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Cerebral palsy (CP) causes neurological disability in early childhood. Hypoxic-ischaemic injury plays a major role in its aetiology, nevertheless, genetic and epigenetic factors may contribute to the clinical presentation. Mutations in ADD3 (encoding γ-adducin) gene have been described in a monogenic form of spastic quadriplegic cerebral palsy (OMIM 601568). We studied a 16-year-old male with spastic diplegia. Several investigations including neurometabolic testing, brain and spine magnetic resonance imaging (MRI) and CGH-Array were normal. Further, clinical genetics assessment and whole exome sequencing (WES) gave the diagnosis. We generated an animal model using Drosophila to study the effects of γ-adducin loss and gain of function. WES revealed a biallelic variant in the ADD3 gene, NM_016824.5(ADD3): c.1100G gt; A, p.(Gly367Asp). Mutations in this gene have been described as an ultra-rare autosomal recessive, which is a known form of inherited cerebral palsy. Molecular modelling suggests that this mutation leads to a loss of structural integrity of γ-adducin and is therefore expected to result in a decreased level of functional protein. Pan-neuronal over-expression or knock-down of the Drosophila ortholog of ADD3 called hts caused a reduction of life span and impaired locomotion thereby phenocopying aspects of the human disease. Our animal experiments present a starting point to understand the biological processes underpinning the clinical phenotype and pathogenic mechanisms, to gain insights into potential future methods for treating or preventing ADD3 related spastic quadriplegic cerebral palsy.

Background Prolonged cellular activity may overload cell function, leading to high rates of protein synthesis and accumulation of misfolded or unassembled proteins, which cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to re-establish normal protein homeostasis. Previous molecular work has demonstrated that sleep deprivation (SD) leads to ER stress in neurons, with a number of ER-specific proteins being upregulated to maintain optimal cellular proteostasis. It is still not clear which cellular processes activated by sleep deprivation lead to ER- stress, but increased cellular metabolism, higher request for protein synthesis, and over production of oxygen radicals have been proposed as potential contributing factors. Here, we investigate the transcriptional and ultrastructural ER and mitochondrial modifications induced by sleep loss. Results We used gene expression analysis in mouse forebrains to show that SD was associated with significant transcriptional modifications of genes involved in ER stress but also in ER-mitochondria interaction, calcium homeostasis, and mitochondrial respiratory activity. Using electron microscopy, we also showed that SD was associated with a general increase in the density of ER cisternae in pyramidal neurons of the motor cortex. Moreover, ER cisternae established new contact sites with mitochondria, the so-called mitochondria associated membranes (MAMs), important hubs for molecule shuttling, such as calcium and lipids, and for the modulation of ATP production and redox state. Finally, we demonstrated that Drosophila male mutant flies (elav > linker), in which the number of MAMs had been genetically increased, showed a reduction in the amount and consolidation of sleep without alterations in the homeostatic sleep response to SD. Conclusions We provide evidence that sleep loss induces ER stress characterized by increased crosstalk between ER and mitochondria. MAMs formation associated with SD could represent a key phenomenon for the modulation of multiple cellular processes that ensure appropriate responses to increased cell metabolism. In addition, MAMs establishment may play a role in the regulation of sleep under baseline conditions.

Insects are ectothermal animals that are constrained in their survival and reproduction by external temperature fluctuations which require either active avoidance of or movement towards a given heat source. In Drosophila, different thermoreceptors and neurons have been identified that mediate temperature sensation to maintain the animal’s thermal preference. However, less is known how thermosensory information is integrated to gate thermoresponsive motor behavior. Here we use transsynaptic tracing together with calcium imaging, electrophysiology and thermogenetic manipulations in freely moving Drosophila exposed to elevated temperature and identify different functions of ellipsoid body ring neurons, R1-R4, in thermoresponsive motor behavior. Our results show that warming of the external surroundings elicits calcium influx specifically in R2-R4 but not in R1, which evokes threshold-dependent neural activity in the outer layer ring neurons. In contrast to R2, R3 and R4d neurons, thermogenetic inactivation of R4m and R1 neurons expressing the temperature-sensitive mutant allele of dynamin, shibireTS, results in impaired thermoresponsive motor behavior at elevated 31 °C. trans-Tango mediated transsynaptic tracing together with physiological and behavioral analyses indicate that integrated sensory information of warming is registered by neural activity of R4m as input layer of the ellipsoid body ring neuropil and relayed on to R1 output neurons that gate an adaptive motor response. Together these findings imply that segregated activities of central complex ring neurons mediate sensory-motor transformation of external temperature changes and gate thermoresponsive motor behavior in Drosophila.

Alzheimer's disease (AD) is the most common neurodegenerative disease which is becoming increasingly prevalent due to ageing populations resulting in huge social, economic, and health costs to the community. Despite the pathological processing of genes such as

Alzheimer’s disease (AD) involves pathological processing of amyloid precursor protein (APP) into amyloid-β and microtubule associated protein Tau (MAPT) into hyperphosphorylated Tau tangles leading to neurodegeneration. Only 5% of AD cases are familial making it difficult to predict who will develop the disease thereby hindering our ability to treat the causes of the disease. A large population who almost certainly will, are those with Down syndrome (DS), who have a 90% lifetime incidence of AD. DS is caused by trisomy of chromosome 21 resulting in three copies of APP and other AD-associated genes, like dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) overexpression. This implies that DYRK1a inhibitors may have therapeutic potential for DS and AD, however It is not clear how overexpression of each of these genes contributes to the pathology of each disease as well as how effective a DYRK1A inhibitor would be at suppressing any of these. To address this knowledge gap, we used Drosophila models with human Tau, human amyloid-β or fly DYRK1A (minibrain (mnb)) neuronal overexpression resulting in photoreceptor neuron degeneration, premature death, decreased locomotion, sleep and memory loss. DYRK1A small molecule Type 1 kinase inhibitors (DYR219 and DYR533) were effective at suppressing these disease relevant phenotypes confirming their therapeutic potential.

As in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. We have characterized and modelled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). We show that currents mediated by the voltage-gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole-cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole-cell biophysical model using Hodgkin-Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, we can rescue the pharmacological block of Shaw and Shal, restore the firing rhythm, and thus demonstrate the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp we can switch the LNvs between morning-like and evening-like states of electrical activity. We conclude that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate.

Down syndrome (DS) is characterised by abnormal cognitive and motor development, and later in life by progressive Alzheimer's disease (AD)-like dementia, neuropathology, declining motor function and shorter life expectancy. It is caused by trisomy of chromosome 21 (Hsa21), but how individual Hsa21 genes contribute to various aspects of the disorder is incompletely understood. Previous work has demonstrated a role for triplication of the Hsa21 gene DYRK1A in cognitive and motor deficits, as well as in altered neurogenesis and neurofibrillary degeneration in the DS brain, but its contribution to other DS phenotypes is unclear. Here we demonstrate that overexpression of minibrain (mnb), the Drosophila ortholog of DYRK1A, in the Drosophila nervous system accelerated age-dependent decline in motor performance and shortened lifespan. Overexpression of mnb in the eye was neurotoxic and overexpression in ellipsoid body neurons in the brain caused age-dependent neurodegeneration. At the larval neuromuscular junction, an established model for mammalian central glutamatergic synapses, neuronal mnb overexpression enhanced spontaneous vesicular transmitter release. It also slowed recovery from short-term depression of evoked transmitter release induced by high-frequency nerve stimulation and increased the number of boutons in one of the two glutamatergic motor neurons innervating the muscle. These results provide further insight into the roles of DYRK1A triplication in abnormal aging and synaptic dysfunction in DS., Highlights • Overexpression of minibrain (DYRK1A) causes Down's relevant phenotypes including: • Age-dependent degeneration of brain neurons • Accelerated age-dependent decline in motor performance and shorted lifespan • Modified presynaptic structure and enhanced spontaneous transmitter release • Slowed recovery from short-term depression of synaptic transmission

Schizophrenia shows high heritability and several of the genes associated with this disorder are involved in calcium (Ca2+) signalling and synaptic function. One of these is the Rab-3 interacting molecule-1 (RIM1), which has recently been associated with schizophrenia by Genome Wide Association Studies (GWAS). However, its contribution to the pathophysiology of this disorder remains unexplored. In this work, we use Drosophila mutants of the orthologue of RIM1, Rim, to model some aspects of the classical and non-classical symptoms of schizophrenia. Rim mutants showed several behavioural features relevant to schizophrenia including social distancing and altered olfactory processing. These defects were accompanied by reduced evoked Ca2+ influx and structural changes in the presynaptic terminals sent by the primary olfactory neurons to higher processing centres. In contrast, expression of Rim-RNAi in the mushroom bodies (MBs), the main memory centre in flies, spared learning and memory suggesting a differential role of Rim in different synapses. Circadian deficits have been reported in schizophrenia. We observed circadian locomotor activity deficits in Rim mutants, revealing a role of Rim in the pacemaker ventral lateral clock neurons (LNvs). These changes were accompanied by impaired day/night remodelling of dorsal terminal synapses from a subpopulation of LNvs and impaired day/night release of the circadian neuropeptide pigment dispersing factor (PDF) from these terminals. Lastly, treatment with the commonly used antipsychotic haloperidol rescued Rim locomotor deficits to wildtype. This work characterises the role of Rim in synaptic functions underlying behaviours disrupted in schizophrenia.

Background Paroxysmal dyskinesias (PxDs) are characterized by involuntary movements and altered pre‐motor circuit activity. Causative mutations provide a means to understand the molecular basis of PxDs. Yet in many cases, animal models harboring corresponding mutations are lacking. Here we utilize the fruit fly, Drosophila, to study a PxD linked to a gain‐of‐function (GOF) mutation in the KCNMA1/hSlo1 BK potassium channel. Objectives We aimed to recreate the equivalent BK (big potassium) channel mutation in Drosophila. We sought to determine how this mutation altered action potentials (APs) and synaptic release in vivo; to test whether this mutation disrupted pre‐motor circuit function and locomotion; and to define neural circuits involved in locomotor disruption. Methods We generated a knock‐in Drosophila model using homologous recombination. We used electrophysiological recordings and calcium‐imaging to assess AP shape, neurotransmission, and the activity of the larval pre‐motor central pattern generator (CPG). We used video‐tracking and automated systems to measure movement, and developed a genetic method to limit BK channel expression to defined circuits. Results Neuronal APs exhibited reduced width and an enhanced afterhyperpolarization in the PxD model. We identified calcium‐dependent reductions in neurotransmitter release, dysfunction of the CPG, and corresponding alterations in movement, in model larvae. Finally, we observed aberrant locomotion and dyskinesia‐like movements in adult model flies, and partially mapped the impact of GOF BK channels on movement to cholinergic neurons. Conclusion Our model supports a link between BK channel GOF and hyperkinetic movements, and provides a platform to dissect the mechanistic basis of PxDs. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Neonicotinoids are the most widely used insecticides in the world and are implicated in the widespread population declines of insects including pollinators. Neonicotinoids target nicotinic acetylcholine receptors which are expressed throughout the insect central nervous system, causing a wide range of sub-lethal effects on non-target insects. Here, we review the potential of the fruit fly Drosophila melanogaster to model the sub-lethal effects of neonicotinoids on pollinators, by utilizing its well-established assays that allow rapid identification and mechanistic characterization of these effects. We compare studies on the effects of neonicotinoids on lethality, reproduction, locomotion, immunity, learning, circadian rhythms and sleep in D. melanogaster and a range of pollinators. We also highlight how the genetic tools available in D. melanogaster, such as GAL4/UAS targeted transgene expression system combined with RNAi lines to any gene in the genome including the different nicotinic acetylcholine receptor subunit genes, are set to elucidate the mechanisms that underlie the sub-lethal effects of these common pesticides. We argue that studying pollinators and D. melanogaster in tandem allows rapid elucidation of mechanisms of action, which translate well from D. melanogaster to pollinators. We focus on the recent identification of novel and important sublethal effects of neonicotinoids on circadian rhythms and sleep. The comparison of effects between D. melanogaster and pollinators and the use of genetic tools to identify mechanisms make a powerful partnership for the future discovery and testing of more specific insecticides.

