InteractiveFly: GeneBrief

insomniac: Biological Overview | References


Gene name - insomniac

Synonyms -

Cytological map position - 2B1-2B1

Function - adaptor protein

Keywords - adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex - mono-ubiquitination - together with Cul3 Inc is essential for normal sleep regulation, compartmentalized at postsynaptic densities of the NMJ - gates retrograde signaling - provides a molecular link between the control of sleep and homeostatic plasticity at the neuromuscular junction

Symbol - inc

FlyBase ID: FBgn0025394

Genetic map position - chrX:1,513,059-1,517,612

Classification - BTB_POZ: BTB (Broad-Complex, Tramtrack and Bric a brac)/POZ (poxvirus and zinc finger) domain superfamily

Cellular location - cytoplasmic



NCBI link: EntrezGene, Nucleotide, Protein
inc orthologs: Biolitmine
BIOLOGICAL OVERVIEW

At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. In this study a forward genetic screen is described to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, it was found that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, it was shown that Peflin, a Ca(2+)-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca(2+) signaling and control of Cul3/Inc activity in the postsynaptic compartment. This study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses (Kikuma, 2019).

By screening >300 genes with putative functions at synapses, this study has identified inc as a key postsynaptic regulator of retrograde homeostatic signaling at the Drosophila NMJ. The data suggest that Inc and Cul3 are recruited to the postsynaptic compartment within minutes of glutamate receptor perturbation, where they promote local mono-ubiquitination. Inc/Cul3 appear to function downstream of or in parallel to CaMKII and upstream of retrograde signaling during PHP. Pef was identified as a putative co-adaptor that may work with Inc/Cul3 to link Ca2+ signaling in the postsynaptic compartment with membrane trafficking and retrograde communication. Altogether, these findings implicate a post translational signaling system involving mono-ubiquitination in the induction of retrograde homeostatic signaling at postsynaptic compartments (Kikuma, 2019).

Although forward genetic screens have been very successful in identifying genes required in the presynaptic neuron for the expression of PHP, these screens have provided less insight into the postsynaptic mechanisms that induce retrograde homeostatic signaling. It seems clear that many genes acting presynaptically are individually required for PHP, with loss of any one completely blocking PHP expression. Indeed, ~25 genes that function in neurons have thus far been implicated in PHP expression. In contrast, forward genetic screens have largely failed to uncover new genes functioning in the postsynaptic muscle during PHP, implying some level of redundancy. The specific postsynaptic induction mechanisms driving retrograde PHP signaling have therefore remained unclear, and are further complicated by cap-dependent translation and metabolic pathways that contribute to sustaining PHP expression over chronic, but not acute, time scales. Therefore, it is perhaps not surprising that despite screening hundreds of mutants, this study found only a single gene, insomniac, to be required for PHP induction. Inc is expressed in the nervous system and can traffic to the presynaptic terminals of motor neuron. In the context of PHP signaling, however, inc was found to be required in the postsynaptic compartment, where it functions downstream of or in parallel to CaMKII. One attractive possibility is that a reduction in CaMKII-dependent phosphorylation of postsynaptic targets enables subsequent ubiquitination by Cul3-Inc complexes, and that this modification ultimately drives retrograde signaling during PHP. Indeed, reciprocal influences of phosphorylation and ubiquitination on shared targets are a common regulatory feature in a variety of signaling systems. The dynamic interplay of phosphorylation and ubiquitination in the postsynaptic compartment may enable a sensitive and tunable mechanism for controlling the timing and calibrating the amplitude of retrograde signaling at the NMJ (Kikuma, 2019).

The substrates targeted by Inc and Cul3 during PHP induction are not known, but the identification of mono-ubiquitination in the postsynaptic compartment during PHP signaling and the putative Cul3 co-adaptor Peflin provides a foundation from which to assess possible candidates and pathways. In mammals, Pef forms a complex with another Ca2+ binding protein, ALG2, to confer Ca2+ regulation to membrane trafficking pathways. Moreover, Pef/ALG2 were recently found to serve as target-specific co-adaptors for Cul3-KLHL12. In particular, SEC31 and other components involved in ER-mediated membrane trafficking pathways were shown to be targeted for mono-ubiquitination, which in turn modulate Collagen secretion. One attractive possibility, therefore, is that Cul3/Inc could respond to changes in Ca2+ in the postsynaptic compartment through regulation by Pef during PHP signaling to control membrane trafficking pathways. Importantly, the subsynaptic reticulum (SSR) is a complex and membraneous network at the Drosophila NMJ, where electrical, Ca2+-dependent, and membrane trafficking pathways in the postsynaptic compartment are integrated (Teodoro, 2013; Nguyen, 2016). Indeed, Multiplexin, a fly homolog of Collagen XV/XVIII and a proposed retrograde signal, is secreted into the synaptic cleft and is required for trans-synaptic retrograde signaling during PHP (Wang, 2014). In addition, another proposed retrograde signal and secreted protein, Semaphorin 2B, was recently shown to function postsynaptically in retrograde PHP signaling (Orr, 2017). However, inc does not appear to be the closest Drosophila ortholog to KLHL12, and it is therefore possible that Pef and Cul3/Inc regulate postsynaptic PHP signaling through a more indirect mechanism (Kikuma, 2019).

