Role of d-serine in intestinal ROS accumulation after sleep deprivation

Abstract

Prolonged sleep deprivation (SD) results in increased accumulation of reactive oxygen species (ROS) in gut, although the underlying mechanisms remain to be elucidated. This study identifies d-serine as a crucial regulator of gut ROS during SD. Knockdown of serine racemase (SR), the enzyme responsible for d-serine production, prevents the enhanced ROS buildup during SD in Drosophila. Gut enterocytes (ECs) respond to γ-aminobutyric acid (GABA) signaling by producing d-serine, which influences downstream N-methyl-d-aspartate receptor (NMDAR) activity and modulates sleep pressure. However, the continuous demand for sleep disrupts this feedback loop. Prolonged SD leads to increased levels of d-serine in the gut, an elevated pyruvate pool in ECs, enhanced mitochondrial oxidative phosphorylation, impaired lipid metabolism in peroxisomes, and the accumulation of harmful ROS. In conclusion, our findings illuminate the metabolic alterations and brain-gut communication pathways that may contribute to the increase in gut d-serine and subsequent ROS accumulation induced by SD.

Fig. 1.. The fruit flies with knockout of SR or DAAO exhibit similar levels of SD at 29°C.

(A) Schematic representation of the genetic manipulation of the SD model. (B) Sleep status of Canton-S, SRko, DAAOko, and CG12338ko at 22°C, with 2 days of sleep data consolidated. (C) Boxplot statistics for the sleep amounts shown in (B). *P < 0.05; ***P < 0.001; ****P < 0.0001. (D) Sleep status of UAS-TrpA1/+;11H05GAL4/+, UAS-TrpA1/+;60D04GAL4/+, UAS-TrpA1/ CG12338ko;11H05GAL4/+, UAS-TrpA1/CG12338ko;60D04GAL4/+, UAS-TrpA1/DAAOko;11H05GAL4/+, UAS-TrpA1/DAAOko;60D04GAL4/+, UAS-TrpA1/+;11H05GAL4/SRkoGAL4, and UAS-TrpA1/+;60D04GAL4/SRkoGAL4 flies at both 22° and 29°C, with consolidated sleep data from day 1 and day 2 at each temperature. (E) Boxplot statistics for the sleep amounts of 11H05GAL4- and 60D04GAL4-associated flies at 22° and 29°C. Significance labels are omitted. Data are presented as medians with the 25th and 75th percentiles. Statistical analysis was performed using the pairwise Wilcoxon test (C and E). Relevant statistical information can be found in data S1. h, hours; n.s., not significant.

Fig. 2.. Fruit flies with SR knockout show reduced accumulation of gut ROS and an extended life span during SD.

(A and B) Representative confocal images of the gut displaying oxidized DHE (DHE OX) in flies subjected to thermogenetic SD for 10 days. Scale bars, 100 μm; pseudo-color “glow” has been applied. (C and D) Quantification of DHE intensity and survival rates of flies undergoing thermogenetic SD. 60D04GAL4 and 11H05GAL4 lines were used a parental control. (E) Representative confocal images of the gut showing DHE OX in flies that experienced consistent mechanical SD for 7 days, compared to 50-day-old flies that did not undergo SD. Scale bars, 100 μm; pseudo-color “glow” has been applied. (F) Quantification of DHE intensity from the images in (E). Data are presented as means ± SEM. Statistical analyses were conducted using the Kruskal-Wallis test with Dunn’s multiple comparisons test (C, D, and F) or the log-rank test (C and D). n.s., not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001. Relevant statistical information can be found in data S1. a.u., arbitrary unit.

Fig. 3.. Increased levels of d-Ser lead to gut ROS accumulation during SD.

(A and B) The negative geotaxis assay indicates that flies lacking d-Ser after SD exhibit significantly greater overall muscle and neuronal functions. The numbers indicate the sample size. (C and D) Survival rates of thermogenetic sleep-deprived flies fed 5% sucrose food supplemented with l-Ser or d-Ser. (E) Reintroduction of SR expression in the gut, but not in the brain, induces gut ROS accumulation during SD. Genotypes:elavGAL4;SRko, SRko,UAS-SR, elavGAL4/+;SRko/SRko,UAS-SR, SRkoGAL4,elavGAL80, SRkoGAL4,elavGAL80/SRko,UAS-SR. a.u., arbitrary unit. (F and G) Relative mRNA expression levels of SR and CG12338 in sleep-deprived flies. Data are presented as means ± SEM. Statistical analyses were performed using the Kruskal-Wallis test with Dunn’s multiple comparisons test (A and B), a two-sample t test with Bonferroni-Dunn correction (F and G), the Mann-Whitney U test (E), or the log-rank test (C and D). n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Relevant statistical information can be found in data S1.

Fig. 4.. ECs activated by Cschrimson require gut d-Ser to induce ROS accumulation during the night phase.