SummaryGlobally, neonicotinoids are still the most used insecticides, despite their well-documented sub-lethal effects on beneficial insects1. Neonicotinoids are agonists at the nicotinic acetylcholine receptors, the main mediator of synaptic transmission in the insect brain2–5, making them highly potent neurotoxins and insecticides6,7. Memory, circadian rhythmicity and sleep are essential for efficient foraging in many pollinating insects, and involve nicotinic acetylcholine receptor signalling2,4,8–10. The effect of field-relevant concentrations of European Union-banned neonicotinoids: imidacloprid, clothianidin and thiamethoxam, as well as the currently unbanned thiacloprid were tested on Drosophila memory, circadian rhythms and sleep. Field-relevant concentrations of imidacloprid, clothianidin and thiamethoxam disrupted learning, behavioural rhythmicity and sleep whilst thiacloprid exposure only affected sleep. Exposure to imidacloprid and clothianidin directly affected neurophysiology, preventing the day/night remodelling and accumulation of pigment dispersing factor neuropeptide in the dorsal terminals of clock neurons. Knockdown of the neonicotinoid susceptible Dα1 and Dβ2 nicotinic acetylcholine receptor subunits in the mushroom bodies or clock neurons recapitulated the neonicotinoid like deficits in memory or circadian/sleep behaviour demonstrating that neonicotinoid effects are likely mediated in the mushroom body and clock circuitry. Disruption to learning, circadian rhythmicity and sleep are likely to have far-reaching detrimental effects on beneficial insects in the field.

Summary Neonicotinoids have been implicated in the large declines observed in insects such as bumblebees, an important group of pollinators. Neonicotinoids are agonists of nicotinic acetylcholine receptors that are found throughout the insect central nervous system and are the main mediators of synaptic neurotransmission. These receptors are important for the function of the insect central clock and circadian rhythms. The clock allows pollinators to coincide their activity with the availability of floral resources and favorable flight temperatures, as well as impact learning, navigation, and communication. Here we show that exposure to the field-relevant concentration of 10 μg/L imidacloprid caused a reduction in bumblebee foraging activity, locomotion, and foraging rhythmicity. Foragers showed an increase in daytime sleep and an increase in the proportion of activity occurring at night. This could reduce foraging and pollination opportunities, reducing the ability of the colony to grow and reproduce, endangering bee populations and crop yields., Graphical Abstract, Highlights • Field-relevant concentrations of imidacloprid disrupt bumblebee foraging rhythms • Drugged foragers are inactive for more of the day and more active at night • Neonicotinoid-treated foragers are more arrhythmic • These effects occurred in naturalistic 12 h light:12 h dark light cycles, Ecology; Biological Sciences; Zoology; Animal Physiology; Behavioral Neuroscience

BackgroundGenetic and in vitro studies have linked a heterozygous gain-of-function mutation (D434G) in the hSlo1 BK (Big potassium) channel to paroxysmal dyskinesia. However, support for this linkage from in vivo models has been lacking.ObjectivesWe aimed to re-create the equivalent mutation to hSlo1 D434G in the fruit fly, Drosophila, and examine how this mutation altered movement and action potential waveforms.MethodsWe generated a knock-in Drosophila model of hSlo1 D434G. We used video-tracking and infra-red beam-break systems to test whether locomotion was altered in this model, and patch-clamp electrophysiology to determine how the mutation affected action potential waveforms.ResultsWe identified profound motor dysfunction and sporadic leg twitches, as well as a reduced width and an enhancement of the afterhyperpolarization phase of action potentials, in the model background.ConclusionOur results support a conserved relationship between enhanced BK channel function and disrupted motor control across distantly related species.

Essential Tremor (ET) is one of the most common neurological diseases, with an estimated 7 million affected individuals in the US; the pathophysiology of the disorder is poorly understood. Recently, we identified a mutation (KCNS2 (Kv9.2), c.1137 T > A, p.(D379E) in an electrically silent voltage-gated K+ channel α-subunit, Kv9.2, in a family with ET, that modulates the activity of Kv2 channels. We have produced transgenic Drosophila lines that express either the human wild type Kv9.2 (hKv9.2) or the ET causing mutant Kv9.2 (hKv9.2-D379E) subunit in all neurons. We show that the hKv9.2 subunit modulates activity of endogenous Drosophila K+ channel Shab. The mutant hKv9.2-D379E subunit showed significantly higher levels of Shab inactivation and a higher frequency of spontaneous firing rate consistent with neuronal hyperexcitibility. We also observed behavioral manifestations of nervous system dysfunction including effects on night time activity and sleep. This functional data further supports the pathogenicity of the KCNS2 (p.D379E) mutation, consistent with our prior observations including co-segregation with ET in a family, a likely pathogenic change in the channel pore domain and absence from population databases. The Drosophila hKv9.2 transgenic model recapitulates several features of ET and may be employed to advance our understanding of ET disease pathogenesis.

Down syndrome (DS) is caused by triplication of chromosome 21 (HSA21). It is characterised by intellectual disability and impaired motor coordination that arise from changes in brain volume, structure and function. However, the contribution of each HSA21 gene to these various phenotypes and to the causal alterations in neuronal and synaptic structure and function are largely unknown. Here we have investigated the effect of overexpression of the HSA21 gene DSCAM (Down syndrome cell adhesion molecule), on glutamatergic synaptic transmission and motor coordination, using Drosophila expressing three copies of Dscam1. Electrophysiological recordings of miniature and evoked excitatory junction potentials at the glutamatergic neuromuscular junction of Drosophila larvae showed that the extra copy of Dscam1 changed the properties of spontaneous and electrically-evoked transmitter release and strengthened short-term synaptic depression during high-frequency firing of the motor nerve. Behavioural analyses uncovered impaired locomotor coordination despite preserved gross motor function. This work identifies DSCAM as a candidate causative gene in DS that is sufficient to modify synaptic transmission and synaptic plasticity and cause a DS behavioural phenotype., Highlights • Drosophila expressing a third copy of Dscam have altered neuromuscular transmission. • Drosophila expressing a third copy of Dscam have deficits in locomotor coordination. • Drosophila are a powerful system for studying single-gene effects in Down syndrome.

The insect central complex and vertebrate basal ganglia are forebrain centres involved in selection and maintenance of behavioural actions. However, little is known about the formation of the underlying circuits, or how they integrate sensory information for motor actions. Here, we show that paired embryonic neuroblasts generate central complex ring neurons that mediate sensory-motor transformation and action selection in Drosophila. Lineage analysis resolves four ring neuron subtypes, R1-R4, that form GABAergic inhibition circuitry among inhibitory sister cells. Genetic manipulations, together with functional imaging, demonstrate subtype-specific R neurons mediate the selection and maintenance of behavioural activity. A computational model substantiates genetic and behavioural observations suggesting that R neuron circuitry functions as salience detector using competitive inhibition to amplify, maintain or switch between activity states. The resultant gating mechanism translates facilitation, inhibition and disinhibition of behavioural activity as R neuron functions into selection of motor actions and their organisation into action sequences.