While the precise relationships between CaMKII, Inc, Cul3, and Pef are currently unclear, the activity of membrane trafficking pathways could ultimately be targeted for modulation by Ca2+- and Cul3/Inc-dependent signaling during PHP induction. First, a role for postsynaptic membrane trafficking and elaboration during PHP signaling has already been suggested. In addition, extracellular Ca2+ does not appear to be involved in rapid PhTx-dependent PHP induction. It is therefore tempting to speculate that Ca2+ release from the postsynaptic SSR during rapid PHP signaling may influence Cul3/Inc activity through Pef-dependent regulation, as transient changes in ER-derived Ca2+-signaling controls Pef-dependent recruitment of Cul3 (McGourty, 2016). Alternatively, postsynaptic scaffolds and/or glutamate receptors themselves may be targeted by Cul3/Inc at the Drosophila NMJ, given that these proteins are involved in ubiquitin-mediated signaling and remodeling at dendritic spines. Consistent with this idea, there is evidence that signaling complexes composed of neurotransmitter receptors, CaMKII, and membrane-associated guanylate kinases are intimately associated at postsynaptic densities in Drosophila, as they are in the mammalian central nervous system. There has been speculation that these complexes are targets for modulation during PHP signaling. Although these models are not mutually exclusive, further studies will be required to determine the specific substrates and signal transduction mechanisms through which Cul3/Inc and Pef initiate and sustain retrograde homeostatic communication in postsynaptic compartments (Kikuma, 2019).

While it is well established that the ubiquitin proteasome system can sculpt and remodel synaptic architecture, the importance of mono-ubiquitination at synapses is less studied. Ubiquitin-dependent pathways play key roles in synaptic structure, function, and degeneration, and also contribute to activity-dependent dendritic growth. However, the fact that some proteins persist for long periods at synapses suggests that modification of these proteins by ubiquitin likely include non-degredative and reversible mechanisms. Indeed, a recent study revealed a remarkable heterogeneity in the stability of synaptic proteins, with some short lived and rapidly turned over, while others persisting for long time scales, with half lives of months or longer. At the Drosophila NMJ, rapid ubiquitin-dependent proteasomal degradation at presynaptic terminals is necessary for the expression of PHP through modulation of the synaptic vesicle pool (Wentzel, 2018). In contrast, postsynaptic proteasomal degradation does not appear to be involved in rapid PHP signaling, suggesting that ubiquitin-dependent pathways in the postsynaptic compartment contribute to PHP signaling by non-degradative mechanisms. The current data demonstrate that Cul3, Inc, and Pef function in muscle to enable retrograde PHP signaling, and suggest that Cul3/Inc rapidly trigger mono-ubiquitination at postsynaptic densities following glutamate receptor perturbation. Interestingly, synaptic proteins can be ubiquitinated in <15 s following depolarization-induced Ca2+ influx at synapses (Chen, 2003) and changes in intracellular Ca2+ can activate Pef and Cul3 signaling with similar rapidity. Therefore, both poly- and mono-ubiquination may function in combination with other rapid and reversible processes, including phosphorylation at postsynaptic compartments to enable robust and diverse signaling outcomes during the induction of homeostatic plasticity (Kikuma, 2019).

A prominent hypothesis postulates that a major function of sleep is to homeostatically regulate synaptic strength following experience-dependent changes that accrue during wakefulness. Several studies have revealed changes in neuronal firing rates and synapses during sleep/wake behavior, yet few molecular mechanisms that directly associate the electrophysiological process of homeostatic synaptic plasticity and sleep have been identified. The finding that inc is required for the homeostatic control of synaptic strength provides an intriguing link to earlier studies, which implicate inc in the regulation of sleep. It remains to be determined to what extent the role of inc in controlling PHP signaling at the NMJ is related to the impact of inc on sleep and, if so, whether Inc targets the same substrates to regulate these processes. Interestingly, virtually all neuropsychiatric disorders are associated with sleep dysfunction, including those associated with homeostatic plasticity and Fragile X Syndrome, and sleep behavior is also disrupted by mutations in the Drosophila homolog of FMRP, dfmr1. Further investigation of this intriguing network of genes involved in the homeostatic control of sleep and synaptic plasticity may help solve the biological mystery that is sleep and also shed light on the etiology of neuropsychiatric diseases (Kikuma, 2019).

