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

Abstract

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.

Keywords: Drosophila neuromuscular junction; Keap1; Nrf2; longevity; redox signaling; sleep; synaptic release.

Figure 1

Suppression of Kelch-like ECH associated protein 1 (Keap1) signaling extends life span and promotes negative geotaxis activity. (A) RNAi silencing of Keap1 prolongs life span, whereas CncC downregulation does not affect longevity (n = 60–98 flies, p < 0.0001). (B) Overexpression of CncC does not affect life span (n = 99–72 flies, p > 0.05). 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.

Figure 2

Upregulation of antioxidant signaling enhances spontaneous quantal release at the neuromuscular junction (NMJ) synapse. (A) Example recordings of spontaneous activity at larval NMJs. (B) 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 w¹¹¹⁸ 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].

Figure 3

Increased antioxidant environment allows for reduction of stimulated vesicular release while maintaining synaptic transmission. (A) Recordings of evoked synaptic currents at the larval NMJ. Graphs show means for evoked EJC (eEJC) amplitudes (B), quantal content (QC; C) and cumulative QC (D), QC and cumulative QC graphs of 50 Hz trains for genotypes indicated (E,F). eEJC amplitudes (G), QC (H) and cumulative QC (I) for treatments indicated. (J,K) Graphs of 50 Hz trains from w¹¹¹⁸ larvae and w¹¹¹⁸ larvae treated with GSH and DTT. (L) Paired-pulse ratios (PPR) were analyzed at 20 ms inter-spike intervals at NMJs from indicated genotypes and/or treatments [ANOVA used for comparison of RNAi expressing strains, Student’s t-test was used to compare elav × CncC with elav Ctrl, n—number of NMJs (from at least three larvae) indicated within bars]. (M) Mean crawling distances of different genotypes over a 10 min imaging period (n—number of larvae indicated within bars). One-way ANOVA with post hoc Tukey-Kramer was used for comparisons with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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].

Figure 5

The CncC/Keap1 signaling impacts on sleep and activity patterns. (A,B) Sleep duration (in min) was reduced following CncC OE and enhanced in CncC knock-down (KD) flies in the day time, during the night, CncC KD reduced sleep length. The overexpression of CncC caused fragmented sleep patterns in the day resulting in more but shorter sleep episodes in the night time (B), whereas CncC KD reduces sleep length with more episodes in the night; (C,D) Keap1 silencing reverses the CncC KD effect in the night but not in the day. (D) During the night, both CncC OE and KD induce similar changes resulting in more but shorter sleep episodes. (E–G) Total and night/day activities of genotypes. One-way ANOVA with post hoc Tukey-Kramer was used for comparisons with *p < 0.05, **p < 0.01, ***p < 0.001 [n—numbers: elav × W, 3M (RNAi Ctrl: 38), elav × CncC RNAi: 21, elav × Keap1 RNAi: 44, elav × W¹¹¹⁸: 32, W¹¹¹⁸ × CncC: 27, elav × CncC: 25].