. We conclude that the temporal regulation of GSH biosynthesis is altered in the aging organism and that age-related loss of circadian modulation of pathways involved in glutathione production is likely to impair temporal redox homeostasis.

Temporal Expression of the Clock Genes in the Water Flea Daphnia pulex (Crustacea: Cladocera).

The timekeeping mechanisms that operate at the core of circadian clocks (oscillators) are based on interacting molecular feedback loops consisting of clock and clock-associated genes. However, there is a lack of comprehensive studies on the expression of clock genes (particularly those forming its core) in single crustacean species at the mRNA and protein levels, and these studies could serve as a basis for constructing a model of the crustacean molecular oscillator. Studies on Daphnia pulex are well suited to fill this gap because this species is the only representative crustacean whose genome has been sequenced. We analyzed the abundance of 20 gene transcripts throughout the day in the whole bodies of D. pulex (single clone); we found that 15 of these genes were transcriptionally active, and most had daily expression level changes. According to the functional classification of their homologues in insects, these genes may represent elements of the Daphnia molecular oscillator core and its input and output pathways. Studies of PERIOD (PER) protein, one of the main clock components, revealed its rhythmic expression pattern in the epidermis, gut, and ovaries. Finally, the cycling levels of many of these clock components observed in animals reared in continuous light led to the conclusion that the Daphnia oscillator, even if it is structurally similar to the oscillators of other arthropods, can be considered a particularly important adaptive mechanism for living in environments with extreme photoperiods.

Unexpected Geographic Variability of the Free Running Period in the Linden Bug Pyrrhocoris apterus

Circadian clocks keep organisms in synchrony with external day-night cycles. The free running period (FRP) of the clock, however, is usually only close to—not exactly—24 h. Here, we explored the geographical variation in the FRP of the linden bug, Pyrrhocoris apterus, in 59 field-lines originating from a wide variety of localities representing geographically different environments. We have identified a remarkable range in the FRPs between field-lines, with the fastest clock at ~21 h and the slowest close to 28 h, a range comparable to the collections of clock mutants in model organisms. Similarly, field-lines differed in the percentage of rhythmic individuals, with a minimum of 13.8% and a maximum of 86.8%. Although the FRP correlates with the latitude and perhaps with the altitude of the locality, the actual function of this FRP diversity is currently unclear. With the recent technological progress of massive parallel sequencing and genome editing, we can expect remarkable progress in elucidating the genetic basis of similar geographic variants in P. apterus or in similar emerging model species of chronobiology.

Circadian regulation of caterpillar feeding and growth

Circadian clocks orchestrate many physiological processes in adult organisms. For example, rhythmic feeding behavior is regulated by the central clock in the nervous system in coordination with metabolic rhythms, which in turn depend mostly on peripheral clocks localized in many tissues. Disruption of the circadian clock leads to metabolic dysregulation both in mammals and in the model insect Drosophila melanogaster. Circadian coordination of feeding and metabolism has been studied mainly in adult insects and not in larval stages, which are dramatically different from adults in species with complete full metamorphosis. The goal of this study was to determine whether feeding and metabolism in lepidopteran larvae are subject to circadian regulation. We show that cotton leafworm caterpillars, Spodoptera littoralis, display rhythmic feeding behavior and that circadian clock genes are expressed in two peripheral tissues, the midgut and fat body. Even though both tissues display rhythmic circadian clock gene expression, the main component of the clock, per, is arrhythmic in the gut and rhythmic in the fat body. In both tissues, the presence of rhythmic physiological processes was observed, which suggested that metabolism is already driven by the circadian clock in the insects juvenile stages.

The role of circadian clock genes in the photoperiodic timer of the linden bug Pyrrhocoris apterus during the nymphal stage

Many insects survive seasonal adversities during diapause, a form of programmed developmental and metabolic arrest. Photoperiodically regulated entry into diapause allows multivoltine insect species to optimize the number of generations. The molecular mechanism of the photoperiodic timer is unknown in insects. In the present study, we take advantage of the robust reproductive diapause response in the linden bug Pyrrhocoris apterus and explore the fifth‐instar nymphal stage, which is the most photoperiod‐sensitive stage. The nymphs display daily changes in locomotor activity during short days; this differs from the activity observed during long days. We find evidence of cyclical expression of the circadian clock genes, per and cyc, in nymphal heads; in addition, per expression is also photoperiod‐dependent. The RNA interference‐mediated knockdown of the two circadian clock genes, Clk and cyc, during the nymphal stage results in reproductive arrest in adult females. Furthermore, Clk and cyc knockdown induces the expression of the storage protein hexamerin in the fat body, whereas the expression of vitellogenin diminishes. Taken together, these data support the involvement of circadian clock genes in photoperiodic timer and/or diapause induction.