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 Alzheimer''s 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.     

Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster

Circadian locomotor rhythms of Drosophila melanogaster are controlled by a neuronal circuit composed of approximately 150 clock neurons that are roughly classified into seven groups. In the circuit, a group of neurons expressing pigment-dispersing factor (PDF) play an important role in organizing the pacemaking system. Recent studies imply that unknown chemical neurotransmitter(s) (UNT) other than PDF is also expressed in the PDF-positive neurons. To explore its role in the circadian pacemaker, we examined the circadian locomotor rhythms of pdf-Gal4/UAS-TNT transgenic flies in which chemical synaptic transmission in PDF-positive neurons was blocked by expressed tetanus toxin light chain (TNT). In constant darkness (DD), the flies showed a free-running rhythm, which was similar to that of wild-type flies but significantly different from pdf null mutants. Under constant light conditions (LL), however, they often showed complex rhythms with a short period and a long period component. The UNT is thus likely involved in the synaptic transmission in the clock network and its release caused by LL leads to arrhythmicity. Immunocytochemistry revealed that LL induced phase separation in TIMELESS (TIM) cycling among some of the PDF-positive and PDF-negative clock neurons in the transgenic flies. These results suggest that both PDF and UNT play important roles in the Drosophila circadian clock, and activation of PDF pathway alone by LL leads to the complex locomotor rhythm through desynchronized oscillation among some of the clock neurons.     

Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms

Intercellular signaling is important for accurate circadian rhythms. In Drosophila, the small ventral lateral neurons (s-LN(v)s) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. Here we show that two distinct G protein signaling pathways are required in LN(v)s for 24?hr rhythms. Reducing signaling in LN(v)s via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which we show signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LN(v)s in culture, we found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LN(v)s and that reduced GABA(B)-R3 expression in?vivo lengthens period. Although no clock neurons produce GABA, hyperexciting GABAergic neurons disrupts behavioral rhythms and s-LN(v) molecular clocks. Therefore, s-LN(v)s require GABAergic inputs for 24?hr rhythms.     

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.     

Insect circadian rhythms and photoperiodism

Two clock-controlled processes, overt circadian rhythmicity and the photoperiodic induction of diapause, are described in the blow fly,Calliphora vicina and the fruit fly,Drosophila melanogaster. Circadian locomotor rhythms of the adult flies reflect endogenous, self-sustained oscillations with a temperature compensated period. The free-running rhythms become synchronised (entrained) to daily light:dark cycles, but become arrhythmic in constant light above a certain intensity. Some flies show fragmented rhythms (internal desynchronisation) suggesting that overt rhythmicity is the product of a multioscillator (multicellular) system. Photoperiodic induction of larval diapause inC. vicina and of ovarian diapause inD. melanogaster is also based on the circadian system but seems, to involve a separate mechanism at both the molecular and neuronal levels. For both processes in both species, the compound eyes and ocelli are neither essential nor necessary for photic entrainment, and the circadian clock mechanism is not within the optic lobes. The central brain is the most likely site for both rhythm generation and extra-optic photoreception. InD. melanogaster, a group of lateral brain neurons has been identified as important circadian pacemaker cells, which are possibly also photo-sensitive. Similar lateral brain neurons, staining for arrestin, a protein in the phototransduction ‘cascade’ and a selective marker for photoreceptors in both vertebrates and invertebrates, have been identified inC. vicina. Much less is known about the cellular substrate of the photoperiodic mechanism, but this may involve thepars intercerebralis region of the mid-brain.     

Old flies have a robust central oscillator but weaker behavioral rhythms that can be improved by genetic and environmental manipulations

Sleep-wake cycles break down with age, but the causes of this degeneration are not clear. Using a Drosophila model, we addressed the contribution of circadian mechanisms to this age-induced deterioration. We found that in old flies, free-running circadian rhythms (behavioral rhythms assayed in constant darkness) have a longer period and an unstable phase before they eventually degenerate. Surprisingly, rhythms are weaker in light-dark cycles and the circadian-regulated morning peak of activity is diminished under these conditions. On a molecular level, aging results in reduced amplitude of circadian clock gene expression in peripheral tissues. However, oscillations of the clock protein PERIOD (PER) are robust and synchronized among different clock neurons, even in very old, arrhythmic flies. To improve rhythms in old flies, we manipulated environmental conditions, which can have direct effects on behavior, and also tested a role for molecules that act downstream of the clock. Coupling temperature cycles with a light-dark schedule or reducing expression of protein kinase A (PKA) improved behavioral rhythms and consolidated sleep. Our data demonstrate that a robust molecular timekeeping mechanism persists in the central pacemaker of aged flies, and reducing PKA can strengthen behavioral rhythms.     