Mutations in the dystrobrevin binding protein 1 (DTNBP1) gene that encodes for the dysbindin-1 protein, are associated with a higher risk for schizophrenia. Interestingly, individuals carrying high-risk alleles in this gene have been associated with an increased incidence of negative symptoms for the disease, which include anhedonia, avolition and social withdrawal. Here we evaluated behavioral and neurochemical changes in a hypomorphic Drosophila mutant for the orthologue of human Dysbindin-1, dysb1. Mutant dysb1 flies exhibit altered social space parameters, suggesting asocial behavior, accompanied by reduced olfactory performance. Moreover, dysb1 mutant flies show poor performance in basal and startle-induced locomotor activity. We also report a reduction in serotonin brain levels and changes in the expression of the Drosophila serotonin transporter (dSERT) in dysb1 flies. Our data show that the serotonin-releasing amphetamine derivative 4-methylthioamphetamine (4-MTA) modulates social spacing and locomotion in control flies, suggesting that serotonergic circuits modulate these behaviors. 4-MTA was unable to modify the behavioral deficiencies in mutant flies, which is consistent with the idea that the efficiency of pharmacological agents acting at dSERT depends on functional serotonergic circuits. Thus, our data show that the dysb1 mutant exhibits behavioral deficits that mirror some aspects of the endophenotypes associated with the negative symptoms of schizophrenia. We argue that at least part of the behavioral aspects associated with these symptoms could be explained by a serotonergic deficit. The dysb1 mutant presents an opportunity to study the molecular underpinnings of schizophrenia negative symptoms and reveals new potential targets for treatment of the disease.

The cellular underpinnings of memory deficits in Alzheimer’s disease (AD) are poorly understood. We utilized the tractable neural circuits sub-serving memory in Drosophila to investigate the role of impaired Ca2+ handling in memory deficits caused by expression of human 0N4R isoform of tau which is associated with AD. Expression of tau in mushroom body neuropils, or a subset of mushroom body output neurons, led to impaired memory. By using the Ca2+ reporter GCaMP6f, we observed changes in Ca2+ signaling when tau was expressed in these neurons, an effect that could be blocked by the L-type Ca2+ channel antagonist nimodipine or reversed by RNAi knock-down of the L-type channel gene. The L-type Ca2+ channel itself is required for memory formation, however, RNAi knock-down of the L-type Ca2+ channel in neurons overexpressing human tau resulted in flies whose memory is restored to levels equivalent to wild-type. Expression data suggest that Drosophila L-type Ca2+ channel mRNA levels are increased upon tau expression in neurons, thus contributing to the effects observed on memory and intracellular Ca2+ homeostasis. Together, our Ca2+ imaging and memory experiments suggest that expression of the 0N4R isoform of human tau increases the number of L-type Ca2+ channels in the membrane resulting in changes in neuronal excitability that can be ameliorated by RNAi knockdown or pharmacological blockade of L-type Ca2+ channels. This highlights a role for L-type Ca2+ channels in tauopathy and their potential as a therapeutic target for AD.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by intracellular neurofibrillary tangles of hyperphosphorylated Tau, including the 0N4R isoform and accumulation of extracellular amyloid beta (Aβ) plaques. However, less than 5% of AD cases are familial, with many additional risk factors contributing to AD including aging, lifestyle, the environment and epigenetics. Recent epigenome-wide association studies (EWAS) of AD have identified a number of loci that are differentially methylated in the AD cortex. Indeed, hypermethylation and reduced expression of the Ankyrin 1 ( ANK1) gene in AD has been reported in the cortex in numerous different post-mortem brain cohorts. Little is known about the normal function of ANK1 in the healthy brain, nor the role it may play in AD. We have generated Drosophila models to allow us to functionally characterize Drosophila Ank2, the ortholog of human ANK1 and to determine its interaction with human Tau and Aβ. We show expression of human Tau 0N4R or the oligomerizing Aβ 42 amino acid peptide caused shortened lifespan, degeneration, disrupted movement, memory loss, and decreased excitability of memory neurons with co-expression tending to make the pathology worse. We find that Drosophila with reduced neuronal Ank2 expression have shortened lifespan, reduced locomotion, reduced memory and reduced neuronal excitability similar to flies overexpressing either human Tau 0N4R or Aβ42. Therefore, we show that the mis-expression of Ank2 can drive disease relevant processes and phenocopy some features of AD. Therefore, we propose targeting human ANK1 may have therapeutic potential. This represents the first study to characterize an AD-relevant gene nominated from EWAS.

Alzheimer’s disease (AD) is the most common form of dementia and is characterized by intracellular neurofibrillary tangles of hyperphosphorylated Tau, including the 0N4R isoform and accumulation of extracellular amyloid beta (Aβ) plaques. However, less than 5% of AD cases are familial, with many additional risk factors contributing to AD including aging, lifestyle, the environment and epigenetics. Recent epigenome-wide association studies (EWAS) of AD have identified a number of loci that are differentially methylated in the AD cortex. Indeed, hypermethylation and reduced expression of the Ankyrin 1 (ANK1) gene in AD has been reported in the cortex in numerous different post-mortem brain cohorts. Little is known about the normal function of ANK1 in the healthy brain, nor the role it may play in AD. We have generated Drosophila models to allow us to functionally characterize Drosophila Ank2, the ortholog of human ANK1 and to determine its interaction with human Tau and Aβ. We show expression of human Tau 0N4R or the oligomerizing Aβ 42 amino acid peptide caused shortened lifespan, degeneration, disrupted movement, memory loss, and decreased excitability of memory neurons with co-expression tending to make the pathology worse. We find that Drosophila with reduced neuronal Ank2 expression have shortened lifespan, reduced locomotion, reduced memory and reduced neuronal excitability similar to flies overexpressing either human Tau 0N4R or Aβ42. Therefore, we show that the mis-expression of Ank2 can drive disease relevant processes and phenocopy some features of AD. Therefore, we propose targeting human ANK1 may have therapeutic potential. This represents the first study to characterize an AD-relevant gene nominated from EWAS.

