Regulation of Sleep and Circadian Rhythms by Metabolic Neuropeptides

The increasing prevalence of metabolic disease in modern society has accelerated our need to understand factors that may be contributing to its development. Both circadian disruption and sleep deprivation are associated with metabolic dysfunction. Thus, for my dissertation I sought to gain insight into this association by studying the genetic and neural basis of interactions between circadian rhythms, sleep and metabolism. The relative simplicity of fly neuroanatomy and physiology, vast array of available genetic tools, and conservation across many organisms, makes Drosophila melanogaster an ideal model to dissect complex interactions between physiological systems. Through our studies we identified a novel role for a molecule that regulates feeding behavior, Neuropeptide F (NPF), in the circadian system. We found that NPF drives circadian gene expression of the detoxification gene sex-specific enzyme 1 in a peripheral metabolic tissue, possibly to coordinate consumption of toxins with their removal. Our results support a role for NPF in synchronizing behavior and metabolism to ensure circadian coherence and promote survival. In addition, we studied the interaction between sleep and metabolism by evaluating whether alterations in sleep cause metabolic dysfunction or are the result of shared molecular pathways. The insect equivalent of norepinephrine, octopamine, promotes wake in flies by signaling through insulin-producing neurons. We determined that although sleep and metabolic neural circuitry intersect, the octopamine signaling pathway regulates sleep and metabolism independently. This dissertation highlights the great power of Drosophila as a model organism to investigate complex interactions between different biological systems

The Central Clock Neurons Regulate Lipid Storage in Drosophila

A proper balance of lipid breakdown and synthesis is essential for achieving energy homeostasis as alterations in either of these processes can lead to pathological states such as obesity. The regulation of lipid metabolism is quite complex with multiple signals integrated to control overall triglyceride levels in metabolic tissues. Based upon studies demonstrating effects of the circadian clock on metabolism, we sought to determine if the central clock cells in the Drosophila brain contribute to lipid levels in the fat body, the main nutrient storage organ of the fly. Here, we show that altering the function of the Drosophila central clock neurons leads to an increase in fat body triglycerides. We also show that although triglyceride levels are not affected by age, they are increased by expression of the amyloid-beta protein in central clock neurons. The effect on lipid storage seems to be independent of circadian clock output as changes in triglycerides are not always observed in genetic manipulations that result in altered locomotor rhythms. These data demonstrate that the activity of the central clock neurons is necessary for proper lipid storage

Circadian and feeding cues integrate to drive rhythms of physiology in Drosophila insulin-producing cells

Circadian clocks regulate much of behavior and physiology, but the mechanisms by which they do so remain poorly understood. While cyclic gene expression is thought to underlie metabolic rhythms, little is known about cycles in cellular physiology. We found that Drosophila insulin-producing cells (IPCs), which are located in the pars intercerebralis and lack an autonomous circadian clock, are functionally connected to the central circadian clock circuit via DN1 neurons. Insulin mediates circadian output by regulating the rhythmic expression of a metabolic gene (sxe2) in the fat body. Patch clamp electrophysiology reveals that IPCs display circadian clock-regulated daily rhythms in firing event frequency and bursting proportion under light:dark conditions. The activity of IPCs and the rhythmic expression of sxe2 are additionally regulated by feeding, as demonstrated by night feeding-induced changes in IPC firing characteristics and sxe2 levels in the fat body. These findings indicate circuit-level regulation of metabolism by clock cells in Drosophila and support a role for the pars intercerebralis in integrating circadian control of behavior and physiology

The effect of manipulating the PDF neurons on food consumption.

<p>Total food consumption over a 24-hour period of 4–7 day old <i>Pdf-Gal4/UAS-ClkΔ</i>; <i>Pdf-Gal4/+</i>, <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/UAS-Clk</i>RNAi, and <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/UAS-</i>Aβ42ArcM female flies and normalized to <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/+</i>controls. Each experiment was performed three times with at least 40 animals for each genotype. Values represent mean ± SEM. * p<0.05 assessed by Student's t-test compared to <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/+</i> controls.</p

The effects of age and neurodegeneration on fat body triglycerides.