Cryptochrome restores dampened circadian rhythms and promotes healthspan in aging Drosophila

Circadian clocks generate daily rhythms in molecular, cellular, and physiological functions providing temporal dimension to organismal homeostasis. Recent evidence suggests two‐way relationship between circadian clocks and aging. While disruption of the circadian clock leads to premature aging in animals, there is also age‐related dampening of output rhythms such as sleep/wake cycles and hormonal fluctuations. Decay in the oscillations of several clock genes was recently reported in aged fruit flies, but mechanisms underlying these age‐related changes are not understood. We report that the circadian light–sensitive protein CRYPTOCHROME (CRY) is significantly reduced at both mRNA and protein levels in heads of old Drosophila melanogaster. Restoration of CRY using the binary GAL4/UAS system in old flies significantly enhanced the mRNA oscillatory amplitude of several genes involved in the clock mechanism. Flies with CRY overexpressed in all clock cells maintained strong rest/activity rhythms in constant darkness late in life when rhythms were disrupted in most control flies. We also observed a remarkable extension of healthspan in flies with elevated CRY. Conversely, CRY‐deficient mutants showed accelerated functional decline and accumulated greater oxidative damage. Interestingly, overexpression of CRY in central clock neurons alone was not sufficient to restore rest/activity rhythms or extend healthspan. Together, these data suggest novel anti‐aging functions of CRY and indicate that peripheral clocks play an active role in delaying behavioral and physiological aging.     

Drosophila cryb mutation reveals two circadian clocks that drive locomotor rhythm and have different responsiveness to light

Cryptochrome (CRY) is a blue-light-absorbing protein involved in the photic entrainment of the circadian clock in Drosophila melanogaster. We have investigated the locomotor activity rhythms of flies carrying cryb mutant and revealed that they have two separate circadian oscillators with different responsiveness to light. When kept in constant light conditions, wild-type flies became arrhythmic, while cryb mutant flies exhibited free-running rhythms with two rhythmic components, one with a shorter and the other with a longer free-running period. The rhythm dissociation was dependent on the light intensities: the higher the light intensities, the greater the proportion of animals exhibiting the two oscillations. External photoreceptors including the compound eyes and the ocelli are the likely photoreceptors for the rhythm dissociation, since rhythm dissociation was prevented in so1;cryb and norpAP41;cryb double mutant flies. Immunohistochemical analysis demonstrated that the PERIOD expression rhythms in ventrally located lateral neurons (LNvs) occurred synchronously with the shorter period component, while those in the dorsally located per-expressing neurons showed PER expression most likely related to the longer period component, in addition to that synchronized to the LNvs. These results suggest that the Drosophila locomotor rhythms are driven by two separate per-dependent clocks, responding differentially to constant light.     

Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease

The accumulation and aggregation of phosphorylated tau proteins in the brain are the hallmarks for the onset of Alzheimer's disease (AD). In addition, disruptions in circadian rhythms (CRs) with altered sleep-wake cycles, dysregulation of locomotion, and increased memory defects have been reported in patients with AD. Drosophila flies that have an overexpression of human tau protein in neurons exhibit most of the symptoms of human patients with AD, including locomotion defects and neurodegeneration. Using the fly model for tauopathy/AD, we investigated the effects of an exposure to dim light at night on AD symptoms. We used a light intensity of 10 lux, which is considered the lower limit of light pollution in many countries. After the tauopathy flies were exposed to the dim light at night for 3 days, the flies showed disrupted CRs, altered sleep-wake cycles due to increased pTau proteins and neurodegeneration, in the brains of the AD flies. The results indicate that the nighttime exposure of tauopathy/AD model Drosophila flies to dim light disrupted CR and sleep-wake behavior and promoted neurodegeneration.     

Role of Bacopa monnieri in the temporal regulation of oxidative stress in clock mutant (cryb) of Drosophila melanogaster

Accruing evidences imply that circadian organization of biochemical, endocrinological, cellular and physiological processes contribute to wellness of organisms and in the development of pathologies such as malignancy, sleep and endocrine disorders. Oxidative stress is known to mediate a number of diseases and it is notable to comprehend the orchestration of circadian clock of a model organism of circadian biology, Drosophila melanogaster, under oxidative stress. We investigated the nexus between circadian clock and oxidative stress susceptibility by exposing D. melanogaster to hydrogen peroxide (H₂O₂) or rotenone; the reversibility of rhythms following exposure to Bacopa monnieri extract (ayurvedic medicine rich in antioxidants) was also investigated. Abolishment of 24 h rhythms in physiological response (negative geotaxis), oxidative stress markers (protein carbonyl and thiobarbituric acid reactive substances) and antioxidants (superoxide dismutase, catalase, glutathione-S-transferase and reduced glutathione) were observed under oxidative stress. Furthermore, abolishment of per mRNA rhythm in H₂O₂ treated wild type flies and augmented susceptibility to oxidative stress in clock mutant (cry^b) flies connotes the role of circadian clock in reactive oxygen species (ROS) homeostasis. Significant reversibility of rhythms was noted following B. monnieri treatment in wild type flies than cry^b flies. Our experimental approach revealed a relationship involving oxidative stress and circadian clock in fruit fly and the utility of Drosophila model in screening putative antioxidative phytomedicines prior to their use in mammalian systems.     