Schizophrenia exhibits up to 80% heritability. A number of genome wide association studies (GWAS) have repeatedly shown common variants in voltage-gated calcium (Cav) channel genes CACNA1C, CACNA1I and CACNA1G have a major contribution to the risk of the disease. More recently, studies using whole exome sequencing have also found that CACNA1B (Cav2.2 N-type) deletions and rare disruptive variants in CACNA1A (Cav2.1 P/Q-type) are associated with schizophrenia. The negative symptoms of schizophrenia include behavioural defects such as impaired memory, sleep and circadian rhythms. It is not known how variants in schizophrenia-associated genes contribute to cognitive and behavioural symptoms, thus hampering the development of treatment for schizophrenia symptoms. In order to address this knowledge gap, we studied behavioural phenotypes in a number of loss of function mutants for the Drosophila ortholog of the Cav2 gene family called cacophony (cac). cac mutants showed several behavioural features including decreased night-time sleep and hyperactivity similar to those reported in human patients. The change in timing of sleep-wake cycles suggested disrupted circadian rhythms, with the loss of night-time sleep being caused by loss of cac just in the circadian clock neurons. These animals also showed a reduction in rhythmic circadian behaviour a phenotype that also could be mapped to the central clock. Furthermore, reduction of cac just in the clock resulted in a lengthening of the 24 h period. In order to understand how loss of Cav2 function may lead to cognitive deficits and underlying cellular pathophysiology we targeted loss of function of cac to the memory centre of the fly, called the mushroom bodies (MB). This manipulation was sufficient to cause reduction in both short- and intermediate-term associative memory. Memory impairment was accompanied by a decrease in Ca2+ transients in response to a depolarizing stimulus, imaged in the MB presynaptic terminals. This work shows loss of cac Cav2 channel function alone causes a number of cognitive and behavioural deficits and underlying reduced neuronal Ca2+ transients, establishing Drosophila as a high-throughput in vivo genetic model to study the Cav channel pathophysiology related to schizophrenia.

Summary Bumblebees are a key pollinator. Understanding the factors that influence the timing of sleep and foraging trips is important for efficient foraging and pollination. Here, we illustrate how individual locomotor activity monitoring and colony-wide radio frequency identification tracking can be combined to analyze the effects of agrochemicals like neonicotinoids on locomotor and foraging rhythmicity and sleep quantity/quality in bumblebees. We also highlight aspects of the design that can be adapted for other invertebrates or agrochemicals, allowing broader application of these techniques. For complete details on the use and execution of this protocol, please refer to Tasman et al. (2020)., Graphical abstract, Highlights • Easy and reliable way of testing circadian rhythmicity and sleep in invertebrates • Covers colony care, equipment adaptation, and setup and experimental protocol • This protocol can be used to study the effects of any water soluble/liquid insecticide • The multiple ways to adapt the protocol for other organisms are highlighted, Bumblebees are a key pollinator. Understanding the factors that influence the timing of sleep and foraging trips is important for efficient foraging and pollination. Here, we illustrate how individual locomotor activity monitoring and colony-wide radio frequency identification tracking can be combined to analyze the effects of agrochemicals like neonicotinoids on locomotor and foraging rhythmicity and sleep quantity/quality in bumblebees. We also highlight aspects of the design that can be adapted for other invertebrates or agrochemicals, allowing broader application of these techniques.

Alzheimer’s disease (AD) is the most common form of dementia and is characterized by the accumulation of extracellular amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles of hyperphosphorylated Tau, including the 4R0N isoform. Recent epigenome-wide association studies (EWAS) of AD have identified a number of loci that are differentially methylated in AD cortex. Indeed, hypermethylation of the Ankyrin 1 (ANK1) gene in AD has been reported in the cortex in numerous different post-mortem brain cohorts. Little is known about the normal function of ANK1 in the healthy brain, nor the role it may play in AD. We have generated Drosophila models to allow us to functionally characterize Drosophila Ank2, the ortholog of human ANK1. These models have targeted reduction in the expression of Ank2 in neurons. We find that Drosophila with reduced neuronal Ank2 expression have shortened lifespan, reduced locomotion, reduced memory and reduced neuronal excitability similar to flies overexpressing either human mutant APP (that leads to Aβ42 production) and MAPT (that leads to 0N4R Tau). Therefore, we show that the mis-expression of Ank2 can drive disease relevant processes and phenocopy some features of AD and we propose targeting ANK1 may have therapeutic potential. This represents the first study to characterize a gene implicated in AD, which was nominated from EWAS.Author summaryThe majority (>95%) of Alzheimer’s disease (AD) cases are sporadic, with their incidence attributed to common genetic mutations, epigenetic variation, aging and the environment. There is no cure for AD and only limited treatment options which only treat the symptoms of AD and only work in some people. Recent epigenome-wide association studies (EWAS) in AD have highlighted hypermethylation of the Ankyrin1 (ANK1) gene in AD cortex. Little is known of the normal role of the gene in the brain. Here, we have demonstrated that Drosophila with reduced neuronal expression of the Drosophila ortholog of human ANK1 (Ank2), can drive AD relevant processes including locomotor difficulties, memory loss and shortened lifespan similar to expression of human amyloid-Beta or tau mutant proteins. Furthermore, increasing Ank2 expression reversed the memory loss caused by expression of human amyloid-Beta or tau mutant proteins, suggesting that targeting ANK1 may have therapeutic potential. This represents the first study to characterize a gene implicated in AD, which was nominated from EWAS.

Down syndrome (DS) is characterised by abnormal cognitive and motor development, and later in life by progressive Alzheimer’s disease (AD)-like dementia, neuropathology, declining motor function and shorter life expectancy. It is caused by trisomy of chromosome 21 (Hsa21), but how individual Hsa21 genes contribute to various aspects of the disorder is incompletely understood. Previous work has demonstrated a role for triplication of the Hsa21 geneDYRK1Ain cognitive and motor deficits, as well as in altered neurogenesis and neurofibrillary degeneration in the DS brain, but its contribution to other DS phenotypes is unclear. Here we demonstrate that overexpression ofminibrain(mnb), theDrosophilaortholog ofDYRK1A, in theDrosophilanervous system accelerated age-dependent decline in motor performance and shortened lifespan. Overexpression ofmnbin the eye was neurotoxic and overexpression in ellipsoid body neurons in the brain caused age-dependent neurodegeneration. At the larval neuromuscular junction, an established model for mammalian central glutamatergic synapses, neuronalmnboverexpression enhanced spontaneous vesicular transmitter release. It also slowed recovery from short-term depression of evoked transmitter release induced by high-frequency nerve stimulation and increased the number of boutons in one of the two glutamatergic motor neurons innervating the muscle. These results provide further insight into the roles ofDYRK1Atriplication in abnormal aging and synaptic dysfunction in DS.Author summaryDown syndrome (DS) is caused by three copies of chromosome 21 instead of the usual two. It is characterised by cognitive and motor deficits, which worsen with age resulting in Alzheimer’s disease (AD). Which genes on chromosome 21 cause these phenotypes is incompletely understood. Here we demonstrate that neuronal overexpression ofminibrain, theDrosophilaortholog of the chromosome 21 geneDYRK1A, causes age-dependent degeneration of brain neurons, accelerates age-dependent decline in motor performance and shortens lifespan. It also modifies presynaptic structure, enhances spontaneous transmitter release and slows recovery from short-term depression of synaptic transmission at a model glutamatergic synapse. These findings give insight into the role ofDYRK1Aoverexpression in aberrant aging and altered information processing in DS and AD.