<p>(A) Triglyceride/protein ratios of fat bodies dissected from 50–55 day old wild type females (old <i>iso³¹</i>) at ZT8 and compared to 4–7 day old wild type females (young <i>iso³¹</i>) at ZT8. (B) Triglyceride/protein ratios of fat bodies dissected from 4–7 day old <i>Pdf-Gal4/+</i>; <i>UAS-</i>Aβ42ArcM<i>/+</i> and <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/UAS-</i>Aβ42ArcM female flies and compared to <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/+</i> and <i>UAS-</i>Aβ42ArcM<i>/+</i>controls. Each experiment was performed at least three times and values represent mean ± SEM. The data in (A) were obtained from 36 animals and the data in (<b>B</b>) from greater than 100 animals. *, # p<0.05 assessed by one-way ANOVA followed by post-hoc Tukey test compared to <i>Pdf-Gal4</i> only and transgene only genotypes, respectively.</p

Fat body triglycerides fail to cycle and are unaffected in circadian clock mutants.

<p>(<b>A</b>) Triglyceride/protein ratios of fat bodies dissected from 4–7 day old female <i>iso³¹</i> (wildtype) flies at Zeitgeber time (ZT) 0, 4, 8, 12, 16, and 20. (<b>B</b>) Triglyceride/protein ratios of fat bodies dissected from 4–7 day old <i>Clk^Jrk</i>, <i>cyc⁰¹</i>, <i>tim⁰¹</i>, and <i>per⁰</i> females and compared to <i>iso³¹</i> controls. Each experiment was performed at least three times with greater than 65 animals assayed in total, and values represent mean ± SEM.</p

Activity rhythms in flies expressing ClkRNAi, Aβ42ArcM, tim, and dTRPA1 in PDF neurons.

<p>Activity rhythm data of 1–2 week old female flies.</p>#<p>Data were collected from flies that were housed at 27°C throughout the entire experiment.</p><p>*p<0.05 assessed by one-way ANOVA followed by post-hoc Tukey test compared to <i>Pdf-Gal4</i> only and <i>UAS-Clk</i>RNAi genotypes.</p

Expression of <i>tim</i> and dTRPA1 in the PDF neurons has no effect on fat body triglycerides.

<p>(A) Triglyceride/protein ratios of fat bodies dissected from 4–7 day old <i>Pdf-Gal4/UAS-tim</i> and <i>Pdf-Gal4/UAS-tim</i>; <i>Pdf-Gal4/+</i> female flies and compared to <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/+</i> controls. Animals from each genotype were dissected at ZT8 and 20 and the values from the two time points were averaged. (B) Triglyceride/protein ratios of fat bodies dissected from 7–10 day old <i>Pdf-Gal4/UAS-dTRPA1</i>; <i>Pdf-Gal4/+</i> female flies at ZT 0–2 and compared to <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/+</i> controls at ZT 0–2. Each experiment was performed at least three times and values represent mean ± SEM. The data in (A) were obtained from 120 animals and the data in (B) from 45 animals for each genotype.</p

Inhibiting <i>Clk</i> gene function in the PDF neurons results in increased fat body triglycerides.

<p>(A) Triglyceride/protein ratios of fat bodies dissected from 4–7 day old <i>Pdf-Gal4/UAS-ClkΔ</i> and <i>Pdf-Gal4/UAS-ClkΔ</i>; <i>Pdf-Gal4/+</i> female flies and compared to <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/+</i> and <i>UAS-ClkΔ/+</i> controls. (B) Triglyceride/protein ratios of fat bodies dissected from 4–7 day old <i>Pdf-Gal4/UAS-ClkΔ</i>; <i>Pdf-Gal4/+</i> male flies and compared to <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/+</i> controls. (C) Triglyceride/protein ratios of fat bodies dissected from 4–7 day old <i>Pdf-Gal4/+</i>; <i>UAS-Clk</i>RNAi<i>/+</i> and <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/UAS-Clk</i>RNAi female flies and compared to <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/+</i> and <i>UAS-Clk</i>RNAi<i>/+</i>controls. (D) Triglyceride/protein ratios of fat bodies dissected from 4–7 day old <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/UAS-Clk</i>RNAi male flies and compared to <i>Pdf-Gal4/+</i>; <i>Pdf-Gal4/+</i> controls. (E) Triglyceride/protein ratios of fat bodies dissected from 4–7 day old <i>y w</i>; <i>Pdf⁰¹</i> female flies and compared to <i>y w</i> controls. (A,C,E) Each experiment was performed at least three times with greater than 100 animals in total and values represent mean ± SEM. *, # p<0.05 assessed by one-way ANOVA followed by post-hoc Tukey test compared to <i>Pdf-Gal4</i> only and transgene only genotypes, respectively. (B,D) Each experiment was performed three times with greater than 40 animals in total, and the animals were dissected between ZT6-9. A representative experiment is shown and values represent mean ± SD. * p<0.05 assessed by Student's t-test.</p

Hidden alternative structures of proline isomerase essential for catalysis.

A long-standing challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a timescale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CYPA, also known as PPIA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme