Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila

Kohlschutter-Tönz syndrome (KTS) is a rare genetic disorder with neurological dysfunctions including seizure and intellectual impairment. Mutations at the Rogdi locus have been linked to development of KTS, yet the underlying mechanisms remain elusive. Here we demonstrate that a Drosophila homolog of Rogdi acts as a novel sleep-promoting factor by supporting a specific subset of gamma-aminobutyric acid (GABA) transmission. Rogdi mutant flies displayed insomnia-like behaviors accompanied by sleep fragmentation and delay in sleep initiation. The sleep suppression phenotypes were rescued by sustaining GABAergic transmission primarily via metabotropic GABA receptors or by blocking wake-promoting dopaminergic pathways. Transgenic rescue further mapped GABAergic neurons as a cell-autonomous locus important for Rogdi-dependent sleep, implying metabotropic GABA transmission upstream of the dopaminergic inhibition of sleep. Consistently, an agonist specific to metabotropic but not ionotropic GABA receptors titrated the wake-promoting effects of dopaminergic neuron excitation. Taken together, these data provide the first genetic evidence that implicates Rogdi in sleep regulation via GABAergic control of dopaminergic signaling. Given the strong relevance of GABA to epilepsy, we propose that similar mechanisms might underlie the neural pathogenesis of Rogdi-associated KTS.

transcription factors 8 . Nonetheless, few or no studies have demonstrated the biological activity of Rogdi 9 and genetic models for Rogdi homologs have not been reported yet. Therefore, how Rogdi exerts its physiological roles particularly in the central nervous system and how its mutation leads to the development of KTS are largely unknown.
In the course of our genetic studies to elucidate genes and regulatory pathways involved in sleep behaviors, we identified novel sleep mutant alleles in the Drosophila Rogdi gene. Here, we employed the sleep-promoting effects of Rogdi as a readout of its neural function and demonstrated that Rogdi acts cell-autonomously in GABAergic neurons to enhance metabotropic GABA transmission and thereby sustain sleep. In addition, dopaminergic rescue of Rogdi mutant sleep revealed a novel sleep-regulatory mechanism that functionally links a specific subset of sleep-promoting GABAergic neurons to a wake-promoting dopaminergic pathway. Since epilepsy, a well penetrated phenotype in KTS patients, implicates GABAergic transmission [10][11][12] and sleep disorders [13][14][15][16] , our findings provide an important genetic clue to understanding the molecular and neural pathogenesis of KTS.

Loss-of-function Mutations in Rogdi Suppress Sleep Behaviors in Drosophila.
Sleep is physiologically essential for animals. Genetic models for sleep and sleep-related disorders have been established in multiple species in order to understand why we sleep and how sleep homeostasis occurs [17][18][19] . In addition, sleep-relevant genes have been successfully established in Drosophila sleep models by isolating novel sleep mutants and characterizing their sleep-modulatory effects [20][21][22][23][24][25] . We took a similar approach to identify a transgenic fly that harbors a genomic insertion of a transposable P element and displays insomnia-like sleep behaviors in 12-hour light: 12-hour dark (LD) cycles (Fig. 1). Sequence mapping of the transgene revealed insertion of the P element in the third intron at the Rogdi locus (Rogdi P1 ) (Fig. 1a). We further excised the Rogdi P1 insertion to generate a ~1 kb genomic deletion allele (Rogdi del ) that caused an in-frame deletion of amino acid residues 16-83 in the gene product. Mutant flies homozygous for either the Rogdi insertion or the deletion allele barely expressed ROGDI proteins in head extracts as assessed by immunoblotting with polyclonal anti-ROGDI antibody (Fig. 1b,  Supplementary Fig. 1). To determine if the in-frame deletion caused by Rogdi del allele could give rise to mutant ROGDI proteins, we overexpressed the cDNAs corresponding to either wild-type or Rogdi del allele in Drosophila S2 cells and examined their protein products using an epitope-tag. Indeed, wild-type and ROGDI del proteins were comparably detectable in immunoblots of total cell extracts but the mutant ROGDI del proteins had much shorter half-life (~2.6 hours) than wild-type (~49.5 hours) (Fig. 1c, Supplementary Fig. 2). Moreover, ROGDI del proteins were localized to distinct, cytoplasmic inclusions ( Supplementary Fig. 3a). This subcellular localization contrasted with that of wild-type ROGDI proteins which were evenly distributed in both nucleus and cytoplasm of S2 cells or adult fly neurons ( Supplementary Fig. 3b). Collectively, these data indicate that the Rogdi del allele substantially affects the proteostasis of ROGDI proteins in general (See Discussion).
