Jan Konietzka

BACKGROUND: Ketone bodies are known to substitute for glucose as brain fuel when glucose availability is low. Ketogenic diets have been described as neuroprotective. Similar data have been reported for triheptanoin, a fatty oil and anaplerotic compound. In this study, we monitored the changes of energy metabolites in liver, blood, and brain after transient brain ischemia to test for ketone body formation induced by experimental stroke. METHODS AND RESULTS: Mice were fed a standard carbohydrate-rich diet or 2 fat-rich diets, 1 enriched in triheptanoin and 1 in soybean oil. Stroke was induced in mice by middle cerebral artery occlusion for 90 minutes, followed by reperfusion. Mice were sacrificed, and blood plasma and liver and brain homogenates were obtained. In 1 experiment, microdialysis was performed. Metabolites (eg glucose, β-hydroxybutyrate, citrate, succinate) were determined by gas chromatography-mass spectrometry. After 90 minutes of brain ischemia, β-hydroxybutyrate levels were dramatically increased in liver, blood, and brain microdialysate and brain homogenate, but only in mice fed fat-rich diets. Glucose levels were changed in the opposite manner in blood and brain. Reperfusion decreased β-hydroxybutyrate and increased glucose within 60 minutes. Stroke-induced ketogenesis was blocked by propranolol, a β-receptor antagonist. Citrate and succinate were moderately increased by fat-rich diets and unchanged after stroke. CONCLUSIONS: We conclude that brain ischemia induces the formation of β-hydroxybutyrate (ketogenesis) in the liver and the consumption of β-hydroxybutyrate in the brain. This effect seems to be mediated by β-adrenergic receptors.

Summary Sleep is induced by sleep-active neurons that depolarize at sleep onset to inhibit wake circuits. Sleep-active neurons are under the control of homeostatic and allostatic mechanisms that determine sleep need. However, little is known about the molecular and circuit mechanisms that translate sleep need into the depolarization of sleep-active neurons. During many conditions in C. elegans sleep induction requires a sleep-active neuron called RIS. Here, we defined the transcriptome of RIS to discover that genes of the Epidermal Growth Factor Receptor (EGFR) signaling pathway are expressed in RIS. With cellular stress, EGFR activates RIS, and RIS induces sleep. Activation of EGFR signaling in the ALA neuron has previously been suggested to promote sleep independently of RIS. Unexpectedly, we found that ALA activation promotes RIS depolarization. Our results suggest that ALA is a sedating neuron with two separable functions. (1) It inhibits specific wakefulness behaviors independently of RIS, (2) and it activates RIS to induce sleep. Whereas ALA plays a strong role in surviving cellular stress, surprisingly, RIS does not. In summary, EGFR signaling can induce sleep-active neuron depolarization by an indirect mechanism through activation of the sedating ALA neuron that acts upstream of the sleep-active RIS neuron as well as through a direct mechanism using EGFR signaling in RIS. Sedation rather than sleep appears to be important for increasing survival following cellular stress, suggesting that sedation and sleep play different roles in restoring health. Highlights - The transcriptome of the sleep-active RIS neuron reveals the presence of the EGFR signaling machinery - EGFR activates RIS directly upon cellular stress to induce sleep bouts - In parallel, EGFR activates RIS indirectly through the sedating ALA neuron - Sedation rather than sleep bouts support survival following cellular stress

Why do we sleep? This question is still unsolved, although sleep is such a fundamental behavioral state in all organism with a nervous system. Several physiological mechanisms, like memory consolidation, metabolic waste clearance, or immune system boosting, depend on sleep but none was sufficient to answer yet, why our consciousness has to shut off every night. The nematode and model organism Caenorhabditis elegans has a minimalistic nervous system of exactly 302 neurons. Still, it provides three different types of sleep, which are linked to either-or one of two sleep neurons. The sleep-active neuron RIS controls developmentally-regulated lethargus sleep and environment-stimulated L1 arrest sleep. Stress-induced sleep (SIS) depends on the interneuron ALA. The clear structure of the nervous system, next to the straightforward genetic accessibility of C. elegans, made it an easy choice to use the worms for exploring sleep on a molecular level. To investigate what defines RIS and ALA on the molecular level, I obtained different transcriptomes for both neurons. I got one transcriptome, which was based on RNA sequencing of fluorescence-activated cell sorted (FACS) RIS neurons. Additionally, Cao et al. (2018) used single-cell combinatorial indexing RNA sequencing to publish a data set of 42’035 single cell transcriptomes, spanning all C. elegans L2 cells. From this data set, clusters representing RIS and ALA could be identified and used for the generation of transcriptomes for both cells, respectively. The transcriptomes provided me with genes enriched in RIS, which were potentially important in sleep control in this neuron. I used mutated alleles of these genes for a behavioral sleep screen. A nonsense-allele of the invertebrate-type lysozyme ilys-4 and a gain-of-function allele of the epidermal growth factor receptor (EGFR) let-23 caused worms to sleep more in L1 arrest. Both were known to express in ALA, but I was able to confirm their additional expression in RIS via fluorescent reporters. I also showed the let- 23(gf) phenotype mainly depends on RIS. SIS was known to be mediated via LET-23 in ALA. I used genetic ablations of ALA and RIS, and a RIS-specific knock-out of let-23 to demonstrate that SIS is also highly vi Introduction dependent on LET-23 signaling in RIS. Calcium imaging revealed that ALA activates broadly over the time span of SIS, while RIS activity correlates with individual sleep bouts of SIS. This is likely mediated via EGF signaling in ALA and RIS, as overexpression of EGF activated both neurons and caused movement quiescence of the worms. Next, I used optogenetic manipulation to show that ALA is able to activate RIS. This may function to some extent via the ALA neuropeptides encoded by flp-24, as shown in an overexpression experiment. I could confirm that worms survival after cellular stress is affected by ALA-induced sedation, but discovered survival does not depend on the RIS-induced sleep bouts. In this thesis, I showed that SIS depends on EGF receptor signaling in RIS, besides the known pathway in ALA. RIS seems to be the major controller of sleep in the worm, as I now discovered that it is involved in all types of sleep in C. elegans. Furthermore, I demonstrated that stress-induced EGF receptor signaling acts parallel in ALA and RIS, which inherit different mechanistic properties and thus provide a discrete response. ALA sedates the worm, while RIS activity causes sleep bouts. This dual system allows the worm to fine-tune the behavioral response to cellular stress. Sedation and sleep representing distinct but interacting pathways in C. elegans might be a general principle, which also holds true in other organisms.