Conserved properties of Drosophila Insomniac link sleep regulation and synaptic function

Sleep is an ancient animal behavior that is regulated similarly in species ranging from flies to humans. Various genes that regulate sleep have been identified in invertebrates, but whether the functions of these genes are conserved in mammals remains poorly explored. Drosophila insomniac (inc) mutants exhibit severely shortened and fragmented sleep. Inc protein physically associates with the Cullin-3 (Cul3) ubiquitin ligase, and neuronal depletion of Inc or Cul3 strongly curtails sleep, suggesting that Inc is a Cul3 adaptor that directs the ubiquitination of neuronal substrates that impact sleep. Three proteins similar to Inc exist in vertebrates-KCTD2, KCTD5, and KCTD17-but are uncharacterized within the nervous system and their functional conservation with Inc has not been addressed. This study shows that Inc and its mouse orthologs exhibit striking biochemical and functional interchangeability within Cul3 complexes. Remarkably, KCTD2 and KCTD5 restore sleep to inc mutants, indicating that they can substitute for Inc in vivo and engage its neuronal targets relevant to sleep. Inc and its orthologs localize similarly within fly and mammalian neurons and can traffic to synapses, suggesting that their substrates may include synaptic proteins. Consistent with such a mechanism, inc mutants exhibit defects in synaptic structure and physiology, indicating that Inc is essential for both sleep and synaptic function. These findings reveal that molecular functions of Inc are conserved through ~600 million years of evolution and support the hypothesis that Inc and its orthologs participate in an evolutionarily conserved ubiquitination pathway that links synaptic function and sleep regulation (Li, 2017).

The presence of sleep states in diverse animals has been suggested to reflect a common purpose for sleep and the conservation of underlying regulatory mechanisms. This study has shown that attributes of the Insomniac protein likely to underlie its impact on sleep in Drosophila-its ability to function as a multimeric Cul3 adaptor and engage neuronal targets that impact sleep-are functionally conserved in its mammalian orthologs. This comparative analysis of Inc family members in vertebrate and invertebrate neurons furthermore reveals that these proteins can traffic to synapses and that Inc itself is essential for normal synaptic structure and excitability. These findings support the hypothesis that Inc family proteins serve as Cul3 adaptors and direct the ubiquitination of conserved neuronal substrates that impact sleep and synaptic function (Li, 2017).

The ability of KCTD2 and KCTD5 to substitute for Inc in the context of sleep is both surprising and notable given the complexity of sleep-wake behavior and the likely functions of these proteins as Cul3 adaptors. Adaptors are multivalent proteins that self-associate, bind Cul3, and recruit substrates, and these interactions are further regulated by additional post-translational mechanisms. The findings indicate that KCTD2 and KCTD5 readily substitute for Inc within oligomeric Inc-Cul3 complexes, and strongly suggest that these proteins recapitulate other aspects of Inc function in vivo including the ability to engage neuronal targets that impact sleep. The simplest explanation for why KCTD2 and KCTD5 have retained the apparent ability to engage Inc targets despite the evolutionary divergence of Drosophila and mammals is that orthologs of Inc targets are themselves conserved in mammals. This inference draws support from manipulations of Drosophila Roadkill/HIB and its mammalian ortholog SPOP, Cul3 adaptors of the MATH-BTB family that regulate the conserved Hedgehog signaling pathway. While the ability of SPOP to substitute for HIB has not been assessed by rescue at an organismal level, clonal analysis in Drosophila indicates that ectopically expressed mouse SPOP can degrade the endogenous HIB substrate Cubitus Interruptus (Ci), and conversely, that HIB can degrade mammalian Gli proteins that are the conserved orthologs of Ci and substrates of SPOP. By analogy, Inc targets that impact sleep are likely to have orthologs in vertebrates that are recruited by KCTD2 and KCTD5 to Cul3 complexes. While the manipulations do not resolve whether KCTD17 can substitute for Inc in vivo, the ability of KCTD17 to assemble with fly Inc and Cul3 suggests that functional divergence among mouse Inc orthologs may arise outside of the BTB domain, and in particular may reflect properties of their C-termini including the ability to recruit substrates (Li, 2017).

The finding that Inc can transit to synapses and is required for normal synaptic function is intriguing in light of hypotheses that invoke synaptic homeostasis as a key function of sleep. While ubiquitin-dependent mechanisms contribute to synaptic function and plasticity and sleep is known to influence synaptic remodeling in both vertebrates and invertebrates, molecular links between ubiquitination, synapses, and sleep remain poorly explored. Other studies in flies have indicated that regulation of RNA metabolism may similarly couple synaptic function and the control of sleep. Alterations in the activity of the Fragile X mental retardation protein (FMR), a regulator of mRNA translation, cause defects in the elaboration of neuronal projections and the formation of synapses as well as changes in sleep duration and consolidation. Loss of Adar, a deaminase that edits RNA, leads to increased sleep through altered glutamatergic synaptic function. Like Inc, these proteins are conserved in mammals, suggesting that further studies in flies may provide insights into diverse mechanisms by which sleep influences synaptic function and conversely, how changes in synapses may impact the regulation of sleep (Li, 2017).