Importantly, the daily amount of sleep was substantially decreased in flies homozygous or trans-heterozygous for the mutant Rogdi alleles (Fig. 1d,e). The two mutant alleles led to distinct sleep profiles, likely due to the difference in their allelic nature. The sleep suppression phenotype was accompanied by sleep fragmentation: the average sleep bout length (ABL) was shorter in Rogdi mutants than in control flies (Fig. 1e). In addition, Rogdi mutants took longer to initiate their first sleep bout after lights off when looking at the sleep latency. Sleep fragmentation was also evident in female flies homozygous for the Rogdi deletion ( Supplementary Fig. 4). However, Rogdi mutant phenotypes regarding total sleep amount and sleep latency were observed in virgins but not in mated females, possibly indicating that reproduction and/or neural processes relevant to post-mating responses might modulate Rogdi effects on sleep in female flies 26,27 . Nonetheless, these data suggest that Rogdi is a novel sleep-promoting gene important for both sleep initiation and maintenance.

Insomnia-like Behaviors in Rogdi Mutants Do Not Implicate Deficits in Circadian Rhythms or
Sleep Homeostasis. Circadian rhythms and sleep homeostasis have long been considered as two essential processes that shape sleep behaviors 28 . To understand how these two factors are implicated in Rogdi-dependent sleep, we examined Rogdi mutant sleep in different environmental or genetic conditions. The sleep suppression by Rogdi mutation persisted either in constant darkness or in a genetic background that harbored a hemizygous loss-of-function mutant allele of the circadian clock gene period 29,30 (Fig. 2). Since the clock-less per mutants do not display daily oscillations in clock gene expression and locomotor behaviors, these data imply that Rogdi effects on sleep require neither LD cycles nor the functionality of circadian rhythms.
To examine whether Rogdi contributes to sleep homeostasis, we employed a mechanical sleep-deprivation protocol 31 . We optimized the visibility of homeostatic sleep regulation by applying 9 hours (rather than overnight) of mechanical stimulus starting from lights-off in the LD cycle. Sleep rebound was then measured for the last 3 hours in the D phase so that lights-on would not mask the compensatory increase in sleep drive after sleep deprivation. Under these conditions, Rogdi mutants displayed higher sleep rebound than control flies as assessed by the percentage of sleep gain during the recovery period (Fig. 3). While we do not exclude the possibility that longer baseline sleep in control flies and possible ceiling effects could have underestimated their relative sleep rebound, these data imply that Rogdi suppresses homeostatic sleep gain after sleep loss. In fact, this phenotype contrasts with sleep-promoting effects of Rogdi on baseline sleep but is consistent with a previous observation that sleep-regulatory pathways for baseline and recovery sleep could be genetically separable (See Discussion) 32 .
