Recurrent Sleep Fragmentation Induces Insulin and Neuroprotective Mechanisms in Middle-Aged Flies

Abstract

Lack of quality sleep increases central nervous system oxidative stress and impairs removal of neurotoxic soluble metabolites from brain parenchyma. During aging poor sleep quality, caused by sleep fragmentation, increases central nervous system cellular stress. Currently, it is not known how organisms offset age-related cytotoxic metabolite increases in order to safeguard neuronal survival. Furthermore, it is not understood how age and sleep fragmentation interact to affect oxidative stress protection pathways. We demonstrate sleep fragmentation increases systems that protect against oxidative damage and neuroprotective endoplasmic reticulum molecular chaperones, as well as neuronal insulin and dopaminergic expression in middle-aged Drosophila males. Interestingly, even after sleep recovery the expression of these genes was still upregulated in middle-aged flies. Finally, sleep fragmentation generates higher levels of reactive oxygen species (ROS) in middle-aged flies and after sleep recovery these levels remain significantly higher than in young flies. The fact that neuroprotective pathways remain upregulated in middle-aged flies beyond sleep fragmentation suggests it might represent a strong stressor for the brain during later life.

Keywords: Nrf2; dopamine; glucagon; insulin; metabolism; molecular chaperone; sleep.

FIGURE 1

Sleep fragmentation induces rebound sleep. (A,C) Averaged locomotor activity over a 24 h period of (A) 5–7 days old or (B) 25–27 days old males kept on either a 12:12 light dark cycle (gray line) or sleep fragmented (purple line). (C,D) Averaged sleep during a 30 min period of 24 h. (C) 5–7 days old or (D) 25–27 days old males on either a 12:12 light dark cycle (gray line) or sleep fragmented (purple line; n = 90). (E) Average activity comparing 30 min light-stimulus phase of sleep fragmented males with the same time periods for males kept on a 12:12 light dark cycle (n = 90; *P < 0.05, compared with controls, one-way ANOVA with Tukey’s post hoc test for multiple comparisons). Error bars indicate SEM.

FIGURE 2

Sleep fragmented flies sleep more than normally maintained flies. (A–D) Analysis of sleep behavior comparing flies on a normal 12:12 light:dark cycle with flies undergoing sleep fragmentation. (A) Total sleep during a 24 h period. (B) Average number of minutes flies slept during the 12 h light or dark period. (C) Average number of sleep bouts during the 12 h light or dark period. (D) Average sleep episode length during the 12 h light or dark period. (A–D: n = 60 flies. **P < 0.01, ***P < 0.005 compared with controls, A non-parametric Kruskal–Wallis test was performed). (E) qPCR was performed to determine if sleep fragmentation was able to induce an inflammatory response. Both AttacinB (AttB) and Drosomycin (Drs) antimicrobial genes where induced by sleep fragmentation. (All assays were repeated at least seven times. n = 25 male heads per treatment. **P < 0.01, ***P < 0.005 compared with controls, one-way ANOVA with Tukey’s post hoc test for multiple comparisons). In all graphs error bars indicate SEM.

FIGURE 3

Sleep fragmentation induces the oxidative stress pathway. (A,B) Either 5–7 or 25–27 days old control, sleep fragmented or males allowed to recover for 4 days after sleep fragmentation were used. (A) The expression of the cellular stress regulating KEAP1-NFE2L2 system was assessed. In Drosophila NFE2L2 is known as cap-n-collar (cnc). (B) Reactive oxygen species (ROS) levels in young and middle aged males. All assays were repeated at least seven times. (A: n = 25 male heads per treatment. B: n = 3 male heads per treatment; *P < 0.05, **P < 0.01 compared with controls, one-way ANOVA with Tukey’s post hoc test for multiple comparisons). In all graphs error bars indicate SEM.

FIGURE 4

Sleep fragmentation induces ER molecular chaperone and dopaminergic pathways. (A–D) Either 5–7 or 25–27 days old control, sleep fragmented or males allowed to recover for 4 days after sleep fragmentation were used. (A) The expression of endoplasmic reticulum molecular chaperones CaBP1, Crc, and ERp60 was assessed. (B) The expression of genes involved in dopamine production or signaling (ple, Vmat, and DAT) were assessed. (A,B: n = 25 male heads per treatment, *P < 0.05, **P < 0.01, ***P < 0.005 compared with controls, one-way ANOVA with Tukey’s post hoc test for multiple comparisons). (C) Brains from with young or middle-aged ple-GAL4;UAS-GFP males that were sleep fragmented for 4 days. Fluorescence indicates ple transcription in dopaminergic neurons. Presented as a Z-project (Z = 2 μm, 75 slices total Size bar = 200 μM). (D) Mean brain fluorescence intensity between young and middle-aged normal sleep and sleep fragmented groups. Normal slept young flies were set as 100%, represented as 1 on the graph. (D: n = 20 male heads per group, ***P < 0.05 compared with controls, one-way ANOVA with Tukey’s post hoc test for multiple comparisons). In all graphs error bars indicate SEM.

FIGURE 5

Sleep fragmentation induces the insulin pathway. (A) Relative expression levels of insulin-like peptides (Ilps). (B) Relative expression level of Insulin-like receptor (InR). (A,B) All assays were repeated at least seven times. Error bars indicate SEM. (For all genes, except Ilp6, n = 25 male heads per treatment; for Ilp6, n = 10 male whole bodies per treatment; *P < 0.05, **P < 0.01 compared with controls, one-way ANOVA with Tukey’s post hoc test for multiple comparisons).