These findings at a model synapse suggest that the impact of Inc on synaptic function may be intimately linked to its influence on sleep but do not yet resolve important aspects of such a mechanism. The synaptic phenotypes of inc mutants-increased synaptic growth, decreased evoked neurotransmitter release, and modest effects on spontaneous neurotransmission-are qualitatively distinct from those of other short sleeping mutants. Shaker (Sh) and Hyperkinetic (Hk) mutations decrease sleep in adults but increase both excitability and synaptic growth at the NMJ, suggesting that synaptic functions of Inc may affect sleep by a mechanism different than broad neuronal hyperexcitability. While a parsimonious model is that Inc directs the ubiquitination of a target critical for synaptic transmission both at the larval NMJ and in neuronal populations that promote sleep, this hypothesis awaits the elucidation of Inc targets, definition of the temporal requirements of Inc activity, and further mapping of the neuronal populations through which Inc impacts sleep. Finally, determining the localization of endogenous Inc within neurons is essential to distinguish possible presynaptic and postsynaptic functions of Inc and whether Inc engages local synaptic proteins or extrasynaptic targets that ultimately influence synaptic function (Li, 2017).

A clear implication of these findings is that neuronal targets and synaptic functions of Inc may be conserved in other animals. While the impact of Inc orthologs on sleep in vertebrates is as yet unknown, findings from C. elegans support the notion that conserved molecular functions of Inc and Cul3 may underlie similar behavioral outputs in diverse organisms. INSO-1/C52B11.2, the only C. elegans ortholog of Inc, interacts with Cul3, and RNAi against Cul3 and INSO-1 reduces the duration of lethargus, a quiescent sleep-like state, suggesting that effects of Cul3- and Inc-dependent ubiquitination on sleep may be evolutionarily conserved. The functions of Inc orthologs and Cul3 in the mammalian nervous system await additional characterization, but emerging data suggest functions relevant to neuronal physiology and disease. Human mutations at the KCTD2/ATP5H locus are associated with Alzheimer's disease, and mutations of KCTD17 with myoclonic dystonia. Cul3 lesions have been associated in several studies with autism spectrum disorders and comorbid sleep disturbances. More generally, autism spectrum disorders are commonly associated with sleep deficits and are thought to arise in many cases from altered synaptic function, but molecular links to sleep remain fragmentary. Studies of Inc family members and their conserved functions in neurons are likely to broaden understanding of how ubiquitination pathways may link synaptic function to the regulation of sleep and other behaviors (Li, 2017).

Proteins involved in sleep homeostasis: Biophysical characterization of INC and its partners

The Insomniac protein ( Inc) of Drosophila melanogaster has a crucial role in sleep homeostasis as flies lacking the inc gene exhibit strikingly reduced and poorly consolidated sleep. Nevertheless, in vitro characterizations of Inc biophysical properties and partnerships have not been yet reported. This study reports the heterologous expression of the protein and its characterization using a number of different techniques. Present data indicate that Inc is endowed with a remarkable stability, which results from the cooperation of the two protein domains. Moreover, this study also demonstrated and quantified the ability of Inc to recognize its potential partners Cul3 and dGRASP. Taking into account the molecular organization of the protein, these two partners may be anchored simultaneously. Although there is no evident relationship between the reported Inc functions and dGRASP binding, these data suggest that Inc may cooperate as ligase adaptor to dGRASP ubiquitination. Small angle x-ray scattering data collected on the complex between Inc and Cul3, which represent the first structural characterization of this type of assemblies, clearly highlight the highly dynamic nature of these complexes. This strongly suggests that the functional behavior of these proteins cannot be understood if dynamic effects are not considered. Finally, the strict analogy of the biochemical/biophysical properties of Inc and of its human homolog KCTD5 may reliably indicate that this latter protein and/or the closely related proteins KCTD2/KCTD17 may play important roles in human sleep regulation (Pirone, 2016).

Cul3 and the BTB adaptor insomniac are key regulators of sleep homeostasis and a dopamine arousal pathway in Drosophila

Sleep is homeostatically regulated, such that sleep drive reflects the duration of prior wakefulness. However, despite the discovery of genes important for sleep, a coherent molecular model for sleep homeostasis has yet to emerge. To better understand the function and regulation of sleep, a reverse-genetics approach was employed in Drosophila. An insertion in the BTB domain protein CG32810/insomniac (inc) exhibited one of the strongest baseline sleep phenotypes thus far observed, a ~10 h sleep reduction. Importantly, this is coupled to a reduced homeostatic response to sleep deprivation, consistent with a disrupted sleep homeostat. Knockdown of the Inc-interacting protein, the E3 ubiquitin ligase Cul3, results in reduced sleep duration, consolidation, and homeostasis, suggesting an important role for protein turnover in mediating Inc effects. Interestingly, inc and Cul3 expression in post-mitotic neurons during development contributes to their adult sleep functions. Similar to flies with increased dopaminergic signaling, loss of inc and Cul3 result in hyper-arousability to a mechanical stimulus in adult flies. Furthermore, the inc sleep duration phenotype can be rescued by pharmacological inhibition of tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis. Taken together, these results establish inc and Cul3 as important new players in setting the sleep homeostat and a dopaminergic arousal pathway in Drosophila (Pfeiffenberger, 2012).

Using genetic backcrossing to an isogenic (iso31) strain, the striking observation was made that the vast majority of the mutants (39/45, nearly 90%) identified in the primary genetic screen did not have a significant phenotype after backcrossing, indicating a remarkably pervasive role for genetic background in mediating sleep phenotypes in a variety of mutant strains. There are two main possibilities for how genetic background influences sleep phenotypes: (1) the tested allele indeed affects sleep; however, there are suppressors of this phenotype present in the iso31 background but absent from the original background. (2) The sleep phenotype is not due to the transposon insertion but instead is caused by one or more flanking mutations present in the original mutant background but absent from the iso31 background. Isolated examples of (1) have been observed in the case of Sh and mutants of the Sh regulatory subunit Hyperkinetic as well as Crc and Sema-5c effects on olfactory, startle, and sleep behavior and of (2) in the discovery of DATfmn mutants in the background of a timeless mutant strain. These are consistent with observations in C. elegans indicating the limitations of backcrossing for removing flanking mutations and in Drosophila indicating the widespread presence of background mutations that can suppress mutant-induced behavioral phenotypes. Experience with RhoGDIEY02738 suggests that scenario 2 may be more common than previously thought. Nonetheless, the sheer number of examples observed in this study indicates that the presence of genetic variation at sleep regulatory loci among laboratory stocks is both prevalent and perhaps even sufficiently important to mask or induce significant sleep phenotypes. Moreover, in the case of Sh a single outcross was sufficient to unmask the short sleep phenotype. Indeed, it is assumed that if mutant alleles were outcrossed to a deficiency strain this would effectively remove the influence of accumulated recessive mutations that flank the allele. However, the backcrossing data indicates that this strategy did not remove those concerns, suggesting that background variants may exert dominant effects. Practically, this experience suggests that outcrossing to deletion stocks alone may not be sufficient to verify the function of a genetic locus in sleep. Overall, this observation has important implications for the role of genetic modifiers in sleep, the conduct and design of sleep genetic screens, and for the interpretation of sleep and other behavioral mutant phenotypes in general. While backcrossing can remove flanking genetic variants that may contribute to an observed phenotype, alone it is not sufficient to definitively establish genotype-phenotype causation (Pfeiffenberger, 2012).

Despite the large modulatory effect of genetic background, it was possible to observe persistent phenotypes with inc, which showed the most robust and reproducible sleep phenotypes, in particular demonstrating an important role in the homeostatic regulation of sleep. Several independent lines of evidence support the role of inc in sleep homeostasis. First, two inc alleles (incf00285 and incmw) were backcrossed for 5 generations into an isogenic background and retained their short sleep and suppressed sleep homeostasis phenotypes, each among the strongest observed, as compared to isogenic control lines. Second, incf00285 was rescued in distinct ways: (1) with genomic duplications encompassing the gene but not those that do not include the gene, and (2) using the GAL4/UAS system, the latter rescuing both baseline and homeostatic phenotypes. Third, failure to complement was demonstrated with a deletion removing the inc genomic locus, or inc transheterozygotes. Fourth, it was demonstrated that two independent RNAi lines that target two different regions of inc phenocopy the inc mutant phenotype (Pfeiffenberger, 2012).

In addition, evidence was provided that the Inc-interacting protein, the E3 ubiquitin ligase CUL3, functions to regulate sleep levels suggesting that inc links protein turnover to sleep homeostasis. Two independent inserts of a Cul3-RNAi line that effectively suppress Cul3 mRNA levels resulted in reduced sleep, and induction of a wild-type Cul3 transgene could rescue these phenotypes. The CUL3/Inc interaction was verified in S2 cells, and synthetic genetic interactions between Cul3 and inc were observed using RNAi, consistent with the model that they operate together to affect sleep (Pfeiffenberger, 2012).

A core concept in understanding sleep behavior is its homeostatic regulation, i.e., the observation that the drive to sleep reflects the duration of prior wakefulness. Sleep homeostasis typically is measured by enforcing wakefulness/depriving sleep for a defined period and assaying the increase in subsequent rebound sleep. Importantly, it was demonstrated that both inc and Cul3 have robust effects on sleep homeostasis where reduced inc or Cul3 was accompanied by suppressed or absent sleep rebound under the conditions used in this study. These results suggest that inc and Cul3, and by extension, protein degradation, are important for the accumulation of sleep need during wake and/or dissipation of sleep need after deprivation (Pfeiffenberger, 2012).

For the large majority of sleep mutants that have been described, assessment of developmental and adult contributions has not formally been addressed, raising questions regarding their precise function in sleep. This study provided evidence that inc induction or Cul3-RNAi knockdown during development, but not exclusively during adulthood, could rescue (in the case of induction) or phenocopy (in the case of knockdown) their respective mutant/RNAi phenotypes. The Cul3 results are consistent with an established role for Cul3 in dendritic and axonal arborization, in which dendritic and axonal arborization are reduced in Cul3 mutants. The data also revealed a stochastic branching defect in MB neurons in 26% of inc mutants, in which they lack a single α- or β-lobe. Based on the incomplete penetrance of this morphological defect, it cannot explain the sleep behavior phenotype; however, it may be reflective of other morphological phenotypes that are causative for behavior. Alternatively, the necessity for developmental expression of Cul3 and inc may be for the appropriate processing, maturation and/or localization of these proteins in the adult. The apparent long half-life/persistence of this protein after induction only during development is consistent with the possibility that developmentally expressed transcription is important for adult protein expression and function. Regardless, it will be of interest to examine the relative adult and developmental requirements of other sleep mutants (Pfeiffenberger, 2012).

