Drosophila Nrf2/Keap1 Mediated Redox Signaling Supports Synaptic Function and Longevity and Impacts on Circadian Activity

Many neurodegenerative conditions and age-related neuropathologies are associated with increased levels of reactive oxygen species (ROS). The cap “n” collar (CncC) family of transcription factors is one of the major cellular system that fights oxidative insults, becoming activated in response to oxidative stress. This transcription factor signaling is conserved from metazoans to human and has a major developmental and disease-associated relevance. An important mammalian member of the CncC family is nuclear factor erythroid 2-related factor 2 (Nrf2) which has been studied in numerous cellular systems and represents an important target for drug discovery in different diseases. CncC is negatively regulated by Kelch-like ECH associated protein 1 (Keap1) and this interaction provides the basis for a homeostatic control of cellular antioxidant defense. We have utilized the Drosophila model system to investigate the roles of CncC signaling on longevity, neuronal function and circadian rhythm. Furthermore, we assessed the effects of CncC function on larvae and adult flies following exposure to stress. Our data reveal that constitutive overexpression of CncC modifies synaptic mechanisms that positively impact on neuronal function, and suppression of CncC inhibitor, Keap1, shows beneficial phenotypes on synaptic function and longevity. Moreover, supplementation of antioxidants mimics the effects of augmenting CncC signaling. Under stress conditions, lack of CncC signaling worsens survival rates and neuronal function whilst silencing Keap1 protects against stress-induced neuronal decline. Interestingly, overexpression and RNAi-mediated downregulation of CncC have differential effects on sleep patterns possibly via interactions with redox-sensitive circadian cycles. Thus, our data illustrate the important regulatory potential of CncC signaling in neuronal function and synaptic release affecting multiple aspects within the nervous system.


INTRODUCTION
One of the major cellular defense mechanisms against oxidative stress is mediated by the mammalian nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH associated protein 1 (Keap1) signaling cascade. The Nrf2/Keap1 pathway regulates gene expression of many cytoprotective and detoxifying enzymes, thus playing a pivotal role in maintaining cellular redox homeostasis. Nrf2 belongs to the cap ''n'' collar (CncC) subfamily of basic region leucine zipper transcription factors and its regulation and importance in cellular defense mechanisms has been studied in numerous physiological and pathological conditions.
Nrf2 plays a key role in neuronal resistance to oxidative stress mediated by reactive oxygen species (ROS) and glutamateinduced excitotoxicity (He et al., 2014). Balancing oxidative stress by up-regulation of Nrf2 antioxidant defense has been demonstrated to be effective in neurodegenerative disease treatment. Aging is one of the main risk factors for neurodegenerative conditions, but it is also closely associated with a loss of Nrf2 activity. Nrf2 and expression of downstream target genes are decreased in the substantia nigra of aged rats, with Nrf2 overexpression exerting a protective response to neurodegeneration (Habas et al., 2013), including in models of amyotrophic lateral sclerosis (ALS), stroke, Alzheimer's disease (AD) and Parkinson's disease (PD). Indeed, Nrf2 activation has been shown to alleviate neurodegenerative symptoms in a Drosophila model of PD (Barone et al., 2011). Furthermore, Nrf2-mediated neuroprotection is primarily conferred by astroglia both in vitro and in vivo (Liddell, 2017) and in AD patients, Nrf2 expression is decreased in both hippocampal neurons and astrocytes (Ramsey et al., 2007) indicating a strong involvement of Nrf2 signaling in neurodegeneration and neuronal function. Previous work has shown that activation of the Nrf2/Keap1 transcriptional pathways can protect hippocampal neurons from Aβ-induced neurodegeneration in an AD mouse model (Lipton et al., 2016) and rescue neuronal deficiencies in various models for PD (Johnson and Johnson, 2015), confirming a protective role in neuronal function with potential for therapeutic treatments. However, the exact targets and mechanisms of the antioxidant activities of Nrf2/Keap1 activation in the modulation of neuronal function are not fully understood.