A Wake-promoting Dopaminergic Pathway Mediates Rogdi Effects on Sleep. To elucidate the neural basis underlying the wake-promoting effects of Rogdi mutation, we first examined whether Rogdi mutants could be rescued by pharmacological treatment targeting specific neurotransmitter pathways. Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of the neurotransmitter, dopamine (DA) (Fig. 4a). Dopaminergic signaling pathways have been well established as having wake-promoting effects in both Drosophila and mammals 21,33,34 . In particular, a subset of TH-expressing dopaminergic neurons and their downstream postsynaptic target (D1-like DA receptor-expressing cells in the dorsal fan-shaped body) constitute a neural circuit that is important for promoting wakefulness in Drosophila [35][36][37] . Importantly, we found that oral administration of a sub-dose of the TH inhibitor alpha-methyl-p-tyrosine (AMPT) fully rescued the total sleep amount in Rogdi mutants (Fig. 4b). AMPT also significantly lengthened ABL in Rogdi mutants but not in control flies, indicating that AMPT treatment substantially restored sleep consolidation in Rogdi mutants. Conversely, L-DOPA administration shortened the total sleep amount and ABL in control flies but not in Rogdi mutants (Fig. 4c), indicating that Rogdi mutants are To further validate the implication of dopaminergic transmission in Rogdi-dependent sleep regulation, we examined if genetic disruption in the DA pathway could mask Rogdi effects on baseline sleep. Transgenic expression of tetanus toxin light chain (TNT) in TH-expressing neurons could block neurotransmission specifically at the dopaminergic synapses. This genetic manipulation indeed suppressed the wake-promoting effects of Rogdi mutation (Fig. 4d, Supplementary Fig. 6). By contrast, synaptic blockade of the wake-promoting octopaminergic  Supplementary Fig. 7). Collectively, our pharmacological and genetic data demonstrate that the wake-promoting DA pathway mediates the sleep suppression observed in Rogdi mutants.

GABAergic Transmission via Metabotropic GABA Receptors Supports Rogdi-dependent
Sleep. Gamma-aminobutyric acid (GABA) is known to be involved in sleep regulation and its sleep-promoting effects are well conserved between flies and mammals 17 . We thus assessed possible roles of the inhibitory neurotransmitter GABA in the wake-promoting effects of Rogdi mutation (Fig. 5a). Mitochondrial GABA transaminase (GABA-T) metabolizes GABA into succinic semialdehyde to suppress GABAergic transmission 38 . A low dose of the GABA-T inhibitor, ethanolamine O-sulfate (EOS) modestly elevated sleep amount in control flies, whereas it fully rescued sleep latency and substantially increased sleep amount in Rogdi mutants (Fig. 5b). Since GABA-T expressed in glial cells has been implicated in promoting wakefulness 38 , we next asked if elevated GABA levels in the synaptic clefts or glia could rescue Rogdi mutant sleep. Synaptic GABA levels are lowered by GABA transporter (GAT) that translocates extracellular GABA back to presynaptic neurons and glial cells, thereby restraining GABAergic transmission 39 . We found that the short sleep phenotypes in Rogdi mutants were also rescued by the GAT inhibitor, DL-2,4-diaminobutyric acid (DABA) (Fig. 5c). These data support that the pharmacological elevation of GABAergic transmission could mask the wake-promoting effects of Rogdi mutation, suggesting that deficits in the sleep-promoting GABAergic pathway might be responsible for the sleep phenotypes in Rogdi mutants.
To independently validate this hypothesis, we examined the sleep-promoting effects of GABA receptor agonists 40,41 . Oral administration of 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP), an agonist of ionotropic GABA receptors, promoted sleep quantity comparably in control and Rogdi mutant flies while it shortened sleep latency in Rogdi mutants (Fig. 5d). By contrast, a low dose of the metabotropic GABA receptor agonist SKF-97541 affected ABL very modestly in control flies while it fully restored normal sleep behaviors in Rogdi mutants. These data suggest that sleep-promoting GABAergic transmission via metabotropic GABA receptors might be prominently compromised by Rogdi mutations. Taken together, our pharmacological and genetic evidence demonstrates that Rogdi-dependent sleep regulation involves two opposing effects of the wake-promoting DA and sleep-promoting GABA pathways.

Rogdi in GABAergic Neurons Acts Upstream of a Wake-promoting Dopaminergic Pathway to
Promote Sleep. We next asked if Rogdi expression in either dopaminergic or GABAergic neurons is sufficient to sustain sleep behaviors. Transgenic ROGDI expression in TH-expressing dopaminergic neurons failed to rescue the short sleep phenotypes in Rogdi mutants (Fig. 6a, Supplementary Fig. 8a). However, ROGDI expression in GABAergic neurons, driven by the glutamate decarboxylase 1 (GAD1)-Gal4 transgene 42 , partially but significantly rescued sleep behaviors in Rogdi mutants (Fig. 6b, Supplementary Fig. 8b). To confirm that Rogdi is endogenously expressed in GABAergic neurons, we examined Rogdi-expressing neurons in adult fly brains. Since our anti-ROGDI antibodies were limited to the detection of endogenous ROGDI proteins in immunoblots, we employed an enhancer trap line that expresses a transgenic Gal4 driver from the Rogdi locus (Rogdi-Gal4), likely reflecting the endogenous expression of ROGDI. Rogdi-Gal4 was broadly expressed in both anterior and posterior regions in the whole-mount brain when its spatial expression was indirectly visualized by green fluorescent proteins (Fig. 6c, Supplementary Fig. 9). Co-immunostaining with anti-GABA antibodies further revealed GABA-positive neurons among other Rogdi-expressing neurons, particularly in the anterior brain.