A central hypothesis for brain evolution is that it might occur via expansion of progenitor cells and subsequent lineage‐dependent formation of neural circuits. Here, we report in vivo amplification and functional integration of lineage‐specific circuitry in Drosophila. Levels of the cell fate determinant Prospero were attenuated in specific brain lineages within a range that expanded not only progenitors but also neuronal progeny, without tumor formation. Resulting supernumerary neural stem cells underwent normal functional transitions, progressed through the temporal patterning cascade, and generated progeny with molecular signatures matching source lineages. Fully differentiated supernumerary gamma‐amino butyric acid (GABA)‐ergic interneurons formed functional connections in the central complex of the adult brain, as revealed by in vivo calcium imaging and open‐field behavioral analysis. Our results show that quantitative control of a single transcription factor is sufficient to tune neuron numbers and clonal circuitry, and provide molecular insight into a likely mechanism of brain evolution.

Summary Mutations in the Golgi SNARE (SNAP [soluble NSF attachment protein] receptor) protein Membrin (encoded by the GOSR2 gene) cause progressive myoclonus epilepsy (PME). Membrin is a ubiquitous and essential protein mediating ER-to-Golgi membrane fusion. Thus, it is unclear how mutations in Membrin result in a disorder restricted to the nervous system. Here, we use a multi-layered strategy to elucidate the consequences of Membrin mutations from protein to neuron. We show that the pathogenic mutations cause partial reductions in SNARE-mediated membrane fusion. Importantly, these alterations were sufficient to profoundly impair dendritic growth in Drosophila models of GOSR2-PME. Furthermore, we show that Membrin mutations cause fragmentation of the presynaptic cytoskeleton coupled with transsynaptic instability and hyperactive neurotransmission. Our study highlights how dendritic growth is vulnerable even to subtle secretory pathway deficits, uncovers a role for Membrin in synaptic function, and provides a comprehensive explanatory basis for genotype-phenotype relationships in GOSR2-PME., Graphical Abstract, Highlights • Epilepsy/ataxia-linked mutations in Membrin confer partial Golgi SNARE defects • Partial reductions in membrane trafficking strongly impair dendritic growth • Synaptic morphology and function tightly depend upon efficient secretory trafficking, In this study, Praschberger et al. utilize in vitro assays, patient-derived cells, and Drosophila models to unravel how mutations in the essential Golgi SNARE protein Membrin cause progressive myoclonus epilepsy and to demonstrate a selective vulnerability of developing neurons to partial impairment of ER-to-Golgi trafficking.

Mutations in proline dehydrogenase (PRODH) are linked to behavioral alterations in schizophrenia and as part of DiGeorge and velo-cardio-facial syndromes, but the role of PRODH in their etiology remains unclear. We here establish a Drosophila model to study the role of PRODH in behavioral disorders. We determine the distribution of the Drosophila PRODH homolog slgA in the brain and show that knock-down and overexpression of human PRODH and slgA in the lateral neurons ventral (LNv) lead to altered aggressive behavior. SlgA acts in an isoform-specific manner and is regulated by casein kinase II (CkII). Our data suggest that these effects are, at least partially, due to effects on mitochondrial function. We thus show that precise regulation of proline metabolism is essential to drive normal behavior and we identify Drosophila aggression as a model behavior relevant for the study of mechanisms impaired in neuropsychiatric disorders. ispartof: Disease Models and Mechanisms vol:10 issue:6 pages:705-716 ispartof: location:England status: published

Alzheimer's disease (AD) is the most common cause of dementia, which is associated with an enormous personal, social and economic burden worldwide. However, there are few current treatments with none of them targeting the underlying causes of the disease. Sleep and circadian rhythm defects are not only distressing symptoms of AD and other tauopathies and are a leading cause of care home admission but are also thought to accelerate AD pathology. Despite this, little is understood about the underlying causes of these behavioural changes. Expression of the 0N4R isoform of tau has been associated with AD pathology and we show that expressing it in the Drosophila clock network gives rise to circadian and sleep phenotypes which closely match the behavioural changes seen in human AD patients. Tauopathic flies exhibited greater locomotor activity throughout the day and night and displayed a loss of sleep, particularly at night. Under constant darkness, the locomotor behaviour of tau-expressing flies was less rhythmic than controls indicating a defect in their intrinsic circadian rhythm. Current clamp recordings from wake-promoting, pigment dispersing factor (PDF)-positive large lateral ventral clock neurons (l-LNvs) revealed elevated spontaneous firing throughout the day and night which likely underlies the observed hyperactive circadian phenotype. Interestingly, expression of tau in only the PDF-positive pacemaker neurons, which are thought to be the most important for behaviour under constant conditions, was not sufficient or even necessary to affect circadian rhythmicity. This work establishes Drosophila as a model to investigate interactions between human pathological versions of tau and the machinery that controls neuronal excitability, allowing the identification of underlying mechanisms of disease that may reveal new therapeutic targets.

Parkinson's disease (PD) is more commonly associated with its motor symptoms and the related degeneration of dopamine (DA) neurons. However, it is becoming increasingly clear that PD patients also display a wide range of non-motor symptoms, including memory deficits and disruptions of their sleep-wake cycles. These have a large impact on their quality of life, and often precede the onset of motor symptoms, but their etiology is poorly understood. The fruit fly Drosophila has already been successfully used to model PD, and has been used extensively to study relevant non-motor behaviours in other contexts, but little attention has yet been paid to modelling non-motor symptoms of PD in this genetically tractable organism. We examined memory performance and circadian rhythms in flies with loss-of-function mutations in two PD genes: PINK1 and parkin. We found learning and memory abnormalities in both mutant genotypes, as well as a weakening of circadian rhythms that is underpinned by electrophysiological changes in clock neurons. Our study paves the way for further work that may help us understand the mechanisms underlying these neglected aspects of PD, thus identifying new targets for treatments to address these non-motor problems specifically and perhaps even to halt disease progression in its prodromal phase., Highlights • Drosophila PINK1 and parkin loss-of-function (LOF) mutants have memory deficits. • Drosophila PINK1 and parkin LOF mutants have weakened circadian rhythms. • Drosophila PINK1 and parkin LOF mutants have clock neuron electrophysiology defects. • Drosophila is a powerful model for studying Parkinson's disease non-motor symptoms.