One important characteristic of neurodegenerative diseases and aging is dysregulation of sleep patterns which has been reported across different species ranging from fly to human (De Lazzari et al., 2018;Vanderheyden et al., 2018). Cumulative evidence demonstrates a close connection between cellular circadian rhythm and redox systems. The circadian clock is involved in the regulation of ROS levels both in vivo and in vitro (Desvergne et al., 2014;Early et al., 2018). In mammals, the circadian clock orchestrates the activities of the antioxidant defense and oxidative stress response systems by mediating Nrf2 signaling. Two proteins involved in circadian rhythm, circadian locomotor output cycles kaput (Clock) and Brain and muscle arnt-like protein-1 (Bmal1) can positively regulate Nrf2 transcription, which in turn drives rhythmic oscillations of antioxidant genes (Xu et al., 2012;Pekovic-Vaughan et al., 2014). Conversely, the cellular redox state is critically important for the regulation of Bmal1 and Clock gene transcriptional activities (Ranieri et al., 2015).
The mechanism underlying the Nrf2 protective response remains obscure and given the limited understanding of Nrf2/Keap1 signaling on neuronal function we utilized the Drosophila model in this study to investigate the effects on aging, synapse function, and circadian activity. Drosophila Keap1 acts as a negative regulator of CncC (Itoh et al., 1999;Sykiotis and Bohmann, 2008;Pitoniak and Bohmann, 2015) and its silencing by RNAi leads to endogenous activation of CncC signaling with flies showing upregulation of the classical antioxidant response element cascade (Sykiotis and Bohmann, 2008), which increases their stress resistance. In particular, CncC activation results in enhanced transcription of the antioxidant and detoxifying enzyme glutathione S-transferase encoded by the Drosophila gstD1 gene (Sawicki et al., 2003;Sykiotis and Bohmann, 2008) which acts in a neuroprotective manner.
We manipulated neuronal antioxidant response ability by either overexpressing CncC or reducing the expression of CncC and Keap1 protein by RNAi. We then investigated longevity, activity, and circadian behavior in adult flies in addition to synaptic function at the larval neuromuscular junction (NMJ). The data show that constitutive overexpression of CncC has important impacts on synaptic release and survival with silencing of the CncC inhibitor, Keap1, inducing beneficial effects on survival and synaptic function. Importantly, application of the antioxidant compounds dithiothreitol (DTT) and glutathione (GSH) produced similar effects to those mediated by CncC overexpression or Keap1 silencing, suggesting that an antioxidant environment boosts synaptic function in a redox-specific manner.

Fly Husbandry
Flies were raised on standard maize media at 25 • C at a 12-h LD cycle. The elav-GAL4[C155] driver was obtained from the Bloomington Stock Center (Indiana, US). The UAS-RNAi lines [Keap1 (CG3962) and CncC (CG43286)] were purchased from the Vienna Drosophila Resource Centre (VDRC). The UAS-CncC line was kindly provided by Dirk Bohmann, University of Rochester, USA (Sykiotis and Bohmann, 2008;Pitoniak and Bohmann, 2015). The UAS/GAL4 bipartite expression system was utilized to drive pan-neuronal expression. The elav-GAL4 driver (female flies) and the UAS responder lines (male flies) were crossed to obtain offspring expressing the genes of interest. As a control for the RNAi strains, a line carrying an empty RNAi vector inserted in the AttP40 site was used and crossed to the elav-GAL4 driver (referred to as RNAi Ctrl). For CncC overexpressing (OE) lines, experimental lines were compared to control obtained by crossing the GAL4 (elav Ctrl) and UAS lines to w 1118 . The homozygote w 1118 line was used as a control in the pharmacology experiments.
Miniature excitatory junctional currents (mEJCs) were recorded in the presence of 0.5 µM tetrodotoxin (Tocris, UK). All synaptic responses were recorded from muscles with input resistances ≥4 MΩ, holding currents <4 nA at −60 mV and resting potentials more negative than −60 mV at 25 • C, as differences in recording temperature cause changes in glutamate receptor kinetics and amplitudes (Postlethwaite et al., 2007). Holding potentials were −60 mV. The extracellular HL-3 contained (in mM): 70 NaCl, 5 KCl, 20 MgCl 2 , 10 NaHCO 3 , 115 sucrose, 5 trehalose, 5 HEPES, and 1.5 CaCl 2 . Average single evoked EJC (eEJC) amplitudes (stimulus: 0.1 ms, 1-5 V) were based on the mean peak eEJC amplitude in response to 10 presynaptic stimuli (recorded at 0.2 Hz). Nerve stimulation was performed with an isolated stimulator (DS2A, Digitimer). All data were digitized at 10 kHz and for miniature recordings, 200-s recordings were analyzed to obtain mean mEJC amplitudes. The quantal content (QC) was estimated for each recording by calculating the ratio of eEJC amplitude/average mEJC amplitude, followed by averaging recordings across all NMJs for a given genotype. mEJC and eEJC recordings were off-line low-pass filtered at 500 Hz and 1 kHz, respectively. Materials were purchased from Sigma-Aldrich (UK).