Spaghetti,a homolog of human RPAP3 (RNA polymerase II‐associated protein 3), determines the fate of germline stem cells in Drosophila ovary

The Drosophila ovary provides an attractive model for studying the extrinsic or intrinsic factors that regulate the fate of germline stem cells (GSCs). Using this model, we identified a new role for Drosophila spaghetti (spag), encoding a homolog of human RNA polymerase II‐associated protein 3 (RPAP3), in regulating the fate of ovarian GSCs. Results from spag knockdown and genetic mosaic studies suggest that spag functions as an intrinsic factor for GSCs maintenance. Loss of Spag by, either spag RNAi or null mutation failed to trigger apoptosis in ovarian GSCs. Overexpression of spag led to negligible increases in the number of GSC/Cystoblast (CB) cells, suggesting that an excess of Spag is not sufficient to accelerate the proliferation of GSCs or delay CBs’ differentiation. Our study provides evidence supporting that spag is involved in adult stem cells maintenance. In addition, the RNAi screen results showed that knockdown of Hsp90, one of known Spag interacting partners, led to loss of ovarian GSCs in Drosophila. Heterozygous mutations in hsp90 (hsp90/+) dramatically accelerated the GSC loss in spag RNAi ovaries, suggesting that the Spag‐contained complex possibly plays an essential role in controlling the GSCs fate.     

Hofbauer-Buchner eyelet affects circadian photosensitivity and coordinates TIM and PER expression in Drosophila clock neurons

Extraretinal photoreception is a common input route for light resetting signals into the circadian clock of animals. In Drosophila melanogaster, substantial circadian light inputs are mediated via the blue light photoreceptor CRYPTOCHROME (CRY) expressed in clock neurons within the brain. The current model predicts that, upon light activation, CRY interacts with the clock proteins TIMELESS (TIM) and PERIOD (PER), thereby inducing their degradation, which in turn leads to a resetting of the molecular oscillations within the circadian clock. Here the authors investigate the function of another putative extraretinal circadian photoreceptor, the Hofbauer-Buchner eyelet (H-B eyelet), located between the retina and the medulla in the fly optic lobes. Blocking synaptic transmission between the H-B eyelet and its potential target cells, the ventral circadian pacemaker neurons, impaired the flies' ability to resynchronize their behavior under jet-lag conditions in the context of nonfunctional retinal photoreception and a mutation in the CRY-encoding gene. The same manipulation also affected synchronized expression of the clock proteins TIM and PER in different subsets of the clock neurons. This shows that synaptic communication between the H-B eyelet and clock neurons contributes to synchronization of molecular and behavioral rhythms and confirms that the H-B eyelet functions as a circadian photoreceptor. Blockage of synaptic transmission from the H-B eyelet in the presence of functional compound eyes and the absence of CRY also results in increased numbers of flies that are unable to synchronize to extreme photoperiods, supplying independent proof for the role of the H-B eyelet as a circadian photoreceptor.     

JNK regulates the photic response of the mammalian circadian clock

The posttranslational regulation of mammalian clock proteins has been assigned a time-keeping function, but seems to have more essential roles. Here we show that c-Jun N-terminal kinase (JNK), identified by inhibitor screening of BMAL1 phosphorylation at Ser 520/Thr 527/Ser 592, confers dynamic regulation on the clock. Knockdown of JNK1 and JNK2 abrogates BMAL1 phosphorylation and lengthens circadian period in fibroblasts. Mice deficient for neuron-specific isoform JNK3 have altered behavioural rhythms, with longer free-running period and compromised phase shifts to light. The locomotor rhythms are insensitive to intensity variance of constant light, deviating from Aschoff's rule. Thus, JNK regulates a core characteristic of the circadian clock by controlling the oscillation speed and the phase in response to light.     

Sexual Interactions Influence the Molecular Oscillations in DN1 Pacemaker Neurons in Drosophila melanogaster

Circadian rhythms can synchronize to environmental time cues, such as light, temperature, humidity, and food availability. Previous studies have suggested that these rhythms can also be entrained by social interactions. Here, we used Drosophila melanogaster as a model to study the influence of socio-sexual interactions on the circadian clock in behavior and pacemaker neurons. If two flies of opposite sex were paired and kept in a small space, the daily activity patterns of the two flies were clearly different from the sum of the activity of single male and female flies. Compared with single flies, paired flies were more active in the night and morning, were more active during females’ active phase, and were less active during males’ active phase. These behavioral phenotypes are related to courtship behavior, but not to the circadian clock. Nevertheless, in male-female pairs of flies with clocks at different speeds (wild-type and per ^S flies), clock protein cycling in the DN1 pacemaker neurons in the male brain were slightly influenced by their partners. These results suggest that sexual interactions between male-female couples can serve as a weak zeitgeber for the DN1 pacemaker neurons, but the effect is not sufficient to alter rhythms of behavioral activity.