Given that Rogdi effects on sleep were suppressed by pharmacological and genetic blockade of DA transmission, we reasoned that the wake-promoting DA pathway may act downstream of the Rogdi-expressing GABAergic neurons via metabotropic GABA receptors. To validate this hypothesis, we first examined if RNA interference-mediated depletion of metabotropic GABA receptors in TH-expressing dopaminergic neurons could phenocopy Rogdi mutation. However, none of the RNAi lines caused short sleep phenotypes comparable to those in Rogdi mutants (Supplementary Fig. 10). While lack of RNAi phenotypes does not necessarily verify the absence of the sleep-promoting metabotropic GABA receptors in dopaminergic neurons, we could not rule out the possibility that the inhibitory GABAergic input to DA pathway might be mediated indirectly by interneurons. To further validate our original hypothesis above, we examined if GABA receptor agonists could suppress short   (Fig. 6d,e) although phenotypic difference between Rogdi mutation and constitutive excitation of TH neurons was observed in sleep latency ( Supplementary Fig. 11). Nonetheless, the short sleep phenotype was partially but significantly rescued by oral administration of SKF-97541 whereas sleep-promoting effects of THIP were comparable between TH > NaChBac flies and their heterozygous controls. These data suggest that GABAergic transmission via metabotropic GABA receptors could titrate the wake-promoting effects of the DA pathway regardless of their dopaminergic expression as consistent with our model for Rogdi-dependent sleep regulation.

Insomnia-like Behaviors in Rogdi Mutants Are Sensitized to Select Anti-epileptic Drugs. GABA
has long been implicated in many aspects of neural dysfunction including seizures 10-12 , a well-penetrated neurological phenotype in KTS patients. Moreover, sleep deficits frequently accompany epilepsy [13][14][15][16] , and their severity is positively correlated in human epileptic patients and animal models [44][45][46] . In fact, sleep deprivation can cause or even aggravate epileptic seizures [46][47][48] . Not surprisingly, many anti-epileptic drugs (AEDs), including those promoting GABAergic transmission, also modulate sleep behaviors 15 . We therefore asked if GABA-relevant AEDs could restore sleep behaviors in Rogdi mutants.
Gabapentin and valproate are two AEDs that enhance GABAergic transmission and have been shown to ameliorate seizure susceptibility in Drosophila 47, 49, 50 . We found that oral administration of valproate but not gabapentin fully rescued Rogdi mutant phenotypes in total sleep amount and sleep latency at a dosage showing negligible effects in control flies (Fig. 7). In contrast, the short sleep phenotypes in Rogdi mutants were aggravated by anticonvulsant carbamazepine (CBZ). A previous study demonstrated that CBZ rather suppresses sleep behaviors in Drosophila and its wake-promoting effects are, in part, attributable to increased sleep latency by the desensitization of Resistant to dieldrin, an ionotropic GABA receptor 51 . We note, however, that these AEDs could modulate neural activities in a GABA-independent manner. For instance, CBZ is known as a blocker of voltage-gated sodium channels 52 whereas mechanisms underlying anticonvulsant effects of gabapentin and valproate still remain elusive. Nonetheless, these data indicate that Rogdi mutant sleep is sensitized to select but not all AEDs relevant to GABAergic transmission and possibly correlate with the observation that seizures in Rogdi-associated KTS patients are often resistant to anti-epileptic drugs 53 .