Cumulative Postsynaptic Current Analysis
The apparent size of the RRP was probed by the method of cumulative eEJC amplitudes (Schneggenburger et al., 1999). Muscles were clamped to −60 mV and eEJC amplitudes during a stimulus train [50 Hz, 500 ms (of a 1-s train)] were calculated as the difference between peak and baseline before stimulus onset of a given eEJC. Receptor desensitization was not blocked as it did not affect eEJC amplitudes, because a comparison of the decay of the first and the last eEJC within a train did not reveal any significant difference in decay kinetics. The number of releaseready vesicles (N) was obtained by back extrapolating a line fit to the linear phase of the 500-ms cumulative eEJC plot (the last 200 ms of the train) to time zero by dividing the cumulative eEJC amplitude at time zero by the mean mEJC amplitude recorded in the same cell. To calculate the QC in the train, we used mean mEJC amplitudes measured before the train.

Heat Shock Protocol
For heat shock survival experiments, methods were adapted from Ishida et al. (2012). Briefly, male adult flies (aged 3-5 days) were transferred to vials containing a moistened filter pad to prevent dehydration. Vials were placed in a 37 • C water bath and live flies were counted every 30 min. In larval experiments, heat shock was induced using previously described methods (Robinson et al., 2017). Briefly, age-matched third instar larvae were incubated at 37 • C for 1 h and used for electrophysiology 24 h later. These experiments were repeated a minimum of three times for each genotype.

Circadian
Circadian activity and sleep analysis were performed as described previously (Ishida et al., 2012). Briefly, adult male flies (aged 3-5 days) were individually transferred into glass tubes containing food. Single tubes were then loaded into Drosophila Activity Monitor system (Trikinetics). Following a 2 days period of entrainment in incubators kept on a 12:12 light/dark regime at 25 • C, locomotor activity was recorded for the consecutive 5 days. Sleep behavior was analyzed by using pySolo software (Gilestro and Cirelli, 2009).

Survival
Groups of 10 newly emerged adult male flies were transferred to new vials containing food and deaths were scored daily. Flies were transferred to new food three times per week and otherwise left undisturbed. Cumulative survival curves are presented and compared using the Log-rank (Mantel-Cox) test.

Geotaxis
Rapid iterative negative geotaxis behavior was performed using methods outlined previously (Rhodenizer et al., 2008;Nichols et al., 2012). Briefly, age-matched adult male flies were collected and groups of 10 were transferred to a clear empty vial without anesthesia at weekly intervals. Tubes were transferred to a quiet room and flies acclimated for 15 min. Tubes were tapped three times on a bench and images were taken after 3 s using a digital camera. A minimum of five trials were conducted per session with an inter-trial interval of 1 min. Average height climbed per vial was calculated from images using Image J software. These experiments were repeated a minimum of three times per genotype.

Crawling Activity
Age-matched third instar male larvae (∼100-120 h) were selected, washed and placed onto a moist, food-free surface at a constant temperature of 20 • C. Crawling activities were imaged over 10 min using AnyMaze software v4.98 (Stoelting Co., Wood Dale, IL, USA) and data were analyzed off-line as reported previously (Robinson et al., 2014).

Statistics
Statistical analysis was performed with Prism 7 (Graphpad Software Inc., San Diego, CA, USA). Statistical tests were carried out using a one-way ANOVA test when applicable with a posteriori test (with Tukey's multiple comparisons) or unpaired Student's t-test [for comparisons between elav × CncC and elav × w 1118 (elav Ctrl)]. Data in figures are expressed as mean ± SEM where n is the number NMJs, flies or larvae as indicated, and significance is shown as * p < 0.05, * * p < 0.01, * * * p < 0.001, and * * * * p < 0.0001.

