Anaplastic Lymphoma Kinase Acts in the Drosophila Mushroom Body to Negatively Regulate Sleep

Abstract

Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. We show that Alk mutants have increased sleep. Using a targeted RNAi screen we localized the negative effects of Alk on sleep to the mushroom body, a structure important for both sleep and memory. We also report that mutations in Nf1 produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes.

Fig 1. Alk mutants have increased sleep.

A) The restrictive temperature of 29°C reversibly increases sleep in Alk ^ts/1 mutants. The averaged sleep profiles, plotted as average amounts of sleep in every 30-minute period, are shown for iso31 and Alk ^ts/1 female flies. The white and the grey columns mark periods of day and night, respectively. N = 16. B) Quantification of average daily sleep of control flies and Alk mutants measured by single beam monitors. Total sleep at 18°C was calculated as the average of 3 days before the temperature shift to 29°C. Total sleep amounts at 29°C were calculated as the average of the 3 days following the temperature shift and are significantly different between genotypes (One-way ANOVA, p<0.0001). Asterisk* signifies difference from iso31 control. In this figure and all following figures, *, p<0.05; **,p<0.01; ****,p<0.0001; ns, not significantly different. Error bars are SEM. N = 13–16. C) Measurement by multi-beam monitors similarly revealed longer sleep in Alk ^ts/1 flies at the restrictive temperature. There is no difference between genotypes at 18°C (p = 0.4722), while at 29°C Alk ^ts/1 sleep significantly longer than iso31 and Alk ^1/+ flies. N = 15–16. D) Waking activity in the multi-beam monitors was measured at the restrictive temperature of 29°C and was defined as averaged number of beam crossings per minute during wake. N = 15–16. E) Normal negative geotaxis response in Alk mutants. The negative geotaxis response is measured as the percentage of flies climbing vertically >4 cm from the bottom of a vial within 10s of being tapped down. N = 5 (groups of 10 flies for each genotype).

Fig 2. Inactivity of Alk ^ts mutants is due to a prolonged sleep state.

To distinguish sleep from quiet wake, we subjected previously sleeping or awake flies to a mechanical stimulus at different times of day—ZT6, ZT20 and ZT22. Across all time points, two-way ANOVA found significant differences in the response to simulation between previous behavior states (p = 0.0071) but not between genotypes (p = 0.189). There was no interaction between behavior states and genotypes (p = 0.368). However, Alk flies were less arousable than wild type at ZT22, by Student’s t test. n = 4–5 trials of 26–32 flies of each genotype for all time points. The responses of previously awake flies were similar between the three time points and thus were pooled.

Fig 3. Homeostatic response to sleep loss in Alk mutants.

A) Sleep profiles for the baseline, deprivation and recovery day were plotted against each other to show sleep deprivation and sleep rebound. The experiment was conducted entirely at 29°C. Iso31 and Alk ^ts similarly show an increase in sleep in the morning following sleep deprivation. B) Time course of recovery of sleep lost following deprivation. C) Minutes of sleep recovered on the first and second recovery day were compared between iso31 and Alk ^ts flies. 2-tailed Student’s t-test shows no significant difference between iso31 and Alk ^ts flies. D) Sleep latency after deprivation on recovery day one. *p<0.05. N = 31 for iso31. N = 29 for Alk ^ts.

Fig 4. Alk is required in a subset of CNS neurons to regulate sleep.

A) A targeted Alk RNAi screen with CNS Gal4 drivers to identify regions where ALK acts to regulate sleep. The graph shows the percentage changes in total sleep as a result of expressing Alk RNAi compared to controls UAS-Alk RNAi/+; Dcr2/+ (black bars) and Gal4/+ (grey bars). The difference in sleep amount between the experimental group and each control group was divided by the amount of control sleep to calculate net percentage change. Bars represent pooled data from 2–4 experiments for each genotype and show means ±SEM (n = 8–58 for experimental groups and each Gal4 control group. N = 131 for the UAS-Alk RNAi control). Daily sleep was averaged over 3 days. One-way ANOVA and post hoc analysis were done to compare total sleep in the Alk RNAi-expressing group to UAS-Alk RNAi and Gal4 control groups. * indicates that total sleep of the RNAi expressing group is significantly different from those of both control group. B) The long sleep phenotype of Alk ^ts at the restrictive temperature can be rescued by expressing Alk in sub-regions of the brain. Genotypes for the four bars: iso31 (white), Alk ^ts, UAS-Alk/Alk ^ts; Tub-Gal80 ^ts/+ (light grey), Alk ^ts ; Gal4/+ (dark grey), Alk ^ts, UAS-Alk/Alk ^ts; Gal4/tub-Gal80 (green). The white and light grey controls are the same in these graphs. N = 21–46. Total sleep amounts were averaged for 3 days at 29°C. One-way ANOVA and post hoc Turkey’s test were performed for all pairwise comparisons. In all groups, grey bars (Alk ^ts controls) are significantly higher than the white bar (****p<0.0001), which is not indicated in the graph.

Fig 5. ALK functions in the mushroom body to inhibit sleep.

A) MB-Gal80 eliminates mushroom body expression from 30Y, 386Y and c309 Gal4s. B) Alk RNAi-induced sleep increase is suppressed by MB-Gal80. GAL4-MB represents combination of GAL4 and MB-Gal80. * above bars indicate significant differences from both UAS-Alk RNAi and MB-Gal80 controls. Brackets show comparisons between Gal4>Alk RNAi and Gal4-MB>Alk RNAi. ns, not significant, ***p<0.001; ****p<0.0001, by One-way ANOVA and Turkey’s post hoc analysis. n = 18–49. In combination with 30y, MB-Gal80 decreases sleep below control levels. With C309, MB-Gal80 further decreases sleep. UAS-RNAi and MB-gal80, and 386y-MB are not statistically different.

Fig 6. Sleep reduction in Nf1 mutants.

A) Averaged sleep profiles of control iso31 (black) and Nf1 ^P1/P2 (blue) mutant flies at 25°C. B) Average daily sleep for control iso31, Nf1 mutants and Nf1 flies with transgenic Nf1 gene rescue at 25°C. Comparisons were done with one-way ANOVA and post-hoc Turkey’s test. * indicates significant difference from iso31 control. Error bars are SEM. N = 5–16. Ns, not significant. ***p<0.001, ****p<0.0001. Nf1 transgenic expression in wild type background was done in a separate experiment and their sleep quantities were compared with the respective controls. C) Nf1 mutants exhibit nocturnal hyperactivity. Diurnal/nocturnal index was calculated as (total activity during the day) − (total activity during the night)/(total activity), averaged over a 3-d period per fly.

Fig 7. Nf1 mutations suppress the long-sleep phenotype of Alk mutants.

Bar graphs show total sleep for iso31 controls, Alk mutants, Nf1 ^P1/P2 mutants, and Alk; Nf1 double mutants. One-way ANOVA and Mann-Whitney post hoc analysis was performed to compare groups in each condition. In each graph, groups with the same alphabet label on top are not statistically different from each other; groups with different labels are statistically different (p<0.05). For experiments at 18°C and 25°C, n = 5–16. For 29°C experiment, n = 10–37. All flies were raised at 18°C. Activities were monitored at 18°C, 25°C and 29°C in independent experiments.

Fig 8. Alk does not interact with Nf1 to control circadian rhythms.

Representative activity graphs for each genotype are shown with activity double-plotted (2 day/night cycles). Gray and black bars at the top indicate subjective days and nights. Flies were raised and entrained at 18°C, monitored for 4 days in constant darkness at 18°C and then at 29°C for 4 days. Upon the temperature shift, iso31 and Alk ^ts/1 flies manifest a phase shift in their activity rhythms. Nf1 ^P1/P2 and Alk ^ts/1 ;Nf1 ^P1/P2 double mutants are arrhythmic.