Anaplastic Lymphoma Kinase Acts in the <i>Drosophila</i> Mushroom Body to Negatively Regulate Sleep

<div><p>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 (<i>Alk</i>), 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, <i>Alk</i> genetically interacts with <i>Neurofibromatosis 1</i> (<i>Nf1</i>) to regulate growth and associative learning in flies. We show that <i>Alk</i> mutants have increased sleep. Using a targeted RNAi screen we localized the negative effects of <i>Alk</i> on sleep to the mushroom body, a structure important for both sleep and memory. We also report that mutations in <i>Nf1</i> produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of <i>Alk</i>. Thus <i>Alk</i> and <i>Nf1</i> interact in both learning and sleep regulation, highlighting a common pathway in these two processes.</p></div

Homeostatic response to sleep loss in <i>Alk</i> mutants.

<p>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. <i>Iso31</i> and <i>Alk</i>^<i>ts</i> 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 <i>iso31</i> and <i>Alk</i>^ts flies. 2-tailed Student’s t-test shows no significant difference between <i>iso31</i> and <i>Alk</i>^<i>ts</i> flies. D) Sleep latency after deprivation on recovery day one. *p<0.05. N = 31 for iso31. N = 29 for <i>Alk</i>^<i>ts</i>.</p

<i>Alk</i> mutants have increased sleep.

<p>A) The restrictive temperature of 29°C reversibly increases sleep in <i>Alk</i>^<i>ts/1</i> mutants. The averaged sleep profiles, plotted as average amounts of sleep in every 30-minute period, are shown for <i>iso31</i> and <i>Alk</i>^<i>ts/1</i> 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 <i>Alk</i> 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 <i>iso31</i> 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 <i>Alk</i>^<i>ts/1</i> flies at the restrictive temperature. There is no difference between genotypes at 18°C (p = 0.4722), while at 29°C <i>Alk</i>^<i>ts/1</i> sleep significantly longer than <i>iso31</i> and <i>Alk</i>^<i>1/+</i> 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 <i>Alk</i> 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).</p

ALK functions in the mushroom body to inhibit sleep.

<p>A) MB-Gal80 eliminates mushroom body expression from 30Y, 386Y and c309 Gal4s. B) <i>Alk</i> 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 <i>post hoc</i> 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.</p

<i>Alk</i> is required in a subset of CNS neurons to regulate sleep.

<p>A) A targeted <i>Alk</i> 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 <i>Alk</i> RNAi compared to controls <i>UAS-Alk RNAi/+; Dcr2/+</i> (black bars) and <i>Gal4/+</i> (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 <i>UAS-Alk RNAi</i> control). Daily sleep was averaged over 3 days. One-way ANOVA and post hoc analysis were done to compare total sleep in the <i>Alk</i> RNAi-expressing group to <i>UAS-Alk RNAi</i> and <i>Gal4</i> 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 <i>Alk</i>^<i>ts</i> at the restrictive temperature can be rescued by expressing <i>Alk</i> in sub-regions of the brain. Genotypes for the four bars: <i>iso31</i> (white), <i>Alk</i>^<i>ts</i>, <i>UAS-Alk</i>/<i>Alk</i>^<i>ts</i>; <i>Tub-Gal80</i>^<i>ts</i>/+ (light grey), <i>Alk</i>^<i>ts</i><i>; Gal4</i>/+ (dark grey), <i>Alk</i>^<i>ts</i>, <i>UAS-Alk</i>/<i>Alk</i>^<i>ts</i>; <i>Gal4</i>/<i>tub-Gal80</i> (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 (<i>Alk</i>^<i>ts</i> controls) are significantly higher than the white bar (****p<0.0001), which is not indicated in the graph.</p

Circadian rhythmicity of Alk and Nf1 mutants.

<p>*Flies were raised and entrained at 18. Their activities were monitored in DD at 18°C for 4 days and at 29°C for another 4 days.</p><p>^†Flies were raised and tested at 25°C.</p><p>^‡<i>Alk</i>^<i>ts/1</i> and <i>Alk</i>^{<i>ts/Def</i>} flies are not viable when raised at 25°C.</p><p>Circadian rhythmicity of Alk and Nf1 mutants.</p

Sleep reduction in <i>Nf1</i> mutants.

<p>A) Averaged sleep profiles of control <i>iso31</i> (black) and <i>Nf1</i>^<i>P1/P2</i> (blue) mutant flies at 25°C. B) Average daily sleep for control <i>iso31</i>, <i>Nf1</i> mutants and <i>Nf1</i> flies with transgenic <i>Nf1</i> 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. <i>Nf1</i> transgenic expression in wild type background was done in a separate experiment and their sleep quantities were compared with the respective controls. C) <i>Nf1</i> 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.</p

<i>Alk</i> does not interact with <i>Nf1</i> to control circadian rhythms.

<p>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, <i>iso31</i> and <i>Alk</i>^<i>ts/1</i> flies manifest a phase shift in their activity rhythms. <i>Nf1</i>^<i>P1/P2</i> and <i>Alk</i>^<i>ts/1</i><i>;Nf1</i>^<i>P1/P2</i> double mutants are arrhythmic.</p

Inactivity of <i>Alk</i>^<i>ts</i> mutants is due to a prolonged sleep state.

<p> 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, <i>Alk</i> 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.</p

Coupling Pyrolysis and Gasification Processes for Methane-Rich Syngas Production: Fundamental Studies on Pyrolysis Behavior and Kinetics of a Calcium-Rich High-Volatile Bituminous Coal

The coupling pyrolysis and gasification (CPG) process in the fluidized bed reactor to produce methane-rich syngas is an attractive technology in efficient and clean utilization of coals. Pyrolysis plays a leading role in this technology and other relevant processes. Pyrolysis behavior, gaseous product evolution, and kinetics of a calcium-rich high-volatile bituminous coal were deeply investigated using thermogravimetry coupled with online Fourier transform infrared spectroscopy. The results showed that inherent minerals in the coal were mainly gypsum, calcite, and quartz. Except for SiO₂, CaO is the most abundant species in the coal ash. The first-stage mass loss of coal before 654 °C was attributed to functional group cleavage and aromatic ring condensation reactions, during which the relative content of CH₄ was the largest among the hydrocarbon gases. The second-stage mass loss was mainly caused by mineral (calcite and gypsum) decomposition. It has demonstrated that the major source of CH₄ formation was from cracking of aryl methyl rather than the long alkyl side chains on aromatics in the coal. Furthermore, pyrolysis of the coal could be divided into three kinetic stages based on the activation energy variation. The first and last stages for chemical bond cleavage and mineral decomposition in coal were both controlled by an internal diffusion mechanism. However, the second stage involving a condensation reaction followed a second-order reaction mechanism