Discussion
Modeling of neurological diseases and disease-relevant genes has greatly advanced our understanding of the fundamental principles that underlie disease pathogenesis as well as brain function. Here, we established the first genetic model of the KTS-associated disease gene Rogdi to demonstrate that Rogdi functions as a novel sleep-promoting factor in GABAergic neurons by promoting GABA transmission. While GABA-dependent sleep regulation via ionotropic GABA receptors have been well documented in Drosophila 41, 51, 54-56 , our data suggest that GABAergic transmission via metabotropic GABA receptors might be primarily compromised by Rogdi mutation. Furthermore, we identified the wake-promoting DA pathway as a neural locus downstream of Rogdi-dependent GABA signaling given that Rogdi mutant sleep could be rescued by pharmacological or genetic manipulation of dopaminergic transmission. This sleep-regulatory pathway was further supported by our observation that wake-promoting effects of TH-expressing dopaminergic neurons could be selectively titrated by an agonist of metabotropic GABA receptors. Rogdi thus defines a novel pathway coupling these two neurotransmitters to promote baseline sleep in Drosophila as exemplified in other behavioral paradigms across species [57][58][59] . On the other hand, Rogdi-dependent GABA transmission might have inhibitory effects on a sleep-promoting neural pathway for sleep homeostasis 60,61 to suppress recovery sleep after sleep loss.
What is the molecular basis by which Rogdi supports GABAergic transmission and promotes sleep? A possible role of ROGDI as a transcription factor has been suggested by the nuclear localization of human ROGDI protein, particularly in the nuclear envelope of blood mononuclear cells and dermal fibroblasts 2 , and by the conservation of a putatively dimerizing leucine zipper (ZIP) motif among ROGDI homologs 5 . Several lines of evidence, however, argue against this possibility. bZIP transcription factors possess basic residues followed by their ZIP domains whereas ROGDI protein lacks the canonical motif (i.e., basic residues) for DNA-binding activity and nuclear localization. Drosophila ROGDI actually displays its subcellular distribution in both nucleus and cytoplasm of cultured cells or adult fly neurons (Supplementary Fig. 3) although the exclusive nuclear localization might not be a prerequisite for transcriptional activities. We recently reported the crystal structure of human ROGDI protein 62 and showed that, unlike other bZIP transcription factors, human ROGDI protein exists as a monomer containing two structurally distinguishable domains (designated as α and β domains, respectively) ( Supplementary Fig. 2a). The α domain exhibits an α-helical bundle that consists of H1, H2, H3, and H6 helices. In fact, the ZIP-like motif in the α domain appears to mediate their intramolecular interactions, contributing to the overall structure and stability of a monomeric ROGDI protein. Based on sequence homology between Drosophila and human ROGDI proteins, we predict that Rogdi[del] allele removes the majority of the first helix including the repeated leucine only) and sleep latency (F[1,449] = 18.61, P < 0.0001 for EOS; F[1,209] = 16.61, P < 0.0001 for DABA). (d) An agonist of GABA B -R (SKF-97541) but not GABA A -R (THIP) fully rescues the short sleep phenotypes in Rogdi mutants (n = 29-92). Two-way ANOVA detected significant interaction of Rogdi mutation with SKF-97541 effects on sleep amount (F[1,228] = 56.39, P < 0.0001), ABL (F[1,228] = 33.81, P < 0.0001) and sleep latency (F[1,228] = 14.38, P = 0.0002) but not with THIP. n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001 to no-drug controls in the same genetic backgrounds as determined by Tukey post hoc test.
Scientific REPORts | 7: 11368 | DOI:10.1038/s41598-017-11941-3 residues in the α domain and the first three strands in the β domain, explaining the instability of ROGDI del proteins (Fig. 1c, Supplementary Fig. 2b). A smaller but comparable deletion of the ZIP-like motif has been reported in a KTS patient with a splicing mutation in human Rogdi gene 5 . In addition, a functional study in cervical cancer cell lines demonstrated Rogdi effects on cell cycle progression and radio-sensitivity 9 . However, further investigations will be required to understand how these cellular phenotypes could be linked to the molecular and neural function of ROGDI protein.