RESULTS
We used this well-characterized expression system and first assessed the effects of down-regulating either CncC or Keap1 protein expression on Drosophila life span as it has long been postulated that oxidative stress contributes to age-related neuronal dysfunction known as the free radical Data were compared using the Log-rank (Mantel-Cox) test. (C) Negative geotaxis performance declines with age in control flies (RNAi Ctrl at 21 days, dark gray), however, CncC RNAi expression induces a strong reduction in climbing activity at 7 days (black) with no further effects at older ages. RNAi silencing of Keap1 augments activity decline relative to CncC silencing at seven (black) and 14 (light gray) days (n-number of flies indicated within bars). Data denote mean ± SEM for all data comparisons in (C). One-way ANOVA with post hoc Tukey-Kramer was used for comparisons with * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001.
To further characterize the effects on survival and geotaxisdriven activity and their connection to neuronal health, we assessed neuronal function in more detail in electrophysiological experiments of the larval NMJ. This well-studied synapse allows direct assessment of neuronal health and synaptic function by recording single action potential-eEJC, spontaneous release events (mEJC), and total vesicular pool size release. Oxidative stress and aging have been related to compromised neuronal function and diminished synaptic release. However, to our knowledge, the direct effects of CncC/Keap1 signaling on synaptic release have not yet been documented (Besson et al., 2000;Fremeau et al., 2004;Escartin et al., 2011;Wu and Cooper, 2012;Cirillo et al., 2015;Ivannikov and Van Remmen, 2015).
To exclude the possibility that genetic manipulation caused Overexpression of CncC and silencing of Keap1 enhances the quantal size, illustrated in amplitude frequency plots for miniature excitatory junctional current (mEJC) amplitudes (left), cumulative amplitude frequency plots (middle) and mean bar graphs (right). (C) Exposure to antioxidants dithiothreitol (DTT) and glutathione (GSH) induced a strong increase in quantal size at NMJs of w 1118 larvae as illustrated in frequency plots for mEJC amplitudes (left), cumulative amplitude frequency plots (middle) and mean bar graphs (right). One-way ANOVA with post hoc Tukey-Kramer was used for comparisons with * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001 [n-number of NMJs (from at least three larvae) indicated within bars].
developmental changes which could interfere with our data interpretation, we again assessed the effects of pharmacological manipulation of redox signaling on release parameters of the synapse. Quantification of NMJ evoked responses and QC revealed similar changes using antioxidant supplementation as following genetic manipulations. DTT application for 45 min induced mild effects and GSH application for the same length led to strong effects on the three parameters [eEJC amplitude: w 1118 : 118 ± 5 nA, DTT: 140 ± 7 nA, GSH: 113 ± 4 nA, QC: w 1118 : 213 ± 25, DTT: 167 ± 12, GSH: 65 ± 3, cumulative QC: w 1118 : 441 ± 40, DTT: 247 ± 49, GSH: 63 ± 35, ANOVA, Figures 3G-K] compared to controls (w 1118 ), similar to the changes observed following genetically-induced increase in antioxidant potential suggesting an acute mechanism mediated by a reduction in basal oxidative stress levels. Importantly, CncC KD was without effects indicating that under these unchallenged conditions the deficiency in potential antioxidant capacity did not alter basal synaptic transmission. One important regulatory mechanism with the ability to modulate synaptic release is the control of synaptic release probabilities. The initial release probability can be adjusted in response to various mechanisms. We assessed potential effects on the initial vesicular release probability (p vr ) by measuring paired-pulse ratios (PPR) at a 20 ms inter-spike interval. Previously we found that the nitrergic regulation of synaptic release at the NMJ is mediated via a reduction of p vr (Robinson et al., 2014) as manifested in an increased PPR. Paired-pulse experiments at larval NMJs revealed a significant increase in PPR following genetic alterations of Keap1 expression suggesting that p vr is reduced at lower oxidative stress levels [RNAi Ctrl: 0.81 ± 0.05, CncC-RNAi: 0.92 ± 