The reduced sleep phenotype depends largely on a single neurotransmitter, dopamine, establishing a transmitter basis to inc/Cul3 function. Dopaminergic signaling is a key regulator of sleep/wake behavior. In humans, sleep deprivation has been associated with increased brain levels of dopamine. Treatment of Parkinson's disease with L-DOPA can alleviate daytime sleepiness, or in the extreme result in insomnia. In Drosophila, genetic loss or pharmacological inhibition of tyrosine hydroxylase increases sleep. Furthermore, flies that lack a functional copy of DopR exhibit increased sleep and general arousal defects, including reduced arousing effects of caffeine. Conversely, in DATfmn flies, or flies fed dopamine-enhancing methamphetamine, sleep levels are severely reduced. Dopamine arousal effects are modulated by light. Moreover, sleep deprivation induced reductions in learning can be suppressed by enhancing dopaminergic signaling. Other than dopamine receptors and Dopamine transporter (DAT), members of the dopaminergic arousal pathway remain largely unknown (Pfeiffenberger, 2012).

This study reports that inc and Cul3 function in the dopaminergic arousal pathway. First, inc mutants, Cul3-RNAi, and DATfmn all showed robust sleep duration and consolidation phenotypes. Second, all three groups were hyper-arousable to mechanical stimuli. Third, disruption of inc, Cul3, and DAT all exhibited suppressed or absent homeostatic responses to sleep deprivation. Fourth, the short-sleep phenotypes of inc and DATfmn were non-additive in double mutants. Fifth, while wild-type flies exhibited reduced sleep when fed the dopamine precursor L-DOPA, inc mutants were resistant to these effects, but not the arousing effects of the Rdl antagonist CBZ. Finally, the sleep duration phenotypes in flies with disrupted inc, Cul3, and DAT could be suppressed by pharmacologically inhibiting dopamine synthesis with 3IY or AMPT, linking short sleep to excess dopamine function. Importantly, this study demonstrated that inhibition of dopamine synthesis via tyrosine hydroxylase inhibition does not affect L-DOPA-induced sleep reductions. It was also observed that 3IY could restore sleep homeostasis to Cul3-RNAi. Similar 3IY effects on homeostasis were only observed in one of the two inc alleles. Nonetheless, these studies do further link dopamine signaling to sleep homeostasis. inc and Cul3 are the first genes that are not known dopamine receptors reported to function in the dopaminergic arousal pathway, further reinforcing the pivotal role of dopamine in sleep homeostasis (Pfeiffenberger, 2012).

These data suggests Cul3/inc function to regulate dopaminergic signaling downstream of dopamine. inc phenotypes did not map to dopaminergic neurons nor was it possible to identify consistent changes in global dopamine levels among Cul3-RNAi and inc mutants. Thus, Cul3/inc may be involved in active turnover of dopamine receptors or their effectors in neurons defined by Cha-GAL4 and 30Y-GAL4. Double mutants were examined of inc and a major dopamine receptor involved in arousal in Drosophila, DopR, and no suppression of inc baseline phenotypes was observed; moreover, it was found that DopR mutant flies were responsive to 3IY consumption (i.e., exhibit increased sleep), suggesting that additional dopamine receptors function in Cul3/inc-based dopamine arousal. Drosophila has 2 other dopamine receptors and partial suppression of inc was observed with DopR and DopR2 RNAi, suggesting that multiple dopamine receptors may contribute to these effects. Alternatively, Cul3/inc may be important for protein turnover of other homeostatically regulated components. For example, extensive and dose-dependent changes in synaptic protein expression throughout the brain with sleep deprivation and recovery may depend on Cul3/inc-dependent turnover of these proteins during sleep (Pfeiffenberger, 2012).

Interestingly, Cul3 has also been linked to sleep behavior via a candidate gene for Restless Leg Syndrome (RLS) and BTB gene, BTBD9 (Freeman, 2012). Unlike the current studies, disruption of the Drosophila BTBD9 is not associated with reduced sleep, a reduced level of waking activity, nor elevated dopaminergic signaling. In addition, the phenotypes map in part to dopaminergic neurons in the case of BTBD9 rather than cholinergic neurons for inc. Thus, Cul3/inc likely represents a distinct pathway regulating sleep. Nonetheless, these studies further highlight the importance of Cul3/BTB adaptor pathways in sleep regulation in both Drosophila and humans. Future work will be required to identify the dopamine and sleep-relevant ubiquitination target(s) of inc and Cul3 (Pfeiffenberger, 2012).

insomniac and Cullin-3 regulate sleep and wakefulness in Drosophila

In a forward genetic screen in Drosophila, insomniac, a mutant that severely reduces the duration and consolidation of sleep, has been isolated. Anatomically restricted genetic manipulations indicate that insomniac functions within neurons to regulate sleep. insomniac expression does not oscillate in a circadian manner, and conversely, the circadian clock is intact in insomniac mutants, suggesting that insomniac regulates sleep by pathways distinct from the circadian clock. The protein encoded by insomniac is a member of the BTB/POZ superfamily, which includes many proteins that function as adaptors for the Cullin-3 (Cul3) ubiquitin ligase complex. It was shown that Insomniac can physically associate with Cul3, and that reduction of Cul3 activity in neurons recapitulates the insomniac phenotype. The extensive evolutionary conservation of insomniac and Cul3 suggests that protein degradation pathways may have a general role in governing the sleep and wakefulness of animals (Stavropoulos, 2011).