We have characterized a novel light-input pathway regulating Drosophila clock neuron excitability. The molecular clock drives rhythmic electrica lexcitability of clock neurons and we show that the recently discovered light input factor Quasimodo (Qsm) regulates this variation presumably via a Na+,K+, Cl- co-transporter (NKCC) and the Shaw K+channel (dKV3.1). Due to light-dependent degradation of the clock protein Timeless (Tim), constant illumination (LL) leads to a breakdown of molecular and behavioral rhythms. Both over-expression (OX) and knock-down (RNAi) of qsm, NKCC or Shaw led to robust LL-rhythmicity. Whole-cell recordings of the large ventral lateral neurons (l-LNv) showed that altering Qsm levels reduced the daily variation in neuronal activity: qsmOX led to a constitutive less active, night-like state, and qsmRNAi to a more active, day-like state. Qsm also affected daily changes in K+ currents and the GABAreversal potential, suggesting a role in modifying membrane currents and GABA responses in a daily fashion, potentially modulating light arousal and input to the clock. When directly challenged with blue light, wild-type l-LNvs responded with an increase in firing at night and no net-response during the day, while altering Qsm, NKKC or Shaw levels abolished these day/night differences. Finally, co-expression of ShawOX and NKCCRNAi in a qsm mutant background restored LL-induced behavioral arrhythmicity and wild-type neuronal activity patterns, suggesting that the three genes operate in the same pathway. We propose that Qsm affects both daily and acute light effects in l-LNvs probably acting on Shaw and NKCC.

A sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. Here we show that the Hofbauer-Buchner eyelets differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, we show that the large LNvs interact with subsets of “evening cells” to adjust the timing of the evening peak of activity in a day length-dependent manner. Our work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms. SIGNIFICANCE STATEMENT In animals, circadian clocks have evolved to orchestrate the timing of behavior and metabolism. Consistent timing requires the entrainment these clocks to the solar day, a process that is critical for an organism9s health. Light cycles are the most important external cue for the entrainment of circadian clocks, and the circadian system uses multiple photoreceptors to link timekeeping to the light/dark cycle. How light information from these photorecptors is integrated into the circadian clock neuron network to support entrainment is not understood. Our results establish that input from the HB eyelets differentially impacts the physiology of neuronal subgroups. This input pathway, together with input from the compound eyes, precisely times the activity of flies under long summer days. Our results provide a mechanistic model of light transduction and integration into the circadian system, identifying new and unexpected network motifs within the circadian clock neuron network.

Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day. Synchronized circadian clocks improve fitness and are crucial for our physical and mental well-being. Visual and non-visual photoreceptors are responsible for synchronizing circadian clocks to light, but clock-resetting is also achieved by alternating day and night temperatures with only 2-4 °C difference. This temperature sensitivity is remarkable considering that the circadian clock period (~24 h) is largely independent of surrounding ambient temperatures. Here we show that Drosophila Ionotropic Receptor 25a (IR25a) is required for behavioural synchronization to low-amplitude temperature cycles. This channel is expressed in sensory neurons of internal stretch receptors previously implicated in temperature synchronization of the circadian clock. IR25a is required for temperature-synchronized clock protein oscillations in subsets of central clock neurons. Extracellular leg nerve recordings reveal temperature- and IR25a-dependent sensory responses, and IR25a misexpression confers temperature-dependent firing of heterologous neurons. We propose that IR25a is part of an input pathway to the circadian clock that detects small temperature differences. This pathway operates in the absence of known 'hot' and 'cold' sensors in the Drosophila antenna, revealing the existence of novel periphery-to-brain temperature signalling channels.

Drosophila Shaw encodes a voltage-insensitive, slowly activating, noninactivating K(+) current. The functional and developmental roles of this channel are unknown. In this study, we use a dominant transgenic strategy to investigate Shaw function and describe a second member of the Shaw family, Shawl. In situ hybridization showed that the two Shaw family genes, Shaw and Shawl, have largely nonoverlapping expression patterns in embryos. Shaw is expressed mainly in excitable cells of the CNS and PNS of late embryos. Shawl is expressed in many nonexcitable cell types: ubiquitously in embryos until the germband extends, then transiently in the developing CNS and PNS, becoming restricted to progressively smaller subsets of the CNS. Ectopic full-length and truncated Shaw localize differently within neurons, and produce uneclosed small pupae and adults with unfurled wings and softened cuticle. This phenotype was mapped to the crustacean cardioactive peptide (CCAP)-neuropeptide circuit. Widespread expression of Shaw in the nervous system results in a reduction in body mass, ether-induced shaking, and lethality. Expression of full-length Shaw had more extreme phenotypic consequences and caused earlier lethality than expression of truncated Shaw in a given GAL4 pattern. Whole cell recordings from ventral ganglion motor neurons expressing the truncated Shaw protein suggest that a major role of Shaw channels in these cells is to contribute to the resting potential.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the regulation of neuronal excitability in many systems. Recent studies suggest that local regulation of membrane potential can have important computational consequences for neuronal function. In Drosophila, CaMKII regulates the eag potassium channel, but if and how this regulation was spatially restricted was unknown. Using coimmunoprecipitation from head extracts and in vitro binding assays, we show that CaMKII and Eag form a stable complex and that association with Eag activates CaMKII independently of CaM and autophosphorylation. Ca(2+)/CaM is necessary to initiate binding of CaMKII to Eag but not to sustain association because binding persists when CaM is removed. The Eag CaMKII-binding domain has homology to the CaMKII autoregulatory region, and the constitutively active CaMKII mutant, T287D, binds Eag Ca(2+)-independently in vitro and in vivo. These results favor a model in which the CaMKII-binding domain of Eag displaces the CaMKII autoinhibitory region. Displacement results in autophosphorylation-independent activation of CaMKII which persists even when Ca(2+) levels have gone down. Activity-dependent binding to this potassium channel substrate allows CaMKII to remain locally active even when Ca(2+) levels have dropped, providing a novel mechanism by which CaMKII can regulate excitability locally over long time scales.

The ability of calcium/calmodulin-dependent protein kinase II (CaMKII) to become calcium independent after autophosphorylation makes this enzyme a temporal marker of neuronal activity. Here we show that the calcium-independent form of CaMKII has unique effects on larval viability, locomotion, and neuronal excitability in Drosophila. Expression of constitutively active T287D, but not calcium-dependent T287A, mutant CaMKII in Drosophila neurons resulted in decreased viability, behavioral defects, and failure of action potential propagation. The actions of T287D may be mediated, at least in part, by increased potassium conductances. Expression of T287D CaMKII also stimulated an increase in the number of boutons at the larval neuromuscular junction, but did not affect the mechanics of release. This study defines a role for autophosphorylation of CaMKII in the regulation of multiple neuronal functions including the intrinsic properties of neurons.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes and in flies to measure the contribution of adult plasticity genes. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal. Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory. Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS(+)) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal. A second conditioned stimulus (CS(-)) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Ca(2+)/CaM serine/threonine kinase II (CaMKII) is a central molecule in mechanisms of synaptic plasticity and memory. A vital feature of CaMKII in plasticity is its ability to switch to a calcium (Ca(2+)) independent constitutively active state after autophosphorylation at threonine 287 (T287). A second pair of sites, T306 T307 in the calmodulin (CaM) binding region once autophosphorylated, prevent subsequent CaM binding and inactivates the kinase during synaptic plasticity and memory. Recently a synaptic molecule called Ca(2+)/CaM-dependent serine protein kinase (CASK) has been shown to control both sets of CaMKII autophosphorylation events and hence is well poised to be a key regulator of memory. We show deletion of full length CASK or just its CaMK-like and L27 domains disrupts middle-term memory (MTM) and long-term memory (LTM), with CASK function in the α'/β' subset of mushroom body neurons being required for memory. Likewise directly changing the levels of CaMKII autophosphorylation in these neurons removed MTM and LTM. The requirement of CASK and CaMKII autophosphorylation was not developmental as their manipulation just in the adult α'/β' neurons was sufficient to remove memory. Overexpression of CASK or CaMKII in the α'/β' neurons also occluded MTM and LTM. Overexpression of either Drosophila or human CASK in the α'/β' neurons of the CASK mutant completely rescued memory, confirming that CASK signaling in α'/β' neurons is necessary and sufficient for Drosophila memory formation and that the neuronal function of CASK is conserved between Drosophila and human. At the cellular level CaMKII overexpression in the α'/β' neurons increased activity dependent Ca(2+) responses while reduction of CaMKII decreased it. Likewise reducing CASK or directly expressing a phosphomimetic CaMKII T287D transgene in the α'/β' similarly decreased Ca(2+) signaling. Our results are consistent with CASK regulating CaMKII autophosphorylation in a pathway required for memory formation that involves activity dependent changes in Ca(2+) signaling in the α'/β' neurons.

BackgroundIn addition to the molecular feedback loops, electrical activity has been shown to be important for the function of networks of clock neurons in generating rhythmic behavior. Most studies have used over-expression of foreign channels or pharmacological manipulations that alter membrane excitability. In order to determine the cellular mechanisms that regulate resting membrane potential (RMP) in the native clock of Drosophila we modulated the function of Shaw, a widely expressed neuronal potassium (K(+)) channel known to regulate RMP in Drosophila central neurons.Methodology/principal findingsWe show that Shaw is endogenously expressed in clock neurons. Differential use of clock gene promoters was employed to express a range of transgenes that either increase or decrease Shaw function in different clusters of clock neurons. Under LD conditions, increasing Shaw levels in all clock neurons (LNv, LNd, DN(1), DN(2) and DN(3)), or in subsets of clock neurons (LNd and DNs or DNs alone) increases locomotor activity at night. In free-running conditions these manipulations result in arrhythmic locomotor activity without disruption of the molecular clock. Reducing Shaw in the DN alone caused a dramatic lengthening of the behavioral period. Changing Shaw levels in all clock neurons also disrupts the rhythmic accumulation and levels of Pigment Dispersing Factor (PDF) in the dorsal projections of LNv neurons. However, changing Shaw levels solely in LNv neurons had little effect on locomotor activity or rhythmic accumulation of PDF.Conclusions/significanceBased on our results it is likely that Shaw modulates pacemaker and output neuronal electrical activity that controls circadian locomotor behavior by affecting rhythmic release of PDF. The results support an important role of the DN clock neurons in Shaw-mediated control of circadian behavior. In conclusion, we have demonstrated a central role of Shaw for coordinated and rhythmic output from clock neurons.

CaMKII is critical for structural and functional plasticity. Here we show that Camguk (Cmg), the Drosophila homolog of CASK/Lin-2, associates in an ATP-regulated manner with CaMKII to catalyze formation of a pool of calcium-insensitive CaMKII. In the presence of Ca(2+)/CaM, CaMKII complexed to Cmg can autophosphorylate at T287 and become constitutively active. In the absence of Ca(2+)/CaM, ATP hydrolysis results in phosphorylation of T306 and inactivation of CaMKII. Cmg coexpression suppresses CaMKII activity in transfected cells, and the level of Cmg expression in Drosophila modulates postsynaptic T306 phosphorylation. These results suggest that Cmg, in the presence of Ca(2+)/CaM, can provide a localized source of active kinase. When Ca(2+)/CaM or synaptic activity is low, Cmg promotes inactivating autophosphorylation, producing CaMKII that requires phosphatase to reactivate. This interaction provides a mechanism by which the active postsynaptic pool of CaMKII can be controlled locally to differentiate active and inactive synapses.

SummaryThe ability of CaMKII to act as a molecular switch, becoming Ca2+ independent after activation and autophosphorylation at T287, is critical for experience-dependent plasticity. Here, we show that the Drosophila homolog of CASK, also known as Camguk, can act as a gain controller on the transition to calcium-independence in vivo. Genetic loss of dCASK significantly increases synapse-specific, activity-dependent autophosphorylation of CaMKII T287. In wild-type adult animals, simple and complex sensory stimuli cause region-specific increases in pT287. dCASK-deficient adults have a reduced dynamic range for activity-dependent T287 phosphorylation and have circuit-level defects that result in inappropriate activation of the kinase. dCASK control of the CaMKII switch occurs via its ability to induce autophosphorylation of T306 in the kinase's CaM binding domain. Phosphorylation of T306 blocks Ca2+/CaM binding, lowering the probability of intersubunit T287 phosphorylation, which requires CaM binding to both the substrate and catalytic subunits. dCASK is the first CaMKII-interacting protein other than CaM found to regulate this plasticity-controlling molecular switch.