0.02, Keap1-RNAi: 0.99 ± 0.02 (ANOVA); CncC OE: 1.00 ± 0.05, elav Ctrl: 0.88 ± 0.11 (Student's t-test), Figure 3L]. However, following pharmacological modulation of redox signaling we detected an increase in PPR only after GSH application [w 1118 : 0.88 ± 0.04, DTT: 0.92 ± 0.04, GSH: 1.12 ± 0.05, ANOVA, Figure 3L]. Importantly, changes in frequency (f ) of spontaneous release indicates direct effects on vesicle fusion mediated by the soluble N-ethyl-maleimidesensitive fusion protein Attachment Protein Receptor (SNARE) and SNARE binding proteins. To investigate the modulation of these mechanisms, we measured mEJC frequencies. The results did not show any differences in f between larvae [RNAi Ctrl: 2.6 ± 0.4 s −1 , CncC-RNAi: 3.2 ± 0.3 s −1 , Keap1-RNAi: 3.4 ± 0.5 s −1 (ANOVA); CncC OE: 1.4 ± 0.2 s −1 , elav Ctrl: 3.14 ± 0.80 (Student's t-test); w 1118 : 2.0 ± 0.2 s −1 , GSH: 1.2 ± 0.2 s −1 , DTT: 3.4 ± 0.3 s −1 (ANOVA) p > 0.05] suggesting that vesicle fusion mechanisms per se are not modulated by changes in the redox level following genetic or pharmacological manipulations. Together, our data show that regulation of the redox environment can alter synaptic function with spontaneous FIGURE 4 | Silencing of Keap1 protects against stress-induced synaptic decline. Life spans were analyzed for indicated lines and survivorship was plotted over time. (A) Survival curves represent an average of three life-span trials (n = 80-106 flies). Data were compared using the Log-rank (Mantel-Cox) test, p < 0.0001). Synaptic function was analyzed showing eEJC amplitudes (B) QC (C), cumulative QC (D) and mEJC amplitudes (E) under control [no heat shock (no HS), gray] and heat shock challenged (24 h HS) conditions. Note that the bars in gray are repeats from Figures 2, 3 and comparisons were made for each genotype before and after HS using the unpaired Student's t-test with * p < 0.05, * * p < 0.01 [n-number of NMJs (from at least three larvae) indicated within bars].
release events being positively modulated in a low oxidative stress environment. We next wondered if the observed regulation of synaptic release could translate into changes of larval activity. To test larval activity, we assessed crawling distances of the different genotypes over a period of 10 min (Robinson et al., 2014). Since motoneuronal transmission during crawling activity is predominately related to single motoneuronal action potential-induced synaptic release, which corresponds to a single eEJC event, we would not expect major effects on larval activity. Indeed, neither activation nor suppression of CncC signaling affected larval crawling distances relative to controls [RNAi Ctrl: 0.5 ± 0.03 cm, CncC-RNAi: 0.4 ± 0.02 cm, Keap1-RNAi: 0.5 ± 0.02 cm, CncC OE: 0.4 ± 0.02 cm, elav × w 1118 : 0.5 ± 0.02 cm, w 1118 × CncC: 0.3 ± 0.02 cm, ANOVA, Figure 3M] but the data showed subtle differences between CncC-RNAi and Keap1-RNAi expressing larvae. Increased oxidative stress levels are caused by altered activities of thiol redox circuits that can result in impaired cell signaling and dysfunctional redox-control (Finkel, 2011). It is linked to several pathological processes including dysfunction of proteostasis and the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER), resulting in ER stress (Braakman and Hebert, 2013). As heat shock is involved in triggering ER stress and ROS signaling, we next wanted to test whether altered expression of CncC and Keap1 would affect Drosophila longevity and synapse function following heat shock challenge. We found that under continual heat stress-challenged conditions, KD of CncC drastically reduced longevity [median life span (in hours): RNAi Ctrl: 4.5, CncC-RNAi: 3, p < 0.0001, Log-rank (Mantel-Cox) test] which can be explained by lack of neuroprotective Nrf2 signaling (Ahmed et al., 2017), while expectedly, Keap1 silencing protected and led to increased survival compared to CncC KD [median life span (in hours): Keap1-RNAi: 4, p < 0.0001, Log-rank (Mantel-Cox) test, Figure 4A]. The neuroprotection mediated by CncC (Nrf2) activation was further assessed in heat shock challenged larvae in which we characterized synaptic responses at the NMJ. The most striking changes at the level of synapse physiology occurred in CncC KD larvae 24 h after a single 1 h of heat shock challenge in which eEJC amplitudes declined drastically. This physiological response was not due to changes in quantal size but rather due to a reduced quantal content [eEJC amplitude: RNAi Ctrl: 112 ± 9 nA, CncC-RNAi: 74 ± 4 nA, Keap1-RNAi: 103 ± 9 nA, QC: RNAi Ctrl: 200 ± 15, CncC-RNAi: 186 ± 33, Keap1-RNAi: 142 ± 15, mEJC amplitude: RNAi Ctrl: 0.56 ± 0.04 nA, CncC-RNAi: 0.54 ± 0.09 nA, Keap1-RNAi: 0.66 ± 0.03 nA, Figures 4B,C,E]. Importantly, control larvae also showed a reduction in QC following challenge with heat shock, with Keap1 KD, however, preventing further neuronal deterioration upon this challenge. These changes were consolidated by measuring the vesicle pool size as cumulative release following synaptic stimulation in trains at 50 Hz [cumulative QC: RNAi Ctrl: 398 ± 64, CncC-RNAi: 423 ± 75, Keap1-RNAi: 272 ± 30, Figure 4D]. The data suggest that following heat shock stimulation, the lack of CncC produces strong phenotypes with regards to longevity and synapse function which were partially observed in controls but abolished following Keap1 KD.
Many neurological conditions including AD and PD exhibit perturbations of the circadian system (sometimes prior to any motor symptoms or clinical manifestation of symptoms), and underlying pathways have been studied in various animal models (Videnovic et al., 2014). Light and temperature are the two most reliable environmental timing cues, referred to as Zeitgeber (ZT), for the resetting of circadian clocks (Pittendrigh, 1960;Buhr et al., 2010;Musiek et al., 2013;Tamaru et al., 2013). Notably, mRNA expression levels for Keap1a/b and Nrf2 vary significantly within 12 h (i.e., between ZT0 and ZT12) implicating their involvement in circadian redox regulation (Zheng et al., 2017). This prompted us to evaluate how changes in ROS levels would impact the circadian behavior of flies with reduced or augmented cellular antioxidant capacity in a 24-h light-dark (LD) cycle. To determine how the circadian system is affected by modulating ROS levels via CncC/Keap1 signaling, we measured activity and sleep pattern as an index of circadian behavior in flies with reduced or augmented cellular antioxidant capacity. Quantification of sleep episodes in the day and night should give the best overall picture of sleep behavior. We quantified the relative length and number of sleep episodes and found that in the light phase CncC OE reduces the length of sleep but enhances the number of sleep episodes, an effect that was reversed with silencing of CncC (Figures 5A,C). Unexpectedly, following Keap1 KD, sleep behavior was similar to CncC KD flies in this phase. In the scotophase (dark), the overexpression of CncC did not cause any change in sleep parameters which were similar to the controls (Figures 5B,D). Interestingly, the behavior of CncC KD flies in the dark phase was different from the one observed in the photophase (light), showing a decreased length of sleep episodes and an increase in the number of these episodes suggesting that driving redox levels in one direction has opposite effects on sleep depending on the time. Conversely, sleep behavior of Keap1 KD flies was more similar to that observed in the photophase (light), showing only a significant increase in the sleep length episodes (Figures 5C,D). Finally, we measured the total activity of adult flies and found that total activity was only reduced in CncC KD flies in comparison to controls and Keap1 KD (Figures 5E,F). Figure 5G separates the total activity profiles for the studied genotypes into day and night phases showing that CncC but also Keap1 KD reduced activity in the dark phase only. These data imply complex interactions between ROS signaling which causes differential effects over the circadian cycle.
In summary, our data provide new evidence of how regulation of the redox homeostasis via modulation of CncC/Keap1 signaling can modulate aging, synapse function and sleep behavior. Specifically, the suppression of Keap1 expression induced beneficial effects on survival and synapse function. Equally, overexpression of CncC and pharmacologically enhancing antioxidant signaling resulted in similar phenotypes, with increases in quantal release being a major result of lowered oxidative stress signaling. The changes in synaptic function can further impact redox-sensitive aspects of sleep behavior which is implicated in disease-associated defects of circadian rhythm in neurodegeneration.

DISCUSSION
Drosophila has been instrumental in studying synapse function but also modeling various neurodegenerative diseases, including polyglutamine expansion diseases, α-synuclein-linked PD, and other prionopathies and tauopathies (see review McGurk et al., 2015). We and others have previously shown that expression of huntingtin with polyglutamine expansions, mutant α-synuclein, Aβ 40/42 toxicity and prion-mediated pathology suppresses glutamatergic function at the NMJ and causes neurodegenerative phenotypes (Outeiro et al., 2007;Romero et al., 2008;Chakraborty et al., 2011;Steinert et al., 2012;Breda et al., 2015;Vicente Miranda et al., 2016;Fernandez-Funez et al., 2017;Martin-Peña et al., 2017). Furthermore, studies in fly have found that dysfunctional superoxide dismutase 1 (SOD1) activity associated with enhanced oxidative stress can impact upon synapse function. In particular, SOD1 mutant flies exhibit signs of neurodegeneration, locomotor deficits, and shortened life span (Sahinahin et al., 2017). The Drosophila NMJ specifically offers a unique model synapse to study regulatory mechanisms of vesicular release. However, the direct effects of the redox signaling mediated by the Nrf2/Keap1 cascade have not yet been fully assessed at the level of synapse function and whole animal behavior. Our data present new evidence of how CncC and Keap1 signaling modulates Drosophila longevity, synapse function, and larval and adult fly activities, including effects on circadian sleep patterns. Furthermore, we determined the protective effects of CncC/Keap1 activity under stresschallenged conditions.
Many neurodegenerative diseases are characterized by a slowly progressive loss of neurons. The etiology of these diseases has still not yet been fully elucidated, although elevated levels of oxidative stress have been suggested as one of the potential common factors. One disease, in particular, is associated with defects in the antioxidant system, with mutations in SOD1 being a strong contributor to ALS. Drosophila has been utilized to characterize the effects of ALS-relevant mutant proteins (Milton et al., 2011;Coyne et al., 2017;Kim et al., 2018) and induction of homeostatic neuronal plasticity can reverse ALS-induced degeneration at the NMJ (Kim et al., 2018).
Data indicate that the abnormal excitotoxic glutamate release in the spinal cord of pre-symptomatic ALS mice is mainly based on the increased size of the readily releasable pool of vesicles and release facilitation, supported by plastic changes of specific presynaptic mechanisms (Bonifacino et al., 2016). As ALS is characterized by enhanced cytotoxic oxidative stress, one could speculate that these conditions favor non-physiological release of neurotransmitter. In light of our data which show a strong reduction in the number of evoked quantal release events with an increased quantal size following augmented antioxidant signaling (GSH, Keap1-RNAi, CncC OE), we suggest that low oxidative stress balances release with a reduction in the number of energy-demanding vesicular release events and simultaneously increased quantal size to sustain physiological action potentialevoked synaptic responses. If one considers that a single vesicular release event requires around 60,000 ATP molecules at a glutamatergic synapse in the mammalian central nervous system (CNS), including neurotransmitter refilling, SNARE protein assemble/dissemble, ion pump activities (Attwell and Laughlin, 2001;Harris et al., 2012) and vesicular release represents the highest energy burden to the presynapse (Rangaraju et al., 2014), it is conceivable to suggest that low oxidative stress levels lead to advantageous low QC/high quantal size release parameters.