Emerging evidence has suggested that the sleep states of diverse animals may be regulated by conserved molecular mechanisms, although many of these mechanisms remain undefined. insomniac, a gene that governs the duration of sleep and wakefulness in Drosophila is likely to engage protein degradation pathways to regulate sleep. Both insomniac and these pathways are well conserved, suggesting that they may be employed generally to regulate sleep in animals (Stavropoulos, 2011).

In rats and Drosophila, chronic sleep deprivation leads to reduced lifespan and lethality. Mutations in Shaker, sleepless, and Hyperkinetic that strongly reduce sleep in Drosophila are also associated with decreased longevity. In each case, longevity has been assessed for classical mutants in which gene function is reduced or absent in all tissues. Two independent insomniac mutants exhibit similarly decreased longevity. However, neuronally restricted depletion of insomniac, which sharply reduces the duration of sleep, has no measurable effect on longevity, demonstrating that the two attributes can be uncoupled. Similarly, fumin mutants affecting the Drosophila dopamine transporter gene display a strong decrease in sleep but normal longevity (Stavropoulos, 2011).

These results do not contradict the notion that sleep has critical physiological functions or that sleep deprivation leads to deficits in waking performance, although they do suggest that certain disruptions of sleep can be tolerated without impacting lifespan. Reductions in sleep duration may need to exceed a certain threshold to affect longevity, and the lethality elicited by chronic sleep deprivation regimens, as well as that of especially severe sleep mutants, may reflect the reduction of sleep to extremely low levels. For mutations with more modest effects on sleep, interpretations that attribute a causal relationship between altered sleep and reduced longevity may be problematic, particularly for those genes that are broadly expressed and whose loss-of-function is likely to have numerous pathological consequences. Additional genetic manipulations that perturb sleep in increasingly specific ways are required to further assess the relationship between sleep and longevity in both Drosophila and vertebrates (Stavropoulos, 2011).

Anatomically restricted manipulations of insomniac indicate that its expression within neurons is essential for normal sleep and wakefulness. The neuronal requirement for insomniac appears to be broad, as drivers that provide panneuronal or broad neuronal expression alter sleep most strongly in depletion and rescue experiments. The possibility cannot however be excluded that insomniac regulates sleep by functioning in a smaller number of neurons that are dispersed within the brain and not effectively represented by individual drivers were assayed. In particular, an insomniac-Gal4 reporter is expressed in regions of the Drosophila brain that are implicated in regulating sleep, including the mushroom bodies and the pars intercerebralis, although driving insomniac expression in these areas individually does not rescue the sleep defect of insomniac mutants, with the exception of a weak rescue provided by the pars-intercerebralis-specific Mai301-Gal4 driver. Further manipulations of insomniac within the nervous system are necessary to understand the neuroanatomical basis by which it regulates sleep (Stavropoulos, 2011).

Several lines of evidence indicate that insomniac exerts its effects on sleep by a mechanism functionally distinct from the circadian clock. The circadian clock is intact in insomniac mutants, and insomniac expression is not regulated in a circadian fashion. Furthermore, the expression of insomniac in clock neurons is unable to restore normal sleep patterns in insomniac mutant backgrounds. Consistent with these data, daily sleep profiles indicate that the circadian control of sleep is intact in insomniac mutants. As is the case for wild-type animals, the highest probability of sleep during the dark phase is observed soon after the onset of darkness, with a decreasing sleep drive as the dark phase proceeds. The profile of sleep probability during the light phase is similarly intact. The principal alteration of sleep in insomniac animals is a reduced likelihood of sleeping throughout the day and night, consistent with the inference that insomniac may contribute to homeostatic mechanisms that regulate sleep need (Stavropoulos, 2011).

Cullins are scaffold proteins that assemble multisubunit E3 ubiquitin ligase complexes that ubiquitinate and degrade a variety of protein substrates in diverse biological contexts. The C termini of cullins interact with RING-domain ubiquitin ligases, while the N termini interact with adaptor proteins that recruit substrates for ubiquitination. Cul3 complexes utilize proteins of the BTB superfamily as their adaptor. In addition to the KCTD proteins that are known to function as Cul3 adaptors, more than half of the non-channel KCTD proteins, including the three vertebrate orthologs of Insomniac, are candidate Cul3 adaptors, as they copurify specifically with Cul3, but not with other cullins. For several of these candidate adaptors, including KCTD5 and TAG-303, the C. elegans ortholog of Insomniac, independent biochemical evidence confirms their ability to associate physically with Cul3. The finding that Insomniac is able to physically interact with Cul3 indicates that this interaction is evolutionarily conserved and supports the hypothesis that Insomniac serves as a Cul3 adaptor (Stavropoulos, 2011).