(A) Representative DAPI and GFP fluorescent images of the gut from SD and non-SD SRkiGAL4>CaLexA flies. Scale bars, 100 μm. (B) Quantification of GFP intensity from the images in (A). (C) Representative fluorescent images of gut H2DCF in ATR-fed and non–ATR-fed flies illuminated on the head or abdomen during the time intervals ZT4-ZT8 or ZT12-ZT16. Scale bars, 100 μm; the pseudo-color “Green Fire Blue” has been applied. (D and E) Quantification of gut H2DCF intensity. Genotypes: UAS-Cschrimson,mcherry/+;SRkiGAL4/+, UAS-Cschrimson,mcherry/+;SRkoGAL4/SRko, UAS-Cschrimson,mcherry/+;SRkoGAL4/SRko,UAS-SR, UAS-Cschrimson,mcherry/+;SRkoGAL4,elav-GAL80/SRko, and UAS-Cschrimson,mcherry/+;SRkoGAL4,elav-GAL80/SRko,UAS-SR. Data are presented as means ± SEM. Statistical analyses were conducted using t tests with Bonferroni-Dunn correction (B) and the Mann-Whitney U test (D and E). n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Relevant statistical information can be found in data S1. a.u., arbitrary unit.

Fig. 5.. Gut GABA-B-R1 in ECs receives GABA signal input from dFB neurons.

(A) Schematic diagram of the RNAi screening aimed at identifying neurotransmitters released by R5 neurons that can extend the life span of sleep-deprived flies. TNT, tetanus toxin; ASTC, allatostatin C; SIFa, neuropeptide SIFamide; DH31, diuretic hormone 31; Gad1, glutamic acid decarboxylase 1; Trh, tryptophan hydroxylase; CHAT, choline acetyltransferase. (B) Statistical plots of survival days indicate that R5 neurotransmitters do not extend life span. Kir2.1, inwardly rectifying potassium channel; ASTA, allatostatin A; Mip, myoinhibiting peptide precursor. (C) Schematic diagram of the RNAi screening for neurotransmitters released by dFB neurons that can extend the life span of sleep-deprived flies. (D) Statistical plots of survival days show that only GABA from dFB neurons extends life span. (E) Schematic diagram of the RNAi screening for neurotransmitter receptors in ECs that can extend the life span of sleep-deprived flies. (F) Statistical plots of survival days demonstrate that RNAi targeting GABA, CHAT receptors, and SR can extend life span. AChR, acetylcholine receptor; SPR, sex peptide receptor; GABA, γ-aminobutyric acid; 5-HT, 5-hydroxytryptamine (serotonin); Dopa, dopamine. Data are presented as means ± SEM. Statistical analyses were performed using the log-rank test (B, D, and F). n.s., not significant; ****P < 0.0001. Relevant statistical and genotypes information can be found in data S1.

Fig. 6.. Downstream NMDAR1 receives gut d-Ser signals to alleviate sleep pressure.

(A) Representative confocal images of the gut displaying oxidized DHE (DHE ox) in wild-type Canton-S, NMDAR1 knockout (NMDAR1ko), and DAAO knockout;NMDAR1 knockout (DAAOko;NMDAR1ko) flies subjected to consistent mechanical SD for 7 days, as well as in 50-day-old flies that had not experienced SD. (B) Quantification of DHE intensity from (A). (C) Relative mRNA expression levels of SR and CG12338 in NMDAR1ko and DAAOko;NMDAR1ko flies subjected to consistent mechanical SD. (D) Negative geotaxis assay results indicate that sleep-deprived flies lacking NMDAR1 exhibit poorer overall muscle and neuronal function. Sample sizes are indicated numerically. The data for Canton-S are the same as shown in Fig. 3B. (E) Enhanced sleep during stimulation of the R72G06LexA driver also necessitates NMDAR1 in dFB neurons and SR. Genotypes: R72G06LexA/+;LexAop-TrpA1/23E10GAL4, R72G06LexA/LexAop-TrpA1;SRko/SRko, and R72G06LexA/UAS-NMDAR1-RNAi;LexAop-TrpA1/23E10GAL4. h, hours. (F) The schematic representation illustrates the potential brain-gut axis connections implicated in the regulation of SD-induced intestinal ROS accumulation by d-Ser. Data are presented as means ± SEM or as medians with the 25th and 75th percentiles. Statistical analyses were performed using the Kruskal-Wallis test with Dunn’s multiple comparisons test (B and C), t tests with Bonferroni-Dunn correction (D), and the pairwise Wilcoxon test (E). n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Relevant statistical information can be found in data S1.

Fig. 7.. Gut d-Ser influences pyruvate levels to enhance ROS accumulation.

(A) The bubble chart displays the top significant enrichment pathways for brain and gut samples, illustrating the interaction between SD and the knockdown of SR. NCAM1, Neural Cell Adhesion Molecule 1; SLC, solute carrier. (B) The interconnections of the enriched pathways and the metabolites identified in (A). The size of each circle corresponds to the significance of the enriched pathway, with differentially expressed molecules highlighted in blue. CoA, coenzyme A. (C) Simplified clustered heatmap showing changes in expression of gut metabolites hit in ASCA analysis of (11H05>TrpA1;SRkoGAL4/+ versus 11H05>TrpA1) group data. Differentially expressed molecules associated with lipid peroxide metabolism highlighted in red, and those associated with the mitochondrial TCA cycle highlighted in yellow. (D) Schematic diagram illustrating the mechanism by which gut d-Ser induces ROS accumulation in the gut. (E and F) Survival rates of thermogenetic sleep-deprived flies fed different concentrations of UK-5099. Data are presented as means ± SEM or as medians with the 25th and 75th percentiles. Statistical analyses were conducted using the log-rank test (E and F). n.s., not significant; *P < 0.05; **P < 0.01; ****P < 0.0001. Relevant statistical information can be found in data S1.