A great variety of factors controls the level of neurotransmitter within the vesicle and changes in vesicle filling thus have great potential to influence synaptic transmission with vesicular glutamate filling determining quantal size (Karunanithi et al., 2002;Wu et al., 2007;Huang and Trussell, 2014;Choudhury et al., 2016). Increases in Drosophila larval quantal size at the NMJ have been reported following >30 min of enhanced activity (Steinert et al., 2006). Conversely, high-frequency stimulation of the NMJ results in a strong decrease in quantal size (Doherty et al., 1984;Naves and Van der Kloot, 2001) illustrating the ability of this particular synapse which is under strong homeostatic control (Newman et al., 2017), but also others (see review Edwards, 2007), to modulate quantal release. It has been shown that for instance, overexpression of vesicular glutamate transporter (vGLUT) leads to larger quantal sizes and resulting reduced QC (Daniels et al., 2004). Further mechanisms include uptake of transmitter via the Na +dependent excitatory amino acid transporters (EAATs; Wang and Floor, 1998;Takayasu et al., 2005;Rose et al., 2018), glutamate recycling which includes the glutamine-glutamate cycle, and transmitter transport into the vesicle which involves the exchange of lumenal H + for cytoplasmic transmitter and hence depends on a H + electrochemical gradient, that is produced by the vesicular (H + )-vATPase (Cotter et al., 2015). In fact, it has been shown that DTT or reduced GSH reverse H 2 O 2 -induced inhibition of the vATPase, suggesting that the mechanism of its inhibition by H 2 O 2 involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of vATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus reduce the quantal size during episodes of oxidative stress (Wang and Floor, 1998).
EAAT transporters contain cysteine-associated sulfhydryl groups sensitive to free radical species. The actions of free radicals result in the formation of cysteine bridges, thereby inhibiting glutamate transport into the cells (Trotti et al., 1998), as demonstrated for superoxide anion, hydrogen peroxide, NO and peroxynitrite (Pogun et al., 1994;Volterra et al., 1994) or into vesicles by reducing vGLUT activities . Conversely, overexpression of SOD1 protected glutamate transporters from inhibition (Chen et al., 2000). Studies in SOD1 knock-out mice NMJs found reduced quantal size (equal to mEJC) following enhanced oxidative stress leading to weakening of the muscle (Ivannikov and Van Remmen, 2015). Conversely, by reducing oxidative stress in a mouse model of peripheral nerve injury, the authors found increases in expression of vGLUT (Cirillo et al., 2015), the predominate vGLUT responsible for vesicular glutamate filling at the Drosophila NMJ (Wu and Cooper, 2012) and mammalian CNS synapses (Fremeau et al., 2004). Previous studies confirmed that activation of the Nrf2 pathway led to upregulation of the neuronal EAAT3 in mice (Escartin et al., 2011). This transporter has a homologue in Drosophila (Besson et al., 2000) and its upregulation enhances antioxidant activity via increases in glutathione production in addition to allowing sustained presynaptic glutamate levels available for release.
Conceivably, modulation of redox levels might impact on any of the above mechanisms and regulate transmitter release in a negative or positive direction and our data describe how reducing redox stress, either genetically or pharmacologically, leads to enhanced quantal size. However, this increase in quantal size resulted in a reduction of QC, likely due to homeostatic feedback regulation of this highly plastic synapse (Frank, 2014;Li et al., 2018) and future studies will have to evaluate the specific mechanisms by which redox signaling can alter vesicular transmitter release.
The observed changes of neuronal function will have wide implications on animal behavior and together with reported redox-mediated regulation of circadian function, determined by Nrf2/CncC-Keap1 signaling, our data show further evidence how this cascade can influence circadian rhythm. Neurodegeneration causes abnormalities in sleep patterns, partially due to neuronal loss but likely also due to specific dysregulation of circadian circuits. We demonstrated that increases in CncC expression or its silencing results in an opposite alteration in sleep episode length in the day. However, both conditions resulted in similar effects on sleep length during the night time. Total 24-h activity following CncC overexpression was not altered, whereas CncC KD reduced overall activity. These observations are further complicated with the expression of Nrf2 falling under the transcriptional regulation of the Clock/Bmal1-complex (Xu et al., 2012;Pekovic-Vaughan et al., 2014). Clock/Bmal1-dependent Nrf2 regulation gives rise to diurnal patterns in Nrf2 signaling, which underlies the rhythmic expression of antioxidant and metabolic enzymes reported in different cellular systems (Xu et al., 2012;Wang et al., 2018;Ishii et al., 2019). In this context it has been reported that Nrf2 gain-and loss-of-function affect circadian gene expression and rhythmicity in mammalian cellular systems, indicating the coupling of Nrf2 and Clock and the role of Nrf2 to integrate cellular redox status into timekeeping (Wible et al., 2018).
Collectively, this study and previous work illustrate a key mechanistic link between circadian oscillations in redox balance and Clock gene expression rhythms. However, key questions on the complex bidirectional regulation of ROS by circadian activity and vice versa remain to be answered in future studies.