What will be the relevance of our findings to KTS pathogenesis? Genetic heterogeneity has been reported among KTS patients, indicating that Rogdi-independent genetic mutations could contribute to KTS pathogenesis 4,63 . A recent study indeed showed that familial mutations in a sodium-citrate transporter gene SLC13A5 are the second genetic cause of KTS 64 . The pathogenic phenotypes commonly found in Rogdi-and SLC13A5-associated KTS gives rise to the intriguing possibility that these two genes might work together to control the intracellular levels of citrate 65 . This idea is further supported by the relevance of citrate metabolism to neurological phenotypes in KTS patients. Neurons are energetically dependent on astrocytes because neurons lack pyruvate carboxylase, an enzyme that converts pyruvate to oxaloacetate in the citric acid cycle 66,67 . SLC13A5 plays an important role in the transport of glial citrate into neurons to supplement the neuronal citric acid cycle and thereby supply cellular energy 68,69 . Furthermore, citrate, an intermediate in the citric acid cycle, acts as a precursor of α-ketoglutarate, which can be metabolized to glutamate and GABA 70 , implicating SLC13A5 in the biogenesis of GABA. Consistently, anti-epileptic drugs that elevate GABAergic transmission rescued the seizure phenotypes in SLC13A-associated KTS patients 65 . In addition, we showed that the pharmacological enhancement of GABAergic transmission by oral administration of GABA-T or GAT inhibitors was sufficient to rescue the short sleep phenotypes in Rogdi mutant flies.  Our genetic studies strongly implicate Rogdi function in GABAergic transmission, providing the first clue to understanding the neurological phenotypes observed in KTS patients. Molecular and neural deficits selectively caused by Rogdi mutation might explain why seizures in Rogdi-associated KTS are often resistant to anti-epileptic drugs 53 , as exemplified by our pharmacological rescue of Rogdi mutant sleep with a specific AED (Fig. 7). Future studies should thus address if Rogdi mutant flies display seizure-like behaviors similarly as in KTS patients and if Rogdi-dependent neural relay of GABAergic transmission controls seizure susceptibility in parallel with baseline sleep. In addition, it will be important to determine whether sleep deficiencies are also observed in KTS patients and whether reduced GABAergic transmission in Rogdi-and, possibly, SLC13A5-associated KTS patients is responsible for their neural dysfunctions, including early-onset seizures. Taken together, our genetic model would constitute an important platform for elucidating the molecular and neural pathogenesis underlying KTS and hint towards a precise development of a therapeutic strategy for KTS in the future.

Materials and Methods
Fly Stocks. All flies were maintained in standard cornmeal-yeast-agar medium under 12 hour light: 12 hour dark cycles at 25 °C. w 1118 (BL5905), TH-Gal4 (BL8848), TDC2-Gal4 (BL9313), GAD1-Gal4 (BL51630), UAS-NaChBac-EGFP (BL9467), UAS-GABA-B-R1 RNAi (BL28353, BL51817), and UAS-GABA-B-R2 RNAi (BL27699) stocks were obtained from the Bloomington Drosophila Stock Center. dumb 2 (f02676) was obtained from the Exelixis Collection at Harvard Medical School. UAS-TNT was described previously 71 . Rogdi-Gal4 (C0113) was a gift from J. Dubnau (Cold Spring Harbor Laboratory). Rogdi P1 (12866R-1) was obtained from the National Institute of Genetics, Japan. The imprecise excision of the P element insertion in Rogdi P1 mutants was induced by genetic crosses to a transgenic line expressing a transposase. Excision lines were individually established and screened for large deletions in the genomic Rogdi locus to isolate the Rogdi del allele. Rogdi mutant stocks were isogenized by outcrossing to w 1118 backgrounds more than six times prior to the behavioral analyses. A full-length cDNA encoding ROGDI-PA was PCR-amplified from head cDNA library and inserted into a modified pUAS-C5 with a C-terminal 3xFLAG tag 72 . The UAS-ROGDI-3xFLAG transgene was then injected into w 1118 to establish several independent UAS-ROGDI lines (BestGene Inc).