The reductions in sleep elicited by neuronal depletion of Cul3, and of its activator Nedd8, show that protein degradation pathways have a vital role in regulating sleep in Drosophila. Although alternative mechanisms cannot be excluded, the simplest hypothesis consistent with the data is that Insomniac engages the Cul3 protein degradation pathway to regulate sleep. One clear implication of this hypothesis is that the increased wakefulness of insomniac and Cul3 mutants may result from the inappropriate accumulation of substrates whose degradation is normally mediated by these proteins. The results suggest that such target substrates promote wakefulness and inhibit sleep, but they do not distinguish the neuronal function of these substrates. Target substrates regulated by Insomniac and Cul3 might function in a developmental manner, for example, in the elaboration of neural circuits that regulate sleep. Indeed, Cul3 has been implicated in regulating axonal and dendritic branching. Alternatively, such substrates might actively promote waking in adult animals, such that their ongoing degradation is part of the homeostatic mechanism contributing to the regulation of sleep-wake cycles (Stavropoulos, 2011).


REFERENCES

Search PubMed for articles about Drosophila Insomniac

Chen, H., Polo, S., Di Fiore, P. P. and De Camilli, P. V. (2003). Rapid Ca2+-dependent decrease of protein ubiquitination at synapses. Proc Natl Acad Sci U S A 100(25): 14908-14913. PubMed ID: 14657369

Freeman, A., Pranski, E., Miller, R. D., Radmard, S., Bernhard, D., Jinnah, H. A., Betarbet, R., Rye, D. B. and Sanyal, S. (2012). Sleep fragmentation and motor restlessness in a Drosophila model of Restless Legs Syndrome. Curr Biol 22(12): 1142-1148. PubMed ID: 22658601

Kikuma, K., Li, X., Perry, S., Li, Q., Goel, P., Chen, C., Kim, D., Stavropoulos, N. and Dickman, D. (2019). Cul3 and insomniac are required for rapid ubiquitination of postsynaptic targets and retrograde homeostatic signaling. Nat Commun 10(1): 2998. PubMed ID: 31278365

Li, Q., Kellner, D. A., Hatch, H. A. M., Yumita, T., Sanchez, S., Machold, R. P., Frank, C. A. and Stavropoulos, N. (2017). Conserved properties of Drosophila Insomniac link sleep regulation and synaptic function. PLoS Genet 13(5): e1006815. PubMed ID: 28558011

McGourty, C. A., Akopian, D., Walsh, C., Gorur, A., Werner, A., Schekman, R., Bautista, D. and Rape, M. (2016). Regulation of the CUL3 ubiquitin ligase by a calcium-dependent co-adaptor. Cell 167(2): 525-538 e514. PubMed ID: 27716508

Nguyen, C. T. and Stewart, B. A. (2016). The influence of postsynaptic structure on missing quanta at the Drosophila neuromuscular junction. BMC Neurosci 17(1): 53. PubMed ID: 27459966

Orr, B. O., Fetter, R. D. and Davis, G. W. (2017). Retrograde semaphorin-plexin signalling drives homeostatic synaptic plasticity. Nature 550(7674): 109-113. PubMed ID: 28953869

Pfeiffenberger, C. and Allada, R. (2012). Cul3 and the BTB adaptor insomniac are key regulators of sleep homeostasis and a dopamine arousal pathway in Drosophila. PLoS Genet 8(10): e1003003. PubMed ID: 23055946

Pirone, L., Smaldone, G., Esposito, C., Balasco, N., Petoukhov, M. V., Spilotros, A., Svergun, D. I., Di Gaetano, S., Vitagliano, L. and Pedone, E. M. (2016). Proteins involved in sleep homeostasis: Biophysical characterization of INC and its partners. Biochimie 131: 106-114. PubMed ID: 27678190

Stavropoulos, N. and Young, M. W. (2011). insomniac and Cullin-3 regulate sleep and wakefulness in Drosophila. Neuron 72(6): 964-76. PubMed Citation: 22196332

Teodoro, R. O., Pekkurnaz, G., Nasser, A., Higashi-Kovtun, M. E., Balakireva, M., McLachlan, I. G., Camonis, J. and Schwarz, T. L. (2013). Ral mediates activity-dependent growth of postsynaptic membranes via recruitment of the exocyst. EMBO J 32(14): 2039-2055. PubMed ID: 23812009

Wang, T., Hauswirth, A. G., Tong, A., Dickman, D. K. and Davis, G. W. (2014). Endostatin is a trans-synaptic signal for homeostatic synaptic plasticity. Neuron 83(3): 616-629. PubMed ID: 25066085

Wentzel, C., Delvendahl, I., Sydlik, S., Georgiev, O. and Muller, M. (2018). Dysbindin links presynaptic proteasome function to homeostatic recruitment of low release probability vesicles. Nat Commun 9(1): 267. PubMed ID: 29348419


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date revised: 25 March 2010

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