Behavioral Analyses. Behavioral data were recorded using the Drosophila Activity Monitor system (Trikinetics) under 12-hour light: 12-hour dark cycles at 25 °C. Each male fly was transferred into a 65 × 5 mm glass tube containing 5% sucrose and 2% agar food. Locomotor activity in individual flies was quantified by counting the number of infrared beam crosses per minute. A sleep bout was defined as a behavioral episode during which flies did not show any activity for 5 minutes or longer. Sleep parameters were analyzed with an Excel macro 73 . Mechanical sleep deprivation was conducted by Sleep Nullifying Apparatus (SNAP) 74 while sleep behaviors in individual flies were continuously monitored on the SNAP device before or after the sleep deprivation. Since Rogdi P1 allele has an unrelated RNA interference transgene and induces its overexpression by Gal4 drivers, most behavioral tests including transgenic rescue experiments were performed in Rogdi del mutant backgrounds. Drug Treatment. AMPT (Sigma), L-DOPA (Sigma), EOS (Tokyo Chemical Industry), DABA (Sigma), and valproic acid (Sigma) were directly dissolved at the indicated concentrations in the food used during behavioral testing, which contained 5% sucrose and 2% agar ('behavior food'). THIP (Tocris), SKF-97541 (Tocris), CBZ (Acros), and gabapentin (Sigma) were first dissolved at 10 mg/ml (THIP, SKF-97541, CBZ) or 100 mg/ml (gabapentin) and then diluted in the behavior food. For AMPT treatment, flies were fed on AMPT-containing behavior food for 12 hours in the dark phase then switched to standard behavior food at the beginning of the next day; subsequent sleep behaviors were monitored for 24 hours. The wake-promoting effects of L-DOPA were similarly assessed except that 25 μg/ml of ascorbic acid was also included in the behavior food and sleep behaviors were monitored on the same day during 24-hour administration of L-DOPA. For EOS, CBZ, and DABA treatments, flies were pre-fed on drug-containing behavior food for 3 days (EOS, CBZ) or 1.5 days (DABA) and their sleep behaviors were monitored for 24 hours while continuing to be fed on drug-containing food. For THIP, SKF-97541, gabapentin, and valproate treatments, flies were pre-fed on drug-containing behavior food for 1.5 days and their sleep behaviors were monitored for 3 days (Rogdi mutants and their control flies) or for 24 hours (TH > NaCh and their control flies) while continuing to be fed on drug-containing food.
Whole-brain Imaging. Adult fly brains were dissected in phosphate-buffered saline (PBS), fixed in PBS containing 3.7% formaldehyde, and then blocked with 0.5% normal goat serum (NGS) in PBS containing 0.3% Tritox X-100 (PBS-T). Dissected brains were incubated with rabbit anti-GABA antibody (diluted in PBS-T containing 0.5% NGS and 0.05% sodium azide at 1:1000, Sigma) for 2 days at 4 °C. After washing with PBS-T, brains were further incubated with anti-rabbit Alexa Flour 594 antibody (diluted at 1:600, Jackson ImmunoResearch) for 1 day at 4 °C, washed with PBS-T, and then mounted in a VECTASHIELD mounting medium (Vector Laboratories).
Quantification of Protein Stability. Drosophila S2 cells were cultured in Shields and Sang M3 insect medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (ThermoFisher Scientific) at 25 °C. Expression vectors for either wild-type ROGDI or ROGDI del protein with a C-terminal 3xFLAG tag were transiently transfected using Effectene Transfection Reagent according to the manufacturer's instructions (QIAGEN). Transfected cells were further incubated with 100 μg/ml of cycloheximide to block protein synthesis for the indicated hours before their harvest at 48 hours after transfection. Total cell extracts were resolved by SDS-PAGE and immunoblotted with anti-FLAG or anti-TUBULIN antibodies. Intensities from immunoblotting signals were quantified using ImageJ software. Relative levels of ROGDI proteins at each time-point were calculated by normalizing to those of TUBULIN proteins. Fitting curves for the time-dependent decay of ROGDI proteins were generated using Excel and used to estimate their half-life in hours.
Data Availability. All data analyzed during this study are included in this published article (and its